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CONTRIBUTORS

Numbers in parenthesis indicated the pages on which the authors’ contributions begin.

Nehad M. Alajez (249), Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261 T. Ball (105), Division of Immunopathology, Department of Pathophysiology, University of Vienna, Medical School, A-1090 Vienna, Austria Christophe Benoist (217), Section of Immunology and Immunogenetics, Joslin Diabetes Center, Boston, Massachusetts 02215, and Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts 02115 Shenghe Cai (331), Harvard Medical School, CBR Institute for Biomedical Research, Boston, Massachusetts 02115 Casey A. Carlos (249), Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261 Alvin E. Davis, III (331), Harvard Medical School, CBR Institute for Biomedical Research, Boston, Massachusetts 02115 Anne Durandy (295), INSERM U429, Hoˆpital Necker-Enfants Malades, 75015 Paris, France Olivera J. Finn (249), Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261 Alain Fischer (295), INSERM U429, Hoˆpital Necker-Enfants Malades, 75015 Paris, France M. Focke (105), Division of Immunopathology, Department of Pathophysiology, University of Vienna, Medical School, A-1090 Vienna, Austria Victor Ghetie (155), Cancer Immunobiology Center, University of Texas Southwestern Medical Center, Dallas, Texas 75390 Jessica C. Kettel (249), Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261 D. Kraft (105), Division of Immunopathology, Department of Pathophysiology, University of Vienna, Medical School, A-1090 Vienna, Austria ix

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CONTRIBUTORS

B. Linhart (105), Division of Immunopathology, Department of Pathophysiology, University of Vienna, Medical School, A-1090 Vienna, Austria Dongxu Liu (331), Harvard Medical School, CBR Institute for Biomedical Research, Boston, Massachusetts 02115 Diane Mathis (217), Section of Immunology and Immunogenetics, Joslin Diabetes Center, Boston, Massachusetts 02215, and Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts 02115 Patrick P. McDonald (1), Pulmonary Division, Faculty of Medicine, Universite´ de Sherbrooke, Sherbrooke, Que´ bec JIH 5N4, Canada Paul A. Monach (217), Section of Immunology and Immunogenetics, Joslin Diabetes Center, Boston, Massachusetts 02215, and Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts 02115 N. Mothes (105), Division of Immunopathology, Department of Pathophysiology, University of Vienna, Medical School, A-1090 Vienna, Austria Roald Nezlin (155), Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel V. Niederberger (105), Department of Otorhinolaryngology, University of Vienna, Medical School, A-1090 Vienna, Austria Patrick Revy (295), INSERM U429, Hoˆ pital Necker-Enfants Malades, 75015 Paris, France Jason Rice (49), Molecular Immunology Group, Tenovus Laboratory, Cancer Sciences Division, Southampton University Hospitals Trust, Southampton SO16 6YD, United Kingdom S. Spitzauer (105), Clinical Institute for Medical and Chemical Laboratory Diagnostics, University of Vienna, Medical School, A-1090 Vienna, Austria Freda K. Stevenson (49), Molecular Immunology Group, Tenovus Laboratory, Cancer Sciences Division, Southampton University Hospitals Trust, Southampton SO16 6YD, United Kingdom I. Swoboda (105), Clinical Institute for Medical and Chemical Laboratory Diagnostics, University of Vienna, Medical School, A-1090 Vienna, Austria R. Valenta (105), Division of Immunopathology, Department of Pathophysiology, University of Vienna, Medical School, A-1090 Vienna, Austria Anda M. Vlad (249), Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261 S. Vrtala (105), Division of Immunopathology, Department of Pathophysiology, University of Vienna, Medical School, A-1090 Vienna, Austria

CONTRIBUTORS

xi

K. Westritschnig (105), Division of Immunopathology, Department of Pathophysiology, University of Vienna, Medical School, A-1090 Vienna, Austria Delin Zhu (49), Molecular Immunology Group, Tenovus Laboratory, Cancer Sciences Division, Southampton University Hospitals Trust, Southampton SO16 6YD, United Kingdom

advances in immunology, vol. 82

Transcriptional Regulation in Neutrophils: Teaching Old Cells New Tricks PATRICK P. MCDONALD Pulmonary Division, Faculty of Medicine, Universite´ de Sherbrooke Sherbrooke, Que´bec JIH 5N4, Canada

I. Introduction

Polymorphonuclear neutrophils are by far the most abundant leukocyte population, representing about 60% of all circulating leukocytes. They are terminally differentiated cells in which the apoptotic program is constitutive—a characteristic that accounts for their limited lifespan, estimated to average between 16 and 24 hr in the bloodstream [1]. Neutrophils are best known for their traditional role as professional phagocytes. Because many neutrophils are present everywhere in the microvasculature at any point in time, they can rapidly extravasate and migrate toward an inflammatory focus in response to infection or injury. Under most circumstances, neutrophils are indeed the first leukocytes to infiltrate inflammatory sites, where they accumulate in large numbers, and deploy a battery of responses against invading microorganisms, foreign particles, or cellular debris. Paramount among these functional responses is the removal and destruction of non-self targets by way of phagocytosis and subsequent intracellular digestion. Neutrophils also mount a comprehensive microbicidal response against their targets by generating many oxygen-derived reactive intermediates, and through the massive release of prestored granular components ranging from bactericidal proteins to various lytic enzymes (including lipases, nucleases, phosphatases, various sugardegrading enzymes, and a wide array of proteases) [2]. Finally, activated neutrophils are known to synthesize large quantities of the important inflammatory lipid mediators, leukotriene B4 (a potent neutrophil chemoattractant) and platelet-activating factor (a powerful vasoactive lipid), which together facilitate the extravasation and recruitment of additional waves of granulocytes to inflamed tissues. As a result, neutrophils are rightfully viewed as a formidable first line of defense against pathogenic agents and other immunogenic material. Because of the indisputable aptitude of neutrophils to function as professional phagocytes, the various aspects of neutrophil function relating to this role have been extensively studied, and remain active fields of investigation to this day. An unforeseen consequence of this intense research activity, however, is that it effectively overshadowed another key facet of neutrophil biology, that is, the ability to express such important inflammatory mediators as cytokines, chemokines, adhesion molecules, and so forth. Up to roughly a decade ago, in 1 Copyright 2004, Elsevier Inc. All rights reserved. 0065-2776/04 $35.00

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fact, most immunologists considered neutrophils as cells that are capable of little, if any, gene transcription or protein synthesis. The notoriously short lifespan of the neutrophils probably contributed to this perception of the neutrophil as a one-trick pony. Even early indications of the ability of neutrophils to transcribe genes and synthesize proteins gave the impression that such a process was not quite developed in these cells. For instance, Tiku et al. showed early that neutrophils can release an interleukin (IL)-1-like activity [3], but it was not clear whether this reflected the processing and secretion of preformed cytokine precursor, or the actual de novo generation of IL-1. In another early study, Lindemann and colleagues had shown that in response to granulocyte-macrophage colony-stimulating factor (GM-CSF), neutrophils accumulate tumor necrosis factor (TNF) mRNA, but that no protein is released [4]. Similarly, early reports had demonstrated that neutrophils could inducibly express the mRNA encoding the protooncogene, c-fos, but whether or not this resulted in the formation of a functional protein remained unclear [5–7]. A host of studies performed since the early 1990s, however, were to forever alter this rather simplified view of the neutrophil, by demonstrating that these granulocytes can be induced to express a number of genes whose products lie at the core of inflammatory and immune responses. These include growth factors, cell surface receptors, adhesion molecules, cytokines such as TNF-a, IL-1b, IL-12, and tumor growth factor (TGF)-b, and a wide array of chemokines such as IL-8, Groa, Mip-1a/b, Mip-3a/b, IP-10, MIG, I-TAC, and others (reviewed in [8]). Among these mediators, chemokines are particularly relevant to inflammatory processes because of their ability to selectively recruit discrete cell populations into sites of injury, thereby effectively regulating leukocyte trafficking. As a result, the ability of neutrophils to produce various inflammatory mediators has far-reaching implications. In view of the fact that neutrophils are usually the first blood cells to infiltrate inflamed tissues, and that they vastly outnumber other leukocytes in a variety of chronic inflammatory disorders, their ability to generate various inflammatory cytokines is likely to have a significant impact on the initiation and evolution of inflammatory reactions. Moreover, their ability to produce a plethora of chemokines raises the possibility that neutrophils might actively contribute to the sequential recruitment of distinct leukocyte populations that is typically observed in many, if not most, inflammatory reactions. The production of inflammatory cytokines and chemokines by neutrophils is typically preceded by (and largely dependent upon) an accumulation of the corresponding mRNA transcripts (reviewed in [8]). In the particular case of IL-1b, IL-8, and Mip-1a, inducible gene expression was further shown by us and others to reflect an increased transcriptional activity, as determined in nuclear run-on assays [9–11]. In short, the ability of neutrophils to inducibly transcribe many genes, and to synthesize the corresponding proteins, is no

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longer a matter of debate. In recent years, a growing number of investigators have begun to elucidate some of the regulated transcriptional processes in neutrophils, and though much remains to be learned, significant progress has already been made. The purpose of this review is to provide an overview of the current state of knowledge regarding the issue of transcription factor activation in the context of inducible gene expression in human neutrophils. II. The STAT Family of Transcription Factors

Originally described as interferon-dependent transcription factors, the signal transducers and activators of transcription (STATs) are a family of cytoplasmic proteins that represent a point of convergence for the signaling events triggered by such diverse biological agents as cytokines, growth factors, and hormones [12–14]. As a result, the inducible expression of a number of genes in response to various stimuli is under the partial or full control of the STAT proteins. Seven STAT genes have thus far been identified in mammals; they are termed STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6 [12]. All STAT transcripts have been shown to be regulated by differential splicing, resulting in the generation of various isoforms for each STAT family member [15–21]. Additional variants are generated by posttranslational proteolytic processing, at least in the case of STAT5; similar variants were also postulated in the case of STAT3 [22–24]. Because these spliced or truncated forms generally lack the transactivation domain, they are thought to act as negative regulators of transcription [18,25–28]. This being said, some investigators have proposed that under certain circumstances, spliced or truncated products could preferentially activate a subset of genes that is distinct from those activated by their full-length counterparts [17,25]. In addition, the shorter isoforms may associate with other transcription factors to either enhance or repress transcription in a manner distinct from the corresponding full-length forms [17,23,29]. It is therefore conceivable that cell-specific expression of truncated isoform could be associated with a different pattern of gene expression for those cells [30]. The STATs are latent cytoplasmic proteins, which upon cell stimulation are rapidly recruited to SH2 domains nested within the cytoplasmic tail of cell surface receptors. The STAT proteins subsequently become tyrosine phosphorylated under the action of Janus kinases (JAKs) in the case of cytokine receptors, whereas the phosphorylation step is usually carried out by an intrinsic receptor kinase activity in the case of growth factor receptors [31]. Noteworthy is that JAK activation has also been observed in growth factorstimulated cells, but does not seem to be a prerequisite for STAT activation in response to epidermal growth factor (EGF) or platelet-derived growth factor (PDGF) [12,32,33]. Once they have become tyrosine phosphorylated, STAT proteins can dimerize into various DNA-binding configurations, which differ

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according to the identity of their constituting STAT protein components. For instance, STAT1, STAT3, and STAT5A/5B can all form homo- or heterodimers, whereas it appears that STAT4 and STAT6 can only homodimerize [19]. By comparison, STAT2 does not homodimerize, but rather forms a complex with STAT1 and p48/IRF-9 [34–37]. Following STAT protein phosphorylation and dimerization, the resulting complexes are swiftly translocated to the nucleus, where they can bind specific DNA sequences in the upstream regulatory region of target genes. Binding to these cognate sequences has been reported to occur with varying affinities, depending on each dimer’s composition. For instance, the STAT1–STAT2–p48 complex recognizes a motif termed interferon-stimulated response element (ISRE), whereas many other STAT dimers bind to variants of another motif, known as g-interferon-activated sequences (GAS) [14]. On a final note, it has been reported that many STAT proteins (namely, STAT1, STAT3, STAT4, and STAT5) can become phosphorylated on serine residues (in addition to tyrosine residues) prior to nuclear translocation, and it appears that this phosphorylation could contribute, at least in part, to STAT-driven gene transactivation [13,38]. Finally, signaling through the JAK /STAT cascade is under negative regulation by at least three distinct mechanisms. First, several protein tyrosine phosphatases, including SHP-1, SHP-2, PTP1b, and PTPeC, were shown to inhibit JAK activity, and likewise, STAT activity was reported to be regulated by cytosolic and nuclear phosphatases [39–46]. Another type of negative regulation is that exerted by a family of cytokine-induced immediate-early genes, which encompasses the cytokine-inducible SH2 protein (CIS), and the seven suppressors of cytokine signaling proteins, SOCS1–SOCS7. The genes encoding CIS, SOCS1, SOCS2, and SOCS3 are rapidly induced following cell stimulation (by cytokines, hormones, or growth factors) through the JAK / STAT pathway, and the resulting proteins inhibit the JAK/STAT in a negative feedback loop, thereby terminating signaling [47–50]. This can result from a direct interaction of SOCS with JAKs, in which case the kinase inhibitory domain (KIR) inhibits JAK kinase activity, or from an association with cytokine receptors through the SH2 domain of the SOCS proteins, in which case the access of STATs to the receptor docking sites is blocked [50,51]. A third class of negative regulators of the JAK /STAT pathway is the protein inhibitors of activated STATs (PIAS) family of proteins. The latter can directly interact with activated STAT dimers, and inhibit STAT-mediated gene activation by interfering with the DNA-binding activity of STATs [52–54]. A. Activation of STAT Proteins by G-CSF in Human Neutrophils In neutrophils, the first two studies to address the issue of STAT protein expression and activation were published 5 months apart in 1995–1996. In one of these studies [55], Tweardy and colleagues investigated the signaling events

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triggered by G-CSF—a critical regulator of granulopoiesis and neutrophil maturation [56]. Whole-cell extracts were prepared from neutrophils disrupted by repeated freeze–thaw cycles, and analyzed by electrophoretic mobility shift assay (EMSA) using a human serum-inducible element (hSIE/m67) oligonucleotide probe. A specific complex was found to be induced in G-CSFtreated neutrophils, whose mobility on native gels was similar to that of a STAT3 homodimer present in extracts from Hep-G2 cells [55]. However, the authors were unable to supershift the complex using an antibody raised against the C-terminal (750–769) portion of STAT3, or using antibodies raised against other STAT proteins. This led them to conclude that a novel STAT-like protein must be expressed in granulocytes, which they tentatively designated STAT-G [55]. At about the same time, Bovolenta et al. similarly reported that cytoplasmic extracts prepared from neutrophils disrupted by nitrogen cavitation contain a G-CSF-inducible complex when analyzed in EMSA using an hSIE/m67 probe [57]. In contrast to the previous study, however, the latter group was able to identify the STAT3 as a major constituent of the inducible complex, with STAT1 as a minor participant. Because the authors were interested in elucidating the mechanisms whereby G-CSF induces the expression of FcgRI /CD64, an important player in phagocytosis and antibody-dependent cellular cytotoxicity (ADCC) mediated by activated polymorphonuclear (PMN) cells [58,59], they also performed EMSA analyses using a 39-bp oligonucleotide probe spanning the g-interferon response region (GRR) of the FcgRI/CD64 promoter, which is necessary and sufficient for inducibility [60]. Again, STAT3 and STAT1 were identified as components of the G-CSFinducible GRR-binding complex; in fact, the complex could be entirely supershifted using a combination of anti-STAT3 and anti-STAT1 antibodies. In agreement with these results, Bovolenta et al. further showed that G-CSF treatment of neutrophils resulted in the tyrosine phosphorylation of STAT3, as determined by immunoblot [57]. A subsequent study independently confirmed that STAT3 indeed becomes phosphorylated on both tyrosine and serine residues in response to G-CSF stimulation of neutrophils [61]. While it is now clear that G-CSF can activate STAT3 and STAT1 in neutrophils, it was initially somewhat unsettling that only one of the two groups who originally described the G-CSF-inducible complexes could identify its components, despite using similar antibodies in supershift assays. This discrepancy was briefly discussed by Bovolenta et al., who attributed the difference to the procedures used to prepare neutrophil extracts, namely, nitrogen cavitation versus freeze–thaw cycles. Indeed, nitrogen cavitation had already been shown to represent a gentle, yet efficient way to disrupt human neutrophils while preserving the integrity of intracellular components such as granules and nuclei [62]. Conversely, repeated freeze–thaw cycles was known as an efficient approach to break open the protease-rich neutrophil granules [62,63].

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Consequently, it was likely that disrupting neutrophils in this manner would result in a large-scale release of granular proteases, which could potentially proteolyze STAT proteins to the point that they can no longer be recognized by antibodies. The issue would be resolved 2 years later by two independent studies. In one instance, the Tweardy laboratory published a follow-up to their original article, in which they reported that the G-CSF-inducible hSIEbinding activity could be partially supershifted using anti-STAT3 antibodies directed toward a different part of STAT3 than the one used in their prior study [24]. Accordingly, immunoblot analysis of nuclear extracts from G-CSFtreated neutrophils revealed that the STAT3a protein was now readily detectable; a weaker 72-kDa band was also detected, which they called STAT3g. Importantly, the abundance of the STAT3g band was dramatically increased when the cells were disrupted in the absence of the potent serine protease inhibitor diisopropyl fluorophosphonate (DFP) (as in their previous study). Together, these results strongly indicated that the STAT3 form(s) present in the neutrophil extracts was truncated. Indeed, the authors concluded that STAT3g was derived from STAT3a by limited proteolysis; they also proposed that STAT3g generation occurs as a result of G-CSF stimulation [24]. At the same time as the latter study was published, and in agreement with its findings, we showed that neutrophil disruption by classic means (such as detergent lysis or successive freeze–thaw cycles) often results in the partial degradation of various STAT proteins, even when an elaborate antiprotease cocktail is used, whereas nitrogen cavitation is the only cell disruption procedure that consistently yields intact (i.e., undergraded) STAT proteins in human neutrophils [64]. In the particular case of STAT3, classic cell disruption procedures affected the protein in such a way that it was no longer detected as a single 92-kDa species on SDS–PAGE, but rather as a two distinct bands, including a faster migrating species reminiscent of the one observed by the Tweardy group. Similarly, the G-CSF-inducible hSIE-binding complex migrated faster in EMSA and was partially reactive with some (but not all) anti-STAT3 antibodies. By contrast, every STAT3 antibody tested readily allowed the detection of STAT3 (both in immunoblot and in EMSA) when neutrophils were disrupted by nitrogen cavitation [64]. Thus, much of the early speculation about the identity of the G-CSF-inducible STAT complexes in neutrophils was related to the manner in which the cells were disrupted. In summary, it has become quite clear that G-CSF stimulation of neutrophils leads to the phosphorylation and nuclear recruitment of STAT3 and of STAT1. In addition, it has been suggested that STAT3g, a cleavage product of STAT3a, might also play a role in the context of G-CSF signaling [24]. In this regard, however, it must be pointed out that other investigators who have studied the expression and/or activation of STAT3 in neutrophils did not report the presence of a fast-migrating isoform of STAT3 [61,65,66]. Similarly,

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we reported that only a single STAT3 band of expected molecular size is detected under conditions preventing the solubilization of neutrophil granule proteases [64]. Thus, further evidence will be needed to determine whether STAT3g is specifically generated following G-CSF stimulation of neutrophils, as opposed to being cleaved during cell disruption/lysis. On a final note, it can be envisaged that STAT activation by G-CSF has at least a few consequences in neutrophils. Among them, the inducible expression of CD64 is known to be largely STAT dependent in other leukocytes, at least in response to agents such as interferon-g (IFN-g) [60,67–69], and the rapid accumulation of CD64 transcripts that follows G-CSF-R engagement and STAT activation [57] in neutrophils certainly suggests a role for G-CSF-activated STATs in this process. Similarly, it was recently reported that in human neutrophils, both SOCS-1 and SOCS-3 are rapidly induced by G-CSF, and that at least SOCS-3 negatively regulates STAT protein activation by G-CSF [70]. This is perhaps one of the mechanisms whereby G-CSF-mediated signaling is terminated. Given that the expression of SOCS proteins is known to be largely STAT driven [71], and that in particular SOCS-1 expression by G-CSF is STAT3 dependent in murine myeloid leukemia cells [47], it is conceivable that SOCS induction by G-CSF in human neutrophils similarly involves STAT activation. B. Activation of STAT Proteins by GM-CSF in Human Neutrophils GM-CSF is another growth factor involved in the regulation of leukocytes of granulocytic and monocytic lineage, and is known to exert numerous and diverse actions in mature neutrophils. In this regard, it was first described to act upon neutrophils as a potent priming agent for most of their classic functions (i.e., chemotaxis, degranulation, reactive oxygen intermediate generation, phagocytosis, and lipid mediator synthesis) [72–79]. GM-CSF was later found to stimulate the gene expression of several inflammatory molecules in neutrophils, including chemotactic receptors, phagocytic receptors, cytokines, and related products [including interleukin (IL)-1b, IL-8, and IL-1ra], key components of the leukotriene biosynthetic pathway, and MHC class II molecules [74,80–86]. Another important action exerted by GM-CSF is its inhibition of neutrophil apoptosis, which also appears to involve the onset of gene expression [87–90]. These various effects of GM-CSF naturally prompted some investigators to start elucidating the signaling events involved in GM-CSF-elicited gene activation. First, Brizzi and colleagues reported that JAK2 becomes tyrosine phosphorylated in GM-CSF-stimulated neutrophils [65]. This was accompanied by the rapid phosphorylation of both STAT1 and STAT3 on tyrosine residues, and evidence for a physical interaction of the two STAT proteins with both JAK2 and the b-chain of the GM-CSF-R was also presented [65]. By

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EMSA, Brizzi et al. further observed that GM-CSF promotes the binding of a complex to an hSIE probe, which was almost completely supershifted using antibodies to either STAT1 or STAT3, suggesting that it mostly represents a heterodimer [65]. This said, it is worthy of mention that this GM-CSFinducible neutrophil complex migrated conspicuously faster than an authentic STAT1/STAT3 heterodimer present in nuclear extracts from IL-6-stimulated HepG2 cells. The authors tentatively attributed this fact to a difference in the composition of the STAT1- and STAT3-containing complex between the two cell types. However, it is also possible that in that study, the STAT proteins were partially degraded as a result of disrupting neutrophils by detergent lysis in the presence of relatively few protease inhibitors. Indeed, we later showed that under such conditions, both STAT1 and STAT3 undergo partial proteolysis, but retain the ability to dimerize and bind an hSIE probe in response to neutrophil stimulation, yielding faster migrating complexes in EMSA [64]. This observation notwithstanding, much of the findings of Brizzi et al. would later be reproduced by other investigators in following years. Not unexpectedly, one of the most prolific laboratories investigating GMCSF/neutrophil interactions (and one of the first to document the effects of GM-CSF on neutrophil responses), that of P.H. Naccache, eventually became interested in the issue of GM-CSF nuclear signaling in these cells. In a 1998 paper, Al-Shami and colleagues confirmed that JAK2 activation does take place following GM-CSF stimulation of neutrophils, and further established that among STAT proteins, both STAT5B and STAT3 become tyrosine phosphorylated in response to GM-CSF [66]. Whereas STAT5 phosphorylation represented a strong and sustained response, that of STAT3 appeared to be a more transient phenomenon. By comparison, no increase in STAT1 phosphorylation was observed in response to GM-CSF, in apparent contradiction to the previous observation of Brizzi et al., who had found that GM-CSF induces STAT1 DNA binding in EMSA [65]. A possible explanation is that STAT1 was constitutively phosphorylated in unstimulated cells in the study of Al-Shami et al.—a rather unusual occurrence. This said, another important finding of the study by Al-Shami et al. was that following GM-CSF stimulation, DNA affinity precipitation of neutrophil cellular extracts using the GAS sequence of the FcgRI promoter led to the detection of STAT5B by immunoblot, indicating that the tyrosine-phosphorylated protein could interact with the GAS motif [66]. That STAT5 activation had not been detected in the earlier study of Brizzi et al. probably reflects the fact that the probe that they used in EMSA (hSIE) does not bind STAT5. We independently confirmed that GM-CSF stimulation of neutrophils results in the nuclear mobilization and DNA-binding activity of STAT5, as demonstrated by the finding that neutrophil nuclear extracts contain an inducible complex that binds the GRR of the FcgRI promoter in EMSA,

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and that this complex is completely supershifted by STAT5 antibodies, but not by antibodies to other STAT proteins [64]. By comparison, EMSA analysis of the same extracts using an hSIE probe (which strongly binds STAT1 and STAT3) revealed a GM-CSF-inducible complex that could be efficiently supershifted by anti-STAT3 and (to a lesser extent) by anti-STAT1 antibodies [64] (our unpublished data). Thus, on the basis of the previous studies, it appears that at least three different STAT proteins can be activated by GM-CSF in human neutrophils. More recent studies mostly confirmed (and sometimes extended) our knowledge of the various aspects of STAT protein activation in GM-CSF-treated neutrophils. In one instance, Kuroki et al. confirmed that tyrosine phosphorylation of STAT3 occurs in GM-CSF-stimulated neutrophils, as initially observed by Al-Shami et al., and further showed that STAT3 also becomes phosphorylated on serine residues under the same conditions [61]. Although the significance of STAT3 serine phosphorylation remains poorly understood, it does appear to play a role in transcriptional activation, since mutations of the serine phosphorylation sites within STAT1 and STAT3 were found to impair their ability to drive gene transcription in the U3A cell line [91]. Another recent study described the inducible DNA binding of a shorter form of STAT5 in GM-CSF-treated neutrophils, which could be supershifted using an Nterminal antibody, but not with antibodies to the carboxy-terminus of STAT5; similar results were obtained in IL-5-treated eosinophils [92]. Importantly, incubating nuclear extracts from GM-CSF-treated neutrophils with nuclear extracts from activated HL-60 cells resulted in the degradation of the fulllength STAT5A and STAT5B present in the latter extracts into the truncated form observed in the neutrophil extracts. Moreover, conducting this experiment in the presence of phenylmethylsulfonyl fluoride (PMSF), a general serine protease inhibitor, partially prevented the generation of the truncated form. The authors proposed that granulocyte extracts contain a STAT5-specific serine protease that can process STAT5 into a novel DNA-binding isoform in GM-CSF-treated mature neutrophils, or in IL-5-treated mature eosinophils [92]. While this possibility is conceptually attractive, an alternative explanation is that neutrophil disruption by detergent lysis in the presence of few protease inhibitors is likely to promote the solubilization of granule proteases, which can then cleave STAT5, as demonstrated in our prior study [64]. Indeed, we showed that while full-length STAT5 was detected in nuclear extracts of GM-CSF-treated neutrophils disrupted by nitrogen cavitation, conventional cell disruption procedures yielded a truncated STAT5 form in immunoblot analyses, and a faster migrating native complex that was no longer recognized by C-terminal STAT5 antibodies in EMSA [64]. Thus, although the data of Caldenhoven et al. [92] confirmed that STAT5 is activated in response to

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GM-CSF in neutrophils, it also reemphasizes the need to overcome the action of endogenous proteases when working with neutrophil extracts. On a final note, Epling-Burnette et al. [93] recently confirmed many previous findings relating to the activation of the JAK /STAT pathway by GM-CSF in neutrophils, but also offered new insights by establishing a link between these findings and the effect of GM-CSF on neutrophil survival. On the one hand, they confirmed that GM-CSF induces STAT-containing DNA-binding activities in neutrophils [93]. Using an hSIE probe, the inducible complex could be mostly supershifted by a STAT3 antibody whereas it was moderately affected by an anti-STAT1 antibody. A GM-CSF-inducible complex was also identified using an MGFe probe (which preferentially binds STAT5), and was mostly supershifted with an anti-STAT5B antibody. These authors similarly confirmed that GM-CSF promotes the tyrosine phosphorylation of STAT3, and to a lesser extent, of JAK2 as well. Accordingly, neutrophil pretreatment with AG-490, a selective inhibitor of JAK2 and JAK3 kinase activities, interfered with the GM-CSF-induced STAT3 tyrosine phosphorylation [93]. Importantly, the antiapoptotic effect of GM-CSF in neutrophils was also attenuated by AG-490, as well as by incubating neutrophils in the presence of STAT3 antisense oligonucleotides [93]. The latter findings therefore suggest a role for the JAK /STAT signaling cascade in GM-CSF-mediated neutrophil survival, even though it is clear that additional mechanisms are involved. In summary, the various studies conducted to date have established that in neutrophils, GM-CSF can activate several STAT proteins, as well as the upstream kinase, JAK2. Indeed, STAT1, STAT3, and STAT5 were all described to form nuclear DNA-binding complexes in response to GM-CSF, and accordingly, at least STAT3 and STAT5 were shown to become tyrosine phosphorylated under these conditions. This being said, several lines of evidence indicate that STAT5 might represent the predominant target of GM-CSF action in neutrophils, with STAT3 a close second. First, it was reported that although both STAT3 and STAT5 become tyrosine phosphorylated in response to GM-CSF, the phosphorylation of STAT5 represents a strong and sustained response, whereas that of STAT3 appears to be more transient [66]. By comparison, STAT1 tyrosine phosphorylation in response to GM-CSF has yet to be demonstrated. Second, only STAT5 binding was detected in EMSA using a probe consisting of the GRR of the FcgRI promoter [64], despite the fact that both STAT1 and STAT3 can efficiently bind this sequence [57,94]. This suggests that STAT1 and STAT3 are present in relatively weaker amounts than STAT5 in nuclear extracts from GM-CSF-treated neutrophils. In the case of STAT1, experiments conducted using hSIE probes have made it clear that it is less efficiently induced than STAT3 [64,93]. These considerations notwithstanding, much work remains to be done to determine the actual impact of individual STAT family members on the various

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responses exerted by GM-CSF in neutrophils, and in particular on the induction of gene expression. In this respect, the identification of potential target genes remains far from obvious, as few of the genes induced by GM-CSF are known to be dependent on STAT proteins for activation. To further complicate matters, the up-regulation by GM-CSF of mRNA transcripts often reflects its known ability to dramatically enhance mRNA stability. On the basis of the previous studies, it could be expected that the gene encoding FcgRI /CD64 might represent a likely candidate, since its expression is known to be largely STAT dependent, and since GM-CSF promotes the binding of STAT5 dimers to a GAS sequence in its promoter [64,66]. Despite this, CD64 is not induced by GM-CSF in neutrophils. This is possibly because unlike G-CSF or IFN-g, which activate CD64 expression and which are potent STAT1 inducers, GM-CSF is a relatively poor STAT1 activator in human neutrophils [64,66,93] and monocytes [95]. This said, other potential candidates are intracellular proteins involved in the regulation of apoptosis, in view of the profound effect of GM-CSF on neutrophil survival, and of the apparent role of the JAK/STAT pathway in this phenomenon [93]. In particular, GM-CSF treatment of neutrophils is known to stimulate the mRNA and protein expression of the antiapoptotic protein Mcl-1 [93,96,97], and it was recently shown that a JAK inhibitor (AG-490) could partially block this process [93]. In keeping with this result, it was reported that a serum-inducible element-like motif present in the murine mcl-1 promoter was needed to confer inducibility [98]. Because this motif closely resembles the hSIE, it is likely that it can bind STAT1 and STAT3 with high afinity. This could therefore represent a way in which STAT protein activation by GM-CSF results in the onset of mcl-1 gene expression, should the human mcl-1 promoter share functional similarities with its murine counterpart. Similarly, GM-CSF has been described as rapidly inducing the expression of CIS1 and SOCS-3 in human neutrophils [99], and this process has been shown to require STAT protein activation in other systems [71], including in response to GM-CSF [100,101]. C. Activation of STAT proteins by IFN-g in human neutrophils The pleiotropic cytokine, IFN-g, is a well known STAT activator in many cell types. In neutrophils, IFN-g has been shown to modulate the expression of numerous genes, especially those encoding inflammatory cytokines and chemokines [8]. In addition, IFN-g can also directly induce several neutrophil genes. In particular, IFN-g was shown to rapidly increase FcgRI/CD64 gene expression in neutrophils [102,103], and to a greater extent than G-CSF [57]; this eventually results in a dramatic up-regulation of FcgRI surface expression [102–104]. A similar effect is exerted by IFN-g toward FcgRI in monocytes, and was described as involving the activation of JAK1 and JAK2, as well as the downstream activation of STAT1 [104–108]. These observations initially led us

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to examine whether a similar phenomenon might take place in human neutrophils. Stimulation of neutrophils with IFN-g resulted in the rapid induction of two specific DNA-binding activities to the GRR, a regulatory element within the FcgRI promoter that is required for transcriptional activation. This response preceded the onset of FcgRI gene expression, and involved STAT1 activation, since the IFN-inducible complexes could both be completely supershifted by anti-STAT1 antibodies, but not by antibodies against other STAT family members [94]. In keeping with these results, the complexes could be substantially displaced using an antiphosphotyrosine antibody [94], and the induction by IFN-g of tyrosine-phosphorylated STAT1 was directly observed in a later study [99]. Because the IFN-g-inducible complexes observed in neutrophils comigrated with the ones induced in autologous peripheral blood mononuclear cells (PBMC), it was concluded that the more intense, faster migrating band represents a STAT1 homodimer, whereas the slower one presumably represents a STAT1 tetramer [94]—based on observations made in other experimental systems [109,110]. Together, these results demonstrated that in IFN-g-treated neutrophils, STAT1 is tyrosine phosphorylated and is able to bind the GRR of the FcgRI promoter, as well as the hSIE. Once again, it must be emphasized that the previous data were obtained in large part because neutrophils were disrupted under conditions that prevent the release of intracellular proteases, as we demonstrated in another study published at about the same time. Indeed, classic cell disruption procedures (namely, detergent lysis, sonication, and freeze–thaw cycles) invariably resulted in the loss of the slower migrating STAT1 complex believed to represent a tetramer, and in partially proteolyzed STAT1 homodimers that migrated faster than the authentic STAT1 homodimer observed in human PBMC [64]. Moreover, the truncated STAT1 complex no longer reacted with antibodies raised against the N-terminal region of STAT1 (amino acids 1–194), consistent with the requirement of an intact N-terminus for tetramer formation [109,110]. Similarly, antibodies recognizing the last 10 amino acids of STAT1 did not react with the truncated complex. By contrast, all antibodies efficiently supershifted the STAT1 homodimer in nitrogen-cavitated neutrophils [64]. Finally, the truncated STAT1 protein was also found to migrate faster on SDS–PAGE when neutrophils were disrupted by conventional means, as opposed to nitrogen cavitation [64]. In keeping with the importance of protecting STAT proteins from the action of endogenous neutrophil proteases, the first study to document that an hSIE-binding activity is induced in IFN-g-treated neutrophils reported a complex that migrated much faster than an authentic STAT1 homodimer present in nuclear extracts from IFN-g-treated HepG2 cells [55]. In that study, the neutrophils were disrupted by detergent lysis in the presence of a few protease inhibitors, and accordingly, the IFN-inducible complex failed to react with a C-terminal anti-STAT1 antibody (amino acids

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716–739), and no band corresponding to a STAT1 tetramer was detected either. This effectively prevented the identification of the IFN-inducible complex, which would only become characterized as a STAT1 homodimer 3 years later [64,94]. A recent study has since independently confirmed that IFN-g indeed has the ability to activate STAT1 in neutrophils [93]. The impact of IFN-g-mediated STAT1 activation on inducible gene expression remains to be formally demonstrated in neutrophils. Nevertheless, it is to be expected that the rapid accumulation of FcgRI/CD64 mRNA following neutrophil exposure to IFN-g is due in large part to STAT1 activation by the latter, based on observations made in other inflammatory cells. Similarly, both CIS1 and SOCS-3 are rapidly induced by IFN-g in neutrophils [99], and the IFN-g-elicited induction of CIS/SOCS proteins has been shown to be STAT dependent in other cell types [111,112]. Finally, the induction of several inflammatory cytokines and chemokines (including IL-12, IP-10, MIG, and I-TAC) in neutrophils requires IFN-g as a costimulus [8], and on the basis of studies conducted in other leukocytes, a role for IFN-activated STAT1 (and perhaps STAT2 as well) can be envisaged. D. Activation of STAT Proteins by IL-10 in Human Neutrophils IL-10 is an important immunomodulatory cytokine whose effects are overwhelmingly antiinflammatory. In neutrophils, IL-10 has been shown to downregulate the inducible expression and release of numerous proinflammatory cytokines and chemokines [113–118]; conversely, it has also been demonstrated to up-regulate the production of antiinflammatory mediators such as IL-1ra [114,119]. Up to a few years ago, however, the issue of whether IL-10 could exert direct effects toward neutrophils (as opposed to modulating ongoing responses) was still largely a matter of speculation. In one of the first studies to address this question, we investigated whether IL-10 might activate STAT proteins and thereby induce FcgRI expression in human neutrophils. The underlying rationale was that in monocytes, IL-10 had been shown to stimulate both processes [120–122]. Somewhat disappointingly, we were unable to demonstrate any up-regulation by IL-10 of FcgRI gene and protein expression in neutrophils [94], a finding that was also reported by another group at about the same time [123]. Similarly, we found that IL-10 failed to activate STAT proteins in neutrophils, whereas it readily activated STAT1 and STAT3 in autologous PBMC [94]. The inability of IL-10 to activate STAT proteins in freshly isolated neutrophils was not likely to reflect the absence of IL-10 receptors, especially in view of the various immunomodulatory effects of the cytokine on neutrophil function. Binding experiments revealed that IL-10R was likely expressed on neutrophils [94]. We concluded by speculating that in neutrophils, IL-10 receptors were either insufficiently expressed, or that they lacked cytoplasmic domains required for the recruitment of JAK /STAT

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proteins, to explain the absence of STAT protein activation in response to IL-10. The issue was eventually resolved as a result of the persistent efforts deployed by the laboratory of M.A. Cassatella, who recently demonstrated by FACS analysis that while IL-10R2 is expressed at similar levels on the surface of both neutrophils and monocytes, IL-10RI surface expression is very low in circulating human neutrophils [124]. By comparison, a 4-hr culture of neutrophils resulted in a significantly higher expression of IL-10R1 mRNA and surface protein, an effect that was greatly amplified if 100 ng/ml lipopolysaccharide (LPS) was also included in the culture medium [124]. More importantly, the authors showed that under conditions that were found to enhance surface IL-10RI expression, neutrophil exposure to IL-10 potently promoted the binding of a complex containing both STAT1 and STAT3 to either a GRR probe or an hSIE probe in EMSA [124]. This was also paralleled by the tyrosine phosphorylation of the STAT proteins by IL-10, again in marked contrast to freshly isolated cells. Thus, IL-10 can be an effective STAT inducer in human neutrophils, provided that both chains of its receptor are expressed. The identity of genes that might represent targets for the action of IL-10 through its ability to activate STAT proteins remains largely elusive, and this owes much to the fact that in neutrophils, IL-10 is known to induce only very few genes. For instance, IL-10 was shown to transiently induce SOCS3 gene expression (but not protein synthesis) in the absence of any detectable STAT activation [99], which indicates that this process can proceed independently of STAT proteins, at least in neutrophils. This being said, the ability of IL-10 to induce the expression of SOCS3 was greatly enhanced in cultured neutrophils already expressing the IL-10RI, and this also resulted in the production of the corresponding protein [124]. Thus, sustained SOCS3 expression in IL-10treated neutrophils correlates well with the ability of IL-10 to induce STAT activation in these cells. Although a direct link between these two processes has yet to be demonstrated, similar observations have also been made in other cellular systems [125]. In any instance, a likely consequence of SOCS3 expression is for the protein to participate in the termination of IL-10 signaling. E. Activation of STAT Proteins by Other Agents in Human Neutrophils Various other stimuli have been tested for their ability to promote STAT activation in neutrophils. For instance, the effect of prolactin was investigated in view of its known ability to activate STAT proteins in other leukocyte populations [126,127]. In this respect, Dogusan et al. reported that STAT1 becomes tyrosine phosphorylated upon prolactin stimulation of neutrophils, albeit to a lesser extent than when IFN-g is used [128]. This action of prolactin is paralleled by the tyrosine phosphorylation of JAK2 and STAT5 in human

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PBMC, but not in neutrophils [127,128]. Prolactin treatment of neutrophils was also found to result in the binding of a complex to a GAS probe; while the complex was shown to be specific in competition experiments, its composition remains unknown, as it failed to react with STAT antibodies [128]. A probable reason for this outcome is that a detergent-based kit was used to prepare neutrophil extracts. Despite this, prolactin was shown to rapidly induce the expression of SOCS-2 and IRF-1 in neutrophils, whereas SOCS-3 was not induced [128]. In another study, pituitary growth hormone (GH) was shown to induce the tyrosine phosphorylation of both JAK2 and STAT3 in neutrophils; these effects of GH could be prevented by pretreating the cells with the general tyrosine kinase inhibitor, genistein [129]. Finally, Kuroki et al. have shown that more classic neutrophil agonists such as N-formylated peptides (fMLP), C5a, and phorbol esters (PMA) were all able to induce the phosphorylation of STAT3 on serine, but not on tyrosin residues [61]. The biological significance of STAT3 serine phosphorylation by these stimuli, however, remains poorly understood. F. Concluding Remarks and Future Directions A recurring theme in many of the studies that have investigated STAT activation in neutrophils has been the observation of truncated or unidentifiable STAT proteins and STAT-containing native complexes, which has repeatedly prompted the conclusion that new isoforms must be expressed in neutrophils. In all cases, however, neutrophils were disrupted by classic procedures, which results in the solubilization of neutrophil granule contents [64]. This in turn can be problematic, given that all STAT proteins are susceptible to cleavage by neutrophil proteases (ref. 64 and our unpublished data). For this reason, the question of whether novel STAT isoforms are expressed in neutrophils remains a matter of debate, and strongly emphasizes the paramount importance of shielding neutrophil proteins from proteolytic degradation during cell disruption. Since the adverse effects of neutrophil protease release can be successfully circumvented (and even prevented altogether), future studies can hardly afford to sidestep this issue. Although much has been accomplished since the first descriptions of STAT activation in neutrophils 6 years ago, further studies are needed to extend and deepen our understanding of this process in neutrophils (Table I). First and foremost, the activation of STAT family members other than STAT1/STAT3/ STAT5 needs to be further investigated, especially since all STAT proteins are expressed in these cells (our unpublished data). In this regard, indirect evidence for STAT4 activation by IL-12 in neutrophils was recently reported [130]. On a related issue, it will be interesting to determine which stimulatory conditions lead to the serine phosphorylation of STAT proteins in primary cells such as neutrophils. What we currently know is limited to the observations that

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TABLE I Effect of Various Agonists Toward STAT Activation (Tyrosine Phosphorylation and/or Nuclear DNA Binding) in Human Neutrophils Agonist

STAT1

STAT3

STAT5

References

C5a fMLP G-CSF GM-CSF GH IL-2 IL-10 IFN-a IFN-g PMA Prolactin

nda nd þþ þ nd  þþ  þþþ nd þ

þb þb þþþ þþ þc  þþ þc (þ)d þb nd

nd nd  þþþ nd (þ)d    nd nd

[61] [61] [24,57,61,99,124,249] [61,64–66,92,93,128] [129] [93] [124] [124] [64,93,94,99,124,128,249] [61,99] [128]

a

nd, not determined. Serine phosphorylation only. c Tyrosine phosphorylation only. d Activation of depicted STATs by these agents is controversial and would necessitate independent confirmation. b

STAT3 becomes serine phosphorylated in response to chemoattractants and PMA [61], whereas neither STAT1 nor STAT3 is serine phosphorylated following neutrophil exposure to IFN-g or IL-10 in freshly isolated neutrophils [99]. Other promising research areas include the activation of JAK / Tyk by neutrophil stimuli, and the negative regulation of the JAK / STAT pathway in neutrophils (by SOCS and PIAS proteins, as well as by phosphatases), which has only begun to be investigated. On a final note, it is likely that STAT activation is relevant to neutrophil gene expression. Indeed, several of the genes that are induced in neutrophils are known to depend, at least in part, on STAT proteins for inducibility. These include surface molecules such as the high-affinity IgG receptor, CD64, whose gene is induced by several STAT activators in neutrophils [57,94,99], and the antiapoptotic protein, Mcl-1, which is inducible by GM-CSF [93,96,97]. In addition, most of the stimuli that can activate STAT proteins in neutrophils were also found to induce the expression of CIS/SOCS family members in these cells [70,99,124]. Finally, a number of inflammatory cytokines/chemokines that are known to require STAT activation for their expression can be induced in neutrophils by IFN-g in concert with another stimulus [8], and there is little doubt that other potential STAT-driven genes expressed by activated neutrophils will be identified in future studies. This said, the implication of STAT activation in inducible gene expression still awaits a direct

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demonstration in neutrophils. Because of the importance of STAT proteins in inflammation and immunity, it is likely that selective STAT inhibitors will become available in coming years that will allow researchers to shed more light on this aspect of neutrophil biology. III. The NF-kB Family of Transcription Factors

Since its discovery 15 years ago, the NF-kB transcription factor has emerged as a central regulator of inflammatory and immune processes, given its key involvement in the induction of a multitude of immediate-early genes, including those that encode cytokines and chemokines. This ubiquitously expressed transcription factor consists of a dimer of NF-kB/Rel proteins, a family of related macromolecules that can be subdivided into two groups. The first group includes the products of the Nfkb1 and Nfkb2 genes, which are large precursors (p105 and p100, respectively) of the mature p50 and p52 proteins. The second group encompasses the p65/RelA, c-Rel, and RelB proteins. All of these proteins have the ability to bind DNA through a conserved motif, the Rel homology domain, but only members of the second group feature a transactivation domain [131]. The Rel homology domain also makes it possible for these proteins to homo- and heterodimerize, resulting in multiple variants of the transcription factor, NF-kB [131]. In unstimulated cells, NF-kB dimers are usually complexed to cytoplasmic inhibitor proteins, collectively termed IkB proteins. Upon cell stimulation by a wide variety of agonists, the various intracellular signals that are engendered eventually converge on a multisubunit IkB kinase complex (IKK), which in turn phosphorylates IkB on discrete N-terminal serine residues [132,133]. This is soon followed by the ubiquitination of two nearby lysine residues, a process that is thought to act as a signal for proteolytic degradation by the proteasome pathway [134]. The degradation of IkB unmasks the nuclear localization signal of NF-kB constituent proteins, making it possible for the dimers to enter the nucleus, where they can bind DNA and induce the transcription of target genes. Additionally, the NF-kB constituent proteins themselves are subject to phosphorylation, a process that is required for the full induction of kB-driven genes [135,136]. Among those genes whose expression is most rapidly enhanced in a kB-dependent manner is the one that encodes IkB-a [137,138]. This, in turn, leads to the rapid resynthesis of the inhibitor protein [137,138], of which a portion accumulates in the nucleus where it is able to dissociate NF-kB dimers from DNA, and thereafter retarget NF-kB to the cytoplasm [139]. A few examples of kB-dependent genes are the ones encoding cytokines such as TNF-a, IL-1a/b, IL-1ra, and IL-12; chemokines such as IL-8, Groa, Mip-1b, Mip-3a/b, and IP-10; or surface receptors such as ICAM [140,141].

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Because all of these genes are rapidly expressed by neutrophils in response to stimuli such as LPS or TNF-a [8], which are known to be potent activators of NF-kB in other systems [140], it was only a matter of time before researchers would start investigating the ability of neutrophils to activate NF-kB. A. Expression and Activation of NF-kB Pathway Components in Human Neutrophils The first study to document the presence of NF-kB/Rel proteins in neutrophils was the demonstration that G-CSF could induce the tyrosine phosphorylation of c-Rel in these cells [142]. The authors also showed that following G-CSF treatment, more c-Rel could be recovered from neutrophil cellular extracts by DNA affinity purification. The consequences of this intriguing phenomenon, which has not been further explored, remain unclear to this date. Nevertheless, this early work had the merit of showing that at least one key transcriptional activator was expressed in neutrophils, which were still viewed as little more than short-lived phagocytes specialized in antibacterial defense. Three years later, this view was further challenged by another article, in which we extensively characterized the expression and activation of various components of the NF-kB system in human neutrophils [143]. We first demonstrated that neutrophils express substantial amounts of NFkB1/p50, p65/ RelA, and c-Rel, whereas the p50 precursor, p105, was far less abundant. By contrast, RelB and NFkB2/p52 (or its precursor, p100) were undetectable in neutrophils. In contrast to most other cell types, in which the bulk of NF-kB/ Rel proteins is located in the cytoplasm of unstimulated cells, the NF-kB/Rel proteins expressed in neutrophils were found to be distributed in roughly equal amounts between the cytoplasmic and nuclear fractions [143]—an observation that was independently confirmed at a later date [144]. By immunoprecipitation, we also showed that the three main NF-kB/Rel proteins present in neutrophils form various heterodimers (i.e., p50/RelA, p50/c-Rel, or p65/c-Rel), and that in resting cells all of these combinations (and perhaps homodimers as well) are physically associated with IkB-a [143]. Additionally, the various NF-kB/Rel proteins were found to be fairly stable, as their cellular levels were not diminished after a 3-hr incubation in the presence of cycloheximide. By comparison, the inhibitor molecule IkB-a was found to turn over rapidly, with an estimated half-life of about 60 min [143]. Together, these observations clearly indicated that most of the prerequisites for a functional NF-kB cascade were fulfilled in human neutrophils. Indeed, activation of neutrophils with stimuli such as LPS, TNF-a, or IL-1b leads to the rapid loss (within minutes) of IkB-a and concurrent accumulation of NF-kB/Rel proteins in the nucleus; this is accompanied by a transient induction of NF-kB DNA-binding activity [143]. By supershift analysis, the major inducible complex was shown to contain p50, RelA, and (to a lesser

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extent), c-Rel. Within 20–30 min of stimulation, an accumulation of IkB-a mRNA transcripts was already detectable, and the IkB-a protein was reexpressed shortly thereafter. The latter event correlated temporally with the termination of nuclear NF-kB DNA-binding activity [143]. Other neutrophil agonists were also found to activate NF-kB in these cells—in particular, chemoattractants such as platelet-activating factor (PAF), the formylated tripeptide, f MLP, and leukotriene B4. However, they proved to be weaker NF-kB activators than LPS or TNF-a, and they also elicited a more delayed response [143]. Because all the aforementioned stimuli can induce the expression of IkB-a and of many other kB-dependent genes in neutrophils (in particular, those encoding inflammatory cytokines and chemokines), and in view of their ability to activate NF-kB in these cells, it was proposed that NF-kB activation must underlie their action toward human neutrophil gene expression. Conversely, such neutrophil activators as GM-CSF, G-CSF, IFN-a, IFN-g, IL-8, and IL-10, which by themselves do not induce inflammatory cytokine expression in these cells, were found to lack the ability to activate NF-kB in neutrophils [143]. At about the same time as the latter study was published, Browning et al. reported that f MLP could activate NF-kB in human monocytes, but not in neutrophils, which led them to conclude that the activation of NF-kB by fMLP is cell specific [145]. In fact, these authors failed to detect any NF-kB DNA binding in neutrophils, even after stimulation with LPS or TNF. Because p50 and RelA were barely detected in neutrophil cellular extracts, the observed lack of NF-kB activation was attributed to insufficient amounts of p50 and RelA in these cells [145]. In that study, however, few precautions were taken to counter the action of endogenous neutrophil proteases, as all extracts were prepared by detergent lysis of the cells in a buffer that contained a very low concentration of PMSF as the sole protease inhibitor. In a paper that was published the following year, we showed that in neutrophils, NF-kB dimers as well as individual Rel proteins are indeed undetectable under these conditions, due to extensive proteolytic degradation [64]. This was in fact a general characteristic of neutrophil extracts prepared by conventional cell disruption procedures (i.e., detergent lysis, successive freeze–thaw cycles, or sonication), and strong evidence that the loss of NF-kB/Rel proteins was due to the solubilization of neutrophil proteases was presented. In particular, coincubation of these neutrophil extracts with nuclear extracts from activated Jurkat cells was shown to result in the degradation of authentic NF-kB dimers present in the latter [64]. Noteworthy is that significant degradation occurred even when an elaborate antiprotease cocktail was included in the various buffers. By contrast, disruption of neutrophils by nitrogen cavitation, a procedure that preserves granule integrity [62], allowed the recovery of intact NF-kB proteins and DNA-binding complexes, which comigrated with authentic Rel

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proteins on SDS–PAGE and with genuine NF-kB dimers on native gels, and which were consistently recognized by antibodies directed at various parts of the proteins [64,143]. Thus, the apparent discrepancy between our initial study and that of Browning et al. stemmed from the different cell disruption procedures used, and the consequences thereof on neutrophil NF-kB components. This explanation also helps reconcile a very interesting observation made by Browning and colleagues with our demonstration of fMLP- and TNF-induced NF-kB in neutrophils. Indeed, these authors had observed that as promyeloid HL-60 cells differentiate into granulocytes, they acquire the ability to activate NF-kB in response to f MLP, while their ability to activate NF-kB in response to TNF remains unchanged [145]. This indeed suggests that f MLP can activate NF-kB in mature neutrophils. A number of subsequent studies, by us and other groups, confirmed the ability of neutrophils to activate NF-kB under various experimental conditions. In addition to the stimuli described above, several other agents were described as NF-kB activators in neutrophils. Because neutrophils had been shown to express both the b- and g-subunits of the IL-2/IL-15 receptor complex [146–150], and because IL-2 receptor engagement results in NF-kB activation in several cell types, we investigated the ability of IL-2 and IL-15 to induce NF-kB activation in neutrophils. We reported that the high-affinity IL-15Ra subunit is expressed in neutrophils, and that accordingly, IL-15 elicits several functional responses in these cells, including the production and release of IL-8 [151]. Moreover, IL-15 was shown to transiently enhance a nuclear NF-kB DNA-binding activity (albeit to a lesser extent than LPS), which could be supershifted by antibodies to p50 or RelA. By contrast, IL-2 exerted none of these effects [151], in keeping with the fact that neutrophils express little, if any, high-affinity IL-2Ra subunits on their surface [146,148– 150]. In another study, we investigated whether phagocytic stimuli might also prove to be NF-kB inducers, on the basis that phagocytosis represents an important physiological trigger for the inducible expression of several kBdependent genes in human neutrophils, including those that encode TNF-a, IL-1, and IL-8 [152–154]. In neutrophils undergoing the phagocytosis of IgGopsonized Saccharomyces cerevisiae, a DNA-binding activity primarily consisting of the classic NF-kB heterodimer, p50/RelA, was rapidly induced, and this was mirrored by a decline in immunoreactive IkB-a protein [155]. The cellular IkB-a pool was replenished by 30 min, and by that time, NF-kB binding had returned to near-basal levels. Similar results were obtained in neutrophils engulfing unopsonized yeast particles. We therefore proposed that NF-kB activation could constitute one of the mechanisms whereby the expression of kB-responsive genes is enhanced in phagocytosing neutrophils [155]. In agreement with the previous data, Wakshull et al. reported the activation of NF-kB in neutrophils ingesting PGG-glucan, a glucose homopolymer derived

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from the cell wall of the yeast S. cerevisiae [156]. The inducible DNA-binding complex was shown to be specific, and to consist of p50 and RelA; an anti-p52 antibody did not affect the complex, in agreement with our earlier finding that the p52 protein is undetectable in neutrophils [143]. Activation of NF-kB by PGG was concentration dependent, and could be prevented by pretreating neutrophils with an anti-lactosylceramide antibody [156]. The ability of neutrophils to activate NF-kB in response to phagocytic activity was also observed during the uptake of opsonized Staphylococcus aureus [157]. Interestingly, these authors noted that disrupting neutrophils by detergent lysis resulted in a NF-kB DNA-binding activity that migrated noticeably faster than genuine p50/RelA dimers present in extracts from Wu¨ rzburg T cells, and that could not be supershifted. Mixing the neutrophil and T cell extracts led to the degradation of the complexes present in the latter extracts, indicating the presence of active proteases in the neutrophil extracts. By contrast, when the neutrophil extracts were prepared in the presence of the naturally occurring serine protease inhibitor, a1-antitrypsin, the NF-kB complexes had an almost normal mobility in EMSA, and were now partially reactive with an anti-RelA antibody [157]. These observations are in full agreement with our data showing that failure to counter the action of endogenous granule proteases results in the partial degradation of NF-kB constituents [64]. The issue of NF-kB activation by LPS in neutrophils was revisited by Sugita et al., who noted that this response could vary significantly depending on the origin of the LPS used, and (to a lesser extent) on the presence or absence of serum [158]. Under serum-free conditions, a time- and concentrationdependent activation of NF-kB was observed in response to LPS from Porphyromonas gingivalis 381, a maximal effect being obtained using 500 ng/ml LPS. By comparison, a similar signal was obtained using a 5-fold lesser concentration of LPS from Escherichia coli O111:B4 (which is the same LPS that we had used in our prior study). Thus, not all LPS preparations can activate NF-kB with the same potency. Activation of NF-kB by both types of LPS was enhanced when the cells were stimulated in the presence of autologous serum, even though it must be emphasized that the extent of that increase was of only about 50% [158]. Under these conditions, preincubation of neutrophils with an anti-CD14 antibody diminished by a half the ability of LPS to activate NF-kB; conversely, the antibody had no effect toward this response when the cells were incubated in the absence of serum. This is in keeping with the fact that serum contains a LPS-binding protein, whose interaction with LPS facilitates the ability of the latter to signal through CD14 engagement [159]. On a related topic, Page and co-workers [160] investigated the ability of bacterial LPS to activate transcriptional events compared to that of eosinophil-derived inflammatory proteins that are released in acute asthma settings where neutrophils are also present, and that may

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therefore contribute to neutrophil activation. For this purpose, they used several eosinophil granule proteins such as eosinophil cationic protein, eosinophil-derived neurotoxin, eosinophil peroxidase, and major basic protein (MBP). Among them, none was found to induce NF-kB, despite the fact that MBP could induce IL-8 gene expression and release, to an extent comparable to the effect of LPS from E. coli O55:B5 [160]. MBP instead proved to be a very potent activator of chemokine mRNA stabilization. By contrast, LPS elicited a weak NF-kB DNA-binding activity within 20 min, and maximal induction took place at 60 min [160]. These characteristics of induction differ somewhat from those that we previously observed using LPS from another strain of E. coli (O111:B4), and may reflect differences in the ability of individual strains to activate transcription factors. Finally, two recent studies by Vancurova and colleagues added valuable new insight to our current understanding of the NF-kB system in neutrophils. In one instance, these authors investigated NF-kB activation in neutrophils from newborn and adult donors, and found that TNF induces NF-kB DNA binding more potently in newborn neutrophils [161]. This correlates well with the elevated production of proinflammatory cytokines such as IL-8 and IL-1b previously reported in neonatal neutrophils [162,163]. The TNFinduced NF-kB complexes consisted of a p50 homodimer and of the classic p50/RelA, and confirm our earlier findings made in TNF-treated neutrophils [143,151]. More importantly, Vancurova et al. also demonstrated that therapeutic concentrations of dexamethasone inhibit the induction of the NF-kB complexes by TNF, both in newborn and in adult neutrophils [161]. We observed a similar effect of dexamethasone in neutrophils from adult donors (our unpublished data). In a subsequent study, the same investigators demonstrated that in neutrophils, and in stark contrast to most other cell types, the IkB-a protein is distributed in approximately equal amounts between the cytoplasm and the nucleus [144]. Moreover, stimulation with TNF-a resulted not only in an accumulation of NF-kB/Rel proteins to the nucleus, as previously shown, but also in the degradation of IkB-a in both cellular compartments. This TNF-induced response was partially inhibited by MG-132, a proteasome inhibitor that is also known to inhibit NF-kB [164], as well as by the PKCd inhibitor, rottlerin [144]. B. Regulation of NF-kB Activity by Reactive Oxygen Derivatives in Neutrophils Endogenously generated reactive oxygen intermediates (ROI) have been implicated in the regulation of NF-kB activation in several cellular models [165,166]. Because neutrophils probably produce more ROI than any other cell type, they must be particularly well protected from the adverse effects of ROI, and indeed, neutrophils were shown to resist particularly well to high

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concentrations of exogenous hydrogen peroxide [167]. As a result, it can be expected that the impact of endogenous ROI on transcription factor activation should be minimal in neutrophils. In the first study to characterize calcineurin activity in neutrophils, Carballo et al. observed that the inhibition of calcineurin using cyclosporin A also resulted in the partial inhibition of LPSinduced NF-kB DNA binding [168]. Because previous studies had shown a role for peroxides in the modulation of calcineurin activity in other cell types [169–171], Carballo et al. examined the effect of H2O2 on calcineurin and NF-kB activation in neutrophils. Following pretreatment of the cells with a catalase inhibitor, 3-amino-1,2,4-triazole (AMT), H2O2 dose-dependently inhibited neutrophil calcineurin activity, as well as the LPS-elicited NF-kB activation [168]. The authors concluded that H2O2 exerted its effect on NF-kB through its ability to inhibit calcineurin activity. However, given that calcineurin was unaffected by exogenous H2O2 if endogenous catalase was not deliberately inactivated [168], the same reasoning would imply that under these conditions, H2O2 does not affect NF-kB either. Unfortunately, this was not addressed experimentally. In this regard, however, Vollebregt and colleagues reported that the activation of NF-kB occurring in neutrophils ingesting opsonized S. aureus was unchanged in the presence of various oxidant scavengers (such as exogenous catalase, superoxide dismutase, or methionine), and that accordingly, exogenous H2O2 failed to activate NF-kB [157]. They concluded that in phagocytosing neutrophils, NF-kB activation is independent of endogenous oxidant generation. In full agreement with these results, we also observed that exogenous H2O2 (up to 1 mM) does not induce NF-kB in neutrophils (our unpublished data). While the previous results lend significant support to the idea that endogenous oxidants have little or no impact on NF-kB in neutrophils, a recent study reached the opposite conclusion. Zouki et al. used a FACS-based assay to detect individual transcription factor subunits in nuclei obtained from mixed leukocytes that had been purified following the stimulation of whole blood [172]. By gating on singlet nuclei (which were stated to represent PBMC nuclei) or on doublet nuclei (which were said to represent neutrophil nuclei), this assay reportedly allows us to discriminate between nuclei originating from these leukocyte populations. In this experimental setting, stimulation of whole blood with TNF-a and IL-1b resulted in the nuclear accumulation of RelA in both neutrophils and PBMC [172]. Pretreatment of whole blood with pyrrolidine dithiocarbamate (PDTC), an antioxidant that can function as an NF-kB inhibitor [173], suppressed the cytokine-induced nuclear mobilization of RelA in all leukocyte populations; PDTC similarly inhibited the release of IL-8 in whole blood under the same conditions [172]. Although these results indicate a potential role for endogenous ROI in the activation of NF-kB in neutrophils, some aspects of the FACS procedure make it difficult to conclude unambiguously.

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First, the nature of each of the two populations of nuclei recovered following the lysis of mixed leukocytes was not ascertained. While it is quite obvious that they must represent neutrophils and PBMC, it must be hoped that they are free from cross-contamination. For instance, should some monocyte or lymphocyte nuclei somehow aggregate, then they would no longer behave as singlet nuclei, but rather as ‘‘doublets’’—a widely distributed population that contains the neutrophil nuclei. Any such contamination by PBMC nuclei could be problematic, in view of the fact that the ability of these cells to activate NF-kB dwarfs that of neutrophils [143]. At any rate, this issue would deserve to be further explored. Similarly, a correlation between results obtained using this FACS-based method, on the one hand, and either immunoblot detection of Rel protein nuclear mobilization or EMSA analysis of specific NF-kB binding, on the other hand, has never been established in neutrophils. A more important difficulty in interpretation comes from the fact that all stimulations (and inhibitor pretreaments) were carried out in whole blood, from which leukocytes are later isolated. Thus, it cannot be excluded that the modulation of a given neutrophil function might result from factors that are released by other cells during stimulation in whole blood. This might explain why, for instance, IL-1 was found to be a stronger activator of RelA nuclear movement than TNF following stimulation of whole blood and subsequent analysis of neutrophil nuclei [172], while the opposite is observed when isolated neutrophils are directly stimulated with TNF or IL-1 [143]. More to the point, it is impossible to rule out that the observed nuclear translocation of neutrophil RelA might be due in large part to soluble mediators generated by another redox-sensitive leukocyte population, which are no longer produced in the presence of antioxidants. Indeed, Niwa et al. reported that the TNF-induced NF-kB activation observed in isolated neutrophils was only slightly inhibited following PDTC pretreatment [174]. In conclusion, while it is quite clear that exogenously provided ROI such as H2O2 do not affect NF-kB activation in neutrophils, there remains a certain confusion as to whether endogenous ROI can affect the ability of neutrophils to activate NF-kB. While the available evidence definitely argues against this possibility in phagocytosing cells (which produce far more ROI than TNF- or LPS-treated neutrophils), one of the studies [172] presented previously still leaves room for reasonable doubt. Clearly, further work is required to definitively settle the issue. C. Regulation of NF-kB by Nitrogen-Derived Oxidants in Neutrophils Morphine is an effective immunomodulator, acting in part by stimulating nitric oxide (NO) production in neutrophils, monocytes, and endothelial cells [175,176]. Nitric oxide, in turn, inhibits gene expression in a variety of cells, and was shown to directly impair NF-kB binding in a cell-free system [177]. This is perhaps one of the mechanisms whereby both morphine and NO can

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inhibit such leukocyte functions as adherence, phagocytosis, and motility [178– 180]. These considerations led Welters et al. to investigate the effect of morphine and NO on the ability of neutrophils to activate NF-kB [181]. Whole blood was pretreated with morphine sulfate or the NO donor, Snitroso-N-acetylpencillamine (SNAP), and stimulated with LPS for 30 min, prior to FACS detection of RelA in leukocyte nuclei as described in the previous section. Expectedly, LPS induced the accumulation of nuclear RelA; pretreatment of whole blood with morphine or SNAP inhibited this action of LPS in a dose-dependent manner, and maximal inhibitions of about 30 and 40% were achieved using 50 mM morphine and 1 mM SNAP, respectively [181]. Two NO synthase inhibitors totally blocked this modulatory effect of morphine, thereby confirming that it procedds via NO generation. Thus, it appears that NO can potentially affect NF-kB activation in neutrophils. By comparison, the LPS-induced nuclear mobilization of RelA was abrogated following whole blood pretreatment with the antioxidant N-acetylcysteine. This result suggests that most of the observed effect of LPS toward neutrophils involved the production of oxidants by some of the cells present in whole blood, perhaps even neutrophils themselves. Using the same FACS-based assay to detect nuclear RelA in neutrophils stimulated in whole blood, Zouki et al. reported that peroxynitrite (which results from the reaction of NO with H2O2) may underlie the action of cytokines toward NF-kB activation and IL-8 gene expression in human leukocytes [172]. Specifically, stimulation of whole blood with either TNF or IL-1 resulted in the production of peroxynitrite by all leukocyte populations; expectedly, NO synthase inhibitors blocked this response. The NO synthase inhibitors also suppressed the cytokine-induced IL-8 mRNA expression by mixed leukocytes, and the cytokine-induced accumulation of RelA in both PBMC and neutrophils nuclei. This indicated a role for endogenously generated peroxynitrite in NF-kB activation and in chemokine expression, and accordingly, addition of peroxynitrite to whole blood yielded the same results [172]. In summary, even though the whole blood/FACS approach used in these two studies makes it difficult to determine whether the various stimuli and inhibitors affect neutrophils directly or via other leukocytes (as discussed in the previous section), the previous results do raise the possibility that endogenously generated nitrogen-derived oxidants could participate in the inducible activation of NF-kB in neutrophils. D. Potential Role of NF-kB in Neutrophil Apoptosis A singular characteristic of neutrophils is their huge turnover rate, with an estimated 80 million being released in the bloodstream every minute. This is necessarily offset by an equivalent loss of neutrophils—a reflection of the fact

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that in these terminally differentiated leukocytes, the apoptotic program is constitutively engaged, resulting in a an average lifespan of about 20 hr (though they can last somewhat longer in culture). As a result, spontaneous apoptosis and its modulation by various inflammatory agents have been quite extensively characterized in neutrophils [182]. By contrast, the molecular events underlying this process remain largely undefined. Among several potential pathways that could participate in the regulation of neutrophil apoptosis, NF-kB stands out in view of its key role in the induction of various antiapoptotic cellular proteins, as described in many experimental models [183]. In the first study to address the possible link between NF-kB activation and apoptosis in neutrophils, Ward et al. demonstrated that several distinct NF-kB inhibitors (i.e., curcumin, PDTC, and the proteasome inhibitor, MG-132) increased the constitutive apoptosis of neutrophils after an overnight incubation [184]. A similar effect was exerted by the fungal metabolite, gliotoxin, which had been shown to be a potent and specific inhibitor of NF-kB [185]. This prompted these authors to examine the effect of gliotoxin on a proapoptotic stimulus, TNF-a (which accelerates neutrophil apoptosis at early time points), and on an antiapoptotic stimulus, LPS. At the 2-hr time point, gliotoxin and TNF synergistically increased apoptosis in neutrophils, whereas the survival effect of LPS was completely suppressed by gliotoxin [184]. Because both LPS and TNF are potent NF-kB activators [143], these results raised the possibility that gliotoxin might interfere with the protective effect of NF-kB on apoptosis. Indeed, preincubation of neutrophils with gliotoxin was found to inhibit NF-kB activation by either LPS or TNF [184]. The authors concluded that NF-kB plays a crucial role in regulating the physiological cell death pathway in granulocytes. In subsequent years, other investigators would follow a similar approach, and reach similar conclusions for the most part. For instance, Niwa et al. also observed that TNF induces NF-kB activation, as well as the concomitant degradation of IkB-a, albeit without affecting spontaneous neutrophil apoptosis at early time points (i.e., within 2 hr of incubation) [174]. Nevertheless, by interfering with the ability of TNF to induce NF-kB, the proapoptotic effect of TNF became apparent. The authors therefore concluded that the NF-kB pathway is crucial for neutrophil survival against TNF-a cell toxicity. Similarly, Nolan et al. reported that the inhibition of neutrophil apoptosis that occurs after 12–18 hr of incubation with either TNF-a or LPS was no longer observed in cells pretreated with the proteasome inhibitor PSI-I [186]. Pretreatment of neutrophils with PSI-I also abolished the ability of TNF-a and LPS to induce the nuclear translocation of RelA [186]. By contrast, Dunican et al. reported that the inhibition of neutrophil apoptosis observed following an overnight incubation in the presence of TNF-a was only slightly affected (by

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about 10%) by prior treatment of the neutrophils with SN50, a specific inhibitor of NF-kB [187]. While this result is in apparent contrast to the findings of other groups, it must be stressed that no data were presented to confirm that SN50 did in fact prevent the activation of NF-kB by TNF in their experiments. Finally, Ward et al. recently examined the effect of prostaglandin D2 and of its metabolites on neutrophil apoptosis. Among the latter, both D12 PGJ2 and 15dPGJ2 were found to be proapoptotic in neutrophils, and to suppress the LPS-elicited inhibition of neutrophil apoptosis [188]. Moreover, both lipids were able to inhibit the LPS-induced degradation of IkB-a in neutrophils. Collectively, these various studies indicate that NF-kB is likely to play a protective role against the onset of neutrophil apoptosis. E. Concluding Remarks and Future Directions Whereas NF-kB activation (and the immediately related events) is a process that has been relatively well characterized in neutrophils stimulated by a wide array of agonists (Table II), many questions remain and deserve to be further explored. For instance, a role for reactive oxygen intermediates in this process

TABLE II Effect of Various Agonists Toward NF-kB Activation in Human Neutrophils Agonist

Effect

Eosinophil granule proteins fMLP G-CSF (up to 103 U/ml) GM-CSF (up to 3 nM) H2O2 (up to 1 mM) IL-1b IL-2 (up to 103 U/ml) IL-8 (up to 100 nM) IL-10 (up to 300 U/ml) IL-15 IFN-a (up to 103 U/ml) IFN-g (up to 300 U/ml) LPS

 þ    þþ    þþ   þþþ

Leukotriene B4 Platelet-activating factor Phorbol ester TNF-a S. cerevisiae IgG-opsonized yeasts IgG-opsonized S. aureus

þ þ þ þþþ þ þþ þþ

References [160] [143] [143] [143] [157; McDonald, unpublished] [143] [151] [143] [143] [151] [143] [143] [64,143,156–158,160,168,172,181,184,186,188,250] [155] [143] [143] [143] [64,143,144,157,161,174,184,186] [155] [155] [157]

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remains unclear, and the potential involvement of nitrogen-derived oxidants in NF-kB activation is equally uncertain. Another potentially important issue that has remained untackled to this day is that of whether NF-kB constituent proteins become phosphorylated in response to various neutrophil agonists. Yet another area that remains mostly uncharted consists in the elucidation of the signaling steps that take place upstream of IkB degradation and/or NF-kB subunit phosphorylation. In view of the particularities of the NF-kB system (and its activation) in human neutrophils, such investigations are likely to be of broad interest. Finally, the collective evidence gathered to date makes it very likely that NF-kB activation plays an important role in inducible gene expression in neutrophils. A particularly telling example is that many genes encoding inflammatory cytokines and chemokines, which have been shown to be kB dependent in other cells, are induced in neutrophils by stimuli that activate NF-kB in these cells. Despite this, the actual impact of NF-kB on neutrophil gene expression has yet to be directly demonstrated in neutrophils. Conversely, there is good evidence that NF-kB plays a protective role in the context of spontaneous as well as modulated neutrophil apoptosis, but the targets of this NF-kB action have not been identified yet. There is little doubt at this stage that the continuing study of NF-kB activation and function in neutrophils should soon yield substantial advances in these areas of research. IV. Other Transcription Factors Potentially Involved in Neutrophil Activation

In addition to the STAT and NF-kB families, other transcription factors have been reported to be expressed in neutrophils, and others still are likely to be present. They will be briefly covered in this section. A. The Ets Family of Transcription Factors Transcription factors of the Ets family share a DNA-binding domain spanning about 85 amino acids, termed the Ets domain. This allows them to specifically bind to a series of related purine-rich DNA sequences, which regulate the expression of numerous cellular (and even viral) genes. The importance of the Ets family is illustrated by the fact that many such genes are critical for biological processes ranging from cellular proliferation and differentiation to apoptosis [189]. Among Ets family members, PU.1 is expressed specifically in hematopoietic lineages. It binds DNA as a monomer via its C-terminal Ets domain, to the consensus site 50 -AAAG(A /C/G)GGAAG-30 [190]. Phosphorylation of serine 148 in the N-terminal transactivating domain of PU.1 also allows it to interact with transcription factors of the interferonregulated factors (IRF) family, including IRF-4 [191]. Such an interaction makes it possible for the resulting complex to bind composite elements on

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DNA, thereby adding an element of diversity to potential PU.1 targets. The interaction of IRF-4 or IRF-8/ICSBP with PU.1 in monocytic cells was indeed shown to enable transactivation via hybrid DNA sequences [192,193]. An essential role for PU.1 in neutrophil differentiation was demonstrated in mice with a targeted disruption of the PU.1 gene [194]. Such animals are born with no detectable leukocytes, and die of systemic bacterial infection within 2 days of birth. Nevertheless, their lifespan could be prolonged for up to 2 weeks with antibiotic therapy; under these conditions, T lymphocytes and neutrophillike cells appeared in the first week. Subsequent studies by the same group showed that neutrophils from PU.1-null mice do not express the genes encoding receptors for G-CSF and GM-CSF [195], or those encoding numerous secondary granule components, including CD11b, lactoferrin, gelatinase, lysozyme, or the NADPH oxidase component, gp91phox [196]. This is consistent with the observed inability of these neutrophils to mount a respiratory burst, and with their poor efficiency at bacterial killing, relative to normal cells [196]. Thus, while PU.1 is not required for early myeloid lineage or neutrophil commitment, it is essential for the full differentiation and maturation of neutrophils. The apparent role of PU.1 for the expression of numerous granule proteins is consistent with its reported requirement for the inducibility of most of the corresponding genes. High levels of PU.1 mRNA have been shown to be expressed in mature human neutrophils, whereas another highly related Ets family protein, Spi-B, is not expressed [197]. Accordingly, constitutive binding of PU.1 to its cognate sequence within the CD11b or gp91phox promoters has been shown in EMSA using neutrophil nuclear extracts [197,198]. Although the resulting complex was shown to be specific, it was observed to migrate much faster than the PU.1 complexes present in monocyte or B-lymphocyte nuclear extracts [197,198], and did not react with an antibody raised against the PU.1 N-terminal domain [197]. That another antibody directed to the DNA-binding domain of PU.1 did supershift the complex [197,198], indicated that the PU.1 recovered from the neutrophil extracts was partially degraded. This was confirmed in mixing experiments, in which incubation of in vitro-translated PU.1 with neutrophil extracts resulted in its truncation into the same fast-migrating complex that did not react with an N-terminal antibody [197]. This is consistent with our demonstration that several distinct transcription factors become degraded when neutrophils are disrupted by detergent lysis or other conventional means [64], as in the above studies [197,198]. Thus, PU.1 is expressed in mature neutrophils, and it constitutively binds DNA. Whether this binding can be further induced, or whether it can be modulated at all, remains to be demonstrated. On a final note, it is possible that Ets proteins other than PU.1 are expressed in neutrophils. Indeed, Tsutsumi-Ishii et al. have reported that in nuclear extracts from resting neutrophils, a DNA-binding activity could be

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detected in EMSA using an oligonucleotide containing the Ets-like motif of the a-defensin (or HNP) promoter [199]. This DNA-binding activity was unaffected by an unlabeled PU.1 probe, but partially abrogated by a cold Ets oligonucleotide; the nature of the complex was not further explored. A role for PU.1 in inducible gene expression by mature neutrophils remains highly speculative at this stage. Nevertheless, there exist cases in which such a role is at least conceivable. For instance, PU.1 was shown to play a role in the induction of the gp91phox gene by IFN-g in monocytic cells, by a mechanism that does not increase PU.1 constitutive DNA binding [200,201]. The possibility therefore exists that a similar mechanism might be involved in the upregulation of gp91phox mRNA by IFN-g in neutrophils. Likewise, many others genes containing sites for PU.1 (or for other Ets family members) in their promoter region, and whose expression can be modulated in mature neutrophils, could represent possible targets for PU.1/Ets regulation. Examples include FcgRIb, MHCII, ICAM-1, IL-1b, IL-1ra, TNF-a, MIP-1a, CXCR1 (the IL-8 receptor), and the antiapoptotic protein, Mcl-1 [189]. Finally, several genes encoding proteins formed during granulocytic differentiation have been shown to be under the control of PU.1 These include myeloperoxidase, neutrophil elastase, lysozyme, defensins, lactoferrin [202], and G-CSF and GMCSF receptor subunits [203,204]. However, the expression of most of the latter genes has not been reported to be modulated in mature neutrophils. B. The C/EBP Family of Transcription Factors The CCAAT/enhancer-binding proteins (C/EBP) are transactivators known for their involvement in the regulation of acute phase protein expression [205], as well as that of numerous inflammatory chemokines, including IL-8, ENA78, Groa, and Mip-1a [206–210]. The family consists of several isoforms, including C/EBPa, C/EBPb, C/EBPd, C/EBPe, and CHOP, which can homo- and heterodimerize through their basic leucine zipper region [211], and even heterodimerize with other transcription factors, such as NF-kB [206,212]. Activation of the C/EBP proteins involves their phosphorylation in a negative regulatory domain, which is thought to relieve the inhibition of their DNA-binding and transactivation domains [213,214]. While various kinases have been proposed to phosphorylate the C/EBP proteins, ERK1/2 stand out among the most likely candidates [215]. In a manner similar to PU.1, certain C/EBP family members have been described as essential for granulopoiesis and terminal neutrophil differentiation [202]. More specifically, C/EBPa null mice lack neutrophils and eosinophils, but retain monocytes and lymphocytes [216], while C/EBPe null animals show important defects in neutrophil function, including the lack of secondary granules [217,218]. By comparison, mature neutrophils from mice with a targeted disruption of the C/EBPb or C/EBPd genes did not appear to suffer

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from any severe defect [219–221]. In addition to their involvement in neutrophil development and maturation, C/EBP proteins can also be expected to participate in the induction of several genes in mature neutrophils, based on their known ability to regulate the expression of many proinflammatory mediators such as IL-1b, IL-12, IL-8, MIP-1a, MIP-1b, and TNF-a [207,222–226], which can all be induced in neutrophils [8]. This, in turn, begs the question of whether C/EBP proteins are expressed in mature human neutrophils, and various pertinent observations suggest that they probably are. Indeed, it has been reported that in murine granulocytecommitted progenitor cells, C/EBPa, b, and d exist as nuclear proteins [227,228]; nuclear C/EBPa has also been detected in human cells assuming a neutrophilic phenotype (i.e., in dimethylsulfoxide [DMSO]-differentiated HL-60 leukemia cells and in some bone marrow cell subsets) [228]. In addition, C/EBPe mRNA is greatly induced during the in vitro granulocytic differentiation of human primary CD34þ cells or of HL-60 cells [229]. While suggestive, these findings will nonetheless have to be confirmed in mature, terminally differentiated human neutrophils. In this respect, it was recently reported that neither C/EBPa nor C/EBPb proteins are detectable in neutrophil nuclear extracts, be it in resting cells or following stimulation with either TNF-a or LPS [199]. Accordingly, no DNA binding to a C/EBP probe is detected in EMSA under the same conditions [160,199]. However, it must be pointed out that these extracts were prepared following cell disruption by detergent lysis in the absence of elastase-class protease inhibitors, a procedure that results in the proteolysis of many neutrophil transcription factors [64]. Thus, it cannot be ruled out that the absence of detectable nuclear C/EBP proteins might have been consequent to proteolytic degradation. Clearly, more studies are required to determine if C/EBP transcription factors are expressed and functional in neutrophils. C. The AP-1 Family of Transcription Factors The AP-1 transcription factor typically consists of combinations of Jun and Fos family proteins, which bind as dimers to a common enhancer sequence on gene promoters. Members of the Jun family of proteins (c-Jun, JunB, JunD) have the ability to homo- and heterodimerize among themselves, or to dimerize with Fos family proteins, resulting in multiple AP-1 variants [230]. By contrast, Fos family proteins (c-Fos, FosB, Fra-1, Fra-2) must associate with Jun proteins to form AP-1 complexes [230]. Dimerization occurs via a basic leucine zipper domain within the Jun/Fos proteins, which also makes it possible for them to associate with members of different transcription factor families, such as NF-kB/Rel proteins [231], ATF/CREB proteins [232,233], and a host of others [234,235]. Most cell types contain some Jun/Fos proteins, and upon cell stimulation, their genes are actively transcribed—an immediate-early

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process that usually yields more Jun/Fos proteins, which are then available to form DNA-binding dimers in the nucleus. Posttranslational modification also plays a key role with regard to AP-1 function. Phosphorylation of c-Jun by c-Jun N-terminal kinase (JNK) and of c-Fos by for Fyn-related kinase (FRK) has been shown to greatly potentiate the transactivation potential of AP-1 complexes [236–238]. Similarly, it has been recently reported that JNKs can phosphorylate JunD and thereby enhance its ability to activate transcription [239], even though JunD was observed to bind JNK with much lower affinity than c-Jun. Several inflammatory mediators generated by activated neutrophils are known to contain AP-1 elements within their upstream regulatory region that are either essential or needed for full promoter activity. A partial list of these mediators includes TNF, IL-1, IL-1ra, IL-8, and ICAM [240–244]. Moreover, these mediators are often induced in response to known AP-1 activators in neutrophils. These observations therefore stress the importance of establishing whether AP-1 components are expressed and activable in neutrophils. In this regard, neutrophils have been reported to constitutively express the genes encoding c-Fos, c-Jun, JunB, and JunD [245–247]. The steady-state level of these transcripts can be rapidly up-regulated in response to formyl peptides, GM-CSF, G-CSF, TNF, or phorbol esters in the case of c-Fos [5–7], or in response to LPS, TNF, and phorbol esters in the case of the Jun family members [247,248]. Whether the corresponding proteins are also susceptible to modulation (or whether they are expressed at all), and whether they can form functional AP-1 complexes, remains to be demonstrated in neutrophils. In the latter case, Page et al. observed a moderate increase in AP-1 binding in EMSA when neutrophils were stimulated for 60 min with LPS [160]. Because no supershifts were performed, and because the inducible AP-1 complex was partially displaced by a nonspecific competitor, it is difficult to determine whether the DNA-binding activity was made up of AP-1 dimers or of other constituents. Nevertheless, these results constitute another hint that AP-1 might be activatable in neutrophils. Again, more studies are needed before any conclusion can be drawn. D. Concluding Remarks and Future Directions Our current knowledge of the transcription factors covered in this section in relation to neutrophil activation is fragmentary at best. In the case of the AP-1 and C/EBP families, it seems reasonable to suggest that they may play a role in the expression of many inflammatory cytokines and chemokines that are inducible in neutrophils, and that have already been shown in other cellular models to depend, at least in part, on these factors. By contrast, it would appear that although the PU-1 factor is expressed in neutrophils, its involvement in the regulation of gene expression is less certain, given that most of the PU.1-driven genes are usually expressed during neutrophil differentiation

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and maturation. Nevertheless, there exist circumstances (such as the induction by IFN-g of the gp91phox gene, as mentioned earlier) in which a role for PU.1 cannot be ruled out. Clearly, further investigation is required to start evaluating the potential impact of the previous transcription factors on gene expression in mature neutrophils. V. Conclusion

Progress made in the last decade has made it clear that the traditional representation of neutrophils as professional phagocytes is outdated, and that in addition to this important role, neutrophils can also influence the course of inflammatory and immune reactions through their ability to express genes that encode numerous prominent inflammatory mediators. In fact, transcriptional control of neutrophil gene expression represents a new facet of neutrophil biology that is gradually emerging as a research field in its own right. Despite the substantial advances that have been made, however, much more remains to be learned. One of the most important questions that remains unaddressed is whether transcription factor activation has an impact on inducible gene expression in neutrophils. Even though this is highly probable, only a direct demonstration can satisfactorily settle the issue. Other research areas that will necessitate further investigation in neutrophils include the identification of transcriptional regulators other than NF-kB and STAT proteins, which are either activated or mobilized upon neutrophil stimulation; the possibility that there can be crosstalk or cooperation between transcription factor families; the characterization of the signaling cascades lying upstream of transcription factors; and the elucidation of the mechanisms responsible for terminating transcriptional activation. In a broader perspective, a better understanding of transcriptional regulation in neutrophils could help unveil new pharmacological targets for the treatment of a number of chronic inflammatory conditions in which neutrophils clearly predominate over other cells types, and therefore constitute a likely source of many inflammatory mediators that are typically found in high levels in these conditions. If the strides made in the past 6 years are any indication of future developments in this new field of investigation, then the years ahead should prove quite exciting indeed. Acknowledgments The author would like to thank Mr. Alexandre Cloutier for some preparatory work done toward the completion of this review, and to address special thanks to Dr. Marco Cassatella for his critical reading of the manuscript. P. P. McDonald is a Scholar of the Canadian Institutes for Health Research.

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202. Friedman, A. D. (2002). Transcriptional regulation of granulocyte and monocyte development. Oncogene 21, 3377–3390. 203. Smith, L. T., Hohaus, S., Gonzalez, D. A., Dziennis, S. E., and Tenen, D. G. (1996). PU.1 (Spi-1) and C/EBP alpha regulate the granulocyte colony-stimulating factor receptor promoter in myeloid cells. Blood 88, 1234–1247. 204. Hohaus, S., Petrovick, M. S., Voso, M. T., Sun, Z., Zhang, D. E., and Tenen, D. G. (1995). PU.1 (Spi-1) and C/EBP alpha regulate expression of the granulocyte-macrophage colonystimulating factor receptor alpha gene. Mol. Cell Biol. 15, 5830–5845. 205. Poli, V. (1998). The role of C/EBP isoforms in the control of inflammatory and native immunity functions. J. Biol. Chem. 273, 29279–29282. 206. Stein, B., and Baldwin, A. S., Jr. (1993). Distinct mechanisms for regulation of the interleukin-8 gene involve synergism and cooperativity between C/EBP and NF-kappa B. Mol. Cell Biol. 13, 7191–7198. 207. Matsusaka, T., Fujikawa, K., Nishio, Y., Mukaida, N., Matsushima, K., Kishimoto, T., and Akira, S. (1993). Transcription factors NF-IL6 and NF-kappa B synergistically activate transcription of the inflammatory cytokines, interleukin 6 and interleukin 8. Proc. Natl. Acad. Sci. USA 90, 10193–10197. 208. Grove, M., and Plumb, M. (1993). C/EBP, NF-kappa B, and c-Ets family members and transcriptional regulation of the cell-specific and inducible macrophage inflammatory protein 1 alpha immediate-early gene. Mol. Cell Biol. 13, 5276–5289. 209. Chang, M. S., McNinch, J., Basu, R., and Simonet, S. (1994). Cloning and characterization of the human neutrophil-activating peptide (ENA-78) gene. J. Biol. Chem. 269, 25277–25282. 210. Wood, L. D., and Richmond, A. (1995). Constitutive and cytokine-induced expression of the melanoma growth stimulatory activity/GRO alpha gene requires both NF-kappa B and novel constitutive factors. J. Biol. Chem. 270, 30619–30626. 211. Descombes, P., Chojkier, M., Lichtsteiner, S., Falvey, E., and Schibler, U. (1990). LAP, a novel member of the C/EBP gene family, encodes a liver-enriched transcriptional activator protein. Genes Dev. 4, 1541–1551. 212. LeClair, K. P., Blanar, M. A., and Sharp, P. A. (1992). The p50 subunit of NF-kappa B associates with the NF-IL6 transcription factor. Proc. Natl. Acad. Sci. USA 89, 8145–8149. 213. Kowenz-Leutz, E., Twamley, G., Ansieau, S., and Leutz, A. (1994). Novel mechanism of C/EBP beta (NF-M) transcriptional control: Activation through derepression. Genes Dev. 8, 2781–2791. 214. Twamley-Stein, G., Kowenz-Leutz, E., Ansieau, S., and Leutz, A. (1996). Regulation of C/EBP beta/NF-M activity by kinase oncogenes. Curr. Top Microb. Immunol. 211, 129–136. 215. Nakajima, T., Kinoshita, S., Sasagawa, T., Sasaki, K., Naruto, M., Kishimoto, T., and Akira, S. (1993). Phosphorylation at threonine-235 by a ras-dependent mitogen-activated protein kinase cascade is essential for transcription factor NF-IL6. Proc. Natl. Acad. Sci. USA 90, 2207–2211. 216. Zhang, D. E., Zhang, P., Wang, N. D., Hetherington, C. J., Darlington, G. J., and Tenen, D. G. (1997). Absence of granulocyte colony-stimulating factor signaling and neutrophil development in CCAAT enhancer binding protein alpha-deficient mice. Proc. Natl. Acad. Sci. USA 94, 569–574. 217. Yamanaka, R., Barlow, C., Lekstrom-Himes, J., Castilla, L. H., Liu, P. P., Eckhaus, M., Decker, T., Wynshaw-Boris, A., and Xanthopoulos, K. G. (1997). Impaired granulopoiesis, myelodysplasia, and early lethality in CCAAT/enhancer binding protein epsilon-deficient mice. Proc. Natl. Acad. Sci. USA 94, 13187–13192. 218. Lekstrom-Himes, J. A. (2001). The role of C/EBP(epsilon) in the terminal stages of granulocyte differentiation. Stem Cells 19, 125–133.

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219. Screpanti, I., Romani, L., Musiani, P., Modesti, A., Fattori, E., Lazzaro, D., Sellitto, C., Scarpa, S., Bellavia, D., Lattanzio, G., et al. (1995). Lymphoproliferative disorder and imbalanced T-helper response in C/EBP beta-deficient mice. EMBO J. 14, 1932–1941. 220. Tanaka, T., Akira, S., Yoshida, K., Umemoto, M., Yoneda, Y., Shirafuji, N., Fujiwara, H., Suematsu, S., Yoshida, N., and Kishimoto, T. (1995). Targeted disruption of the NF-IL6 gene discloses its essential role in bacteria killing and tumor cytotoxicity by macrophages. Cell 80, 353–361. 221. Tanaka, T., Yoshida, N., Kishimoto, T., and Akira, S. (1997). Defective adipocyte differentiation in mice lacking the C/EBPbeta and/or C/EBPdelta gene. EMBO J. 16, 7432–7443. 222. Wedel, A., Sulski, G., and Ziegler-Heitbrock, H. W. (1996). CCAAT/enhancer binding protein is involved in the expression of the tumor necrosis factor gene in human monocytes. Cytokine 8, 335–341. 223. Pope, R. M., Leutz, A., and Ness, S. A. (1994). C/EBP beta regulation of the tumor necrosis factor alpha gene. J. Clin. Invest. 94, 1449–1455. 224. Shirakawa, F., Saito, K., Bonagura, C. A., Galson, D. L., Fenton, M. J., Webb, A. C., and Auron, P. E. (1993). The human prointerleukin 1 beta gene requires DNA sequences both proximal and distal to the transcription start site for tissue-specific induction. Mol. Cell Biol. 13, 1332–1344. 225. Plevy, S. E., Gemberling, J. H., Hsu, S., Dorner, A. J., and Smale, S. T. (1997). Multiple control elements mediate activation of the murine and human interleukin 12 p40 promoters: Evidence of functional synergy between C/EBP and Rel proteins. Mol. Cell Biol. 17, 4572–4588. 226. Williams, S. C., Du, Y., Schwartz, R. C., Weiler, S. R., Ortiz, M., Keller, J. R., and Johnson, P. F. (1998). C/EBPepsilon is a myeloid-specific activator of cytokine, chemokine, and macrophage-colony-stimulating factor receptor genes. J. Biol. Chem. 273, 13493–13501. 227. Ford, A. M., Bennett, C. A., Healy, L. E., Towatari, M., Greaves, M. F., and Enver, T. (1996). Regulation of the myeloperoxidase enhancer binding proteins Pu1, C-EBP alpha, -beta, and -delta during granulocyte-lineage specification. Proc. Natl. Acad. Sci. USA 93, 10838–10843. 228. Scott, L. M., Civin, C. I., Rorth, P., and Friedman, A. D. (1992). A novel temporal expression pattern of three C/EBP family members in differentiating myelomonocytic cells. Blood 80, 1725–1735. 229. Yamanaka, R., Kim, G. D., Radomska, H. S., Lekstrom-Himes, J., Smith, L. T., Antonson, P., Tenen, D. G., and Xanthopoulos, K. G. (1997). CCAAT/enhancer binding protein epsilon is preferentially up-regulated during granulocytic differentiation and its functional versatility is determined by alternative use of promoters and differential splicing. Proc. Natl. Acad. Sci. USA 94, 6462–6467. 230. Karin, M., Liu, Z., and Zandi, E. (1997). AP-1 function and regulation. Curr. Opin. Cell Biol. 9, 240–246. 231. Stein, B., Baldwin, A. S., Jr., Ballard, D. W., Greene, W. C., Angel, P., and Herrlich, P. (1993). Cross-coupling of the NF-kappa B p65 and Fos/Jun transcription factors produces potentiated biological function. EMBO J. 12, 3879–3891. 232. Hai, T., and Curran, T. (1991). Cross-family dimerization of transcription factors Fos/Jun and ATF/CREB alters DNA binding specificity. Proc. Natl. Acad. Sci. USA 88, 3720–3724. 233. Kerppola, T. K., and Curran, T. (1993). Selective DNA bending by a variety of bZIP proteins. Mol. Cell Biol. 13, 5479–5489. 234. Mechta-Grigoriou, F., Gerald, D., and Yaniv, M. (2001). The mammalian Jun proteins: Redundancy and specificity. Oncogene 20, 2378–2389. 235. Chinenov, Y., and Kerppola, T. K. (2001). Close encounters of many kinds: Fos-Jun interactions that mediate transcription regulatory specificity. Oncogene 20, 2438–2452.

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advances in immunology, vol. 82

Tumor Vaccines FREDA K. STEVENSON, JASON RICE, AND DELIN ZHU Molecular Immunology Group, Tenovus Laboratory, Cancer Sciences Division Southampton University Hospitals Trust, Southampton SO16 6YD, United Kingdom

I. Introduction

Following the development of a vaccine against smallpox by Edward Jenner, the impact of preventive vaccination against infectious diseases on public health has been clearly demonstrated. The incidence of common infections has fallen dramatically, confirming the effectiveness of this strategy of activating immunity against pathogens. For most infections, the goal of vaccination has been to induce an antibody response, which will remove the invading organism before it has had a chance to enter its favored niche. The situation is different for vaccination against cancer where the tumor cells are already in place. The challenge then is to activate immune pathways capable of attacking tumor cells in situ. While this approach of therapeutic vaccination has rarely been used against infection, it was the setting faced by Louis Pasteur in 1885, who successfully vaccinated individuals infected with rabies virus by injecting spinal cord from an infected rabbit. It is also the situation for patients with chronic viral infections such as human immunodeficiency virus (HIV), where not only is virus already in place, but there is accompanying damage to the immune system. Many parallels can be drawn between infection and cancer, the most obvious being that 10–20% of cancers are known to be associated with infectious organisms (Fig. 1). It is clearly possible to vaccinate against those organisms and thereby prevent the associated cancer. The success of this approach is already evident in Taiwan where vaccination of children against hepatitis B is reducing the incidence of hepatocellular carcinoma (Chang et al., 1997). However, most vaccines against hepatitis B involve the surface antigen, which is relatively simple to prepare, and is now available as a recombinant protein. Vaccines against other cancer-associated organisms are less straightforward, although candidates are emerging. A. Passive Immunotherapy A common approach to treatment of infection and cancer lies in the use of passive immunotherapy, with antibody playing an important role in both these settings. The importance of natural passive antibodies, acquired from maternal transfer, in protecting newborn human babies from infection is clear (Zinkernagel, 2001). The use of passively administered immune serum for 49 Copyright 2004, Elsevier Inc. All rights reserved. 0065-2776/04 $35.00

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Fig 1 Cancers associated with potentially preventable infections.

treatment of infections has a long history, being first developed by Paul Ehrlich for diphtheria and by O. T. Avery for pneumonia. This strategy still has a place for certain infections (Krause, 1999). Treatment of cancer with passive monoclonal antibodies is also finally succeeding, with the therapeutic use of anti-CD20 for patients with lymphoma being a shining example. One reason for the success of anti-CD20 may be that a relatively large dose of antibody was used in the first exploratory tests (Maloney et al., 1994). The importance of administering a large dose of passive antibody, and the requirement for standardization, had been made clear by O. T. Avery in his treatment of pneumonia. Standardization should be more straightforward for monoclonal antibodies, and dose levels remain relevant for cancer. Passive immunotherapy with specific T cells also is gaining a place in the treatment of infectious disease and cancer. For the former, it has been applied mainly to treat immunosuppressed patients at risk of reactivation or infection with Epstein–Barr virus (EBV) or cytomegalovirus (CMV). The clinical setting usually involves patients who have undergone intensive immunosuppressive treatment for leukemia, and who will be receiving an allogeneic transplant from an MHC class I matched donor. In the case of CMV, reactivation can occur during the vulnerable period of immune reconstitution, and adoptive transfer of CD8þ T cells cultured in vitro can provide protection (Riddell and Greenberg, 2000). An alternative approach is to purify the CMV-specific

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CD8þ T cells from the transplant by tetramer selection prior to transfer (Keenan et al., 2001). Similar adoptive immunotherapeutic strategies appear to be effective against EBV, and can suppress growth of EBV-associated posttransplant lymphomas (Rooney et al., 2001). Although the results are encouraging, there are many complex questions surrounding the fate and survival of the transferred effector T cells. A particular question concerns the requirement for CD4þ Th cells to maintain the transferred CD8þ T cell population. Application of adoptive transfer to treat patients with HIV, this time using autologous CD8þ HIV-specific T cell clones expanded in vitro, has revealed a rapid loss of transferred cells (Riddell and Greenberg, 2000). The likely reason for this loss is the deficiency of IL-2-producing recipient CD4þ Th cells, underlining the requirement for this support. Similar questions apply to the passive transfer of T cells aimed against antigens expressed by tumor cells in the recipient. Once again the clinical setting is commonly that of allogeneic transplantation for patients who have undergone high-dose chemotherapy for leukemia. The potential for T cells in the allograft to do more than simply reconstitute immune capacity was revealed by the clinical observation of a graft-versus-leukemia (GvL) effect. The antileukemic activity was particularly evident when recipients in relapse were being treated by donor lymphocyte infusion (DLI). In many cases, DLI led to long-term remission and recovery of the recipients, although the everpresent danger of graft-versus-host (GvH) disease had to be managed. The antigenic target molecules, known as minor histocompatibility antigens, can be expressed at a higher level by the leukemic cells as compared to normal cells, resulting in the desired separation of GvL from GvH. They appear to be polymorphic antigens that differ between donor and recipient and some, such as HA-1 and HA-2, are limited to the hematopoietic system (Goulmy, 1997). There is clear potential for the tactic of transferring specific cellular immunity to patients, in order to combat both infection and cancer. A refinement of the approach is to engineer T cell receptors in the transferred cells to recognize specific target peptides (Stanislawski et al., 2001). Two important general points have been highlighted by the recent observation that transferred CD8þ T cells aimed against melanoma antigens perform optimally both when accompanied by CD4þ T cells and when transfer follows a nonmyeloablative conditioning regimen (Dudley et al., 2002). Creating ‘‘space’’ for transferred cells was also found to be important in a mouse model (Borrello et al., 2000). Cellular therapy, at least for leukemia, has grown out of clinical observation and is now being refined and improved, but it is more technically demanding than antibody therapy and at present not possible to standardize. Although passive immunotherapy can be effective against infection or cancer, monoclonal antibodies alone are unlikely to cure cancer, and the

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potential for adoptively transferred cells remains undetermined. For antibody, combination with drug treatment is one way of increasing efficacy, and many clinical trials of combinatorial strategies are underway for lymphoma and leukemias (Thieblemont and Coiffier, 2002). However, the attraction of active vaccination, with induction of a variety of immune effector pathways, and a memory response able to continually monitor emergent infection or cancer cell growth is obvious. For infection, this is the way both to prevent invasion and to attack resident organisms. For cancer, residual tumor cells remaining after conventional treatment are the major target. It is a curiosity that even while the strategy of antibody therapy has been expanding, it has been assumed by many of those involved in vaccine design that only CD8þ cytotoxic T cells have relevance for removing tumor cells. Clearly, the effector mechanism depends on the molecular nature of the expressed target antigen, and multiple vaccine designs will be required. Dissection of the effector pathways involved in passive therapy will be helpful in designing vaccines to induce appropriate responses. B. Active Vaccination There are several problems facing the use of vaccination as a strategy to treat cancer. The first is that apart from the cancer-associated viral antigens, most tumor antigens are relatively weak immunogens. Many candidates are, in fact, autoantigens, which may be overexpressed by tumor cells, but are evident only at low levels in normal cells. It is a truism that no effective immunity has been activated spontaneously in a patient with a tumor, although low levels of antibody and T cell responses can be measured in some cases (see later). Induction of a natural immune response to tumor antigens is likely to depend on the site of the tumor (Ochsenbein et al., 2001) and on its ability to evade or suppress immune recognition. It is possible that immune control of tumors does occur in some cases and that we see only the failures of this control. This point was made by Burnett who commented that ‘‘if there were tumor immunity, it would be invisible’’ (Burnett, 1957). In human subjects, it is evident for virus-associated tumors such as EBV-associated posttransplant lymphomas, which develop in patients treated with immunosuppressive drugs that lower levels of antiviral cytotoxic T cells. It has always been more difficult to reveal a role for the immune system in tumors not associated with viral infection, but an intriguing set of observations pointing to immune control has been recently summarized (Darnell and Posner, 2003). The tumors include breast, ovarian, and lung malignancies, which are revealed at an early stage by an accompanying paraneoplastic neurological degeneration due to autoimmune attack. Intriguingly, antibodies and cytotoxic T lymphocytes (CTL) against ectopically expressed neuronal antigens on the tumor cells cause the autoimmune manifestations. Importantly the strength of these responses correlates with the

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suppression of tumor growth, offering direct evidence of naturally occurring successful tumor immunity in humans (Darnell and Posner, 2003). In mice, a role for adaptive immunity is indicated by the increased incidence of cancer in aged Rag-2/ knockout models (Shankaran et al., 2001). However, disentangling the relative abilities of the innate and adaptive immune systems to control tumor growth can be difficult. In the double mutant mouse, Rag-2/ STAT1/, which is also deficient in interferon (IFN) signaling, the incidence of carcinomas is increased, but the types of tumor differ from the single Rag-2/ mutant, indicating an influence of innate immunity, possibly mediated by natural killer (NK) cells (Shankaran et al., 2001). Certainly some tumors can downregulate MHC class I molecules, or express stress-associated molecules such as MHC class I chain-related proteins A and B (MICA/B) (human), or Rae1 family members (mouse), which should be recognized by NKG2D-activating receptors on NK or gd T cells (Diefenbach and Raulet, 2002). Knockout mice are a powerful resource to dissect out the role of molecules involved in innate or adaptive immunity in controlling tumor development. However, caution is required before extrapolating to human cancer. Many mouse strains and most murine tumor lines carry retroviruses, and retroviral proteins can act as strong tumor-associated antigens (Huang et al., 1996). Involvement of innate and adaptive immunity in controlling these tumors may differ from immune control of spontaneous human cancer, which, as far as we understand at present, is not commonly associated with retroviruses (Nelson et al., 2003). A further problem for vaccination against cancer is that patients may have a reduced immune capacity, due either to disease or to chemotherapy/radiotherapy. Increasing age per se is associated with a fall in immune capacity (Ershler, 1993), and surgery can induce a transient immunosuppression (Faist et al., 1986; Hensler et al., 1997). Hematological malignancies are a special case since they occupy the immune system and can cause damage. The level of damage from the tumor varies according to its nature and duration. The reduction in normal serum immunoglobulin observed in untreated chronic lymphocytic leukemia (CLL) and myeloma is clear evidence of disease-related effects on antibody production. In CLL this may be due in part to production of growth inhibitory factors such as transforming growth factor-b (TGF-b) by the tumor cells (Lotz et al., 1994). Similar cytokine-mediated effects may operate in myeloma, but patients with non-Hodgkin’s lymphoma appear less immunosuppressed unless treated (King et al., 1979). New treatments of B cell malignancies with antibodies such as anti-CD20, while capable of removing tumor cells, are also likely to affect immune status. Other antibodies such as CamPath1 are even more immunosuppressive (Rebello et al., 2001). Interestingly, anti-CD20 apparently does not affect serum immunoglobulin (lg) levels, but does impair secondary humoral immune responses (van der Kolk et al., 2002). One question for vaccination concerns the period of immune deficit following treatment, and

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whether recovery precedes relapse. The fact that anti-CD20 is now being used in combination with chemotherapy makes assessments of its effect on immunity more difficult. For hematological malignancies, the success of autologous and allogeneic transplantation has facilitated the use of more immunosuppressive regimens in patients. Because the recipients are highly vulnerable to infection after conditioning treatment, there has been concern about the rate of reconstitution of immunity (Singhal and Mehta, 1999), and this has relevance for vaccination as a strategy to prevent resurgence of tumor. Based largely on recovery of antibody responses, the recommended time to vaccinate against infectious organisms in children is 1 year posttransplantation (Singhal and Mehta, 1999). However, investigations in mice suggest that vaccination should be early in order to capture the expanding T cells that are filling the space created by chemotherapy (Borrello et al., 2000). For patients, the level of immunity in the donor, the cell populations transferred, and the conditioning used all influence the rate of recovery of responsiveness. Objective criteria of immune status would be useful to inform the selection of cancer patients for vaccination protocols. However, studies have been generally limited to the practical clinical questions of how to ensure that patients are protected against infection. More subtle questions on the ability of patients to mount CD4þ or CD8þ T cell responses against specific antigen are rarely posed. Perhaps that is not surprising, given that there is no bedrock of information on immune responses in normal human subjects for comparison. In most studies of conventional vaccination against infectious diseases, the effect on the incidence of infection is the primary measure. Since antibody is important in protection, assessment of efficacy has also centered on serum antibody levels, commonly taken at 4–6 weeks following vaccination. There have been few studies of cellular responses in normal adults or in children undergoing routine vaccination, partly because of the technical difficulties involved, but also because the relevance was unclear. The paucity of information on the nature and kinetics of T cell responses in normal subjects undermines our ability to select cancer patients for vaccination. It also creates difficulties for assessing performance of new vaccination protocols. Again, some information is available from vaccination against infectious disease, with one example being clinical testing of a new DNA vaccine against hepatitis B. A surprisingly slow development of immune responses over a period of up to a year was observed (Roy et al., 2000). Whether this was due to the mode of delivery, or reflected a feature of human immune responses remains unclear. However, the fact that comparably slow responses are being observed in monkeys and in clinical trials with a variety of vaccines (Robinson and Pertmer, 2000), suggests that this may be the usual response to vaccination in primates.

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C. Tolerance and Evasion Another potential problem for vaccination against cancer is that of tolerance or evasion of immunity. While the two terms have distinct meanings to immunologists, both processes lead to the same outcome of failure of immune cells to clear tumor. This failure can contribute to tumor progression by allowing local invasion and metastasis. The mechanisms involved in mediating tolerance and evasion are complex and overlapping. Central tolerance reflects deletional loss of responding T cells in the thymus, but in adult mice the efficiency of deletion depends on the avidity of T cells, and may not be complete. Partial tolerance to disparate skin grafts, induced by intrathymic injection of peptide, can, however, be maintained by CD25þ , CD4þ immunoregulatory T cells, demonstrating a multiplicity of tolerizing processes (Trani et al., 2003). Apart from the cancer-associated viral antigens, there are few tumor antigens that can be considered to be new to the immune system. For many, tolerance induction in the thymus may have occurred prior to cancer development. Proteins arising only in the cancer cell include idiotypic determinants expressed by B cell or T cell tumors, mutated protooncogene products, splice variants, and novel peptides generated by chromosomal translocations. Antigens with expression restricted to the testis and the cancer cell, the so-called cancer testis antigens should also have not been exposed to early thymic tolerogenic processes. However, expression by tumor cells during the course of the disease could lead to purging of specific T cells in the residual thymus or in the periphery. This could occur by clonal exhaustion of effector cells similar to that induced by infection with noncytopathic viruses, retroviral superantigens, or bacterial superantigens. The same process may also be involved in tolerance to transplantation antigens (Starzl and Zinkernagel, 1998). For cancer, an important question is whether there has been engagement between the tumor cells and the immune system. If the cancer is localized, and tumor cells do not express immune recognition molecular patterns or stress signals, they may not attract the attention of patrolling dendritic cells, and will therefore fail to reach the lymphoid tissue (Starzl and Zinkernagel, 1998). Under these circumstances, it is unlikely that naı¨ve T cells will be activated. Consequently, tolerance will not result and the T cell repertoire will be retained. Unless this setting is perturbed either by progressive growth, outstripping oxygen or nutrients, or by surgical or radio/chemotherapeutic intervention, these tumors may remain invisible. If so, they could represent the best targets for vaccination. Hematological malignancies occupy the lymphoid system and are therefore exposed, but, even here unless stress proteins or other alerting proteins are expressed and recognized, tumor cells may not be sensed by dendritic cells. For all tumors, changes are likely during progression, and genetic dysregulation should expose new antigenic

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targets. The question is whether these are naturally immunogenic, requiring tumor cells to escape from immunosurveillance (Khong and Restifo, 2002). If tolerance does occur, it may involve deletion, anergy, or regulation of responding T cells (Goodnow, 1997). Transgenic (tg) mouse models are vital in revealing the process of tolerance induction, although each model is necessarily limited to analysis of the fate of a specific Tcell receptor. In a tg mouse expressing antiidiotypic CD4þ T cells, thymic deletion was evident when the level of serum idiotypic immunoglobulin (Id-Ig) was raised to 300 mg/ml (Bogen et al., 1993). It could be concluded from this that patients with myeloma accompanied by a high level of Id-Ig would have deleted the T cell repertoire. However, the data apply only to a single T cell receptor (TCR) of a particular avidity, and leave open the question of the fate of T cells of lower avidity that could be enrolled following vaccination (Bogen, 1996; Bogen et al., 1993). In fact, considerations of tolerance have not stopped the testing of the ability of Id-Ig to activate immunity in patients with myeloma, and T cell responses are being reported (Li et al., 2000; Osterborg et al., 1998). The same is true for follicular lymphoma, where again it might be expected that tumor cells could have deleted the T cell repertoire. In clinical trials of these patients, vaccination with Id-Ig linked to keyhole limpet hemocyanin (KLH) was able to induce anti-Id antibody and proliferative T cells, consistent with retention or regeneration of Id-specific T cells (Hsu et al., 1997). It is certainly possible that tumor in contact with immune cells can prevent induction of immunity via multiple strategies, including low expression of costimulatory molecules, tumor-induced dysfunction of antigen-presenting cells, and secretion of immunosuppressive cytokines (Khong and Restifo, 2002). These blocks could be circumvented by a range of vaccination strategies, particularly if applied in a setting of minimal residual disease. Once immune effector mechanisms are induced, the next problem is that tumor cells may also evade attack. There is highly suggestive evidence that some of the apparent evasion of tumor-infiltrating T cells may simply be due to the fact that there are insufficient numbers of effector T cells (Perez-Diez et al., 2002). This is reminiscent of the situation for antibody therapy where critical levels have to be attained to successfully attack either pathogens or tumor cells (PerezDiez et al., 2002). Tumor cells are dynamic targets, and are capable of evasion, especially if they are given time during the course of development in the host. This is an argument for early treatment, which requires improved methods for detection, together with an increased understanding of tumor behavior. Evasion strategies include down-regulation of MHC class I molecules (Algarra et al., 1997), expression of receptors such as CD94/NKG2A able to inhibit attack by NK or CD8þ cells (Lukacher, 2002), expression of FasL, production of immunosuppressive cytokines, and antiapoptotic mechanisms (reviewed in Khong and Restifo, 2002). Interestingly, NKG2A receptors have been detected in patients on a high proportion of CD8þ T cells infiltrating melanomas and astrocytomas

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(Perrin et al., 2002; Vetter et al., 2000). For tumors associated with g-herpes viruses, such as EBV, a role for viral proteins in tumor development, or in evasion of immune recognition, is strongly indicated (Rickinson and Kieff, 1996). Whether viral immune evasion molecules or cellular analogues contribute to tumor escape from immune effector pathways is not known. However, we showed that introduction of a viral immune evasion gene coding for a chemokine receptor homologue into tumor cells blocked attack by specific CTL in vivo (Rice et al., 2002b). Not only does this illustrate the requirements for CTL movement to tumor sites, but it does point to potential evasion tactics in tumors. There may also be a role for regulatory T cells in suppressing natural or induced immunity against tumor cells. The concept of ‘‘suppressor’’ cells has been resuscitated with the observation that a subset of CD4þ 25þ T cells is involved in maintaining immune homeostasis, and in protecting the host against autoimmune disease (Shevach, 2001). This poorly proliferative population constitutively expresses CD45RO and CD152 (CTLA4), inhibits proliferation of CD4þ 25 and CD8þ T cells, and secretes TGF-b and interleukin (IL)-10 (Shevach, 2001). Several subsets of regulatory T cells with different phenotypes and properties are now being defined (Weiner, 2001). There is evidence in mouse models that regulatory T cells are involved in suppression of natural immunity against transplanted tumors (Golgher et al., 2002; Shimizu et al., 1999; Sutmuller et al., 2001). While their role in modulating tumor immunity in mice is complicated by the fact that natural immunity may be directed against retroviral antigens, the reality of involvement of regulatory T cells in cancer is confirmed by human studies. The prevalence of CD4þ 25þ T cells with suppressor function was found to be increased in the blood of patients with pancreatic and breast adenocarcinoma, and these cells were also present in the tumor microenvironment (Liyanage et al., 2002). Similarly increased numbers of functional CD4þ 25þ T cells have been detected in the blood of patients with other epithelial cancers (Wolf et al., 2003). In one case, it was possible to show that regulatory T cells could inhibit growth of an autologous cytotoxic T cell line specific for tumor cells (Somasundaram et al., 2002). It is interesting that human primary and memory CTL responses can be induced in vitro using either dendritic cells or activated B cells loaded with peptide (von Bergwelt-Baildon et al., 2002). Successful generation of CTL responses against autoantigens such as telomerase suggests that deletional tolerance is not complete. Perhaps surprisingly, the avidity of the CTL was sufficient to kill target telomerase-expressing tumor cells. It appears therefore that conditions in vitro have allowed expansion of the CTL, and it remains unclear which regulatory processes were involved in controlling these cells in vivo. We are now in a position to exploit our knowledge of immunology, and our command of molecular technology, to design strategies to engage the powerful immune system against cancer. As has been found for drug therapy, there will

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be a variety of approaches, depending on the nature and behavior of the cancer. Encouragingly, the immune repertoire appears to be available or restorable, and our task is to deliver tumor antigens in a form that will induce effector pathways able to recognize tumor cells without significant collateral damage to normal cells. The next sections will describe selected strategies that focus on defined target molecules. We will not discuss in any detail the alternative approach of activating immunity against unknown tumor antigens by manipulating tumor cells or by using whole cell extracts, since that has been described recently elsewhere (Rivoltini et al., 2002). Sections will be concerned with methods of delivering candidate antigens and the outcome, using examples taken from different tumors. Although mouse models will provide the rational basis for vaccine designs, we are conscious of the fact that translation to the clinic has to cross unknown territory. Data from pilot clinical trials are therefore of immense importance and will be introduced where possible. II. Tumor Antigens

Tumor antigens include proteins, commonly glycoproteins, peptides, carbohydrates, and glycolipids, expressed by tumor cells and able to act as targets for immune effector mechanisms. Carbohydrate and lipid antigens provide important specialized targets on certain cancers, and have been reviewed elsewhere (Bitton et al., 2002). This review will focus mainly on (glyco)protein and peptide targets. Apart from virus-associated tumor antigens, descriptions of tumor antigens sometimes refer only to expression, without comment on immunogenicity. However, in many cases, immunogenicity has been tested by the detection of naturally occurring antibodies or by natural or expanded T cell responses in patients, indicative of at least some immune recognition (Rosenberg, 1998). The presence of serum antibodies in patients against potential tumor antigens has led to the technology of SEREX (serological identification of antigens by recombinant expression cloning) whereby target antigens are identified by reacting the sera with cDNA libraries derived from tumor cells (Preuss et al., 2002). For antigens recognized by T cells, isolation and cloning of tumor-infiltrating T cells has facilitated identification of expressed tumor-derived peptides that have apparently induced a local response (Rosenberg, 2001). Glycoprotein antigens may be expressed at the surface of the tumor cell, or be secreted, while the peptides will be bound to either the MHC class I or class II molecules. For certain molecules, such as idiotypic Ig, all three ways of expression may be possible. Each molecular form will require a different mode of attack, with surface glycoproteins potentially accessible to antibody, and peptides to T cell attack. Within these three categories, there is a range of specificity of expression, from those detected only on tumor cells to those that are also expressed by normal cells, but that may be up-regulated in tumor cells.

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One promising category of antigen arises from products of specialized cells, with melanocytes being a good example. Tumor cells of melanoma preserve expression of a range of melanocyte lineage proteins, including tyrosinase, gp100, MART-1/Melan-A, TRP-1 (gp75), and TRP-2 (reviewed in Van den Eynde and van der Bruggen, 1997; Rosenberg, 1999). Lineage-specific proteins are also expressed by tumor cells of prostate, breast, and other cancers. These proteins can be targeted by vaccination with minimal side effects anticipated. For melanoma, vaccines successful in inducing antitumor immunity have been noted also to induce vitiligo, indicative of an attack on normal melanocytes, which may be tolerable for patients (Rosenberg et al., 1996). Hematological malignancies express clonally restricted products of the specialized cells of origin, including idiotypic Ig or clonal T cell receptors (George and Stevenson, 1989; Stevenson et al., 1990). These are perfect tumor-specific antigens with no autoimmune outcome expected. Partly for this reason, the Id-Ig of B cell malignancies has been a focus of investigation of immunotherapeutic strategies (Stevenson et al., 1990). However, a disadvantage of clone-specific targets is that vaccines have to be individual for each patient. Cells of hematological malignancies have the advantage that they are generally available for study from blood or accessible tissues. In addition, the clinical success of allogeneic stem cell transplantation for leukemia has shown that the tumor cells are susceptible to attack by passively administered T cells specific for target peptides (see earlier). Candidate tumor antigens expressed by hematological malignancies, several of which are also expressed by other nonhematological tumors, are illustrated in Fig. 2. Cell surface glycoproteins potentially accessible to antibody attack include clone-specific idiotypic Ig of B cell neoplasms and clonotypic T cell receptors of T cell tumors. Mucins and differentiation antigens such as CD20 are also possible targets for induced antibodies. Cell surface glycoproteins may also be expressed as MHC-associated peptides and therefore could also be susceptible to T cell attack. Proteins confined to the intracellular compartment can be attacked only by this route, and these include a large number of candidates (Fig. 2). For B cell malignancies, MHC Class IIassociated peptides could be targets for direct attack by CD4þ T cells. Peptides derived from mutated protooncogenes, such as p53 (Vierboom et al., 1997), are mainly clone specific raising the possibility that patient-specific vaccines may be required. For the vast array of peptide antigens expressed in the context of MHC class I molecules, there is the complication of haplotype specificity, with most knowledge of human peptide targets being derived from the relatively common HLA-A*0201 allele. Prediction of the ability of peptide sequences to bind to human or murine MHC class I molecules is derived from algorithms based on anchor residues and known peptide sequences (Rammensee et al., 1999). Certain tumor antigens may be expressed by groups of patients who share MHC class I haplotypes, with one example being the chimeric gene,

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Fig 2 Target tumor antigens of hematological malignancies.

BCR-ABL, generated by the reciprocal translocation t(9;22)(q34;q11) and characteristic of patients with chronic myeloid leukemia (CML) (reviewed in Melo, 1996). For this junctional peptide, several restriction elements have been identified, and CTL able to kill tumor cells have been detected in vivo and expanded in vitro (Yotnda et al., 1998). CML is also susceptible to infused donor lymphocytes via a GvL effect (Kolb et al., 1990, 1995), directed against minor histocompatibility antigens (Fig. 2). Additional target peptides for CTL attack are likely to be identified by the extensive analysis of gene expression using microarray technology (Staudt and Brown, 2000). It is already clear that tumor cells, particularly at late stage, have genomic instability and this may generate new candidate antigens. The search for a ‘‘universal’’ tumor antigen is intense, since the ability to target a single protein expressed by a wide range of tumors would be very attractive. Cancer-testis antigens approach this category. Although they were first identified in melanoma (Boon et al., 1994), some at least have also been detected in other tumors, including hematological malignancies (van Baren et al., 1999). The relative tumor specificity arises from the fact that they are expressed only by normal testis or placenta. They include the MAGE, BAGE, GAGE, RAGE, and LAGE families, NY-ESO-1 and PRAME, and their properties and patterns of expression in melanoma and other tumors have been widely reviewed (Boon et al., 1994; Van den Eynde and van der Bruggen, 1997; Wang and Rosenberg, 1999). There has been considerable success in identifying peptides associated

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with several human MHC class I haplotypes, and clinical trials of vaccines against them, mainly for melanoma, are in progress (see later). Another tumor antigen with restricted expression in normal tissues is the mucin, MUC1, a transmembrane type I molecule found on almost all human epithelial adenocarcinomas, including breast, pancreas, ovary, lung, urinary bladder, prostate, and endometrium (Finn et al., 1995). Tumor cells express high levels of MUC1, with changes in glycosylation patterns that reveal core protein epitopes, masked in normal mucin. MUC1 is also expressed by myeloma cells, with levels apparently up-regulated by certain drug therapies (Treon et al., 1999), opening the possibility of enhancing the efficacy of immune attack. Cytotoxic T cells specific for the mucin have been detected in cancer patients, and can apparently act in an MHC-unrestricted manner (Magarian-Blander et al., 1998). There have been several attempts to develop vaccines against MUC-1, using a variety of vaccine vehicles (see later). Carcinoembryonic antigen (CEA), a molecule involved in cell adhesion, and related molecules also represent widely expressed potential tumor antigenic targets. CEA is a 180-kD glycoprotein expressed via a glycosylphosphatidylinositol linkage on the surface of adenocarcinomas of colon, breast, lung, and other epithelial cancers. There are suggestive data pointing to expression of CEA on a proportion of tumor cells in patients with chronic lymphocytic leukemia (Penezina et al., 1998). CEA represents an example of the category of oncofetal antigens (Hammarstrom, 1999), and antiCEA monoclonal antibodies (MAbs) and derivatives have been widely used in imaging and treatment (Murray and Unger, 1988). Vaccines containing CEA in a range of delivery systems have been tested in preclinical models and in clinical trials (see later) (Berinstein, 2002). The final category of tumor antigen includes proteins that are expressed by normal cells, but that may be overexpressed by tumor cells. A large proportion of potential tumor antigens lies in this group, and, if powerful vaccines are able to break tolerance and induce effective immunity, care will need to be taken to exclude damaging autoimmunity. They include antiapoptotic proteins such as survivin (Altieri, 2001, 2003), transcription factors such as WTI (Huang et al., 1990; Inoue et al., 1997), and telomerase reverse transcriptase (hTERT) (Vonderheide, 2002), all of which are expressed by both hematological and other tumors.

III. Vaccination Strategies

A. Proteins and Glycoproteins The widely held view that tumor cells can be attacked most efficiently by CTL (Melief and Kast, 1995), and that antibody is not only useless, but may in fact inhibit T cell attack (Prehn, 1994), has diverted attention away from using proteins as vaccines against tumors. The exceptions to this view include

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cell-surface carbohydrate antigens, such as TF, Tn, STn, and globo H antigens expressed in breast cancer, and glycolipids and gangliosides preferentially expressed on the cell sufaces of a range of cancers (Bitton et al., 2002). Exogenous protein or carbohydrate vaccines would be expected to induce antibody responses rather than CTL, and this may be appropriate for cell surface antigenic targets. However, CD4þ T cells can be induced by exogenous protein vaccines, and diversion of the response to Th1 or CD8þ T cell responses may be achievable using appropriate adjuvants. Clearly, CTL have immense potential to attack cancer cells, but the ability of passive monoclonal antibodies to clear malignant B cells points to a role for antibody against certain target antigens. One of these is HER2 (also known as Neu, ErbB2), a member of the epidermal growth factor receptor family of receptor tyrosine kinases, which is overexpressed by a subset of breast cancer. A monoclonal antibody, Herceptin, is currently showing effectiveness in combination treatment (Thomssen, 2001). A second example is Id-Ig expressed by B cell tumors, where protein vaccines, commonly linked to KLH, can induce protective and therapeutic immunity (Hsu et al., 1997; Stevenson et al., 1990). In this case, protection appears to be mediated largely by antibody, and vaccine efficacy parallels the success of passively administered monoclonal anti-Id antibody. As for all antibodies, the mechanism of attack may be multiple and remains unclear. However, there is intriguing evidence for a direct inhibitory signal via the B cell receptor (Uhr et al., 1996; Vuist et al., 1994). Another more recently raised possibility for follicular lymphoma is that anti-Id antibody may dislodge tumor cells from the site in the germinal center where maintenance signaling via the B cell receptor is occurring (Zhu et al., 2002). The finding that specific protective immunity against a T cell lymphoma is mediated by anticlonotypic antibodies against the T cell receptor indicates that Id Ig is not exceptional in being susceptible to antibody attack (Thirdborough et al., 2002). Adjuvants are of obvious importance for protein antigens, and there are several safe choices available of variable efficacy (Hunter, 2002; Schijns, 2001). It was always frustrating that the powerful immunostimulatory effect of complete Freund’s adjuvant (CFA) could not be easily harnessed for human use due to the accompanying damaging inflammatory response. More recently it has become clear that the major active component of CFA is bacterial DNA (Krieg, 2002). Within the DNA sequence there is a prevalence of unmethylated CpG motifs, which activate innate immunity in mammalian hosts. These findings have opened a new area of research into the potential of CpG as an adjuvant for vaccines against infectious diseases or cancer and have been well reviewed (Krieg, 2002). Most investigations are centered on using synthetic stabilized phosphorothioate oligonucleotides, which may behave differently from the natural diester-linked oligonucleotides. Studies of the influence of surrounding base sequence patterns have revealed

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differences in activity with CpG-A motifs activating NK cells while CpG-B are potent B cell activators (Krieg, 2002). The cellular receptor for CpG appears to be the TLR-9 expressed by most mouse dendritic cells (DCs), but in humans expressed only by plasmacytoid DC (Krug et al., 2001). Mouse DCs are therefore directly activated by CpG to produce an array of cytokines, including TNFa (Sparwasser et al., 1998). CpG treatment of human blood secondarily activates monocytes to produce IL-6 and TNF-a, and CpG acts as a stimulatory or costimulatory molecule for a wide range of cells, including NK, B, and T cells (Wagner, 1999). Production of IFN-g and IL12 tends to drive induction of Th1 CD4þ T cells. CpG can promote antibody and CTL responses against diverse antigens in human and in mouse models, with the exception of polysaccharide antigens (Krieg and Davis, 2001). When compared with CFA for the ability to induce a response against a lymphoma Id-Ig, CpG performed at least as well (Weiner et al., 1997). Interestingly, CpG tends to induce higher levels of IgG2a than CFA, consistent with the activation of Th1 CD4þ T cells. In terms of virus-associated cancer, CpG was able to activate immunity against E7 protein from human papilloma virus (HPV), and this provided protection against challenge with HPV-immortalized tumor cells (Kim et al., 2002). A possible role for alternative adjuvant molecules in redirecting induced immune effector function has also been suggested by the data from a clinical trial of patients with follicular lymphoma given Id-Ig KLH protein vaccines delivered with local subcutaneous injection of granulocyte macrophage colony-stimulating factor (GM-CSF) (Bendandi et al., 1999). GM-CSF has multiple effects on hematopoietic and nonhematopoietic cells, with one important function being to stimulate differentiation and outgrowth of DCs. In this trial, patients developed T cell responses, both CD4þ and CD8þ , which appeared to be Id specific, and immune responses were accompanied by eradication of minimal residual disease (Bendandi et al., 1999). The recognition of the pivotal role of DCs in taking up antigen and presenting it to naı¨ve T cells has led to attempts to improve performance of protein vaccines by direct loading of DCs in vitro, followed by maturation and reinfusion (Dhodapkar et al., 1999; Inaba et al., 1998). The complexity and heterogeneity of DCs both within one species and between species make modeling of DCbased vaccine strategies difficult, and argues for testing in pilot clinical trials. Trials of autologous DCs loaded with Id-Ig or Id-Ig KLH have been carried out in patients with lymphoma and myeloma, with evidence for modest beneficial immune responses (Timmerman et al., 2002). A similar approach has been used to vaccinate patients with myeloma, a considerably more challenging disease since patients have high levels of circulating Id-Ig and are immunosuppressed by disease and treatment. Responses as measured by Id-specific T cell proliferation varied from a low (15% of patients) (Liso

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et al., 2000) to a high incidence (83%). A more optimistic setting for myeloma is to vaccinate the donors of allogeneic stem cell transplants and then to transfer immune cells. This was carried out initially using an Id-Ig KLH vaccine with demonstration of transfer of Id-specific T cell immunity (Kwak et al., 1995). Further donors were vaccinated with Id-Ig KLH plus GM-CSF with apparent induction of Id-specific CD4þ and CD8þ T cell responses (Li et al., 2000). CD8þ T cells were also induced from a patient with myeloma by stimulation with autologous DCs pulsed with Id-Ig. In mouse models, it is difficult to induce significant levels of CD8þ T cells using protein, even if loaded onto DCs (Shibagaki and Udey, 2002). Using ovalbumin as an exogenous protein, it was found that in order to gain entry to the processing pathway for MHC class I-restricted peptides, it was necessary to add a transduction domain from HIV to the protein. However, certain proteins can apparently enter the MHC class I processing pathway from an exogenous route, since b–galactosidase-loaded DCs induced CTL able to lyse transfected target cells (Paglia et al., 1996). The heterogeneity of these findings and the lack of comparability between human and mouse may reflect different DC subsets, or the different behavior of proteins. MUC1 has also been used as an exogenous protein vaccine, in one trial consisting of identical tandem repeats of its repetitive peptide sequence linked to KLH and conjugated to oxidized mannan (Vaughan et al., 2000). In mice, this vaccine induces CD8þ responses and protection against tumor challenge. However, in primates, and apparently in human subjects, only a humoral response could be induced, possibly because of preexisting natural antibodies against a 1-3-linked galactose (Apostolopoulos et al., 1998). This result emphasizes the importance of species-specific oligosaccharide patterns on the behavior of glycoproteins or carbohydrate adjuvants present in exogenous vaccines. B. Peptides Peptides represent a simple source of tumor antigen, and can be readily manufactured under good manufacturing practice (GMP) conditions. Candidate epitopes for binding to murine or human MHC class I molecules can be predicted by algorithms of increasing power (Rammensee et al., 1999). For human subjects, early attention was focused on the HLA-A*0201 haplotye expressed by 40% of the white population, but binding motifs for other alleles are becoming increasingly available. Importantly, the ability to bind to MHC molecules can then be tested, followed by scrutiny for immunogenicity based on the induction and expansion of functional CTL in vitro (Schultze and Vonderheide, 2001). The next hurdle is whether the CTL generated can kill target tumor cells. This requires processing of the candidate peptide via the proteasomal degradative pathways in the tumor cell. Again, prediction of

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cleavage sites is available via the databases (http://www.uni-tuebingen.de/uni/kxi/). However, it is becoming clear that degradative pathways can be mediated via either the proteasome or the immunoproteasome, depending on the cytokine environment, adding another complication to the selection procedure (Schultze and Vonderheide, 2001). There are many traps in peptide selection, since peptides may not only be cryptic, but analogue peptides may also be antagonistic (Grey et al., 1993). In contrast, modification of anchor residues to improve binding toMHC class I molecules can create agonistic peptides, which are better able to induce CTL (Abrams and Schlom, 2000). The problem for peptide vaccines is their inherent lack of immunogenicity and their biodegradability (Celis, 2002). In spite of this, vaccination of 25 melanoma patients with the MAGE-3A1 nonapeptide with no additions or adjuvants led to significant tumor regression in seven (Marchand et al., 1999). However, in the four patients analyzed, which included two with regression, no CTL activity was detectable. To raise measurable CTL activity, adjuvants may be desirable both to provide stimulation of innate immunity and possibly to create a protected depot. CpG oligonucleotides (see earlier) may also provide useful adjuvants. Delivery of peptide antigens via heat shock proteins is another intriguing possibility, and has been shown to induce protective peptide-specific immunity against tumor cells in models (Blachere et al., 1997). However, the real strength of this approach is in delivering unknown peptides from the intracellular milieu of the tumor cells as vaccines (Przepiorka and Srivastava, 1998). Alternatively, specific peptides can be loaded onto DCs in vitro and cells reinfused. This strategy has been effective in mouse models (Celluzzi et al., 1996; Porgador and Gilboa, 1995). What is remarkable in human subjects is the relative ease of inducing primary and memory CTL responses in vitro using either DCs or CD40-activated B cells as stimulators (von BergweltBaildon et al., 2002). CTL of significant avidity can be generated from both healthy donors and cancer patients, indicating that a repertoire is available. The potential for activating this repertoire by injection of loaded DCs is clear, but there are pitfalls. There are obvious problems in selecting the DCs and in maturing and loading them optimally in vitro under conditions of GMP. The route of injection and the migratory potential of the DCs are other variables, and modeling in mice is not always helpful. A further question concerns the concentration of peptide, which may be critical, since too high a concentration could lead to ‘‘low-quality’’ CTL unable to kill tumor cells (Celis, 2002). However, in spite of these difficulties, clinical trials of peptide-loaded DCs have already been carried out in patients with cancer, including melanoma (Nestle et al., 1998; Thurner et al., 1999), with encouraging immune responses, and possible clinical responses, detected. Patients with advanced breast and ovarian cancer have also been vaccinated with DCs loaded with

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peptides from HER-2/neu or MUC1 (Brossart et al., 2000). Peptide-specific IFN-g-producing CTL were detected in 5 of 10 patients, and cytolytic activity against a tumor cell line could be mediated by expanded CTL. It will take time to assess if the immense effort and cost involved in ex vivo culture and loading of DCs justify the outcome. However, the approach has clear potential, and, as knowledge accumulates, it should be possible to harness the power of these antigen-presenting cells to activate useful immunity. C. Gene-Based Vaccines The availability of genomic information from normal and tumor cells, together with the development of molecular biological technology able to exploit this information, has opened the possibility of gene-based vaccine designs. The simplicity of the concept of injecting selected gene sequences into patients so that the encoded proteins can be presented to the immune system in situ is attractive. As always, initial enthusiasm has been tempered by the reality of performance. Clearly, time is needed to understand the mechanisms by which the encoded proteins activate immunity, so that the potential of the approach can be fully realized. Translation of observations in preclinical models to patients will also require careful modifications. However, the opportunities to manipulate design and delivery for the whole range of antigens are immense. 1. Naked DNA Vaccines Wolff et al. (1990) first discovered that intramuscular injection of naked plasmid DNA resulted in transfection of muscle cells and production of the plasmid encoded protein, b-galactosidase. It was subsequently demonstrated that vaccination by intramuscular delivery of plasmid DNA encoding the nucleoprotein of influenza A virus could induce specific humoral and cellular immune responses able to protect animals from influenza challenge (Ulmer et al., 1993). In an independent study, Tang et al. (1992) showed that delivery of plasmid DNA coated onto gold particles directly into the skin of mice using a biolistic device (the ‘‘gene gun’’) could elicit a specific immune response against the encoded protein. These pioneering studies have paved the way for the development of a simple and potentially very powerful vaccination technology. Extensive studies in various preclinical disease models have since established that DNA vaccines have the ability to induce a full spectrum of immunological activities and are capable of generating protective immunity in a prophylactic setting. In some cases, DNA vaccines have also been shown to be therapeutically effective against established diseases. The mechanisms by which DNA vaccines elicit immune responses are gradually being revealed (Fig. 3). It is now clear that the bacterial plasmid DNA not only ensures antigen production in transfected host cells, but also

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Fig 3 Induction of immunity by DNA vaccines.

has intrinsic adjuvant properties owing to the presence of immunostimulatory sequences (ISSs) (Krieg, 2002). These ISSs, also known as CpG motifs, are composed of unmethylated CpG dinucleotides with specific flanking nucleotides. Such motifs are present in microbial DNA at a 20-fold greater frequency than in vertebrate genomes and represent one form of pathogen-associated molecular patterns. Recently, the Toll-like receptor 9 (TLR9) has been identified as the ‘‘pattern recognition receptor’’ for bacterial DNA in both mice and humans (Bauer et al., 2001; Hemmi et al., 2000; Takeshita et al., 2001). TLR9 is expressed by B cells, dendritic cells, and other cells of the innate immune system and CpG DNA–TLR9 interaction activates the Toll/IL-1 signaling pathway and triggers an inflammatory responses characterized by the production of IL-6, IL-12, IL-18, TNF-a, IFN-a and -g, and chemokines. The ISSs also induce polyclonal B cell proliferation and stimulate DC maturation with up-regulated expression of MHC class II and costimulatory molecules. In addition, it was recently shown that IL-6 and some other factor(s) produced by CpG- or LPS-stimulated DCs could block the suppressive effect of CD4þ CD25þ regulatory T cells on T cell activation (Pasare and Medzhitov, 2003). It appears that the vertebrate immune system has evolved the ability to recognize the presence of bacterial DNA and activate a coordinated innate and adaptive immune response. DNA vaccines are usually administered by intramuscular (i.m.) or intradermal (i.d.) injection, or into skin cells with a gene gun, and antigens are produced endogenously by transfected cells. When DNA vaccines are

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delivered through the i.d. route, the skin-derived DCs (Langerhans cells) are directly transfected and probably play a key role in antigen presentation to T cells (Akbari et al., 1999; Condon et al., 1996; Porgador et al., 1998). Vaccine delivery by i.m injection predominantly results in transfection of myocytes (Fig. 3). However, muscle cells do not express MHC class II and costimulatory molecules that are critical for effective T cell priming. Instead, there is compelling evidence that the bone marrow-derived antigen-presenting cells, presumably DCs, play a predominant role in the activation of T cells through a process termed ‘‘cross-priming,’’ whereby the antigen released by muscle cells is taken up, processed, and presented by the bone marrow-derived professional APCs (Corr et al., 1996, 1999; Fu et al., 1997; Iwasaki et al., 1997). The mechanism of this transfer is unclear but could involve ferrying of proteins or peptides via heat shock proteins (Srivastava, 2000). However, the i.m. delivery route may also result in transfection of a small number of bone marrow-derived APCs, which could directly activate T cells (Casares et al., 1997). The outcome of DNA vaccination is to induce all arms of the immune response with a tendency for dominance of the Th1 subset of the CD4þ T cell population. 2. DNA Tumor Vaccines The efficacy of DNA vaccines against infectious diseases has clearly been demonstrated in a variety of animal species for a large number of preclinical models (Gurunathan et al., 2000; Robinson and Pertmer, 2000). Several recent clinical trials have further shown that DNA vaccination is safe and capable of inducing humoral and/or cellular immune responses in humans (MacGregor et al., 1998; Roy et al., 2000; Wang et al., 1998, 2001). It is evident that DNA vaccines are capable of inducing high levels of CTL responses, but are generally weaker than protein vaccines in inducing antibody responses. This may be due to the low levels of antigen produced, but it also may derive from the preferential activation of CD4þ Th1 cells leading to production of IgG2a rather than IgG1 antibodies, as noted in mouse models. Development of effective DNA vaccines to attack cancer cells is a much greater challenge. As discussed earlier, most tumor-associated antigens are nonmutated selfproteins of low immunogenicity, and immunological tolerance or evasion may be real issues. It is therefore not surprising that vaccination simply with a eukaryotic expression plasmid encoding the tumor antigen of interest delivered in saline is rarely effective. Efforts are therefore being made to develop various strategies to overcome these hurdles. One widely exploited and simple approach is coadministration of plasmids encoding cytokines, chemokines, or costimulatory molecules. The usage of these genetic adjuvants can not only increase the magnitude but also modulate the type of immune responses to DNA vaccines. However, a dynamic immune response is likely

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to involve these molecules at different stages, and it may be difficult to deliver coactivators at the time required. The strategy of codelivery has recently been reviewed in detail elsewhere (Berzofsky et al., 2001; Scheerlinck, 2001) and will not be further discussed here. We will focus on various DNA fusion vaccine strategies that have been designed to break tolerance and to enhance antigen presentation and immune cell activation. In our view, provision of additional T cell help for weak or tolerizing antigens is a key component for successful induction of antitumor immunity. We will also discuss recent developments in strategies for improving in vivo antigen delivery and production. In addition, DNA vaccines can be combined with various viral delivery systems in a primeboost fashion. Although the latter strategies are largely being investigated for DNA vaccines against infectious diseases, they should also be applicable to cancer. 3. Genetic Fusion Vaccine Strategies The ease of DNA manipulation has facilitated construction and evaluation of various DNA vaccines that genetically link antigens to a range of immunomodulating molecules. These fusion vaccine strategies have been designed to provide cognate CD4þ T cell help critical for antigen-specific B and T cell activation, to activate the innate immune system, to target antigen delivery to professional antigen-presenting cells, and to direct antigen to more relevant processing and presentation pathways. a. Provision of T Cell Help. Conjugation of an immunogenic carrier protein or a Th epitope has proved to be an effective strategy to improve antibody responses to polysaccharide and peptide/protein-based vaccines through provision of cognate CD4þ T cell help. It is also clear that CD4þ T cells play a key role in the induction and maintenance of CD8þ cytotoxic T cell responses (Bourgeois et al., 2002; Clarke, 2000; Janssen et al., 2003). For effective activation of antitumor immunity, it is essential to incorporate strategies capable of breaking tolerance in the CD4þ T cell compartment that likely have occurred in the tumor-bearing host (Bogen, 1996). DNA fusion vaccine strategies designed to provide cognate CD4 T cell help have been shown to be effective in promoting the induction of B cell and T cell responses in a number of preclinical models. In murine B cell lymphomas, vaccination using DNA constructs encoding Id of immunoglobulin antigens genetically linked to immunogenic carrier sequences, either xenogeneic human Ig constant regions (Syrengelas et al., 1996) or a pathogen-derived sequence, fragment C (FrC) of tetanus toxin (King et al., 1998), can generate protective anti-Id antibody responses against lethal tumor challenge. The linkage of immunogenic carrier sequences is absolutely required, because immunization with vaccines containing Id sequence only or fused to autologous mouse Ig constant regions fails

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to induce an anti-Id antibody response (King et al., 1998; Spellerberg et al., 1997; Syrengelas et al., 1996). Importantly, fusion was required because separate genes, even within the same plasmid construct, were ineffective (King et al., 1998). The FrC gene fusion strategy also dramatically increased anti-Id immunity against T cell lymphoma using a DNA vaccine encoding the TCRa/b idiotypic sequences (Thirdborough et al., 2002). Recently, a DNA vaccine trial in patients with stage III or IV follicular B cell lymphoma has been reported (Timmerman et al., 2002). Patients were immunized with plasmid constructs encoding a chimeric immunoglobulin molecule consisting of a tumor-specific Id sequence linked to xenogeneic (murine) constant regions with and without codelivery of GM-CSF-encoding plasmid. A majority of patients mounted B and/or T cell responses to the murine Ig component, while one patient had an Id-specific T cell response and several patients had immune responses that were cross-reactive with other patients’ Id proteins. The data showed that DNA vaccination was safe and potentially a useful approach to anti-Id immunotherapy. The modest antitumor immune responses may be improved by inclusion of more potent carrier sequences. Our laboratory is currently conducting a Phase I/II clinical trial in patients with follicular lymphoma, using idiotype-encoding variable region gene sequences fused to the FrC sequence. Preliminary data show that this strategy is capable of inducing strong proliferative and antibody responses against the FrC component and proliferative IFN-gproducing T cell responses specific to the idiotypic determinants in a majority of patients (unpublished observation). It is possible that the high level of T cell help against FrC is particularly effective in amplifying antitumor immunity in human patients. Importantly, both the clinical trials of DNA idiotypic fusion vaccines show that naked DNA is capable of inducing immune responses even in patients with lymphoma, thus providing a platform for further clinical development of the approach. Cognate T cell help provided by fusion to FrC (King et al., 1998) or to the coat protein of potato virus X (PVXCP) (Savelyeva et al., 2001) also promotes anti-Id immune responses that are protective against an Ig secreting but surface Ig-negative myeloma, with CD4þ T cells as major mediators of protection. The induction of this enhanced Id-specific CD4þ T cell response is probably by a Th–Th cooperation mechanism as proposed by Gerloni et al. (2000); namely, CD4þ T cells reactive with a dominant determinant provide help to other CD4þ T cells recognizing weaker epitopes through up-regulating the costimulatory ability of APCs. Interestingly, the Id–PVXCP fusion vaccine design is also able to induce protective immunity against a surface Ig-positive lymphoma, but unlike the Id–FrC fusion vaccine, the protection is mediated by CD4þ T cells (Savelyeva et al., 2001). Moreover, PVXCP appears to preferentially direct anti-Id antibody response to the IgG2a subclass, indicating a Th-1-type response, whereas FrC also induces IgG1 (King et al., 1998), a

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Th-2-type response. It appears therefore that the molecular nature of the encoded immunoenhancing protein influences the immune pathways activated, and therefore the protective mechanism. Fusion of immunogenic carrier sequences also promotes induction of CD8þ CTL responses targeted to HIV envelope protein (Le Borgne et al., 1998), influenza NP and HPV E7 CTL epitopes (Wolkers et al., 2002), and to a tumor rejection antigen from an endogenous retrovirus (Takeda et al., 2000). However, for amplifying CTL responses, care is required when adding immunoenhancing sequences to avoid the introduction of potentially competitive MHC class I-binding epitopes. For our vaccines that exploit FrC of tetanus toxin as an immunoenhancer, we have engineered a truncated FrC single domain sequence to remove potentially immunodominant CTL epitopes identified in the second domain, while still retaining the known Th epitope within the first domain (Rice et al., 2001, 2002a). Another modification was to place the encoding CTL epitope sequences at the 30 end of the FrC domain. This design led to accelerated and increased CTL responses to the fused epitopes, and protective or therapeutic immunity against tumor. In contrast, the response is less if unmodified, full-length two domain FrC is present, consistent with the phenomenon of immunodominance (Rice et al., 2001). Interestingly, reduction of the truncated FrC to a single helper epitope provided only a modest increase of the CTL response against the fused peptide sequence, indicating a requirement for the full domain. It is unclear if this is due to additional unknown CD4þ epitopes in the domain, or to another property of the domain that could affect the cross-priming process. These results suggest that it is possible to activate an optimal effector mechanism for the target antigen by selecting an appropriate carrier sequence. b. Complement. The complement system plays a key role in innate immunity and also represents an important link between innate and acquired immunity (Fearon, 1998; Ochsenbein and Zinkernagel, 2000). A recombinant protein vaccine consisting of the hen egg lysozyme (HEL) fused to murine C3d component of complement has been shown to dramatically increase (>1000-fold) the efficiency of antibody response against HEL in mice (Dempsey et al., 1996). Ross et al. (2000, 2001) have further demonstrated that the fusion of C3d can also augment the humoral immune response against hemagglutinin (HA) of influenza and against HIV gp120 delivered as DNA vaccines. Immunization with the HA–C3d fusion vaccine induces HA-specific antibodies with higher avidities more rapidly. The accelerated antibody response correlates with a faster appearance of protective immunity. Remarkably, a single vaccination with a low dose of fusion vaccine is able to confer complete protection against live virus challenge (Ross et al., 2000). The mechanism by which C3d promotes the humoral response is likely to be by enhanced stimulation and expansion of antigen-specific B cells. C3d binds to its receptor, CD21, that is expressed and

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associated with the costimulatory molecule CD19 on B cells and follicular dendritic cells. The fusion of an Ag to C3d should allow for a cross-linking of Ag-specific B cell receptor and CD21, thereby delivering a more effective signal for activation. CD21 is also expressed on follicular dendritic cells (FDCs) in germinal centers where affinity maturation takes place. It is conceivable that C3d could facilitate Ag binding to FDCs, and the trapped Ag then attracts and stimulates affinity maturation in responding B cells (Fearon and Carroll, 2000). The humoral immune response plays an important role in the control of certain tumors. Enhancing B cell signaling by the C3d fusion strategy may therefore be a useful approach for developing cancer vaccines with improved humoral response. c. Defensins and Chemokines. Defensins are a family of small, cationic peptides that directly contributes to the killing of microbes, and a recent study by Zhang et al. (2002) has identified human a-defensins as the major components of the antiviral factor secreted by CD8 T cells of HIV-infected patients. Moreover, it has been shown that human b-defensins are also chemotactic for immature DCs and memory T cells (Biragyn et al., 2001; Yang et al., 1999), while murine b-defensin 2 acts directly on immature DCs via TLR-4, inducing up-regulation of costimulatory molecules and DC maturation (Biragyn et al., 2002b). Thus defensins also play a role in bridging innate immunity and adaptive immune responses. Biragyn et al. (2001) show that murine b-defensins 2 and 3 bind CCR6 preferentially expressed on immature DCs and that DNA fusion vaccines consisting of nonimmunogenic B cell idiotypic antigens and murine b-defensin 2 induce protective and therapeutic immunity against two different syngeneic mouse lymphomas. The same study shows that fusion vaccines consisting of tumor Id and inflammatory chemokines MIP-3a or MCP-3, which are chemotactic for immature DCs, are also effective. The same fusion vaccine strategy also enhances immune responses against HIV-1 gp120 (Biragyn et al., 2002a). Importantly, both chemotactic activities and the covalent linkage are required, since neither fusion of inactive pro-b-defensin or mutant chemokine nor coimmunization with a mixture of plasmids encoding unlinked Id antigens and defensin or chemokines is effective. These results suggest that targeting the delivery of antigens to immature DCs is a general applicable approach to increasing the efficacy of DNA vaccines. d. Targeting to Antigen-Presenting Cells. One problem for naked DNA vaccination is that only a small number of host cells is transfected and a relatively low level of antigen is produced. The small amount of antigen available for processing and presentation by APCs could therefore be a limiting factor for the potency of DNA vaccines (Heath and Carbone, 2001).

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This may be a particularly important issue for intramuscular DNA vaccination where antigen transfer from transfected muscle cells to professional APCs is required for effective induction of immune responses. Therefore, various strategies for targeting antigen delivery to professional APCs are currently being explored. Boyle et al. (1998) have shown that i.m. vaccination in mice with a DNA construct encoding a human IgG (hIgG) and the extracellular domain of CTLA-4 fusion protein can generate a faster and higher (up to 10,000-fold) anti-hIgG antibody response than DNA vaccines encoding hIgG alone. The CTLA-4 fusion also enhances the T cell proliferative response (up to 8-fold). In the same study the authors showed that fusion to L-selectin, which binds to CD34 on high endothelial venule cells of lymph node, could also improve the immune response but to a lesser extent. Subsequent studies further show that CTLA-4 DNA fusion vaccines are able to induce accelerated and increased antibody responses against HA of influenza, chicken ovalbumin, and malaria apical membrane antigen (AMA1) (Deliyannis et al., 2000; Lew et al., 2000). More importantly, the CTLA-4 fusion designs are capable of providing more effective protection against influenza virus challenge in a mouse model (Deliyannis et al., 2000) and against Corynebacterium pseudotuberculosis challenge in sheep (Chaplin et al., 1999). A recent report showed that the CTLA-4 fusion protein vaccination approach was also able to generate protective immunity against a self-tumor antigen (Huang et al., 2000). CTLA-4 is expressed by activated T cells and binds with high affinity to CD80/CD86 surface receptors expressed by APCs. The findings that CTLA-4–HA retains the ability to bind to CD80 and CTLA-4-Ig fusion protein and is at a 4-fold higher level than nonfused protein in the draining lymph nodes within 2–24 hr after immunization (Deliyannis et al., 2000) suggest that the mechanism of enhanced immune responses by the CTLA-4 fusion constructs is more efficient antigen capture and presentation by APCs. Another approach for improving antigen delivery to APCs is to target the Fcg receptor. A DNA vaccine encoding a fusion protein consisting of a hepatitis B virus e antigen (HBeAg) and a human IgG Fc has been shown to induce enhanced antigen-specific B cell, CD4þ Th and CD8þ CTL responses in mice as compared to a DNA vaccine encoding HBeAg alone (You et al., 2001). DCs express IgG Fc receptors (FcgRs), and interactions between FcgRs and antigen–Ig complexes result in antigen internalization. Soluble antigen uptake by receptor-mediated endocytosis is 1000- to 10,000-fold more efficient than fluid phase pinocytosis (Regnault et al., 1999; Sallusto and Lanzavecchia, 1994), and the endocytosed antigen is processed via both the MHC class II pathway for CD4þ T cell induction and via MHC class I pathway for CD8þ CTL induction (Regnault et al., 1999). Furthermore, the interaction of Fc with FcgRs stimulates DC maturation by up-regulating expression of surface molecules that are important for antigen presentation

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(Regnault et al., 1999). It should be noted that in this study a human IgG Fc fragment is used; it is therefore possible that the xenogeneic sequence has contributed to the increased immune responses by providing cognate T cell help as discussed earlier. Since cross-presentation is an important pathway for DNA vaccination, increasing antigen transfer from transfected somatic cells to professional APCs may improve the potency of DNA vaccines. This hypothesis has been tested by fusing antigens to the herpes simplex virus type 1 (HSV1) VP22 protein that possesses an intercellular trafficking property (Elliott and O’Hare, 1997). Hung et al. (2001) constructed DNA vaccines with the HPV E7 antigen fused either to the full-length VP22 or a C-terminally truncated VP22 (DVP22) that is defective for intercellular spreading. Vaccination with VP22-E7 induced a markedly improved CD8þ CTL response and tumor immunity compared to the DNA vaccine encoding E7 only. The DVP22-E7 vaccine did not show improvement. In a similar study, Michel et al. (2002) also showed that fusion of VP22 to E7 led to dramatic enhancement of CTL induction. However, the latter study found that fusion to DVP22 was equally effective, indicating that other mechanism(s) may also contribute to the enhancement, such as increased stability of E7 and more efficient secretion. In addition, as discussed earlier, the virus-derived VP22 protein may contain Th epitope(s) that would provide cognate help to the fused antigen. Further investigations will be required to elucidate the exact mechanism(s). One mechanism of cross-priming by professional APCs is acquisition of exogenous antigens by phagocytosis of apoptotic cells (Albert et al., 1998). There is also evidence that DNA vaccination causes injury, local inflammation, and cell death, which may account, at least in part, for the ability of DNA vaccines to induce T cell activation. Two recent reports have reinforced this notion. First, Chattergoon et al. (2000) showed that intramuscular codelivery of a Fas-expressing plasmid with DNA vaccines encoding HIV-1 env or gag/pol generated an increased antigen-specific CTL response, as compared with vaccines without Fas coexpression. They found that anti-Fas antibody treatment of cells transfected in vitro with Fas-expressing plasmid underwent rapid apoptosis, and Fas expression in situ resulted in significant infiltration into the muscle of CD4þ and CD8þ T cells and CD11cþ phagocytic cells. Furthermore, coinjection of Fas and EGFP plasmids led to an 5-fold increase in the number of APCs (macrophages and DCs) containing apoptotic EGFP positive cells in the proximal draining lymph node. Second, Sasaki et al. (2001) constructed dual-expression plasmids encoding influenza virus HA or NP antigens and partially inactivated caspases 2 and 3. These dual vectors were still able to induce apoptosis in vitro but at a level about 10-fold lower than their fully active counterparts. Vaccination with the dual vectors-encoding

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mutant, but not intact caspases, could elicit markedly enhanced T cell responses, indicating that sufficient antigen production in transfected cells before cell death was required. One difference between the two studies is that Fas can be delivered in a separate plasmid from the vaccine, whereas expression of mutant caspases and immunogens in the same cells is required. Nevertheless, the two studies suggest that deliberate induction of apoptosis of antigen-producing cells can enhance antigen presentation by professional APCs and increase the potency of DNA vaccines. e. Somatic Transgene Immunization. Perhaps the most direct approach to optimize antigen presentation by professional APCs within organized lymphoid organs is injection of DNA vaccines into spleen or into lymph nodes. The intrasplenic approach was first described by Zanetti and colleagues (Gerloni et al., 1997), who showed that inoculation of a plasmid containing a chimeric immunoglobulin heavy-chain gene under the control of immunoglobulin promoter and enhancer elements resulted in chromosomal integration in B cells of the plasmid, expression of the transgene, and induction of a specific antibody response. This report of integration from a DNA vaccine is unique, since no significant integration has been found in exhaustive studies of more conventional injection sites (Manam et al., 2000). In this procedure, termed somatic transgene immunization, antigen presentation and T cell priming are dependent on the transgenic B cells that produce the antigen, and apparently do not require bone marrow-derived DCs (Castiglioni et al., 2003). In a further study they inserted a weak CD4þ T cell epitope from MUC1 and a strong epitope from a malaria antigen in the CDRs of the VH gene. Immunization with this construct could induce a strong CD4þ T cell response against the MUC1 epitope (Gerloni et al., 2000). In the study by White et al. (2000), a plasmid encoding CEA under the control of the CMV promoter/enhancer was used for intrasplenic (i.spl) or intramuscular immunization, and antibody responses and tumor protection were assessed. Immunization by the i.spl route induced CEA-specific immune responses comparable to that elicited by i.m. inoculation. Vaccination by intra-lymph node (i.ln) delivery of plasmid DNA was reported by Maloy et al. (2001) to be more effective than conventional i.m. or i.d injection. They immunized mice with a plasmid containing an LCMV gp33 CTL epitope linked to GFP by various routes, including i.m., i.d., i.ln, and i.spl injection. They found that i.ln injection enhanced immunogenicity by 100- to 1000-fold and induced superior CT8þ CTL immunity against viral challenge, and could eradicate the peripherally transplanted tumor pieces expressing the gp33 epitope. There was evidence that a small fraction of CD11cþ lymph node cells expressed GFP protein 24 hr after i.ln vaccination,

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suggesting that the enhancement could be attributed, at least in part, to increased uptake and expression of naked DNA vaccines in DCs and more efficient priming of T cell responses. 4. Manipulating Antigen Presentation and Processing Pathways The ease of recombinant DNA manipulation provides an opportunity to test various strategies to enhance antigen processing via particular pathways. For example, optimal antibody responses are often achieved by linking antigens to secretory signal sequences. There is also evidence that targeting of proteins to the endoplasmic reticulum may increase levels of induced CTL, possibly via retrograde transport into the cytosol (Ciernik et al., 1996). Alternatively, increasing intracellular antigen degradation may enhance CTL induction. Fusion of ubiquitin to several antigens reduces or abolishes antibody responses but enhances cell-mediated immunity with increased CTL activities and IFNg production, and results in improved protection against viral, bacterial, and tumor challenge (Delogu et al., 2000; Rodriguez et al., 1998; Velders et al., 2001b). Ubiquitin plays an important role in proteasome-dependent degradation of intracellular proteins. In some of these reports in vitro studies have confirmed that linkage of ubiquitin leads to more rapid protein degradation, suggesting that the enhanced cellular immune responses are the results of more efficient intracellular antigen processing and presentation via the MHC Class I pathway. However, the influence of ubiquitination on protein degradation and induction of a CTL response appears to be antigen dependent, as the stability of influenza nuclear protein (NP) is not affected by conjugation to ubiquitin in vitro (Fu et al., 1998), and vaccination with ubiquitin fusion vaccines does not increase induction of CTL responses against NP (Fu et al., 1998) or against hepatitis C core antigen in vivo (Vidalin et al., 1999). Heat shock proteins (HSPs), including HSP70, HSP90, gp96, and calreticulin (CRT), play multiple roles in antigen presentation by MHC class I molecules (Basu and Srivastava, 2000). These chaperone proteins form complexes with antigenic peptides, and are involved both in the transport of these peptides to the endoplasmic reticulum (ER) and in the folding and assembly of MHC class I molecules. Furthermore, complexes of HSPs and antigenic peptides released by dying cells, such as virus-infected cells or tumor cells, are efficiently taken up by APCs via the a2-macroglobulin receptor CD91 and re-presented by MHC class I molecules (Basu et al., 2001). Considering the importance of cross-presentation in the induction of CD8þ T cell response by DNA vaccines, linking an antigen to HSP may potentially be an effective approach for enhancing the induction of a CTL response. The CD8þ CTL response specific for HPV16 E7 antigen could be improved by linkage to HSP70 of Mycobacterium tuberculosis (Chen et al., 2000) and to CRT (Cheng et al., 2001a). Similarly, E7-HSP70 fusion vaccines based on

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Sindbis virus self-replicating RNA vector and Semliki Forest virus RNA vector also generated enhanced E7-specific Tcell-mediated immune responses (Cheng et al., 2001b; Hsu et al., 2001). In addition, CRT possesses antiangiogenic activity that inhibits tumor growth in vivo (Pike et al., 1999). Intradermal delivery of the CRT–HPV17 E7 fusion vaccine not only induced increased CTL activities, but also showed antiangiogenesis activities that further enhanced the vaccine potency (Cheng et al., 2001a). In addition to their role in antigen presentation, HSPs are considered as endogenous ‘‘danger’’ signals (Gallucci and Matzinger, 2001), in that they can activate DCs and macrophages, and stimulate production of proinflammatory cytokines (Asea et al., 2000; Basu et al., 2000; Chen et al., 1999; Kol et al., 2000; Singh-Jasuja et al., 2000). The powerful immunostimulatory activities of HSPs make them potentially excellent adjuvants for DNA vaccines. It is increasingly clear that CD4þ T cells are critical in controlling tumors in vivo, mediating direct and indirect killing of tumor cells, as well as providing help for B cells and CD8þ CTLs. Rational vaccine design can also direct antigen processing and presentation to the MHC class II pathway in order to enhance CD4þ T cell responses. The MHC class II-associated invariant chain (li) has recently emerged as an attractive molecule for enhancing MHC class II presentation (Koch et al., 2000; van Bergen et al., 1999). The li ensures correct assembly of MHC class II a- and b-chains in the ER, directs them to the endosomal pathway, and facilitates loading of antigenic peptides (Pieters, 2000). In one DNA vaccine design, the MHC class II-associated invariant chain peptide (CLIP) sequence was replaced by an MHC class II-restricted epitope derived from HSP60 (van Tienhoven et al., 2001). Immunization of rats using this fusion construct induced an HSP-specific T cell proliferative response, while immunization with a plasmid encoding full-length HSP60 failed. The ability of the fusion construct to induce a CD4þ T cell response was correlated with improved antigen presentation by MHC class II molecules as demonstrated by in vitro studies. The CLIP substitution approach has previously been shown to increase MHC class II presentation of other antigens in vitro (Fujii et al., 1998; van Bergen et al., 1997). More recently, a cell-based vaccine with similar CLIP substitution design has been shown to induce a strong CD4þ T cell response that is protective against a highly aggressive tumor (van Bergen et al., 2000). Enhancement of MHC class II presentation has also been achieved by targeting antigen to specific organelles. In one approach, antigens were linked to lysosomal-associated membrane protein LAMP-1 (Rowell et al., 1995; Wu et al., 1995). This approach uses the sorting signal in the cytoplasmic tail of LAMP-1 to target antigens to the endosomal/lysosome compartment where MHC class II-restricted peptides are generated. DNA fusion vaccines consisting of HPV E7 and LAMP-1 conferred superior tumor protection in a mouse

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model (Ji et al., 1999; Smahel et al., 2001), and increased CD4þ and CD8þ T cell responses (Ji et al., 1999). In another approach, minigenes encoding Th epitopes from LCMV were linked to the 20-amino acid C-terminal tail of lysosomal integral membrane protein-II (LIMP-II) (Rodriguez et al., 2001). The potential advantage of LIMP-II is that it moves directly from ER to the lysosomal compartment, directed by residues in its C-terminal tail. Using this strategy, induction of a CD4þ T cell response to the NP309–328 epitope of LCMV was greatly enhanced as assessed by IFN-g production. However, the same design did not improve the CD4þ T cell response to the GP61–80 epitope of LCMV, possibly due to rapid degradation of this epitope by cathepsin D in lysosomes. 5. Technologies for Enhanced DNA Vaccine Delivery Conventional DNA vaccine delivery by injection of naked plasmid DNA in saline solution results in inefficient uptake of DNA by myocytes and poor transfection of APCs, especially in large animals and in primates (Dupuis et al., 2000). One approach for improving in vivo transfection is to use electroporation following inoculation of plasmid DNA. Electroporation has two consequences: first, formation of inverted hydrophilic pores in the cell membrane, and second, electrophoresis of the DNA into the cell (Satkauskas et al., 2002). The outcome is to increase DNA uptake and protein expression by cells of skin, muscle, liver, and tumors (Bigey et al., 2002; Smith and Nordstrom, 2000). When applied to DNA vaccination, electroporation improves vaccine potency with faster induction and a higher level of immune response against viral and bacterial antigens in mice, guinea pigs, rabbits, and pigs (Babiuk et al., 2002; Drabick et al., 2001; Dupuis et al., 2000; Selby et al., 2000; Tollefsen et al., 2002; Widera et al., 2000). Electroporation also increases the efficacy of antimelanoma DNA vaccines in murine models (Kalat et al., 2002; Mendiratta et al., 2001). Generally, there is a correlation between the level of gene expression and the magnitude of induced immunity. However, application of electrical current per se may contribute to the immune enhancement. Electroporation may induce some inflammatory responses with release of cytokines and migration of antigen-presenting cells to the site of increased antigen production, thereby acting synergistically to enhance immune responses. While electroporation is being used clinically to increase uptake of chemotherapeutic drugs (Mir, 2001), so far it has not been used in a clinical trial of DNA vaccination. Another strategy for improving in vivo plasmid DNA uptake is to use biodegradable microparticle (<10 mM) delivery technology. Plasmid DNA can be either encapsulated in a poly(lactide-co-glycolide) (PLG) microsphere or adsorbed onto the surface of cationic PLG microparticles. Both procedures are able to augment the antibody and cellular immune responses of DNA vaccines delivered via mucosal (Jones et al., 1997; Singh et al., 2001) or i.m.

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routes (McKeever et al., 2002; O’Hagan et al., 2001; Singh et al., 1997) in both mice and in larger animal species such as guinea pigs and rhesus macaques (O’Hagan et al., 2001). Adsorption of DNA to the microparticle surface appears to be less damaging to DNA and may be a preferable option (O’Hagan et al., 2001). Possible mechanisms for the enhancement include reduced enzymatic DNA degradation and prolonged antigen expression, and perhaps more importantly, phagocytosis of plasmid/PLG microparticles and subsequent antigen presentation by professional APCs (Denis-Mize et al., 2000; Lunsford et al., 2000). PLG is a biocompatible and biodegradable material with an extensive record of safe use in humans; DNA vaccine delivery via microparticles may thus offer a promising technology for clinical application. 6. Alphavirus-Based Cancer Vaccines Another strategy to increase performance of DNA vaccines is to deliver the genes via viral vectors. Alphaviruses are positive-strand RNA viruses capable of replicating in many vertebrate and invertebrate cells. The alphavirus genome is 12 kb in length and is divided into two open reading frames (ORFs). The first ORF located in the 50 two thirds of the genome encodes four nonstructural proteins (nsP1–nsP4) that function as a replicase to transcribe negativestrand as well as positive-strand viral RNA. The second ORF is located in the 30 one third of the genome and encodes the viral structural proteins. A subgenomic (26S) promoter is located between the two ORFs in the negative strand. RNA replication occurs via synthesis of a negative strand of the genomic RNA. This is used as the template for transcription of additional full-length genomic RNA, and for transcription of a positive-strand subgenomic RNA encompassing the second ORF by the nonstructural proteins from the 26S promoter. The subgenomic RNA can accumulate to levels as high as 106 copies per cell. For vaccine purpose, alphavirus replicon vectors retain the sequence coding for the nonstructural proteins, but a heterologous gene replaces the genes encoding the viral structural proteins. Most alphaviral replicon vaccines currently being developed are based on Sindbis (SIN) virus, Semliki Forest virus (SFV), or Venezuelan equine encephalitis (VEE) (Rayner et al., 2002). The recombinant replicon vaccines can be delivered as recombinant viral particles, in vitro transcribed RNA, or plasmid DNA with initial transcription of the RNA genome under the control of a eukaryotic promoter. Many studies have shown that replicon vaccines encoding tumor as well as microbial antigens are significantly more potent than conventional plasmid DNA vaccines. For example, Cheng et al. (2001b, 2002) showed, using HPV–E7 tumor antigen as a model and various gene fusion strategies, that delivery of the tumor antigen as Sindbis replicon RNA, DNA, or viral particles can markedly enhance antitumor immunity. The enhancement included increased CD4þ , CD8þ , and NK cell activities,

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depending on the form of the vaccines. Daemen et al. (2000) constructed recombinant SFV expressing HPV E6 and E7 antigens (SFV–E6E7). They found that immunization of mice with SFV–E6E7 recombinant viral particles resulted in efficient activation of HPV-specific CTL activities, and protection from HPV–E7 expressing tumor cells. Recombinant SFV vectors expressing the P815A antigen (a murine cancer-testis antigen) were also able to induce strong CTL responses and protect mice against P815 tumor challenge (Colmenero et al., 1999). Similarly, a recombinant VEE expressing HPV–E7 was constructed and tested in mice (Velders et al., 2001a). Vaccination with the VEE–HPV–E7 vector induced CD8þ T cell responses as assessed by IFN-g production, tetramer staining, and cytotoxicity assays. Furthermore, vaccination completely prevented tumor growth and was even effective in eliminating 7-day established tumors. When b-galactosidase was used as a model tumor antigen, it was shown that vaccination with replicon vaccines in either RNA form (SFV–LacZ) (Ying et al., 1999) or as DNA plasmid (pSIN–LacZ) (Leitner et al., 2000) could elicit strong b-gal-specific humoral and cytotoxic T cell responses. Prevaccination protected mice from tumor challenge, and therapeutic treatment could significantly prolong the survival of mice with established tumors. Compared to conventional plasmid vaccines, DNA replicons were as effective at doses 100- to 1000-fold lower. More significantly, a recent report by Leitner et al. (2003) further showed that alphavirus-based DNA vaccination could effectively induce protective immunity against a nonmutated, tumor-associated self-antigen. Vaccination with a pSIN plasmid expressing the melanoma-associated antigen, tyrosinase-related protein 1 (TRP-1), was as effective as a plasmid encoding the xenogeneic human TRP-1 in breaking immunological tolerance (Leitner et al., 2003). It was initially thought that the self-replicating nature of alphaviruses would drive vigorous self-amplification of the viral RNA genome, leading to highlevel antigen production. There is indeed evidence for substantial amplification of viral RNA in in vitro transfected cells (Ying et al., 1999). However, antigen expression in vitro or in vivo was not necessarily increased (Hariharan et al., 1998; Leitner et al., 2003). The elegant study of Leitner et al. (2003) instead indicated that production of double-stranded RNA, a potent inducer of innate immunity, and increased apoptosis of transfected cells contributed more significantly to the enhanced vaccine potency. As discussed previously, apoptosis may enhance antigen presentation by dendritic cells and significantly improve the efficacy of DNA vaccines (Chattergoon et al., 2000; Sasaki et al., 2001). 7. Prime-Boost It is generally accepted that current immunization strategies, with protein or attenuated/inactivated virus, require booster vaccinations to maintain or improve immunity. In the context of gene-based vaccines there is now a

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large body of evidence in animal models demonstrating the success of heterologous prime-boost protocols (Ramshaw and Ramsay, 2000). This strategy entails priming with DNA and then boosting via sequential delivery of the same recombinant antigen delivered in a different vaccine vector (such as a replication-restricted recombinant virus), or as recombinant protein. In this way it is possible to generate higher levels of immunity when compared with homologous vaccination with DNA, recombinant viral vectors, or protein. Following DNA vaccination, improved humoral responses have been demonstrated when boosting with either recombinant protein (Babiuk et al., 2002; Jones et al., 2001; Tanghe et al., 2001) or recombinant viral vectors (Eo et al., 2001; Meseda et al., 2002; Ramshaw and Ramsay, 2000). Boosting with replication-restricted recombinant virus can also lead to enhanced T cell responses, including CD8þ CTL (Hanke and McMichael, 1999; Hanke et al., 1998, 1999; Robinson et al., 1999; Schneider et al., 1998). This may be particularly significant for the immunotherapy of cancer because the intensity of the immune response appears to determine tumor clearance (Perez-Diez et al., 2002). This vaccination strategy has led to enhanced immunogenicity and protective efficacy in a range of disease models, including HIV (Amara et al., 2001; Kent et al., 1998; Robinson et al., 1999), malaria (Jones et al., 2001; Schneider et al., 1998; Sedegah et al., 2000), and cancer (Greiner et al., 2002; Grosenbach et al., 2001). While the precise immunological mechanisms responsible for the efficacy of this approach have yet to be elucidated, several explanations have been proposed to account for the synergistic effect of this strategy. The simple, nonreplicating nature of DNA vaccines ensures that the immune response will become focused on vaccine-encoded antigens (Harrington et al., 2002). In addition, the immunostimulatory nature of CpG elements within the bacterial plasmid backbone will lead to preferential activation of CD4þ Th1 cells, thereby orienting the immune response in a particular direction. In the mouse, for example, this orientation will have a propensity to induce immunoglobulin of the IgG2a subclass and will also favor CD8þ CTL induction. It has also been suggested that the low, but persistent nature of antigen expression following DNA vaccination might select for responding T cells with higher affinity TCRpeptide/MHC interactions (Busch and Pamer, 1999; Estcourt et al., 2002; Rees et al., 1999). Subsequent immunization with a recombinant nonreplicating viral vector, expressing the same antigen, boosts this immunity. Again, the immunological mechanisms responsible for this effect are unclear, although they probably include a combination of increased antigen expression, effective delivery of antigen to the processing and presentation machinery of transfected or infected cells, and the inflammatory response associated with delivery of an immunogenic viral vector (Ramshaw and Ramsay, 2000; Zavala et al., 2001).

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While this approach shows promise in the field of infectious diseases, with several immunization strategies entering clinical trials (Doolan and Hoffman, 2001; Hanke and McMichael, 2000), it is not without its limitations. DNA vaccines are considered to be safe for use in humans; however, the safety of many recombinant viral vaccines has not been sufficiently tested. Certain vectors (e.g., modified vaccinia virus Ankara [MVA]), do appear to be suitable for use in humans. MVA is replication restricted and appears to be safe following immunization of approximately 120,000 humans, including high-risk groups, during the global program to eradicate smallpox (Blanchard et al., 1998; Mahnel and Mayr, 1994; Mayr et al., 1978); testing in animal models, including irradiated and neonatal mice confirm the avirulence and safety of this vector (Mayr et al., 1978). Such safety concerns will be paramount in the setting of cancer immunotherapy, since many patients will be immunocompromised due to tumor or myeloablative treatment regimes. Schneider et al. (1998) demonstrated enhanced boosting of a DNA-primed CTL response when using recombinant MVA, compared with a virulent strain of recombinant vaccinia virus. This may be due to the deletion of a number of viral immune evasion genes from the MVA genome, including soluble cytokine receptors (e.g., IFN-g, TNF, and IFN-a/b) and the CC chemokine-binding receptor (Alcami and Koszinowski, 2000; Blanchard et al., 1998). Despite an inability to replicate in most mammalian cells, this could explain the enhanced immunogenicity of MVA, making it the vector of choice for many prime-boost studies in the field of infectious diseases. However, the immunogenicity of many viral vectors can act as a double-edged sword. The first problem is that preexisting immunity to vector-encoded antigens can neutralize the virus, impairing responses to vaccine-encoded antigen (Cooney et al., 1991; Kundig et al., 1993; Palmowski et al., 2002; Rooney et al., 1988; Schneider et al., 1998). This is particularly pertinent in the current climate, with many countries prepared to immunize their citizens against smallpox, to counter the threat of bioterrorism. This could negate the use of vaccinia-based vectors for human prime-boost vaccination programs of infectious diseases and cancer. Even where human subjects do not have preexisting immunity to a particular viral vector, its administration could negate further use. This factor could limit application of the prime-boost approach to cancer immunotherapy, where the immunosuppression of many patients and the low immunogenicity of many tumor antigens suggest that it will probably be necessary to give patients multiple booster vaccinations in order to generate and maintain tumor-specific responses. An additional concern is that highly immunogenic viral vectors may drive coexpression of viral proteins containing potentially immunodominant T cell epitopes. This could prevent the immune response from focusing exclusively on ‘‘weak’’ vaccine-encoded tumor antigens (Harrington et al., 2002; Yewdell and Bennink, 1999).

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For infectious diseases, a recent strategy to induce an even more effective CD8þ T cell response has been to prime with one recombinant viral vector and then boost with another, replacing the DNA vaccine component altogether (Gilbert et al., 2002; Hanke et al., 2003). Although successful, this strategy again relies on immunization with different recombinant viruses to overcome the problem of preexisting immunity to viral antigens. However, the development of alphaviral vectors may offer one solution, since removal of genes encoding viral structural proteins reduces vector immunogenicity, allowing the same vaccine to be used on multiple occasions, for both the priming and boosting phases (Hanke et al., 2003) (see earlier). 8. RNA Vaccines Vaccination with antigen-encoding RNA is a relatively new concept. It is possible to induce immune responses by direct in vivo delivery of RNA-encoding single antigens (Conry et al., 1995; Hoerr et al., 2000; Martinon et al., 1993; Qiu et al., 1996; Wolff et al., 1990; Zhou et al., 1999), entire virus (Vassilev et al., 2001), or in vitro-generated self-replicating recombinant RNA of alphaviruses (discussed earlier). Most recent work has, however, focused on using RNA to transfect DCs in vitro for subsequent vaccination (Mitchell and Nair, 2000; Sullenger and Gilboa, 2002). Dendritic cells are the most potent professional antigen-presenting cells, and clinical grade DCs are readily available (Fong and Engleman, 2000). To deliver antigens to DCs, peptides, proteins, DNA, tumor lysates, or even cell fusion have been used. Transfecting DCs with antigenencoding mRNA offers another simple but efficient method to load DCs with antigens (Boczkowski et al., 1996). RNA transfection can be achieved by incubation of DCs with RNA as cationic lipid complex or in naked form, and electroporation can further improve transfection efficiency (Saeboe-Larssen et al., 2002). mRNA can be isolated from tumor cells or synthesized in vitro from cDNA templates. Vaccination with tumor RNA-transfected DCs can induce CTL responses and antitumor immunity in several preclinical murine models (Ashley et al., 1997; Boczkowski et al., 1996, 2000; Granstein et al., 2000; Koido et al., 2000; Nair et al., 2000; Zhang et al., 1999). DCs generated from healthy human subjects or from cancer patients and transfected with tumor RNA can also activate CTLs and kill tumor cells in vitro (Boczkowski et al., 2000; Heiser et al., 2000, 2001a, 2001b; Nair et al., 1998, 2000; Saeboe-Larssen et al., 2002; Strobel et al., 2000; Su et al., 2001; Thornburg et al., 2000; Van Tendeloo et al., 2001; Weissman et al., 2000). A recent phase I clinical trial in patients with metastatic prostate cancer showed that vaccination with prostatespecific antigen (PSA) RNA-transfected DCs elicited PSA-specific T cell responses in all patients, and significantly reduced serum PSA velocities in some patients (Heiser et al., 2002). Apart from efficient antigen loading, another potential advantage of RNA transfection of DCs is that RNA encoding single

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or multiple tumor antigens can be isolated and amplified from a small number of tumor cells. RNA transfection may thus represent a convenient and powerful technology for DC-based tumor vaccines.

IV. Concluding Summary

Tumor vaccines able to deliver specific antigen to the immune system are now available and are beginning to stand the test of performance in human subjects. Target antigens are being defined, and sophisticated methods to measure induced specific responses are available. Initially, therefore, outcome is measurable in terms of immune parameters. This allows rapid evaluation of one level of efficacy prior to assessing clinical value. Our task is to rouse immunity against weak or tolerizing tumor antigens, and, in order to succeed, we have to build vaccines that contain not only antigen, but also additional essential components. Adjuvants to activate innate immunity are clearly required, and the definition of the Toll-like receptors is opening possibilities for selective stimulation. Delivery of antigen to dendritic cells is a second goal and can be achieved either directly or via gene-based vaccines. Finally, there is a perceived need to activate high levels of T cell help to promote and maintain antibody, CD4þ , or CD8þ effector pathways. The easiest way to build components into a vaccine is to use genetic technology, and therefore gene-based vaccines are likely to have a real future. Physical means to improve delivery and potential combinations with viral vector-mediated delivery may be required to optimize performance. Opportunities to codeliver genes encoding cytokines, chemokines, and other molecules are there. It will take time to exploit the new genetic information and technology, and tailoring of vaccines for specific target antigens will be required. Provided flexible and rapid evaluation in pilot clinical trials is allowed at reasonable cost, design will progress to the stage where tumor vaccines will be a reality. Vaccination of patients is likely to succeed mainly in the setting of minimal residual disease. However, transfer of immunity from vaccinated donors of transplants, or from cells expanded in vitro, should also have a place. Vaccination could enhance the already successful passive immunotherapies being used to attack residual leukemic cells or dangerous viruses in immunosuppressed patients. A successful immune response should provide continuous surveillance against emergent tumor, and this would be a major contribution to the treatment of cancer.

Acknowledgments We thank the Leukaemia Research Fund, Tenovus, and Cancer Research UK for their support.

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advances in immunology, vol. 82

Immunotherapy of Allergic Disease R. VALENTA,* T. BALL,* M. FOCKE,* B. LINHART,* N. MOTHES,* V. NIEDERBERGER,{ S. SPITZAUER,{ I. SWOBODA,{ S. VRTALA,* K. WESTRITSCHNIG,* AND D. KRAFT* *Division of Immunopathology, Department of Pathophysiology { Department of Otorhinolaryngology {Clinical Institute for Medical and Chemical Laboratory Diagnostics University of Vienna, Medical School, A-1090 Vienna, Austria

I. Introduction

The term allergy was introduced by Clemens von Pirquet in 1906 to describe overwhelming pathological reactions of the body due to intercurrent contact with antigens (von Pirquet, 1906). More than 50 years later Coombs and Gell (1975) proposed a first detailed classification of allergic reactions in four types based on defined underlying pathomechanisms. We know today that the four principal types of allergic reactions (Types I–IV) described by Coombs and Gell do not occur in a mutually exclusive manner and there is now much more detailed information available about the molecular and cellular players in allergic reactions. However, the classification of Coombs and Gell still summarizes essential features of allergic reactions leading to tissue damage and hence is extremely useful for describing the mechanisms of human hypersensitivity disease. When we discuss the immunotherapy of allergic diseases in this chapter we will restrict ourselves to the most common form of allergic disease in humans, which is actually characterized by Type I reactions as described by Coombs and Gell. The hallmark of Type I allergy is the formation of a unique class of antibodies, that is, immunoglobulin E (IgE) antibodies against harmless antigens (i.e., allergens). IgE antibodies were not characterized until 1966 because of their extremely low concentration in serum and other body fluids (Ishizaka et al., 1966; Johansson and Bennich, 1967). However, upon contact with incremental doses of the corresponding allergens, they can cause severe inflammatory reactions through the activation of various cells of the immune system, especially mast cells and basophils (Kawakami and Galli, 2002; Marone et al., 2002; Turner and Kinet, 1999). In 1921, long before the characterization of IgE antibodies, Prausnitz and Ku¨stner (1921) demonstrated that the allergic reaction depends on three factors: the disease-eliciting allergen; a transferable serum factor, later identified as IgE antibodies, that distinguishes allergic patients from healthy persons; and a tissue factor (i.e., mast cells) that can be found in all individuals. Type I allergy affects more than 25% of the population and shows a continuously increasing prevalence (Wu¨thrich et al., 1995). The manifestations of Type I 105 Copyright 2004, Elsevier Inc. All rights reserved. 0065-2776/04 $35.00

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allergy can be very diverse, ranging from mild to severe forms (e.g., allergic rhinoconjunctivitis, food allergy, dermatitis, asthma, fatal anaphylactic shock), and may occur locally and/or systemically. All of the diverse phenotypes of Type I allergy are, however, associated with the production of allergen-specific IgE antibodies and hence can be precisely diagnosed and distinguished from other forms of allergic diseases by the demonstration of allergen-specific IgE antibodies in serum or other body fluids and by the elicitation of immediate tissue reactions by provocation testing with allergens (Johansson et al., 2001). In this chapter we will discuss recent advances in the field of immunotherapy of IgE-mediated allergies because IgE-mediated allergies belong to the most common forms of allergic diseases with relatively well-defined immunological mechanisms. This chapter will focus on data obtained in allergic patients but, on certain occasions, will also refer to results from animal models with a close connection to human disease. First we will review the current knowledge regarding the immunological mechanisms operative in IgE-mediated allergy with special reference to disease manifestations and the time course of disease development. In addition to allergen-specific immunotherapy, several other forms of immunotherapy have been recently developed for the treatment of allergy. We will therefore summarize the general forms of immunotherapy without antigen specificity according to their molecular and cellular targets. Special emphasis will be given to results that add to our current knowledge about the mechanisms of human allergic disease. Next we will summarize the development of allergen-specific immunotherapy and provide an overview of immunological mechanisms that have been described in clinical studies performed in allergic patients. These observations suggest that different, but not mutually exclusive, immunological mechanisms may be operative in allergenspecific immunotherapy. Based on these mechanisms, several concepts have been developed to improve allergen-specific immunotherapy. These concepts are based on modifications of the mode of administration, of the adjuvant, and of the antigen. Due to recent rapid developments in the field of allergen characterization, we will finally focus on possible improvements of allergen-specific immunotherapy through the use of modified antigens that can be obtained by recombinant DNA technology and synthetic peptide chemistry. In the end, a possible scenario for the development of prophylactic vaccination concepts based on the use of recombinant and synthetic allergy vaccines will be created. II. Time Course and Pathophysiology of Type I Allergy

A. The Concept of Allergic Sensitization Sensitization to allergens is the initial key event leading to the development of allergic disease. Table I summarizes some of the crucial factors involved in the sensitization process. These factors can be classified as environmental

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TABLE I Factors in Allergic Sensitization a Environment Eliciting factors

Promoting/inhibiting factors

Allergens . Immunogenicity . Dose . Route/site

Adjuvant effect Hygiene Endotoxin Bacterial infection Parasites Vaccination . Allergen specific

Atopic predisposition . Tendency for IgE production

Age Host a

Several environmental factors can promote or inhibit the development of an allergic immune response. Properties of allergens may influence their disease-eliciting potency, and numerous host factors may determine the susceptibility of a subject to develop allergy.

factors, which are required for the induction of sensitization (e.g., allergens), and those that act as promoting or inhibiting factors. The susceptibility of the host may be dictated by allergen-specific factors (e.g., HLA restriction of allergen-specific immune responses), by factors influencing the individual’s tendency to mount IgE responses, and by age. Analysis of immune responses in allergic children, as well as in experimental animal models of allergy, suggests that allergic sensitization is induced by early, postnatal allergen contact (Niederberger et al., 2002; Schiessl et al., 2003; Wahn et al., 1997). Some evidence also points to the possibility that prenatal maternal allergen contact may lead to the onset of allergy in offspring, but this is controversial (Edelbauer et al., 2003; Herz et al., 2001; Lange et al., 2002; Melkild et al., 2002; Platts-Mills et al., 2001, 2003; Victor et al., 2003). The susceptibility of the host to develop allergy, termed atopic predisposition, is controlled by a variety of genetic factors, of which some seem to be allergen related (i.e., allergen-specific HLA restrictions), whereas others affect the host’s general ability to mount IgE responses upon antigen contact (e.g., cytokine expression) (Ansari et al., 1989; Daniels et al., 1996; Kim et al., 2002; Lee et al., 2000; Lonjou et al., 2000; Marsh et al., 1982a, 1982b, 1994; Sandford et al., 1993; Texier et al., 2002). In addition to the previously-mentioned host factors, several environmental factors may influence whether an allergic immune response develops. However, controversial evidence exists on how factors affecting the innate immune system (e.g., bacillis Calmette–Gue´ rin [BCG] vaccination, contact

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with endotoxins, bacterial infection, parasitic infestation, hygiene) influence the onset of allergic immune responses (Wills-Karp et al., 2001). For example, the hygiene hypothesis would predict that poor hygienic conditions and endotoxin contact in early life would prevent the development of allergic immune responses, but recent data suggest that in countries with poor hygienic conditions (e.g., Central Africa), allergies are a common problem (BraunFahrla¨ nder et al., 2002; Westritschnig et al., 2003). Likewise, greatly varying and sometimes opposite effects of BCG vaccination or parasite infestation on the development of allergy have been reported (Alm et al., 1997; Herz et al., 1998, 2000, 2001; Shirakawa et al., 1997; Sibanda 2003; Van den Biggelaar et al., 2000). In this context it will be interesting to obtain, in addition to the epidemiological data, more results from defined experimental animal models that precisely distinguish factors promoting allergy from those preventing it. Considerable progress has been made regarding the characterization of those antigens (i.e., allergens) that induce and maintain allergic diseases in humans (Valenta and Kraft, 2002). It has been shown that most allergens fall into the class of proteins or glycoproteins with molecular masses between 5 and 80 kDa (Valenta and Kraft, 2001). IgE antibodies react primarily with proteins, whereas carbohydrate epitopes represent rather rare targets for IgE antibodies of uncertain biological significance (van Ree, 2002). Using recombinant DNA technology, cDNAs coding for the most common allergens have recently been isolated and allowed to reveal their identities, as well as their structures. Moreover, recombinant allergens mimicking their natural counterparts have allowed us to study in detail allergen-specific immune responses and to understand the structural basis for allergic cross-reactivity. The three-dimensional structures of many important allergens have been determined, and epitope mapping studies provide evidence that those allergens causing respiratory allergies contain mainly conformational IgE epitopes (reviewed in Valenta and Kraft, 2001). It has not been possible as yet to determine structural motifs or biological properties that predispose the allergenic nature of an allergen. Allergens may be composed of all a-helical, mixed a-helical and b-sheet structure, or all b-sheet structure and include storage proteins, cytoskeletal proteins, enzymes, calcium-binding proteins, or defense proteins. Results obtained from the molecular and epitope analysis of respiratory allergens indicate that allergic sensitization occurs mainly against intact, soluble, folded, and immunogenic proteins that are released from respirable particles in very low concentrations. Allergens that are released from their primary allergen source may also bind to nonallergenic environmental particles with a possible adjuvant effect. In this context it was reported that allergens coupled to diesel exhaust particles can induce strong IgE responses in animals (Fujieda et al., 1998). IgE recognition of many respiratory allergens not only requires the presence of conformational epitopes (Laffer et al., 1996; Vrtala et al., 1997), but was

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even found to depend critically on a certain conformational state of the allergen (Akdis et al., 1998), emphasizing the importance of conformational IgE epitopes. For example, it has been shown that certain calcium-binding allergens are primarily recognized in their calcium-bound, but not in their calcium-free, apoform, suggesting that the initial sensitization has occurred against a certain allergen conformation (Seiberler et al., 1994; reviewed in Valenta et al., 1998; Verdino et al., 2002). The preferential recognition of certain conformational allergen epitopes by IgE antibodies hence allows us to reconstruct some of the molecular events during primary sensitization in patients. On the other hand, it seems possible to construct allergen derivatives with reduced IgE binding capacity and low allergenic activity for immunotherapy by rational disruption of the allergen’s structure, a subject that will be discussed in the context of allergen-specific immunotherapy. Other important allergen-related factors for sensitization are the dose, route, and mode of allergen contact (Constant et al., 2000; Kolbe et al., 1991; McCusker et al., 2002). In principle, there are at least three possibilities for allergic sensitization of patients, depending on the organs involved. They include respiratory, gastrointestinal, and skin-mediated sensitization. Since the induction of robust IgE responses in experimental animal models requires the injection of adjuvant-bound allergen, primary sensitization against low doses of soluble allergen as it occurs in patients is difficult to study in animals. The question of whether respiratory, gastrointestinal, or skin-mediated sensitization represents the predominant event in allergic sensitization can therefore be reconstructed only indirectly. It is known that allergy to typical food allergens precedes respiratory allergy in children, but almost no experimental data from allergic patients are available that demonstrate that dietary intake of food allergens can induce and increase allergen-specific IgE responses (Kulig et al., 1999; Reininger et al., 2003). Interestingly, many allergic children grow out of food allergies and develop respiratory allergy later, raising the possibility that early intake of large amounts of food allergens may induce tolerance. Evidence for the possibility of skin-mediated sensitization is scarce (Beck and Leung, 2000). Moreover, it has been demonstrated that allergic immune responses in the skin are frequently directed against respiratory or food allergens and that skin symptoms either occur after dietary allergen intake or after primary respiratory sensitization (Reekers et al., 1999; van Reijsen et al., 1998; Werfel et al., 1999). The most important and frequent manifestations of IgE-mediated allergies affect the respiratory tract, and a large body of experimental evidence highlights the importance of the respiratory tract (especially the upper respiratory tract—the nose) in allergic sensitization, maintenance, and boosting of the allergic immune response (Durham et al., 1997; Henderson et al., 1975; McCusker et al., 2002; Naclerio et al., 1997; Simons,

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1999; Smurthwaite et al., 2001; Ying et al., 2001). We will therefore discuss allergic sensitization using respiratory allergens as examples. Figure 1 gives an overview of the allergen-specific immune response during sensitization and memory. Furthermore, it describes mechanisms of allergic inflammation in sensitized allergic patients. Respiratory allergens (e.g., allergens from pollen, dust mites, animals, and molds) are released from airborne particles (e.g., pollen) either after contact with mucosal surfaces by simple aqueous elution or via the release of small allergen-bearing and hence respirable particles (Grote, 1999; Grote et al., 2000; Suphioglu et al., 1992; Taylor et al., 2002). According to measurements of allergen concentrations in the environment, only tiny amounts of antigen are sufficient for the initiation of allergic sensitization (Baur et al., 1998). Highly efficient mechanisms of allergen presentation must therefore be operative during initial allergic sensitization. Experimental data from patients are rare because sensitization occurs very early in childhood and hence was not studied in detail in humans, and data from animal studies are difficult to transfer to the human situation. In principle, at least two molecular and cellular scenarios are possible. Studies in mouse models suggest that antigenpresenting cells (APC; e.g., dendritic cells) take up allergens and prime allergen-specific T cells, which may then activate B cells by cognate T cell–B cell interactions (Constant et al., 2000; Lambrecht, 2001; Masten and Lipscomb, 1999). However, since allergen-specific T and B cells recognize entirely different epitopes of the allergen, the APC involved in the initial sensitization process must present simultaneously T cell as well as B cell epitopes to act as a communication platform for B cells and T cells with specificity for the same allergen. In an alternative scenario in which allergenspecific B cells pick up the allergen via specific immunoglobulin, direct presentation to specific T cells would be possible and allow immediate interaction between the specific B cell and the specific T cell for the development of IgE and T cell responses (Constant et al., 2000). In atopic individuals, CD4þ Th2 cells produce preferentially cytokines such as interleukin (IL)-4 and IL-13 that promote the immunoglobulin class switch of B cells to IgE (Paul, 1987; Romagnani, 1997; Vercelli and Geha, 1992). As a consequence of sensitization, allergic patients produce allergen-specific IgE antibodies, whereas low levels of allergen-specific IgG antibodies can be found in allergic as well as in nonallergic individuals (Valenta and Ball, 1997). The presence of low levels of allergen-specific IgE antibodies and skin sensitivity without manifestation of clinical disease may precede allergic disease (Bodtger et al., 2003). The evolution of the allergen-specific antibody responses has recently been determined in children becoming allergic and provided evidence for a nonsequential, direct class switch mechanism to IgE (Niederberger et al., 2002). This observation and the fact that the hypervariable regions of the few allergen-specific

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IgE antibodies sequenced today showed relatively few somatic mutations suggest that IgE antibodies represent perhaps a rather early and primitive class of antibodies that may precede the development of allergen-specific IgG responses (Edwards et al., 2002; Flicker et al., 2002; Steinberger et al., 1996; S. Flicker and R. Valenta, unpublished data). The latter assumption is indeed supported by data obtained in mouse models and by the monitoring of the development of humoral immune responses in allergic children (Niederberger et al., 2002; Vrtala et al., 1998). B. Pathophysiology of the Established Allergic Memory Immune Response The previously described process of allergic sensitization leads to the establishment of an allergen-specific memory response on the humoral and cellular level (Fig. 1). Cells producing allergen-specific IgE antibodies can be detected in the peripheral blood of allergic patients (Dolecek et al., 1995; Steinberger et al., 1995, 1996). Interestingly, the allergen-specific IgE antibody production of these cells seems to be rather insensitive to cytokine stimulation (e.g., IL-4, IL-13) and cannot be abrogated by cytokine antagonists inhibiting a class switch to IgE (Dolecek et al., 1995; Steinberger et al., 1995). IgE-producing cells are also found in the mucosa of the respiratory tract (e.g., nasal and bronchial mucosa), and it has been demonstrated that seasonal allergen contact and nasal allergen exposure lead to strong increases of systemic allergen-specific IgE antibody levels, suggesting that allergen contact is a strong, if not the most important, stimulus for specific IgE production (Durham et al., 1997; Henderson et al., 1975; Naclerio et al., 1997; Simons, 1999; Smurthwaite et al., 2001; Ying et al., 2001). In addition to IgE-producing cells, allergen-specific T cells also seem to form a pool of long-lived memory T cells that respond to repeated allergen contact (Mojtabavi et al., 2002). These T cells can be detected specifically in the skin and peripheral blood of allergic patients via their T cell receptor sequences (Bohle et al., 1998; van Reijsen et al., 1997). Although the classical Th1/Th2 paradigm would predict a clear-cut distinction between allergic patients and nonallergic subjects based on their T helper cell and cytokine profile (Mosmann and Sad, 1996; Parronchi et al., 1991; Romagnani, 1997; Wierenga et al., 1991), several investigations demonstrate that allergen-specific T cells from allergic individuals also mount considerable interferon (IFN)-g responses (Byron et al., 1994; Hales et al., 2000; Looney et al., 1994; Oldfield et al., 2001; van Neerven et al., 1994a). Likewise, allergen-specific Th2 cells have been found in nonallergic patients, suggesting a less clear-cut distinction of the allergen-specific T cell responses in atopic versus nonatopic persons as originally anticipated (Ebner et al., 1995; van Neerven et al., 1994b). In accordance with the classification of Coombs and Gell, the immediate reaction represents the hallmark of Type I allergy (Fig. 1). Allergen-induced

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Fig 1 The allergic immune response (sensitization and memory) and mechanisms of allergic inflammation. The allergic immune response (top and middle panels). During initial sensitization, contact with minute amounts of intact, soluble allergen on mucosal surfaces, particularly of the respiratory tract, leads to allergen uptake by antigen-presenting cells (e.g., dendritic cells) and/or immunoglobulin-mediated recognition of allergens by B cells. In allergic individuals, T cells differentiate preferentially toward the TH2 phenotype, which may be promoted by microenvironmental factors of the mucosa, the genetic background of the individual, the site of allergen contact,

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cross-linking of IgE antibodies bound via high-affinity receptors to mast cells and basophils leads to the rapid release of inflammatory mediators (e.g., histamine, leukotrienes), but mast cells and basophils are also potent sources of Th2 cytokines (e.g., IL-4) (Bradding et al., 1993; Brunner et al., 1993; Burd et al., 1989). Most of the immediate symptoms occurring a few minutes after allergen contact in sensitized patients (e.g., rhinitis, conjunctivitis, asthma, edema, urticaria) can be attributed to the rapid release of inflammatory mediators from mast cells and basophils. It is, however, becoming increasingly clear that Type IV-like T cell-mediated reactions can also occur in allergic individuals (Haselden et al., 1999; Oldfield et al., 2001). In this context it has been reported that patients suffering from severe and chronic forms of allergy (e.g., atopic dermatitis, chronic asthma) exhibit more pronounced T cell responses to allergens compared with patients suffering only from allergic rhinoconjunctivitis (Rawle et al., 1984). The occurrence of T cell-dependent late responses is thus perceived as a sign of severe and chronic allergic disease, whereas in most allergic patients, classic immediate, Type I-like reactions dominate. It is well established that cross-linking of FceRI-bound IgE antibodies on mast cells and basophils by allergens induces the rapid release of inflammatory mediators and Th2 cytokines (Turner and Kinet, 1999). However, recently additional effects of monomeric IgE on mast cells have been reported. In this context it has been demonstrated that IgE promotes the survival of mast cells and also of FceRI-bearing antigen-presenting cells by preventing apoptosis (Asai et al., 2001; Kalesnikoff et al., 2001; Katoh et al., 2000) and it was also demonstrated that high IgE concentrations upregulate the expression of FceRI (Kubo et al., 2001; MacGlashan et al., 2001; Saini et al., 2000; Yamaguchi et al., 1999). A close linkage between IgE and T cells has been established by demonstrating that various cells express (e.g., B cells, monocytes) the low-affinity receptor for IgE, FceRII, or the high-affinity allergen dose and conformation, as well as by other factors. The TH2 cells secrete factors (e.g., IL-4, IL-13) that favor the immunoglobulin switch of specific B cells to immunoglobulin E. The process of allergic sensitization leads to the establishment of an allergen-specific memory T cell pool, as well as of an IgE memory B cell pool, both of which can be strongly activated by contact with allergens. Mechanisms of allergic inflammation (bottom panel). The manifestations of allergic disease occur primarily as immediate and, under certain circumstances, as late/chronic symptoms. Immediate reactions are caused by the cross-linking of effector-cell (i.e., mast cell, basophil)bound IgE by allergens, leading to the release of biologically active mediators (e.g., histamine, leukotrienes) and proinflammatory cytokines (e.g., IL-4). Late and chronic reactions are caused by the presentation of allergens to T cells, which then become activated, proliferate, and release cytokines. Allergen presentation to T cells can occur in a highly efficient manner by IgE-dependent mechanisms using FceRI or FceRII on APCs. In addition, allergens can be taken up by APCs in an IgE-independent manner and then be presented to T cells. Activated TH2 cells release IL-4, IL-13, and IL-5 and thus cause tissue eosinophilia, but also TH1-cells secreting IFN-g may be activated by allergens, especially during late phase reactions. APC, antigen-presenting cell; DC, dendritic cell; TCR, T cell receptor.

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IgE receptor, FceRI (e.g., moncytes, dendritic cells, eosinophils, thrombocytes, epithelial cells) (Campbell et al., 1998; Hasegawa et al., 1999; Gounni et al., 1994, 2001; Kita et al., 1999; Smith et al., 2000) and can use receptor-bound IgE for highly efficient presentation of allergens to T cells (Kraft et al., 1998; Maurer et al., 1996; Mudde et al., 1990; van der Heijden et al., 1993). In addition, it has been found that histamine, a mast cell and basophil-derived mediator, can regulate T cell responses (Jutel et al., 2001) and that T cell-derived cytokines can prime mast cells and basophils for enhanced degranulation (Bischoff et al., 1990a,b). All these findings demonstrate that IgE-mediated effects do not exclusively comprise the classic Type I reaction described by Coombs and Gell (i.e., allergen-induced mast cell degranulation) but also have effects on T cell activation and thus may be of importance for the late responses and chronic manifestations of allergic disease. The chronic manifestations of allergic disease (e.g., chronic allergic asthma, atopic dermatitis) are dominated by T cells and are characterized by the influx of eosinophils (Larche´ et al., 2003; Leung, 2000). Interestingly chronic allergen exposure of the skin in patients with atopic dermatitis induces not only the influx of CD4þ Th2 cells, but also of Th1 cells secreting IFN-g (Thepen et al., 1996). The latter finding is interesting in the context of recent findings, suggesting that IFN-g may induce keratinocyte apoptosis, a hallmark of eczematous diseases (Trautmann et al., 2000). The activation of allergenspecific T cells is strongly enhanced when allergens are presented via IgE antibodies bound to APCs via FceRI or FceRII, but it has also been demonstrated that IgE-independent activation of allergen-specific T cells can occur during late-phase reactions. The injection of T cell epitopecontaining peptides of the major cat allergen Fel d 1, which lacked IgE binding sites, was shown to induce late-phase reactions in cat-allergic patients in an MHCII-restricted manner (Haselden et al., 1999; Oldfield et al., 2001). These data demonstrate that non–IgE-dependent, T cell-mediated mechanisms can also play a role in late-phase allergic inflammation. T cell-mediated late reactions predominate in certain severe and chronic manifestations of allergic disease (e.g., atopic dermatitis) and are frequently associated with sensitization to a great variety of different allergens (i.e., polysensitization) (Leung, 2000). It should, however, be kept in mind that the frequent manifestations of atopy (e.g., allergic rhinoconjunctivitis, acute allergic asthma, urticaria, oral allergy syndrome) are caused preferentially by immediate reactions, whereas late-phase allergic reactions occur in fewer allergic patients. On the other hand, there is considerable evidence that untreated acute manifestations of allergy, especially under chronic allergen exposure, can progress into chronic forms and that manifestations in the upper respiratory tract may lead to symptoms in the deeper airways (Fuhlbrigge and Adams, 2003; Simons, 1999).

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III. Possible Targets for Immunotherapy of Allergic Disease

The major subject of this chapter is allergen-specific immunotherapy, an active vaccination approach based on the therapeutic administration of the disease-eliciting allergens. Before we discuss allergen-specific immunotherapy, we would like to briefly discuss other therapies for the treatment of IgEmediated allergies that are based on immunological strategies and hence may be referred to as immunotherapies. Table II summarizes these therapy forms according to the targeted structures and mechanisms. The aim of allergen-specific passive forms of immunotherapy is the inhibition of the interaction between allergens and IgE antibodies, which may be achieved at least in two ways. First, it is possible to produce allergen-specific antibodies or antibody fragments that may capture allergens before they can form complexes with IgEs. Such therapeutic allergen-specific antibodies may be administered locally into the target organs of allergy (e.g., nasal or bronchial mucosa, conjunctiva) to form a first line of defense against intruding allergens (reviewed in Valenta et al., 1997). Human antibodies with therapeutic potential have been isolated by classic tissue culture and combinatorial cloning technology using B cells from allergic patients as a source (Flicker et al., 2002; Lebecque et al., 1997; Sun et al., 1995; Visco et al., 1996). These antibodies were shown to inhibit the allergen–IgE interaction and to prevent allergen-induced basophil degranulation. Second, it has been shown that allergen-derived IgE-reactive haptens obtained by proteolytic digestion of allergen extracts, by recombinant DNA technology, by chemical approaches, or by synthetic peptide chemistry (e.g., mimotopes) bind to IgE, but due to monovalent IgE binding, fail to cross-link and activate effector cells (Attallah and Sehon, 1969; Ball et al., 1994; de Weck and Schneider, 1972; Jensen-Jarolim et al., 1998). When effector cell-bound IgE is saturated with IgE-reactive haptenic structures, complete allergens should fail to induce the release of biologically active mediators and thus inflammation will be suppressed. The studies mentioned document that allergen-specific passive therapy concepts are effective in reducing effector cell activation (e.g., basophil degranulation), but several technical problems need to be overcome before these strategies may enter clinical applications. For example, it will be necessary to review the complexity of the natural allergen and epitope repertoire in order to obtain a representative panel of blocking antibodies or hapten-like structures, which may be achieved by the application of combinatorial cloning and peptide library technology. In addition, it is likely that passive therapy strategies will be preferentially applied in the target organs of atopy and it will hence be necessary to develop technologies for preventing the rapid wash-out of therapeutic components and to avoid

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TABLE II Molecular and Cellular Targets for Immunotherapy of Allergy Target structure Allergens

Mechanism Active immunotherapy Passive immunotherapy Blocking antibodies

Haptens

IgE

Mimotopes Therapeutic administration of aIgE Induction of aIgE antibodies by immunization

Removal of IgE Receptors

Blockade of IgE/FceRI interaction

aFceRl aCD23

Cytokines

Co–cross-linking of FceRI and FcgRII IL-4 antagonists aIL-5, IL-12

IgEþ B cells T cell

Mast cell/basophil APC

Immune response modifier Inhibition of IgE production Anti-CD4 antibody Immunosuppressors

Inhibition of mediator release Immunosuppressors

Referencesa Bousquet et al., 1998 Flicker et al., 2002; Lebecque et al., 1997; Sun et al., 1995; Valenta et al., 1997; Visco et al., 1996 Attalah and Sehon, 1969; Ball et al., 1994; de Weck and Schneider, 1972; Hedin and Richter, 1982 Jensen-Jarolim et al., 1998 Heusser and Jardieu, 1997; MacGlashan et al., 1997; Milgrom et al., 1999 Haba and Nisonoff, 1987, 1990; Hellman et al., 1994; Rudolf et al., 1998; Vemersson et al., 2002; Zuercher et al., 2000 Dau, 1988; Laffer et al., 2001; Lebedin et al., 1991 Hamburger, 1975; Helm et al., 1988, 1997; Kelly et al., 1998; McDonnell et al., 1996; Naito et al., 1996; Vangelista et al., 1999 Nechansky et al., 2001 Dasic et al., 1999; Sherr et al., 1989 Daeron et al., 1995; Zhu et al., 2002 Borish et al., 2001; Grunewald et al., 1998 Bryan et al., 2000; Flood-Page et al., 2003; Leckie et al., 2000 Brugnolo et al., 2003 Yanagihara et al., 1994 Kon et al., 1998 Alexander et al., 1992; Fleischer, 1999; Kapp et al., 2002; Lock and Kay, 1996 Sperr et al., 1997; Triggiani et al., 1989; Majlesi et al., 2003; Zuberbier et al., 2001 Panhans-Gross et al., 2001 (continues)

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a

References for Table II: Alexander, A. G., Barnes, N. C., and Kay, A. B. (1992). Lancet 339, 324–328. Attallah, N. A., and Sehon, A. H. (1969). Immunochemistry 6, 609–619. Ball, T., Vrtala, S., Sperr, W. R., Valent, P., Susani, M., Kraft, D., and Valenta, R. (1994). J. Biol. Chem. 269, 28323–28328. Borish, L. C., Nelson, H. S., Corren, J., Bensch, G., Busse, W. W., Whitmore, J. B., and Agosti, J. M. (2001). J. Allergy Clin. Immunol. 107, 963–970. Bousquet, J. (1999). Allergy 54, 37–38. Brugnolo, F., Sampognaro, S., Liotta, F., Cosmi, L., Annunziato, F., Manuelli, C., Campi, P., Maggi, E., Romagnani, S., and Parronchi, P. (2003). J. Allergy Clin. Immunol. 111, 380–388. Bryan, S. A., O’Connor, B. J., Matti, S., Leckie, M. J., Kanabar, V., Khan, J., Warrington, S. J., Renzetti, L., Rames, A., Bock, J. A., Boyce, M. J., Hansel, T. T., Holgate, S. T., and Barnes, P. J. (2000). Lancet 356, 2149–2153. Daeron, M., Malbec, O., Latour, S., Arock, M., and Fridman, W. H. (1995). J. Clin. Invest. 95, 577–585. Dasic, G., Juillard, P., Graber, P., Herren, S., Angell, T., Knowles, R., Bonnefoy, J. Y., Kosco-Vilbois, M. H., and Chvatchko, Y. (1999). Eur. J. Immunol. 29, 2957–2967. Dau, P. C. (1988). J. Clin. Apheresis 4, 8–12. De Weck, A. L., and Schneider, C. H. (1972). Int. Arch. Allergy Appl. Immunol. 42, 782–797. Fleischer, A. B., Jr. (1999). J. Allergy Clin. Immunol. 104, 126–130. Flicker, S., Steinberger, P., Norderhaug, L., Sperr, W. R., Majlesi, Y., Valent, Plk Kraft, D., and Valenta, R. (2002). Eur. J. Immunol. 32, 2156–2162. Flood-Page, P. T., Menzies-Gow, A. N., Kay, A. B., and Robinson, D. S. (2003). Am. J. Respir. Crit. Care Med. 167, 199–204. Grunewald, S. M., Werthmann, A., Schnarr, B., Klein, C. E., Brocker, E. B., Mohrs, M., Brombacher, F., Sebald, W., and Duschl, A. (1998). J. Immunol. 160, 4004–4009. Haba, S., and Nisonoff, A. (1987). Proc. Natl. Acad. Sci. USA 84, 5009–5013. Haba, S., and Nisonoff, A. (1990). Proc. Natl. Acad. Sci. USA 87, 3363–3367. Hamburger, R. N. (1975). Science 189, 389–390. Hedin, H., and Richter, W. (1982). Int. Arch. Allergy Appl. Immunol. 68, 122–126. Hellman L. (1994). Eur. J. Immunol. 24, 415–420. Helm, B., Marsh, P., Vercelli, D., Padlan, E., Gould, H., and Geha, R. (1988). Nature 331, 180–183. Helm, B. A., Spivey, A. C., and Padlan, E. A. (1997). Allergy 52, 1155–1169. Heusser, C., and Jardieu, P. (1997). Curr. Opin. Immunol. 9, 805–814. Jensen-Jarolim, E., Leitner, A., Kalchhauser, H., Zurcher, A., Ganglberger, E., Bohle, B., Scheiner, O., BoltzNitulescu, G., and Breiteneder, H. (1998). FASEB J. 12, 1635–1642. Kapp, A., Papp, K., Bingham, A., Folster-Holst, R., Ortonne, J. P., Potter, P. C., Gulliver, W., Paul, C., Molloy, S., Barbier, N., Thurston, M., and de Prost, Y. (2002). J. Allergy Clin. Immunol. 110, 277–284. Kelly, A. E., Woodward, E. C., Chen, B. H., and Conrad, D. H. (1998). J. Immunol. 161, 6696–6704. Kon, O. M., Sihra, B. S., Compton, C. H., Leonard, T. B., Kay, A. B., and Barnes, N. C. (1998). Lancet 352, 1109–1113. Laffer, S., Hogbom, E., Roux, K. H., Sperr, W. R., Valent, P., Bankl, H. C., Vangelista, L., Kricek, F., Kraft, D., Gronlund, H., and Valenta, R. (2001). J. Allergy Clin. Immunol. 108, 409–416. Lebecque, S., Dolecek, C., Laffer, S., Visco, V., Denepoux, S., Pin, J. J., Guret, C., Boltz-Nitulescu, G., Weyer, A., and Valenta, R. (1997). J. Allergy Clin. Immunol. 99, 374–384. Lebedin, Y. S., Gorchakov, V. D., Petrova, E. N., Kobylyansky, A. G., Raudla, L. A., Tatarsky, A. R., Bobkov, E. V., Adamova, I. Y., Vasilov, R. G., Nasonov, E. L. et al. (1991). Int. J. Artif. Organs 14, 508–514. Leckie, M. J., Ten-Brinke, A., Khan, J., Diamant, Z., O’Connor, B. J., Walls, C. M., Mathur, A. K., Cowley, H. C., Chung, K. F., Djukanovic, R., Hansel, T. T., Holgate, S. T., Sterk, P. J., and Barnes, P. J. (2000). Lancet 356, 2144–2148. Lock, S. H., and Kay, A. B. (1996). Am. J. Respir. Crit. Care Med. 153, 509–514. MacGlashan, D. W., Jr., Bochner, B. S., Adelman, D. C., Jardieu, P. M., Togias, A., and Lichtenstein, L. M. (1997). J. Immunol. 158, 1438–1445. Majlesi, Y., Samorapoompichit, P., Hauswirth, A. W., Schernthaner, G. H., Ghannadan, M., Baghestanian, M., Rezaie-Majd, A., Valenta, R., Sperr, W. R., Buhring, H. J., and Valent, P. (2003). J. Leukoc. Biol. 73, 107–117. McDonnell, J. M., Beavil, A. J., Mackay, G. A., Jameson, B. A., Korngold, R., Gould, H. J., and Sutton, B. J. (1996). Nat. Struct. Biol. 3, 419–426. Milgrom, H., Fick, R. B., Jr., Su, J. Q., Reimann, J. D., Bush, R. K., Watrous, M. L., and Metzger, W. R. (1999).

(continues)

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TABLE II (continued) N. Engl. J. Med. 341, 1966–1973. Naito, K., Hirama, M., Okumura, K., and Ra, C. (1996). J. Allergy Clin. Immunol. 97, 773–780. Nechansky, A., Robertson, M. W., Albrecht, B. B., Apgar, J. R., and Kricek, F. (2001). J. Immunol. 166, 5979–5990. Panhans-Gross, A., Novak, N., Kraft, S., and Bieber, T. (2001). J. Allergy Clin. Immunol. 107, 345–352. Rudolf, M. P., Vogel, M., Kricek, F., Ruf, C., Zurcher, A. W., Reuschel, R., Auer, M., Miescher, S., and Stadler, B. M. (1998). J. Immunol. 160, 3315–3321. Sherr, E., Macy, E., Kimata, H., Gilly, M., and Saxon A. (1989). Eur. J. Immunol. 142, 481–489. Sperr, W. R., Agis, H., Semper, H., Valenta, R., Susani, M., Sperr, M., Willheim, M., Scheiner, O., Liehl, E., Lechner, K., and Valent, P. (1997). Int. Arch. Allergy Immunol. 114, 68–73. Sun, L. K., Fung, M. S., Sun, W. N., Sun, C. R., Chang, W. I., and Chang, T. W. (1995). Biotechnology 13, 779–786. Triggiani, M., Cirillo, R., Lichtenstein, L. M., and Marone, G. (1989). Int. Arch. Allergy Appl. Immunol. 88, 253–255. Valenta, R., Almo, S., Ball, T., Dolecek, C., Steinberger, P., Laffer, S., Eibensteiner, P., Flicker, S., Vrtala, S., Spitzauer, S., Valent, P., Denepoux, S., Kraft, D., Banchereau, J., and Lebecque, S. (1998). Int. Arch. Allergy Immunol. 116, 167–176. Vangelista, L., Laffer, S., Turek, R., Gronlund, H., Sperr, W. R., Valent, P., Pastore, A., and Valenta, R. (1999). J. Clin. Invest. 103, 1571–1578. Vernersson, M. Ledin, A., Johansson, J., and Hellman, L. (2002). FASEB J. 16, 875–877. Visco, V., Dolecek, C., Denepoux, S., Le Mao, J., Guret, C., Rousset, F., Guinnepain, M. T., Kraft, D., Valenta, R., Weyer, A., Banchereau, J., and Lebecque, S. (1996). J. Immunol. 15, 956–962. Yanagihara, Y., Kajiwara, K., Ikizawa, K., Koshio, T., Okumura, K., and Ra, C. (1994). J. Clin. Invest. 94, 2162–2165. Zhu, D., Kepley, C. L., Zhang, M., Zhang, K., and Saxon, A. (2002). Nat. Med. 8, 518–521. Zuberbier, T., Chong, S. U., Grunow, K., Guhl, S., Welker, P., Grassberger, M., and Henz, B. M. (2001). J. Allergy Clin. Immunol. 108, 275–280. Zuercher, A. W., Miescher, S. M., Vogel, M., Rudolf, M. P., and Stadler, B. M. (2000). Eur. J. Immunol. 30, 128–135.

the development of unwanted immune responses against the therapeutic competitors. Another possible target for therapy of allergy is the IgE molecule. It has been debated whether IgE may have beneficial roles as protective antibodies that defend against parasites and perhaps cancer (El Ridi et al., 1998; Gounni et al., 1994; Reali et al., 2001), but no disease related to a defect in IgE antibodies has been described in humans or in mouse strains that are deficient in IgE responses (Oettgen et al., 1994). Therefore different therapeutic strategies for the removal of IgE antibodies have been developed. One approach evaluated in clinical studies in a large number of patients is the systemic administration of anti-IgE antibodies to allergic patients with the aim to capture and complex IgE antibodies so that they cannot bind to effector cells (Heusser and Jardieu, 1997; MacGlashan et al., 1997; Milgrom et al., 1999). For this purpose, anti-IgE antibodies preventing the binding of IgE to the high-affinity receptor have been produced and humanized for therapeutic application. Successful clinical outcomes have been reported, but it is not clear

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whether the approach of injecting anti-IgE antibodies is suitable to treat patients containing high levels of IgE antibodies (Milgrom et al., 1999). Furthermore, results from long-term applications that would allow longterm side effects to be evaluated are not yet available. One interesting finding made in these studies was that the removal of IgE antibodies led to a strong reduction of the expression of the high-affinity IgE receptor on mast cells and basophils, suggesting that IgE up-regulates its own receptors (MacGlashan et al., 2001). Another possible way to prevent IgE binding to the high-affinity receptor and to remove IgE from the circulation by the formation of IgE immune complexes is the induction of anti-IgE antibodies by active immunization (Haba and Nisonoff, 1987, 1990; Hellman, 1994; Rudolf et al., 1998; Vernersson et al., 2002; Zuercher et al., 2000). For this purpose, complete IgE antibodies, recombinant fragments of the IgE constant region implicated in the FceRI binding and peptides mimicking the receptor-binding domains of IgE, have been used for immunization in animals (Table II). It could be shown that immunized animals developed anti-IgE antibodies that inhibited the IgE– FceRI interaction and that these immunized animals exhibited reduced immediate-type skin reactions (Hellman, 1994). Furthermore, it has been shown that the therapeutic removal of IgE by selective plasmapheresis can reduce allergic symptoms (Dau, 1988; Lebedin et al., 1991), and antibodies have been developed that are suitable for the depletion of IgE antibodies, as well as of IgE-bearing effector cells from the blood of allergic patients (Laffer et al., 2001). The effect of IgE on immune cells is mediated by two different receptors (i.e., the high-affinity receptor, FceRI and the low-affinity IgE receptor, FceRII [CD23]) (Bonnefoy et al., 1997; Heyman, 2000; Kinet, 1999; Novak et al., 2003). FceRI is the key structure mediating immediate-type inflammation via the IgE-dependent degranulation of mast cells and basophils and, more recently, has been found to be important for IgE-mediated activation of eosinophils and IgE-mediated allergen presentation (Gounni et al., 1994; Kraft et al., 1998; Maurer et al., 1996). FceRII seems to have regulatory functions on IgE synthesis and is involved in allergen presentation to T cells (Mudde et al., 1990; van der Heijden et al., 1993). Therapeutic approaches to prevent the interaction of IgE and FceRI include IgE-derived peptides or recombinant fragments, as well as the FceRI a-chain or portions thereof (Hamburger, 1975; Helm et al., 1988, 1997; Kelly et al., 1998; McDonnell et al., 1996; Naito et al., 1996; Vangelista et al., 1999). Furthermore, antibodies against FceRI have been shown to inhibit IgE-mediated basophil activation (Nechansky et al., 2001). Antibodies against FceRII have been shown to inhibit IgE-allergen presentation and IgE synthesis (Dasic et al., 1999; Sherr et al., 1989).

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The interesting observation that co–cross-linking of FceRI and FcgRII causes a reduction of effector cell activation has led to the construction of a chimeric molecule consisting of the receptor-binding portions of human IgG and IgE that inhibited mast cell and basophil function (Daeron et al., 1995; Zhu et al., 2002). Therapeutic approaches aiming to antagonize the effects of Th2 cytokines have mainly focused on IL-4, the key cytokine involved in the class-switch to IgE production, and IL-5, a potent activator of eosinophils. In this context it has been shown that the administration of IL-4 antagonists (e.g., an IL-4 mutant protein) during primary sensitization prevented the development of IgE responses in mice, and continuing clinical trials investigate the potential of IL-4 antagonists for the treatment of allergic patients (Borish et al., 2001; Grunewald et al., 1998). In recent clinical studies, recombinant IL-12 and anti-IL-5 monoclonal antibodies, both known to suppress eosinophilic inflammation, have been used for the treatment of patients with allergic asthma (Bryan et al., 2000; Flood-Page et al., 2003; Leckie et al., 2000). Although a strong reduction of tissue eosinophilia was observed in treated patients, no effects on airway hyperresponsiveness and late asthmatic responses were found, thus questioning the pathological role of eosinophils in asthma. A synthetic immune response modifier capable of shifting T cells from Th2 to Th1 cytokine production has recently been evaluated in vitro and may be a therapeutic substance for the treatment of allergies (Brugnolo et al., 2003). The inhibition of IgE production in B cells using recombinant portions of FceRI indicates that it may be possible to target IgE-producing cells for therapeutic intervention (Yanagihara et al., 1994). Support for a critical role of T cells in late allergic reactions comes from a number of studies in which T cell-specific reagents (e.g., anti-CD4 antibodies) or immunosuppressive drugs with a strong focus on T cell reactivity (e.g., cyclosporine, tacrolimus, pimecrolimus) were shown to be effective in the treatment of chronic forms of atopy (e.g., chronic asthma, atopic dermatitis) (Alexander et al., 1992; Fleischer, 1999; Kapp et al., 2002; Kon et al., 1998; Lock and Kay, 1996). It should, however, be mentioned that several in vitro findings indicate that cyclosporine, as well as tacrolimus, is also effective in reducing allergeninduced immediate inflammation due to degranulation of mast cells and basophils and may affect antigen-presenting cells (Panhans-Gross et al., 2001; Sperr et al., 1997; Triggiani et al., 1989; Zuberbier et al., 2001). Furthermore, previously unknown suppressive effects of already known drugs on allergen-specific mast cell and basophil activation have been reported (Majlesi et al., 2003). The risk of side effects limits the systemic application of immunosuppressive drugs for the treatment of allergy, but the macrolactams tacrolismus and pimecrolimus seem to be very effective and safe for the local treatment of atopic dermatitis.

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IV. Allergen-Specific Immunotherapy

Allergen-specific immunotherapy is the administration of increasing doses of the disease-eliciting allergens to allergic patients with the aim to induce a state of allergen-specific nonresponsiveness. An overview regarding immunological and clinical studies related to allergen-specific immunotherapy, together with recommendations for the use of this treatment, has been prepared by an international expert consortium (Bousquet et al., 1998). More than 90 years ago, when allergen-specific immunotherapy was applied for the first time to treat grass pollen–allergic patients, nothing was known about the immunological mechanisms operative in Type I allergy (Noon, 1911). At that time pollen-induced hay fever was erroneously interpreted as an intoxication caused by a grass pollen toxin. Nevertheless, patients having received injections containing grass pollen extract showed improvement, and this protection was found to last for at least 1 year after treatment was discontinued. After the first successful trials, the treatment was also applied for desensitization to other allergen sources. In 1935 Cooke and co-workers provided the first evidence for mechanisms operative in allergen-specific immunotherapy by demonstrating that symptoms of allergy could be suppressed in untreated patients by the transfer of blood from successfully treated patients (Cooke et al., 1935). As a protective factor, ‘‘blocking antibodies’’ were identified by Mary Loveless, who showed that the injection of serum from successfully treated patients into the skin of untreated patients suppressed allergen-induced immediate skin reactions (Loveless, 1940). A major improvement of safety of allergen-specific immunotherapy was achieved by the use of adjuvant-bound allergen extracts, which caused fewer systemic allergenic side effects than the injection of aqueous allergen extracts (Sledge, 1938). Analyzing the development of allergen-specific immunotherapy, it becomes evident that this treatment has been practiced in patients with considerable success for a long time, although the molecular and cellular mechanisms involved in the pathogenesis of Type I allergy were not known. For example, IgE antibodies were discovered in the late 1960s, when immunotherapy had already been routinely used for more than 50 years (Ishizaka et al., 1966; Johansson and Bennich, 1967). Likewise, many immune cells and cytokines involved in the regulation of IgE synthesis were characterized in the 1980s (reviewed in Romagnani, 1997; Paul, 1987), and the nature of the most common disease-eliciting allergens was revealed by molecular cloning techniques only in the past 15 years (reviewed in Valenta and Kraft, 2002). It is therefore not surprising that the operative immunological mechanisms behind allergen-specific immunotherapy are not fully understood even today, although it is a frequently practiced allergy treatment and perhaps one of the few causative treatment forms (Bousquet et al., 1998).

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V. Possible Mechanisms Underlying Allergen-Specific Immunotherapy

Currently, the most common form of immunotherapy is injection immunotherapy, which is based on the repeated subcutaneous injection of gradually increasing amounts of adjuvant-bound allergen extracts (Bousquet et al., 1998). In Table III we summarize possible mechanisms and effects observed during the treatment of allergic patients according to the targeted structure, cell, or process. Viewing the various immunological and clinical effects allows us to propose at least three mutually nonexclusive models for the immunological mechanisms underlying allergen-specific immunotherapy, which will be briefly described and discussed. A. Model 1: Allergen-Specific Immunotherapy Shifts the Th2-Dominated Allergen-Specific Immune Response toward a Th1 Response The identification of Th2 cytokines as the classic factors initiating IgE antibody production and thus Type I allergic immune responses, together with the finding that in allergic patients allergen-specific T cell clones, but not T cell clones specific for bacterial antigens exhibit a preferential Th2 cytokine profile, has led to the Th2/Th1 paradigm (Romagnani, 1997). This paradigm is extremely useful in explaining major features of allergic diseases as being due to a predominance of Th2 cytokines (e.g., IL-4, IL-13, IL-5) that are responsible for the predisposition of atopic individuals to mount IgE antibody responses against allergens. According to this paradigm, it has been suggested that it would be desirable to shift the allergen-specific immune response in allergic patients from the dominating Th2 response toward a preferential Th1 response. Support for the assumption that allergen-specific immunotherapy may indeed be capable of inducing such a shift came from observations that among allergen-specific T cell clones isolated from the peripheral blood of allergic patients after immunotherapy, the number of Th1 clones increased and there was also an increase in Th1 cytokine production (Ebner et al., 1997; Jutel et al., 1995; McHugh et al., 1995; Secrist et al., 1993). The reduction of eosinophils and IL-5 production after immunotherapy has been interpreted as another sign of the proposed shift toward Th1 immunity (Rak et al., 2001; Wilson et al., 2001). The Th2 to Th1 shift model, however, does not explain the increase of IgE antibodies during the initial phase of immunotherapy and the heavy production of allergen-specific IgG4 and IgG1 antibodies while the Th1indicative antibody subclasses (IgG2, IgG3) fail to increase after treatment. Furthermore, the enthusiasm about shifting allergen-specific immune responses toward a Th1 profile has been reduced by the following considerations. First, it turned out that allergen-specific T cell responses in allergic versus nonallergic persons are not as strictly polarized as was originally

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anticipated, and there is even evidence that allergen-specific Th1 responses may be responsible for pathogenetic effects in patients suffering from chronic forms of atopy (Byron et al., 1994; Hales et al., 2000; Looney et al., 1994; Oldfield et al., 2001; van Neerven et al., 1994a, 1994b). Second, the finding that overwhelming Th1 responses are implicated in a number of autoimmune diseases (Del Prete, 1998; Ghoreschi et al., 2003; Kaufman et al., 1993; Mirakian et al., 2001) has raised the question of whether it will be desirable to shift Th2 responses toward a strong Th1 response in allergic patients. B. Model 2: Allergen-Specific Immunotherapy Induces Allergen-Specific T Cell Tolerance The idea that induction of T cell tolerance may be a relevant mechanism in allergen-specific immunotherapy is supported by several observations. For example, one early study reported that allergen-specific suppressor cells are induced by immunotherapy (Rocklin et al., 1980). Based on the idea that a down-regulation of allergen-specific T cell activity may be beneficial, allergenspecific T cell epitope-containing peptides may be used for the induction of tolerance/anergy by specific immunotherapy. Studies in mice have shown that it is possible indeed to induce T cell tolerance/anergy with T cell epitopecontaining peptides (Briner et al., 1993; Hoyne et al., 1993), but clinical trials conducted in patients with allergen-derived peptides provided controversial results (Mu¨ ller et al., 1998; Oldfield et al., 2001; Pene et al., 1998; Simons et al., 1996). It has been proposed that induction of T cell anergy may also play a role in the currently used allergen-specific immunotherapy (i.e., injection immunotherapy) because reduced proliferation of allergen-specific T cells was demonstrated after immunotherapy (Akdis and Blaser, 1999; Ebner et al., 1997). In this context IL-10 has been suggested as a crucial factor that may be produced in an autocrine manner by allergen-specific T cells and may down-regulate T cell responses (Akdis et al., 1996). However, several other immunological changes observed during allergenspecific immunotherapy are difficult to explain with the tolerance model. First, it is difficult to explain the strong therapy-induced increase of allergen-specific IgG1 and IgG4 antibody levels by an induction of allergen-specific tolerance. Furthermore, it is difficult to explain how T cell tolerance/anergy may be linked to the immunotherapy-associated reduction of immediate reactions. Allergen-specific effects on immediate reactions cannot be easily understood by general cytokine effects (e.g., IL-10) because they are caused by strictly allergen- and IgE-dependent degranulation of mast cells and basophils. Moreover, there is considerable evidence that the down-regulation of allergen- and IgE-dependent effector cell degranulation is mediated mainly by therapy-induced allergen-specific blocking antibodies (Ball et al., 1999a, 1999b; Clinton et al., 1989; Mothes et al., 2003).

TABLE III Possible Mechanisms of Allergen-Specific Immunotherapy 124

Targeted structure/process Mast cell and basophil degranulation

Mast cell number IgE-mediated allergen presentation to T cells, T cell activation, and cytokine release B cells T cell proliferation

Th1/Th2 cell ratio

Mechanism Mast cell and basophil degranulation Reduction of histamine releasability Reduction by T cell-derived IL-10 and IFN-g Reduction by unknown mechanism Blocking IgG competing with IgE

Down-regulation of CD23 Generation of suppressor cells; T cell tolerance/anergy

Shift from Th2 to Th1 pattern

References Ball et al., 1999a, 1999b; Clinton et al., 1989; Loveless, 1940; Mothes et al., 2003 Shim et al., 2003 Pierkes et al., 1999 Durham et al., 1999 van Neerven et al., 1999

Hakansson et al., 1998; Roever et al., 2002 Akdis and Blaser, 1999; Akdis et al., 1996; Baskar et al., 1997 Briner et al., 1993; Ebner et al., 1997; Hoyne et al., 1993; Rocklin et al., 1980; Mu¨ ller et al., 1998; Oldfield et al., 2001, 2002 Ebner et al., 1997; Hakansson et al., 1998; Jutel et al., 1995; Kammerer et al., 1997; McHugh et al., 1995; Pene et al., 1998; Secrist et al., 1993

Th2 cytokine production

Preferential apoptosis of Th2 cells Reduction of T cell-derived IL-4 and IL-13

Th1 cytokine production

Preferential apoptosis of Th2 cells Induction of Th1 cells

Eosinophil number/activation

Eosinophil reduction and IL-5 reduction by unknown mechanism Possible suppression by blocking antibodies Unknown mechanism

IgE production by memory cells; allergen-induced boost of IgE production Prevention of bronchial hyperresponsiveness Prevention of disease progression

Unknown mechanism

Guerra et al., 2001 Ebner et al., 1997; Gabrielsson et al., 2001 Jutel et al., 1995; McHugh et al., 1995; Pene et al., 1998; Secrist et al., 1993 Guerra et al., 2001 Ebner et al., 1997; Jutel et al., 1995; Kammerer et al., 1997; McHugh et al., 1995 Rak et al., 2001; Wilson et al., 2001 Mothes et al., 2003 Rak et al., 2001 Moller et al., 2002

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C. Model 3: Allergen-Specific Immunotherapy has Vaccination Character and Induces an Immune Response that Counteracts the Allergic Immune Response The third model suggests that allergen-specific immunotherapy has a vaccination character. In this model the injection of adjuvant-bound allergens induces a new type of allergen-specific immunity, which, in the case of the most frequently used adjuvant (i.e., aluminium hydroxide) has features of a Th2-like immunity. This immune response is dominated by the induction of strong allergen-specific IgG1 and IgG4 responses and an intial boost of allergen-specific IgE responses (Ball et al., 1999a, 1999b; Mothes et al., 2003). The therapy-induced humoral immune response may be directed against new epitopes as well as against IgE epitopes, depending on the allergen preparation used. If immunotherapy is tolerated and can be continued by injection of increasing allergen doses, the initial IgE response is overwhelmed by strong IgG responses (Ball et al., 1999a, 1999b). Furthermore, if allergen-specific IgG antibodies recognize IgE epitopes, they can compete with allergen-specific IgE antibodies and accordingly inhibit allergen-induced mast cell, as well as basophil activation (Ball et al., 1999a, 1999b; Clinton et al., 1989; Mothes et al., 2003). The latter effect explains the suppression of immediate, IgE-dependent allergic reactions. In addition to a simple blocking of IgE allergen recognition, it is also possible that allergen-specific IgG leads to a co–cross-linking of FceRI and FcgRII receptors, which may down-regulate effector cell activation (Daeron et al., 1995). Using recombinant allergen fragments, it has been demonstrated that allergen-specific immunotherapy induces antibody responses against epitopes that were not recognized before treatment, suggesting that the treatment does have a vaccination character (Ball et al., 1999b). The newly induced IgG responses not only may have beneficial effects regarding the suppression of mast cell degranulation, but also may inhibit IgE-mediated allergen presentation to T cells and thus suppress T cell activation and the release of proinflammatory cytokines (van Neerven et al., 1999). Furthermore, it has been shown that patients developing allergen-specific IgG antibodies after immunotherapy exhibit a reduced boost of allergen-specific IgE antibodies due to allergen exposure compared with placebo-treated patients lacking allergen-specific IgG (Mothes et al., 2003). The latter result suggests that immunotherapy-induced allergen-specific IgG antibodies may also suppress the boost of allergen-specific IgE production in memory cells caused by allergen exposure. The vaccination model thus would explain the reduction of immediate, latephase responses and possibly the long-term effects observed during injection immunotherapy. According to the vaccination model, immunotherapy would induce a new allergen-specific immune response that competes with the

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established allergic immune response and is characterized by the formation of ‘‘blocking antibodies’’ as already suggested by Loveless (1940) and Cooke et al. (1935). These blocking antibodies exhibit their protective role simply by antagonizing the effects of IgE. Due to the fact that aluminium hydroxide almost exclusively is permitted for use as adjuvant for allergen-specific immunotherapy, the therapy-induced immune response is characterized by Th2 features (i.e., initial boost of IgE responses, induction of Th2-indicative IgG4 and IgG1 responses). However, the protective role of therapy-induced antibodies has been questioned because it has been observed that clinical success of immunotherapy does not always correlate with the induction of IgG antibodies (Birkner et al., 1990; Djurup and Malling, 1987). In this context it must, however, be stated that these immunotherapy studies and the corresponding clinical, as well as immunological, analyses were performed with crude allergen extracts. Using such allergen extracts, it is not possible to determine whether therapy-induced IgG antibodies react with allergens and inhibit IgE recognition of allergens. It will hence be necessary to reinvestigate the influence of blocking antibodies on clinical outcome parameters with defined allergens and epitopes. VI. Modifications of Traditional, Allergen Extract-Based Immunotherapy

Several variations of traditional allergen extract-based injection immunotherapy have been introduced and evaluated (Table IV). The aim of these variations has been to improve the disadvantages of immunotherapy (e.g., risk of anaphylactic side effects). We have summarized these variations according to the target structure (i.e., the administered antigen, the mode of administration, and the adjuvant used to formulate the vaccine) (Table IV). Major disadvantages of allergen-specific immunotherapy have been the risk of inducing anaphylactic side effects in sensitized patients, the inconvenience of frequent injection treatments, and difficulties in obtaining defined allergen preparations. Successful attempts to reduce the allergenic activity of the vaccines were based on the coupling of allergen extracts to adjuvants that were thought to prevent the spreading and systemic release of the administered allergens (Sledge, 1938). Compared to the injection of aqueous allergen extracts, the rate of systemic anaphylactic side effects observed in the course of immunotherapy could indeed be drastically reduced by using aluminium hydroxide-adsorbed allergen extracts (reviewed in Bousquet et al., 1998). Although aluminium hydroxide is known to induce antigen-specific Th2 responses it is still today the most frequently, if not the only adjuvant permitted for vaccination treatment and allergen-specific immunotherapy. Other adjuvants such as liposomes or monophosphoryl lipid A (MPL) are rarely used (Drachenberg et al., 2001; Santeliz et al., 2002).

TABLE IV Modifications of Traditional Allergen-Specific Immunotherapy Target Antigen

Type of variation

Mechanism/advantage

Chemically modified allergen Reduction of allergenic activity; extracts (haptens, PEG, allergoids) induction of tolerance; induction of Th1 responses

128

Recombinant allergens and modified recombinant allergens

T cell peptides

B cell peptides

Mimotopes

DN Vaccines

Allergen specificity; induction of blocking antibodies; increase of safety; immunomodulation; induction of T cell tolerance Allergen specificity; increase of safety; induction of T cell tolerance Allergen specificity; increase of safety; induction of blocking antibodies Allergen specificity; induction of blocking antibodies DNA vaccines Allergen specificity; induction of Th1 responses

References Attallah and Sehon, 1969; HayGlass and Stefura, 1991; Lee and Sehon, 1977; Litwin et al., 1988, 1991; Malley et al., 1976; Marsh et al., 1970; Norman et al., 1982 Valenta, 2002; Valenta and Kraft, 2002

Briner et al., 1993; Hoyne et al., 1993; Mu¨ ller et al., 1998; Oldfield et al., 2001, 2002; Simons et al., 1996 Ball et al., 1999a; Focke et al., 2001

Jensen-Jarolim et al., 1998

Hartl et al., 1999; Hochreiter et al., 2003; Hsu et al., 1996; Raz et al., 1996; Slater et al., 1998

Mode of Oral/sublingual administration administration

Adjuvant

Safety; induction of T cell anergy; easy to perform; convenient for patients

Andre et al., 2000; Bjorksten et al., 1986; Clavel et al., 1998; Fanta et al., 1999; Quirino et al., 1996; Taudorf et al., 1989; Urbanek et al., 1990 Andri et al., 1996; Passalacqua et al., 1997; Welsh et al., 1983 Sledge, 1938

Nasal administration

Safety; convenient for patients

Al(OH)3 CpG, MPL, liposomes

Reduction of anaphylactic side effects Induction of Th1 responses; reduction of allergenic activity (CpG) Alvarez et al., 2002; Drachenberg et al., 2001; Horner et al., 2002; Marshall et al., 2001; Mothes et al., 2003; Santeliz et al., 2002; Tighe et al., 2000 Induction of T cell tolerance Roy et al., 1999 Reduced tissue damage; Gro¨ nlund et al., 2002 ease of production Induction of Th1 responses Vrtala et al., 1995 Induction of Th1 responses Jahn-Schmid et al., 1996

Chitosan-nanoparticles Carbohydrate-based particles

129

Live vaccines Surface layers

130

R. VALENTA ET AL.

Modifications of allergen extracts were already performed more than 30 years ago in order to reduce anaphylactic side effects (Attallah and Sehon, 1969; Lee and Sehon, 1977; Litwin et al., 1988; Malley et al., 1976; Marsh et al., 1970). These modifications were achieved by chemical modifications of the allergen extracts and their proteolytic digestion, by coupling of allergen extracts to polyethyleneglycol (PEG), or by chemical denaturation of allergen extracts using various aldehydes. Proteolytic digestion of allergen extracts and coupling to PEG have indeed yielded material with reduced allergenic activity. Allergoids produced by chemical denaturation had a strongly reduced allergenic activity and even induced strong Th1 immune responses (HayGlass and Stefura, 1991). Due to the difficulties in manufacturing chemically modified allergen extracts of consistent quality, today only allergoids are frequently used for routine treatment. Another major problem of allergen-specific immunotherapy is that allergen extracts are mixtures of allergenic and nonallergenic components that cannot be tailored according to the individual patient’s sensitization profile (reviewed in Bousquet et al., 1998). Since great variations regarding allergen content have been found for various allergen extracts, antibody-based assays have been developed that at least have allowed us to detect and quantify some of the common allergens in the therapeutic extracts (van Ree, 1997). However, the composition of allergen extracts of natural allergen sources depends on the representation of the individual allergen molecules in the sources and cannot be changed during manufacturing. Furthermore, the extraction process and many other factors (e.g., proteolysis) may influence the presence of allergenic and nonallergenic components in natural allergen extracts. Due to the progress made in the field of allergen characterization, the cDNAs coding for the most common environmental allergens have been isolated, and it has become possible to produce recombinant allergens that closely mimic the properties of the corresponding natural allergens (reviewed in Valenta and Kraft, 2002; Valenta et al., 1999a). Moreover, it has been demonstrated that the complete allergen/ epitope repertoire of natural allergen sources can be replaced by recombinant allergens. The finding that recombinant allergens can resemble the epitope repertoire of natural allergen sources represents a prerequisite for the development of new diagnostic and therapeutic strategies for Type I allergy (reviewed in Valenta, 2002). The advantages of using recombinant allergens for diagnosis and therapy will, however, be discussed separately. Based on the DNA and deduced amino acid sequences obtained for the most common allergens by molecular cloning, highly specialized and experimental forms of therapy strategies have been developed. They include, for example, the use of allergen-derived T cell epitope-containing peptides for immunotherapy. Studies performed in mouse models have indicated that it is possible to induce T cell tolerance against the major allergens from mites and

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cats by using only a few major T cell epitope-containing peptides (Briner et al., 1993; Hoyne et al., 1993). However, T cell peptide-based therapy evaluated in cat and bee venom allergic patients yielded controversial results (Mu¨ ller et al., 1998; Oldfield et al., 2001; Pene et al., 1998; Simons et al., 1996). Another concept suggests using recombinant fragments or synthetic peptides derived from B cell epitopes of allergens or mimotopes (i.e., peptides mimicking B cell epitopes) for the induction of blocking antibodies (Ball et al., 1999a; Focke et al., 2001; Jensen-Jarolim et al., 1998). Based on the finding that injection of allergen-encoding DNA can induce Th1-prone immune responses in animals, it was also suggested that DNA vaccination be applied for the treatment of Type I allergy (Hartl et al., 1999; Hsu et al., 1996; Raz et al., 1996). A major problem limiting the clinical application of DNA vaccination to allergic patients was the finding that it leads to a rather uncontrolled spreading of allergen-encoding DNA in various organs, bearing the risk that active allergen may be synthesized at these sites (Slater et al., 1998). The latter problem may, however, be overcome if DNA coding for hypoallergenic allergen versions is used for vaccination (Hochreiter et al., 2003). More than 10 years ago oral or nasal administration of allergens was studied as a possible alternative to injection immunotherapy (Bjorksten et al., 1986; Taudorf et al., 1989; Urbanek et al., 1990; Welsh et al., 1983). Recently, a renaissance of clinical studies investigating again whether oral, sublingual, or nasal application of allergens may represent an alternative to current forms of injection immunotherapy has been observed (Andri et al., 1996; Andre et al., 2000; Clavel et al., 1998; Fanta et al., 1999; Passalacqua et al., 1997; Quirino et al., 1996). These forms of administration appear safe and convenient, and evidence for clinical efficacy has been provided in certain studies. However, it appears that these forms of immunotherapy seem to have little detectable immunological effects. While it has been speculated that they may induce tolerance preferentially, the immunological mechanisms of these alternative forms of treatment are currently completely elusive. The discovery that certain bacterial DNA sequences, that is, immunostimulatory oligodeoxynucleotides (ISS-ODN, CpG oligonucleotides) promote strong Th1 immune responses has lead to several in vitro and animal studies exploring the usefulness of immunostimulatory DNA sequences as general immunomodulators and as adjuvants for allergen-specific immunotherapy (Horner et al., 2002; Marshall et al., 2001; Tighe et al., 2000). Recently, a clinical study with a CpG-coupled version of the major ragweed allergen, Amb a 1, has been initiated in patients. Likewise, chitosannanoparticles, bacterial surface layers, and carbohydratebased particles have been suggested as possible adjuvants for allergy vaccination (Gro¨ nlund et al., 2002; Jahn-Schmid et al., 1996; Roy et al.,

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1999). In addition, attempts have been made to express allergens in bacterial strains that can be used as live vaccines (Vrtala et al., 1995). VII. Possible Improvement of Immunotherapy by Using Recombinant Allergens

A. The Development of Recombinant Allergens New possibilities for the improvement of diagnosis and allergen-specific immunotherapy evolved from the molecular and structural characterization of allergens (reviewed in Valenta, 2002; Valenta and Kraft, 2002). Our knowledge about the molecular nature of the disease-eliciting allergens has dramatically increased through the application of molecular cloning techniques to the field of allergen characterization. By the end of the 1980s, several research groups started to isolate cDNAs coding for allergens, and soon thereafter recombinant allergens mimicking the natural allergens became available. The characterization of allergens by molecular cloning was initially thought to reveal the molecular nature of paradigmatic allergen molecules and to allow studies of the allergen-specific immune responses using well-defined allergens that are frequently recognized by allergic patients. Unexpectedly, immunological studies (i.e., IgE reactivity, T cell proliferations) showed that only a few recombinant allergens were required to cover the majority of disease-eliciting epitopes that are present in natural allergen extracts (reviewed in Valenta et al., 1999a, 1999b). More than 10 years ago, two studies demonstrated that allergy to birch pollen and grass pollen could be diagnosed with a few recombinant allergens and suggested replacing diagnostic allergy tests based on crude allergens with recombinant allergen tests (Valenta et al., 1991, 1992). Due to the work of several research groups, the allergen repertoires of the most common allergen sources have now been reconstructed with recombinant allergens and new types of multiallergen tests based on microarrayed recombinant allergens have been developed (Harwanegg et al., 2003; Hiller et al., 2002). There is a fundamental difference between previously used diagnostic tests based on crude allergen extracts and the new test systems using recombinant allergen molecules. Allergen extract-based tests only determine if a patient is sensitized against a particular allergen source, but provide no information about the immune reactivity to the individual allergenic molecules in the allergen source. The analysis of the reactivity profiles of allergic patients with recombinant allergens, termed component-resolved diagnosis (CRD), has revealed that allergic patients are characterized by individual reactivity to the allergens in a given allergen source (Valenta et al., 1999a, 1999b). This IgE reactivity profile evolves in the first years of life and seems to remain unchanged in the further natural course of the disease. Studies analyzing the sensitization profiles in different populations with recombinant allergens have demonstrated that

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the IgE reactivity profiles of patients also vary depending on the locally predominating allergens and thus are an imprint of the local allergen exposure. For example, it was found that individuals living in the northern parts of Europe, where birch predominates, are more frequently and exclusively sensitized against Bet v 1, the major birch pollen allergen, suggesting that these patients were genuinely sensitized to birch pollen, whereas patients from the more southern parts of Europe more frequently reacted with birch pollen due to sensitization to cross-reactive allergens from sources other than birch (Moverare et al., 2002a). Similarly, it was found that allergic individuals from Central Africa were preferentially sensitized against marker allergens that reflect the predominance of certain grass species in their local environment (Westritschnig et al., 2003). The differences of reactivity profiles in populations may have considerable implications for selecting the suitable composition of allergy vaccines. B. The Impact of Recombinant Allergen-Based Diagnostic Testing on Traditional Allergen-Specific Immunotherapy Adequate diagnosis of IgE-mediated allergy is a prerequisite for the inclusion of patients for allergen-specific immunotherapy. However, as mentioned earlier, the currently used allergen extract-based diagnostic tests cannot identify the disease-eliciting allergen molecules and therefore do not allow a precise selection of patients for immunotherapy. It has therefore been suggested that recombinant allergen-based tests be used for an improved selection of patients for immunotherapy (Kazemi-Shirazi et al., 2002). For example, it has been shown that certain allergen molecules occur abundantly and rather exclusively in a given allergen source and therefore can be used as diagnostic markers for a genuine sensitization toward this allergen source. On the other hand, sensitization to highly cross-reactive allergens that occur in many different unrelated allergen sources may give rise to positive test reactions to all these sources when diagnostic tests are performed with allergen extracts. On the basis of these findings, it has been suggested that speciesspecific marker allergens and highly cross-reactive allergens be used to differentiate patients with a genuine sensitization to a given allergen source from cross-sensitized patients who appear polysensitized to many allergen sources and hence may benefit less from allergen-specific immunotherapy (Bousquet et al., 1991; Kazemi-Shirazi et al., 2002). To accommodate the wealth of individual allergen molecules, chip-based allergy tests that utilize microarrayed recombinant allergens have recently been developed (Harwanegg et al., 2003; Hiller et al., 2002). They allow rapid screening of the sensitization profile toward numerous allergen molecules and epitopes with minute amounts of serum in single determinations. Using recombinant allergen-based diagnostic tests, it has thus become

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possible to obtain additional diagnostic information for the selection of patients for traditional immunotherapy and also to monitor the development of immune responses toward individual allergen molecules in the course of immunotherapy (Ball et al., 1999a, 1999b; Mothes et al., 2003). Analyses of patients undergoing allergen extract-based immunotherapy with purified recombinant allergens and epitopes have provided evidence for several potential weaknesses of traditional immunotherapy that are related to the use of allergen extracts instead of defined allergen molecules. For example, it was found that the injection of allergen extracts can induce de novo IgE sensitizations against allergens that were not recognized before the therapy (Ball et al., 1999b; Moverare et al., 2002b). Although the clinical relevance of these new IgE sensitizations has not yet been demonstrated in all of the reported studies, these findings strongly suggest that treatment with defined allergen molecules selected according to the patient’s sensitization profile should be preferred to treatment with allergen extracts. Another weakness of therapeutic allergen extracts is that these extracts may lack important allergens or that certain allergens are not present as immunogenic molecules and hence fail to induce protective immune responses (Mothes et al., 2003). Since it is technically impossible to influence the composition of natural allergen extracts, the replacement of these extracts by recombinant allergens seems to be a logical next step toward the improvement of allergen-specific immunotherapy. C. Reconstructing the Natural Allergen Repertoire by Recombinant Allergens: A Prerequisite for New Therapeutic Concepts The potential replacement of natural allergen extracts by recombinant allergens depends on whether it is possible to replace the majority of disease-eliciting epitopes of a given allergen source by recombinant molecules. For this purpose, the immunological equivalence of natural and recombinant allergens has to be demonstrated, and, as a next step, a panel of recombinant allergens resembling the relevant IgE epitopes and T cell epitopes of the natural allergen source has to be defined. For the most common allergen sources (e.g., pollens, mites, animal dander), recombinant allergens containing most of the IgE and T cell epitopes present in the natural allergen sources have now been identified (reviewed in Valenta et al., 1999a, 1999b). In principle, it is therefore possible to formulate recombinant allergen-based vaccines that can be tailored according to the patient’s sensitization profiles. However, recombinant allergens that equal the natural allergens will exhibit allergenic activity and hence may induce anaphylactic side effects. It has therefore been suggested that the safety and immunological features of recombinant allergenbased vaccines be improved by the use of genetically engineered recombinant allergen derivatives (reviewed in Valenta et al., 1999a, 1999b; Singh et al.,

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1999; Valenta, 2002). A major aim of allergen engineering work has been reduction of the allergenic activity of recombinant allergen derivatives, but additional unexpected results have been obtained in the course of the experiments. They include changes of the immunological properties of the engineered allergen derivatives (e.g., alterations of immunogenicity, cytokine profiles) and the possibility of fusing unrelated allergens/epitopes to obtain combination vaccines for the treatment of complex sensitization profiles. VIII. Genetic Engineering of Modified Allergens for Immunotherapy

A. Reduction of the Allergenic Activity by Allergen Engineering For most of the common respiratory allergens, IgE recognition depends on the presence of conformational epitopes on intact and structurally folded allergens (reviewed in Valenta and Kraft, 2001). Using allergen-encoding cDNAs as templates, it has now become possible to engineer defined recombinant allergen derivatives that can be produced in a standardized manner. Table V displays a summary and brief characterization of hypoallergenic allergen derivatives obtained by genetic engineering for several important allergen sources. A reduction of IgE reactivity and allergenic activity of allergens can be obtained by splitting them into defined recombinant fragments or by the deletion of IgE-reactive portions. The reduced allergenic activity of recombinant fragments of the major timothy grass pollen allergen, Phl p 1, was demonstrated by basophil histamine release studies 9 years ago (Ball et al., 1994). A similar reduction of IgE reactivity was also noted for several allergens from pollen, mites, and molds when truncated proteins were produced that lacked IgE-reactive portions (Hayek et al., 1998; Tamborini et al., 1997; Tang et al., 2000; Twardosz et al., 1997; Vrtala et al., 1997, 1999; Zeiler et al., 1997). Advantages of the fragmentation approach are that a profound reduction of allergenic activity can be obtained for most allergens by this technology and that different fragments from one molecule can be combined so that no primary sequence information, and hence T cell epitope, is lost. Furthermore, it is possible to reassemble fragments derived from a given molecule in a different order in the form of a new mosaic protein that preserves the repertoire of T cell epitopes of the wild-type allergen (Mothes and Valenta, unpublished data). The second type of modifications is based on the observation that natural isoforms of allergens exist in allergen sources that exhibit a strongly reduced IgE reactivity (Breiteneder et al., 1993; Ferreira et al., 1996). Such isoforms with reduced IgE reactivity were found to differ from the highly IgE-reactive versions in only a few amino acids. The reduction of IgE reactivity of such

TABLE V Modified Recombinant Allergens for Immunotherapy

Allergen source

136

Pollen Trees Birch

Allergen

IgE T cell Basophil Cytokine Animal reactivity reactivity activation profile model

References

Cor a 1 Aln g 4

Oligomerization Fragmentation Mutation Isoform Deletion Mutation Isoform Fragmentation

þ       

þ þ þ þ n.d. n.d. n.d. n.d.

  n.d.a n.d. n.d. n.d. n.d. n.d.

Th1 Th1 n.d. n.d. n.d. n.d. n.d. n.d.

þ þ n.d. n.d. n.d. n.d. n.d. n.d.

Vrtala et al., 2001 Vrtala et al., 1997, 2000 Ferreira et al., 1998 Ferreira et al., 1996 Twardosz et al., 1997 Engel et al., 1997 Breiteneder et al., 1993 Hayek et al., 1998

Phl p 1 Phl p 2

Fragmentation Mosaic

þ/ 

n.d. n.d.

n.d. 

n.d. n.d.

n.d. þ

  þ þ þ

þ n.d. þ þ þ

  þ þ þ

n.d. n.d. n.d. n.d. n.d.

n.d. n.d. þ þ þ

Ball et al., 1999a Mothes et al. (unpublished data) Schramm et al., 1999 Vrtala et al., 1999 Linhart et al., 2002 Linhart et al., 2002 Linhart et al., submitted

Bet v 1

Bet v 4 Hazel Alder Grasses Timothy grass

Type of modificator

Phl p 5b Deletion Phl p 6 Deletion Phl p 1þPhl p 5 Hybrid molecule Phl p 2þPhl p 6 Hybrid molecule Phl p 1þPhl p 2þPhl p Hybrid molecule 5þPhl p 6

Ryegrass Weeds Pellitory Oil seed rape Mites House dust mites

Storage mites Mould Aspergillus fumigatus Animals Cow 137

Lol p 1 Lol p 5

Deletion Mutation

 

n.d. þ

 

n.d. n.d.

n.d. n.d.

Tamborini et al., 1997 Swoboda et al., 2002

Par j 1 Bra r 1

Mutation Mutation

 

þ n.d.

n.d. n.d.

n.d. n.d.

n.d. þ

Bonura et al., 2001 Okada et al., 1998

Der p 2 Der f 2

Mutation Mutation

 

n.d. þ

n.d. 

n.d. Th1

n.d. þ

Lep d 2

Deletion Mutation

 

n.d. þ

n.d. n.d.

n.d. n.d.

n.d. n.d.

Smith and Chapman, 1996 Korematsu et al., 2000; Noguchi et al., 1996; Takai et al., 1997 Takai et al., 1999 Olsson et al., 1998

Asp f 2

Deletion



n.d.

n.d.

n.d.

n.d.

Tang et al., 2000

Bos d 2

Fragmentation Mutation

 

þ þ

n.d. n.d.

n.d. n.d.

n.d. n.d.

Zeiler et al., 1997 Kauppinen et al., 1999

Hybrid molecule



þ



n.d.

þ

King et al., 2001

Mutation Mutation Mutation

  

þ þ þ

n.d. n.d. n.d.

n.d. n.d. n.d.

þ þ þ

Bannon et al., 2001 Bannon et al., 2001 Bannon et al., 2001; Rabjohn et al., 2002

Venoms Yellow jacket/paper wasp Ves v 5þPol a 5 Food Peanut Ara h 1 Ara h 2 Ara h 3 a

n.d., not done.

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R. VALENTA ET AL.

isoforms may be explained either by effects on the general fold of the molecule or by their direct involvement in IgE recognition. Based on rational considerations mutations can be introduced by sitedirected mutagenesis that may profoundly alter the IgE reactivity of the mutated protein. For example, it has been shown that the mutation of cysteine residues in the group 2 mite allergens leads to a disruption of disulfide bonds and thus causes reduced IgE reactivity (Olsson et al., 1998; Smith and Chapman, 1996; Takai et al., 1997). Likewise, it was shown that mutation of the acidic amino acids in the calcium-binding domains of the birch pollen allergen, Bet v 4, reduced its IgE-binding capacity (Engel et al., 1997). A rather unexpected finding was that oligomerization (i.e., formation of recombinant dimers, trimers) of the major birch pollen allergen, Bet v 1, led to a strong reduction of the allergenic activity of this molecule, but preserved its immunogenic activity and even part of its IgE reactivity (Vrtala et al., 2001). Table V summarizes recombinant allergen derivatives with altered allergenic and/or immunological properties. A major advantage of recombinant allergen derivatives is that they can be engineered in a manner to preserve most of the allergens and T cell epitopes and to retain the immunogenicity of the wild-type allergen that is required for the induction of protective antibody responses. As indicated in Table V, T cell reactivity, reduced IgE reactivity and allergenic activity, cytokine profile, and immunogenic activity (animal models) need to be evaluated before the application of genetically modified allergens in patients should be considered. The molecular engineering of allergens has, however, not only provided us with derivatives with reduced allergenic activity but also demonstrated that changes in a particular allergen can profoundly alter its immunological properties. B. Genetic Engineering of Allergens can alter their Immunological Properties To illustrate unexpected changes of immunological properties of genetically engineered allergens, we will briefly discuss a few concrete examples. A recently described recombinant trimer consisting of three covalently linked copies of the major birch pollen allergen, Bet v 1, breaks the rule that IgE reactivity must be completely abrogated if a strong reduction of allergenic activity needs to be achieved (Vrtala et al., 2001). The described recombinant Bet v 1 trimer exhibited a structural fold similar to the allergenic Bet v 1 monomer and maintained its IgE reactivity. Despite these characteristics, the trimer had strongly reduced activity in inducing basophil degranulation and skin reactions in Bet v 1-allergic patients. The mechanism for the reduced allergenic activity remains unknown, but the study points to the possibility that either partial hiding of IgE epitopes or perhaps their reorientation has reduced

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the allergenic activity of the engineered molecule. Another striking feature of the hypoallergenic trimeric form of Bet v 1 was its altered capacity to induce lymphoproliferative and cytokine responses. T cells from Bet v 1-allergic patients showed increased lymphoproliferative responses to the trimer, and their cytokine release profile was strongly diverted toward a Th1 profile (Vrtala et al., 2001). That the engineering of allergens may also have profound effects on their ability to induce cytokine responses has been also demonstrated by the finding that a recombinant Bet v 1 fragment and mutated versions of the major house dust mite allergen, Der f 2, more strongly induced the release of Th1 cytokines from patient’s peripheral blood mononuclear cells (PBMC) than the allergenic wild-type allergens (Korematsu et al., 2000; Vrtala et al., 2000). Whether the altered immunological properties of the engineered allergen versions may be due to a different presentation of folded versus unfolded proteins depends on the type of APC, the MHC molecule repertoire, or is due to differences between presentation with or without antibodies remains to be answered. Although there are by now only a few examples demonstrating profound alterations of the immunological properties of engineered allergen variants, these findings lend support to the idea that molecular engineering of a given allergen can strongly alter its immunological characteristics without requiring the addition of adjuvants. C. Hybrid Allergens with Increased Immunogenicity for the Treatment of Complex Sensitization Profiles Another unexpected example of a profound alteration of the immunological features of genetically engineered allergens was discovered when hybrid allergens were constructed out of two or more immunologically unrelated allergens. The latter approach was chosen to investigate whether it is possible to engineer hybrid molecules that can cover the allergen/epitope repertoire of complex allergen sources (Linhart et al., 2002, submitted). For this purpose, unrelated grass pollen allergens were fused by polymerase chain reaction (PCR) based on their corresponding cDNAs, and the resulting hybrid allergens were then characterized. The unexpected finding in this study was that the fusion of weakly immunogenic allergens yielded hybrid molecules with strongly enhanced immunogenicity (Linhart et al., 2002). The grass pollen hybrid allergens induced stronger IgG antibody responses in animals and lymphoproliferative responses in PBMC cultures than equimolar mixtures of the individual allergens. This observation may have important implications for vaccine development in general, as it suggests that it is possible to increase the immunogenicity of weakly immunogenic antigens by using another unrelated antigen as a kind of scaffold. It will thus be possible to engineer improved

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allergy vaccines, especially for complex allergen sources where certain allergens are present in low immunogenic form. IX. Clinical Results with Modified Recombinant Allergens

The immunological features of genetically engineered recombinant allergen derivatives can be evaluated using different types of in vitro assays. These assays include the measurement of IgE and antibody reactivity, the induction of lymphoproliferative and cytokine responses, and especially the induction of basophil degranulation as a very sensitive marker for the allergenic activity of the molecule. Small allergen fragments and, in particular, allergen-derived peptides may either preferentially target allergen-specific T cells or induce antibody responses against new epitopes and thus favor only certain immunological mechanisms, as described in the three possible models explaining the effects of immunotherapy. A major advantage of genetically modified allergens containing most of the primary sequence information of the originating wildtype allergens is that they will comprise most of the T cell epitopes of the wild-type allergen, exhibit reduced anaphylactic activity (i.e., reduced basophil degranulation), and preserve the immunogenicity/tolerogenicity of the wildtype allergens in experimental animal models. These molecules may therefore be used simultaneously for different therapy strategies (e.g., modulation of T cells, induction of protective antibody responses) (Valenta, 2002). However, before genetically engineered allergen derivatives are used in immunotherapy trials, a clinical evaluation regarding their in vivo allergenic activity must be performed by provocation testing. Table VI shows that many recombinant allergen derivatives have already been compared regarding their allergenic activity with the corresponding wild-type allergens in patients. The induction of immediate inflammatory reactions can be precisely determined and quantified by skin testing and by nasal and conjunctival provocation testing. Since skin testing allows us to perform simultaneous end point titrations for several antigens and dilutions, it has been the preferred method used for the evaluation of genetically modified allergens. A strong reduction of allergenic activity was found for genetically modified versions of the major birch pollen allergen, Bet v 1, in representative numbers of patients (Arquint et al., 1999; Pauli et al., 2000; van Hage-Hamsten et al., 1999). Similar data were obtained for genetically modified mite, weed, and cow dander allergens (Bonura et al., 2001; Kauppinen et al., 1999; Kronqvist et al., 2001; Kusunoki et al., 2000; Ruoppi et al., 2001). For other genetically modified allergens, only a few patients have been tested so far. The results obtained by nasal and conjunctival provocation obtained for genetically engineered versions of the major birch pollen allergen, Bet v 1, were similar to those obtained by skin testing and hence suggest that skin

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TABLE VI Clinical Evaluation of Modified Recombinant Allergens Type of modification

Allergen

Evaluationa

Number of patients

References

Fragments

Bet v 1

SPT, IDT SPT, IDT SC NPT NPT SPT, IDT, CPT SPT SPT SPT SPT SPT SPT SPT SPT SPT, IDT SPT SPT SPT SPT, IDT SPT, IDT SC NPT

23 36 9 10 10 48 4 11 2 5 10 3 1 20 4 1 17 9 23 36 9 10

van Hage-Hamsten et al., 1999 Pauli et al., 2000 Nopp et al., 2000 van Hage-Hamsten et al., 2002 Ruoppi et al., 2001 Arquint et al., 1999 Gehlhar et al., 1997 Ferreira et al., 1998 Swoboda et al., 2002 Schramm et al., 1999 Bonura et al., 2001 Takai et al., 1997 Takai et al., 1999 Kusunoki et al., 2000 Smith and Chapman, 1996 Olsson et al., 1998 Kronqvist et al., 2001 Kauppinen et al., 1999 van Hage-Hamsten et al., 1999 Pauli et al., 2000 Nopp et al., 2000 van Hage-Hamsten et al., 2002

Isoforms Mutants

Oligomers

Bos d 2 Bet v 1 Phl p 5 Bet v 1 Lol p 5 Phl p 5 Par j 1 Der f 2 Der f 2 Der f 2 Der p 2 Lep d 2 Bos d 2 Bet v 1

a CPT, conjunctival provocation test; IDT, intradermal test; NPT, nasal provocation test; SC, skin chamber; SPT, skin prick test.

testing is an appropriate method for the evaluation of their allergenic activity (Arquint et al., 1999; van Hage-Hamsten et al., 2002). The fact that genetically modified Bet v 1 derivatives could be safely administered to the nasal mucosa suggests that these derivatives are also suitable for therapeutic nasal application (van Hage-Hamsten et al., 2002). Using an in vivo skin chamber model that allows us to investigate allergenmediated late effects on cell migration, cell activation, cytokine, and mediator release, it could be demonstrated that genetically modified Bet v 1 derivatives not only exhibited reduced allergenic activity, but also caused reduced eosinophil activation and release of proinflammatory cytokines (Nopp et al., 2000). Most of the genetically engineered allergen derivatives described in Tables V and VI contain many of the T cell epitopes of the corresponding wild-type allergens, and several of them were found to induce protective antibody responses in experimental animal models. They should therefore be useful for conventional injection immunotherapy in an adjuvant-bound form or,

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alternatively, may be used for T cell immunomodulation strategies (e.g., tolerance induction) by mucosal administration. As yet one double-blind, placebo-controlled multicenter immunotherapy trial has been carried out with recombinant Bet v 1 fragments and the Bet v 1 trimer (Vrtala et al., 1997, 2001) in more than 100 patients so that data about clinical efficacy, safety, and immunological mechanisms will be available in the near future.

X. Formulating Prophylactic Allergy Vaccines

The fact that prophylactic vaccination against a great variety of infectious diseases is performed routinely and safely very early in childhood raises the question of whether specific prophylactic vaccinations against the most common allergies might become possible in the future. The advantage of early prophylactic treatment would be that the pathological allergic immune response has not yet started or has not led to severe clinical disease. It may thus be easier to prevent or counteract the development of the allergic response early in childhood. To achieve this goal, several prerequisites need to be fulfilled and a number of critical issues must be addressed. The requirement for defined allergens is nearly fulfilled due to molecular allergen characterization work. Today most of the common allergens covering most of the relevant allergen sources have been reconstructed by recombinant DNA technology. However, these recombinant allergens are almost equivalent to the natural allergens and, hence, if given for prophylactic vaccination, may also induce IgE responses. Therefore, either alternative routes of administration or adjuvants preventing the induction of allergen-specific immune responses need to be used if prophylactic vaccination with wild-type recombinant allergens is considered. The danger of inducing unwanted allergic sensitization may be overcome by the use of genetically modified allergen derivatives that differ substantially from the naturally occurring allergens. However, regardless of the type of allergen/allergen modification, route of administration, or adjuvant used, it must be demonstrated in animal and later in clinical studies that prophylactic vaccination does not induce allergic sensitization and that it is capable of preventing allergic sensitization. The genetic blueprints of the most common environmental allergens are now available to approach prophylactic vaccination strategies for allergic diseases. With such an approach, it may be possible to prevent the development of allergic diseases. Acknowledgments This work was supported by grants from the Austrian Science Fund (Y078GEN, F01801, F01803, F01804, F01811, Hertha Firnberg Award T163, Schroedinger Award to T. B.), from the CeMM project of the Austrian Academy of Sciences, and from BIOMAY, Vienna, Austria.

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Trautmann, A., Akdis, M., Kleemann, D., Altznauer, F., Simon, H. U., Graeve, T., Noll, M., Brocker, E. B., Blaser, K., and Akdis, C. A. (2000). J. Clin. Invest. 106, 25–35. Triggiani, M., Cirillo, R., Lichtenstein, L. M., and Marone, G. (1989). Int. Arch. Allergy Appl. Immunol. 88, 253–255. Turner, H., and Kinet, J. P. (1999). Nature 402, B24–30. Twardosz, A., Hayek, B., Seiberler, S., Vangelista, L., Elfman, L., Gronlund, H., Kraft, D., and Valenta, R. (1997). Biochem. Biophys. Res. Commun. 239, 197–204. Urbanek, R., Burgelin, K. H., Kahle, S., Kuhn, W., and Wahn, U. (1990). Eur. J. Pediatr. 149, 545–550. Valenta, R. (2002). Nat. Rev. Immunol. 2, 446–453. Valenta, R., and Ball, T. (1997). In ‘‘IgE Regulation: Molecular Mechanisms’’ (D. Vercelli, Ed.), p. 225. John Wiley & Sons, Chichester, UK. Valenta, R., and Kraft, D. (2001). Immunol. Rev. 179, 119–127. Valenta, R., and Kraft, D. (2002). Curr. Opin. Immunol. 14, 718–727. Valenta, R., Duchene, M., Vrtala, S., Birkner, T., Ebner, C., Hirschwehr, R., Breitenbach, M., Rumpold, H., Scheiner, O., and Kraft, D. (1991). J. Allergy Clin. Immunol. 88, 889–894. Valenta, R., Vrtala, S., Ebner, C., Kraft, D., and Scheiner, O. (1992). Int. Arch. Allergy Immunol. 97, 287–294. Valenta, R., Flicker, S., Eibensteiner, P. B., Steinberger, P., Laffer, S., Dolecek, C., and Kraft, D. (1997). Biol. Chem. 378, 745–749. Valenta, R., Hayek, B., Seiberler, S., Bugajska-Schretter, A., Niederberger, V., Twardosz, A., Natter, S., Vangelista, L., Pastore, A., Spitzauer, S., and Kraft, D. (1998). Int. Arch. Allergy Immunol. 117, 160–166. Valenta, R., Lidholm, J., Niederberger, V., Hayek, B., Kraft, D., and Gronlund, H. (1999a). Clin. Exp. Allergy 29, 896–904. Valenta, R., Vrtala, S., Focke-Tejkl, M., Bugajska-Schretter, A., Ball, T., Twardosz, A., Spitzauer, S., Gronlund, H., and Kraft, D. (1999b). Biol. Chem. 380, 815–824. Van den Biggelaar, A. H., van Ree, R., Rodrigues, L. C., Lell, B., Deelder, A., Kremsner, P. G., and Yazdanbakhsh, M. (den Biggelaar 2000). Lancet 356, 1723–1727. van der Heijden, F. L., van Neerven, R. J., van Katwijk, M., Bos, J. D., and Kapsenberg, M. L. (1993). J. Immunol. 150, 3643–3650. van Hage-Hamsten, M., Kronqvist, M., Zetterstrom, O., Johansson, E., Niederberger, V., Vrtala, S., Gronlund, H., Gronneberg, R., and Valenta, R. (1999). J. Allergy Clin. Immunol. 104, 969–977. van Hage-Hamsten, M., Johansson, E., Roquet, A., Peterson, C., Andersson, M., Greiff, L., Vrtala, S., Valenta, R., and Gronneberg, R. (2002). Clin. Exp. Allergy 32, 1448–1453. van Neerven, R., van de Pol, van Milligan, F. J., Jansen, H. M., Aalberse, R. C., and Kapsenberg, M. L. (1994a). J. Immunol. 152, 4203–4210. van Neerven, R., van de Pol, M. M., Wierenga, E. A., Aalberse, R. C., Jansen, H. M., and Kapsenberg, M. L. (1994b). Immunology 82, 351–356. van Neerven, R., Wikborg, T., Lund, G., Jacobsen, B., Brinch-Nielsen, A., Arnved, J., and Ipsen, H. (1999). J. Immunol. 163, 2944–2952. van Ree, R. (1997). Allergy 52, 795–805. van Ree, R. (2002). Int. Arch. Allergy Immunol. 129, 189–197. van Reijsen, F. C., Bruijnzeel-Koomen, C. A., de Wega, R. A., and Mudde, G. C. (1997). J. Invest. Dermatol. 108, 530. van Reijsen, F. C., Felius, A., Wauters, E. A., Bruijnzeel-Koomen, C. A., and Koppelman, S. J. (1998). J. Allergy Clin. Immunol. 101, 207–209. Vangelista, L., Laffer, S., Turek, R., Gronlund, H., Sperr, W. R., Valent, P., Pastore, A., and Valenta, R. (1999). J. Clin. Invest. 103, 1571–1578.

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Vercelli, D., and Geha, R. S. (1992). Curr. Opin. Immunol. 4, 794–797. Verdino, P., Westritschnig, K., Valenta, R., and Keller, W. (2002). EMBO J. 21, 5007–5016. Vernersson, M., Ledin, A., Johansson, J., and Hellman, L. (2002). FASEB J. 16, 875–877. Victor, J. R., Jr., Fusaro, A. E., Duarte, A. J., and Sato, M. N. (2003). J. Allergy Clin. Immunol. 111, 269–277. Visco, V., Dolecek, C., Denepoux, S., Le Mao, J., Guret, C., Rousset, F., Guinnepain, M. T., Kraft, D., Valenta, R., Weyer, A., Banchereau, J., and Labecque, S. (1996). J. Immunol. 157, 956–962. von Pirquet, C. (1906). Mnch. Med. Wochenschr 30, 1457–1461. Vrtala, S., Grote, M., Ferreira, F., Susani, M., Stocker, B., Kraft, D., and Valenta, R. (1995). Int. Arch. Allergy Immunol. 107, 290–294. Vrtala, S., Hirtenlehner, K., Vangelista, L., Pastore, A., Eichler, H. G., Sperr, W. R., Valent, P., Ebner, C., Kraft, D., and Valenta, R. (1997). J. Clin. Invest. 99, 1673–1681. Vrtala, S., Ball, T., Spitzauer, S., Pandjaitan, B., Suphioglu, C., Knox, B., Sperr, W. R., Valent, P., Kraft, D., and Valenta, R. (1998). J. Immunol. 160, 6137–6144. Vrtala, S., Fischer, S., Grote, M., Vangelista, L., Pastore, A., Sperr, W. R., Valent, P., Reichelt, R., Kraft, D., and Valenta, R. (1999). J. Immunol. 163, 5489–5496. Vrtala, S., Akdis, C. A., Budak, F., Akdis, M., Blaser, K., Kraft, D., and Valenta, R. (2000). J. Immunol. 165, 6653–6659. Vrtala, S., Hirtenlehner, K., Susani, M., Akdis, M., Kussebi, F., Akdis, C. A., Blaser, K., Hufnagl, P., Binder, B. R., Politou, A., Pastore, A., Vangelista, L., Sperr, W. R., Semper, H., Valent, P., Ebner, C., Kraft, D., and Valenta, R. (2001). FASEB J. 15, 2045–2047. Wahn, U., Lau, S., Bergmann, R., Kulig, M., Forster, J., Bergmann, K., Bauer, C. P., and Guggenmoos-Holzmann, I. (1997). J. Allergy Clin. Immunol. 99, 763–769. Welsh, P. W., Butterfield, J. H., Yunginger, J. W., Agarwal, M. K., and Gleich, G. J. (1983). J. Allergy Clin. Immunol. 71, 454–460. Werfel, T., Reekers, R., Busche, M., Schmidt, P., Constien, A., Wittmann, M., and Kapp, A. (1999). Curr. Probl. Dermatol. 28, 18–28. Westritschnig, K., Sibanda, E., Thomas, W., Auer, H., Aspock, H., Pittner, G., Vrtala, S., Spitzauer, S., Kraft, D., and Valenta, R. (2003). Clin. Exp. Allergy 33, 22–27. Wierenga, E. A., Snoek, M., Jansen, H. M., Bos, J. D., van Lier, R. A., and Kapsenberg, M. L. (1991). J. Immunol. 147, 2942–2949. Wills-Karp, M., Santeliz, J., and Karp, C. L. (2001). Nat. Rev. Immunol. 1, 69–75. Wilson, D. R., Nouri-Aria, K. T., Walker, S. M., Pajno, G. B., O’Brien, F., Jacobson, M. R., Mackay, I. S., and Durham, S. R. (2001). J. Allergy Clin. Immunol. 107, 971–976. Wu¨ thrich, B., Schindler, C., Leuenberger, P., and Ackermann-Liebrich, U. (1995). Int. Arch. Allergy Immunol. 106, 149–156. Yamaguchi, M., Sayama, K., Yano, K., Lantz, C. S., Noben-Trauth, N., Ra, C., Costa, J. J., and Galli, S. J. (1999). J. Immunol. 162, 5455–5465. Yanagihara, Y., Kajiwara, K., Ikizawa, K., Koshio, T., Okumura, K., and Ra, C. (1994). J. Clin. Invest. 94, 2162–2165. Ying, S., Humbert, M., Meng, Q., Pfister, R., Menz, G., Gould, H. J., Kay, A. B., and Durham, S. R. (2001). J. Allergy Clin. Immunol. 107, 686–692. Zeiler, T., Taivainen, A., Rytkonen, M., Rautiainen, J., Karjalainen, H., Mantyjarvi, R., Tuomisto, L., and Virtanen, T. (1997). J. Allergy Clin. Immunol. 100, 721–727. Zhu, D., Kepley, C. L., Zhang, M., Zhang, K., and Saxon, A. (2002). Nat. Med. 8, 518–521. Zuberbier, T., Chong, S. U., Grunow, K., Guhl, S., Welker, P., Grassberger, M., and Henz, B. M. (2001). J. Allergy Clin. Immunol. 108, 275–280. Zuercher, A. W., Miescher, S. M., Vogel, M., Rudolf, M. P., and Stadler, B. M. (2000). Eur. J. Immunol. 30, 128–135.

advances in immunology, vol. 82

Interactions of Immunoglobulins Outside the Antigen-Combining Site ROALD NEZLIN* AND VICTOR GHETIE{ *Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel { Cancer Immunobiology Center, University of Texas Southwestern Medical Center Dallas, Texas 75390

I. Introduction

Immunoglobulin (Ig) molecules possess two main types of interactions. First, they can form specific immune complexes (IC) with antigens using the antigen-combining site located on the tips of Fab regions. Just as important is the second type of interactions, with sites localized on Ig constant domains. Some of these interactions relate to the effector functions of antibodies, which are the essential part of the immune response. They include such well-known reactions as the activation of the complement cascade and the activation of cells after binding the cell Fc receptors. Both these processes, which are stimulated by the formation of antigen–antibody complexes, enhance significantly the response against infections. Other effector functions are related to the transportation of Ig molecules through cell membranes and IgG homeostasis, both operating independently of antigen binding. Also important are Ig interactions with proteins of bacterial and virus origin, which can significantly influence the course of infection diseases. Many past methodological advances were based on studies on binding bacterial proteins to Ig molecules. The main structural unit of immunoglobulins is an Ig fold or domain, a compact globule, which is formed by antiparallel strands arranged in two b-pleated sheets. The sheets are packed face to face and linked by a disulfide bond (Fig. 1). The amino acid residues that comprised interactions sites belong to the b-strands, as well as to the loops connecting the strands and the interdomain linkers. In this chapter, we discuss structural and functional aspects of interactions of Ig molecules with various ligands of animal, plant, bacterial, viral, or synthetic origin that may occur in the circulation or on the cell surfaces. II. Fc Receptor-Binding Sites

A. Fc Cell Receptors Involved in Effector Functions 1. Fcg Receptors The Fcg receptor (FcgR) family consists of three representatives, FcgRI (CD64), FcgII (CD32), and FcgRIII (CD16) expressed on almost all cells of the immune system (Hulett and Hogarth, 1994). FcgRI has a high affinity for 155 Copyright 2004, Elsevier Inc. All rights reserved. 0065-2776/04 $35.00

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Fig 1 Structure of the C2 immunoglobulin domain. Reprinted from Barclay et al. (1997) with permission from Elsevier.

monomeric IgG (108 M
1), whereas the FcgRII and FcgRIII have a low affinity for monomeric IgG (106 M
1) but strongly bind IgG-containing IC. These FcgRs exist in two or three isoforms, denoted a, b, and c. All FcgR are transmembrane glycoproteins belonging to the Ig superfamily, with the IgGbinding subunit consisting of two (FcgRIIb and c and IIIa and b) and three (FcgRIa, b, and c) domains. There are activating (FcgRI, FcgRIIa, and FcgRIIIa and b) and inhibitory (FcgRIIb) receptors coexpressed on the same effector cells. All FcgR show a high degree of sequence homology in their extracellular portion but differ in their cytoplasmic portion, carrying either the tyrosine-based activator (ITAM) (FcgRI, FcgRIIa, and FcgRIII) or inhibitor (ITIM) (FcgRIIb) motif (Ravetch and Bolland, 2001). The cross-linking of IgG antibodies bound to FcgRI by multivalent antigens or the interaction of IC with FcgRII and FcgRIII induces clustering of these receptors. As a result, the activation of cells and the initiation of a variety of effector mechanisms such as antibody-dependent cellular cytotoxicity, phagocytosis of IC, or release of inflammatory mediators are followed (Hogarth, 2002; Takai, 2002).

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a. The Localization of the Fc-Binding Site on FcgR. The crystal structures of the two Ig-like domains of FcgRII/III (D1 and D2) have been solved (Maxwell et al., 1999; Sondermann et al., 1999; Zhang et al., 2000). It showed an acute hinge angle of about 508 between the two domains. The amino acid residues of the D2 domain are directly involved in the binding of the Fcg region, while the D1 domain is important for maintaining the D2 domain conformation (Radaev and Sun, 2001). The FcgRI also contains the D1 and D2 domains, which are highly homologous to their FcgRII/III counterparts, plus the third domain (D3) attached to the C-terminal end of domain D2 (Hulett and Hogarth, 1998). The significantly higher affinity of FcgRI for monomeric IgG is considered to be mediated by its third domain since both D1 and D2 displayed an affinity as low as that of the FcgRII/III (Harrison and Allen, 1998). Crystallographic data on the structure of FcgRI alone or in complex with Fc are absent, and the D2 domain of FcgRI may also be the key domain involved in the direct binding of IgG. The D3 domain may play a critical role in conferring high affinity and specificity by allowing receptor dimerization (Harrison and Allen, 1998). The D2 domain consists of two b sheets arranged like a sandwich, one containing three antiparallel b strands (A, B, and E) and the other containing five (C0 , C, F, G, and A0 ), a feature showed by the V domains of immunoglobulins. The Fc-binding sites are localized in the C0 strand and in the three main loops joining the BC, C0 E, and FG strands. These loops and the C0 strand are adjacent and accessible to the Fc region. The replacement of most residues residing in the BC (Trp-113 to Ala-117), C0 E (Asp-130 to His-134), and FG (Arg-155 to Lys-161) loops (alignment for FcgRIIIb) by alanine results in a decrease of the affinities for IgG (Radaev and Sun, 2001). b. FcgR Interaction Sites of IgG. Earlier mutational analyses of the Fc residues involved in the binding of domain D2 pointed out that the main interaction site is localized in the lower hinge region and in the adjacent sites of the Cg2 domain (Tamm and Schmidt, 1997). The results of these experiments were confirmed by crystallographic studies of the human FcgRIII complexed with the Fc fragment of human IgG1 (Radaev et al., 2001; Sondermann et al., 2000). The residues present in the interface between FcgRIII and both chains of the Fc fragment (denoted A and B) are shown in Table I (Radaev et al., 2001). The differences in the amino acid sequence of the lower hinge region (234–239) of various isotypes of human and mouse IgGs may explain the hierarchy of their binding to FcR. Thus human IgG3 and IgG1 are binding to all FcRs with higher affinities than the IgG4 and IgG2, the latter devoid of any binding capacity to FcRI (Hulett and Hogarth, 1994). The position of the D1 and D2 domains of FcgRIII into the opening of both Fc chains, which resembles a horseshoe, is shown in Fig. 2. The FcgRIII/Fc

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TABLE I Interface Contacts between Human FcgRIII and Fcg Fragment a Fcg

Location

Chain

FcgRIII

Loop/strand

Contacts

Leu-235 Leu-235 Leu-235 Leu-235 Gly-236 Gly-236 Gly-236 Gly-236 Gly-237 Gly-237 Leu-238 Ser-239 Asp-265 Pro-329 Pro-329 Ala-330

LHb LH LH LH LH LH LH LH LH LH LH LH CH2 CH2 CH2 CH2

B A B B A B B B A A B A A B B B

Thr-116 His-135 Val-158 Gly-159 His-134 Val-156 Lys-161 Tyr-90 Lys-120 His-134 Gly-159 Lys-120 Lys-120 Trp-90 Trp-113 Ile-88

BC C0 E FG FG C0 E FG FG A C0 C0 E FG C0 C0 A BC A

H-Sc H-S HC HC H-S H-S HC HC H-S H-S HC H-S H-S HC HC HC

a

Radaev et al. (2001). LH, low hinge. c H-S, hydrogen and salt bridges; HC, hydrophobic contact. b

complex burried 1450 A˚ 2 of solvent-accessible area, from which approximately 60% is contributed by chains A and B of the lower hinge region (Radaev and Sun, 2001). This region is not visualized in the unbound Fc fragment, but becomes visible after cocrystallization with FcgRIII, indicating that the binding of the receptor stabilizes the conformation of the lower hinge region. The interface between FcgRIII and chain A is dominated by hydrogen bonding interactions, whereas chain B is involved in hydrophobic interactions (Table I). A hydrophobic proline ‘‘sandwich’’ is formed between Trp-90 and Trp-113 of FcgRIII and Pro-329 (chain B), which is extended, including Val-158 and Lys-161 (the aliphatic portion) from the receptor and Leu-235 (chain B) of Fc (Radaev et al., 2001). The side chain of the Leu-235 residue also interacts with Gly-159 of the receptor so tightly that no other residue larger than glycine can be accommodated in this space. The steric constraint imposed by the Gly-159 and Leu-235 tight contact is reflected in the complete disruption of the interaction of FcgR with Fc by mutations of either one of these two residues (Hulett et al., 1994). The mutation of Trp-113!Phe resulted in the loss of the binding of Fc, not only by disrupting the interaction with Pro-329 (Table I), but also by altering the orientation between D1 and D2 (Radaev and Sun, 2001).

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Fig 2 Model of the FcgR III complexed with IgG1. Reprinted from Radaev and Sun (2001) with permission from Elsevier.

The presence of an N-linked oligosaccharide at Asp-297 in Cg2 is critical for binding to all FcgRs. The mutation of Asn-297 to Ala results in a considerably reduced affinity of the FcgRs binding, as the carbohydrate located between the Cg2 domains is critical for the conformation of the region. The replacement of the amino acid residues interacting with the N-acetylglucosamine residues (e.g., Asp-265) altered both the composition of the carbohydrate and the binding to FcgRI (Jefferis and Lund, 2002). In addition to these amino acid residues of the Fc region directly or indirectly involved in the binding to FcgRs, some other residues of the Cg2 domain

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(Asp-270, Arg-292, Ala-327, Pro-331, and Lys-338) and Cg3 domain (Lys-414) are important for binding to FcgRs (Canfield and Morrison, 1991; Shields et al., 2001) (Fig. 3). Some mutations induced decreased binding to FcgRII and FcgRIII, but not to FcgRI (e.g., Asp-270, Ala-327), or reduced binding to FcgRI, but not FcgRIII (e.g., Arg-292, Lys-414). This was also true for a residue from the lower hinge (Ser-239), which, after mutation to alanine, affected only the binding to FcgRIII, but not to FcgRI and FcgRII. The fact that some mutations did not affect equally well the binding of IgG to all FcgRs suggests that the contact interface between Fcg and the three FcgRs may consist of some common (overlapping) residues, as well as a few distinct ones (Shields et al., 2001). c. The Stoichiometry of the FcgR/Fc Interaction. Stoichiometry values of 1:1 for the FcgR/Fc interaction have been obtained by ultracentrifugation and equilibrium size exclusion chromatography. X-ray structural studies of FcgRIII in the complex with Fc have shown that identical residues from the lower hinge A and B interact with different unrelated regions of the receptor. This excludes the possibility of the interaction of another receptor molecule with the same Fcg, thus confirming the 1:1 stoichiometry (Radaev et al., 2001). Using nuclear magnetic resonance (NMR) spectroscopy, it was shown that mouse FcgRII binds to one of the two lower hinge regions of Fc, inducing a conformational change in the other site that precludes the binding of the second receptor. Precluding the FcgR aggregation by a divalent IgG molecule avoids permanent stimulation of the immune system by IgG monomers present in high concentration in serum (Kato et al., 2000a, 2000b). d. Functional Implications. FcgRs link the humoral and cellular function of the immune system, being the key player in the activation and inhibition of the immune response (Hogarth, 2002; Takai, 2002). The engagement of the activating type of FcgRs to targets coated by IgG antibodies triggers phagocytosis, cytolysis, and release of inflammatory cytokines. This activity can be downregulated by inhibitory FcgRIIb, thus keeping the immune response within its normal limits (Ravetch and Bolland, 2001). Another important function of FcgR is the uptake of the IgG containing IC followed by intracellular degradation of antigen. The resulting peptides are directed either to the MHC antigenpresenting pathway or are completely destroyed. It is believed that defects in the activating/inhibitory function of FcgRs or in the IC handling are linked to many autoimmune diseases (e.g., Goodpasture’s syndrome, SLE, etc.) (Takai, 2002). 2. Fca and Fcm Receptors Receptors for IgA, the second abundant Ig class, are expressed by different myeloid cells, including eosinophils, neutrophils, and monocytes (Monteiro and van de Winkel, 2003). The most studied receptor, FcaRI (CD89), is a

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Fig 3 Amino acid positions in the Cg2 domain involved in the binding of FcgR and C1q (prepared by J. Smallshaw). The participation of the amino acid residues from the lower hinge region in binding IgG to FcgR is presented in Table I. Ligands

Cg2 residues

All FcgRs (human IgG1) FcgRII, FcgRIII (human IgG1); C1q (human IgG1) FcgRII (human IgG1) C1q (mouse IgG2b); FcgRII (mouse IgG2b) C1q (mouse IgG2b) C1q (mouse IgG2b, human IgG1) C1q (human IgG1) C1q (mouse IgG2a,b); FcgRIII (human IgG1) C1q (human IgG1); FcgRI (human IgG3) FcgRIII (human IgG1)

265 270 292 318 320 322 329 330 331 338

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heavily glycosylated transmembrane protein, the extracellular part of which comprises two Ig–like domains that are homologous to the proteins coded by the leukocyte receptor cluster, including the killer-inhibitory receptors. It is able to interact with both IgA subclasses with low affinity (Ka  106 M
1). Poly-IgA and IgA-IC are bound more effectively than monomeric IgA. Secretory IgA is able to bind FcaRI only in the presence of the integrin coreceptor Mac-1. The affinity of interaction is increased significantly after the immobilization of FcaRI (Wines et al., 2001). The localization of the FcaRI interaction site involved in binding IgA is different from the interaction sites of the other FcRs, as it locates in the membrane-proximal D1 domain. Studies of mutant FcaRI molecules and X-ray crystallographic studies of the FcaRI–IgA1 complex indicated that residues of four regions of D1, which build a single continuous region, participate in the interaction with IgA (Herr et al., 2003; Wines et al., 2001). The IgA residues essential for interactions with human FcaRI were located at the Ca2–Ca3 interface. Using site-directed mutants, it was shown that two loops participate in the formation of the binding site (Pleass et al., 1999, 2001). The first one belongs to the Ca2 AB helix/loop and comprises residues 257–259, which are conserved among human, mouse, and bovine IgA. The second region comprises residues 440–443 from the Ca3 FG loop. The critical role of these residues in binding FcaRI is supported by comparison of the reactivity of human IgA with bovine and mouse IgA. In bovine IgA, which is able to bind human FcaRI, residues 440–443 are identical to those of human IgA. Mouse IgA with substitutions in positions 441 and 442 have no binding affinity. X-Ray crystallographic studies reveal that six residues of FcaRI and seven residues of Fca comprise a central hydrophobic core of the interface, which is flanked by several charged residues. Totally 19 conserved residues of Fca comprise the FcaRI-binding site. The FcaRI–IgA1 interface buries 1656 A˚ 2 (Herr et al., 2003). The position of the IgA-binding site is markedly different from the receptorbinding sites of IgG and IgE, which are located correspondingly at Cg2 near the hinge region or at the top of the Ce3 domain. As a result, the IgA molecule bound to FcaRI located on the cell surface is in an ‘‘upright’’ position. IgG and IgE molecules bound to their receptors are in an opposite orientation (Herr et al., 2003). A single Fca is able to bind two FcaRI molecules. The bivalent interaction of IgA with two FcaRI on the cell surface would lead to significantly higher binding activity and more effective stimulation of cell activities. Several IgM-binding proteins were identified on B cells. One of them is anchored to the cell surface via a glycosylphosphatidylinositol linkage (Ohno et al., 1990). Another one with both IgM- and IgA-binding activity (Fca/mR) is expressed on human and mouse mature B cells and monocytes (Shibuya et al., 2000). Its extracellular part consists of only one Ig-like domain, in contrast with

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other Ig cell receptors. Fca/mR mediates endocytosis of IC composed of IgM antibody and staphylococcal cells. 3. Fce Receptors For the development of allergic reactions, the most important step is the interaction of IgE antibodies with the high-affinity IgE receptor, FceRI, expressed on mast cells, basophils, and some other cells. Multivalent allergens reacting with the bound IgE antibodies aggregate the IgE–FceRI complex. The interaction results in signal transduction, leading to cellular activation and release of pharmacologically active substances mediating immediate hypersensitivity, thus triggering allergic reactions (Gould et al., 2003; Metzger, 2002; Turner and Kinet, 1999). FceRI is a highly glycosylated heterodimer and contains an a chain, a b chain, and two g chains. In a trimeric FceRI molecule, the b chain is absent. The b and g chains contain tyrosine activation motive (ITAM) and participate in intracellular signaling. The a chain is responsible for the interaction with IgE molecules. Its extracellular part is composed of two closely packed small Ig-like domains, a-1 (D1) and a-2 (D2), forming a convex surface adapted for interaction with a complimentary arch of Fce. Mutational and structural studies had indicated that the a-2 domain and the linker between both domains are responsible for the contacts with Fce fragment (Garman et al., 2000). The general structure of IgE is different from that of IgG. According to earlier fluorescent polarization studies, IgE molecules are less flexible and more compact (Nezlin, 1990). Fluorescence energy transfer experiments have shown that the IgE molecules are bent (Baird et al., 1993), which could be explained by the bending of Fc domains in the domain linker regions. The results of X-ray structural studies correspond to a compact structure of Fce. According to a suggested model, the Ce3–Ce4 domain two-fold axis is nearly perpendicular to that of the Ce2 domain and Fce has an acutely bent structure (Wan et al., 2002). IgE reacts with FceRI with high affinity (Kd  109–1010 M
1). Because the binding stoichiometry of the interaction is 1:1, monomeric IgE molecules cannot cross-link two receptors and induce cell activation. The receptor binds across the Fce two-fold symmetry axis between the two Fc chains (Fig. 4) and therefore the interaction with the second receptor molecule is blocked. By genetic manipulation it was shown that the Ce3 domains of IgE molecule are involved in interaction with the receptor (Nissim et al., 1993). According to X-ray crystallographic studies, residues of both Ce3 domains and of both Ce2–Ce3 linkers participate in contact with two binding sites on the FceRI surface (Garman et al., 1998, 2001). Each of the Ce3 domains interacts with a distinct binding site on the surface of the FceRI molecule. The first one is related to the C0 C region of the a-2

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Fig 4 Model of the FceRIa–Fce complex. Reprinted from Garman et al. (2001) with permission from Elsevier.

domain, and Tyr-131 is a central residue of the site, which projects into a shallow pocket of one Fce chain. The cluster of four exposed tryptophan residues located on the top of both receptor a domains is an important part of the second site. Two of these residues form a hydrophobic ‘‘sandwich’’ with Pro426 of the second Fce chain. Two other tryptophan residues interact with the Ce2–Ce3 linker. Site 1 is formed by 12 IgE residues and eight receptor residues burying about 860 A˚ 2 of surface area, and site 2 is formed by 10 IgE residues and eight receptor residues burying about 970 A˚ 2. Of the 15 receptor residues, seven are aromatic, which may contribute to the high stability of the IgE–receptor complex. Of 18 IgE residues interacting with FceRI, none is aromatic (Garman et al., 2000; Wurzburg and Jardetzky, 2001). These structural studies are important for understanding the mechanisms of allergy. The 1:1 stoichiometry of the FceRI–IgE interaction indicates that the essential step in the initiation of allergic reactions is the cross-linking of the receptor-bound IgE antibodies by multivalent allergens. X-ray crystallographic studies help indicate why IgE molecules react with the receptor with such a high affinity. Structural data are also very important for designing new types of inhibitory drugs useful for treatment of allergic diseases.

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The second Fce receptor, the low-affinity FceRII (CD23), belongs to the Ctype (calcium-dependent) lectins. It is expressed on a variety of cells in humans and reacts with IgE with an affinity 106–107 M
1, as well as with several other proteins (Delespesse et al., 1992). The binding stoichiometry of the CD23–IgE interaction is 2:1. It has been proposed that the receptor in an oligomeric form regulates IgE production (Kilmon et al., 2001). B. Fc Receptors Involved in Transcytosis and Catabolism 1. Neonatal Fc Receptor The neonatal Fc receptor (FcRn) delivers IgG across the maternofetal barrier during gestation and is responsible for the maintenance of serum IgG levels by controlling its catabolism (Ghetie and Ward, 2000). The transfer of IgG from mother to young provides the newborn with humoral immunity. For maternal IgG to be transferred, several cellular barriers containing FcRn have to be crossed, first from the maternal blood across the placenta’s trophoblasts (humans) or yolk sac cells (rodents) to the fetus, and second from the maternal blood into milk and then across the neonatal intestine following ingestion (rodents and ruminants) (Simister, 1998). The persistence of IgG in the circulation is also regulated by FcRn present in the endothelial cells of adults (Borvak et al., 1998; Ward et al., 2003). FcRn as an IgG homeostat binds to IgG that is taken up by endothelial cells by nonspecific pinocytosis. The IgG molecules bound to FcRn are salvaged from degradation and recycle into the circulation. This process maintains the constant serum levels of IgG (Ghetie and Ward, 2000). A model of transcytosis and catabolism of IgG mediated by FcRn is presented in Fig. 5. The key mechanism controlling both transport and recycling of IgG is its ability to react with FcRn in a pH-dependent manner. The receptor binds IgG at slightly acidic pH 6.0–6.5 and releases it at pH 7.0–7.4 (Martin et al., 2001). After internalization, the binding of IgG to FcRn takes place mainly in the endosomal compartment, while the release takes place on the plasma membrane in contact with the slightly alkaline pH of the environment (blood) (Ghetie and Ward, 2000). IgG molecules in excess of the FcRn or unable to bind to FcRn are directed toward lysosomes and destroyed. a. The Amino Acid Residues of FcRn Involved in the Binding of IgG. FcRn is a heterodimer of an a chain (homologous to MHC class I a chain) and b2microglobulin (Simister and Mostov, 1989). FcRn a-chain genes have been isolated and characterized. The FcRns of various species have a high degree of homology in the a-2 domain, which is involved in binding Fcg (Kacskovics et al., 2000). The contact residues have been determined by X-ray crystallography and site-directed mutagenesis (Burmeister et al., 1994a, 1994b; Martin

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Fig 5 A model of transcytosis and catabolism of IgG mediated by FcRn.

et al., 2001; Vaughn et al., 1997; West and Bjorkman, 2000). The residues that play a direct role in the interaction between IgG and FcRn are located in close proximity at the C end of the a-2 domain and are conserved in both human and mouse with the exception of residues 132 and 137 (Table II). b. The Amino Acid Residues of IgG Involved in the Binding to FcRn. The delineation of IgG residues involved in regulation of transcytosis and catabolism was determined by site-directed mutagenesis (Kim et al., 1994a; Medesan et al., 1997) and confirmed by X-ray crystallography (Burmeister et al., 1994b; Martin et al., 2001). The resulting mutants were tested for binding recombinant FcRn and the ability to be transported through the neonatal intestine, yolk sac, placenta, and mammary gland (Cianga et al., 1999; Firan et al., 2001; Kim et al., 1994b; Medesan et al., 1996, 1997). The results demonstrated that

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TABLE II Amino Acid Residues Involved in the FcRn–IgG Interaction a Mouse FcRnb

Mouse IgG

Human FcRnb

Human IgG

Glu-117 Glu-118 Asp-132 Trp-133 Glu-137e

His-310 Gln-311 His-435c Ile-253 His-436c

Glu-117 Glu-118 Glu-132 Trp-133 Leu-137

His-310 Gln-311 His-435d Ile-253 Tyr-436 f

a

Ober et al. (2001). Homology alignment of mouse and human FcRn. In all IgGs except IgG2b, where the residue is Tyr. d In all IgGs except the IgG3 allotype, where the residue is Arg. e Asp in rat FcRn. f In all IgGs except the IgG3 allotype, where the residue is Phe. b c

TABLE III Delineation of the Amino Acid Residues of IgG Involved in the Binding of FcRn, Transcytosis and Catabolism in Mousea Transcytosis through b

Residue

Affinity for FcRn

Catabolism

Intestine

Yolk sac

Wild typec Ile-253 His-285d His-310 His-433 His-435 His-436

100 18 98 13 75 5 20

100 22 89 14 97 15 41

100 10 98 12 71 25 51

100 22 83 17 78 11 83

a

Medesan et al. (1997). All residues were mutated to Ala. c The activities are expressed as percentage of the activity of the wild type. d The His-285 is located in a loop on the external surface of the CH2 domain, which is distal to the CH2–CH3 cleft (negative control). b

residues located at the Cg2 (Ile-253, His-310) and Cg3 (His-435) domain interface are essential for transcytosis and regulation of IgG catabolism (Table III and Fig. 6). His-436 plays a minor but significant role in the mouse FcRn–IgG interaction. Titration of His with a pKa identical to the pH of the Fc–FcRn interaction (pH 6.5) accounts for the acid pH dependence of the binding. Thus there are

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Fig 6 Structure of the Cg2–Cg3 cleft (prepared by P. Sondermann). The amino acid residues are those of human IgG1. The following positions are involved in the binding: FcRn (253, 254, 309, 310, 433, 435, 436); RF (252, 253, 254, 433, 434, 435, 436); SpA (251, 252, 253, 254, 310, 311, 434, 435); SpG (251, 252, 253, 254, 311, 433, 434, 436, 438); and HSV-1 (435).

three pairs of salt bridges between rat Fc and FcRn, namely, His-310–Glu-117, His-435–Glu-132, and His-436–Asp-137 (Martin et al., 2001). FcRn binds to Fc at pH < 6.5 (when the His residues are partially charged) and releases Fc after deprotonization at pH > 7.0. The acidic nature of the residues in FcRn and the presence of the His residues in IgG (Table II) strongly suggest that electrostatic forces may play a predominant role in mediating the FcRn–IgG interaction. Hydrophobic interactions between the Trp-133 in FcRn and Ile-253 of IgG are also certainly involved (Table II). In addition to these residues, others, at least in human IgG1, are important for binding human FcRn. These include Ser-254, Lys-288 (in Cg2) and Ser-415, His-433 (in Cg3) (Shields et al., 2001). However, in the absence of data on transcytosis and catabolism, it is difficult to estimate how IgG1 mutants with these residues will be handled in vivo. The close correlation between the effect of a given mutation of the mouse or human Fc on catabolism, transcytosis, and affinity for FcRn supports the concept that FcRn is involved in all three processes in mice and humans (Kim et al., 1999; Medesan et al., 1997). However, the ultimate proof that

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FcRn plays a key role in both transcytosis and catabolism of IgG was provided by experiments using mice that do not express functional FcRn due to homozygous deletion of the gene encoding b2-microglobulin. Thus the intestinal transfer of IgG was ablated in neonate mice with b2 deficiency (Israel et al., 1995). Similarly, the persistence (half-life) of IgG was considerably decreased in b2-microglobulin knockout mice relative to wild-type mice (Ghetie et al., 1996; Junghans and Anderson, 1996; Israel et al., 1996) (Table IV). These results were recently confirmed by using mice, with a defective FcRn gene. Similar to b2-microglobulin-deficient mice, these animals have a low perinatal IgG transport and a very short persistence of IgG in circulation (Roopenian et al., 2003). Human FcRn binds to all human IgG isotypes, but not to rat and mouse IgG, while mouse FcRn binds equally well to human, mouse, and rat IgG (Ober et al., 2001). The lack of affinity of human FcRn for murine IgG explains the short persistence of mouse monoclonal IgG antibodies in humans as compared with human IgG. Conversely in mice, both human and mouse IgG have the same half-life, since they have a comparable affinity for mouse FcRn (Ghetie et al., 2003). c. The Conformational Dependence of the FcRn Interaction Site. The IgG residues that are critical for FcRn binding are located on three proximal loops of the Cg2–Cg3 cleft that are noncontiguous in the primary sequence (Table III and Fig. 6). This suggests that the relative position of residues might be dependent on the conformation of the b strands that support them and also on the topology of the Cg2 and Cg3 domain relative to each other. Thus the detrimental effect of mutating Pro to Ala at position 257 on the serum half-life and affinity for FcRn is most likely due to perturbation of the conformation of the loop encompassing Ile-253. The same sequence is most probably responsible for the shorter half-life of rat IgG2b (Ala-257) relative to rat IgG1 and IgG2a (Pro-257) (Medesan et al., 1998). The FcRn–IgG interaction can be also modulated by conformational effects of residues distal to the Cg2–Cg3 cleft TABLE IV Intestinal Transfera and Catabolismb of IgG in FcRn-Deficient Mice

Mice

Intestinal transfer of mouse IgG2a (mg equivalents/ml)

Catabolism of mouse IgG1 (half-life, h)

Wild type Deficient

1.28 0

97.7 17.6

a

Israel et al. (1995). Ghetie et al. (1996).

b

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(Zuckier et al., 1998). Thus the removal of the hinge region from Fc results in a shorter half-life, indicating that this region determines the relative spatial orientation of Cg2 and Cg3 and the configuration of their interface. In fact, the replacement of the hinge sequence with a synthetic hinge containing an S–S bridge in the context of a different sequence generates an Fc fragment with a half-life similar to that of the wild type (Kim et al., 1995). d. The Stoichiometry of IgG–FcRn Interaction. The symmetry of the IgG molecule with two potential FcRn interaction sites suggests that one IgG molecule may bind two FcRn molecules simultaneously. The observation that a hybrid Fc fragment with only one functional FcRn interaction site per molecule is not transcytosed across the neonatal gut and has a considerably shorter half-life than the Fc fragment with two virtual FcRn interaction sites clearly suggested that two functional sites per IgG molecule are necessary for transcytosis and catabolism (Kim et al., 1994b, 1994c). Although the FcRn–Fcg complexes with 2:1 stoichiometry have been reported to form in solution (Martin and Bjorkman, 1999; Sanchez et al., 1999), other studies indicated that the stoichiometry is 1:1 (Popov et al., 1996). This observation suggests that the Fc or IgG molecules may be asymmetric with respect to FcRn binding. Sedimentation equilibrium clearly showed the existence of a high and low interaction site of the mouse Fc fragment for the mouse FcRn. A high-affinity 1:1 complex is first formed (binding constant <0.13 mM), and subsequently a second FcRn molecule binds with lower affinity (binding constant 6 mM) (Schuck et al., 1999). Recently it was shown that although soluble human FcRn does not dimerize, the membrane-anchored receptor can form dimers even in the absence of its IgG ligand (Praetor et al., 2002). The reaction of the FcRn dimer with IgG may involve a symmetric (stand up) and asymmetric (lay down) configuration as suggested by the ‘‘ribbon’’ model of interaction (Raghavan and Bjorkman, 1996) (Fig. 7). e. Engineering of the FcRn-Binding Site of IgG. The good correlation between the affinity of IgG for FcRn and IgG persistence in the circulation (Table III) has been exploited by engineering IgG molecules with increased affinity for FcRn and better pharmacokinetics. This was carried out by randomly mutating a recombinant murine Fcg at three residues located in proximity to the FcRn interaction site. Residues Thr-252, Thr-254, and Thr-256 were chosen because they are exposed and not highly conserved across species. One of these Fcg fragments with mutations to Leu-252, Ser-254, and Phe-256 had an affinity for FcRn three times higher than the wild type and a half-life of 153 h versus 93 h for the wild type (Ghetie et al., 1997). Analysis of the affinity of human IgG1 with multiple mutation in the Cg2–Cg3

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Fig 7 Schematic representation of the FcRn–Fc oligomeric ‘‘ribbon’’ model (according to Raghavan and Bjorkman, 1996). Both symmetric and asymmetric complexes have a 2:1 ratio between the FcRn dimer and the Fc fragment.

interface for human FcRn has shown that the affinity can be increased even higher (over 10 times) (Shields et al., 2001). However, if the increase in affinity for FcRn at pH 6 is accompanied by a simultaneous increase at pH 7.4, this affects the release of IgG into the serum and offsets the benefits of the enhanced binding at pH 6. Consequently, human Fcg mutants with a 10- to 20-fold increase of their affinity at pH 6 for mouse FcRn and with a parallel increase at pH 7.4 have a significantly shorter half-life than the wild type. However, since these Fcg mutants do not have a parallel increase in their affinity for human FcRn at pH 7.4, it is possible that in humans their half-life would be longer than that of the wild type (Dall’Acqua et al., 2002). Engineered humanized monoclonal antibody with a longer persistence in the circulation might have a better antitumor effect. Thus a long half-life results in a higher concentration gradient across cellular barriers with better penetration into the targeted tumor tissue (Ghetie et al., 2004). f. Therapeutic Implications. The administration of high doses of intravenous immunoglobulins (IVIG) leads to the enhanced catabolism of endogenous IgG (including autoantibodies). This is the result of saturation of FcRn in patients and leads to the destruction of all IgG present in excess (Masson, 1993; Yu and Lennon, 1999). The contribution of enhanced IgG catabolism to the beneficial effects of IVIG in the treatment of some autoimmune diseases is also suggested by the observation that the severity of experimental systemic lupus erythematosus (SLE) is greatly attenuated in b2-microglobulin-deficient mice with a nonfunctional FcRn. The very low levels of IgG and antibodies in

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these mice are the cause of the protection from the disease. Hence, since the mice lack a functional FcRn, they rapidly catabolize normal IgG and pathogenic autoantibody (Christianson et al., 1997). It was also shown that b2-microglobulin knockout mice are protected against some other antibody-mediated diseases (e.g., bullous pemphigus), since the amount of IgG antibodies reaching the epithelial target is greatly reduced due to their hypercatabolism (Liu et al., 1997). Accordingly, the development of SLE in MLR-1pr mice can be blocked in the majority of cases by treatment with a synthetic peptide interacting with the FcRn-binding site of IgG (Marino et al., 2000). 2. Polymeric Immunoglobulin Receptor The transport of dimeric IgA (dIgA) and pentameric IgM (both associated with the J chain) to the mucosal lumen is mediated by the polymeric immunoglobulin receptor (pIgR). The receptor is localized on the basolateral face of the epithelial cells that have to be crossed over by pIg to reach the mucosal surface (e.g., gastrointestinal or respiratory tract). At the basolateral surface pIgR binds noncovalently polymeric immunoglobulins (pIgs) through the Fc regions, and after endocytosis it subsequently translocates pIgs to the apical surface of the cells. During this translocation, some pIgs (e.g., human dimeric IgA) are also covalently bound through their Fc region to pIgR (Norderhaug et al., 1999). The release of the transcytosed pIgs from the plasma membrane takes place by proteolytic cleavage of the extracellular portion of pIgR (the secretory component) that remains attached to pIgs. The majority of investigations focused on the interaction of dIgA with pIgR or with the secretory component (SC), because the secretory IgA is the main immunoglobulin that mediates the humoral defense of mucosal surfaces. The secretory IgA consists of two molecules of IgA dimerized tail to tail with one J chain molecule inserted between the penultimate cysteine residue of the two a-chain tail pieces (Corthesy, 2002). To the Fc region of both IgA molecules, the SC is attached by noncovalent (rabbit) or noncovalent and covalent (human) bonds (Fallgreen-Gebauer et al., 1993; Frutiger et al., 1986). SC contains all five Ig-like homologous extracellular domains of pIgR (I–V) (Norderhaug et al., 1999). The first four are structurally similar to the V region of Ig molecules (with all three CDR-like loops), while the fifth is similar to the C region (Coyne et al., 1994). Each domain consists of 110 residues and is stabilized by one (domain II) and two (domains I, III, IV, and V) disulfide bonds. Only two domains of pIgR/SC are directly involved in binding the Fc region of dIgA. Domain I carries the site for the noncovalent interaction with both dIgA and IgM (Bakos et al., 1994). The mapping of the dIgA-binding site has revealed that a highly conserved sequence comprising residues 15–37 (in a CDR1-like loop) may be one of the binding site of domain I (Bakos et al., 1991). Other sites located in CDR2-like and CDR3-like loops of domain I may

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also be involved in the noncovalent binding to dIgA (Coyne et al., 1994). The binding of domain I of pIgR/SC to dIgA involves the Ca3 domain. The residues between positions 402 and 410 (Gln-Glu-Pro-Ser-Gln-Gly-Thr-ThrThr), located in an exposed loop of Ca3, are considered to be a potential binding site (Hexham et al., 1999). Domain V carries the site for the covalent interaction with dIgA belonging to some species (human, murine, bovine, etc.). SC is bound to the dIgA–J complex by a single disulfide bond between Cys-463 of domain V and Cys-311 of Ca2 (Fallgreen-Gebauer et al., 1993). A putative model of the interaction between dIgA and SC involves noncovalent binding of domain I with the Ca3 domain of one IgA molecule and a disulfide bond between domain V and the Ca2 domain of the other IgA molecule. According to this model, only one of the a-chains of both IgA molecules is involved in the binding of SC, as the other pairs remain unoccupied (Norderhaug et al., 1999). Domains II and III of SC do not interact directly with Fc of dIgA, but they are necessary for the covalent binding of domain V to Ca3 of IgA (Crottet and Corthesy, 1999). The fact that dIgA and IgM, devoid of the J chain, do not bind SC (Vaerman et al., 1998) suggests that pIgR/SC needs to bind not only to the Fc region of IgA, but also to the J chain to form a stable IgA2–SC–J complex. It was speculated that such J chain-binding sites might be located on domain I of pIgR/SC (Norderhaug et al., 1999). Thus the formation of the SC/dIgA complex is the result of the simultaneous noncovalent binding of pIgR/SC not only to Ca3 of IgA, but also to the J chain via binding sites located on domain I. The local humoral immune response is mainly mediated by secretory IgA, which plays a major role in protecting the mucosal surface against the invasion of pathogenic agents. SC present in the molecules of secretory IgA antibodies has a double role. First, it enhances the stability of the antibody by conferring resistance to the proteolytic attack of bacteria or local proteases (Crottet and Corthesy, 1998), and second, it ensures, through its multiple carbohydrate residues, appropriate tissue localization by anchoring the antibody to mucus lining the epithelial surface (Phalipon et al., 2002). III. Complement-Binding Sites

The complement system is an important mediator of innate immunity, linking it to the adaptive immune system. An array of complement proteins plays a significant role in host defense against infections and in inflammation (Reid, 1996). For the activation and function of the complement system, one of the most important steps is interaction of Ig molecules and IC with complement components. Several complement proteins interact with immunoglobulins (Miletic and Frank, 1995). The major Ig molecules, IgG and IgM, bind the C1q complement component, which initiates the classic complement pathway.

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The C3b and C4b complement components form a covalent linkage with IgG, and IgG molecules can noncovalently bind C3a, C4a, and C5a anaphylatoxins. A. C1q Binding The efficient activation of the complement pathway begins by the interaction of Ig molecules with the C1q glycoprotein, which is one of three components of the first complement component C1 (Kishore and Reid, 1999). C1q is a large molecule (460 kDa) composed of 18 polypeptide chains (six A, six B, and six C), each built from about 220 residues. A pair of A–B dimers and one C–C dimer linked by covalent and noncovalent bonds compose a subunit, and three such subunits associate into a hexameric molecule resembling a ‘‘bunch of tulips’’ with six globular regions, which contain the binding site for Ig molecules. Such a specific C1q structure with multiple binding sites allows multivalent high-affinity interactions with Ig molecules to be realized, leading to complement activation. Only IgG and IgM are capable of activating the classical complement pathway in humans. Monomers of these molecules have weak affinity to C1q, and they coexist in the circulation together with C1q without activating the complement cascade. The low affinity for C1q binding to monomeric IgG (Ka ¼ 5  104 M
1) increases dramatically upon IgG aggregation to Ka ¼ 108 M
1 due to polyvalent interactions. Human IgG3 and IgG1 have significantly higher binding activity than IgG2, while IgG4 cannot fix complement. The C1q binding hierarchy in mice is IgG2a > IgG2b > IgG1 and in rats IgG2b > IgG2c > IgG1 > IgG2a (Bru¨ ggemann et al., 1989). 1. The Amino Acid Residues of IgG and IgM Involved in Binding C1q The C1q binding to IgG is determined primarily by the structure of the Cg2 domain. Multiple amino acid residues build the C1q binding site. Residues Glu-318, Lys-320, and Lys-322 containing charged side chains compose the mouse IgG2b-binding motif (Duncan and Winter, 1988). Glu-318 can be replaced by Thr or by Asp and Lys by Arg without loss of the lytic capacity of IgG2b. The residues of this motif are conserved in all subclasses of human IgG and in most IgG subclasses of other mammals, which varied in their reactivity with C1q. These differences in C1q binding are determined by other Cg2 residues (Table V and Fig. 3). The substitution Leu-235!Glu in human IgG1 abolishes human complement lysis, whereas the same mutation has no effect on the human C1q affinity of murine IgG2b (Morgan et al., 1995). Furthermore, Ala substitutions at positions 318 and 320 of human IgG1 have little or no effect on C1q binding, whereas Ala substitutions at positions Asp-270, Lys-322, Pro-329, and Pro-331 considerably reduced C1q binding and activation of complement. The Lys-322!Arg mutation does not alter C1q binding, and probably a positive charge in this position is required for

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TABLE V Amino Acid Residues of IgG and IgM Important for the Binding of C1q Complement Component Immunoglobulin

Ig domain

Residues

References

Mouse IgG2b

Cg2

Duncan and Winter (1988)

Human IgG1

Cg2

Human IgG3

Cg2

Human IgM

Cm3

Glu-318 Lys-320 Lys-322 Leu-235 Asp-270 Lys-322 Pro-329 Pro-331 Gly-233 Leu-234 Leu-235 Gly-236 Lys-322 Asn-432 Pro-434 Pro-436

Morgan et al. (1995) Idusogie et al. (2000)

Sensel et al. (1997)

Thommesen et al. (2000) Arya et al. (1994)

the IgG1 – C1q interaction (Idusogie et al., 2000). The substitutions of prolines could change the configuration of the site. The binding site of human IgG3 for C1q is also different from that of murine IgG2b as only IgG3 mutants that lacked Lys-322 show strong reduction in antibody-dependent complement lysis (Thommesen et al., 2000). It seems likely that the C1q-binding sites of murine and human IgG are structurally different. A position 331, which locates near the murine IgG2b key binding motif, was identified as a critical element responsible for differences between the human IgG subclasses to activate complement (Tao et al., 1993). Human IgG4, which is unable to fix complement, has Ser-331, but molecules of other subclasses have proline at the same position. Substitution of Ser-331 in IgG4 with Pro partly restores complement activity, and the Pro-331!Ser substitution decreases or even abolishes the capacity to activate complement of other IgG subclasses. According to studies of human IgG2 and IgG3, mutants for optimal C1q binding the presence of residues Glu-233, Leu-234, Leu-235, and Gly-236 in the N-terminus of Cg2 (low hinge) is of importance and determines the relative capacity of IgG2 and IgG3 to bind C1q (Sensel et al., 1997). All these data suggest that C1q-binding activity and complement lysis are dependent on multiple residues, which are not conserved. Some of the residues can support the specific conformation of the interaction site(s) at a distance.

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The absence of the oligosaccharide at Asn-297 in Cg2 or alteration of its structure changes the ability of IgG to activate complement. Aglycosylated IgG2b with mutation Asn-297!Ala has reduced the C1 activation capacity (Duncan and Winter, 1988), and IgG with truncated Cg2 oligosaccharides are deficient in C1q binding (Wright and Morrison, 1994). However, the polymerization of aglycosylated IgG1 and IgG3 significantly increases the affinity for C1q, and such polymers are able to bind C1q, although less efficiently than corresponding wild-type polymers (Coloma et al., 2000). The C1q-binding site on the lower hinge and Cg2 overlap that of FcR (Fig. 3), and both ligands show a diminished ability to attach aglycosylated IgG. This suggests that C1q fixation and antibody-dependent cellular cytotoxicity (ADCC) are two mutually exclusive functions, and an antibody molecule bound to the target can bind either C1q or FcR-bearing cells, but not both simultaneously. The IgM monomers do not activate complement, and their affinity to C1q is low (Ka ¼ 2.5  104 to 5  105 M
1). However, after interaction of IgM antibodies with antigen, the affinity for C1q increases 103- to 104-fold. The binding site for C1q localizes on Cm3. The mutations of Cm3 residues Asn-432, Pro-434, and Pro-436 decrease the complement-dependent cytolytic activity of mouse IgM (Arya et al., 1994). Pro-436 of Cm3 is homologous to Pro-331 of Cg2, which is involved in C1q binding by IgG. However, most probably the IgG and IgM sites for C1q binding are not identical. The intact interheavy S–S bonds and oligosaccharides at Cm3 are important for the interactions with C1q (Wright et al., 1990). After binding to an antigen with multiple epitopes, IgM molecules become five-legged table-like structures exposing sites for complement binding (Perkins et al., 1991). B. C3b and C4b, Binding C3 and C4 complement components become covalently bound to antibody after activation by IC (Law and Dodds, 1997). These interactions play an important role in the elimination of antigens from the circulation. The C3and C4-binding sites are formed after enzymatic removal of the N-terminal parts of chains of C3 and C4. Due to a major conformational rearrangement resulting from proteolytic cleavage, the internal thioester is exposed to the solvent and its carbonyl group forms an ester bond with hydroxyl groups on receptive molecules. After incubation of C3 with heat-denatured IgG, C3b is covalently linked to the Cg1 domain of aggregated IgG in the region composed of residues 123–156 (Shohet et al., 1993). However, in experiments with IC of ovalbumin and antiovalbumin rabbit antibodies, it was found that C3b binds not only to Fab, but also to Fc and even with similar efficiency (Anto´ n et al., 1994). Deleting the Cg1 domain does not alter the ability of IgG either to bind C3 or to activate the alternative pathway (Mun˜ oz et al., 1998a). SpG inhibits the

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177

C3b covalent binding to the Fc region of human IgG1. As the Fc-binding site for the B fragment of SpG is located in the cleft between Cg2 and Cg3, it is highly probable that the C3b interaction site is also positioned there. The SpG B domain does not interfere with the C3b binding to the Fab fragment (Mun˜ oz et al., 1998b), which could be explained by the fact that the Fab binding sites for SpG (residues 209–216) and C3b (residues 123–156) are placed on opposite faces of Cg1 (Vivanco et al., 1999). All the previouslymentioned data support the view that C3b can attach to multiple sites on IgG, which favors the important functional role of this complement component. C3b bound to IgG retains its capacity to react with the C3 cell receptors. Therefore IC can react with cells not only through their Fc receptors, but also through the C3 receptors, which facilitates the interactions of IC with cells. C. Anaphylatoxin Binding Upon activation of the complement cascade, the C3a, C4a, and C5a anaphylatoxins are released from the C3, C4, and C5 components, respectively. These small molecules are responsible for many inflammatory and anaphylactoid reactions, even at very low concentrations, contributing to various pathological conditions (Hugly, 1984). Anaphylatoxins form noncovalent complexes with human IgG in serum at an approximately 1:1 molar ratio. They can be separated from IgG in strong denaturing conditions. The separated C3a and C4a purified by high-performance liquid chromatography (HPLC) are able to reassociate with intact IgG molecules (Nezlin and Freywald, 1992). The binding site for C3a was localized by immunoblotting at the Fab region (Nezlin et al., 1993). The reaction of IgG with C3a is specific, as C3a complexes with serum albumin were not found. C3a are cationic molecules with pI above 8.5 (Hugly and Mu¨ ller-Eberhard, 1978), and electrostatic interactions with negatively charged groups have to play an important role for the formation of complexes with Fab. As anaphylatoxins are very stable molecules, it is quite possible that they retain their biological activity after combining with IgG. The complexes could probably react simultaneously with cells by the Fc region (with FcR), as well as by anaphylatoxin molecules bound to the Fab region (with corresponding cell surface receptors). Such bivalent interactions would augment the cell responses. It was proposed that IgG molecules could serve as scavengers of free anaphylatoxins by eliminating them from the circulation, especially when the level of these highly active complement fragments increases in serum significantly due to complement activation (Nezlin et al., 1993). This view was recently supported by Basta et al. (2003). C3a bound to IgG was also found in commercial g-globulin preparations (Nezlin, 1993).

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IV. Proteins and Peptides Reacting with Fc

Many functionally important ligands interact with the Fc region of Ig molecules. Some of these interactions were studied in detail, such as binding Fc receptors and bacterial proteins, and are covered in corresponding sections of the review. Several other interactions are discussed below. A. Clusterin A conserved, highly glycosylated protein, clusterin (75–80 kDa) is constitutively synthesized in various tissues and presents in the circulation in concentration of about 0.1 mg/ml (Jones and Jomary, 2002). Precise functions of clusterin are still disputable. Clusterin interacts with many native or partly unfolded proteins using different binding sites (Lakins et al., 2002). Clusterin binds to all isotypes of human IgG and to other Ig classes. It reacts with both Fab and Fc fragments. Fc-binding site(s) do not overlap with C1q- or SpAbinding sites, as these proteins do not inhibit clusterin binding. Clusterin can promote the formation of insoluble IC probably by cross-linking soluble complexes (Wilson and Easterbrook-Smith, 1992; Wilson et al., 1991). B. Fibronectin Fibronectin (Fn), a large glycoprotein molecule, is found in soluble form in blood and other body fluids. Fn molecules are deposited as insoluble fibrils in the extracellular matrix and on the surface of many cells. Aggregated human immunoglobulins of all major classes bind Fn with the following hierarchy of affinity: IgG > IgM > IgA and IgG1 > IgG3 ¼ IgG4 > IgG2 (Rostagno et al., 1991, 1996). Fn–Ig complexes can be detected in normal plasma (Bray et al., 1994). The Fc region of IgG is responsible for the interactions with Fn, and the affinity of the Fn binding to Fc (Kd ¼ 3.69  109 M1) is nearly identical to that of its binding to intact IgG. Fn, as a constituent of the extracellular matrix, can associate with soluble circulating IC that are present in the blood of patients with autoimmune, rheumatic, and myeloproliferative disorders. It also interacts with Ig aggregates such as heat-denatured IgG or cold aggregated cryoglobulins, but not with monomeric IgG. The reaction is specific, as the blocking of Fn by anti-Fn antibodies inhibits the reaction with aggregated IgG. The matrix-associated Fn may participate in removing IC and aggregates of cryoglobulins from the circulation, causing their deposition in basement membranes of different organs. Such localization of IC could lead to the development of fibrotic diseases such as pulmonary fibrosis and glomerulonephritis, as well as occlusive vasculopathies and other pathology. Peptides from the Ig-binding site of Fn could be effective in blocking deposition of IC in tissues (Rostagno et al., 2002).

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C. Fc-Binding Peptides A number of polypeptides of various length and affinity are bound to the Fc region of IgG. Information on peptide–Fcg complexes was obtained by fluorescence polarization experiments with IgG and Fcg labeled with fluorescent dye (Dudich et al., 1978). It was observed that Fc-binding peptides could be separated either by dilution of IgG or Fc solutions or by decreasing their pH below pH 6. The dissociation of the labeled peptides from Fcg is responsible for the concentration and pH dependence of the rotational relaxation time (rh) of IgG or the isolated Fcg. The rh value is dropped after acidification of the IgG or Fcg solutions or after their dilution below 2 mM. As the molecular mass of the peptides is about 2 kDa, they have a small rh value. This dependence of rh on concentration was not found for Fabg. The separated peptides rotate independently and decrease the experimentally obtained mean values of the rh of IgG or Fcg. Nearly half of the peptides separated from Fcg can reassociate with the fragment. The peptide-binding site is probably located in the Cg2 domain (Dudich and Dudich, 1983). D. Rheumatoid Factor Rheumatoid factors (RF) are IgM or IgG autoantibodies directed toward the Fc region of IgG and are present in the sera and synovia of patients with rheumatoid arthritis (RA) (Levinson, 1989). There are several hypotheses for the origin of RF explaining some features of RF from RA patients. (1) RF-producing B cells are activated by T helper cells following the binding and processing of IC with IgG (Sutton et al., 2000). In the activation of B cells, Toll-like receptors also participate (Leadbetter et al., 2002). The activation of B cells through the Fcg region would be expected to determine all known features of RF, such as monoclonality, heterogeneous V-gene usage, idiotypical and isotypical diversity, and antigen-driven somatic mutation (Youngblood et al., 1994). (2) The Fc oligosaccharides are altered, producing new epitopes that could elicit RF production (Newkirk, 1996). (3) There is antiidiotypic mimicking with some Fc-binding protein (Oppliger et al., 1987; Tsuchiya et al., 1990). 1. Contact Amino Acid Residues between Fcg and RF The residues involved in binding RF are localized at the Cg2–Cg3 interface as determined by site-directed mutagenesis and confirmed by X-ray crystallography. The residues making contact with the RF-binding site of IgG were localized by X-ray crystallography using the Fab fragment of an IgM RF complexed with the Fc fragment of human IgG4 (Corper et al., 1997). The region of Fcg that is recognized by Fab of RF involves two segments of the Cg2–Cg3 cleft, namely, residues 251–255 in Cg2 and 422–440 in Cg3. Both

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segments are also involved in binding FcRn, SpA, and SpG, for which the crystal structure of their complexes with Fcg was also determined (Sections II.B and VII.A). As with SpA and FcRn, the essential residues of these segments are Ile-253 (in Cg2) and His-435 (in Cg3) (Bonagura et al., 1992, 1993; Corper et al., 1997). If His-435 is substituted by Arg (as in an IgG3 allotype), RF, as well as SpA and FcRn, fail to react with this IgG allotype (Kim et al., 1999; Matsumoto et al., 1983). The loop containing residues 309–311 involved in binding FcRn to IgG does not seem to play a significant role in the binding of RF. The heterogeneity of RF from various patients was reflected in the ability of some polyclonal RF to react with the allotype of IgG3 with Arg-435, as well as the inability of SpA to inhibit the RF–IgG interaction for some RFs (Artandi et al., 1991). Although some of the residues recognized by RF are also involved in the binding of SpA (or SpG), there is only partial identity. Each of the three Fcg residues, which form hydrogen bonds to RF (Ile-253, Ser-254, and Asp-434), also forms hydrogen bonds to SpA and SpG, but the orientation of these bonds cannot be spatially superimposed. Furthermore, the salt bridge formed between Arg-255 (in Cg2) and Asp-31 (in RF) has no correspondence in the SpA/SpG–IgG complexes (Corper et al., 1997). 2. The Topology of the Fab RF–Fc IgG4 Interaction The residues involved in the binding of Fab RF to human Fc IgG4 are completely different from those of SpA/SpG or FcRn and support the idea that there is little if any correspondence at the atomic level between the interactions of these Fcg ligands (Corper et al., 1997). The Fab RF fragment is bound symmetrically on both side of Fcg, revealing a 2:1 stoichiometry. The bivalency of Fcg suggests that the mechanism of activation of RF-producing B cells may occur by cross-linking their surface antigen receptors by IgG and driving the autoimmune response (Sutton et al., 2000). The topology of this particular Fab RF–Fcg interaction is significantly different from all other familiar Fab–antigen complexes. In the Fab RF–Fcg complex, only one side of the combining site surface participates in the interaction, and the number of contacts are fewer than in other antigen–antibody complexes due to the lack of contribution from L1 and L3 CDRs. The principal contact region of RF is only H3 CDR, which protrudes into the Cg2–Cg3 cleft. The consequence of this particular topology is that the conventional binding area remains unoccupied and is therefore accessible to other unidentified antigens, which may in fact be responsible for the induction of RF (Corper et al., 1997). The identification of the residues of Fab RF that are in contact with those of the Cg2–Cg3 interface of IgG4 may not be relevant for other RFs due to their well-known idiotypic heterogeneity (Chen and Carson, 1994). However, as far as the Fcg residues involved in the binding of various RFs are concerned, the

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available data clearly point to the participation of the residues from the two main loops of the Cg2–Cg3 cleft centered around Ile-253 and Ser-254 in the Cg2 domain and Asp-434 and His-435 in the Cg3 domain. V. Proteins Reacting with Fab

Proteins that are able to react with the Fab portion of Ig molecules in a nonantigen way can activate cells bearing a particular V-domain family of the B cell Ig receptors. A requirement for cell activation is the functional multivalency of Fab-reacting molecules, which is essential for cross-linking of cellbound Ig molecules. The B cell activation could lead to serious pathological events, especially if the stimulated B cells are producers of autoantibodies (Silverman, 1997). The Fab-reactive proteins can also bind to Ig molecules, which are complexed with the Fc cell receptors, and stimulate cell activities. A. Prolactin Prolactin (Prl), a stimulator of lactation, is a polypeptide hormone secreted by the anterior pituitary gland. In humans it presents as a 23-kDa monomer and also as dimers and polymers. Prl receptors are located mainly on cells of the mammary gland, but also on human B and T lymphocytes, monocytes, and NK cells. There is evidence that Prl has an immunomodulatory role. Prl can form complexes with IgG, interacting with the Fd part of all four human IgG subclasses (Walker et al., 1995). The IgG–Prl complex has about 1 mol of Ne(gglutamyl)lysine cross-links per mole of the complex, which indicated a possible role of enzyme transglutaminase in the formation of linkages between IgG and Prl. About 0.8% of all IgG molecules in the circulation are complexed with one or two Prl molecules. The Prl–IgG complexes can stimulate lymphocytes of some patients with chronic lymphocytic leukemia. However, Prl alone is not active in this respect, and the proliferating activity of the complex involves the engagement of both Prl and IgG. The effect is probably due to the coligation of the receptors specific to both these proteins. B. Protein Fv Protein Fv (PrFv), a 175-kDa hexavalent sialoprotein, is synthesized in the liver and released into the digestive tract during liver diseases. This protein is also present in healthy persons in complexes with luminal immunoglobulins (Bouvet et al., 1996). PrFv is absent in the gut of axenic rats, but it appears after colonization by human gut microflora, which seems to be a major factor in the release of PrFv (Andrieux et al., 1998). In feces, two types of PrFv complexes with F(ab0 )2 of secretory IgA were found with molecular masses of 1800 and 800 kDa. The last complex consists of six F(ab0 )2 and one PrFv dimer. PrFv binds to VH domains of human immunoglobulins (Ka ¼ 6.7  108 M
1), as well

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as of Ig molecules of most animal species studied at sites that are highly conserved throughout evolution. It preferentially interacts with the H chains of the VH3 Ig family in areas of FR1 and 3 exposed to solvents and additionally can recognize some VH6 and VH4 immunoglobulins. PrFv competes with SpA for the same binding site on VH3 (Silverman et al., 1996). However, the binding sites for these two proteins are not identical. There are VH3 molecules, which do not react with SpA, but bind PrFv. VH6 and VH4 immunoglobulins with PrFv-binding activity have no binding specificity for SpA. CDR sequences do not correlate with the PrFv-binding activity, and the interaction with PrFv does not interfere with the reaction of antibodies with antigen. PrFv is capable of inducing or enhancing several important effector functions of immunoglobulins. This functional activity of PrFv could be explained by its ability to bridge Ig molecules and thus to form nonimmune complexes. Due to its constant presence in the intestinal tract, PrFv is an important factor in mucosal immunity. Large complexes of secretory IgA or its F(ab0 )2 fragments are formed by PrFv in the digestive lumen, which greatly increases the protective capacity of antibacterial and antiviral agglutinating IgA antibodies and improves the excretion of bacteria from the gut. The Ig–PrFv complexes activate the classical complement pathway by mimicking IC, which could induce local intestinal lesions (Ruffet et al., 1994). PrFv also acts as an activator of human basophils and mast cells by interacting with the VH domain of IgE molecules bound to the cell Fce receptors. Such reactions stimulate the synthesis and release of proinflammatory mediators such as histamine and leukotrienes, as well as cytokines interleukin (IL)-4 and IL-13 from these cells. It is likely that this PrFv activity could desensitize mast cells by continued interactions and prevent hypersensitivity reactions induced by allergens (Patella et al., 1998). The VH3–PrFv interactions could contribute to the development of malignancy. Nearly all studied VH genes of mucosal-associated lymphoid tissue lymphoma cells are the VH3 type (Hashimoto et al., 2001). It was hypothesized that PrFv could react with B lymphocytes, which possess VH3 Ig receptors and enhance the replication of these cells. The increased number of cell divisions of the VH3 memory B cells could stimulate their transformation into lymphoma cells. C. T Cell Protein CD4 CD4 is a 55-kDa membrane glycoprotein expressed on helper T lymphocytes and composed of four Ig-like domains (Brady and Barclay, 1996). It binds to the nonpolymorphic regions of MHC II molecules, to the human immunodeficiency virus (HIV) gp 120 protein, and also interacts with Ig molecules of nearly all classes with relatively low affinity. Two distinct sites on the CD4 first

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V-set Ig-like domain (residues 21–28 and 35–38) are involved in the reaction with immunoglobulins, and one CD4 molecule is able to cross-link two Ig molecules (Lenert et al., 1990, 1995). The similar CD4 sites on two juxtaposed antiparallel C0 C00 loops are responsible for HIV gp 120 binding. This fact explains the inhibition of the CD4–Ig interaction by gp 120. Polianionic substances such as sulfated dextrans and heparin inhibit CD4 binding, which indicates participation of charged residues of CD4 in the reaction. The VH FR residues participate in the interaction with soluble recombinant CD4. The interaction with CD4 enhances the antigen–antibody reaction, which could explain the antibody-mediated enhancement of the HIV infection.

D. Histidine-Rich Glycoprotein Histidine-rich glycoprotein (HRG) is a 75-kDa protein composed of four domains. It is synthesized in liver and is contained in plasma at a relatively high concentration (0.11 mg/ml). The precise functions of HRG are still unknown, but its modular structure suggests the presence of several binding sites with the ability to interact with various ligands simultaneously. Indeed, HRG could bind to heparin, fibrinogen, complement components, immunoglobulins, and some other ligands. The N-terminal HRG domain (30 kDa) is involved in binding C1q and immunoglobulins. HRG binds to IgG of all subclasses with relatively higher affinity compared to its other ligands (Gorgani et al., 1999a). The type of L chains affects the kinetics of the reaction. The on rate for HRG binding to IgG1k and IgG2k is significantly faster than that for the binding to IgG1l and IgG2l. Surprisingly, the on rate for HRG binding to IgG3k and IgG4k is slower than that for IgG3l and IgG4l. HRG is also able to bind to Bence–Jones proteins with an affinity higher for the k type of these proteins. The binding site for HRG locates on Fab regions, as HRG binds to F(ab0 )2 with the same affinity as to the whole IgG molecule (Gorgani et al., 1997). The interaction of HRG with IgMk is much weaker than with IgGk. HRG or its N-terminal domain can inhibit in vitro the formation of insoluble IC between ovalbumin and antiovalbumin IgG or between human IgG and rheumatoid factor. The addition of whole human plasma prevents the formation of insoluble IC, whereas plasma with reduced HRG concentration even enhances the formation of such complexes. The addition of HRG to plasma with reduced HRG concentrations restores its inhibitory activity. These data suggest that inhibition of the formation of IC by HRG is physiologically significant and could be relevant to the process of clearance of circulating IC. HRG also promotes the solubilization of preformed IC in a dose-dependent manner and inhibits interactions between monomeric IgG, as well as IC with mononuclear cells (Gorgani et al., 1999b, 1999c).

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VI. Lectins

Lectins are widely distributed proteins that interact noncovalently with carbohydrates, both simple and complex, as glycoproteins and glycolipids (Sharon and Lis, 2003). Each lectin has two or more combining sites and could cause cross-linking of glycoproteins or cells. They interact with immunoglobulins through sugar residues.

A. Plant Lectins Jacalin, a 65-kDa protein isolated from seeds of a tropical plant, jackfruit, has a strong affinity for galactose b1!3 linked to N-acetylgalactosamine (Kabir, 1998). There are four aromatic residues in the sugar-combining site of jacalin. The lectin binds to serum or secretory human IgA1, but not to IgA2 of both allotypic variants or to other Ig classes or IgA of other mammals (with the exception of chimpanzee and rabbit secretory IgA). Jacalin interacts with human IgD, which contains galactose b1!3 linked to N-acetylgalactosamine in the hinge region. The jacalin specificity for IgA1 is due to the presence of five nonsialylated galactose-containing O-linked oligosaccharides located in the IgA1 hinge. The IgA2 molecules have no glycans in this region due to a deletion of 13 residues. Jacalin is used effectively for the purification of human IgA1 and its separation from IgA2 (Haun et al., 1989). A small fraction of rabbit IgG is also bound by jacalin. The interaction is probably mediated through O-linked glycans present on the H chain (Kabir et al., 1995). A technique was developed to measure IgA1 concentration in serum and saliva using jacalin. Jacalin is also a useful tool in studies of IgA nephropathy, which is characterized by the deposition of IgA1 in renal tissue and by elevated IgA1 concentration in blood. The galactose-specific lectin from castor beans, Ricinus communis, reacts specifically with accessible terminal galactose residues of the Fc and Fab portions of IgG molecules. The affinity of the lectin is higher after the aggregation of IgG, and increases with the mass of IgG aggregates. The lentil, Lens culinaris lectin, specific for a-mannose residues, binds to IgM molecules and can be used for purification of monoclonal IgM antibodies from ascites fluid. Several lectins were isolated from Malaysian champedak seeds. The first of them is a 52-kDa galactose-binding lectin-C that reacted with human IgA1 and colostral IgA, but not with IgA2 and other immunoglobulins (Hashim et al., 1991). The second lectin, a 64-kDa mannose-binding lectin-M, interacts strongly with a number of human serum proteins and IgA, as well as with g- and m-chains, but not with intact IgG molecules (Hashim et al., 2001).

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B. Animal Lectins 1. Galectin-3 and Galectin-1 A 31-kDa IgE-binding protein, galectin-3 (Gal-3), is expressed on the surface, as well as in the cytoplasm and nucleus, of different kinds of cells, including mast cells, macrophages, neutrophils, and eosinophils. It also exists in the extracellular medium. Gal-3 is a highly conserved multifunctional protein with b-galactoside specificity belonging to the galectin (S-type) animal lectin family (Liu, 1990, 1993; Rabinovich et al., 2002). The receptors for Gal-3 on IgE are N-linked oligosaccharides, and the interaction depends on sialylation of their terminal residue (Robertson and Liu, 1991). Gal-3 could act as an amplifier of the inflammatory cascade because it recognizes not only IgE, but also the Fce receptor on mast cells. The activation of these cells by Gal-3 is achieved by cross-linking either the Fce receptors or receptor-bound IgE molecules or both. Another member of the galectin family, galectin-1 (Gal-1), is a multifunctional homodimer protein with b-galactoside binding activity (Rabinovich et al., 2002). Gal-1 is expressed on the surface of various cells, including cells of the immune system, and reacts with a number of cell surface receptors, initiating signal-transduction events. It was found that the reaction of human pre-B cells with stromal cells is accounted for by the ability of the pre-B cell receptor to interact with Gal-1, as well as with some other stromal cell receptors (Gauthier et al., 2002). The interactions result in pre-B cell triggering. NH2-terminal part of the invariant l-like peptide, a part of the surrogate L chains of the human pre-B cell receptor, is responsible for the interaction with Gal-1 (Ka~106M1). In contrast, binding of a murine pre-B cell receptor to stromal cells depends on interactions of l-like peptide (l5) of the receptor with stroma cell-associated heparan sulfate (Bradl et al., 2003). 2. Mannose-Binding Protein A and Mannose Macrophage Receptor A 650-kDa serum lectin, mannose-binding protein A (MBP-A), is a hexamer of trimeric units forming a ‘‘bouquet’’-type structure, which structurally resembles C1q. Its carbohydrate recognition domains bind not only mannose, but also N-acetylglucosamine and some other sugar residues (Holmskov et al., 1994). MBP-A is a member of the collectin family and plays an important role in innate immunity, as it mediates the activation of the classical complement pathway in an antibody independent way (Epstein et al., 1996). MBP-A binds to IgG molecules, the N-terminal sugar residue of which is N-acetylglucosamine, but not galactose. The MBP-A interaction with IgG results in complement activation. As the levels of agalacto-IgG are markedly increased in patients with rheumatoid arthritis, the interaction can induce chronic inflammatory reactions of the synovial membranes of injured joints (Malhotra et al.,

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1995). IgM molecules have a high content of oligomannose, and MBP-A could be used for the isolation and detection of this immunoglobulin (Koppel and Solomon, 2001; Nevens et al., 1992). Mannose receptor, a C-type lectin expressed on macrophages, dendritic cells, and epithelial cells, binds to agalacto-IgG in a manner similar to MBP-A (Dong et al., 1999). 3. Galactosyltransferase An enzyme involved in the biosynthesis of oligosaccharides, b-1,4-galactosyltransferase (GT), either in soluble or membrane-bound form, binds to molecules of main Ig classes and acts as a lectin (Tomana et al., 1993a). The interactions with immunoglobulins can be explained by incomplete galactosylation of their oligosaccharides and the ability of GT to bind galactose residues. The most effective binding is to polymeric IgA2 molecules and less effective to IgG. Isolated Ig peptide chains interact more efficiently with GT than intact Ig molecules, which could be explained by the higher accessibility of terminal residues of oligosaccharide moieties. The more effective interactions of GT with IgA, IgM, and their H chains (as compared with IgG) could be explained by the presence of several carbohydrate chains on a-, m-, and J-chains, which in higher proportion lack terminal galactose or sialic acid residues and therefore are substrates for galactosylation. The functional significance of GT–Ig complexes is not so clear, but they could probably prevent the interactions of immunoglobulins with cell receptors. The membrane-bound GT found on the surface of various types of cells, including human lymphocytes, is also able to bind immunoglobulins and Ig peptide chains and acts as a lectin-like receptor (Tomana et al., 1993b). VII. Proteins from Pathogens Reacting with Immunoglobulins

A. Bacterial Immunoglobulin-Binding Proteins Proteins with the ability to bind Ig molecules in a nonimmune fashion are expressed on the surface of many microorganisms. They are involved in the process of infection and are able to weaken the immune response. Bacterial Ig-binding proteins have been widely applied for isolation and quantitation of immunoglobulins and their fragments. 1. Staphylococcal Protein A a. Structure and Fc-Binding Site. Protein A (SpA) is a component of the Staphylococcus aureus cell wall, consisting of an extracellular part, which reacts with the Fcg region, and a cell wall-binding part (Forsgren et al., 1983). The extracellular part contains a tandem of five highly homologous monovalent Fc-binding domains designated E, D, A, B, and C. Each domain is composed

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of about 60 residues (6.6 kDa). Domains A–D have similar affinities for Fcg but the affinity of the E domain is lower (Moks et al., 1986). The B fragment (or its recombinant counterpart, Z) is composed of a bundle of three a-helices, all of them being retained in the B–Fcg complex, but only two (helices I and II) making contact with Fcg (Kato et al., 1993). The threedimensional structure of the B–Fcg complex has been resolved by X-ray diffraction analysis (Deisenhofer, 1981), and the contact residues between B and Fc were confirmed by an NMR study (Kato et al., 1993), as well as by mutation analysis (Cedergren et al., 1993). The residues of the Z fragment involved in contact with Fc are located in helix I (Phe-5, Glu-9, Asn-11, Phe-13, Tyr-14, and Leu-17) and helix II (Asn-28, Phe-30, Ile-31, Glu-32, and Lys-35). From these residues, Leu-17, Asn-28, Ile-31, and Lys-35 are all clustered at the Cg2–Cg3 interface, establishing hydrophobic and hydrogen bond contacts (Tashiro and Montelione, 1995). b. Amino Acid Residues of the Fcg Region Involved in SpA Binding. By X-ray diffraction analysis, it was shown that the previously-mentioned residues of the B fragment may establish contact with some residues of human Fcg, which are located in three separate loops of the Cg2–Cg3 interface (252–254, 308–312, and 433–436) (Deisenhofer, 1981). The participation of some of these residues in binding the B fragment was confirmed by NMR analysis, indicating the key role of Ile-253, Ser-254, His-310, and Gln-311 in Cg2 and His-433, His-435, and His-436 in Cg3 (Kato et al., 1993). These findings were confirmed by site-directed mutagenesis of mouse Fcg showing that the mutations of Ile-253, His-310, His-435, and, to a lesser extent, His-433, Asn-434, and His-436 diminish the reactivity with immobilized SpA (Kim et al., 1994a) (Table VI). The mutation of His-435 (in human IgG1) by Arg (as in a human IgG3 allotype) decreased SpA binding, shortened the IgG half-life in mice (Kim et al., 1999), and reduced in situ transmission through the placenta (Firan et al., 2001). The residues of the three loops of the Cg2–Cg3 cleft show species variation (with the exception of Ile-253, His-310, Gln-311, and Asn-434) that may explain the variable affinity of SpA for IgG belonging to different species and isotypes (Forsgren et al., 1983). The reaction of SpA and SpG with IgG was extensively exploited for the measurement and purification of IgG antibodies and various antigens (Boyle, 1990), for the isolation of IC (Nezlin, 2000), as well as for the detection, isolation, and purification of a variety of cell populations (Ghetie and Sjo¨ quist, 1984a, 1984b). c. The Stoichiometry of the IgG–SpA Interaction. The reaction of the monovalent B fragment of SpA with human Fc and rabbit and mouse Fcg and IgG yields soluble complexes B–Fcg or B–IgG with a molar ratio of 2:1,

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indicating that both Cg2–Cg3 clefts are equally available for interactions with the B fragment (Deisenhofer, 1981; Dima et al., 1984). The stoichiometry of the reaction between IgG and intact SpA is difficult to estimate, because IgGs of many species form insoluble or weak complexes that cannot be properly characterized. The only exception is rabbit IgG, which forms stable soluble complexes with SpA and some of its fragments. In a wide range of molar ratios (from 1:4 to 4:1), only two main types of soluble rabbit IgG–SpA complexes were identified (Dima et al., 1984; Langone et al., 1978; Mihaescu et al., 1979; Mota et al., 1978). One with a molecular mass of 190 kDa and a molecular formula of IgG1–SpA1 forms in excess of SpA, and the other is obtained in excess of IgG with a molecular mass of 680 kDa and a molecular formula of IgG4–SpA2. Since the IgG1–SpA2 complex was not identified, it was concluded that the reaction of SpA with one side of Fcg exerts a steric constraint on the other site or masks it (Hanson and Schumacher, 1984). The apparent monovalency of IgG in the reaction with SpA is reminiscent to the behavior of human IgG, which was able to interact with two Fab fragments of RF (Corper et al., 1997), but only with one intact RF molecule (Nardella et al., 1981). Evidently, the reaction of intact RF with one of the available Cg2–Cg3 clefts makes the other cleft sterically inaccessible. d. The Binding of SpA to the Fab Region of IgG. In addition to the Fcg binding site, SpA has another distinct site with the ability to interact with the Fab region of IgG and some other Ig classes (Romagnani et al., 1982). Through this interaction, a significant percent of human IgM, IgA, IgE, and IgG binds

TABLE VI Amino Acid Residues of Murine Fc Fragment Involved in the Binding of Immobilized SpAa Amino acid residueb

Binding to SpAc

Wild type Ile-253 His-310 His-433 Asn-434 His-435 His-436

100 23 12 38 62 10 79

a

Kim et al. (1994) and Medesan et al. (1997). All residues were mutated to Ala. c Radiolabeled wild-type mouse Fc (from IgG1) and its mutants were reacted with SpASepharose, and the amount of the bound Fc was measured. The amount of the bound wildtype Fc was considered as 100%. b

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to SpA (Inganas, 1981; Inganas et al., 1980). The Fab-binding site seems to be located on all of the five domains (Ljungberg et al., 1993; Roben et al., 1995). Human and mouse immunoglobulins that interact with SpA in the Fab region are encoded by gene segments belonging to the VH3 or J606 and S107 VH families (Potter et al., 1996; Roben et al., 1995; Sasso et al., 1991; Seppa¨la¨ et al., 1990). The pattern of SpA binding to human immunoglobulins indicates not only that residues in FR1, CDR2, and FR3 are involved in the interaction, but also that all these three regions are required for binding to occur. The interaction with SpA does not block the antigen-combining site, leading to the conclusion that the contact occurs outside this site and the SpA molecule could be considered as a B cell superantigen (Silverman, 1997). The crystal structure of the complex between fragment D and the Fab fragment of a human IgM antibody indicated that a-helices II and III of fragment D interact with the VH3 region through framework residues without any involvement of the CDRs (Graille et al., 2000). The contact residues are highly conserved in both the V region of the antibody and in the D domain of SpA. The interacting surfaces are mainly hydrophilic, involving polar side chains and salt linkages, as also indicated by an NMR study of the reaction of SpA fragment E with a Fv fragment derived from a human antibody (Meininger et al., 2000).

e. SpA Mimetics. Several peptidyl-SpA mimetics with molecular sizes smaller than the B(Z) domain were obtained (Braisted and Wells, 1996; Nilson et al., 1987). From a combinatorial phage display library, a decapeptide was selected that resembles residues of SpA responsible for interaction with Fc (FCRLVSSIRY). Since it was eluted from an immobilized Fc fragment with SpA, it is quite possible that some of these decapeptides react with the same sites on Fc (CH2–CH3 cleft) as SpA does (Krook et al., 1998). Another SpA mimetic (PAM) consisting of a tetrameric tripeptide (Tyr-Thr-Arg linked to polylysine) was able to inhibit the binding of SpA to IgG and to react with mammalian IgGs and chicken IgY. It was used effectively for the isolation of immunoglobulins from serum and the purification of monoclonal antibodies from ascites (Fassina, 2000; Fassina et al., 1996). Nonpeptidyl ligands that mimic SpA were also synthetized (Kabir, 2002). Using a computer modeling, a series of nonpeptidyl biomimetic molecules around residues Phe-132/Tyr-133 involved in the reaction with Fc were designed (Li et al., 1998). One of them (ApA) was used for the purification of IgG. The SpA mimetics could also be valuable for the development of a new family of candidate drugs for the control of IgG overproduction by accelerating the autoantibodies, catabolism (Marino et al., 2000). Similar to SpA or its B fragment, these drugs may block the interaction of IgG with FcRn (Dima et al., 1984; Raghavan et al., 1994) and as a consequence may accelerate

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the catabolism of IgG (including autoantibodies) and its removal from the circulation. f. Therapeutic Implications. Immobilized SpA (e.g., Prosorba) was used for the extracorporeal selective removal of plasma proteins to achieve immune modulation and a therapeutic effect. This procedure has a beneficial clinical effect in various diseases such as cancer (Messerschmidt et al., 1989), idiopathic thrombocytopenia (Guthrie and Oral, 1989; Muroi et al., 1989), rheumatoid arthritis (Felson et al., 1999), SLE (Braun et al., 2000), and various renal diseases (Belson et al., 2001; Esnault et al., 1999). It also causes the modulation of the immune response in patients with autoimmune diseases (Braun and Riesler, 1999; Snyder et al., 1989). 2. Streptococcal Protein G a. Structure and Fcg-Binding Site. Protein G (SpG) originates from two main streptococcal strains, C and G (Boyle, 1990). SpG from strain G (65 kDa) consists of several repetitive domains. Three of them placed at the C-terminal half of the molecule, with very similar if not identical structures and designated C1, C2, and C3 or B1, B2, and B3, are involved in the binding to Fcg (Sjobring et al., 1991). The two or three other domains located in the N-terminal half of the molecule have been found to bind human serum albumin (A˚ ckerstro¨ m et al., 1987). Each of the B/C domains of SpG consists of approximately 60 residues and presents a high degree of homology, if not identity (Sjobring et al., 1991; Tashiro and Montelione, 1995). The structure of the three recombinant B/C domains reveals the presence of one single a-helix positioned diagonally across four b sheets (b-1 to b-4) (Achari et al., 1992; Gallagher et al., 1994; Lian et al., 1992; Sauer-Eriksson et al., 1995). The residues involved in the binding to Fcg are localized in two section of the B/C domain, namely, in the central part of the a-helix and in the N-terminal end of the b-3 sheet. The a-helix contacts contain a tandem of two pairs of residues (Lys-28/Glu-32 and Glu-27/Lys-31) whose side chains are exposed on the opposite side of the a-helix. The Nterminal end of the b-3 sheet contains Glu-42 and Trp-43 in close spatial proximity to Asp-35, and Asp-40 situated at the end of the a-helix and in the loop connecting the a-helix with the b-3 sheet, respectively. These findings resulted from NMR and crystallographic studies and are also supported by the fact that a short fragment containing residues 34–44 is able to inhibit binding SpG to Fcg (Frick et al., 1992). b. Amino Acid Residues of the Fc Region Involved in the Binding of SpG. The residues of Fcg that interact with the B/C residues are localized in all three hydrophobic loops of the Cg2–Cg3 cleft, and some of them are also involved in

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TABLE VII Interaction of Amino Acid Residues of Fc Region with SpG and SpAa Fc residues Loop 1 (CH2 domain) Leu-251 (MC)b Met-252 (H) Ile-253 (H) Ser-254 (SC) Loop 2 (CH2 domain) Glu-311 (SC) Leu-314 (H) Loop 3 (CH3 domain) Met-428 (H) His-433 (SC) Asn-434 (SC)

SpG (C2 domain residues)

SpA (B domain residues)

Lys-31 (SC) Lys-28 (H) Trp-43 (H) Glu-27 (SC)

Gln-10 (SC) Phe-5 (H) Phe-13 (H) Gln-9 (SC)

Glu-42 (SC) —

Asn-18 (SC) Leu-17 (H)

Lys-28 (H) Asn-35 (SC) Asn-35 (SC) Val-39 (SC) Trp-43 (MC)

— — Asn-11 (SC)

His-435 (H) His-436 (MC) Gln-438 (SC)

Lys-28 (H) Asn-35 (SC) Gln-32 (SC)

Tyr-14 (H) Leu-17 (H) — —

a

Adapted from Sauer-Eriksson et al. (1995). MC, polar interaction with main-chain atoms; SC, polar or charged interaction; H, hydrophobic interaction.

b

binding SpA. A comparison of the Fcg residues involved in binding SpG and SpA is presented in Table VII. In addition to these residues, there are two residues (Gln-380 and Glu-382) in the Cg3 domain outside the Cg2–Cg3 cleft that establish a bridge with Lys-28 of the C2 domain of SpG (Sauer-Eriksson et al., 1995). These two Cg3 residues are not involved in binding SpA. His-310, which plays a key role in binding FcRn (Table III) and SpA (Table VI), seems not involved in binding SpG and RF (Corper et al., 1997; Deisenhofer, 1981; Medesan et al., 1997). Four of the Cg2–Cg3 interface residues (Ile-253, Ser-254, Glu-311, and Asn-434) are of particular importance because their side chains interact with the side chains of both SpA and SpG (Table VII). Since there is no structural homology between SpG and SpA, it is not surprising that these two proteins use particular modes of interaction with Fcg. Thus SpG makes contact with His-433, Glu-380, Gln-382, and Glu-438 (the last three residues being outside the Cg2–Cg3 cleft), whereas SpA interacts with the side chains of Leu-314 and His-435. The lack of interaction of position 435 with SpG but not with SpA explains why the latter cannot interact with an allotype of human IgG3 with Arg-435. In fact, the introduction of Arg in position 435 can

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be accommodated into the SpG C2–Fcg complex, but not in the SpA B–Fcg complex, where His-435 is in close proximity to several hydrophobic residues of the B fragment (Deisenhofer, 1981). All these differences in the reaction of Fcg with SpG and SpA are reflected in the remarkable differences in their affinity and pH dependence for IgG. Thus the affinity of SpG for many IgGs is 10-fold higher than that of SpA (1–10 nM) (Boyle, 1990). The optimal binding of Fcg to SpG takes place at around pH 5.0, while SpA requires a neutral or, for some classes and subclasses of IgG, an alkaline pH, both complexes dissociating at lower pH than 5.0. Interestingly, some IgGs bound to SpG-Sepharose at pH 5.0 can be eluted by raising the pH in the alkaline region (A˚ ckerstro¨ m and Bjo¨ rk, 1986), a behavior reminiscent of the ability of Fcg to bind FcRn at acid pH and release it at a slightly alkaline pH (Ghetie and Ward, 2000). c. Interaction of SpG with the Fab Region. Different portions of the B/C domains are involved in binding the Fcg and Fabg regions (Derrick and Wigley, 1994; Lian et al., 1994). The affinity for Fabg is, however, 10 times lower (Bjo¨ rck and Kronval, 1981). The SpG–Fabg interaction involves exclusively the b-2 strand of the B/C domain. This strand interacts with the last, surface-exposed b strand of the Cg1 domain. The Cg1 residues that are in contact with the B/C domain are highly conserved in human and mouse IgG (Pro-126, Val-128, Tyr-129, Ser-209, Ser-210, Thr-211, and Lys-215). These residues establish main chain/main chain hydrogen bonds and van der Waals contacts with residues 11–17 of the b-2 strand of the B/C domain (Derrick and Wigley, 1994). The antiparallel pairing of these two b sheets from Fabg and the B/C domain is a novel protein–protein recognition system different from that of the key/lock type of the SpG–Fcg interaction (Kuehn et al., 1993). The highly conserved nature of Cg1 residues explains the reaction of SpG with Fab of all Ig isotypes of human, mouse, and probably other species. This is in contrast with SpA, which recognizes only particular sequences of some human and mouse IgG belonging to restricted VH families (Tashiro and Montelione, 1995). 3. Streptococcal Protein H Protein H (SpH) is a polyreactive molecule expressed on the surface of some strains of Streptococcus pyogenes. It belongs to the large family of fibrous surface M proteins, which have structural similarity to a number of host proteins such as tropomyosin, myosin, and laminin. SpH interacts by its Nterminal domains with the Fc region of human IgG with a high affinity (Ka ¼ 1.6  109 M
1). The Fc-binding site is located at the Cg2–Cg3 interface, nearly in the same region as the binding sites for SpA and SpG (Frick et al., 1994, 1995). However, the Fcg-binding sites for these proteins have different structures. The SpH–Fcg interaction is temperature dependent with high

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affinity at 4 8C and 22 8C, but no reaction is registered near 37 8C when SpH is in an unfolded inactive form (Nilson et al., 1995). At low temperatures, SpH exists as a dimer, but at 378C it can be found only as monomer. Most likely, SpH has to be in a dimeric form for effective binding to IgG. 4. Peptostreptococcal Protein L Protein L (PpL) (76–106 kDa), expressed by some strains of Peptostreptococcus magnus, binds exclusively to the V regions of k L chains of human and other mammalian immunoglobulins with high affinity. The binding activity is located at four or five small homologous extracellular domains in the Nterminal end of PpL. The three-dimensional structure of these domains is in general very similar to that of the SpG domains despite the fact that SpG and PpL have no significant sequence homology. Both types of domains are composed of a-helix on top of two antiparallel b-stranded sheets (Wikstro¨ m et al., 1994). The interaction between human L chains and PpL (Ka ¼ 109 M
1) involves only V regions of three k subgroups, VkI, VkIII, and VkIV (i.e., about a half of the Vk repertoire), but not of VkII or Vl (Nilson et al., 1992). The PpL–k chain complex has a structure similar to the SpG–Cg1 complex and involves b sheet interactions (Wikstro¨ m et al., 1995). An X-ray crystallographic study was performed on the complex between a single domain of PpL (61 residues) and Fab fragments isolated from an IgM rheumatoid factor with VkI chains (Graille et al., 2001). The complex contains two Fabs and one PpL domain located between them. Contact areas include similar VL framework areas, which are located distantly from VL hypervariable loops and are remote from H chains. The interaction does not block antigenbinding activity because contact areas locate away from the antigen-combining site. However, both Fab–PpL interactions are characterized by significantly different affinities despite the fact that total buried solvent-accessible areas are nearly similar (1300 and 1400 A˚ 2). The first Fab–PpL interaction is dominated and encompasses 13 VL residues, 10 of which are located in FR1. Twelve residues of the PpL domain participate in these contacts. The second VL contact area is built from 15 residues, and 10 of them are similar to those that participate in the first interaction. Fourteen residues construct the second contact area of the PpL domain. The first interface is characterized by six hydrogen bonds. The second interaction is also mediated by six hydrogen bonds and, in addition, by two salt bridges. No conformational changes were registered in the backbone of Fab and the PpL domain upon binding. The first type of contact between human VkI and PpL is conserved and similar to that of mouse Vk and PpL (Graille et al., 2002). According to these data, the Fab–PpL complex could be assigned to the major group of protein–protein complexes, which are characterized by ‘‘standard-size’’ interfaces with a total buried area in the recognition site equal to 1600 ( 400) A˚ 2. Such interactions are sufficient

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to form stable specific complexes without large conformational changes (Lo Conte et al., 1999). PpL and bacterial cells bearing this protein on their surface can activate mast cells, which are effector cells synthesizing and releasing vasoactive and proinflammatory molecules. After the addition of PpL or bacterial cells, mast cells release presynthesized active mediators and also begin to synthesize leukotriene C4 de novo (Genovese et al., 2000, 2003). Monoclonal IgE with k chains completely blocks PpL activity, whereas IgEl has no such effect. The activation of mast cells by PpL could be caused by cross-linking IgEk molecules bound to the Fce receptors, which are anchored to the surface of mast cells. Bacterial cells that have SpA or PpL on their surface could be important factors in the pathogenesis of inflammatory processes, particularly in heart and cardiovascular diseases. 5. IgA- and IgD-Binding Bacterial Protein A major human pathogen, Streptococcus pyogenes, expresses IgA-binding proteins, which are members of the M protein family. A 37-kDa protein, Sir22, interacts with IgA and IgG molecules with similar affinity (Stenberg et al., 1994). A 50-residue peptide Sap was derived from this protein with specific IgA-binding activity. Sap reacts with both human IgA subclasses, as well as with secretory IgA, but does not bind mouse IgA. The immobilized Sap peptide can be used for purification of IgA, as well as for detection of IgA antibodies in complexes with antigen (Sandin et al., 2002). A 40-kDa protein, Arp, was isolated from group A Streptococcus (A˚ ckerstro¨ m et al., 1994) and a 124-kDa b protein from group B Streptococcus (Hede´ n et al., 1991). Protein Arp is homologous to protein Sir and has higher affinity for serum IgA than for secretory IgA, as the secretory component interferes with Arp binding. The oligomerization of IgA has no influence on the affinity of the interaction with Arp, and the formation of the Arp–IgA complex is independent of the J chain. The Arp protein has only one binding site and cannot cross-link IgA molecules. The b protein binds serum IgA of both subclasses, but poorly interacts with secretory IgA. The previously-mentioned IgA-binding proteins interact with the closely related binding sites at the Ca2–Ca3 interface, which overlap that used by the human FcaRI cell receptor. Therefore the IgA-binding proteins can block the interactions of IgA molecules with FcaRI and in this way interfere with IgA effector functions, thereby contributing to bacterial virulence. Diplococcus Moraxella catarrhalis cells that frequently colonize the respiratory tract express a 200-kDa IgD-binding protein, MID (Forsgren et al., 2001). MID protein is bound only by IgD, but not by molecules of other Ig classes. It is able to induce a proliferative response in peripheral B cells.

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B. Viral Immunoglobulin-Binding Proteins 1. Herpes Simplex Virus Proteins Virus particles of herpes simplex virus (HSV-1) bind to the Fc region of human IgG molecules. Two viral glycoproteins, gE and gI, together form an Fc receptor on the virion envelope. The cells infected by this virus express the same two proteins on their surfaces and are able to bind IgG molecules. Soluble extracellular domains of gE and gI proteins (42 and 36 kDa, correspondingly) assemble in a stable heterodimer with a 1:1 stoichiometry. This complex interacts with IgG also in a 1:1 ratio with relatively high affinity (Chapman et al., 1999). Protein gE alone binds only to IgG aggregates, but not to IgG monomers, whereas soluble gI has no binding activity. Rodent IgGs do not react with HSV-1. A part of protein gE (residues 323–359) participating in Fc binding has sequence similarity with Ig chains and shares homology with human Fc receptors (Dubin et al., 1994). A mutation at gE position 339 causes loss of IgG binding, as the binding site was destroyed (Nagashunmugam et al., 1998). Human IgG1, IgG2, and IgG4 bind to the viral Fc receptor, but IgG3 binding is dependent on the IgG3 phenotype. Only IgG3 molecules from Oriental populations with an Fc phenotype different from that of white populations have HSV-1 Fcg-binding activity (Johansson et al., 1994). The Fcg-binding site for HSV-1 is located at the Cg2–Cg3 region (Johansson et al., 1988). For interaction of the gE–gI complex with Fcg residue, His-435 is critical, as IgG3 allotypes with Arg-435 are unable to bind HSV-1 proteins (Chapman et al., 1999). His-435 located at the Cg2–Cg3 interface is also a contact residue for interactions with SpA and some RFs. Nonreactive IgG3 variants can be converted into HSV-binding molecules after a single residue change Arg435!His-435, and the His-435!Arg-435 change results in loss of binding activity. Several other residues important for IgG–gE–gI interactions were found in experiments on interactions of various human IgG1 allotypes with the cell membrane gE–gI complex (Armour et al., 2002). Molecules G1m(3), common in white populations, have Arg in position 214 of the Cg1 domain, and molecules G1m(null) have Thr-214. The latter molecules have good binding activity, but the G1m(3) molecules possess only weak affinity, which indicates the importance of the residue in position 214 for IgG binding. As IgG4 molecules with high binding activity also have Arg-214, the context of residues in position 214 seems very important. Molecules G1m(17) and Gm(1,17), which varied in binding affinity, have different residues at positions 356 and 358 of the Cg3 domain. Most probably these residues also contribute to binding specificity. Therefore residues located in the Cg1 and Cg3 domains influence binding

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to gE–gI, in addition to the Cg2–Cg3 interface residues. The IgG-binding hierarchy for this interaction is IgG4 > IgG1  IgG2 (Atherton et al., 2000). a. Functional Implications. Specific anti-HSV antibodies are relatively ineffective against HSV infection, and the ability of the virus to react with IgG with relatively high affinity explains this phenomenon. The IgG molecules bind to virion particles in the circulation and inhibit the neutralizing activity of antiHSV antibodies. The expression of gE–gI virus proteins on the surface of infected cells protects them from destruction by effector mechanisms that require the presence of the free Fc region. Anti-HSV antibodies readily destroy cells infected by mutant HSV virions lacking the IgG-binding site, whereas the same antibodies were ineffective against cells infected with wild-type virus (Nagashunmugam et al., 1998). The presence of an Fc-binding site protects cells infected by HSV from antibody-dependent cell-mediated cytotoxicity (Dubin et al., 1991). The mechanism of the protection could be explained by antibody bipolar bridging (i.e., by binding antiviral antibodies to cell surface antigens by the antigen-combining site and simultaneous binding the Fc region by the gE–gI complex). Such a ‘‘head and tail’’ interaction is possible due to the pronounced flexibility of IgG molecules (Nezlin, 1990). As a result, the Fc region of antiviral antibodies is blocked, which prevents the Fc-dependent immune attack. Indeed, antibodies bound to noninfected cells have free Fc, as they can react with SpA. In contrast, antibodies bound to the HSV-infected cells have the blocked Fc region and are unable to react with SpA (Van Vliet et al., 1992). The antibody bipolar bridging also blocks binding of C1q to Fc of antiviral IgG antibodies on infected cells and protects from complement-mediated cytolysis. The protection from the host immune attack by expression of Fc receptors on infected cells is a common strategy among some other viruses. Cells infected by varicella-zoster virus (Litwin et al., 1992), pseudorabies virus (Favorell et al., 1997), and human cytomegalovirus (Antonsson and Johansson, 2001) also express viral Fcg-binding proteins on the surface. Studies of Fcg viral receptors could be important for selecting the strategy for the preparation of virus vaccines. 2. HIV-1 Protein gp120 The major envelope protein, gp120, of HIV-1 virus is bound by Ig molecules, the VH domains of which are coded preferentially by the VH3 gene family, the largest family of the V regions genes (Berberian et al., 1993). Ig molecules with other VH segments do not interact with gp120. The isolated gp120 reacts with a monoclonal IgM, which possesses the VH3 region with a high affinity (Kd ¼ 8.6  109 M
1), whereas the binding activity of IgG with the VH3 region for gp120 is significantly lower. However, not all VH3 Ig molecules are able to react with

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gp120, as only molecules with VH3–23, VH3–30, VH3–15, and VH3–73 gene segments, comprising 25% of all VH3, have gp120-binding activity (Karray et al., 1998). These VH3 segments have germline sequences, whereas nonreactive VH3 encompass between 2 and 15 amino acid substitutions. Apparently the ability to bind gp120 is very sensitive to somatic hypermutations of VH3. SpA also reacts with VH3 segments, but binding sites for SpA and gp120 are not identical. It was found that about 16 nonsequential residues of FR1 and FR3 and CDR1 and CDR2 loops correlate with gp120 binding. These residues locate in the solvent-exposed regions mainly outside the antigen-combining site on the face of the VH region opposite to the contact area with L chains. Only three of 16 residues are common for SpA and gp120-binding sites (Karray et al., 1998). The ability of the gp120 protein to react with VH3 immunoglobulins could lead to serious pathological consequences. In AIDS patients there is a clonal deficit of VH3-expressing nonimmune B lymphocytes, which is preceded by a stimulation and expansion of these B cells on earlier steps of the disease. Such progressive cell depletion could be accounted for by the fact that the gp120 protein can target the membrane anchored VH3 immunoglobulins and activate B lymphocytes that bear these surface Ig molecules as cell receptors. The VH3 Ig family comprises nearly half of the expressed human antibody repertoire, and the elimination of VH3 B cells could cause significant lymphocyte deficiency and, as a consequence, diminished antibody response in HIV-infected patients. VIII. The Promiscuity of the Cg2–Cg3 Interface

The promiscuous ability of the Cg2–Cg3 interface to bind at least six proteins (SpA, SpG, SpH, RF, HSV-1 proteins, and FcRn) and some synthetic peptides (Braisted and Wells, 1996; DeLano et al., 2000), all of them lacking any structural similarity, is mainly determined by its physical and chemical features. The junction between the Cg2 and Cg3 domains creates a large cavity in the Fcg region consisting of three loops, one on the face of Cg3 (residues 433–436) and two on the opposite side of the Cg2 face, one proximal to the Cg3 loop (residues 252–254) and the second distal (residues 309–311) (Fig. 6). The surface area of this junction cavity (2000 A˚ 2) can be expanded and diminished by the independent and free movement of two Cg2 domains, which do not interact with each other (Edmundson et al., 1995). The Cg2 motion pivots around a helical loop (Pro-257 to Pro-270) that forms the principal contact interface between the Cg2 and Cg3 domains (Harris et al., 1997). This segmental flexibility of the Cg2–Cg3 cleft was proven by comparing the angle between the Cg2 and Cg3 domains with or without the hinge region (e.g., IgG Mcg) (Guddat et al., 1993). IgG Mcg, with a lower angle between the two domains (678 vs. 908), has an enlarged Cg2–Cg3 cavity, as shown by the

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increased distance between Met-252 and His-435 from 7.9 to 13.6 A˚ . If the disulfide bonds of the hinge region are cleaved or missing, the Fab arms can move freely along with their attached Cg2 domains, the junction with the Cg3 domain behaving like an alternative hinge (Seegan et al., 1979). This intrinsic physical property of the Cg2–Cg3 cleft makes it adaptable for interaction with a variety of proteins with different sizes and shapes, offering them a surface area of approximately one third of the whole surface area of the cavity (700–800 A˚ 2 ). The chemical accessibility of the Cg2–Cg3 cleft by so many unrelated ligands is determined by the unique combination of the amino acid residues, with distinct characteristics present in all three binding loops. The majority of these 10 residues is well conserved across species and is able to establish a variety of noncovalent interactions, some common but others distinct for each ligand. Thus Ile-253 makes hydrophobic interactions with all tested ligands such as SpA (Phe-13), SpG (Trp-43), RF (Tyr-98), FcRn (Trp-133), and Val-10 in a small synthetic peptide (DeLano et al., 2000). In contrast, His-310 is important in the binding of FcRn (Glu-117) and possibly SpA, probably by salt bridges, but has no reported role in binding RF or SpG. However, the adjacent residue (Glu-311) does not participate in the binding of FcRn and RF, but is essential for the interaction of SpA (Asn-18) and SpG (Glu-42). In the Cg3 loop, His-435 is necessary for binding FcRn (Glu-132) and SpA (Tyr-14) but not SpG, while the neighbor residue (Asn-434) has no involvement in binding FcRn, but is essential for the polar or charged interaction with SpA (Asn-11), SpG (Asn-35), and RF. These few examples suggest that the interaction of multiple ligands with the residues of the Cg2–Cg3 binding loops is elective, ‘‘choosing’’ only the residues of the ligands that allow the appropriate noncovalent pairing. The fact that the reaction of the multiple ligands with IgGs has a characteristic pH dependency and binding affinity clearly indicates that the Cg2–Cg3 cleft has not only physical but also chemical flexibility. The flexibility and accessibility of this region are also supported by experiments showing that from a vast library of peptides selected to bind any region of the Fcg fragment, the dominating peptides were those reacting with the Cg2–Cg3 interface (DeLano et al., 2000). Therefore it will not be surprising to find in the future new natural proteins and synthetic peptides with affinity for this region of IgG. IX. Concluding Remarks

Many Ig interactions with various ligands occur in the circulation, when both components are in soluble form, whereas others take place on the cell surface, as one of the components is localized on the cell membrane (Table VIII). Both types of interactions have functional consequences essential for the development

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TABLE VIII Immunoglobulin Interaction Sites Ligands Cell receptors FcgRI, II, III FceRI FcaRI PolyIgR FcRn Complement components C3b C1q Anaphylatoxins Bacterial proteins SpA SpG SpH PpL Arp, Sir22 Viral proteins HSV-1 HIV-1 Lectins Prolactin Fibronectin Rheumatoid factor CD4 Histidine-rich glycoprotein Clusterin Peptides Protein Fv

Localization of Ig sites Low hinge, Cg2 Ce3 and Ce2–Ce3 linkers Ca2–Ca3 cleft Ca3 Cg2–Cg3 cleft Cg1, Cg2–Cg3 cleft Low hinge and Cg2; Cm3 Fabg Cg2–Cg3 cleft, VH3 Cg2–Cg3 cleft, Cg1 Cg2–Cg3 cleft Vk Ca2–Ca3 cleft Cg2–Cg3 cleft VH 3 Ig glycans Fdg Fc Cg2–Cg3 cleft VH Fab Fab and Fc Fcg VH

of the immune response, as well as for the progress of various disorders. The initiation of the complement cascade follows the reaction of the complement component C1q with IC, and the covalent binding of C3 and C4 is important for the elimination of antigens from the circulation. The formation of the complexes can close some functionally important sites. An example of such reactions is shielding virion particles with Fc receptors by Ig molecules, which inhibits inactivation of viruses by neutralizing antibodies. Histidine-rich glycoprotein can inhibit the formation of insoluble IC and also affects the binding of IgG and IC by cells. Biologically active substances (for example, anaphylatoxins) could be eliminated from the circulation after the binding to immunoglobulins. The interactions with cell-bound ligands are just as important. Various Fc receptors are expressed on the surface of many types of cells, and binding Ig molecules to these receptors could activate or inhibit cell activity or stimulate

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the transportation of Ig molecules through cell membranes. A complex of an Ig molecule with a ligand can react simultaneously with two corresponding types of cell receptors—with Fc receptors by the Fc moiety and with specific receptors for the Ig-bound ligand. Such ‘‘double’’ interactions can amplify signaling and change the cell response (the effect of coligation). A special case of the receptor coligation is the antibody bipolar bridging, when virus-neutralizing antibodies react with antigens expressed on cells infected with some viruses. Due to the pronounced flexibility, antibody molecules bind by the antigencombining sites to cell-surface viral antigens and interact simultaneously by their Fc regions, with Fc viral receptors expressed on the cell surface. Such ‘‘head and tail’’ antibody binding results in blocking the antibody Fc region and preventing the host Fc-dependent immune attack. Ig interactions with cellbound ligands could have important consequences for the development of some disorders. For example, fibronectin associated with matrix participates in removing IC from the circulation and deposing them in tissues, which could cause serious pathological complications. Studies of Ig–ligand interactions also have a methodological aspect. Ligands with specificity for immunoglobulins, such as SpA and SpG, are effectively used for detection and isolation of Ig molecules. Lectins could be helpful for structural studies of oligosaccharides linked to Ig molecules. IVIG preparations, which contain mainly IgG, are widely used for prophylaxis and treatment of various pathologies. Therefore it is important to know whether these preparations also contain complexes of IgG with some biologically active substances, which could be harmful for the body. Ig peptide chains have a modular structure and are built from several compact folds or domains homologous to those of proteins, which are members of the large Ig superfamily (Nezlin, 1998). A very stable structure of Ig fold has been conserved in evolution and can be found in protein molecules of a wide variety of species, including invertebrates and ancient vertebrates (Barclay et al., 1997). Molecules of the Ig superfamily are able to bind ligands of various structures and dimensions from complex proteins to small molecules. Therefore the Ig fold can be considered a scaffold on which arrays of various binding sites are displayed either on b strands or at the loops connected to the strands or on both. Amino acid residues of nearly all Ig domains participate in the construction of binding sites for interactions with various ligands. Some ligands can bind to only one Ig domain, whereas others interact with binding sites localized on different Ig domains. A large number of ligands react with the CH2–CH3 interface. The promiscuity of this location could be at least partly explained by the flexible structure of the Fc region and, as a consequence, by the ability of Fc to adapt more easily to various spatial configurations important for the interactions with different ligands.

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The Role of Antibodies in Mouse Models of Rheumatoid Arthritis, and Relevance to Human Disease PAUL A. MONACH, CHRISTOPHE BENOIST, AND DIANE MATHIS Section of Immunology and Immunogenetics, Joslin Diabetes Center, Boston, Massachusetts 02215, and Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts 02115

I. Rheumatoid Arthritis: Clinical and Pathological Features

Rheumatoid arthritis (RA) is a chronic inflammatory disease affecting approximately 1% of the world’s population (Lee and Weinblatt, 2001). Although inflammatory lesions in the skin, lungs, and other organs are common, the disease hallmark is severe, often destructive, inflammation of peripheral joints. Although manifestations vary among patients, RA is usually a symmetric polyarthritis affecting distal [metacarpophalangeal (MCP), metatarsophalangeal (MTP), proximal interphalangeal (PIP), wrist, and ankle] more than intermediate (knee, elbow) and more than proximal (hip, shoulder) joints, and sparing the distal interphalangeal (DIP) joints. The cervical spine is often affected, but not the remainder of the axial skeleton. Synovial tendon sheaths and bursae are frequently involved. Microscopically, the characteristic lesion of RA is a novel tissue called pannus, composed of a greatly expanded number of both type 1 (macrophage-like) and type 2 (fibroblast-like) synoviocytes, as well as new blood vessels and a mononuclear cell infiltrate that is often follicular and can contain germinal centers. Although neutrophils are the predominant cell in inflamed synovial fluid, they are sparse in pannus. Pannus overgrows and erodes articular cartilage; destroys bone, especially at the junction of bone and cartilage; and erodes through tendons and ligaments. Together, these processes often destroy joint function, usually over the course of many years. II. RA: Theories of Pathogenesis

Broadly speaking, RA has been conceived of as fundamentally an autoimmune, infectious, or neoplastic disease. The pathogenesis has not been established despite considerable advances in understanding made through genetics, cell biology, and biochemistry, summarized as follows. A. MHC and T cells Genetic linkage studies have identified the major histocompatibility complex (MHC) class II gene DR as the locus most closely associated with RA (Jawaheer et al., 2003; Stasny, 1978). These findings strongly implicate CD4þ 217 Copyright 2004, Elsevier Inc. All rights reserved. 0065-2776/04 $35.00

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T cells somehow in pathogenesis, but do nothing to differentiate between immunity to self versus a pathogen, nor among potential downstream effector mechanisms. T cells are found in large numbers in RA lesions. Efforts to identify skewed T cell receptor (TCR) usage suggestive of clonal expansion have been inconclusive (Jenkins et al., 1993; Uematsu et al., 1991): attempts to identify antigenic peptides have also been unsuccessful. T cell-depleting therapies with anti-CD4 or anti-CD152 (CAMPATH-1 h) have been only transiently effective (Matteson et al., 1995; Moreland et al., 1995; van der Lubbe et al., 1995; Weinblatt et al., 1995), but depletion within the lesions was poor or transient (Ruderman et al., 1995), so these trials do not really shed much light on the role of T cells in disease progression. B. Immune Complexes and Autoantibodies Complexes containing immunoglobulin (Ig) and the C3 component of complement are present in the synovium and articular cartilage of rheumatoid lesions (Cooke et al., 1975; Vetto et al., 1990) and within infiltrating phagocytes (Britton and Schur, 1971). Hemolytic complement is depleted in the synovial fluid of RA patients, in contrast to individuals with other varieties of inflammatory arthritis (Pekin and Zvaifler, 1964; Ruddy et al., 1969); breakdown products of C3 provide further evidence of local complement activation in the RA joint (Mollnes et al., 1986; Olmez et al., 1991). Various autoantibodies are found at higher levels in RA sera than in control sera (Morgan et al., 1987; Souto-Carneiro et al., 2001; Verheijden et al., 1997), but their role in disease is unclear. The most specific yet found are those recognizing peptides in which arginine has been modified to citrulline (Schellekens et al., 1998; Vincent et al., 1999). Support for a central role of antibodies (Ab) in RA has come recently from studies showing clinical effectiveness of the B cell-depleting (anti-CD20) monoclonal Ab (mAb) rituximab; in addition, disease has tended to recur only with the return of detectable B cells and auto-Ab (Cambridge et al., 2002; Edwards et al., 2002). However, it is not yet clear whether these findings reflect a role for Ab or some other B cell function. Rheumatoid factors (RF)—Ab that bind to the Fc portion of IgG—are found in the sera of about 80% of RA patients, but are also found in association with other inflammatory diseases and in perhaps 5% of healthy people. RF can be recovered from RA lesions (Jasin, 1985), appear to be synthesized locally based on greater levels in synovial fluid relative to blood (Jones et al., 1984), and can fix complement in vitro (Bianco et al., 1974). RF in RA are present in higher concentrations, are of higher avidity, are more frequently of IgG isotypes, and have altered glycosylation relative to RF from control

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patients (reviewed in Firestein, 2001). Nevertheless, their role, if any, in the pathophysiology of RA is still unclear. C. Cytokines and Macrophages Numerous cytokines and other mediators are found at elevated levels in RA lesions, including both pro- and antiinflammatory molecules. Macrophagederived mediators are much more prevalent than those secreted by lymphocytes (reviewed in Firestein, 2001). As before, macrophage-like synoviocytes are a prominent feature of pannus. The effectiveness of treatments (mAb, soluble receptors, and natural receptor antagonists) that specifically block tumor necrosis factor-a (TNF-a) or interleukin (IL)-1 have confirmed the importance of these cytokines in RA (Bresnihan et al., 1998; Maini et al., 1999; Weinblatt et al., 1999). The usually rapid return of disease when anti-TNF treatment is discontinued shows that this treatment does little to interrupt the underlying pathophysiology, and must be interfering with a downstream effector mechanism, such as TNF production by macrophages, neutrophils, or mast cells. D. Synovial Fibroblasts Fibroblasts from RA synovium show some properties characteristic of transformed cells (reviewed in Firestein, 2001), as well as the ability to invade and destroy cartilage when cotransplanted into severe combined immunodeficient (SCID) mice (Muller-Ladner et al., 1996). Fibroblast-like synoviocytes can also produce some of the cytokines that are elevated in RA lesions. E. Innate Immunity Many of the previous elements do not strictly require the participation of the adaptive immune system in order to be activated. For this reason, and because nonspecific adjuvants can cause synovial thickening prior to or in the absence of recognizable autoimmunity, it has been proposed that RA can be explained without a role for antigen-specific immunity (Firestein and Zvaifler, 2002). The relative importance of these components has been the subject of much debate, often unnecessarily polarized, although some authors have made an effort to promote balanced views (Panayi et al., 1992). Interestingly, and in our estimation for no compelling reason, the previously popular concept of RA as an immune-complex-mediated disease (Zvaifler, 1974) was discarded by all but a few (Edwards and Cambridge, 1998) theorists. Following a brief review of effector mechanisms associated with Ab and immune complexes (IC), we will summarize in the remainder of this chapter the evidence that IC produce RAlike pathology in a variety of mouse models and that these findings may be relevant to the human disease.

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III. Effector Mechanisms of Antibody-Mediated Disease

A. Complement As reviewed in more detail by Walport (2001), the complement cascade is activated by three major means: the classical, alternative, and mannose-binding-lectin (MBL) pathways (Fig. 1). The classical pathway is activated by the Fc portions of certain isotypes of Ig if present in sufficient density to cross-link the components of C1. The alternative pathway is activated by foreign surfaces (i.e., those lacking the complement inhibitors present on host cells) and also plays an important role in amplifying the cascade initiated via other pathways. The MBL pathway is activated by terminal mannose residues found on various bacteria, and also by agalactosyl IgG, a form that is, interestingly, often found in rheumatoid joints (Malhotra et al., 1995). All pathways cleave C3 and initiate the effector functions of complement. The subsequent cleavage of C5 leads to the formation of the pore-forming membrane attack complex (MAC). The released fragments C3a and especially C5a promote inflammation by binding receptors on a variety of cell types. The surface-bound or IC-bound

Fig 1 Outline of the complement cascade. Thick arrows denote transitions from one protein or complex into another. Thin arrows denote catalysis of such transitions by the products at the origins of the arrows. Note that the mechanism(s) by which mannose-binding lectin (MBL) can activate C3 are not fully known. MAC, membrane attack complex.

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fragments of C3 interact with different cell-surface receptors (CR1, CR2, CR3) to effect phagocytosis, clearance of ICs, and immunoregulation. B. Fc Receptors As reviewed by Takai (2002), the Fc portions of IgG isotypes interact with multiple cell types via different cell-surface Fc receptors (FcR). Mice have three known FcR. All three types are found on macrophages, neutrophils, eosinophils, and dendritic cells, with further differences in expression as follows. FcgRI is of relatively high affinity and is also found on monocytes. FcgRII and FcgRIII are of lower affinity and therefore bind better to larger complexes with multivalent ligands. FcgRII is also found on mast cells and B cells; FcgRIII is found on monocytes, mast cells, and NK cells (reviewed in Takai, 2002). FcgRI and FcgRIII both use a common chain (FcRg) that delivers an activating signal; FcgRII, in contrast, delivers an inhibitory signal. Human FcR are analogous but more diverse: FcgRIA, B, and C; FcgRIIA, B, and C, of which only B is inhibitory; and FcgRIIIA and B, where B has only extracellular domains attached to the plasma membrane by a GPI tail (reviewed in Takai, 2002). C. Cells of the Innate Immune System Neutrophils are bone-marrow-derived cells that circulate in the blood and exit into tissues at sites of incipient inflammation. Various adhesion molecules and chemotactic factors are involved in the multistep exodus (rolling, adhesion, transmigration) of these cells from the bloodstream. As reviewed by Burg and Pillinger (2001), most of the functions of neutrophils can be teleologically associated with defense against bacterial infection (phagocytosis, production of toxic oxygen radicals and bacteriocidal peptides, and secretion of proinflammatory mediators), but their products also contribute to tissue damage in both infectious and noninfectious inflammation. Mast cells (reviewed in Gurish and Austen, 2001) are also bone-marrowderived cells, but they circulate as committed progenitors and mature only within peripheral tissues. The phenotypic diversity of these cells appears to go well beyond the traditional subtypes of mucosal and connective tissue mast cells, but is only beginning to be described. Mast cells promote vascular permeability and influx and activation of inflammatory cells by secretion of histamine, serotonin, prostaglandins, leukotrienes, cytokines, chemokines, proteases, and proteoglycans. Mast cells are readily activated by cross-linking of the high-affinity receptor for IgE (FceR) and are best known for their prominent role in allergy and anaphylaxis. However, interest in these cells has broadened recently with the demonstration of their importance in several animal models of non–IgE-mediated autoimmune disease (reviewed in Benoist and Mathis, 2002).

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Macrophages are derived from circulating monocytes and exist in peripheral tissues in two general forms: (1) tissue macrophages, which populate normal tissues to varying degrees and can have quite specialized functions (such as osteoclasts in bone, Kuppfer cells in liver, type 1 synoviocytes in joint, microglia in brain, and alveolar macrophages in lung), and (2) inflammatory macrophages, which invade inflamed tissues, generally after neutrophils do. Macrophages are phagocytes and produce oxygen radicals, but they are involved in much more complex functions than neutrophils, including antigen presentation to T cells, removal of debris, tissue remodeling, and modulation of immune responses through the secretion of numerous cytokines and other mediators. The production of TNF and IL-1 by macrophages has been of particular interest to those studying their role in RA (reviewed in Kinne et al., 2000). IV. Animal Models: General Considerations

The major models discussed as follows have several features that differ from human RA. They are dependent upon either deliberate immunization or transgenic manipulation. Yet susceptibility to RA clearly has a genetic component and, furthermore, might be initiated by inadvertent exposure to a pathogen. Joint destruction proceeds more rapidly in animal models, over weeks rather than months to years. However, it is plausible that animal models represent more overt expression of mechanisms that occur with more subtlety in RA. Patterns of joints involvement can differ. However, the mechanical stresses on joints in bipeds and quadripeds differ, there is great heterogeneity in joint involvement even within the human disease, and distal peripheral joints tend to be affected more than proximal joints in both RA and numerous animal models. Finally, only a few models feature detectable RF. Yet, as discussed previously and later, the role of RF in RA is unclear, and in both RA and those models in which RF is found, the levels correlate imperfectly with the presence or severity of disease. Animal models were developed in rabbits and rats before mice. The availability of numerous genetically-modified mouse strains, more extensive genetic information, and tools with which to evaluate immune responses has allowed mouse models to be explored in greater detail. The remainder of this chapter will focus on mouse models of RA, especially on elements that have been tested in multiple models, and on inflammation rather than the destruction of cartilage and bone. Special attention will be paid to those models in which the effector phase can be evaluated separately by adoptive transfer; recent studies in these models have helped produce a resurgence of interest in considering RA as a disease mediated by auto-Ab (Firestein, 2003). We will first discuss those models in which arthritis is induced by immunization, then those in which it occurs spontaneously in mutant or engineered strains.

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V. Collagen-Induced Arthritis

Originally developed in rats (Trentham et al., 1977) and soon after in mice (Courtenay et al., 1980), collagen-induced arthritis (CIA) is produced by immunizing animals with xenogeneic type II (cartilage-specific) collagen in complete Freund’s adjuvant (CFA). In susceptible mouse strains, the most commonly used of which is DBA/1, arthritis appears in the majority of mice 3–5 weeks after immunization (Courtenay et al., 1980). Often a booster immunization in incomplete adjuvant (IFA), CFA or saline is used but is not strictly required. Disease primarily affects the front and rear paws, with occasional involvement of the spinal column, tail, and ear (Courtenay et al., 1980). Investigators generally examine the tarsal joints histologically, and we are not aware of a study documenting the degree of inflammation in proximal versus distal joints. Histologically, synovial hyperplasia is a relatively early finding, followed by infiltration of the synovium, subsynovial connective tissue, and joint space with neutrophils, then mononuclear cells. Subsequently, pannus develops, with erosion of cartilage and bone (Courtenay et al., 1980). IgG and C3 accumulate on the cartilage surface, but not in the synovium (Wang et al., 2000). Susceptibility to CIA in mice is linked to the MHC class II region (Wooley et al., 1981). CD4þ T cells (Ranges et al., 1985) and B cells (Svensson et al., 1998) are required for the full spectrum of disease, although DBA/1 mice deficient in the RAG1 gene (and thus lacking mature B and T lymphocytes) still develop some synovial hyperplasia, pannus, and erosion of cartilage and bone (Plows et al., 1999). Depletion of macrophage-like synoviocytes by local injection of clodronate-containing liposomes decreases inflammation (van Lent et al., 1996) and cartilage loss (van Lent et al., 1998b). Various cytokines are important in CIA. Blockade of TNF-a markedly decreases inflammation and joint destruction when given early (Williams et al., 1992), but its effectiveness in established disease has been less clear (Joosten et al., 1996). Blockade of IL-1 also prevents arthritis and is particularly protective against destruction of cartilage and bone (Joosten et al., 1996, 1999). Mice lacking IL-6 are resistant to CIA (Alonzi et al., 1998), and blockade of IL-18 reduces disease (Plater-Zyberk et al., 2001). Mice lacking IL-10 develop more severe arthritis (Cuzzocrea et al., 2001), and exogenous IL-10 ameliorates disease (Joosten et al., 1997a). The roles of IL-4 and IL-12 are complex, apparently different in different phases of disease (Joosten et al., 1997b; Svensson et al., 2002). Susceptible strains generate Ab binding to both the immunizing (xenogeneic) and autologous type II collagen. In support of these Ab playing an important role in disease, mice deficient in C3 (Hietala et al., 2002), factor B (Hietala et al., 2002), or C5 (Wang et al., 2000) are resistant to CIA, and

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anti-C5 mAb prevents CIA and ameliorates established disease (Wang et al., 1995). Mice lacking the shared FcRg chain (therefore lacking FcgRI, FcgRIII, and FceR) (Kleinau et al., 2000) or only FcgRIII (Diaz et al., 2002) are highly resistant to CIA, whereas mice lacking the inhibitory receptor FcgRII develop more severe disease (Kleinau et al., 2000). Most importantly, arthritis can be transferred to naı¨ve mice using serum from arthritic mice (Stuart and Dixon, 1983), or a mixture or mAb to type II collagen (Terato et al., 1992). Arthritis produced by passive transfer of anticollagen Ab resembles actively induced CIA, but is milder, much more rapid in onset, and transient (Stuart and Dixon, 1983). Disease is evident 2–3 days after an injection of Ab, is maximal a day later, and gradually resolves over the next 4–5 days. Deposits of IgG and C3 are found, as in the case of active immunization (Stuart and Dixon, 1983). Disease susceptibility is independent of MHC alleles (Stuart and Dixon, 1983), and T and B cells are dispensable (Kagari et al., 2002). IL-1 and TNF-a are required, but IL-6 is not (Kagari et al., 2002). C5 (Watson et al., 1987) and the C5aR (Grant et al., 2002) are required in recipient mice; such mice still accumulate IgG and C3 on the articular surface, but without inflammation. Mice lacking the common FcRg chain are highly resistant and those lacking FcgRIII partially so. Absence of FcgRII does not appear to exacerbate disease (Kagari et al., 2003). Thus the ability to isolate the effector phase has allowed a more precise assignment of roles for complement and cytokines in CIA, although these findings do not preclude roles for these factors in the induction phase. Likewise, an Ab-independent role for T cells in the effector phase is not precluded, but supporting data are lacking. VI. Antigen-Induced Arthritis

Originally described in rabbits (Cooke et al., 1972) and later in mice, (Brackertz et al., 1977a), antigen (Ag)-induced arthritis (AIA) is produced by immunizing an animal systemically with an Ag and challenging locally, typically 21 days later, with the same Ag in a knee joint. Since inflammation is confined to the injected joint, AIA is not precisely a model of RA, but it is plausible that the principles operating in this model could apply to a symmetric polyarthritis in which the target Ag resides in multiple joints. Methylated bovine serum albumin (mBSA) has been the most frequently used Ag, although others have been employed. Cationic Ags work better than neutral or anionic ones, correlating with the greater retention of cationic proteins in articular cartilage (van den Berg et al. 1984; van den Berg and Van de Putte, 1985). Histopathologically, affected joints develop mixed inflammatory infiltrates (predominantly mononuclear cells, with neutrophils in synovial effusions), synovial hyperplasia, pannus, and destruction of cartilage and

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bone; a smoldering synovitis persists for at least 3 months (Brackertz et al., 1977a). Ab, Ag, and C3 are colocalized on the articular surface (Cooke et al., 1972; van den Berg and Van de Putte, 1985). Not all mouse strains are susceptible (Brackertz et al., 1977a), but linkage to the MHC has not been described. T cells are required, based on the absence of disease in athymic nude mice (Brackertz et al., 1977a). T cells from immunized mice can transfer disease susceptibility to naı¨ve mice (Brackertz et al., 1977b), but no results in B celldeficient mice have been reported. Antiserum has been reported to cause only mild synovial hyperplasia and mononuclear cell infiltration of subsynovial connective tissue (Brackertz et al., 1977b). Mast cells are not required for inflammation, but may promote cartilage destruction (van den Broek et al., 1988). In a modification of AIA, in which an intravenous injection of Ag causes acute reactivation of chronic disease locally, depletion of local macrophage-like synoviocytes with clodronate-impregnated liposomes markedly decreases disease (van Lent et al., 1998a). In this same ‘‘flare-up’’ model, depletion of complement with cobra venom factor has no effect (Lens et al., 1984). Blockade of either IL-1, TNF-a, or IL-6 has no effect on acute inflammation in AIA, although IL-1 blockade markedly reduces chronic inflammation (van de Loo et al., 1995). The common FcRg chain is important in acute and chronic inflammation (van Lent et al., 2000), but the absence of FcgRI or FcgRIII individually has little effect (van Lent et al., 2001); FcgRI is important, however, in destruction of cartilage (van Lent et al., 2001). Disease is more severe in mice lacking FcgRII (van Lent et al., 2001). Thus there is only indirect evidence for an important role for Ab in AIA, and no evidence for such in the flare-up reaction. However, a similar disease can be induced by injection of a different cationic Ag, lysozyme-poly-l-lysine, into the knees of mice passively immunized with Ag-specific rabbit Ab (van Lent et al., 1992). Arthritis, featuring a massive influx of neutrophils, is evident within 1 day and wanes over the course of a week (van Lent et al., 1992). Ag is deposited on the articular surface, presumably in complex with specific Ab (van Lent et al., 1992). Local depletion of macrophage-like synoviocytes prevents disease (van Lent et al., 1993), as in the flare-up reaction of active AIA. No role for T or B cells has been reported; such would be unlikely in light of the rapidity of the response. IL-1 is required for inflammation and cartilage destruction (van Lent et al., 1992, 1995), but TNF-a may be dispensable (van Lent et al., 1995). FcgRIII is required for inflammation and cartilage breakdown, whereas FcgRI seems to be important only in cartilage loss (Nabbe et al., 2003). FcgRII plays a suppressive role, since inflammation and cartilage breakdown are enhanced in FcgRII-deficient mice (Nabbe et al., 2003). The complement system is also required for disease, since treatment with cobra venom factor largely prevents arthritis (van Lent et al., 1992); the roles of individual components have not been reported.

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VII. Proteoglycan-Induced Arthritis

BALB/c mice immunized with human fetal cartilage proteoglycan (PG) in CFA uniformly develop arthritis of gradual onset (Glant et al., 1987). Redness and some swelling are noted as early as 9–12 days postinjection. Swelling is maximal at 7–9 weeks, usually affects all four paws, and progresses to joint destruction. Distal joints, knees, elbows, lumbar spine, and tail are affected (Glant et al., 1987). Histologically, synovium, subsynovial connective tissue, and other periarticular tissue are infiltrated by mononuclear cells and, to a lesser extent, neutrophils, beginning in perivascular areas. Pannus forms and erodes cartilage and bone, with progression to ankylosis (Glant et al., 1987). Ig is deposited in the synovium and articular cartilage (Mikecz et al., 1987). Proteoglycan-induced arthritis (PGIA) has been found only in BALB/c mice, although other strains, including those of the same MHC haplotype, can make T cell and Ab responses to PG (Mikecz et al., 1987). Based on studies with subset-depleting mAb, CD4þ cells are required, but CD8þ cells are not (Banerjee, et al., 1992). PGIA can be transferred to irradiated BALB/c mice using restimulated lymphocytes; both T and B cells are needed (Mikecz et al., 1990). B cell-deficient mice, as well as mice with B cells bearing surface IgM but unable to secrete Ig, are completely resistant (O’Neill et al., 2001). Development of PGIA is preceded by production of Ab binding both the xenogeneic and autologous PG, and many immunized mice produce RF (Mikecz et al., 1987), but disease has not been transferred with antiserum (Mikecz et al., 1990). PGIA is more severe in IL-4-deficient and less severe in IFNg-deficient mice (Kaplan et al., 2002a). The roles of TNF-a, IL-1, and complement have not yet been reported. Mice lacking FcRg (and therefore both FcgRI and FcgRIII) are completely resistant, despite producing effective immunity, as shown by the ability of cells from such mice to cause disease upon transfer into FcRgþ but lymphocyte-deficient SCID mice (Kaplan et al., 2002b). Mice lacking FcgRII develop more severe PGIA (Kaplan et al., 2002b). Thus, although PGIA has not been transferred to naı¨ve mice using Ab, there is considerable indirect evidence that it is an Ab-mediated disease.

VIII. Streptococcal Cell Wall Arthritis

Rats given a single intraperitoneal (i.p.) injection of a sonicate of streptococcal cell walls (SCW) develop progressive polyarthritis shortly thereafter (Cromartie et al., 1977). Mice do not produce such a response, but have been reported to develop a transient polyarthritis (Koga et al., 1985). SCW have been used to induce murine arthritis in two other ways (reviewed in Joosten et al., 2000a). First, in a manner analogous to AIA, mice immunized

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systemically with SCW develop chronic, destructive arthritis in a knee joint after intraarticular injection of SCW. Second, injection of SCW into the knee joints of naı¨ve mice produces an acute, transient arthritis. In the latter model, neutrophil influx is apparent on Day 1 and is maximal between Days 2 and 4; macrophage infiltration is evident on Days 4–7, and inflammation subsides thereafter. PG is depleted from cartilage. According to the timing of the response, acute SCW arthritis is thought not to involve the adaptive immune system. Based on blockade of cytokines with mAb, TNF-a plays an important role in joint swelling, and IL-1 is important in both inflammation and cartilage destruction (Kuiper et al., 1998). Blockade of IL-18 suppresses swelling, inflammation, and cartilage loss (Joosten et al., 2000b). IL-4 and IL-10 appear to play protective roles (Lubberts et al., 1998). IX. Pristane-Induced Arthritis

Injection of the hydrocarbon pristane (2,6,10,14-tetramethylpentodecane) i.p. into mice of susceptible strains leads to chronic arthritis, with an incidence of 22–100%, beginning 2–10 months after injection (Potter and Wax, 1981; Wooley et al., 1989). Ankles and wrists are most prominently affected (Wooley et al., 1989). The histological picture is dominated by mononuclear cell infiltration, later forming pannus with erosion of cartilage and bone. Nodules are found with central necrosis and surrounding macrophages, analogous to rheumatoid nodules (Wooley et al., 1989). A similar disease occurs in rats. The role of MHC and other genes is unclear (Wooley et al., 1989). T cells are involved, based on resistance of athymic nude mice (Wooley et al., 1989). Depletion of CD4þ cells with mAb decreases severity of disease (Levitt et al., 1992). Susceptibility can be reconstituted in sublethally irradiated mice using CD4þ cells in the absence of CD8þ or B cells (Stasiuk et al., 1997). Mice treated with pristane develop anti-type-II-collagen and IgM RF auto-Ab, but there is only a modest correlation between disease and titer of either of these Ab (Wooley et al., 1989). Two different C5-deficient strains are resistant to disease (Wooley et al., 1989). Anti-TNF treatment reduces incidence and severity (Beech and Thompson, 1997). X. Zymosan-Induced Arthritis

Zymosan, a glycan derived from yeast cell walls, activates complement by the alternative pathway and produces inflammatory arthritis within 48 h of intraarticular injection in mice (Keystone et al., 1977). The arthritis resolves over about 2 weeks. An infiltration of neutrophils is seen first, followed by synovial hyperplasia and macrophage infiltration, with pannus formation (Keystone et al., 1977). Neutralization of IL-1 or TNF-a has only modest

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effects on inflammation (van de Loo et al., 1995), although mice lacking these cytokines by gene disruption have not been tested. XI. Adjuvant Arthritis

Adjuvant arthritis (AA) is a disease produced in rats by immunization with killed Mycobacterium tuberculosis. This disease has been difficult to recapitulate in mice (Knight et al., 1992; Yoshino et al., 1998). XII. K/BxN (Anti-GPI-Mediated) Arthritis

The K/BxN (or KRN or anti-GPI) model of RA was discovered fortuitously when a mouse bearing transgenes encoding the KRN TCR (reactive with bovine RNase presented by Ak) was bred to an NOD mouse (Kouskoff et al., 1996). Subsequent studies have revealed that the disease is caused by T and B cell autoimmunity to the glycolytic enzyme glucose-6-phosphate isomerase (GPI). Mice expressing the KRN TCR and the MHC class II allele Ag7 (K/BxN) invariably and spontaneously develop severe peripheral arthritis beginning at about 3 weeks of age. Distal joints, including tarsal, carpal, and all IP joints, are severely affected; knees and elbows are involved but less severely; hips, shoulders, and spine are spared (Kouskoff et al., 1996). Histologically, a mixed infiltrate of neutrophils and mononuclear cells is seen in the synovium and subsynovium, with neutrophils predominant in the joint space. The mononuclear infiltrate becomes more prominent over time, develops into pannus, and erodes cartilage and bone; joint damage progresses to ankylosis (Kouskoff et al., 1996). Development of disease is dependent on the MHC molecule Ag7, but independent of other genes from the autoimmunity-prone NOD background (Kouskoff et al., 1996). CD4þ T cells and B cells are required (Korganow et al., 1999). Blockade of TNF-a with mAb (starting at 3 weeks of age) does not prevent disease (Kyburz et al., 2000), although the role of this cytokine in K/BxN mice has not been tested more definitively in TNF knockout mice. Most importantly, disease can be transferred to naı¨ve mice using 100–300 ml of K/BxN serum (Korganow et al., 1999). This serum contains large amounts (>10 mg/ml) of Ab recognizing GPI (Matsumoto et al., 1999); affinity-purified anti-GPI Ab (Matsumoto et al., 1999) or a combination of two or more antiGPI mAb (Maccioni et al., 2002) can cause arthritis. The KRN TCR recognizes a peptide derived from GPI, in the context of Ag7 (Matsumoto et al., 1999). Only one finding supports a role for KRN T cells in arthritis independent of B cells: a single injection of anti-GPI Ab causes prolonged and more severe arthritis in B cell-deficient (m/) K/BxN mice, whereas the arthritis

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is transient in nontransgenic mice (Korganow et al., 1999). However, the basis for this apparent difference remains to be explored. The arthritis produced by transfer of anti-GPI serum affects the carpal and tarsal joints predominantly; the MCP, MTP, IP, knee, and elbow variably; and spares the hip, shoulder, and spine (Korganow et al., 1999). Arthritis is clinically apparent 1–4 days after injection, peaks at 10–14 days, and resolves slowly over the next 2 weeks. Severe arthritis is maintained if repeated injections of serum are given (Korganow et al., 1999). Histologically, degranulation of mast cells is apparent within an hour (Lee et al., 2002a), and influx of neutrophils is prominent within 1–2 days (Wipke and Allen, 2001); synovial hyperplasia and mononuclear cell infiltration, with pannus formation and erosion of bone and cartilage, begin within a week (Korganow et al., 1999; Wipke and Allen, 2001). Affected joints have colocalizing deposits of Ig, Ag, and C3 (Matsumoto et al., 2002). Arthritis caused by Ab transfer is independent of MHC haplotype and occurs readily in RAG1/ mice, which lack mature T and B cells (Korganow et al., 1999). Mice lacking neutrophils (by treatment with anti-Gr-1 Ab) are resistant (Wipke and Allen, 2001); those lacking macrophage-like synoviocytes (op/op mice) are susceptible (Bruhns et al., 2003; Lee et al., 2002b). Mice lacking c-kit or its ligand, and therefore profoundly deficient in mast cells, are resistant, and susceptibility can be restored by reconstitution with mast cell precursors (Corr and Crain, 2002; Lee et al., 2002a). Disease caused by serum transfer is diminished in mice lacking TNF-a and absent in mice lacking IL-1R1 (Ji et al., 2002b). IL-4 (Ohmura et al., 2002) and IL-6 (Ji et al., 2002b) are dispensable. The roles of complement components have been evaluated in detail (see Fig. 1 for a diagram of the cascade) using gene-disrupted or congenic strains: factor B, C3, and C5 are required, whereas C1q, C4, MBL-1, and C6 are not (Ji et al., 2002a). The C5aR is required, but CR1, 2, and 3 are not (Ji et al., 2002a; Solomon et al., 2002). Thus the critical role of complement seems to be activation through the alternative pathway leading to the generation of C5a. In contrast to CIA, nonarthritic mice lacking C5 do not have deposits of Ab, Ag, and C3 on the articular surface (Ji et al., 2002a). FcgRIII-deficient mice are resistant, whereas FcgRI-deficient mice are susceptible; mice lacking the common chain FcRg were reported to be more resistant than those lacking only FcgRIII (Ji et al., 2002a), although an independent strain with disruption of FcgRIII appears to be completely resistant (J. Ravetch, personal communication). In our laboratory, absence of FcgRII had no effect (Ji et al., 2002a), but others have found an earlier onset and/or greater severity of disease in such mice (Corr and Crain, 2002). The ‘‘neonatal,’’ MHC-like FcR (FcRn) is also required for Ab-transferred disease; resistance is associated with a very short circulating half-life of the

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transferred Ab, with the site of clearance uncertain (D. Roopenian, personal communication). XIII. Arthritis in the Ipr Mouse

The MRL/lpr mouse gets a disease very similar to human systemic lupus, as well as a lymphoproliferative disorder, due to absence of the proapoptotic Fas molecule in combination with undefined gene products of the MRL background (Andrews et al., 1978). Arthritis is among the common manifestations. In the first description, 75% of 5-month-old female mice had inflamed joints, and half of those had significant joint destruction (Hang et al., 1982); however, the frequency and severity of arthritis seem to vary among laboratories (Gilkeson et al., 1992; Hang et al., 1982; Kamogawa et al., 2002; O’Sullivan et al., 1985). In the initial report, synovial hyperplasia and subsynovial infiltration (mononuclear cells more than neutrophils) were noted at 3–4 months, followed by pannus formation and destruction of cartilage and bone. Subcutaneous fibrinoid nodules similar to rheumatoid nodules were noted, as well as inflammation of periarticular structures and vasculitis (Hang et al., 1982). A second study, however, reported joint destruction with synovial hyperplasia, but only a modest inflammatory infiltrate (O’Sullivan et al., 1985). Arthritis in the lpr mouse appears to be a complex trait influenced by multiple undefined genes (Kamogawa et al., 2002). Depletion of CD4þ cells inhibits development of arthritis (Gilkeson et al., 1992); there have been no reports on B cell-deficient mice. MRL/lpr mice have numerous auto-Ab of uncertain relevance to arthritis, including RF (Hang et al., 1982) and Ab to collagens and other extracellular matrix proteins (Gay et al., 1987; Ratkay et al., 1991). In the first report, levels of IgM RF correlated well with arthritis (Hang et al., 1982). Immune complexes with features of cryoglobulins (precipitating spontaneously in the cold) isolated from lpr serum cause arthritis in MRL (non-lpr) mice when injected intraarticularly; the transient inflammation can be prolonged by repeated injection (Itoh et al., 1991). Arthritis in lpr mice can be enhanced by administration of IL-1 (Hom et al., 1990) or CFA (Ratkay et al., 1993). No findings have been reported for mice lacking particular cytokines, complement factors, or FcR. XIV. HTLV Transgenic Mouse

On several genetic backgrounds, mice transgenic for the pX region of the human T cell leukemia virus type 1 (HTLV-1) develop a chronic inflammatory arthritis (Iwakura et al., 1991). Incidence in susceptible strains varies from

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about 30% to 70% (Iwakura et al., 1998; Yamamoto et al., 1993). The ankle is most prominently affected; the hindlimb more than the forelimb; and the shoulder, knee, and elbow more than the wrist, finger, and toe (Iwakura et al., 1991). Histologically, synovial proliferation is seen first, followed by formation of synovial villi, cell infiltration (neutrophilic more than mononuclear), pannus formation, destruction of cartilage and bone, formation of lymphoid follicles, and vascular changes (Yamamoto et al., 1993). Genetic background is important to susceptibility, since BALB/c mice have 72% incidence and C57BL/6 mice only 2%. This difference is not due to MHC alleles, based on information from H-2 congenic strains (Iwakura et al., 1998). The importance of different cell types has not been reported. However, several auto-Ab are produced, including RF, anti-type-II collagen, and Abs to heat shock proteins (HSP) (Iwakura et al., 1995). There is also evidence for expansion of T cells specific for type II collagen, and HTLV-1 transgenic mice are highly susceptible to CIA, inviting the hypothesis that arthritis in these mice is mediated at least in part by autoimmunity to type II collagen (Kotani et al., 1999). Mice lacking IL-1 are relatively resistant to arthritis in this model (Saijo et al., 2002). The roles of other cytokines, complement, and FcR have not been reported.

XV. Human TNF Transgenic Mouse

Mice transgenic for a modified human TNF (huTNF) gene develop a chronic inflammatory polyarthritis (Keffer et al., 1991). Truncation of the 30 end of the gene leads to deregulated expression, including expression in synovium (Douni et al. 1995; Keffer et al., 1991). One hundred percent of transgenic mice are affected, beginning at 3–4 weeks of age with ankle swelling and progressing to joint destruction by 9–10 weeks. Histologically, synovial hyperplasia and mixed (neutrophilic and mononuclear) infiltrates are seen early, followed by pannus formation, destruction of cartilage and bone, and fibrosis (Keffer et al., 1991). Arthritis is more severe on the DBA/1 than on the C57BL/6  CBA background (Butler et al., 1997), but little else is known about susceptibility genes. Arthritis is independent of T and B cells, as it still occurs in RAG1deficient mice (Douni et al., 1995). Development of arthritis is, predictably, completely blocked by anti-huTNF Ab, but it is also prevented by Ab to the murine IL-1R1; such mice also have decreased levels of circulating huTNF (Probert et al., 1995).

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XVI. IL-1ra Knockout Mouse

Mice with targeted disruption of the IL-1 receptor antagonist (IL-1ra) on the BALB/c, but not on the C57BL/6, background invariably develop chronic inflammatory polyarthritis, primarily of the ankles (Horai et al., 2000). The usual features of synovial hyperplasia, mixed inflammatory infiltration, pannus formation, and erosion of cartilage and bone are seen, starting between 5 and 8 weeks of age (Horai et al., 2000). This gene deletion does not lead to arthritis in RAG2-deficient mice (Iwakura, 2002), indicating an essential role for T and/or B lymphocytes. Auto-Ab, including RF (IgG but not IgM subclass), anti–type II collagen, and anti-dsDNA, are induced, but their role is unknown (Horai et al., 2000).

XVII. Mutant IL-6 Receptor Mouse

Mice engineered to have a point mutation in a phosphatase-binding site of the gp130 subunit of the IL-6 receptor develop inflammatory arthritis (Atsumi et al., 2002). Lymphocytes are required for disease to occur. Thymic and peripheral T cell tolerance are impaired, and autoAb are made. No other mechanistic details are known as yet.

XVIII. Other Models

Arthritis can be produced by immunization with components of cartilage other than type II collagen or PG, namely, type IX collagen, type XI collagen (Boissier et al., 1990), cartilage link protein (Zhang et al., 1998), and YKL-39 (Sakata et al., 2002). A transient but destructive AIA can be induced in the knees of nonimmunized mice if IL-1 is given subcutaneously concurrently (Staite et al., 1990). Some inbred strains, such as DBA/1 (males only), develop spontaneous arthritis at an advanced age (Bouvet et al., 1990; Nordling et al., 1992). These models have not been dissected in detail; in DBA/1 males, disease is not dependent on T cells and more closely resembles ankylosing spondylitis than RA (Corthay et al., 2000). Intraarticular injection of DNA sequences containing unmethylated CpG motifs (characteristic of bacterial DNA) causes an inflammatory arthritis lasting about 14 days (Deng et al., 1999). Macrophages are the dominant infiltrating cell, and neutrophils and lymphocytes appear to be dispensable (Deng et al., 2000). The disease is greatly attenuated in mice lacking TNF-a (Deng et al., 1999). Although this model is primarily one for septic arthritis and reactive arthritis, its findings may be relevant to RA, since the role of infection and stimulation of cells via toll-like receptors (TLRs) is unclear.

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Human synovial tissue and cartilage can be transplanted into SCID mice without any apparent host reaction (Geiler et al., 1994; Rendt et al., 1993). Synovial tissue or isolated synovial fibroblasts from RA patients erode normal human cartilage in this model, showing an intrinsic destructive behavior of these cells independent of ongoing immunity (Geiler et al., 1994; MullerLadner et al., 1996). The model has been very useful for evaluating synovial fibroblasts and is beginning to be used for immunological studies; for example, activation of T cells requires HLA matching and cotransplanation of B cells (Takemura et al., 2001). Mouse models that resemble ankylosing spondylitis (Khare et al., 1995) and psoriatic arthritis (Bardos et al., 2002) have been developed. Abs have not been implicated in these models, and, notably, there is no compelling evidence for auto-Ab involvement in the human diseases they resemble. XIX. Summary of Mouse Models

As described earlier and summarized in Table I, among those models in which numerous aspects of immune function have been evaluated, there is far more similarity than difference. A requirement for both B and CD4þ T cells is prominent in models induced by active immunization or autoreactive transgene-encoded TCRs, but is absent in models induced by passive immunization with Ab. There is substantial agreement on the roles of different FcR, especially on the common FcRg chain as an effector and on FcgRII as a suppressor of arthritis. The importance of TNF-a and IL-1 is more evident in passive models, perhaps because it is easier to fully block the effects of mediators in the setting of milder disease. Notably, most of the active models have not been evaluated in mice lacking cytokine or cytokine receptor genes due to gene disruption. A general scheme for the development of arthritis in mice is proposed in Fig. 2. In most of the models discussed earlier, T cell reactivity, whether produced by intentional immunization or spontaneous loss of tolerance, leads to the production of Ab. A role for these Ab in disease is established by either the ability to transfer disease using Ab or by a dependence on FcR. The formation of IC leads to activation of complement and of various effector cells of the innate immune system (macrophages, mast cells, and neutrophils); the importance of particular cell types varies among models. These innate effector cells produce many mediators, most prominently TNF and IL-1, which provide an important positive feedback to promote further inflammation; in addition, IL-1 stimulates joint destruction. Most models in which T and/or B cells are dispensable are, nevertheless, consistent with this pathway, initiating pathology at different points. All models in which Ab is given passively subvert the need for T cell or B cell reactivity in

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TABLE I Characteristics of Mouse Models and Human RAa Active

Deposits: Ig/C3 MHC II restriction T cells CD4+ cells B cells Macrophages Neutrophils Mast cells Transfer by Ab Complement: CVF Complement: C5 Complement: C5aR FcRg FcgRI FcgRIII FcgRII TNF-a IL-1 IL-6 IL-10 IL-18

Passive

CIA

K/BxN

AIA

PGIA

CIA

Anti-GPI

AIA/ICA

RA

+ + + + + + nr nr + nr + nr + – + –– +(Ab) + + –– +

+ + + + + nr nr nr + nr nr nr nr nr nr nr –(Ab) nr nr nr nr

+ nr + nr nr (+)b nr – +/– (–)a nr nr + – – –– –(Ab) +/– – nr nr

+ nr + + + nr nr nr – nr nr nr + nr nr –– nr nr nr nr nr

+ – – – – nr nr nr (na) nr + + + +/– + – +(Ab) + – nr nr

+ – – – – (–)c + + (na) nr + + + – + –– +(KO) + – nr nr

+ nr nr nr nr + nr nr (na) + nr nr + +/– + –– –(Ab) + nr nr nr

+ + +/– +/– + nr nr nr – nr +/– nr nr nr nr nr + + + nr nr

a +, required; –, not required; – –, protective; nr, not reported; na, not applicable; CVF, cobra venom factor; (Ab), anti-TNF mAb; (KO), knockout mouse. b ‘‘Flare-up’’ reaction. c Method different from others.

the host. CpG directly activates innate immune system cells, and zymosan directly activates complement. Local injection of SCW induces TNF in the joint, and the huTNF transgenic mouse produces high levels spontaneously, particularly in the joint. Only two models cannot be readily incorporated into this scheme: pristane-induced arthritis and the flare-up reaction of AIA, in which T cells are required but B cells are not. Of course, many complex issues are touched upon only lightly in this scheme: the breaking of tolerance; the rich variety of inflammatory responses; and the means by which IL-1, TNF-a, and probably numerous other mediators contribute to joint destruction. Models that rely on immunization involve immunity to antigens that are joint specific either innately (CIA, PGIA) or experimentally (AIA). However, in the K/BxN model, in which the factors that govern disease are similar to these

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Fig 2 General mechanism for the development of inflammatory arthritis in mice. Elements of the mechanism are shown in blue, the names of various mouse models boxed in red, and Abindependent pathways in black. Thick arrows connect the common mechanistic components. Thin arrows show diversity of mechanism at particular points, as well as the entry points of the mouse models. Dotted lines show hypothetical or less-well-documented pathways; for example, it is unclear whether loss of tolerance in the lpr and HTLV-1 models is at the level of B cells as well as T cells. ‘‘Other mediators’’ include angiogenic factors, arachidonic acid derivatives, cytokines, and chemokines. CIA, collagen-induced arthritis; AIA, antigen-induced arthritis; PGIA, proteoglycan-induced arthritis; SCW, staphylococcal cell wall-induced arthritis; TgM, transgenic mouse; HTLV-1, HTLV-1 transgenic mouse; IC, immune complexes; ICA, immune-complex arthritis; C, complement; anti-GPI, arthritis from passive transfer of anti-GPI Ab; IL-1ra KO, IL-1ra gene knockout on BALB/c background; CpG, arthritis from injection of hypomethylated CpG-rich DNA; mf, macrophages; nf, neutrophils; huTNF-TgM, mouse transgenic for deregulated human TNF. (See Color Insert.)

other models, immunity is directed against an Ag that is found in all cells and circulates at low levels in the blood (Matsumoto et al., 1999, 2002). Thus a joint-specific autoimmune disease need not involve immunity to a jointspecific Ag; it is, nevertheless, likely that the distribution of GPI in the normal joint (on the articular surface or otherwise) is important in its arthritogenic properties (Matsumoto et al., 2002). Thus noting the compatibility among many mouse models, the question of how closely they resemble human RA must be addressed. XX. Relevance to RA

The pathology and time course of RA were described at the outset of this chapter. All of the models mentioned previously have been described as having pathology that closely resembles RA. Although we cannot argue the fine points

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of histopathology in the various models and in RA, it seems fair to conclude that RA-like pathology can be produced by a variety of insults. Therefore it is reasonable to propose that human RA could be integrated into the pathway shown in Fig. 2 (and described earlier) at any point. The question then arises: at which point or points? Susceptibility to RA is more closely linked to the MHC class II locus HLADR than to other, as yet undefined, genes (Jawaheer et al., 2003; Stasny, 1978). Thus CD4þ cells are implicated, although their importance has not been formally demonstrated in clinical trials (Moreland et al., 1995; van der Lubbe et al., 1995). A role for B cells in RA has been shown by the clinical improvement in patients depleted of B cells with regimens including the antiCD20 mAb rituximab (Edwards et al., 2002). Roles for CD4þ and B cells have been more definitively shown in many mouse models. Blockade of three different cytokines—TNF-a, IL-1, and IL-6—has been shown to be effective in ameliorating established RA (Bresnihan et al., 1998; Maini et al., 1999; Nishimoto et al., 2002; Weinblatt et al., 1999). These findings are in line with most mouse models, except that data on IL-6 are limited (see Table I). It is important to note, however, that these mediators are pleiotropic and likely operate in a wide variety of otherwise dissimilar inflammatory diseases. Auto-Abs, most notably RF and anticyclic-citrullinated-peptide Ab, are found in RA but are of uncertain pathological significance (Morgan et al., 1987; Souto-Carneiro et al., 2001; Verheijden et al., 1997). RA has not been transferred to naı¨ve (in more ways than one) humans using serum (Harris and Vaughan, 1958), and rarely have human auto-Abs been shown to cause arthritis in mice (Wooley et al., 1984). However, the same is true for murine PGIA, in which the indirect evidence for the importance of auto-Abs is strong. There is little direct evidence for involvement of complement in RA. Patients deficient in C3 get severe infections, and there are no data on the incidence of RA in such patients (reviewed in Schur, 1986). Patients deficient in C1q, C2, or C4 frequently suffer from systemic lupus, and patients lacking C5 are highly susceptible to infection with Neisseria bacteria (reviewed in Schur, 1986); again, there are no data on the incidence of RA. A C5-blocking mAb has shown some clinical benefit in established RA in an early trial (Jain et al., 1999). Additional, although indirect, evidence for involvement of complement in RA comes from the findings, apparently unique among inflammatory arthritides, that total hemolytic complement (CH50) is depleted in the synovial fluid of active rheumatoid joints (Pekin and Zvaifler, 1964; Ruddy et al., 1969), and that levels of complement breakdown products are concomitantly elevated (Mollnes et al., 1986; Olmez et al., 1991). Deposits of Ig and C3 are found in the articular cartilage of affected joints (Cooke et al., 1975; Vetto et al., 1990), as in auto-Ab-mediated mouse models. The roles of FcRs have

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not yet been directly evaluated in RA, but polymorphisms in the genes for FcgRIIIA and FcgRIIA may be linked to susceptibility or severity (Brun et al., 2002; Morgan et al., 2000; Nieto et al., 2000). Mixed results have been obtained with the administration of high doses of intravenous Ig (Maksymowych et al., 1996; Muscat et al., 1995; Tumiati et al., 1992), which may modulate FcR function, but likely has other effects (reviewed in Kazatchkine and Kaveri, 2001). Thus, apart from the inevitably murkier results that come from working with heterogeneous human populations rather than homogeneous inbred mouse strains, RA most resembles the ‘‘active’’ models shown in Table I and on the left side of Fig. 2. We propose that RA—like the CIA, K/BxN, AIA, and PGIA models—is initiated by the breakdown of T cell tolerance to auto-Ag that reside in (but may not be specific to) the joint, leading to production of auto-Ab, formation of ICs, activation of cells of the innate immune system, and cytokine-mediated pathologic remodeling of joint tissues. The process may be initiated, and proceeds to a certain point, systemically rather than locally. As in the mouse models, roles for Ab-independent pathways are not excluded. Any theory for the pathogenesis of RA must provide an explanation for RF. We will discuss two hypotheses, both of which are consistent with RA as an ICinitiated or IC-mediated disease, but that put RF in very different roles. First, RF could be an epiphenomenon, the common, nearly inevitable, consequence of chronic IC disease. B cells bearing surface IgM with RF activity are able to take up Ab-rich ICs with great efficiency and present peptides from the associated Ag to CD4þ T cells (Roosnek and Lanzavecchia, 1991). Thus these RF B cells could in turn receive cognate T cell help for the production of RF. It is not unreasonable to propose, as Roosnek and Lanzavecchia (1991) have, that such a mechanism normally plays a useful role in facilitating the clearance of ICs and the appropriate down-regulation of Ab responses that have served their purpose. Second, RF could serve as an amplifier of Ab-mediated disease by enhancing the activation of complement or the activation of cells through FcgRIII. Indeed, in the presence of IgG, RF can activate complement in vitro (Tanimoto et al., 1975; Zvaifler and Schur, 1968). Thus one can envision a ‘‘two-hit’’ mechanism for the initiation of RA: first, an auto-Ab response to a joint-associated Ab; second, an RF response that magnifies Ab-associated effector mechanisms. Such a two-hit mechanism could explain many of the troublesome findings related to RF in both RA and mouse models. People with RF, whether with other inflammatory diseases or in good health, could remain free of RA because they do not have the primary arthritogenic auto-Ab. The 20% of RA patients who lack RF may be those whose auto-Ab response is sufficient to cause arthritis, as in the CIA and K/BxN models. It is perhaps not a coincidence that in the models in which disease can be transferred using serum (CIA, K/BxN), RF is not found, but in

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models that cannot be transferred with serum, it often is (PGIA, HTLV-1, lpr). An important prediction of this hypothesis is that the addition of RF to Ab-mediated models of RA should make disease worse. To date, there is only one such report, showing a greater severity of disease with the addition of monoclonal human RF (IgM or IgG) to mice immunized with type II collagen (Ezaki et al., 1996). Similar findings would need to be made in passive models of arthritis, however, to support the notion of RF as an amplifier of the effector phase. Another prediction is that rituximab and other B cell-depleting therapies should be equally effective in RF-positive and RF-negative patients, as long as production of the relevant primary auto-Ab declines. With renewed interest in auto-Ab in RA, this prediction will likely be testable in the near future. The development of mAb-based immunological therapies, in addition to providing great benefit to patients, is allowing more precise testing of hypotheses about the pathogenesis of human RA. As was the case in the development of TNF-blocking agents, it will probably be by a fruitful combination of research on mouse models and on human patients that the reemerging paradigm of RA as an Ab-mediated disease will be assessed. References Alonzi, T., Fattori, E., Lazzaro, D., Costa, P., Probert, L., Kollias, G., De Benedetti, F., Poli, V., and Ciliberto, G. (1998). Interleukin 6 is required for the development of collagen-induced arthritis. J. Exp. Med. 187, 461–468. Andrews, B. S., Eisenberg, R. A., Theofilopoulos, A. N., Izui, S., Wilson, C. B., McConahey, P. J., Murphy, E. D., Roths, J. B., and Dixon, F. J. (1978). Spontaneous murine lupus-like syndromes. Clinical and immunopathological manifestations in several strains. J. Exp. Med. 148, 1198–1215. Atsumi, T., Ishihara, K., Kamimura, D., Ikushima, H., Ohtani, T., Hirota, S., Kobayashi, H., Park, S. J., Saeki, Y., Kitamura, Y., and Hirano, T. (2002). A point mutation of Tyr-759 in interleukin 6 family cytokine receptor subunit gp130 causes autoimmune arthritis. J. Exp. Med. 196(7), 979–990. Banerjee, S., Webber, C., and Poole, A. R. (1992). The induction of arthritis in mice by the cartilage proteoglycan aggrecan: Roles of CD4þ and CD8þ T cells. Cell. Immunol. 144(2), 347–357. Bardos, T., Zhang, J., Mikecz, K., David, C. S., and Glant, T. T. (2002). Mice lacking endogenous major histocompatibility complex class II develop arthritis resembling psoriatic arthritis at an advanced age. Arthritis Rheum. 46(9), 2465–2475. Beech, J. T., and Thompson, S. J. (1997). Anti-tumour necrosis factor therapy ameliorates joint disease in a chronic model of inflammatory arthritis. Br. J. Rheumatol. 36(10), 1129. Benoist, C., and Mathis, D. (2002). Mast cells in autoimmune disease. Nature 420(6917), 875–878. Bianco, N. E., Dobkin, L. W., and Schur, P. H. (1974). Immunological properties of isolated IgG and IgM anti-gamma-globulins (rheumatoid factors). Clin. Exp. Immunol. 17(1), 91–101. Boissier, M. C., Chiocchia, G., Ronziere, M. C., Herbage, D., and Fournier, C. (1990). Arthritogenicity of minor cartilage collagens (types IX and XI) in mice. Arthritis Rheum. 33(1), 1–8.

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advances in immunology, vol. 82

MUC1 Immunobiology: From Discovery to Clinical Applications ANDA M. VLAD, JESSICA C. KETTEL, NEHAD M. ALAJEZ, CASEY A. CARLOS, AND OLIVERA J. FINN Department of Immunology, University of Pittsburgh School of Medicine Pittsburgh, Pennsylvania 15261

I. Introduction

For more than a decade, tumor immunologists have focused their efforts on discovering tumor-associated antigens, as a first step toward the design of an effective cancer vaccine. To date, approximately 70 MHC class I and IIassociated tumor antigens have been described, while more than 1700 have been identified by antibodies in cancer patients (Yu and Restifo, 2002). However, it has become increasingly evident that this growing list of putative tumor-associated antigens will need to be supplemented with greater understanding of their molecular nature and mechanisms of action in order to validate them as suitable targets for tumor immunotherapy. In this chapter we will highlight studies on MUC1, one of the first tumor antigens shown to be a target for human tumor-specific T cells and thus a valid target for immunotherapy. MUC1 is a member of the mucin family of molecules. It is expressed on the luminal surface of most polarized epithelial cells and overexpressed over the entire cell surface of most adenocarcinomas. Cancer-associated MUC1 is different from MUC1 on normal cells. During tumor progression there are changes in glycosylation that result in the synthesis of tumor-specific glycoforms bearing novel T and B cell epitopes. Thus MUC1 glycoprotein meets the criteria of a tumor-specific antigen and is currently employed in vaccines under investigation in several clinical trials. Research on MUC1 has been reported in over 700 publications in the past 5 years, with the majority of these publications being focused on MUC1 immunobiology. These numbers, illustrating the interest in this molecule as an important tool in cancer research, also indicate the amplitude of the ongoing efforts to further explore the basic mechanisms behind its immunogenicity and its suitability as a target antigen for cancer treatment and prevention. We will briefly describe here the key research efforts that elucidate MUC1 structure and biosynthesis pathways; however, our emphasis will be on the most recent studies that mark progress toward a better understanding of what makes MUC1 a tumor antigen, what kind of immune responses this molecule can trigger, and how various immune effector mechanisms can be manipulated for therapeutic purposes. 249 Copyright 2004, Elsevier Inc. All rights reserved. 0065-2776/04 $35.00

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II. History of MUC1, the Pioneer Member of the Mucin Family

MUC1 was first identified in the milk fat globule membrane fraction and described as a protein rich in serine, threonine, proline, glycine, and alanine (Shimizu and Yamauchi, 1982). It was found to contain a high percentage of O-linked carbohydrates that accounted for about 50% of its molecular weight. In 1987, Gendler and colleagues (1987) were able to clone a fragment from this first human mucin gene by screening a mammary tumor cell line MCF-7 cDNA library using antibodies raised against a chemically deglycosylated form of milk mucin. The cloned gene, located on chromosome 1q21 (Dekker et al., 2002), was sequenced and found to consist of numerous 60-base pair tandem repeats (Gendler et al., 1987; Siddiqui et al., 1988). Subsequently, cDNA encoding for splice variants of mucin were cloned from breast carcinoma cell lines (Ligtenberg et al., 1990), from human breast tumor tissue (Gendler et al., 1990; Wreschner et al., 1990), and from pancreatic tumors (Lan et al., 1990). This first human mucin gene was given the name MUC1 to replace preexisting names that included polymorphic epithelial mucin (PEM), polymorphic urinary mucin (PUM), epithelial membrane antigen (EMA), episialin, and MAM-6 DF3 antigen. The other members of the mucin gene family were numbered in the order they were identified: MUC2, MUC3A, MUC3B, MUC4, MUC5AC, MUC5B, MUC6–9, MUC11–13, and MUC15–17. All mucins have certain structural features in common. They all consist of a peptide core with O-linked glycans attached to serine and threonine residues. The protein core consists of a variable number of repeated sequences (tandem repeats) distinct to each mucin. Mucins can exist in a secreted form (gel-forming), membrane-bound form, or both. MUC1, MUC3–4 (Moniaux et al., 2000; Williams et al., 1999b), MUC12–13 (Williams et al., 1999a, 2001), and MUC15–17 (Gum et al., 2002; Pallesen et al., 2002; Yin et al., 2002) can be expressed as membrane-bound glycoproteins. These membrane-bound mucins have a transmembrane domain that facilitates their anchoring in the membrane lipid bilayer. The rest of the mucins can only be expressed in soluble form. MUC1 is normally present on the apical surface of most polarized epithelial tissues of the respiratory tract, genitourinary tract, and digestive system. It is also expressed on normal breast ducts. MUC1 is overexpressed on the majority of adenocarcinomas of the breast, lung, colon, pancreas, stomach, prostate, and ovary (Ho et al., 1993). MUC1-expressing cancers account for about 70% of new cancer cases expected in the year 2003 (Jemal et al., 2003). The forms of MUC1 produced by tumor cells differ in many ways from normal MUC1. As an epithelial cell undergoes malignant transformation, it loses the normal apical-basolateral polarity and begins to express MUC1 on the entire cell surface. The level of expression also increases, and a soluble form of MUC1 can be found in the serum of cancer patients.

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Similar to MUC1, other members of the mucin family also have an altered expression on different tumors. MUC4 is overexpressed in adenocarcinomas of the lung and expressed de novo in pancreatic and gastric cancers (Balague et al., 1994; Buisine et al., 2000; Nguyen et al., 1996). In one report, about 29% of lung cancer patients had high titers of anti-MUC4 IgG and IgM antibodies (Hanaoka et al., 2001). Overexpression of MUC6 and de novo expression of MUC2, MUC4, and MUC5AC has been demonstrated on the surface of adenocarcinomas of the pancreas and on pancreatic tumor cell lines (Balague et al., 1994). However, there is only limited information about the immunogenicity of these other mucins, and prognostic significance of their altered expression is not known.

III. Structure, Biosynthesis, and Physiology of MUC1 in Health and Disease

A. MUC1 Structure and Biosynthesis Unlike the majority of mucins that are secreted from cells, MUC1 is expressed as both a transmembrane and secreted form. Though it is encoded as a single protein, it is expressed as a type I transmembrane heterodimer. The two proteins that make up MUC1 differ greatly in size, with most of the larger MUC1 fragment being composed of a tandemly repeated 20 amino acid sequence, PDTRPAPGSTAPPAHGVTSA. This serine-, threonine-, and proline-rich sequence can be repeated up to 125 times in a single MUC1 molecule, commonly occurring between 41 and 85 times (Carvalho et al., 1997; Gendler et al., 1990). This region of the molecule is referred to as variable number of tandem repeats (VNTR). The biosynthesis of MUC1 proceeds via distinct steps (Hilkens and Buijs, 1988). The newly synthesized protein receives several N-glycans adjacent to its transmembrane region following cotranslational transfer of high-mannose glycans during synthesis in the endoplasmic reticulum. Within 1–2 min, while still in the endoplasmic reticulum, MUC1 undergoes proteolytic cleavage. Ligtenberg et al. showed in 1992 that the two cleavage products remain noncovalently associated so that the smaller transmembrane fragment anchors the larger piece. A proteolytic cleavage site, FRPG/SVW, located 65 amino acids upstream of the transmembrane domain, was identified recently (Parry et al., 2001). After cleavage, the precursors move through the Golgi, where the N-glycans become more complex and O-glycosylation is started on the VNTR region. O-Glycosylation increases the molecular weight dramatically within the first 30 min of synthesis. MUC1 becomes partially sialylated on its O-linked oligosaccharides before leaving the Golgi as a premature form. Completely and incompletely sialylated MUC1 are both expressed on the cell surface (Litvinov and Hilkens, 1993). Trafficking of MUC1 to the cell surface is

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thought to be controlled by at least two signals, one contained in the CysGln-Cys motif at the junction of the MUC1 tail and transmembrane domains, and a second in the extracellular domain but outside of the VNTR region (Pemberton et al., 1996). To become fully sialylated, the premature form recycles several times from the cell surface to the trans-Golgi and back to the surface. Complete sialylation occurs within 3 h (Hilkens and Buijs, 1988). The recycling of MUC1 is constitutive so that even after full sialylation, a mature MUC1 molecule completes 10 cycles before being released from the cell, approximately 24 h after synthesis. MUC1 on the surface of normal cells is completely sialylated, while on tumor cells the surface MUC1 is a combination of completely and incompletely sialylated molecules. It was suggested that this is due to the greater abundance of MUC1 on tumor cells and/or a less efficient sialylation process compared to normal cells (Litvinov and Hilkens, 1993). Nuclear magnetic resonance (NMR) studies using peptides composed of one to three tandem repeats have shown that as the number of repeats increases, the structure of MUC1 becomes more ordered. Indeed, intrinsic viscosity measurements indicate that the peptide composed of three repeats has a rod-like structure (Fontenot et al., 1993), suggesting that MUC1 on the cell surface would project outward rather than exist in a globular shape. Further NMR studies established that in each repeat, the APDTR sequence, to which antibodies have been raised, exists on a protruding knob-like structure on the MUC1 backbone (Fontenot et al., 1995b). When multiple repeats are examined, the overall effect is a rod with evenly spaced knobs throughout the entire VNTR region. Most antibodies against MUC1 bind to this epitope, making it immunodominant on the native MUC1 molecule (Price et al., 1998). Because of the large number of repeats in the VNTR region, MUC1 can extend 300–500 nm above the cell surface, towering over other cell surface molecules. Twenty-five percent of the amino acids in the VNTR region are either serine or threonine that can be O-glycosylated. On either side of the VNTR region are several degenerate repeats (Engelmann et al., 2001; Ligtenberg et al., 1990). Recently, Engelmann et al. (2001) provided genetic evidence of variation in the 20 amino acid sequence within the VNTR domain. By sequencing polymerase chain reaction (PCR) products followed by minisatellite variant repeat analysis of the 50 and 30 peripheral areas of the VNTR region in 33 samples taken from normal and cancerous cells, they found that the same sequence variation consistently occurred in the same repeats. This indicates that the variation predates the duplication event that has led to the elongated VNTR domain. The proline (cca) in position 13 of the tandem repeat sequence PDTRPAPGSTAPPAHGVTSA could be altered to glutamine (caa), alanine (gca), or threonine (aca), possibly generating an additional glycosylation site. The other location of sequence change is in the immunodominant epitope, APDTR, in which the DT (gacacc)

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is substituted with ES (gagagc). This was the most commonly seen sequence variation within the diverse population studied. However, in the majority of samples this variation was found in only four of the 24 repeats sequenced from each of 33 samples. This particular variation within the immunodominant peptide sequence could be regarded as a source of additional epitopes with immunogenic potential; nevertheless, since this mutated (ES) sequence is less commonly seen than the conserved DT sequence found in the majority of repeats, the majority of responses should to be directed toward the highly conserved and overwhelmingly abundant tandem repeat sequences. The smaller piece ( 20 kDa) of MUC1 contains a short extracellular portion, a transmembrane region, and a short intracellular tail. In its extracellular domain are sites for N-linked glycosylation (Gendler et al., 1990; Wreschner et al., 1990). The transmembrane region carries cysteines that may be used for fatty acid acetylation to help anchor MUC1 in a cell’s membrane (Ligtenberg et al., 1990). In the cytosolic tail are potential sites of phosphorylation and intracellular protein binding that prompted research into the possibility that MUC1 could be a signaling molecule. This was of interest because the exact function of MUC1 is still not known. Alternative splicing of MUC1 mRNA can lead to multiple forms being expressed by a single cell type. When the fulllength cDNA and genomic organization of MUC1 were initially published, they showed that two different amino-terminal signal sequences could be produced. The longer form, referred to as MUC1/A, has an additional 27 base pairs when compared with MUC1/B (Ligtenberg et al., 1990; Wreschner et al., 1990). Whether MUC1/A or MUC1/B is produced depends on whether a guanine or adenine is present eight nucleotides downstream of exon 1, in the first intron. When guanine is present, the longer MUC1/A is synthesized and the number of repeats is higher. Conversely, when adenine is present there are fewer repeats and the shorter isoform MUC1/B is made (Ligtenberg et al., 1991). Soluble MUC1 is found in human milk (Patton, 2001; Peterson et al., 2001) and in barely detectable amounts in the serum of healthy men and women (Croce et al., 2001b; McGuckin et al., 1994). This form may be produced when a splice donor site downstream of the VNTR region is not used during transcription, allowing translation of a stop codon prior to the transmembrane region (Wreschner et al., 1990). In 1996, a monoclonal antibody was generated against this spliced out peptide sequence (Smorodinsky et al., 1996). With this antibody, soluble MUC1 was detected in supernatants of cancer cell lines and in sera of cancer patients. However, mouse mammary epithelial cells transfected with full-length human MUC1 in which alternative splicing could not occur (Boshell et al., 1992) still produced soluble MUC1 lacking the cytosolic tail. This supports a second mechanism for production of soluble MUC1 that proposes that MUC1 is released from the surface of cells by proteolytic cleavage (Hilkens et al., 1991). TACE [tumor necrosis factor-a (TNF-a)

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converting enzyme] is considered the likely protease responsible for the cleavage (Thathiah et al., 2003). Other potential mechanisms are cleavage by external proteases or simple dissociation of the heterodimeric complex. The involvement of external proteases is not likely, given that addition of proteolytic inhibitors has no effect on the amount of soluble MUC1 (Julian and Carson, 2002). Simple dissociation seems unlikely as well, given that MUC1 remains a stable heterodimer during repeated recycling through the cell for further glycosylation and sialylation (Ligtenberg et al., 1991; Litvinov and Hilkens, 1993). Furthermore, when a mutated form of MUC1 that lacks the site of initial cleavage is expressed as a single protein, it is still released from the cell (Ligtenberg et al., 1992). 1. O-Linked Glycosylation of MUC1 in Normal Epithelia Because of the differences in MUC1 glycoforms expressed on normal and cancerous epithelium, there has been a great effort to understand MUC1 O-linked glycosylation. The majority of MUC1 glycosylation occurs in the VNTR region on the two serines and/or three threonines in each repeat. The most common carbohydrate additions to these amino acids is a core 2 structure, an N-acetylgalactose that has a galactose branching from its third carbon and N-acetylglucose branching from its sixth carbon. In normal MUC1, these branches are elongated and effectively cloak the peptide backbone. Only a minor fraction of normal MUC1 glycosylation consists of core 1 additions (Hanisch and Muller, 2000). The core 1 structure is an N-acetylgalactose that has only the galactose branching from its third carbon, no addition to carbon 6. This yields a less effective cloaking of the peptide backbone and is predominantly seen on the tumor form of MUC1. While N-linked glycosylation occurs at known consensus sites, O-linked glycosylation motifs have not been identified. However, human GalNAc transferases responsible for initiating O-linked glycosylation on MUC1 have been studied in vitro (Wandall et al., 1997) and their in vivo products analyzed (Muller et al., 1997) using recombinant enzymes and MUC1 peptides. Regardless of whether the peptide contained one or five repeats PDTRPAPGSTAPPAHGVTSA, only three of the five Ser/Thr sites per repeat were glycosylated. No glycosylation was seen on the Ser in GVTSA or Thr in DTR. Interestingly, the enzyme kinetics varied for the site being glycosylated (e.g., GalNAc-T2 being the fastest to glycosylate ST in GSTAP but slowest on the T in GVTSA) (Wandall et al., 1997). In human milk, however, all five potential sites could be glycosylated, with an average of 2.7 sites per repeat (Muller et al., 1997). The discrepancy between in vitro and in vivo work could be attributed to additional GalNAc transferases working in vivo and an enhancing effect of previous glycosylation on subsequent glycosylation. This was demonstrated with a recombinant GalNAc-T4 transferase that could

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glycosylate Ser in GVTSA and Thr in PDTR, but only if the peptide had been previously glycosylated (Bennett et al., 1998). Furthermore, studies with transferases GalNAc-T1, -T2, and -T3 showed that in vitro glycosylation occurred differently on single MUC1 tandem repeat peptides, depending on how many sites were previously glycosylated. Distant and neighboring effects on subsequent glycosylation, as well as enzymatic competition between core synthesizing enzymes and transferases, could explain the MUC1 glycosylation differences between normal and cancer cells (Hanisch et al., 1999). Further studies are continuing to explore this highly dynamic regulation of O-glycosylation (Dalziel et al., 2001; Hanisch et al., 2001). 2. O-Linked Glycosylation of MUC1 in Tumor Cells The most striking difference between normal MUC1 and tumor MUC1 is in their glycosylation (Burchell et al., 2001; Hanisch and Muller, 2000). Changes in the relative and total levels of glycosyltransferases in tumor cells are largely responsible for this aberration (Beum et al., 1999). Prematurely terminated carbohydrates found on tumor MUC1 include the Thomsen– Friedenreich antigen (Galb1-3GalNAc-Thr/Ser), Tn antigen (GalNAc-Thr/ Ser), and sialyl-Tn antigen (Sialyla2-6GalNAc-Thr/Ser). Many of the antibodies generated by cancer patients have been found to be specific for these short carbohydrates linked to the MUC1 backbone. Shorter carbohydrate chains also allow the peptide backbone of the VNTR region, and especially the immunodominant knobs, to be exposed and recognized by MHC-unrestricted T cells and antibodies (Barnd et al., 1989; Fontenot et al., 1995b). In 2001, Obermair et al., looking at cervical carcinoma cells, found two novel MUC1 splice variants. These were shorter than the variants described for normal MUC1 and were named MUC1/C and MUC1/D. Both are the result of alternative splice acceptor sites when joining exons one and two. Splice variants (MUC1/Y, MUC1/X, and MUC1/Z) of MUC1 lacking the VNTR region have also been reported. MUC1/Y transcripts and protein were found in primary breast cancer tissue (Zrihan-Licht et al., 1994). MUC1/X (Baruch et al., 1997) and MUC1/Z (Oosterkamp et al., 1997), both larger than MUC1/Y by 18 amino acids, were reported in cancer cell lines. Polymorphisms in the length of the VNTR region have also been studied in patients with gastric carcinoma and two of the premalignant states associated with this disease. The allele that encodes the short VNTR region was found to be highly associated with both premalignant conditions and with susceptibility to gastric carcinoma. Homozygosity for the long allele was associated with one of the premalignant states but not with carcinoma. The heterozygous state was found to offer the most protection from disease (Carvalho et al., 1997; Silva et al., 2001).

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B. MUC1 Physiology The physiological role of normal MUC1 is still undetermined. As a member of the mucin family, its assumed role, both in its secreted and membranous forms, is the lubrication of epithelial and ocular surfaces (Gipson and Inatomi, 1998). MUC1 can bind to pathogens at the epithelial surface through its O-linked carbohydrates (DeSouza et al., 1999; Lillehoj et al., 2001; Schroten et al., 1992; Yolken et al., 1992). This interaction has been hypothesized to either prevent the pathogen access to the cell membrane or, conversely, to aid in the adherence and subsequent infection of the host tissue. It acts as a barrier to embryo implantation in multiple species and may play a part in the maintenance of pregnancy (Bowen et al., 1996; Croy et al., 1997; DeSouza et al., 1998; Hewetson and Chilton, 1997; Hild-Petito et al., 1996; Hoffman et al., 1998; Meseguer et al., 1998; Surveyor et al., 1995). During embryogenesis, MUC1 is induced in developing epithelial tissue, but not in squamous tissue (Braga et al., 1992; Guzman et al., 1996; Shin et al., 2000). The yeast homologue of MUC1 is necessary for pseudohyphal differentiation and invasive growth in yeast (Lambrechts et al., 1996). Human MUC1 induces alterations in cellular morphology of transfected mammalian cells (Hudson et al., 2001). However, whether this expression is simply correlated with epithelial development in mammalian tissue or whether it plays a role in the spatial development of glandular tissue is still under investigation. More recently, it has been suggested that MUC1 may affect erythropoiesis, since it is temporally expressed in erythroblasts (Rughetti et al., 2003). This list is by no means comprehensive, and new roles for MUC1 are still being defined. One of the best ways to gain a global understanding of the importance of a molecule is through the study of knockout animal models. A muc1 (the mouse MUC1 homologue) knockout mouse was created on the C57BL/6 background (Spicer et al., 1995). Interestingly, in a transgenic germ-free environment, these mice developed normally into fertile and healthy adults. However, primary breast tumors induced by polyoma middle T antigen grew significantly slower in these muc1 knockout mice (Danjo et al., 2000; Spicer et al., 1995). The same investigators had previously shown that muc1 plays a role in the formation of intestinal mucus in a cystic fibrosis mouse/muc1 double knockout model (Parmley and Gendler, 1998). The muc1 knockout mouse has been reported to have increased susceptibility to bacterial conjunctivitis, vulvovaginitis, and decreased litter size, but only when housed in a specific pathogenfree vivarium with exposure to endogenous mouse flora (Croy et al., 1997; DeSouza et al., 1999; Kardon et al., 1999). From this work it is clear that although muc1 has some unique functions, there must also be other proteins that can compensate for its role in development.

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Recent studies have shown that MUC1 is expressed on the surface of T cells after activation. MUC1 expression by T cells has been documented by immunohistochemistry (Delsol et al., 1984), flow cytometry (Agrawal et al., 1998a; Chadburn et al., 1992; Chang et al., 2000; Correa et al., 2003; Fattorossi et al., 2002; Wykes et al., 2002), RT-PCR (Agrawal et al., 1998a; Chang et al., 2000; Correa et al., 2003; Fattorossi et al., 2002), Northern blotting (Chang et al., 2000), and confocal microscopy (Correa et al., 2003). Most studies have shown MUC1 expression on activated and not resting T cells (Agrawal et al., 1998a; Chadburn et al., 1992; Chang et al., 2000; Correa et al., 2003; Fattorossi et al., 2002; Wykes et al., 2002). The function of MUC1 on activated T cells has not been clearly defined, but it has been suggested that it may play a role in immune regulation (Agrawal et al., 1998a) and modulation of cell–cell interaction (Correa et al., 2003). Indications of its role in vivo may come from determining where MUC1 expressing T cells are found in the body. Correa et al. (2003) detected MUC1 on 10% of T cells in the synovial fluid of patients with rheumatoid arthritis. No MUC1þ T cells could be detected in the patient’s blood. This suggests the possibility that MUC1 on activated T cells is used during migration into the inflamed joint. This finding opens up a novel application for anti-MUC1 vaccination. Antibodies against MUC1 that would be generated through vaccination could be expected to hinder T cell entry into the arthritic joint. Since activated memory T cells are the dominant cell type present in synovial tissue (Kohem et al., 1996) and memory T cells express MUC1 (Correa et al., 2003), MUC1 vaccination could reduce inflammation in a T cell-specific manner. A frequent question regarding the expression of MUC1 on activated T cells is whether an immune response elicited by MUC1 cancer vaccines would target activated T cells. This is highly unlikely to happen when immune responses elicited by vaccines are focused on tumor-specific forms of MUC1. These immunogens generate immune cells specific for epitopes present only on tumor cells. Activated T cells express the glucosyltranferase enzymes that lead to long, highly branched polysaccharides on MUC1 (Correa et al., 2003; Piller et al., 1988) and do not present the same MUC1 epitopes found on tumor cells. Most of the studies on the function of MUC1 involve the extracellular domain of the molecule; however, its cytosolic tail is also important. The cytosolic tail of MUC1 is well conserved among many species (Pemberton et al., 1996). Seven tyrosines are present in that region (Wreschner et al., 1990) and available for phosphorylation. According to work done with tumor cells, MUC1 transfected cells, or CD8/MUC1 chimeric fusion protein-expressing cells, these tyrosines can be phosphorylated (Meerzaman et al., 2000; Pandey et al., 1995; Quin and McGuckin, 2000; Zrihan-Licht et al., 1994). In a variety

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of cells and conditions, MUC1 coimmunoprecipitates several intracellular protein(s) (Pandey et al., 1995; Quin and McGuckin, 2000; Schroeder et al., 2001; Yamamoto et al., 1997; Zrihan-Licht et al., 1994). Associations between MUC1 and the c-Src tyrosine kinase have been reported (Gonzaez-Guerrico et al., 2002; A. Li et al., 2001; Y. Li et al., 2001), as well as activation of ERK1/2 in vivo (Schroeder et al., 2001) and indirect activation of ERK2 via Ras and MEK in vitro (Meerzaman et al., 2000). MUC1 also interacts with catenin, p120, increasing the nuclear localization of p120 (Li and Kufe, 2001). Association of MUC1 with transmembrane tyrosine kinases, epidermal growth factor receptor, erbB2, erbB3, and erbB4 has been shown in vivo (Schroeder et al., 2001). MUC1 in tumor cells has been associated with b-catenin (Schroeder et al., 2001; Yamamoto et al., 1997), a protein involved in cadherin-mediated cell adhesion. Further studies have shown that binding to b-catenin is affected by phosphorylation of the MUC1 tail. There is increased binding following MUC1 phosphorylation by protein kinase C d (Ren et al., 2002), but decreased binding to b-catenin following the action of glycogen synthase kinase 3b (Li et al., 1998). Though the interaction between MUC1 and b-catenin has been proposed to explain the inhibitory effect of MUC1 expression on cadherin-mediated adhesion (Carraway et al., 2003), this is highly unlikely, since tailless mutants of MUC1 equally hinder binding (Wesseling et al., 1995). Rather, inhibition is more likely due to the high degree of steric hindrance that MUC1 provides on the cell surface (Ligtenberg et al., 1992), illustrated by experiments using MUC1 with varying numbers of repeats (Wesseling et al., 1995, 1996). In hope of better understanding the role of MUC1 in metastasis of tumor cells, the study of tumor MUC1 in cell adhesion continues to be an active and important area of research. Tumor cell adhesion has been associated with the extent of phosphorylation of the MUC1 tail (Quin and McGuckin, 2000). As cells begin to adhere, MUC1 phosphorylation decreases over time, indicating that motility and adherence are associated with MUC1 phosphorylation. How this occurs is debatable. Pandey et al. (1995) showed that phosphorylated MUC1 associates with Grb2, an adapter protein involved in signaling pathways. However, this association could not be replicated by Quin and McGuckin (2000). The latter group did, however, coimmunoprecipitate with MUC1 a 60-kDa phosphorylated molecule as yet unidentified. Further work is needed to elucidate these interesting associations between MUC phosphorylation, interactions with other proteins inside the cell, as well as the effect on adhesion. 1. Expression of MUC1 by Tumor Cells and Its Role in Carcinogensis MUC1 has been identified as a marker of preneoplastic conditions and of several chronic inflammatory diseases. Changes that occur during chronic inflammation include increases in the serum level of MUC1, the generation

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of antibodies to MUC1, and increases in the cell surface level of MUC1 on affected cells (Kohno, 1999; Nakajima et al., 1998; Takaishi et al., 2000). The antibodies to MUC1 found in patients with ulcerative colitis are found in chronic but not acute ulcerative colitis. Interestingly, these antibodies are specific for the peptide backbone of MUC1 and suggest that changes in MUC1 glycosylation may be occurring early in chronic inflammatory conditions, as well as in cancer (Hinoda et al., 1993). The surface expression of MUC1 is reported to be up-regulated in preneoplastic conditions of virtually every tissue that gives rise to a MUC1 positive neoplasm (Adsay et al., 2002; Arul et al., 2000; Boman et al., 2001; Buisine et al., 2001; Cao et al., 1999; Copin et al., 2000; Jarrard et al., 1998; Lopez-Ferrer et al., 2001; Luttges et al., 2002; Masaki et al., 1999; Reis et al., 1999). Whether the expression of MUC1 is part of the pathogenesis of cancer or inflammatory disease or whether it is simply a marker of the disease state is still under investigation. MUC1 is expressed on virtually all adenocarcinomas, as well as several other malignancies, and numerous groups have been able to use MUC1 or the immune response to MUC1 as a marker of disease state (Brossart et al., 2001). Its expression has been linked to a worse prognosis for many of these tumors (Ajioka et al., 1996; Baldus et al., 2002a, 2002b; Kraus et al., 2002; Leroy et al., 2002a, 2002b; Pinto-de-Sousa et al., 2002; Sagara et al., 1999; Sivridis et al., 2002; Yamato et al., 1999). There is also evidence that MUC1 enhances the metastatic abilities of tumor cells (Aoki et al., 1998; Guddo et al., 1998; Hiraga et al., 1998; Tanimoto et al., 1999; Utsunomiya et al., 1998). Cancer patients often have an immune response to MUC1 manifested by lowaffinity cytotoxic T cells and low titer antibodies to MUC1. An immune response to MUC1, manifested through antibodies to MUC1 or MUC1-specific T cells, has been linked to a better overall prognosis. Progression from chronic inflammation to preneoplastic and then neoplastic disease is sometimes a lengthy process. During this process, MUC1 undergoes changes that can render the molecule capable of triggering immune effector mechanisms. Identifying these changes and understanding how the immune effector mechanisms could be manipulated at preneoplastic stages through vaccination may constitute a first and important step toward the design of a vaccine for tumor prevention. 2. MUC1 Expression and Function on Tumor Cells Since MUC1 is up-regulated in preneoplastic and neoplastic conditions, it has been postulated that it may play a role in the growth and/or dissemination of tumor cells. One of the ways that MUC1 contributes to tumor growth is through its ability to affect cell–cell and cell–matrix adhesion (Ciborowski and Finn, 2002; Ligtenberg et al., 1990; Wesseling et al., 1995, 1996). MUC1 can also hinder the function of the shorter adhesion molecules on the tumor cell

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surface through its large and rigid structure and thus serve as an antiadhesive molecule. This inhibition of adhesion may be critical to the metastasis of cells from the primary tumor site. It could also explain the correlation between MUC1 expression and increased metastatic potential seen in some cancers. Conversely, MUC1 is able to bind to adhesion molecules, through its carbohydrate residues and its backbone, which may be important for tumor migration (McDermott et al., 2001; Regimbald et al., 1996; Tomlinson et al., 2000). MUC1 also affects the efficiency of various antitumor immune responses by preventing NK cell binding to tumor cells, suppressing T cell function, and affecting the ability of dendritic cells to function as antigen-presenting cells (Agrawal et al., 1998b; Fung and Longenecker, 1991; van de Wiel-van Kemenade et al., 1993; Zhang et al., 1997). Our group has shown that a circulating form of MUC1 purified from cancer patients can bind to dendritic cells through the mannose receptor and most likely through other lectin receptors (Hiltbold et al., 2000). We are currently investigating the functional consequences of this interaction with the dendritic cell. IV. MUC1 Immunobiology

A. Naturally Occurring Immune Responses to MUC1 1. In Healthy Humans The presence of anti-MUC1 antibodies of IgM and IgG isotypes, as well as of circulating MUC1 antigen in sera from normal healthy women, is well documented (Richards et al., 1998). Agrawal et al. (1995) have shown that MUC1-specific T cells can be primed during pregnancy, as T cells from biparous but not nulliparous women proliferated specifically in response to core MUC1 peptides. These findings could be explained by the fact that anatomical and physiological changes of MUC1-expressing organs (like the uterus and breast) during normal processes (like pregnancy and lactation) can prompt subtle changes in MUC1 production and can eventually trigger priming to MUC1 of immune effectors, like B cells and possibly T cells. Two recent studies (Croce et al., 2001a, 2001b) provide a good analysis of antibody responses in healthy women, correlated with their current or previous pregnancy/lactation status. Plasma measurements of free circulating MUC1, as well as of MUC1 complexed with antibodies in immune complexes, showed elevated levels in pregnant women, compared with nonpregnant women. During pregnancy, there is a dramatic increase in MUC1 during the second trimester up to puerperium; by contrast, although the levels of immune complexes are gradually increasing, there is a drop in the levels of free anti-MUC1 IgG and IgM antibodies, which reach their lowest value at puerperium and then gradually increase after delivery. Lactation can also influence anti-MUC1

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antibody production, since the titer of the IgG isotype was significantly higher in the lactating group when compared with nonlactating women. Despite the fact that these studies provide a good description of the spectrum of anti-MUC1 immune responses arising spontaneously to a selfmolecule, they do not identify any of the reacting epitopes. Further analyses of the (expectedly polyclonal) MUC1 epitopes are still needed. Moreover, the significance of this natural immunization with MUC1 remains to be elucidated. Epidemiological studies performed to date suggest a correlation between pregnancy and lower risk of developing breast cancer (Kalache et al., 1993; MacMahon et al., 1982). In that regard, we have reported a case of a long-term breast cancer survivor whose pregnancy might have triggered a MUC1-specific immune response that prevented recurrence of tumor (Jerome et al., 1997). The patient was diagnosed with a breast tumor that was successfully removed. Five years later, she became pregnant and developed acute inflammatory cellulitis in her breast. Breast tissue from this patient expressed the same MUC1 immunodominant epitope as presented by the original tumor. The subject had a high titer of circulating anti-MUC1 IgM and IgG antibodies and a high frequency of MUC1-specific cytotoxic T lymphocytes in the blood. She remained tumor-free for an additional 5 years of follow-up. It is possible that secondary immune responses against MUC1 were precipitated by pregnancy and prevented the recurrence of breast cancer. The importance of such natural immunity to MUC1 on the incidence of other cancers (like uterine and ovarian carcinomas) also remains to be addressed. 2. In Cancer Patients In addition to the previous findings that suggest immunization to a selfantigen under physiological conditions, we and others have shown that antiMUC1 responses could also be triggered in cancer patients during growth of MUC1-positive tumors. In general, tumor cells are poorly recognized by the immune system due to tolerance to self-antigens. Moreover, tumor cells can utilize a variety due to mechanisms to evade recognition and to suppress cells of the immune system: down-regulation of MHC class I (Zheng et al., 1999), lack of costimulation (Banat et al., 2001), loss of antigenic variants (Riker et al., 1999), expression of FasL (Strand et al., 1996), secretion of inhibitory cytokines (Beck et al., 2001; Shurin et al., 2002; Yang et al., 2003), and so on. Despite these inhibitory mechanisms, given its characteristics that differentiate it from self, MUC1 made by tumor cells can still trigger, in cancer patients, humoral and cellular responses, although of low efficiency. Kotera and colleagues (1994) reported anti-MUC1 IgM antibodies in more than 10% of sera from breast, colon, and pancreatic cancer patients. The presence of only the IgM isotype in these sera indicated a T helper-independent antiMUC1 immune response. Petrarca and colleagues (1999) were able to isolate

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in vivo-primed B cells from the draining lymph nodes of 6 of 12 patients with epithelial tumors. These B cells secreted anti-MUC1 IgM and IgG antibodies when cultured in vitro. There was a strong association between the ability to isolate B cells from these patients and the presence of anti-MUC1 IgM antibodies in their sera. A number of other reports demonstrated the presence of anti-MUC1 IgM and IgG antibodies in sera from patients with ulcerative colitis (Hinoda et al., 1993), ovarian cancer (Snijdewint et al., 1999), and colorectal cancer (Nakamura et al., 1998). A strong correlation between the presence of anti-MUC1 antibodies in sera from cancer patients and better prognosis and patient survival has been reported in patients with pancreatic (Hamanaka et al., 2003) and breast tumors (von Mensdorff-Pouilly et al., 1996). Antibodies developed in cancer patients could bind to tumor antigens on the tumor cell surface and mediate complement-dependent cytotoxicity and/or antibody-dependent cell-mediated cytotoxicity. Such mechanisms are able to eliminate circulating tumor cells and micrometastases, as shown in preclinical and clinical studies by Zhang et al. (1998). MUC1 is also recognized by T cells. Cytotoxic T lymphocytes (CTLs) that recognized MUC1 on the surface of epithelial tumors were found in the draining lymph nodes of pancreatic cancer patients (Barnd et al., 1989). It was then demonstrated that these CTLs recognize MUC1 on tumor cells in an MHC-unrestricted manner. These T cells have an a/b T cell receptor (TCR) and have CD3þCD8þCD4 phenotype. This MHC-unrestricted recognition of MUC1 could be blocked using an antibody against the immunodominant APDTRP epitope in the tandem repeat of the extracellular domain of tumor MUC1. CTLs that recognize MUC1 in an MHC-unrestricted manner were also established from draining lymph nodes of breast cancer patients (Jerome et al., 1991) and from peripheral blood mononuclear cells (PBMCs) from patients with multiple myeloma (Takahashi et al., 1994). Extensive studies of these T cells showed that they undergo intracellular signaling events similar to T cells that recognize the conventional MHC/peptide complex (MagarianBlander et al., 1998). In fact, these T cells showed a large calcium influx when stimulated with beads coated with a MUC1 synthetic 100-mer peptide carrying five repeats from the VNTR region and thus carrying five APDTR epitopes. This phenomenon of MHC-unrestricted recognition of MUC1 can be explained by the fact that MUC1 has multiple repeated epitopes that can cross-link the TCR on T cells. This hypothesis was further supported when the NMR structure of the unglycosylated synthetic MUC1 peptide was determined by Fontenot and colleagues (1995a). Their data revealed the presence of a knob-like structure protruding away from the backbone of each MUC1 tandem repeat with the sequence APDTR at the tip of this knob (described earlier in this chapter). MUC1-specific antibodies and T cells have increased accessibility to the immunogenic peptide backbone, which

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exhibits shorter carbohydrate side chains on tumor cells, and are otherwise masked by heavy glycosylation in normal epithelial tissues (Hinoda et al., 1998; Noto et al., 1997). CTLs that recognize MUC1 peptides presented by MHC class I molecules have also been detected in patients. Antigenic peptides bound to MHC class I molecules are between 8 and 10 residues in length and are enclosed in a binding groove formed between a1 and a2 helices and the b-sheet platform of the MHC class I heavy chain. MHC alleles have preferences for particular amino acids (called anchors) at certain positions in the peptide (Rock and Goldberg, 1999). These anchor residues, most often described for high-affinity binding peptides, are necessary for stabilization of the peptide–MHC complex. Many peptides lacking the canonical anchor residues can also bind to MHC class I molecules, albeit with lower affinity, and be recognized by CD8þ T cells. Most MHC class I-restricted MUC1 peptides are of that type. Eight to 10 amino acid peptides from the VNTR region were reported to bind to most H-2 and HLA alleles ( Apostolopoulos et al., 1997a, 1997b). For example, the 9-mer SAPDTRPAP and 8-mer SAPDTRPA bind to and are presented by H-2Kb, even though they do not contain the Kb consensus anchor motifs (Phe/Tyr) at position 5/6 and (Leu) at position 8/9. Their binding is such that the N-terminus of the peptide is buried in the groove, the middle portion protrudes outward, and the C-terminus is free and can react with antibodies specific for the peptides. The high-resolution crystal structure of H-2Kb complexed with the 8-mer peptide shows that, by contrast with high-affinity H-2Kb-binding peptides, this peptide is less buried in the MHC and two water molecules that are expected to occupy vacated or nonoptimally filled pockets are missing. This leaves a large cavity in one of the peptidebinding pockets that contributes to the low affinity of binding (Apostolopoulos et al., 2002). The general consensus to date is that T lymphocytes are the major mediators of antitumor immunity. Based on the fact that the majority of tumors are MHC class I positive but lack MHC class II molecules, numerous studies have focused primarily on CD8þ CTLs, which are considered the ultimate effectors at the tumor site. Although CTLs can directly kill tumor cells and their effectiveness in vivo has been demonstrated, it has now become evident that long-lasting antitumor immunity depends on successful activation of tumorspecific CD4þ T helper cells. CD4þ T cells can be divided into T helper 1 (Th1) and T helper 2 (Th2) cells based on their cytokine secretion profile (Morel and Oriss, 1998). Th1 cells help prime CD8þ T cell responses, while Th2 cells help establish humoral immune responses. In addition to their role in CTL priming, Th1 cells also secrete cytokines [such as interleukin (IL)-2] required for maintaining CD8þ T cell growth and proliferation (Greenberg, 1991; Rosenberg, 1999).

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Taking all these observations into consideration, an ideal MUC1 vaccine would be defined as one that induces strong helper and cytolytic MUC1specific cellular responses, stimulates antibody production, and also provides long-term immunological memory to the tumor. In an attempt to achieve these goals, groups working on the immunobiology of MUC1 have tested numerous antigenic formulations in several animal models. We will now discuss past and current vaccination strategies that have been tested against a variety of tumors in preclinical animal models or in clinical trials in patients. B. MUC1 Immunogenicity in Animal Models 1. Nonhuman Primates The majority of studies on MUC1 immunogenicity, performed in experimental animals, used wild-type mice or rats as recipients of various MUC1 vaccines. Since human MUC1 is xenogeneic to these animals, such studies do not provide a realistic evaluation of its immunogenicity and potential for immunotherapy in humans. We have conducted a number of studies in healthy chimpanzees, a more relevant animal model in which MUC1 is highly homologous to human MUC1 (Barratt-Boyes et al., 1998, 1999; Pecher and Finn, 1996). These studies show that immunization with soluble glycoprotein or synthetic peptides leads to antibody responses but no CTL. By contrast, Pecher and Finn (1996) showed that when irradiated autologous EpsteinBarr virus (EBV) immortalized B cells transfected with MUC1 cDNA, and expressing tumor-like MUC1, are used as a vaccine, they elicit CTLs. We have also tested a vaccine based on in vitro-derived chimpanzee dendritic cells pulsed with MUC1 peptide and have shown that these can elicit T cell responses after a single intravenous injection (Barratt-Boyes et al., 1998). The lack of a tumor model in chimpanzees did not allow evaluation of the efficacy of these responses in tumor rejection. Macaques express MUC1 that differs by five amino acids out of 20 in each repeat (75% identity) from the human MUC1 VNTR. Using oxidized mannan as a delivery system for 100-mer (five repeats) human MUC1 peptide, Vaughan et al. (1999) showed that both humoral and cellular anti-MUC1 immune responses could be induced in immunized macaques. The antibody response was predominant, in contrast to responses previously seen in MUC1 in wild-type mice where cellular responses were predominant. In a later report, the same group (Vaughan et al., 2000) isolated, sequenced, and expressed macaque MUC1. They then vaccinated both mice and macaques with a fusion protein of the monkey MUC1 VNTR conjugated to oxidized mannan. The response elicited by the macaque MUC1 mannan vaccine in two immunized monkeys showed a Th2 type of response, with low titers of anti-MUC1 antibodies of IgG1 and IgM isotypes and no proliferative or CTL responses in

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the spleen or draining lymph nodes of the immunized animals. The animals remained healthy and showed no signs of autoimmunity throughout the vaccination period. By contrast, wild-type mice immunized with mannan–macaque MUC1 (a xenoantigen) exhibited a strong cellular response and protection to tumor challenge, indicative of Th1 (low antibody, strong CTL) immunity. Moreover, mice immunized with self-murine muc1 conjugated to mannan (Vaughan et al., 2000) and cancer patients immunized with mannan-conjugated human MUC1 display Th2 responses. These studies reiterate our findings in chimpanzees and show that nonhuman primates such as macaques are more suitable preclinical models, reflecting more closely human responses than the murine model, where MUC1 is a xenoantigen. Nevertheless, as with the chimpanzees, this model is expensive and does not allow evaluation of in vivo responses to tumor challenge. 2. Murine Models There is only slight homology between the human MUC1 and mouse muc1 molecules. Because of this, any vaccines based on the human MUC1 administered to wild-type mice will trigger immune responses that will not reflect the intrinsic immunogenic properties of MUC1 but rather its foreignness. However, mouse models are the only systems in which tumor rejection studies can be performed. Mice transgenic for human HLA class I molecules represent an attractive model system to study immunogenicity of CTL epitopes and immunodominance in immune responses. While MUC1 immunization of these mice does not circumvent the problems associated with MUC1 seen as ‘‘foreign’’ by the murine immune system, it could lead to identification of MHC class I-restricted CTL epitopes with potential for vaccine design. As discussed earlier, immune recognition of MUC1 peptide sequences within the tandem repeats can occur in both an MHC-restricted (discussed earlier) and MHC-unrestricted manner. We and others (Apostolopoulos et al., 1997b; Domenech et al., 1995) have shown that the STAPPAHGV peptide sequence derived from the VNTR region of MUC1 constitutes a target for both HLA-A11- and HLA-A2-restricted CTLs. HLA-A11 and HLA-A2 are two of the more frequently expressed MHC class I alleles. Prediction algorithms using computer software show that the tandemly repeated 20-mer MUC1 sequence generates epitopes that do not fully comply with the classical binding motifs to human MHC class I molecules and are considered to bind in a nonconventional manner (Apostolopoulos et al., 1997b). In contrast, MUC1 regions outside VNTR display many epitopes with potential to bind to HLA-A molecules. The combined results from three research groups (Brossart et al., 1999; Carmon et al., 2000; Heukamp et al., 2001) show that there are six HLA-A2-restricted and naturally processed

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non-VNTR epitopes identified to date that can generate human CTLs able to recognize MUC1-expressing tumors. Three of these have been tested in vivo in HLA-A2/Kb transgenic mice (Heukamp et al., 2001). Mice were injected with peptide in incomplete Freund’s adjuvant (IFA) and subsequently challenged with B16 murine melanoma cells transfected with human MUC1 cDNA and the HLA-A2/Kb gene. The tumor-rejection experiments suggested that vaccination with peptides from the N-terminal region of MUC1 that bind HLA-A2 in vitro renders protection against tumor challenge. Similarly, vaccination with dendritic cells pulsed with the same set of peptides leads to protection against tumor growth. VNTR-derived peptide epitopes could potentially be generated in large numbers by the tumors, as well as by normal epithelial and antigen-presenting cells. As much as their abundance could be considered an advantage in generating robust antitumor responses, it could also be regarded as a strong stimulus for autoimmunity. Epitopes generated from sequences outside the MUC1 VNTR represent a less abundant source of antigen, at least in normal MUC1expressing cells. Vaccination with non-VNTR HLA-A2-restricted MUC1 epitopes might circumvent autoimmunity; however, such an approach could lead to emergence of antigen loss variants of the tumor. Transgenic (Tg) mouse models expressing human tumor antigens provide a suitable model for testing their immunogenicity, given the fact that they are endogenous self-proteins to which both B and T cell tolerance should develop. Several lines of human MUC1-transgenic mice have been generated on the C57BL/6 (Rowse et al., 1998), BALB/c (Carr-Brendel et al., 2000), and DBA (Acres et al., 2000) backgrounds. These mice show MUC1 expression with a distribution pattern similar to that in humans and better reflect immunopathology of human tumors in which MUC1 is a self-antigen subjected to the mechanisms of immunological tolerance. MUC1 transgenic mice challenged with MUC1-bearing syngeneic tumors fail to develop effective antitumor responses (Rowse et al., 1998), in contrast to wild-type mice that reject all MUC1-positive tumors. In addition, there is no antibody class switching in Tg mice immunized with MUC1 peptide, suggesting that these animals are tolerant to MUC1 in both the T and B cell compartments. With the advent of the MUC1 Tg mouse model, we and others have focused our attention on vaccination strategies that could break tolerance and elicit efficient antitumor immune responses. A very important aspect of such immunizations, besides their effectiveness to rejecting tumors, is the possibility of eliciting undesired autoimmune responses during the course of tumor rejection. Soares et al. (2001b) have shown that three vaccination protocols based on a peptide composed of seven tandem repeats of MUC1 elicit different immune effectors in the Tg versus wild-type mouse, with different potential for tumor

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rejection. The vaccines were either MUC1 peptide plus murine granulocytemacrophage colony-stimulating factor (GM-CSF) as an adjuvant, MUC1 peptide plus the adjuvant SB-AS2, or MUC1 peptide pulsed on DC. Cytokine and antibody production by T and B cells, respectively, ability of mice to reject tumors, and signs of autoimmunity were monitored in both Tg and wild-type mice vaccinated with either of the three vaccines. The only vaccination protocol that led to tumor rejection in both types of mice was the peptide-pulsed DC vaccine. In Tg mice the tumor rejection was solely attributed to interferon (IFN)-g-producing CD8þ T cells. Importantly, although a high degree of tolerance to MUC1 was expected, it was surprising that tolerance in the helper T cell compartment could not be overcome by the DC-based vaccine. We found this to be intriguing, especially since we have shown that helper responses to the same MUC1 peptide can be elicited in immunized healthy chimpanzees (Barratt-Boyes et al., 1999) and that MUC1 loaded onto human DC can prime MUC1-specific CD4þ T cell responses in vitro (Hiltbold et al., 1998). However, in a subsequent study, Soares (2001) was the first to demonstrate that CD4þ T cell tolerance in MUC1 Tg mice could be overcome upon vaccination with MUC1 peptide microencapsulated in poly-d,l-lactic-coglycolic acid (PLGA), administered in the absence of adjuvant. The MUC1 microsphere-based vaccine triggered Th1 helper responses, elicited MUC1specific CD8þ responses, and caused tumor rejection of MUC1-expressing tumors. Importantly, no autoimmune responses in MUC1 expressing tissues were detected in immunized mice. Administering MUC1 in biodegradable microparticles could result in enhanced immunity through several possible mechanisms. First, the antigen, incorporated within the copolymer matrix, can be delivered in high concentration directly to antigen-presenting cells (APC) (such as Langerhans cells in the skin or resident dendritic cells and macrophages in the periphery). These microparticles are taken up via phagocytosis by APC, and the internalized particles will release the antigen for processing in the MHC class II pathway. Some of the encapsulated antigen will also enter the cytoplasm, where it will be processed for binding to MHC class I molecules. Thus PLGA microparticles could also elicit both MHC class II- (helper) and I- (cytotoxic) restricted responses. Finally, the polymer coating of the antigen provides a depot-like system for sustained release of MUC1, thus boosting the efficiency of vaccination by increasing the persistence of antigen. Mukherjee et al. (2000) reported a double transgenic mouse model (called MET) that is a result of a cross between MUC1 Tg mice and ET mice bearing an oncogene that causes spontaneous tumors in the pancreas. MET mice express human MUC1 as a self-molecule in the pancreas, hence their spontaneous pancreatic adenocarcinomas also express MUC1. The animals develop pancreatic pathology that resembles the progression of human pancreatic

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cancer. Thus MET mice are valuable tools for preclinical testing of MUC1 vaccines for pancreatic cancer. For patients with adenocarcinomas of the pancreas, the treatment options currently available (pancreatectomy, radiation, and chemotherapy) are aggressive, toxic, and most of the times inefficient, as they do not decrease the mortality rate. In fact, pancreatic adenocarcinoma is the fourth leading cause of cancer death in the United States, with a 5-year survival rate of only 3%. Moreover, even for the 15–20% of patients who are diagnosed early and undergo potentially curative resection, followed by radiation and chemotherapy, the 5-year survival is only 20%, due to persistence of residual tumor cells that can then proliferate at metastatic sites. We and others consider that eliciting effective immune responses that could identify and eliminate the transformed cells left over after surgical resection of the primary tumor could provide additional help to enhance survival. As with most cancer types, various genetic modifications have been linked to increased incidence of pancreatic adenocarcinomas. Predisposed patients undergo local inflammatory changes that result in local production of growth factors, cytokines, and reactive oxygen species. These changes induce cell proliferation that, associated with inherited genomic instability, leads in time to tumorigenesis. Identifying patients at risk and treating them early with a preventive vaccine could be more beneficial for long-term survival. Most of the mouse models, prior to development of the MET mouse, used direct injections into the pancreas of either tumor cell lines or xenografts of primary human pancreatic tumors. Morikane et al. (2001) have shown that it is more difficult to induce immune responses to tumors transplanted to the pancreatic site than to pancreatic tumor cells injected at the subcutaneous site. While CD8þ T cells are required for rejection of Panc02-MUC1 pancreatic tumors at a subcutaneous site, both CD4þ and CD8þ T cells are required for rejecting tumors at a pancreatic site, confirming the fact that local environment (cytokines, hormones, expression of MHC complexes and of adhesion molecules) can dramatically influence the outcome of antitumor immune responses. Pancreatic tumors that spontaneously develop in MET mice overexpress underglycosylated MUC1. As the tumors progress, the MET mice exhibit, similarly to cancer patients, increased levels of circulating MUC1. Mukherjee et al. (2000) have shown that nonimmunized MET mice developed MUC1specific CTLs that could lyse as much as 80% of MUC1-transfected B16 murine melanoma target cells (compared with only 20% of the control MUC1-negative B16 cells). The killing was MHC class I restricted, and one of the peptide epitopes recognized by some of the CTLs matched the epitope identified in humans, the STAPPAHGV from the MUC1 VNTR. Despite the fact that MET mice develop identifiable CTL responses, those CTLs are inefficient in

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preventing spontaneous growth of tumors in vivo, and 90% of mice die of pancreatic cancer by 16 weeks of age. However, when adoptively transferred in tumor-challenged MUC1 Tg mice, the MUC1-specific CTLs are able to prevent growth of MUC1-expressing B16 tumor cells and not of MUC1 negative control tumors. Evasion of CTLs by the pancreatic tumors in MET mice can be explained by many factors, generally applicable to immune effector cells in a tumor environment: down-regulation of surface MHC I, antigenic modulation, up-regulation of FasL, secretion of inhibitory cytokines, low avidity CTLs, and so on. This proves once again that the presence of CTL responses cannot be used as the sole indicator of clinical outcome and that broader immune responses from helper T cells, as well as B cells, should also be examined. Furthermore, MET mice with fully developed aggressive pancreatic tumors might not provide the right setting for therapeutic vaccination. However, we consider (for reasons discussed later) that early MUC1 vaccination of MET mice might provide a more appropriate approach for modeling cancer prevention, rather than therapy. The pancreas of MET mice contains displastic acinar cells producing underglycosylated MUC1 by 3 weeks of age. In situ carcinomas can be diagnosed by week 13, and by 15 weeks the mice grow well-differentiated pancreatic tumors. This progression resembles (on a different time scale) what has been described in humans, and by attempting to vaccinate MET mice early with an efficient and safe vaccine capable of eliciting strong B and T cell responses, a robust level of protection to cancer development might be achieved. However, despite the relatively slow rate of tumor progression seen in MET mice, it could still be too quick to fully allow tumor-specific immune responses to develop. Thus this model may not accurately reflect the kinetics of tumor development seen in cancer patients, who might live with premalignant lesions for long periods of time before developing pancreatic carcinomas, and who might have even a better potential to destroy incipient tumors if properly vaccinated. Consequently, vaccination approaches inducing immune responses capable of slowing down tumor progression in animal models of spontaneous tumor development should be regarded as encouraging and should prompt further exploration of such vaccines for cancer prevention in humans.

V. MUC1 Vaccines

A. Peptide Vaccines Vaccines based on synthetic peptides have the advantage of being readily available, although they require the identification of exact epitopes recognized by T or B cells. Most peptide vaccines have been tested for their ability to elicit strong CTL responses; however, optimal vaccine formulations should also

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include one or more antigen-specific T helper epitopes. Helper responses to MUC1 have not been detected to date in cancer patients. Therefore identification of MUC1-derived helper epitopes and testing such epitopes in vivo are of crucial importance and need to be further addressed. We conducted one of the first peptide cancer vaccine clinical trials. The vaccine consisted of a 105-mer synthetic MUC1 peptide admixed with bacillus Calmette-Gue´ rin (BCG) and was administered to 63 patients with adenocarcinomas of the breast, pancreas, or colon (Goydos et al., 1996). BCG is an attenuated form of Mycobacterium bovis and has well-known adjuvant properties. The vaccine was associated with some toxicity that involved skin breakdown at the vaccination site and various degrees of other symptoms such as fever, chills, nausea, and vomiting. Skin biopsies at the injection sites showed delayed type hypersensitivity (DTH) reactions to MUC1 peptides and intense T cell infiltration. Seven of 22 patients tested showed a 2- to 4-fold increase in MUC1-specific CTL precursor frequency. BCG can also be genetically engineered to deliver various antigens in humans. Very recently, Chung et al. (2003) engineered BCG to express a 22-tandem-repeat-long MUC1 cDNA and to simultaneously secrete human IL-2. The BCG hIL-2MUC1 vaccine was able to inhibit tumor growth in SCID mice reconstituted with human peripheral blood lymphocytes (PBLs) and xenografted with the MUC1-positive ZR75-1 human breast cancer line. Musselli et al. (2002) tested six breast cancer patients vaccinated four times each with a 106-mer MUC1 peptide covalently linked to keyhole limpet hemocyanin (KLH) and administered in the presence of QS21 adjuvant. The patients developed strong anti-MUC1 IgM and IgG responses. The IgG antibodies were of the IgG1 and IgG3 isotypes. PBMCs were tested to detect MUC1-specific T cell responses using proliferation assays and ELISPOT assays for IFN-g production. Only sporadic increases in T cell precursors specific for MUC1 were detected. Reddish et al. (1998) used 16-mer MUC1 peptide conjugated to KLH and administered in the presence of DETOX as an adjuvant to 16 metastatic breast cancer patients. Three patients developed anti-MUC1 IgG responses, and 7 of the 11 patients tested developed MHC class I (HLA-A2, A1, and A11)-restricted CTLs. Acres et al. (2000) tested the ability of peptide vaccines to elicit MUC1specific immune responses in MUC1 Tg mice. One of the vaccines was a 100-amino acid-long MUC1 peptide, corresponding to five tandem repeats, expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST). This fusion protein stimulates weak CTL activity (Apostolopoulos et al., 1995b). Coupling of mannan to the GST part of the fusion protein [MFP, also used in clinical trials (Karanikas et al., 1997)] significantly increased the CTL precursor frequency in immunized MUC1 Tg mice. There were no signs of autoimmunity in vaccinated mice

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B. DNA Vaccines DNA vaccination with a MUC1-encoding plasmid offers yet another method to target antigen to DC and muscle cells for antigen presentation. This method has been reported to induce both cellular and humoral immunity; moreover, due to prolonged expression of the antigen, it also elicits immunological memory. Graham et al. (1996) and later Johnen et al. (2001) administered intramuscular immunizations to C57/BL/6 wild-type mice using 50–100 mg MUC1 cDNA. Mice were then monitored for their ability to reject human MUC1expressing syngeneic tumors. As expected, approximately 80% of the mice were protected against tumor challenge and both humoral and cellularmediated immune responses were detected. Although the relevance of these findings is limited to the fact that wild-type mice develop strong antihuman MUC1 immune responses, which they see as ‘‘foreign,’’ they confirmed that injected DNA was expressed and that the animals developed antibody responses to the protein after immunization with DNA alone. One important aspect of MUC1 DNA vaccination that needs to be considered is the fact that MUC1 produced by in vivo-transfected muscle cells or APC would be the normal rather than the tumor form. Moreover, MUC1 molecules released from the surface of such expressing cells are heavily glycosylated and not likely to be processed by adjacent DC, due to defective intracellular trafficking (Hiltbold et al., 2000). Furthermore, persistent expression of a self-molecule could also lead to a state of autoimmunity or a state of immunological unresponsiveness. C. Dendritic Cell-Based Vaccines Loading DC with tumor antigens for presentation to CD4þ and CD8þ T lymphocytes has been employed by many scientists in their approach to induce strong antitumor responses (for review see Zhou et al., 2002). DC have the ability to process the antigen intracellularly and to present peptide epitopes to both CD4þ (direct priming) and CD8þ T cells (cross-priming/crosspresentation). DC also have the exclusive ability to prime naı¨ve CD4þ and CD8þ T cells. They express high levels of MHC class I and II complexes, adhesion (CD11a, CD11c, ICAM-1, -2, and -3, etc.) and costimulatory molecules CD80 and CD86, as well as molecules regulating costimulation such as CD40. Expression of many of these molecules varies with different stages of DC maturation. For example, adhesion molecules, CD80, CD86, and MHC complexes are up-regulated upon maturation, especially following CD40 ligation. DC function is carried out in three interrelated stages: antigen uptake (through various mechanisms), intracellular processing (in either MHC class I or II compartments), and epitope presentation / T cell priming. In peripheral

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tissues, immature DC are highly endocytic. Following activation and migration from the tissues to lymph nodes, they become highly efficient at antigen processing and presentation. Numerous animal models, to date have demonstrated the ability of tumor antigen-loaded DC to prime both CD4þ and CD8þ T cells and to confer protection from tumor challenge. DC-based vaccines have been tested lately in cancer patients, and encouraging phase I/II results are emerging (Fong and Engleman, 2000). Several approaches have been used to arm DC with tumor antigens for use in clinical trials (Zhou et al., 2002). DC pulsed with peptides derived from antigens such as MART-1, MAGE-1, CEA, and PSA have all been used to elicit in vitro CTL responses. Domenech et al. (1995) and others (Apostolopoulos et al., 1997b; Brossart et al., 1999) have shown that DC pulsed with MUC1 peptides able to bind HLA can elicit MUC1-specific CTL responses restricted by that haplotype. This approach has the advantage of focusing on short peptides (8–11 amino acids in length) that can be easily synthesized for large-scale immunizations. Most importantly, if the peptide epitopes are tumor specific, the CD8þ CTLs target cancer cells exclusively, thus limiting the possibility of inducing autoimmunity. However, MUC1 has an identical peptide sequence in normal and tumor cells. HLA molecules at the basolateral surface of normal MUC1-expressing epithelial cells can present MUC1 peptides. Following immunization, these peptide–MHC complexes could be recognized by ‘‘armed’’ CD8þ effectors, possibly leading to destruction of normal MUC1-positive tissues. In addition, several other major disadvantages of vaccines based on MHC class I peptide-loaded DC may limit their clinical applications. First, elicitation of significant antitumor responses requires T cell help, strong humoral responses, as well as tumor-specific immunological memory. Thus peptide vaccines must include longer peptides that can bind to human MHC class II molecules and elicit helper CD4+-restricted T cell responses. Due to the paucity of defined MHC class II-restricted peptides, heterologous proteins have often replaced tumor-specific helper epitopes. While these proteins can sometimes be effective in eliciting antibodies and CTLs, they do not provide the molecular basis for long-term tumor-specific memory responses. Second, peptides used for DC loading are restricted to specific HLA haplotypes and are applicable only to MHC-matched patients. Given the tremendous variability in HLA haplotypes among cancer patients, it is not feasible to attempt identification of all peptide candidates for vaccine formulations. Third, targeting effector mechanisms to a narrow spectrum of antigenic epitopes could lead to the emergence of antigen loss variants of the tumor (Ikeda et al., 1997; Kerkmann-Tucek et al., 1998; Slingluff et al., 2000). Longer protein tumor antigens are alternatives to short peptides and are considered to have certain advantages. Their use in vaccine formulations does not require identification of specific epitope sequences. Processing of whole

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proteins by DC generates multiple peptide epitopes presented by the host’s MHC that are not restricted to single alleles. Soluble proteins pulsed onto immature DC are endocytosed and processed in the MHC class II pathway leading to presentation of antigenic epitopes to CD4þ T cells, thus ensuring elicitation of tumor-specific helper responses. Finally, endocytosed proteins can gain access to the MHC class I pathway, where they undergo proteasomal processing for presentation on MHC class I complexes (cross-presentation) (Brossart et al., 1997; Mitchell et al., 1998; Paglia et al., 1996; Porgador et al., 1996). MUC1 is a tumor antigen that differs quantitatively (due to overexpression) and qualitatively (due to changes in glycosylation) on tumor versus normal cells. If whole MUC1 antigen was to be used for loading onto DC, large amounts of tumor-like MUC1 glycoprotein would be needed for vaccine preparation. Tumor mucin can be obtained from either tumor cells grown in vitro or ascites fluid obtained from cancer patients. Purification procedures from tumor cells are labor intensive and generate low amounts of tumor MUC1 glycoprotein. Expression of recombinant MUC1 in E. coli results in its rapid degradation by bacterial proteolytic enzymes (Dolby et al., 1999). MUC1 made by insect cells infected with a recombinant baculovirus vector (Soares et al., 2001a) is a good source of tumor-like MUC1, especially since insect cells glycosylate MUC1 only at very low levels. This baculovirusencoded MUC1 (BV-MUC1) is immunogenic in BALB/c mice that generate antibodies that cross-react with human tumors. Due to its very low glycosylation, BV MUC1 has similar immunogenicity to the synthetic 100-mer MUC1 peptide and might not provide additional benefit to this form, currently employed in clinical trials. The most ‘‘authentic’’ tumor-like MUC1 glycoprotein is MUC1 purified from ascites fluid of cancer patients (ASC-MUC1). Tumor cells release soluble MUC1, which can drain to regional lymph nodes, enter the peripheral circulation, and be found circulating in patients’ sera. Beatty et al. (2001) reported the biochemical structure of ASC-MUC1 that was isolated from sera and ascites fluid of patients with late stage breast and pancreatic cancers. This circulating form of MUC1 is of major interest, since this form, possibly the only circulating form of MUC1 available to patients’ APC (especially DC) in vivo, would be expected to be taken up, processed, and presented by APC to helper T cells. Hiltbold et al. (2000) have shown that ASC-MUC1 is effectively endocytosed by DC through mannose receptors. However, following uptake, the protein is retained long term in early endosomes and is not transported to late endosomes or MHC class II compartments for proteolytic processing. This block in intracellular trafficking of ASC-MUC1 and its inefficient processing in the MHC class II pathway represents a specific mechanism by which cancer patients’ T cells are kept unresponsive to this tumor antigen. Interestingly,

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long-term retention in early endosomes of the internalized Ag by DC (which does not interfere with their ability to process and present other antigens) was also observed for another glycoprotein tumor antigen, Her-2/neu, which also fails to elicit Th responses in vivo. Understanding how whole protein antigens are handled by DC and how their structure influences processing and presentation to immune effectors is critical for any effort directed toward manipulating the immune response against those antigens and an important consideration in the design of cancer vaccines. The obstacle in intracellular processing of ASC-MUC1 by DC can be overcome by MUC1 peptide vaccines that present DC with the form of MUC1 that can be efficiently processed. This vaccine is currently in clinical trials. In addition to developing MUC1 peptide vaccines, we have more recently focused on the immunogenic properties of MUC1 glycopeptides. MUC1 made by normal cells is highly glycosylated with branched O-linked oligosaccharides (Gendler and Spicer, 1995). By contrast, in tumor cells, MUC1 O-glycosylation is prematurely terminated, leading to the accumulation of short carbohydrate precursors such as the monosaccharide Tn (GalNAca1-O-S/T) or disaccharide T (Galb1-3GalNAca1-O-S/T), and their sialylated forms sTn and sT, respectively (reviewed in Baldus and Hanisch, 2000). These tumor-specific carbohydrates are O-linked to serines and threonines in the tandem repeat domain of the MUC1 molecule. Using synthetic MUC1 glycopeptides bearing tumor-like Tn and T saccharide epitopes, we addressed the question of whether such saccharide residues are removed during antigen processing by DC when exogenously fed MUC1 glycoproteins and whether DC are able to generate glycopeptide epitopes for presentation on MHC class II. Our results (Vlad et al., 2002) show that DC endocytose glycopeptides, transport them to acidic compartments, process them into smaller peptides, and present them on MHC class II molecules without removing the carbohydrates. Glycopeptides that are presented on DC are recognized by T cells, suggesting that a much broader repertoire of T cells could be elicited against MUC1 than expected, based solely on peptide sequences. Further in vivo studies, aimed at measuring the efficiency of MUC1 glycopeptide-specific T cell responses against tumors, need to be performed. An alternative method of providing DC with whole tumor antigens is by creating DC–tumor cell hybrids, such as fusions of DC with MUC1-expressing carcinoma cells. The fused cells express MHC class I and II and costimulatory molecules, normally present on DC. In addition, unlike DC, the hybrid cells also display MUC1 (and possibly other tumor-specific antigens) on their cell surface. Upon injection, these hybrids migrate to draining lymph nodes, where they distribute to T cell areas in a manner similar to normal DC (Koido et al., 2002). As shown by Gong et al. (1998), the hybrid cells are able to break T cell

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tolerance to MUC1 in MUC1 Tg mice and to trigger CTLs that are able to reject MUC1-expressing pulmonary metastases. Immunization with DC–tumor cell hybrid vaccines has the potential of eliciting unwanted autoimmunity. No autoimmunity was detected in vaccinated MUC1 Tg mice. However, the preclinical mouse MUC1 Tg model displays an unexpectedly high tolerance to human MUC1 and may not parallel the clinical situation. Breaking tolerance in humans might require a less intense immune response; therefore, lack of autoimmunity in mice after immunization with DC–tumor hybrids should be carefully considered before translating it into clinical practice. Induction of antitumor immunity with DC transfected with tumor antigenencoding RNA represents another approach for DC-based vaccines. This approach, pioneered in Eli Gilboa’s laboratory (Boczkowski et al., 1996), is based on the findings that vaccination of mice with DC pulsed with RNA from OVA-expressing tumor cells confers protection against challenge with OVAexpressing tumors. Moreover, monocyte-derived human DC transfected with RNA encoding the CEA tumor antigen (Gilboa et al., 1998; Nair et al., 1998) stimulate potent CEA-specific CTL responses in vitro. Similarly, Koido et al. (2000) showed that vaccination of wild-type mice with MUC1 RNAtransfected DC elicits MUC1-specific CTL responses, resulting in rejection of MUC1-transfected MC38 tumor cells but not of untransfected cells. The same immunization protocol, effective in wild-type mice, failed to protect MUC1 Tg mice from tumor challenge. However, coadministration of IL-12 with RNA-transfected DC resulted in increased MUC1-specific CTL activity and rejection of MUC1/MC38 tumors. MUC1 expression by DC can by achieved by gene transduction using MUC1encoding recombinant adenovirus. DC endogenously expressing MUC1 present MHC class I-restricted MUC1 peptides that could trigger naı¨ve CD8þ T lymphocytes. Maruyama et al. (2001) have shown that despite the efficiency seen in murine systems, adenoviral gene transduction of human DC has limited efficiency, with only 39% of transduced DC showing MUC1 protein expression. Nevertheless, the transduced DC were able to induce MUC1-specific CTLs in vitro, although not better than peptide-pulsed DC. This approach has the same problem as DNA because normal MUC1 is being made. Despite the fact that DC vaccines have shown encouraging results, the strategy of using in vitro-generated DC from each cancer patient for reinjection after antigen loading has major technical limitations. All experimental steps performed ex vivo (culture of large numbers of precursors in the presence of cytokines, purification of immature DC, loading of DC with desired antigen) are costly, time consuming, and pose the risk of contamination. As an alternative approach, in vivo loading of DC using antigenic formulations with increased uptake efficiency [such as liposome-delivered tumor peptides

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(Ludewig et al., 2000) or injections of Fas-expressing apoptosing autologous tumor cells (Chattergoon et al., 2000)] eliminates the need for ex vivo manipulation and could be more applicable on a larger scale. D. Recombinant Vaccinia Virus Vaccines Immunization of mice (Acres et al., 1993) or rats (Hareuveni et al., 1990) with a recombinant vaccinia virus that expresses MUC1 upon infection of mammalian cells protects animals against challenge with MUC1-expressing but not control, MUC1-negative syngeneic tumor cells. The vaccinia genome is subjected to high-frequency homologous recombination and therefore has unstable expression of the tandem repeats. Akagi et al. (1997) generated a recombinant vaccinia virus containing a modified ‘‘mini’’ MUC1 gene containing only 10 tandem repeat sequences to minimize vaccinia-mediated rearrangement. The construct was used in combination with recombinant vaccinia virus containing the gene for the murine T cell costimulatory molecule B7-1. Vaccine efficacy was tested in a MUC1-expressing pulmonary metastases prevention model and showed that mice inoculated two times with vacciniaencoded MUC1 were protected from the establishment of metastases. Recombinant constructs that express both MUC1 and IL-2 (Balloul et al., 1994) were also successful in generating detectable CTL responses and blocking tumor growth upon challenge. This later construct has been used (Scholl et al., 2000), together with two other peptide-based vaccines (Acres et al., 2000), to immunize MUC1 Tg mice. VI. MUC1 Vaccines in Clinical Trials

Vaccines based on MUC1 are currently tested in cancer patients with advanced tumors for therapeutic purposes. In recent years, advances in basic immunology and biotechnology have contributed to the design of vaccines with better immunogenic properties. Development of MUC1 antigens with increased immunogenicity, discovery of more potent adjuvants, and design of efficient antigen delivery systems are all important contributions for clinical applications of MUC1 vaccines. Some of these contributions and their impact on patients in clinical trials will be mentioned later. Various MUC1 phase 1 clinical trials have recently been performed in two joint medical centers in Victoria and Queensland, Australia. The first vaccine formulation employed by McKenzie and co-workers was a MUC1 peptide coupled to diphtheria toxin. MUC1 antigen comprising five MUC1 tandem repeats was linked to a GST fusion protein and administered with oxidized mannan. In its oxidized form, mannan targets antigen to the mannose receptors on the surface of APC and favors antigen processing in the MHC class I pathway and cross-presentation to CTLs, as shown in preclinical studies

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on mice (Apostolopoulos et al., 1995a, 1995b). By contrast, MUC1 conjugated to reduced mannan favors antigen presentation in the MHC class II pathway and elicits primarily humoral responses in vaccinated mice. Efficiency of this oxidized mannan–MUC1 GST fusion protein vaccine was tested on 25 patients with metastatic carcinomas of the breast, colon, stomach, or rectum (Karanikas et al., 1997). The patients received increasing doses of antigen, administered subcutaneously. Contrary to the expectation that such a vaccine formulation would favor cellular rather than antibody responses, as seen in vaccinated wild-type mice, patients immunized to oxidized mannan– MUC1 fusion protein generated strong antibody responses and only moderate cellular proliferative and cytotoxic responses. These results parallel the findings (discussed earlier) in monkeys vaccinated with either human or macaque MUC1 conjugated to mannan. More recently, Karanikas et al. (2001) tested the efficacy of mannan–MUC1 vaccine when administered intraperitoneally in the presence of cyclophosphamide. The intraperitoneal route was chosen based on findings in mice showing better CTL responses to this route of injection. Similarly, the addition of cyclophosphamide increased the frequency of CTL precursors (Apostolopoulos et al., 1998). Of 41 patients with adenocarcinomas of the breast, colon, stomach, rectum, prostate, and ovaries, 60% developed high titer antibody responses of the IgG1 isotype and only 28% developed cellular responses, as detected by proliferation and cytotoxicity assays or intracellular detection of TNF-a and IFN-g in response to MUC1 stimulation. Cytotoxic T lymphocytes detected in 20% of the patients were of low frequency and potency. While the route of administration made a difference, with antibody titers 10-fold higher following intraperitoneal (versus intramuscular) vaccination, the presence of cyclophosphamide failed to skew the immune response from humoral toward cellular immunity. In summary, MUC1 linked to the GST fusion protein and delivered with oxidized mannan could be considered potent for antibody production but of limited efficiency for the induction of cellular antitumor responses. The vaccine is generally well tolerated, with the only toxic effect being erythema at the injection site. Scientists from Sloan Kettering Cancer Center in New York have tested MUC1 peptide vaccines in various clinical trials. According to preclinical studies, covalent attachment of MUC1 (or of other cancer antigens like gangliosides GD3 and GM2) to KLH plus the use of a potent immunological adjuvant showed efficient induction of antibody responses (Livingston, 1995; Zhang et al., 1996). Out of 19 different adjuvants tested by Kim et al. (1999) in mice, QS-21, a natural saponin, exhibited the best adjuvant properties. Furthermore (Kim et al., 2000), combinations of QS-21 with several other adjuvants (of different chemical structures) resulted in significant increases in

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anti-MUC1 antibody titers compared with QS-21 alone. A newly identified adjuvant, GPI-0100, a semisynthetic saponin adjuvant (Marciani et al., 2000), was at least as potent as any of the adjuvant combinations and significantly more potent than QS-21 alone. Subsequent to their studies in mice, Livingston et al. (1994) tested the MUC1-KLH plus QS-21 vaccine in patients. In 2000, Gilewski et al. reported the first results of subcutaneous immunization of nine patients with a history of breast cancer but without evidence of disease, using 30-mer MUC1–KLH conjugate plus QS-21 as an adjuvant. The combination of antigen-KLH and QS-21 was effective in inducing antibody responses against other antigens, as seen with gangliosides KLH conjugates that trigger high antibody responses in vaccinated melanoma patients (Livingston et al., 1994; Livingston and Ragupathi, 1997). The 30-mer (1.5 repeat) MUC1 peptide vaccine plus QS-21 was well tolerated and resulted, as expected, in significant production of both IgM and IgG antibodies against synthetic MUC1. No T lymphocyte responses were detected. The anti-MUC1 IgM antibodies isolated from seven of nine patients were able to stain MUC1 on surface of MCF-7 tumor cells, but only minimal staining was observed with the IgG. The epitope recognized by these antibodies lies within the APDTRPA sequence. Within the 30-mer MUC1 peptide used as an immunogen, the APDTRPA eptitope was located in both a medial and C-terminal position, with the antibodies being able to ‘‘see’’ the epitope only when located at the C-terminus. It was hypothesized that modest cell surface antibody reactivity (despite the high titers of anti-MUC1 IgGs) could be attributed to dependency on epitope conformation and that the original epitope in the synthetic immunogen may differ from the ones encountered on natural, surface-bound MUC1. In a later report, Musselli et al. (2002) showed that 106-mer MUC1 conjugated to KLH and administered with QS-21 triggered IgM and IgG. The IgG antibodies were of IgG1 and IgG3 isotypes. In 1999, Brossart et al. (1999) reported the identification of two immunogenic peptides derived from MUC1 and restricted to HLA-A2. While one of the epitopes (M1.1 peptide) was derived from the tandem repeat region of the protein, the second epitope (LLLLTVLTV identified as M1.2) is derived from the signal sequence of the protein, demonstrating that immunogenicity of MUC1 is not limited to responses against epitopes within the tandem repeats. Also of interest is the fact that the M1.1 peptide is a 9-mer whose sequence (STAPPVHNV) differs from the classical STAPPAHGV by two amino acids: V in position 6 and N in position 8. These substitutions occur naturally on tumor MUC1 (Brossart et al., 1999), and the epitopes can be presented by HLA-A2 on tumors and antigen-presenting cells. Moreover, the presence of valine at position 6 increases the binding of the M1.1 peptide to the HLA-A2 molecule.

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CTLs induced by in vitro priming using autologous DC pulsed with the M1.1 and M1.2 peptides can efficiently lyse target cells pulsed with the peptides or HLA-A2-positive tumor cells (Brossart et al., 1999). Based on this finding, Brossart et al. (2000) administered the peptide-pulsed DC vaccine to 10 patients with advanced breast and ovarian cancers, enrolled in a phase I/II study at the University of Tubingen Medical Center. Out of 10 patients, six received DC pulsed with Her-2-neu-derived peptides and four received MUC1 peptide-pulsed DC, according to the expression of the tumor marker on the tumor. Prepulsed DC were subcutaneously injected close to the inguinal lymph nodes, every 14 days for the first 6 weeks and repeated afterward every 28 days. The authors showed peptide-specific CTL activity detected in the peripheral blood of five of the vaccinated patients, after three rounds of vaccinations. Interestingly, one patient treated with MUC1-pulsed DC also developed MAGE-3- and CEA-peptide-specific CD8þ T cells, while another patient developed MUC1-specific CTLs after vaccination with Her-2-neu peptide-pulsed DC, suggestive of epitope spreading. Induction of CTLs in vivo can lead to destruction of tumor cells, which are then taken up by APC-like DC. Their tumor-derived antigens can then be processed inside the DC and cross-presented to MHC class I-restricted and tumor antigen-specific CD8þ T cells. Overall, these findings show that cancer patients diagnosed with advanced tumors and pretreated with high doses of chemotherapy could still mount efficient antigen-specific cellular responses following vaccinations with peptide-pulsed DC and that such a vaccine is well tolerated. Brossart et al. (2001) have recently shown that the list of MUC1-expressing malignancies, comprising mostly epithelial solid tumors and (as previously shown) multiple myelomas, could be extended to other hematopoietic malignancies such as acute myeloid leukemia (AML, showing positivity for MUC1 in 67% of cases), follicular lymphoma, chronic lymphocytic leukemia (CLLs), and hairy cell leukemia, all showing varying degrees of MUC1 expression. Using DC derived from PBMCs from normal HLA-A2-positive donors, pulsed with M1.1 and M1.2 MUC1 peptides, it was shown that in vitro-primed CTLs were able to recognize, in an HLA-A2-restricted manner, primary leukemic blasts, as well as multiple myeloma cells and several other AML cell lines endogenously expressing MUC1 protein. Expanding vaccination with MUC1 to patients with hematological malignancies needs to be further explored. Immunizations of mice using a recombinant vaccinia virus (which encodes for human MUC1 as well as for IL-2) can stimulate CTL responses at levels protective against tumor challenge (Acres et al., 1993). The efficacy of the vaccine was later tested on nine patients with advanced breast cancer in a Phase 1 and 2 clinical trial conducted in France. The patients, diagnosed with MUC1-positive advanced breast tumors with chest wall recurrences, were injected intramuscularly in the deltoid muscle with recombinant viral

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suspension. The vaccination resulted in no significant systemic adverse effects. However, only modest MUC1-specific immune responses in vaccinated patients were reported. None of the vaccine recipients was able to mount MUC1-specific antibody responses, as detected by ELISA testing of patients’ sera using a MUC1 peptide from the tandem repeat sequence. Based on our experience using MUC1-encoding vaccinia vectors (Bu et al., 1993), it is probable that the lack of antibodies to the MUC1 tandem repeats is due to the fact that vaccinia is recombining out the tandem repeats of the molecule, thus preventing humoral responses against this region. Two of the nine recipients showed MUC1-specific CTL activity against EBV-transformed B lymphoblastoid cell lines established from each tested patient. The lytic activity was specific against the EBV B cells previously infected with MUC1-encoding vaccinia virus and not against the cells infected with control vaccinia vector. Vaccinia viral vectors display several advantages that make them attractive vaccine candidates: ability to incorporate large amounts of foreign DNA, wide host cell range, stability, enhanced immunogenicity due to live attenuated vaccinia virus, and so on. However, there are also disadvantages and they are mostly related to the potential toxicity triggered in patients by the extremely immunogenic vaccinia viral proteins. In addition, we consider that although the stability of the encoded protein may not be a problem for many other antigens, it should be carefully addressed when using MUC1-encoding vaccinia vectors. The MUC1 extracellular domain comprises a large number of tandem repeats, with each repeat displaying immunodominant epitopes. Thus the inability of the virus to preserve transcription of the repeats may result in decreased immunogenicity of the vaccine. Lastly, vaccinia-infected cells produce normal forms of MUC1 that might, as discussed previously, elicit immunity to normal rather than tumor tissues expressing MUC1. The quest for the best method of genetically engineering DC to express MUC1 is continuing, as scientists try to identify the most efficient transfection method that is also easy to handle and applicable for use in clinical trials. Recently, Pecher et al. (2002) tested a DC-based vaccine in a phase I/II clinical trial using autologous DC transfected with MUC1 cDNA using liposomes. A group of 10 patients with advanced breast, pancreatic, or papillary cancer received two or three subcutaneous immunizations with transfected DC. The efficiency of transfection varied widely with as little as 2% and as high as 53% of DC showing MUC1 expression. While the study demonstrated the feasibility and safety of this vaccination approach, weak immune responses were seen in only four patients, of which only one showed stable disease for 3 months after starting vaccination. The other nine patients showed clinical progression of the disease. Although liposomal transfection is technically easy to execute, it shows high variability in transfection efficiency and might not be suitable for large-scale clinical trials.

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One important aspect when considering vaccines based on DC that are genetically manipulated to express MUC1 (following either retroviral, adenoviral, or simply naked DNA vaccination) is the nature of the MUC1 protein that will be produced. As we have shown (Henderson et al., 1996), MUC1 on DC is fully glycosylated and will not exhibit any tumor-like (such as Tn or T) epitopes. DC could still prime CTLs to naked peptides generated from the normal processing in the MHC class I pathway. However, transfected cells generate fully glycosylated, normal MUC1 and possibly become, upon uptake by adjacent APC, a source of large and heavily glycosylated MUC1 glycoprotein that is difficult to process. Thus genetic manipulation for MUC1 production should ensure production of a MUC1 form that is tumor-like and therefore more suitable for vaccination. Hybrid cells created by DC–tumor cell fusions provide a tumor-like source of MUC1 and efficient costimulatory properties. Using electrofusion techniques, Kugler et al. (2000) generated hybrids of allogeneic DC and autologous tumors from 17 patients with metastatic renal cell carcinoma. Following electrofusion (from a total of 50 million DC and 50 million tumor cells) and irradiation, the cells were immediately injected subcutaneously in close proximity to inguinal lymph nodes. All patients had their tumors removed prior to vaccination. Immune responses against MUC1 (expressed on 82% of the primary tumors included) and Her-2-neu were used to monitor induction of antitumor immunity. Out of the 17 patients tested, four had complete tumor remissions, two underwent partial remissions, and one had a ‘‘mixed’’ response. Additionally, two patients with multiple lung metastatic lesions were stabilized for 17 and 15 months, respectively. Three of the four patients with complete remissions successfully rejected metastases of the lung, bones, lymph nodes, and soft tissues. Eight of 17 patients showed progressive disease. The vaccine was well tolerated, and there were no clinical signs of autoimmunity. Despite its partial success, hybrid cell-based vaccines are difficult to generate. Availability of the primary tumor, in vitro processing of tumor cells for reinjection, generation of allogeneic DC of different haplotypes to avoid predominance of allogeneic responses, low efficiency of fusion (only 10%), risk of contamination during in vitro manipulation, and high cost are all technical factors that limit applicability of this vaccine for large-scale immunization. VII. Conclusions and Future Perspectives

Important developments in MUC1 research have provided new insights to our understanding of how MUC1 glycoprotein changes during premalignant and later malignant transformation and how these changes are recognized by various immune effector mechanisms. It has been demonstrated by us and others that MUC1 is antigenic: cancer patients with MUC1-positive tumors

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generate anti-MUC1 antibodies and MUC1-specific CTLs. However, in a tumor environment, these immune responses are inefficient at controlling tumor growth. The aim of current studies by several research groups is therefore to potentiate existing antitumor responses against autologous cancer cells, using MUC1 vaccines. The mechanisms that form the basis of immunotherapeutic MUC1 vaccines are outlined in Fig. 1. MUC1 antigens can be administered in a variety of forms: as soluble peptides or proteins in the presence of adjuvants, coated on beads, as MUC1-encoding cDNA, via preloaded DC, or on DC–tumor cell hybrids. Following vaccination, MUC1-specific T and B cellular immune responses are triggered in secondary lymphoid organs. The armed effectors need to migrate to the tumor site, where they are expected to induce antitumor defense mechanisms. Ideally, successful vaccination should elicit IgG antibodies, strong helper and cytotoxic responses, and long-lasting antitumor immunity. Major efforts have been made toward the design of better MUC1 vaccines; for example, synthesis of MUC1 peptides and glycopeptides with improved immunogenic properties, discovery of more potent adjuvants, and efficient delivery systems have advanced the field of MUC1 vaccination. Vaccines that showed promising results in preclinical models were later tested on patients in Phase I/II clinical trials. However, none of the vaccines tested to date shows an ability to successfully trigger both humoral and cellular MUC1specific responses and to reverse the clinical course of disease in cancer patients. Translating encouraging results from mouse models to patients often leads to disappointing results, partly due to the fact that patients enrolled in clinical trials usually have large, well-established and/or disseminated tumors. These late-stage patients, with impaired immunity and large tumor burdens, might display insurmountable barriers to activating immune effector mechanisms. Many important aspects of MUC1 vaccination still need to be addressed, such as identifying the optimal dose of antigen and optimal frequency of vaccination in order to create a therapeutic window of antigen concentration able to induce efficient T and B cell responses. In addition, we need to identify what anti-MUC1 antibody titers are associated with antitumor effects and what parameters better define the right type of T lymphocyte responses. CTLs, most studied to date, are only one of many players in the antitumor response, and induction of cytotoxic responses is essential, but not sufficient, to control tumor progression. In addition to chemo- and radioresistance, tumor cells can evade CTLs through various mechanisms such as release of inhibitory cytokines or developing resistance to perforin (Lehmann et al., 2000) or to the granzyme B pathway (Medema et al., 2001). In this context, we need to further explore the functional interactions between the immune effectors and the tumor and its microenvironment. Thus vaccination approaches that combine

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Fig 1 Vaccination strategies using MUC1 tumor antigen. MUC1 antigens can be administered as soluble peptides or proteins in the presence of adjuvants, coated on beads, as MUC1-encoding cDNA, via preloaded dendritic cells (DC) or on DC–tumor cell hybrids. Antigen-presenting cells such as DC process the MUC1 antigen and present MUC1-derived peptides on MHC class I and II molecules to CD8þ and CD4þ T cells, respectively. Following antigen recognition, MUC1specific T and B cellular immune responses are triggered and expanded in secondary lymphoid organs. The activated effector cells then migrate to the tumor site, where they are expected to induce antitumor responses. Ideally, successful vaccination should elicit specific antibodies, strong helper and cytotoxic responses, and long-lasting immune memory.

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potent immunogens with modulators of tumor cell resistance to killing could prove to be beneficial. Successful vaccine development also depends on our ability to detect immune responses. Currently, a variety of options exist for monitoring MUC1-specific immunity, and each technique has its own set of advantages and disadvantages (serological techniques for antibody responses or DTH reactions, ELISPOT assays, tetramer staining, cytotoxic assays, and so on, for T cell function). However, the challenge still remains to develop immune monitoring systems that are highly reproducible, show enhanced sensitivity and specificity, and better correlate with clinical outcome. Many of the experimental systems (i.e., mice challenged with tumor cells following vaccination) show that immunity can be activated to prevent tumors. Thus we can focus in the future on strategies using MUC1 for prevention rather than therapy. Individuals with high genetic risk of developing cancer, or patients diagnosed with MUC1-positive preneoplastic lesions, have an immune system that is not yet suppressed by the tumor and are likely to mount stronger, protective responses upon vaccination. Similarly, vaccination after surgical resection or in the presence of minimal residual disease following standard therapy might show a better rate of success in preventing tumor recurrence. We provided here a broad view of MUC1 immunobiology and its impact on cancer vaccine research. MUC1 vaccines can prevent tumors in animal models and are safe to test in cancer patients. The creation of the MUC1 Tg mouse model will further advance our efforts to design better vaccines, be able to break immune tolerance, protect against tumors, and avoid unwanted autoimmunity. However, the real challenge for MUC1 immunologists in the future will be to decide whether to continue to test these cancer vaccines for therapeutic purposes in cancer patients, or to begin testing them for their ability to prevent cancer development in patients with premalignant lesions.

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advances in immunology, vol. 82

Human Models of Inherited Immunoglobulin Class Switch Recombination and Somatic Hypermutation Defects (Hyper-IgM Syndromes) ANNE DURANDY, PATRICK REVY, AND ALAIN FISCHER INSERM U429, Hoˆpital Necker-Enfants Malades, 75015 Paris, France

I. Introduction

Maturation of the antibody repertoire results in the production of efficient antibodies of various isotypes, via a two-step process. The first step is stochastic recombination, resulting in the expression of rearranged IgH(m) and L genes by mature B cells (Tonegawa, 1983). The second is driven by antigen encounter and involves two processes: immunoglobulin class switch recombination (CSR) and the generation of somatic hypermutations (SHM), which precedes the selection of B cells expressing a B cell receptor (BCR) with a high affinity for antigen. A. Generation of the Primary Antibody Repertoire Generation of the primary repertoire is antigen and T cell independent and occurs in the primary lymphoid organs: the fetal liver and bone marrow. B cell precursors are engaged in the B cell differentiation pathway once they have produced the transcription factor Pax5 and express CD19 molecules on their membrane (Souabni et al., 2002). They undergo sequential rearrangements of the V(D)J regions of the immunoglobulin (Ig) genes. Rearrangement begins in the D to J segments, then extends to the V to DJ segments of the heavy chain, followed by the V to J regions of the k or l light chain (Bassing et al., 2002b; Ghia et al., 1996). V(D)J rearrangements are site specific and are mediated by the recognition of recombination signal sequences (RSS), which consist of conserved heptamers and nonamers separated by 12- and 23-bp spacers, respectively. V(D)J recombination is initiated by the Rag1–Rag2 complex, which is lymphoid specific and tightly regulated (Gellert, 2002; Mombaerts et al., 1992; Shinkai et al., 1992). The Rag proteins recognize and bind to RSS regions, introducing a DNA double-strand break (DNA DSB) at the junction between the heptamer and the coding sequence, leaving hairpinsealed coding ends on the chromosome and phosphorylated, blunt signal ends excised from the chromosome. The resulting DNA damage is repaired by the general DNA repair machinery of the cell, including, in particular, the nonhomologous end-joining (NHEJ) pathway (Haber, 2000). In this pathway, DSB are recognized by the DNA-dependent protein kinase (DNA–PK) 295 Copyright 2004, Elsevier Inc. All rights reserved. 0065-2776/04 $35.00

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complex, which is formed from the Ku70/Ku80 heterodimer (Lee and Kim, 2002). The DNA-PK complex probably activates Artemis, a ubiquitous repair enzyme (Moshous et al., 2001), which opens hairpins. Further DNA processing at coding ends occurs prior to ligation (Ma et al., 2002). Finally, the XRCC4/DNA–ligaseIV complex catalyzes the ligation step (Chen et al., 2000). During the final phase of V(D)J recombination, the lymphoid-specific terminal deoxynucleotidyltransferase (TdT) enzyme further increases the diversity of joins by adding nontemplated (N) nucleotides (Hofle et al., 2000). Immature B cells express functional IgM on their cell membranes. Mature B cells coexpress IgM and IgD, with IgD generated by differential splicing (Melchers et al., 1995). This results in the production of a primary antibody repertoire of IgM antibodies, which is limited by the number of germinal V, D, and J regions undergoing stochastic recombination. B. Generation of the Secondary Repertoire Mature B cells (IgMþ/IgDþ) emigrate and populate the secondary lymphoid organs (spleen, tonsils, lymph nodes, and gut-associated lymphoid tissue), in which terminal antibody maturation occurs in an antigen- and T cell-dependent manner to generate the predominant B2 cell subset. When a B cell encounters an antigen specifically recognized by its IgM BCR, it proliferates, resulting in the formation of a germinal center within the secondary lymphoid organs. In this unique anatomical formation, two major antibody maturation events occur in close contact with T cells: CSR and the generation of SHM (Berek et al., 1991; Kuppers et al., 1993; Liu et al., 1989; Rajewsky, 1996). However, a minor B1 cell population, involved in the T cell-independent antibody response, may mature in the absence of T cells and germinal centers, probably in the splenic marginal zone (Mond et al., 1995; Szomolanyi-Tsuda et al., 2001; Vos et al., 2000). This population may then undergo CSR and SHM. CSR is a process of recombination between two different switch (S) regions located upstream from constant C regions, in which the intervening DNA is deleted (Iwasato et al., 1990; Kinoshita and Honjo, 2000; Manis et al., 2002b; Matsuoka et al., 1990; von Schwedler et al., 1990). Replacement of the Cm region by a C region from another class of Ig (Cg1–4, Ca1–2, or Ce in humans) results in the production of antibodies of different isotypes (IgG, IgA, and IgE) with the same V specificity, and therefore the same antigen affinity. This is a three-step process. 1. Induction of transcription of the targeted DNA by cytokines secreted by activated T cells (Hein et al., 1998; Nagaoka et al., 2002). Each cytokine specifically activates an I promoter located upstream from the S region, leading to the production of germline I-Cx transcripts after splicing of the

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intervening S regions. Cytokine signaling probably opens the chromatin of S regions, rendering them accessible. The specificity of each intervening cytokine is strictly restricted: interleukin (IL)-4 and IL-13 for Se or Sg4, IL-10 for Sg1, Sg3, and Sa, and transforming growth factor-b (TGF-b) for Sa in humans (Tangye et al., 2002). 2. DNA breaks occur in S regions. It has been shown that Sm and downstream S regions are essential for CSR because they act as targets for DNA cleavage (Petersen et al., 2001; Wuerffel et al., 1997). S regions contain short stretches of inverted repeats (Davis et al., 1980; Tashiro et al., 2001). Following the production of germline transcripts, RNA/DNA hybrids form on the template DNA strand, leaving the single nontemplate strand open to cleavage (Bransteitter et al., 2003; Chaudhuri et al., 2003; Dickerson et al., 2003; Ramiro et al., 2003; Yu et al., 2003). The mechanism by which the DSB required for CSR are produced is currently unknown. The template DNA strand may be attacked in transcription bubbles (Bransteitter et al., 2003) or, alternatively, other, currently unidentified factors may be involved (Fugmann and Schatz, 2003). 3. The DNA repair machinery joins Sm and Sx sequences, probably by means of the widespread, constitutively expressed NHEJ, including the KU proteins that act on DNA DSB (Casellas et al., 1998; Manis et al., 1998; Rolink et al., 1996). However, NHEJ are not required for the generation of IgG1 (Manis et al., 2002a) and their role has recently been called into question after the observation of normal CSR in SCID mice, which lack DNA–PK activity (Bosma et al., 2002). Other DNA repair factors may therefore be required for CSR. The different isotypes have various activities (half-life, binding to Fc receptors, ability to activate the complement system) and tissue localization. Thus CSR is necessary for an optimal humoral response toward pathogens. SHM introduces missense mutations, and more rarely deletions or insertions, into the V regions of immunoglobulins. This process is triggered by activation of the BCR and CD40 (Jacobs et al., 2001; Storb et al., 1998). These mutations occur at high frequency in the V regions and their proximate flanks (1  103 bases/generation). Mutations overlap the complementary determining regions (CDR) and specifically target RGYW motifs (R ¼ G or A, Y ¼ T or C, W ¼ A or T). They also affect other genes (first intron of bcl-6, and the CD95 gene), but at a much lower frequency (Muschen et al., 2000; Shen et al., 1998; Zan et al., 2000). SHM is required for the selection of B cells expressing a BCR with a high affinity for antigen and the negative selection of B cells expressing a BCR recognizing an autoantigen or carrying BCR with low antigen affinity. Close interactions with follicular dendritic cells occur during the selection process (Frazer et al., 1997; Rajewsky, 1996). As in CSR, the first essential step in SHM is transcription of the targeted DNA because the intronic enhancer and promoter are required (Betz et al., 1994). The rate of

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mutation is correlated with transcription efficiency (Bachl et al., 2001; Fukita et al., 1998). DNA cleavage is also required. DNA breaks probably occur in single-stranded DNA (Kong and Maizels, 2001; Sale and Neuberger, 1998). It has been suggested that DSB are required for SHM (Bross et al., 2000; Papavasiliou and Schatz, 2000), but these data have been challenged (Bross et al., 2002; Faili et al., 2002). The final step, DNA repair, probably involves the mismatch repair enzymes Pms2, Msh2, and Mlh1 (Cascalho et al., 1998; Evans and Alani, 2000; Kenter, 1999; Schrader et al., 1999; Wiesendanger et al., 1998) and/or the error-prone polymerases i, h, z, and m (Dominguez et al., 2000; Faili et al., 2002; Zan et al., 2001; Zeng et al., 2001). Mutations are introduced during this DNA repair phase. NHEJ have been shown to be dispensable for SHM (Bemark et al., 2000). Thus CSR and SHM share several important features, such as signaling via the BCR/CD40-activation pathway, transcription of the targeted DNA, and DNA cleavage. These processes occur simultaneously in germinal centers, but neither is a prerequisite for the other because IgM may be mutated, while IgG or IgA can remain unmutated (Jacob and Kelsoe, 1992; Kaartinen et al., 1983; Liu et al., 1996). Interestingly, both processes have been shown to occur even in the absence of T cells or germinal centers, probably in the splenic marginal zone for the B1 population (Mond et al., 1995; Szomolanyi-Tsuda et al., 2001; Vos et al., 2000). The recent elucidation of the molecular basis of inherited immune deficiencies—hyper-IgM syndromes (HIGM)—has made it possible to delineate some of the molecular events involved in antibody maturation in humans. Patients with HIGM have normal or high serum IgM levels, but little or none of the other isotypes, strongly suggesting a defect in CSR. Most HIGM patients also display impaired SHM generation (Fig. 1 and Table I). II. Hyper-IgM Syndrome Caused by a CD40-L/CD40 Activation Pathway Defect

In a subset of HIGM syndromes, there is a defect in the CD40-L/CD40 activation pathway leading to a deficiency that affects both humoral and cellular immunity because CD40-L/CD40 interaction is required for efficient B and T cell responses. Associated impairment of the T cell-mediated immune response is responsible for the severity of these diseases because this defect cannot be compensated by Ig substitution and can be cured only by allogeneic bone marrow transplantation (Thomas et al., 1995). A. Biology of CD40 Ligand/CD40 Molecules The murine CD40L maps to the X chromosome. It was first identified as an EL4 thymoma membrane molecule (Armitage et al., 1992), and the human cDNA for CD40L was then cloned from activated T cells (Graf et al., 1992;

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Fig 1 Generation of the antibody repertoire and its abnormalities. The antibody repertoire is generated by means of a two-step process. The first step occurs in primary lymphoid organs, is antigen and T cell independent, and generates the primary repertoire (IgM). It involves stochastic recombination events between the various regions, V(D)J, of the immunoglobulin, under control of the lymphoid-specific Rag1 and Rag2 proteins and the ubiquitous DNA repair molecule, Artemis. Defects in the Rag or Artemis molecules lead to severe combined immunodeficiency (SCID) because T and B cells use the same mechanisms to generate their antigen receptors. The second step occurs in the secondary lymphoid organs, is antigen and T cell dependent, and leads to generation of the secondary repertoire, characterized by the production of efficient antibodies of different isotypes. This maturation is achieved by the generation of somatic hypermutations in the V region of the immunoglobulin (followed by the selection of B cells carrying a receptor with high affinity for antigen) and the class switch recombination process (production of IgG, IgA, and IgE). A defect in the class switch recombination process (with or without defective generation of somatic hypermutation) results in a hyper-IgM syndrome (HIGM). CD40L, CD40 ligand; AID, activation-induced cytidine deaminase; UNG, uracil-N glycosylase.

Hollenbaugh et al., 1992; Spriggs et al., 1992). The human CD40L cDNA contains an open reading frame of 783 bp that encodes a 261-amino acid type II membrane protein belonging to the TNF superfamily. CD40L is a highly inducible gene and is principally expressed in activated CD4þ T cells. The transcription of this gene is regulated by the NF-AT transcription factor (Schubert et al., 1995). The T cells of newborns have been found to be deficient in CD40L expression and are unable to induce CSR (Durandy et al., 1995; Nonoyama et al., 1995). CD40L is not specific to T cells; it has been shown to be expressed by other cells, including NK cells, basophils, eosinophils, mast cells, platelets, endothelial cells, and even activated B cells (Cocks et al., 1993; Gauchat et al., 1993, 1995; Grammer et al., 1998; Mach et al., 1997).

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TABLE I HIGM Syndromes HIGM1 Gene Transmission Clinical manifestations Bacterial infections Opportunistic infections Lymphadenopathies Autoimmunity HIGM syndromes (%) IgM normal > 2 SD above N IgG < 2 SD below N Undetectable IgA < 2 SD below N Undetectable In vitro CD40-induced CSR SHM

HIGM3

HIGM2

UNG deficiency

HIGM4

CD40L XL

CD40 AR

AID AR

UNG AR

? AR

þ þ  þ

þ þ  ?

þ  þ þ

þ  þ ?

þ  þ/ þ

53 47 100 93 90 80 Normal #

0 100 100 75 100 75 Absent #

5 95 100 95 100 95 Absent # or absent

0 100 100 75 100 75 Absent Biased

10 90 100 65 100 90 Absent Normal

CD40L binds to CD40, a member of the TNF receptor (TNFR) superfamily. The gene encoding CD40 maps to chromosome 20 in humans and encodes a 289-amino acid type 1 protein. CD40 is constitutively expressed on B cells (Rousset et al., 1991), but also on monocytes, dendritic cells, follicular dendritic cells, myeloid progenitors, and epithelial, endothelial, and neuronal cells (Dubois et al., 1997; Gaspari et al., 1996; Kotowicz et al., 2000; Solanilla et al., 2000; Tan et al., 2002). The activation of B cells via the CD40 pathway has several consequences: rescue from apoptotic signals, homotypic cell adhesion, and, in response to cytokines, proliferation, up-regulation of activation molecules, and CSR (Armitage et al., 1993; Banchereau et al., 1991; Gordon et al., 1988; Ranheim and Kipps, 1993; Rousset et al., 1991; Saeland et al., 1993; Wang et al., 1995). CD40 activation of monocytes/dendritic cells leads to the production of proinflammatory and antiinfectious cytokines, including IL-12 (Caux et al., 1994; Kato et al., 1996; Kennedy et al., 1996). The biochemical pathway underlying CD40 activation has not been completely elucidated. Cross-linking of CD40 activates the lyn tyrosine kinase, leading to the activation of Pi-3K and PLCg2 (Ren et al., 1994; Uckun et al., 1991). As CD40 has no intrinsic tyrosine kinase activity, it probably associates with other signaling molecules, including jak3, leading to the activation of transcription factors of the STAT family (Hanissian and Geha, 1997). A role for btk and its adaptor BLNK in CD40 signaling has also recently been

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described (Brunne et al., 2002; Brunner et al., 2002; Hayashi et al., 2000). The CD40 activation pathway also induces the activation of kinases of the MAPK family (Aicher et al., 1999; Berberich et al., 1996). The intracytoplasmic domain of CD40 also binds to TNFR-associated factors (TRAF), resulting in activation of the transcription factor NF-kB (Ishida et al., 1996a, 1996b; Rothe et al., 1995), through activation of the NF-kB-inducing kinase (NIK) (Luftig et al., 2001). CD40 activation induces two NF-kB pathways: the NF-kB1 (p50 and its precursor p106) and the NF-kB2 (p52 and its precursor p100) pathways. The NF-kB1 pathway requires IKKa, IKKb, and IKKg (NEMO) activation, whereas NEMO is not required for NF-kB2 activation (Coope et al., 2002). CD40-induced NF-kB activation has pleiotropic effects, including proliferation and CSR (Brady et al., 2000). Interestingly, the Jak/STAT pathway was recently shown to be involved in the induction of activation-induced cytidine deaminase (AID), a B cell-specific molecule required for CSR and SHM (Zhou et al., 2003). B. Hyper-IgM Syndrome Caused by CD40 Ligand Deficiency (HIGM1) The first HIGM syndrome to be described (HIGM1) was the X-linked form caused by mutations in the gene encoding the CD40 ligand (CD40L, CD154) (Allen et al., 1993; Aruffo et al., 1993; DiSanto et al., 1993; Korthauer et al., 1993; Narayanaswamy, 1999). CD40L is transiently expressed on activated helper T cells and interacts with CD40 molecules constitutively expressed on B cells, monocytes, and dendritic cells (Alderson et al., 1993; Castle et al., 1993; Fuleihan et al., 1993; Nonoyama et al., 1993). HIGM1 patients display significantly lower than normal levels of membrane CD40L expression on activated CD4þ T cells, or no expression at all, making diagnosis of this syndrome straightforward. The mutations responsible for this condition are scattered throughout the gene, with no particular hot spot (Fig. 2). Due to a CD40 trans-activation defect, the B cells of patients with this syndrome cannot proliferate or form germinal centers in secondary lymphoid organs in vivo. Humoral immunodeficiency is characterized by defective CSR. Impaired production of IgG and IgA is responsible for specific susceptibility to recurrent bacterial infections caused by encapsulated bacteria, as observed in other severe B cell deficiencies. No antibodies against infectious agents or vaccines are produced, but isohemagglutinins are normally detected. B cells are intrinsically normal, as they can be induced to proliferate and to undergo CSR to generate IgG, IgA, and IgE upon in vitro activation by CD40 agonists and appropriate cytokines (Durandy et al., 1993). The B cells of all patients coexpress IgM and IgD, reflecting the lack of CSR (Durandy et al., 1993). Most, but not all patients present smaller than normal

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Fig 2

Mutations in CD40L result in HIGM1. From Narayanaswamy (1999).

numbers of ‘‘memory’’ CD27þ B cells and a low frequency of SHM (Agematsu et al., 1998; Razanajaona et al., 1997). Significant amounts of IgA or IgE are present in the sera of some patients, even in the total absence of CD40L expression. The residual CSR responsible for generating these isotypes may result from the generation of germinal centers in the absence of T cells (Lentz and Manser, 2001) or may indicate the involvement of environmental or other mechanisms, even in the absence of CD40L signaling. CD40L-deficient T cell lines can induce CSR in normal B cells, suggesting that another pathway may occur, at least in vitro (Life et al., 1994). Viral glycoproteins and bacterial polysaccharides are also known to stimulate the production of IgG and IgA in the absence of CD40L-expressing CD4þ T cells (Mond et al., 1995; Szomolanyi-Tsuda et al., 2001; Vos et al., 2000). CSR was recently shown to occur via a CD40-independent pathway, through the overexpression of BlyS/BAFF and APRIL on dendritic cells activated by interferon (IFN)-a or -g or lipopolysaccharide (LPS). Following exposure to BlyS/BAFF or APRIL, and in the presence of appropriate cytokines and BCR engagement, B cells undergo an entirely CD40-independent CSR leading to the production of IgG and IgA (Litinskiy et al., 2002). This T cell-independent CSR occurs in the splenic marginal zone or intestinal lamina propria. Repeated stimulation of the gastrointestinal tract in HIGM1 patients may therefore lead to the production of IgA, probably in gutassociated lymphoid tissue. Similarly, the presence of unswitched mutated B cells has been reported in a few patients, suggesting that SHM may also

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occur in the absence of a classic CD40L/CD40 interaction (Weller et al., 2001). This B cell population may be related to IgM memory B cells generated in the spleen, producing antibodies against encapsulated bacteria (Kruetzmann et al., 2003). This subset of cells displays similarity to murine B-1a B cells, which produce natural antibodies and are required for the T cell-independent antibody response to polysaccharide antigens (Martin and Kearney, 2000; Wardemann et al., 2002). Pyk2-deficient mice, which lack marginal zone B cells, have an impaired antibody response to polysaccharides (Fagarasan and Honjo, 2000; Guinamard et al., 2000). Unswitched mutated B cells probably act as a defensive barrier against certain invading extracellular pathogens that are not readily engulfed by phagocytic cells because of their thick polysaccharide capsules. Impaired CD40L expression also leads to defective T cell interactions with monocytes/dendritic cells, resulting in an abnormal cellular immune response, the consequence of which is specific susceptibility to opportunistic infections with Pneumocystis cariniii, Cryptosporidia, and Toxoplasma gondii, which cannot be controlled by Ig substitution therapy (Kutukculer et al., 2003; Notarangelo et al., 1992). These severe infections result in a much poorer clinical prognosis. This defect results from impaired T cell priming, defective production of IFN-g and the type 1 cytokine IL-2, and impaired IL-2 secretion by monocytes (Jain et al., 1999; Subauste et al., 1999). The necessity of a CD40L / CD40 interaction in defenses against opportunistic infections has been confirmed in an experimental model of Cryptosporidia infection (Hayward et al., 2001). Neutropenia is also a common feature of HIGM1, and may result from the expression of CD40 on myeloid progenitors. There is no strict correlation between genotype and phenotype, as patients with identical mutations may present very different disease severities. This suggests that other genetic or environmental factors are also involved in the pathogenesis of this syndrome. However, some patients who carry missense mutations affecting the N-terminal part of the molecule, resulting in low-level CD40L expression, present less severe disease, with no opportunistic infections and residual levels of IgG or IgA. In rare cases, this syndrome has been observed in female patients presenting extreme lyonization of the X chromosome (de Saint Basile et al., 1999). C. Hyper-IgM Syndrome Caused by a Defect in CD40 (HIGM3) An HIGM syndrome with a phenotype identical to that of HIGM1, but autosomal recessive (AR) inheritance, has been reported in a few cases (three patients in two consanguineous families) as secondary to mutations in the CD40 gene (HIGM3) (Ferrari et al., 2001) (Fig. 3). The genetic defect leads to the defective expression of CD40 molecules on B cells and monocytes. As a

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Fig 3

Mutations in CD40 result in HIGM3. From Ferrari et al. (2001).

consequence, B cells and monocytes cannot be activated via the CD40 pathway, and patients suffer from a profound CSR defect and susceptibility to both bacterial and opportunistic infections. One patient was found to have very few ‘‘memory’’ B cells and the level of SHM generation in these few CD27þ B cells was extremely low, although this process was not completely abolished. This finding is reminiscent of the occurrence of SHM in some HIGM1 patients. D. Hyper-IgM Syndrome Caused by Defective NF-kB Activation (XHM–ED) Three groups have unraveled the molecular basis of another X-linked form of HIGM syndrome, associated with anhydrotic ectodermal dysplasia (XHM– ED), showing this condition to be secondary to mutations affecting the zinc finger domain of the nuclear factor kB essential modulator gene (also called NEMO or IKKg) (Doffinger et al., 2001; Jain et al., 2001; Zonana et al., 2000). This syndrome is characterized by a dermopathy (no sweat glands, sparse hair, and conical teeth) and is associated with an immune deficiency. Some patients have been reported to suffer life-threatening opportunistic bacterial and mycobacterial infections. Serum Ig concentrations vary, but hypo-IgG and hyper-IgM are often observed in patients with this syndrome. Antibody responses, particularly those directed against polysaccharide antigens, have been found to be impaired (Carrol et al., 2003; Smahi et al., 2002). CD40-mediated B cell proliferation and CSR are partially impaired in vitro, but the B cell activation marker CD23 is normally up-regulated following CD40 activation. Patients with this condition have very few ‘‘memory’’ B cells, but no data are available concerning SHM in these patients. B cell abnormalities result from impaired NF-kB signaling following CD40 activation, as NEMO serves as a scaffolding protein, binding two kinase proteins, IKKa and IKKb, necessary for NF-kB activation. In normal conditions, CD40 stimulation leads to the activation of these kinases, phosphorylation and degradation of the NF-kB inhibitory protein (I-kB), and thus to the activation and nuclear translocation

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of NF-kB components (Ghosh et al., 1998; Israel, 2000; Karin and Ben-Neriah, 2000; Rothwarf et al., 1998; Yamaoka et al., 1998). The B cell defect observed in XHM–ED patients, similar to the phenotype of mice lacking both the p50 and p52 components of NF-kB, provides evidence that the NF-kB transcription factor is involved in CD40-mediated B cell activation and Ig switching (Franzoso et al., 1997). The molecular mechanism by which CD40 activates the IKK complex is unclear. A defect in BCR signaling is probably involved because this activation pathway requires NF-kB translocation (Bajpai et al., 2000). However, the CD40-induced NF-kB2 pathway is unaffected because NEMO is dispensable for nuclear translocation (Coope et al., 2002). Ectodermal dysplasia, a key feature of this syndrome, may also be due to the defect in NEMO because the DL receptor expressed on tissues derived from the ectoderm also belongs to the TNF receptor superfamily and has been shown to activate NF-kB via the IKKa/b NEMO complex (Doffinger et al., 2001). These three HIGM syndromes demonstrate the essential role played by CD40 activation, including the NF-kB pathway, in antibody maturation. Both CSR and SHM require CD40 activation for full efficiency, although a CD40/ CD40L-independent pathway can trigger antibody maturation. III. Hyper-IgM Syndromes Associated with an Intrinsic Functional B Cell Deficiency

AR forms of HIGM are characterized by a B cell-specific functional deficiency (Callard et al., 1994; Durandy et al., 1997). Patients with these syndromes are therefore prone to infections with bacterial pathogens but not to opportunistic infections, in contrast to the situation for patients with HIGM in which the CD40 activation pathway is disturbed. The prognosis is therefore much better for these patients, although continuous Ig substitution treatment is required. Patients with AR forms of HIGM, unlike those with HIGM1 or HIGM3, frequently present enlargement of lymphoid organs such as the spleen, tonsils, and lymph nodes. As in the other HIGM syndromes, patients have normal or high serum IgM levels, contrasting with markedly low serum IgG and IgA levels. Consistent with serum Ig levels, IgG antibodies against infectious agents or immunization antigens are not detectable, whereas IgM isohemagglutinins are detected. In these patients, in contrast to HIGM1 patients, B cells are intrinsically defective because they do not undergo class switch recombination in vitro in the presence of CD40 agonists and appropriate cytokines (Durandy et al., 1997), whereas monocytes and dendritic cells are normally activated by CD40 agonists (Revy et al., 1998). The dissection of B cell abnormalities and molecular definition has led to the conclusion that this AR HIGM group can be separated into several entities.

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A. Hyper-IgM Syndrome Caused by Activation-Induced Cytidine Deaminase Deficiency (HIGM2) This HIGM syndrome was the second to be described, hence its designation (HIGM2). It is caused by deleterious mutations in the gene encoding activation-induced cytidine deaminase (AID) (Revy et al., 2000). AID was first identified in mice (Muramatsu et al., 1999) and was cloned by subtractive hybridization between murine lymphoma CH12F-2 B cells with and without induction of class switch recombination in vitro. The AID protein is detected only in B cells undergoing CSR or SHM in vivo (in germinal center B cells) and in vitro (Diaz and Casali, 2002; Faili et al., 2002; Muramatsu et al., 1999; Papavasiliou and Schatz, 2002). This protein is structurally similar (34% amino acid sequence identity) to the apoB RNA-editing enzyme APOBEC-1. RNA editing is widely used to create new functional RNAs from a restricted genome in plants and protozoa (Scott, 1995; Simpson and Thiemann, 1995). An increasing number of mammalian mRNAs are also known to be edited by cytidine or adenosine deaminases, including apolipoprotein B mRNA, which is edited by APOBEC-1. ApoB mRNA editing involves the site-specific C to U deamination of the first base of a CAA codon encoding glutamine at residue 2158 in apoB100, and produces an in-frame UAA stop codon in apoB48 mRNA (Mehta et al., 2000; Navaratnam et al., 1993; Teng et al., 1993). ApoB100 and ApoB48 are the translation products of the unedited and edited apoB mRNAs, respectively. These proteins have entirely different functions and expression profiles. APOBEC-1 requires an auxiliary factor (ACF) for the site-specific editing of apoB mRNA. This auxiliary factor is widely expressed, including some tissues that do not express APOBEC-1 (Navaratnam et al., 1993; Teng et al., 1993; Yamanaka et al., 1994). The open reading frame of the AID cDNA encodes a 198-residue protein with a molecular mass of approximately 24 kDa. AID contains an active site for cytidine deamination, the sequence of which is conserved throughout the large cytidine deaminase family, and has been shown to display cytidine deaminase activity in vitro (Muramatsu et al., 1999). Its C-terminal domain also contains a leucine-rich region that is probably important for protein– protein interaction. This region may bind accessory factors required for AID activity or may be important in AID tetramerization (Dickerson et al., 2003). The human counterpart of the murine AID gene has been mapped to chromosome 12 and cloned. The two proteins display a high level of sequence identity (96%), especially in the cytidine deaminase domain (100%). Human AID is expressed only in the germinal centers of secondary lymphoid organs (spleen, lymph nodes, and tonsils), in peripheral blood B cells in which CSR has been induced by stimulation with soluble CD40 ligand and interleukins

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(IL-4 or IL-10) and in Epstein-Barr virus (EBV)-induced B cell lines (Muto et al., 2000; Revy et al., 2000). AID deficiency leads to HIGM2 syndrome. Recessive mutations have been identified throughout the gene, including the deaminase activity domain (Fig. 4). HIGM2 patients, like other patients with HIGM, are susceptible to infections caused by bacterial pathogens, especially those involving the respiratory and digestive tracts. A hallmark of this syndrome is frequent and impressive enlargement of the lymphoid organs. Histological examination reveals the presence of giant germinal centers (5–10 times larger than normal) filled with highly proliferating B cells. Proliferating B cells coexpress IgM, IgD, and CD38, a phenotype previously described for a small B cell subset corresponding to germinal center (GC) founder cells (Lebecque et al., 1997). These cells are thought to correspond to a transitional stage between follicular mantle and GC B cells, at the onset of somatic mutation of the Ig variable region gene and antigen-driven selection. The normal expression of CD95 on B cells and the presence of numerous macrophages filled with apoptotic bodies within the germinal centers of AID-deficient patients appear to rule out a defect in apoptosis. One possible explanation for the intense proliferation observed is that in the absence of functional AID, antigens continuously induce the proliferation of B cells (which normally express the BCR), provided that no successful antibody maturation has occurred (Fagarasan et al., 2001). Alternatively, AID may exert direct control over GC B cell proliferation.

Fig 4

Mutations in AID result in HIGM2.

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There is a profound defect in CSR, as IgG, IgA, and IgE levels are barely detectable in serum in vivo, in contrast to IgM levels that are high. Isohemagglutinins are present in normal amounts, contrasting with the absence of IgG antibody production in response to vaccine antigens or microorganisms. In vitro, B cells proliferate, produce large amounts of IgM, and upregulate the CD23 activation marker when activated by sCD40Lþ appropriate cytokines, but cannot undergo CSR to generate IgG, IgA, or IgE. This is demonstrated by the complete lack of Ig production in culture supernatants and by the absence of Ig excision circles and functional transcripts (Revy et al., 2000). All of the B cells in peripheral blood coexpress IgM and IgD, and a fraction of these cells carry the CD27 marker (20–50%). SHM either does not occur at all, or occurs at very low levels in the CD27þ B cell subset. Thus AID deficiency leads to defects in both CSR and SHM, consistent with observations in AID-deficient mice (Muramatsu et al., 2000). Thus these two B cell maturation processes must share a common mechanism requiring AID—a hypothesis supported by the observation of frequent mutations in S regions (Chen et al., 2001; Lee et al., 1998; Li et al., 1996). AID has also been shown to be required for the gene conversion process used by chickens to diversify the antibody repertoire (Arakawa et al., 2002; Harris et al., 2002b). The mode of action of AID and its target in CSR and SHM have recently been determined. The first step of CSR and SHM is transcription of the targeted DNA. Levels of germline transcripts induced during CSR have been checked in AID-deficient patients (HIGM2) and mice and found to be normal (Muramatsu et al., 2000; Revy et al., 2000). The second step, the formation of DNA strand breaks, has been investigated by ligated mediated polymerase chain reaction (PCR), in which a blunt-ended linker was ligated to the DNA of interest. This technique was applied to Sm regions in CSRactivated B cells from age-matched controls and patients. Double-strand DNA breaks (DSBs) were readily detected in the Sm regions of B cells from controls, but were absent or rare in AID-deficient B cells (Catalan et al., 2003). This observation clearly indicates that AID acts upstream from DSB generation in CSR. This is consistent with previous data showing that phosphorylated histone H2AX and the Nbs1 repair foci associated with DSB repair are not properly recruited to Ig loci during CSR in AID-deficient mice (Petersen et al., 2001). AID is the only B cell-specific factor required, as it has been shown to be sufficient to induce CSR and SHM in fibroblasts on artificial substrates (Okazaki et al., 2002; Yoshikawa et al., 2002). AID exerts its activity downstream from the transcription step and upstream from DSB formation during CSR. AID probably acts in a similar manner in SHM generation, although this has not been demonstrated experimentally. A recent study suggests that AID could act differently in SHM, downstream of the DBS occurring spontaneously in V regions (Zan et al., 2003). The

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mechanism of action of AID, however, remains open to debate. As the sequence of AID is similar to that of the RNA-editing enzyme APOBEC-1, it has been suggested that AID edits an mRNA encoding a substrate common to CSR and SHM, probably an endonuclease (Chen et al., 2001; Honjo et al., 2002; Kinoshita and Honjo, 2001). The recently described requirement for de novo protein synthesis downstream from AID expression in CSR could suggest the synthesis of a recombinase, and is consistent with the RNA-editing model (Doi et al., 2003). Alternatively, recent data strongly suggest that AID may have DNA-editing activity. Following the transfection of Escherichia coli with a construct encoding AID, the AID produced deaminates deoxycytosine (dC) residues to deoxyuracil (dU) on DNA (Petersen-Mahrt et al., 2002). Several groups have demonstrated a direct role for AID in DNA dC deamination in cell-free assays. Moreover, it has been shown that AID acts on single-stranded DNA (ssDNA) but not on double-stranded DNA, RNA–DNA hybrids, or RNA (Bransteitter et al., 2003; Chaudhuri et al., 2003; Dickerson et al., 2003; Ramiro et al., 2003). Interestingly, AID exerts its activity on single-stranded DNA only in the presence of RNase, suggesting that some RNAs may inhibit this activity (Bransteitter et al., 2003). The transcription of S regions increases AID activity (Ramiro et al., 2003; Shinkura et al., 2003), probably by generating the secondary structures required for this activity (Yu et al., 2003). R-loops are generated by the formation of RNA–DNA hybrids on the template DNA strand, rendering the single nontemplate strand a target for AID. Nicks occurring on this single-stranded DNA are probably sufficient for the introduction of mutations during DNA repair in SHM, although it has been suggested that DSBs are involved in this process (Bross et al., 2000; Papavasiliou and Schatz, 2000). Double-stranded DNA breaks are required for inter-S region recombination in CSR (Kessler et al., 1996). It is unclear how single-stranded DNA breaks lead to double-stranded DNA breaks (DSB). However, it has been shown that AID is active against double-stranded DNA substrates containing transcription-like bubbles of at least nine nucleotides (Bransteitter et al., 2003). It is also possible that additional, as yet unknown factors, are involved (Fugmann and Schatz, 2003). These data provide strong evidence that AID has DNA-editing activity, but were obtained in nonphysiological conditions, involving overproduction in E. coli or in in vitro assays, in which the well-known RNA-editing protein APOBEC-1 has a similar effect (Harris et al., 2002a; Petersen-Mahrt and Neuberger, 2003). Elements targeting AID to S regions (in CSR) or V regions (in SHM) remain to be identified. The recent description of a partial CSR defect and a skewed pattern of SHM (despite a normal frequency of SHM) in uracil DNA glycosylase (UNG)deficient mice (Rada et al., 2002) and of a new hyper IgM syndrome characterized in humans by a profound CSR defect with the same SHM alterations

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caused by deleterious mutations in the UNG gene (see later) (Imai et al., 2003b) provides further evidence that AID acts on DNA. Hence, in the proposed model, AID deaminates dC on one strand of DNA to generate dU residues (Di Noia and Neuberger, 2002; Rada et al., 2002). Following the deglycosylation and removal of the misintegrated dU residues by UNG, an abasic site is created, which can be attacked by an apyrimidinic endonuclease, resulting in single-stranded DNA breaks (Fig. 5). According to this hypothesis, UNG deficiency leads to the partial impairment of CSR, particularly in young mice, and this impairment is best demonstrated in in vitro CSR experiments. The residual CSR and normal frequency of SHM may be accounted for by compensatory mechanisms exerted by other uracil DNA glycosylases,

Fig 5 AID as a DNA-editing enzyme in the class switch recombination process. A schematic representation of the action of activation-induced cytidine deaminase (AID) in class switch recombination (CSR) is shown. After the induction of germline immunoglobulin transcripts by cytokines, RNA–DNA hybrids form on the template DNA strand, rendering the nontemplate single strand an accessible target for AID, induced by CD40 activation. AID deaminates cytosine to generate uracil residues. Uracil-N glycosylase (UNG) deglycosylates and removes these uracil residues from DNA, creating an abasic site attacked by an apyrimidinic (AP)-endonuclease. It is unclear how the single-stranded DNA breaks thus generated the double-stranded DNA breaks necessary for CSR. DNA is then repaired by the nonhomologous end-joining complex (NHEJ). This is the classic hypothetical process, which has recently been called into question.

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including SMUG1, TDG, and MBD4 (Hendrich et al., 1999; Neddermann et al., 1996; Nilsen et al., 2001), although MBD4 has been shown to be dispensable in antibody maturation (Bardwell et al., 2003). They may also be attributable to mismatch repair enzymes, which are known to play a role in CSR and SHM in mice (Cascalho et al., 1998; Ehrenstein and Neuberger, 1999; Schrader et al., 1999, 2003). The skewed pattern of SHM in UNGdeficient mice, involving predominantly transitions at dG/dC residues, may arise from replication of the dU:dG lesions without uracil removal. A similar skewed pattern of SHM has also been observed in B cell lines following UNG inactivation (Di Noia and Neuberger, 2002). This observation strongly reinforces a role of AID as a DNA-editing enzyme. However, a distinct role of UNG in CSR cannot be definitively excluded. UNG might act in conjunction with mismatch repair enzymes in the base excision repair process. It has been suggested that UNG is part of a complex retaining cleaved DNA ends, or of a recombinase complex mediating a later step of CSR (Doi et al., 2003). It is also unclear how AID activity targets Ig loci. This targeting presumably depends on cofactors, which are probably widely expressed as AID is sufficient to induce CSR and SHM on artifical substrates in fibroblasts (Okazaki et al., 2002; Yoshikawa et al., 2002). There are neither consensus nor homologous sequences around the S junctions, in contrast to the RSS regions recognized specifically by Rag enzymes during V(D)J recombination (Hiom et al., 1998; Oettinger, 1999). However, Sm and downstream S regions contain short stretches of inverted repeats, which may facilitate the formation of the secondary structures after transcription, providing AID with access to the single strand of nontemplate DNA (Honjo et al., 2002; Yu et al., 2003). B. Hyper-IgM Syndrome Caused by Uracil-N Glycosylase Deficiency Recently, a new HIGM entity has been described caused by deleterious recessive mutations of the uracil-N glycosylase (UNG) gene (Imai et al., 2003b), reinforcing in physiological conditions the role of AID as a DNA-editing enzyme. Three patients are reported to be affected by a severe HIGM with high levels of IgM and very low levels or the absence of IgG and IgA, leading to recurrent bacterial infections. Two of the three patients present lymphadenopathies. The CSR defect is also observed in vitro by a complete lack of production of IgG, IgA, or IgE by patients’ B cells after activation by CD40Lþ appropriate cytokines, although B cells do normally proliferate in the same culture conditions. As observed in HIGM2, the CSR defect occurs downstream from the germline transcript induction and upstream from the DSB occurrence. However, in contrast with what is observed in HIGM2, the frequency of SHM is found to be normal in CD19þCD27þ B cells.

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Strikingly, SHM exhibit a skewed pattern with transitions almost only at dG/dC residues. The HIGM phenotype observed in these patients resembles the phenotype described as a consequence of homozygous Ung gene inactivation in mice (Rada et al., 2002), although in the latter a much milder CSR defect was found. We therefore explored the possibility of UNG deficiency in these three patients. Two patients present drastic mutations leading to premature stop codons, and one patient has a homozygous missense mutation in the catalytic domain of UNG. As in mice, the human UNG gene has two promoters (Pb for UNG1 and Pa for UNG2) and two alternative splice forms, that is, the mitochondrial isoform UNG1, which is ubiquitously expressed and the nuclear isoform UNG2, which is strongly expressed in proliferating cells (Nilsen et al., 2000; Otterlei et al., 1998). All mutations affect both UNG1 and UNG2 (Fig. 6). In patients’ EBV cell lines, no protein was detected even in the patient with the missense mutation, suggesting an instability of the protein. No uracil-DNA glycosylase activity was found, providing evidence that there is no compensatory activity in human B cells. Since this observation shows that UNG is required for CSR, the expression of the mitochondrial UNG1 and the nuclear UNG2 isoforms was checked in CD19þ B cells before and after activation by sCD40Lþ IL-4. Resting and activated CD19þ and CD19 cells do equally express UNG1 transcripts. In contrast, UNG2 transcripts were detected only in CSR-induced CD19þ B cells, in correlation with AID expression, an observation that reinforces the crucial role of UNG2 in CSR in humans. The de novo protein synthesis-dependent step downstream from AID activity in CSR

Fig 6

Mutations in UNG result in a new HIGM entity.

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recently described in murine splenic B cells (Doi et al., 2003) could possibly correspond to UNG2 induction. C. Hyper-IgM Syndrome Involving Defective Class Switch Recombination and Normal Somatic Hypermutation (HIGM4) Most patients affected by an AR form of HIGM do not carry mutations in the AID gene (Rapalus et al., 2001). Although no molecular defect has been identified in this subset of patients, this condition appears to be fairly homogeneous and is referred to herein as HIGM4. The clinical phenotype is very similar to that of HIGM2 and includes lymphoid hyperplasia. However, none of the giant germinal centers typical of HIGM2 is detected on histological examination, which reveals only moderate follicular hyperplasia. The CSR defect also appears to be slightly milder, with some patients presenting residual levels of IgG. Isohemagglutinins are present, but no IgG antibodies against immunization agents or infectious antigens are detected (Imai et al., 2003a). In vitro, B cells from these patients (all of which coexpress IgM and IgD) do not undergo CSR after activation by the CD40þ cytokine pathway. However, these cells proliferate normally, produce IgM, and up-regulate the CD23 activation marker. As in AID deficiency, the blockade occurs downstream from S region transcription, because Ig germline transcripts are induced normally, whereas Ig excision circles and functional transcripts are undetectable. Even B cells from patients with residual levels of IgG in the serum lack functional transcripts, indicating that the in vitro test is less sensitive than tests in vivo. These results also suggest that a residual CSR may occur in vivo as a result of repeated stimulation, or via another activation pathway. The overexpression of BlyS/BAFF or APRIL on activated dendritic cells stimulates marginal zone B cells to undergo CSR in a CD40-independent pathway—a compensatory mechanism absent from patients with AID deficiency. However, the main difference between this condition and AID deficiency is that the CSR defect occurs after DNA cleavage, as CSR-induced double-stranded DNA breaks occur in Sm regions in CD40Lþ IL-4-activated B cells from HIGM4 patients. The number of CD27þ B cells is either normal or slightly low, but SHM is within the normal range and displays a pattern similar to that of the control. Thus HIGM4 differs from HIGM2 principally in terms of the post DNA-cleavage CSR deficiency and normal SHM generation observed in patients with HIGM4. Indeed, AID deficiency can be excluded in patients with normal AID gene sequences and normal AID RNA transcript levels after CD40 activation. UNG deficiency has also been excluded by gene sequencing, although it was unlikely in any case due to the normal pattern of SHM (Imai et al., 2003a).

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Thus HIGM4 is characterized by defective CSR after cleavage, associated with normal SHM. This phenotype may result from a defect in the survival signals delivered to switched B cells. Some molecular interactions are known to be required for B cell survival, including the interaction between BlyS/ BAFF and its B cell-specific receptor, BR3. The natural mutant mouse strain A/WySnJ, BR3 knockout mice, and mice treated with the BAFF-neutralizing BR3-Fc protein display B cell depletion and an impaired T cell-dependent antibody response (Kayagaki et al., 2002; Schiemann et al., 2001; Thompson et al., 2001). However, a BAFF/BR3 defect is unlikely because HIGM4 patients have normal numbers of circulating B cells and defective CSR in vitro that cannot be accounted for by a BR3 or BlyS/BAFF abnormality. Other, as yet unknown, factors may be involved. A defect in the activationinducible Tcell molecule, ICOS, which was recently shown to be responsible for an AR form of common variable immunodeficiency (Grimbacher et al., 2003), is also unlikely, given the intrinsic B cell abnormality observed in HIGM4. Alternatively, HIGM4 could be the consequence of a CSR-specific DNA repair defect. NHEJ enzymes, known to repair double-stranded DNA breaks, have been shown to be involved in CSR-DNA repair (Casellas et al., 1998; Rolink et al., 1996), except for the IgG1 isotype (Manis et al., 2002a). Nevertheless, a defect in one of these proteins seems unlikely because NHEJ proteins are required for V(D)J recombination and HIGM4 patients have normal numbers of T and B cells, with functional antigen receptors. Other DNA repair enzymes may also play an indirect role, as suggested by the recent observation of normal CSR in scid mice, despite the lack of DNA– PK activity (Bosma et al., 2002). A defect in one such repair enzyme could be responsible for HIGM4. It has been shown that CSR induces the formation of DSB DNA repair foci involving histone H2AX and MRE11/hRad50/Nbs1 protein complex at Ig loci (Petersen et al., 2001). Indeed, defective CSR has been reported in NBS1-deficient patients (Gregorek et al., 2002; van Engelen et al., 2001) and H2AX knockout mice (Petersen et al., 2001). However, a defect in one of these proteins results in chromosomal instability (Bassing et al., 2002a; Bender et al., 2002; Jaspers et al., 1988; Stewart et al., 1999; Thompson and Schild, 2002), but no such instability is observed in HIGM4. Furthermore, mutations in the NBS1 and MRE11 genes are responsible for Nijmegen breakage syndrome (Varon et al., 1998) and ataxia-like disease (Stewart et al., 1999), respectively. Mismatch repair enzymes do not seem to be involved in humans, as indicated by the phenotype of patients carrying mutations in the MLH1 and MSH2 genes. Indeed, these patients are prone to cancer, but no particular susceptibility to infections has yet been reported (Bougeard et al., 2003; Wang et al., 1999; Whiteside et al., 2002). Nevertheless, no precise data concerning CSR or SHM are available for these patients.

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IV. Other Hyper-IgM Syndromes

A. HIGM Associated with T Cell Deficiency Other HIGM conditions, for which the underlying molecular defect has yet to be identified, have been reported (Imai et al., 2003a). These conditions are severe forms of HIGM, with defective cellular and humoral responses. The phenotype of one of these forms of HIGM consists of a defective B cell proliferative response to CD40 and BCR activation, despite normal induction of the activation marker CD23 after CD40 activation. B cell count decreases with age, but serum IgM levels remain high. The B cell defect involves impairment of both CSR and SHM. Patients also suffer from a mild in vitro impairment of T cell responses to mitogens and antigens. A defect in the CD40-induced NF-kB activation pathway in fibroblasts has been ruled out. Another of these conditions has a similar clinical phenotype, but with an absence of B cells in peripheral blood. However, high serum IgM concentrations indicate that B cells are present but are probably sequestered in secondary lymphoid organs. Although no in vitro data concerning T cell functions are available, patients are prone to opportunistic infections, probably reflecting an additional T cell defect. Two lymphomas have been reported among eight patients evaluated, suggesting genomic instability. We cannot exclude the possibility that these two phenotypes overlap and share a common causal mechanism. As patients carrying hypomorphic mutations in Artemis present susceptibility to lymphoma and hypogammaglobulinemia affecting IgG and IgA (Moshous et al., 2003), mutations in this gene were sought, but not found. Nevertheless, a defect in DNA repair is strongly suspected. B. HIGM and Ataxia Telangiectasia (AT) Ataxia-telangiectasia (AT) is a complex disease caused by recessive mutations in the ATM gene and characterized by cerebellar degeneration with ataxia, ocular, and cutaneous telangiectasias, chromosomal instability, and predisposition to cancer (McFarlin et al., 1972; Telatar et al., 1996). Patients display both cellular and humoral immunodeficiency, and HIGM is a frequent feature (Weemaes et al., 1984). HIGM in these patients has been reported to be due to defective T cell activation, leading to the impairment of CD40L expression (Gatti et al., 1991). However, an intrinsic functional B cell defect has recently been described. Indeed, Switch (S) region junctions in B cells from AT patients have been shown to be abnormal, dependent on short sequence microhomologies, and devoid of mutations, which is not the case in Nijmegen breakage syndrome. (Pan et al., 2002). In contrast, SHM is normal in the variable region of Ig genes (Pan-Hammarstrom et al., 2003).

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This finding, consistent with the observed role of ATM in double-stranded DNA repair, shows that ATM is a CSR-specific DNA repair molecule that is dispensable for SHM.

V. Concluding Remarks

The study of inherited immunodeficiencies has provided insight into the differentiation and maturation of cells of the immune system in humans. All known hyper-IgM syndromes involve defects in CSR, and most of these syndromes also display defective generation of SHM. The first description of the X-linked form (HIGM1), followed by description of HIGM3 and NEMO deficiency, provided clear evidence that the CD40 activation pathway, including the activation and nuclear translocation of the NF-kB transcription complex, was essential. Additional data indicated that a separate B cell maturation pathway, independent of the CD40L/CD40 pathway and occurring outside the germinal centers, possibly in the spleen marginal zone, might attenuate the infectious consequences of defective CD40 signaling in B cells. The role of a specific molecule transiently produced in B cells undergoing CSR or SHM—activation-induced cytidine deaminase—was then demonstrated in studies in a subset of patients with an autosomal recessive form of HIGM and in AID/ mice. This led to the conclusion that AID is absolutely required for both CSR and SHM, linking these two B cell recombination processes. Description of the immune defect indicated that AID acts upstream from the generation of DNA breaks at least in S regions, a common prerequisite for both CSR and SHM. Thus AID, which deaminates cytosine to give uracil residues, exerts its activity either by editing an mRNA encoding an endonuclease or, alternatively, by directly modifying DNA. Several lines of evidence, originating from experiments in E. coli or from cell-free assays, are consistent with a DNA-editing role for AID, but no definitive evidence has yet been provided in vivo. The recent description of a CSR deficiency in mice and humans lacking uracil-N glycosylase, which removes misintegrated uracil residues from DNA, provides further support for the DNA-editing hypothesis. Additional studies of human HIGM could help to resolve this issue. The most frequent autosomal recessive form of HIGM is characterized by defective CSR and normal generation of SHM. The defect is located downstream from AID activity and AID-induced DNA modification, suggesting a probable DNA repair defect. Elucidation of the molecular basis of this HIGM syndrome should increase our understanding of the CSR process, including its DNA repair phase, which is as yet poorly characterized. The findings of these studies suggest that there may be a B cell-specific DNA repair pathway necessary for CSR implementation.

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advances in immunology, vol. 82

The Biological Role of the C1 Inhibitor in Regulation of Vascular Permeability and Modulation of Inflammation ALVIN E. DAVIS, III, SHENGHE CAI, AND DONGXU LIU Harvard Medical School, CBR Institute for Biomedical Research, Boston, Massachusetts 02115

I. Introduction

The C1 inhibitor (C1INH) was first described as a heat-labile factor in human plasma that inhibited the esterolytic activity of the first component of complement (Ratnoff and Lepow, 1957). Although it is the only plasma protease inhibitor that regulates complement system activation (Sim et al., 1979b; Ziccardi, 1981), it is at least as important as a regulator of activation of the contact system (de Agostini et al., 1984; Harpel et al., 1985; Pixley et al., 1985; Schapira et al., 1982b; van der Graaf et al., 1983). Because the description of C1INH as an inhibitor of complement system activation preceded the demonstration of inhibition of contact system activation, there probably was a somewhat greater emphasis on complement regulatory activity than on its effects on the contact system, particularly among immunologists. In addition, the contact system proteases can be inactivated by other protease inhibitors, such as a2-macroglobulin or a1-antitrypsin, which contributed to the assumption that C1INH was a less important regulator of contact system activation than of complement system activation. However, using a variety of different experimental approaches, a great deal of data accumulated during the 1970s and 1980s clearly demonstrated that C1INH provided the majority of the plasma inhibitory capacity toward both factor XIIa and plasma kallikrein, and provided evidence that it is the primary regulator of contact system activation (de Agostini et al., 1984; Gallimore et al., 1979; Gigli et al., 1970; Harpel et al., 1985; Lewin et al., 1983; McConnell, 1972; Pixley et al., 1985; Schapira et al., 1982b; van der Graaf et al., 1983). In addition to the complement and contact system proteases, C1INH is also able to inactivate a variety of other proteases, including plasmin and tissue plasminogen activator (tPA) (Harpel and Cooper, 1975; Huisman et al., 1995; Ranby et al., 1982; Ratnoff et al., 1969; Sulikowski and Patston, 2001). C1INH does not appear to play a major role in the inactivation of plasmin in vivo; its primary inhibitor is a2-antiplasmin (Aoki et al., 1977; Harpel, 1981). However, some evidence suggests that it may contribute to the inactivation of tPA. C1INH–tPA complexes were detected in the plasma of some normal subjects at rest and after exercise, desmopressin infusion, or venous occlusion, and in peritoneal fluid from patients with peritoneal inflammatory disease (Huisman et al., 1995). Complexes were also detected in the plasma of patients following 331 Copyright 2004, Elsevier Inc. All rights reserved. 0065-2776/04 $35.00

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tPA infusion. Lastly, there is also evidence for activation of both the coagulation cascade and the fibrinolytic pathway during attacks of hereditary angioedema (HAE) (Cugno et al., 1993, 1997). This may suggest either that C1INH plays a role in the regulation of these pathways in vivo or that activation of these pathways by other exogenous factors triggers attacks of angioedema. Partial deficiency of C1INH results in hereditary angioedema, a disease characterized by recurrent episodes of localized edema of the skin or of the mucosa of the gastrointestinal tract or upper airway. Patients with HAE are heterozygous for deficiency of C1INH. The disease, therefore, has an autosomal dominant inheritance and may result from lack of expression of C1INH from one allele (type 1 HAE) or from expression of a nonfunctional C1INH protein (type 2 HAE). A major role of C1INH, therefore, is to prevent the development of excessive vascular permeability. Angioedema in HAE results from increased permeability at the level of the postcapillary venule. It is not associated with symptoms or signs of inflammation or of an allergic component. Accordingly, the fluid accumulation is not accompanied by transmigration of leukocytes from the vascular to the extravascular space and, as would be expected, therapy with antihistamines, epinephrine, or antiinflammatory agents is not effective. Because C1INH regulates activation of both the complement and contact systems, it seemed likely that angioedema might be mediated by products of activation of either (or both) system(s). The majority of data accumulated over the past several years now strongly support the hypothesis that the major mediator is bradykinin generated via the contact system (see Section II.A). Patients with HAE are susceptible to the development of autoimmune disease, particularly sytemic lupus erythematosus (Brickman et al., 1986a, 1986b; Donaldson et al., 1977). Insufficient regulation of classic pathway activation results in a secondary depletion of the second and fourth components of complement, which are the natural substrates of C1s. Genetic deficiencies of these complement proteins are associated with the development of autoimmunity. In both cases, this association probably is a product of resulting abnormalities in the clearance/catabolism of immune complexes and/or to abnormalities in B lymphocyte maturation and activation secondary to inadequate complement receptor-mediated signaling (Boackle and Holers, 2003; Prodeus et al., 1998). Surprisingly, C1INH has also proven to be protective in a variety of animal models of inflammatory disease (see Section VI). These raise the question as to whether this protection is mediated strictly via the known activities of C1INH, that is, via regulation of complement and contact system activation, or if it might have other, not previously identified antiinflammatory effects. Analysis of the mechanism of inhibition of alternative pathway activation by C1INH (Jiang et al., 2001) and the protective effect of C1INH in endotoxin shock suggest that this may be the case (Liu et al., 2003) (see Section VI.D).

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II. C1 Inhibitor Structure and Function

A. The Serpin Mechanism of Inhibition C1INH is a member of the serpin family, most members of which are serine proteinase inhibitors. The members of this family share amino acid sequence homology and a similar three-dimensional structure. Those that are inhibitors share the same inhibitory mechanism. Protease inactivation depends on a trapping mechanism that is activated following recognition of the pseudosubstrate reactive center loop by protease. This reactive center loop is displayed above the surface of the molecule. Cleavage of the reactive center peptide bond (P1–P10 ) triggers insertion of the reactive center loop into the fivestranded b sheet A to convert it to a six-stranded sheet (Fig. 1). This moves the bound protease from one pole of the molecule to the other, distorts the protease catalytic triad, and results in covalent bond formation between the P1 residue of the inhibitor and the active site serine of the protease (Huntington et al., 2000). The protease within the complex, because of the distortion, is also more susceptible to proteolytic attack. Although the crystal structure of C1INH has not been determined, the number of conserved structurally important sequence elements, confirmed by molecular modeling, indicates that the three-dimensional structure of the serpin domain of C1INH is very similar to other serpins that have been crystallized (Bos et al., 2002). Many serpin–protease complexes, including C1INH–C1s complexes, are removed from the circulation primarily via the low-density lipoprotein receptor-related protein (Kasza et al., 1997; Storm et al., 1997). These complexes, as expected, are removed from the circulation much more rapidly than is the native, active C1INH (Kasza et al., 1997; Malek et al., 1996; Storm et al., 1997). B. Function of the Amino-Terminal Nonserpin Domain C1INH consists of the serpin domain together with an amino terminal nonserpin domain of approximately 120 amino acids. This domain does not share any homology with other serpin family members. It is heavily glycosylated with three N-linked and at least seven O-linked carbohydrate groups; three additional N-linked groups are contained within the serpin domain (Fig. 2). The amino-terminal domain is mucin-like, with a repeating peptide unit containing the sequence Glx-Pro-Thr-Thr (Bock et al., 1986). This sequence, with minor variations, is repeated seven times, and several other similar sequences are also present. Deglycosylation of C1INH with N-glycanase, O-glycanase, or both has no significant effect on protease inhibitory function (Reboul et al., 1987). Carbohydrate, or, more precisely, sialic acid does appear to play a role in the in vivo stability of C1INH. Removal of sialic acid enhances the clearance of C1INH in the rabbit, presumably via binding to the asialoglycoprotein receptor (Minta, 1981).

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Fig 1 Serpin–protease complex formation. Ribbon diagram of native a1-antitrypsin with trypsin aligned above in the docking orientation (left). The complex (right) shows the 71 A˚ shift of the P1 residue of a1-antitrypsin, with insertion of the reactive center loop into b sheet A. Red, a1-antitrypsin b sheet A; yellow, reactive center loop; green ball-and-stick, P1 methionine; cyan, trypsin (with helices in magenta for orientation); red ball-and-stick, active site Ser-195. Reprinted with permission from Huntington et al. (2000).

The major portion of the amino-terminal domain is not required for complex formation with target proteases (Coutinho et al., 1994). A recombinant C1INH with an amino-terminal truncation at amino acid 97 complexed with C1s, plasma kallikrein, and factor XIIa to the same extent and at the same rate as wild-type recombinant C1INH. Recently these observations were confirmed and extended by the demonstration that an identical truncated inhibitor displayed normal inhibition kinetics (Bos et al., 2003). However, Bos et al. (2003) also showed that the absence of the connecting segment between the serpin and nonserpin domains (amino acids 100 through 116) resulted in a nonfunctional C1INH molecule that polymerized, probably because Cys-101

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Fig 2 Domain structure and glycosylation of C1 inhibitor. The amino-terminal heavily glycosylated domain consists of approximately the first 100–120 amino acids and contains N-linked carbohydrate attachment sites at Asn-3, -47, and -59, and O-linked sites at Ser-42 and Thr-26, -49, -61, -66, -70, and -74. The serpin domain contains N-linked sites at Asn-216, -231, and -330.

and Cys-108 are required to disulfide bond with Cys-406 and Cys-183, respectively, in order to maintain stability of b sheet A. This conclusion was derived from analysis of the mutation in an individual with HAE who was found to have a deletion of Asp-62 through Thr-116. This mutant and a mutant in which the amino-terminal 116 amino acids were deleted were nonfunctional due to spontaneous multimer formation. It was suggested that this segment in C1INH plays a similar role to that of heparin in antithrombin III, in that it maintains C1INH in the active serpin conformation with a completely exposed reactive center loop and that it prevents the formation of an inactive latent form (Bos et al., 2003). No other convincing function for the amino-terminal domain has been demonstrated. We had previously hypothesized that the extended rod-like nature of this domain (Odermatt et al., 1981) might restrict access to complex proteases, such as C1r and C1s within the C1 macromolecule, or plasma prekallikrein complexed with high-molecular-weight kininogen (Coutinho et al., 1994). However, comparison of the ability of the truncated and recombinant full-length C1INH to prevent activation of intact C1 (unpublished data) combined with the observation that wild-type and the truncated C1INH are equally active in reversing increased vascular permeability in C1INH-deficient mice (see Section V.B) (Han Lee et al., 2003) suggest that the amino-terminal domain does not play a role in limiting access to complexed proteases. Recent data, however, indicate that the amino-terminal domain may play an important role in C1INH-mediated protection from endotoxin shock (see Section VI.D). C. Potentiation of C1INH Activity by Heparin The activity of C1INH against some target proteases, but not others, is potentiated by heparin. The rate of reactivity with C1s and, to a lesser extent, C1r, is enhanced by heparin (Lennick et al., 1986; Sim et al., 1980). This

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enhancement is the primary explanation for the anticomplementary effect of heparin (Caughman et al., 1982). Activity against C1s is enhanced 30- to 60-fold, and is also enhanced against coagulation factor XI, but heparin has no effect on the inhibition of factor XIIa or kallikrein (Caldwell et al., 1999; Wuillemin et al., 1996). The mechanism of enhancement of C1INH by heparin is not clear, but it almost certainly differs from the heparin-mediated enhancement of antithrombin III activity against thrombin. In this instance, heparin induces a conformational change in antithrombin in which the major b sheet A is converted from a six-stranded form in which the reactive center loop is partially inserted to one in which the loop is exposed, with sheet A remaining in a five-stranded form (Jin et al., 1997). As pointed out by Bos et al. (2002), it is unlikely that enhancement of C1INH activity is via a similar mechanism because the C1INH reactive center loop, being five amino acids shorter than that of antithrombin III, is probably too short for the partially inserted form to exist. However, other data, including the fact that both low-molecular-weight heparin and low-molecular-weight dextran enhance C1INH activity as well as their higher molecular weight counterparts, suggest that the mechanism is via a conformational change rather than a simple bridging mechanism (Mauron et al., 1998; Wuillemin et al., 1997). The observation that C1s does not bind heparin supports this suggestion (Sahu and Pangburn, 1993). However, the finding that activity against some proteases is enhanced (C1r, C1s, factor XI), while activity against others is unaffected (factor XII, kallikrein), would seem to be more consistent with a bridging mechanism. These questions, for now, remain unanswered. The heparin-binding site in antithrombin III consists of positively charged residues within helix D, with contributions by residues near the amino-terminus and within helices A and C (Jin et al., 1997). Based on homology modeling, Bos et al. (2002) suggested that Lys residues 189 and 194, within helix D, are candidates for a potential C1INH heparin-binding site. Preliminary data from our laboratory suggest that these residues do participate in heparin binding, although other sites are likely to contribute also (unpublished data). Because heparin enhances C1INH activity against C1r and C1s, but not against factor XIIa and kallikrein, it relatively specifically enhances complement system inhibition while having no effect on kinin generation via the contact system. It is possible that this property may be used therapeutically in situations in which relatively specific inhibition of complement system activation is desired. For example, in an ex vivo pig lung-to-human xenotransplantation model, in which hyperacute rejection is complement mediated, low-molecular-weight dextran prolonged the time until hyperacute rejection by greater than 2-fold (Fiorante et al., 2001). However, the utility of such approaches is limited by the anticoagulant effects of glycosaminoglycans, including low-molecular-weight dextran (Zeerleder et al., 2002).

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III. Regulation of Complement Activation

A. Inhibition of Classic Complement Pathway Activation As pointed out earlier, C1INH was discovered in the mid-1950s during studies that characterized and isolated the first component of complement (Ratnoff and Lepow, 1957). C1INH very likely also regulates the lectin pathway of complement activation and also may be involved in regulation of the alternative pathway. C1INH is the only plasma protease inhibitor that inactivates the C1r and C1s subcomponents of C1 (Sim et al., 1979b; Ziccardi, 1981) and is, therefore, the primary regulator of classical pathway activation (Fig. 3). Upon complex formation with the activated C1s–C1r–C1r–C1s tetramer within the C1 macromolecule, two C1INH–C1r–C1s–C1INH complexes dissociate from C1q (Laurell et al., 1976; Sim et al., 1979a; Ziccardi and Cooper, 1979). The interaction with C1r is the primary determinant of this dissociation (Sim et al., 1979a). The mechanism of regulation of activation is primarily via inactivation of proteolytically active C1r and C1s following activation of C1 by an immune complex, which thereby limits the consumption of C2 and C4. However, C1INH also has been reported to prevent spontaneous activation of circulating C1 via a reversible interaction with the zymogen forms of C1r and C1s (Folkerd et al., 1980; Ziccardi, 1985). The mechanism of and the relative importance of this interaction in the regulation of C1 activation, however, have not been investigated further. B. Inhibition of Lectin Pathway Activation Lectin pathway activation is similar to that of the classic complement pathway. Mannan-binding lectin (MBL) binds to specific carbohydrates on the surface of a variety of microorganisms. This results in the subsequent activation of MBL-associated proteases (MASPs) 1 and 2, both of which are highly homologous to C1r and C1s (Petersen et al., 2001; Wong et al., 1999). However, the MASPs do not function sequentially, as do C1r and C1s. MASP-2 is activated following the binding of MBL, and it mediates the cleavage of C2 and C4 (Thiel et al., 1997) (Fig. 3). It therefore is C1s-like in its function. MASP-1, on the other hand, does not have activities that are similar to either C1r or C1s, but it can cleave and activate C3, albeit quite inefficiently (Hajela et al., 2002). This activity, therefore, seems unlikely to represent its biological function. In addition, a third protease, MASP-3, is an alternatively spliced product of the same gene as MASP-1 (Dahl et al., 2001). Finally, a related protein with no proteolytic activity, Map19, is an alternatively spliced product of the MASP-2 gene (Stover et al., 1999). The functions of MASP-1 and -3 and of Map19 remain to be determined. Furthermore, the stoichiometry of the MBL –MASP complexes have not been determined, although the data suggest that one complex consists of MBL with MASP-2 and MASP-3 and another

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Fig 3 The pathways of complement activation. In the classical pathway, immune complexes (Ag:Ab) bind and activate C1, resulting in cleavage and activation of C4 and C2, which form the C4b2a enzyme (classic pathway C3 convertase), which cleaves C3 to C3a and C3b. Association of C3b with the convertase allows the cleavage of C5 and activation of the terminal pathway. The lectin pathway is activated directly by the binding of mannan-binding lectin (MBL) to microbial surfaces. This results in activation of MBL-associated proteases (MASP)-1 and -2. MASP-2 cleaves C4 and C2, which then activate the remainder of the pathway as with classic pathway activation. C1INH regulates activation of both of these pathways by covalent complex formation with activated C1r, C1s, and the MASPs. The alternative pathway differs in that it is constantly activated in the fluid phase. C3b resulting from this low-grade activation is usually inactivated. However, in the presence of a suitable microbial surface with chemical characteristics that protect C3b from inactivation, alternative pathway activation will proceed. C3b binds factor B, which is then cleaved by the active protease factor D to form the alternative pathway convertase, C3bBb, the activity of which is stabilized by properdin (P). The major plasma regulator of this enzyme is factor H, which binds to C3b and enhances the decay of the C3bBb complex. Factor H bound to C3b serves as a cofactor for factor I-mediated enzymatic inactivation of C3b. C1INH recently has been shown to have alternative pathway-inhibiting activity, apparently very similar to that of factor H (Jiang et al., 2001). Analogous to the classic pathway, association of a second C3b with the C3bBb complex converts this complex to a C5-cleaving enzyme, which results in activation of the terminal pathway.

consists of MBL with MASP-1 and MAp19 (Dahl et al., 2001). MASP-1 and MASP-2 both form complexes with, and their proteolytic activities are inactivated by, C1INH, both as the isolated proteases and within the MBL complex (Matsushita et al., 2000; Petersen et al., 2000). However, appropriate experiments to determine whether C1INH regulates lectin pathway activation have not been performed. In particular, the kinetics of the reaction with MASP-2

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have not been determined. It has also been reported that a2-macroglobulin associates with and inhibits the MBL complex (Storgaard et al., 1995; Terai et al., 1995). However, Petersen et al. (2000), using highly purified a2-macroglobulin, did not find inhibition, although the a2-macroglobulin did associate with the MBL complex. Interestingly, Peterson et al. (2000) also found that the Kunitz domain inhibitor, aprotinin, was able to inhibit MASP-2 and MBL pathway activation. Therefore, although the data currently available indicate that C1INH may be a regulator of lectin pathway activation, it remains possible that other inhibitors also play a role.

C. Inhibition of Alternative Pathway Activation C1INH inhibits the lysis of paroxysmal nocturnal hemoglobinuria erythrocytes in human serum (Jiang et al., 2001). This lysis is mediated via activation of the alternative complement pathway. This inhibition appears to be the result of binding of C1INH to C3b, which was demonstrated using a dot blot assay. We have confirmed this binding using an enzyme-linked immunosorbent assay (ELISA) in which C3b is in the solid phase and C1INH is in the liquid phase (unpublished data). This interaction with C3b interferes with the binding of factor B to C3b, and thereby prevents alternative pathway activation. No binding to either factor B or factor D was observed. In addition to the direct binding of C1INH to C3b, experiments were performed demonstrating that incubation of C1INH with C3b interfered with the subsequent formation of the alternative pathway C3 convertase. Because C3b is not a protease, it seems likely that this inhibition does not require the protease inhibitory activity of C1INH, although this has not been formally proven. The removal of C1INH from serum by immunoabsorption increased the extent of alternative pathway activation, which suggests that under normal conditions, it functions to down-regulate alternative pathway activation. This inhibition of alternative pathway activation is only the second example of a C1INH activity that does not depend on stable complex formation with protease. The first was the reversible inhibition of spontaneous activation of macromolecular zymogen C1 described in Section III.A (Folkerd et al., 1980; Ziccardi, 1985). The mechanism for this inhibition has never been determined, although it is presumed to result from some type of reversible interaction with the active site of the protease. In the case of alternative pathway inhibition, even though the ultimate effect is to prevent protease activation, the immediate effect of the binding to C3b is to prevent the formation of a bimolecular complex, not to inhibit a protease. It will be important to characterize the mechanism and molecular determinants of this inhibition, and the relative role of C1INH in comparison with other mechanisms of alternative pathway regulation.

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IV. Regulation of Contact System Activation

A. Activation IN VITRO by Negatively Charged Surfaces The contact system was originally described as a group of plasma proteins activated in vitro following the addition of an artificial negatively charged substance such as glass, kaolin, elagic acid, dextran sulfate, and possibly endotoxin lipopolysaccharide (Cochrane et al., 1973; Colman and Schmaier, 1997; Griffin, 1978; Kaplan et al., 1997; Kirby and McDevitt, 1983; Revak et al., 1977). This leads both to the generation of bradykinin and to activation of the intrinsic coagulation pathway via factor XIIa-mediated activation of factor XI (Fig. 4). The pivotal reaction is the binding of factor XII, a serine protease zymogen, to the negatively charged surface, which leads to its autoactivation to factor XIIa (Dunn et al. 1982; Espana and Ratnoff, 1983; Miller et al., 1980; Silverberg et al., 1980; Tankersley and Finlayson, 1984; Wiggins and Cochrane, 1979). Factor XIIa, in turn, activates factor XI and plasma prekallikrein, also serine protease zymogens, to their active forms, factor XIa and kallikrein. Approximately 80–90% of prekallikrein circulates in plasma in an equimolar complex with its substrate, high-molecular-weight kininogen (HK). Kallikrein cleaves HK at two sites to release the nine amino acid residue peptide, bradykinin, which mediates a variety of activities, including vasodilation, increased vascular permeability, constriction of uterine and gastrointestinal

Fig 4 Contact pathway activation in vitro. The addition of a negatively charged substance such as glass, kaolin, or elagic acid to plasma results in the autoactivation of coagulation factor XII. Factor XIIa, in turn, cleaves and activates both factor XI to XIa to trigger the intrinsic coagulation pathway and prekallikrein (PK) to the active protease, kallikrein (Kal). Kallikrein cleaves highmolecular-weight kininogen (HK) at two sites to release bradykinin (Bk).

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smooth muscle, constriction of coronary and pulmonary vasculature, and bronchoconstriction (Kaplan et al., 1997). Unfortunately, an in vivo counterpart to this pathway utilizing activation initiated by a negatively charged surface has not been convincingly demonstrated. In particular, there is now general agreement that the intrinsic coagulation pathway as described previously is unlikely to be physiologically important. The best evidence for this is the fact that although deficiencies of factor XII, prekallikrein, or highmolecular-weight kininogen lead to abnormal coagulation in vitro, individuals with these deficiencies have no hemorrhagic disorders. B. Activation IN VIVO on Endothelial Cell Surfaces The contact system, in vivo, almost certainly is activated on cell surfaces, including particularly neutrophils, platelets, and endothelial cells (Greengard and Griffin, 1984; Gustafson et al., 1986, 1989; Meloni et al., 1992). The activation mechanism has been best characterized on the endothelial cell surface, although some of the details of this mechanism remain to be elucidated and some controversy remains to be resolved (Fig. 5). The first progress toward characterization of this pathway came from the demonstration of zincdependent binding of HK to the surface of endothelial cells (Schmaier et al., 1988; Van Iwaarden et al., 1988). Next, factor XII was also shown to bind to endothelial cells and its binding was competitive with that of HK (Reddigari et al., 1993). It therefore appeared likely that HK and factor XII were binding at or near the same site. Subsequently, at least three different proteins on the endothelial cell surface, cytokeratin 1 (CK1), the receptor for the globular domain of C1q (gC1qR), and urokinase plasminogen activator receptor (uPAR), have been identified as HK-binding proteins (Colman et al., 1997; Hasan et al., 1998; Herwald et al., 1996; Joseph et al., 1996; Shariat-Madar et al., 1999). Endothelial cell glycosaminoglycans also are able to bind HK (Renne et al., 2000). The following observations led to the hypothesis that CK1, gC1qR, and uPAR make up a multiprotein receptor complex: (1) antibodies to the HK-binding domains of each protein completely block binding of HK to endothelial cells; (2) CK1 and uPAR colocalize on the cell surface, while CK1 and gC1qR partially colocalize (Mahdi et al., 2001; Schmaier et al., 1999). The explanation for this is that there are many more gC1qR per cell than there are either CK1 or uPAR (Barnathan et al., 1990; Mahdi et al., 2001; Peerschke et al., 1996). CK1 is able to bind to both gC1qR and uPAR, while the latter two do not bind to one another (Kaplan et al., 2002). The binding of the HK–prekallikrein complex to the receptor complex on the endothelial cell results in activation of prekallikrein to kallikrein, and the release of bradykinin from HK. The data from different groups who have investigated the activation of the contact system on endothelial cell surfaces differ in the apparent relative importance of factor XII and in whether it is

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Fig 5 Proposed mechanisms of contact pathway activation on endothelial cell surfaces. (A) Activation of prekallikrein by factor XII (Joseph et al., 2001; Reddigari et al., 1993). In this proposed mechanism, efficient activation requires factor XII, which, following interaction with the gC1qR, autoactivates to XIIa. The HK–prekallikrein (PK) complex binds to the endothelial surface, probably via the trimolecular complex consisting of the gC1qR, urokinase plasminogen activator receptor (uPAR), and cytokeratin 1 (CK1). The active factor XIIa then cleaves PK within the complex, which in turn cleaves the HK with which it is associated to release bradykinin (Bk). (B) Activation of prekallikrein by prolylcarboxypeptidase (PCRP) and/or Hsp90 (Mahdi et al., 2001, 2002; Shariat-Madar et al., 2002a, 2002b; Zhao et al., 2001). This mechanism proposes that rather than factor XII, cleavage of PK is mediated by PCRP. Adapted with permission from Schmaier (2002).

required for efficient activation. Kaplan and colleagues report that efficient activation requires the presence of factor XII and that factor XII autoactivates to factor XIIa on the endothelial cell surface as a result of its interaction with gC1qR (Fig. 5A) (Joseph et al., 2001; Reddigari et al., 1993). The data from

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Schmaier’s group indicate that factor XII is not required, that it does not autoactivate when bound to the endothelial cell surface, and, in fact, that in the presence of bound HK–prekallikrein, factor XII does not bind to the endothelial cell (Fig. 5B) (Shariat-Madar et al., 2002a). The data supporting this contention are that factor XII binding requires a 30-fold higher concentration of zinc than that of HK, and that at physiological concentrations of HK and vitronectin, factor XII binding to endothelial cells is inhibited 100% and 50%, respectively (Mahdi et al., 2002; Zhao et al., 2001). This group reports that the enzyme responsible for the activation of prekallikrein to kallikrein is endothelial cell prolylcarboxypeptidase, a serine protease that also can convert angiotensin I and angiotensin II to angiotensin II1–7, which causes vasodilation by inducing NO formation (Ren et al., 2002; Santos et al., 1992). Furthermore, the data from Schmaier’s group indicate that factor XII activation on endothelial cells requires prior prekallikrein activation; this factor XII activation in turn can increase the rate and extent of kallikrein formation on the endothelial cell and also can initiate factor XI activation (Rojkjaer et al., 1998). Kaplan’s group, while agreeing that a factor XII-independent mechanism for prekallikrein activation does exist on the endothelial cell, suggests that this process is extremely slow and that the factor required for activation is heat shock protein 90 (Hsp90) (Joseph et al., 2002a, 2002b). Hsp90 has not been demonstrated to possess proteolytic activity, so the mechanism by which it catalyzes activation remains to be defined. It also is not known if it works in concert with, or independently from, prolylcarboxypeptidase. The differences in results and interpretation from these two groups remain to be resolved. However, although there are important differences in the precise mechanism, all groups are in general agreement that activation on the endothelial surface (and probably other cell types, in addition) is the biologically important mode of activation of the contact system. In addition, it has been quite clear for a number of years that C1INH is the major regulator of activation of the contact system. The first evidence that this was the case, although it was not apparent at the time, came from the observation of Landerman et al. (1962) that plasma from patients with HAE was deficient in kallikrein inhibitory capacity. The following year, Donaldson and Evans (1963) discovered that the deficient plasma protein in HAE is C1INH. Early studies suggested that C1INH was the major inhibitor of plasma kallikrein (Gigli et al., 1970; McConnell, 1972). Subsequent studies suggested that C1INH provided from about half to greater than three quarters of the plasma inhibitory activity toward kallikrein, while most of the remainder was provided by a2-macroglobulin (Harpel et al., 1985; Schapira et al., 1982b; van der Graaf et al., 1983). Of particular relevance was the observation that the rate of kallikrein inactivation was dramatically slower in C1INH-deficient plasma, but was only slightly slower in a2-macroglobulin-deficient plasma (van der Graaf et al., 1983).

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Similarly, factor XIIa (and XIIf, a smaller proteolytically active fragment of factor XII) is inhibited almost solely by C1INH (de Agostini et al., 1984; Pixley et al., 1985). It should also be kept in mind that the ability of C1INH to inactivate kallikrein or factor XIIa on the surface of endothelial cells has not been tested. In this regard, it may be relevant that Bergamaschini et al. (2001b) have shown that C1INH binds to endothelial cells when incubated in the cold. The molecular mechanism responsible for this binding has not been characterized. We have found that C1INH binds to human umbilical vein endothelial cells treated with tumor necrosis factor (TNF)-a and that this binding appears to be mediated via binding to selectins (unpublished data). These observations may suggest a mechanism for the concentration of C1INH at sites of inflammation and contact system activation. C. Contact System Function Activation of the contact system results in a variety of activities, many of which can be ascribed to liberated bradykinin. However, a number of potentially important activities also are mediated by activated kallikrein, factor XIIa, and HK. Table I lists many of these functions. The most obvious and dramatic effect of activation is vasodilation with a consequent decrease in blood pressure secondary to the effects of bradykinin on endothelial cells and vascular smooth muscle. Aside from its role in blood pressure regulation, activation of the contact system also results in a variety of antithrombotic and fibrinolytic effects. These include inhibition of thrombin-induced platelet aggregation, both by bradykinin and by HK (Hasan et al., 1996; Jiang et al., 1992; Meloni and Schmaier, 1991; Puri et al., 1991), the induction of tissue plasminogen activator release by bradykinin (Brown et al., 1997; Smith et al., 1985), and activation of urokinase plasminogen activator by kallikrein (Hauert et al., 1989). In addition to the increase in vascular permeability mediated by bradykinin, a variety of other proinflammatory effects result, including enhanced prostacyclin and superoxide generation by bradykinin-stimulated endothelial cells (Holland et al., 1990; Hong, 1980), neutrophil/monocyte chemotaxis and neutrophil aggregation and elastase release induced by kallikrein, and neutrophil aggregation and degranulation induced by factor XIIa/XIIf (Gallin and Kaplan, 1974; Kaplan et al., 1972; Schapira et al., 1982a; Wachtfogel et al., 1983). The contact system, therefore, interacts both directly and indirectly with a variety of other systems. In addition to those described earlier, as discussed by Schmaier (2002), the contact system interacts with the renin–angiotensin system in a variety of ways. First, plasma kallikrein, in addition to cleaving HK to release bradykinin, can activate prorenin to renin (Derkx et al., 1979; Sealey et al., 1978, 1979). Second, angiotensin-converting enzyme (ACE) converts angiotensin I to angiotensin II, but also inactivates bradykinin, both of which have the effect of increasing blood pressure, and the

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TABLE I Activities Resulting from Contact System Activation Bradykinin Vasodilation Increased vascular permeability Induction of NO formation Release of tissue plasminogen activator resulting in plasmin generation Stimulation of prostacyclin synthesis Stimulation of superoxide formation Induction of NO formation Contraction of uterine and gastrointestinal smooth muscle Bronchoconstriction Inhibition of thrombin-induced platelet aggregation Kallikrein Urokinase plasminogen activator activation Activation of prorenin to renin Neutrophil/monocyte chemotaxis Neutrophil aggregation/elastase release Complement factor B activation Factor XIIa/XIIf C1r/C1s activation Neutrophil aggregation/degranulation High-molecular-weight kininogen Bradykinin release Procoagulant activity secondary to binding of prekallikrein and factor XI Cysteine proteinase inhibition Inhibition of thrombin-induced platelet aggregation

bradykinin(1–5) resulting from this cleavage inhibits thrombin-induced platelet aggregation (Hasan et al., 1996). In addition to its newly described role in the activation of prekallikrein on endothelial cells, prolylcarboxypeptidase converts angiotensin II to angiotensin II(1–7) (Odya et al., 1978). Both of these actions ultimately result in vasodilation with a decrease in blood pressure. C1INH, by virtue of its regulation of contact system activation, therefore, indirectly may have a variety of far-reaching biological effects, in addition to the antiinflammatory effects resulting from inhibition of complement system activation. V. Regulation of Vascular Permeability

A. Mediation of Angioedema As demonstrated by the fact that C1INH deficiency is characterized by angioedema, C1INH clearly is required for regulation of normal vascular permeability. Angioedema is the result of loss of endothelial barrier function

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within the postcapillary venule with leakage of fluid and protein, but not cells, from the intravascular to the extravascular space. During attacks of angioedema, C1INH levels decrease from the approximately 30% of normal levels observed during symptom-free intervals, and activation of both the complement and contact systems takes place. Decreased levels of both C4 and C2, together with the presence of circulating complexes of C1INH with C1r and C1s, indicated that complement system activation takes place during episodes of angioedema (Laurell et al., 1976). Similarly, cleavage of HK has been demonstrated in the plasma of patients with HAE (Berrettini et al., 1986; Buhler et al., 1995; Cugno et al., 1996; Nielsen et al., 1996). Because both systems are activated, it has been difficult to determine which system was responsible for the mediation of symptoms. Bradykinin was an obvious candidate, and a number of studies over the years strongly suggested that it might be the mediator (Curd et al., 1980, 1982; Fields et al., 1983). A kinin-like peptide is generated in HAE plasma during incubation at 37 8C. Several early studies suggested that production of this peptide was dependent on complement system activation (Donaldson, 1973; Donaldson et al., 1969, 1970). In addition, C2 digested with C1s and plasmin resulted in release of a peptide that had vascular permeability-increasing activity (Strang et al., 1988). Using C1INHdepleted plasma rather than HAE plasma, a later study showed that vascular permeability-increasing activity could be generated from plasma depleted of C1INH and from C1INH-depleted C2-deficient plasma, but not from plasma deficient in any contact system proteins (Shoemaker et al., 1994). The factor in plasma responsible for this activity was isolated and sequenced and found to be bradykinin. Nussberger et al. (1998) subsequently demonstrated elevated levels of bradykinin in the plasma of HAE patients during attacks of angioedema. A different approach toward identification of the mediator of vascular permeability was provided by the fortuitous observation of a patient with systemic lupus erythematosus who had low levels of C4, who was ultimately shown to express a dysfunctional C1INH in which Ala-443, which is at the P2 position, was replaced with a Val (Wisnieski et al., 1994; Zahedi et al., 1995). A recombinant C1INH protein with this substitution was shown to have an altered specificity toward target proteases with greatly diminished activity against C1r and C1s but normal activity against plasma kallikrein and factor XIIa (Zahedi et al., 1995, 1997). Several members of this family had the same amino acid substitution, but no family member had ever experienced an episode of angioedema. Analysis of this family, therefore, strongly suggested that insufficient regulation of complement system activation alone does not result in angioedema and supported the hypothesis that the mediator is bradykinin.

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B. Analysis of the C1INH-Deficient Mouse One of the primary goals in developing a C1INH-deficient (C1INH/) mouse was to test the previous hypothesis. Both the C1INHþ/ and C1INH/ mice had no obvious abnormal phenotype. They appeared normal at birth, and subsequently developed and reproduced normally (Han et al., 2002). Heterozygous matings resulted in no increased fetal death among either the C1INHþ/ or the C1INH/ mice. The plasma from C1INH/ mice contained no C1INH protein, while that from the heterozygous deficient animals contained somewhat less than 50% of C1INH levels as compared with C1INHþ/þ mice. C4 levels were variable, but were decreased in most of the C1INH/ mice, which suggested unregulated complement system activation, as occurs in humans with C1INH deficiency. The mice do not have episodes of cutaneous angioedema. However, eight mice from two litters developed gastrointestinal edema with obstuction and one of these mice also developed laryngeal edema. These were isolated incidents that involved C1INHþ/ and C1INH/ mice; no C1INHþ/þ littermates were affected. The triggers, if any, for these attacks are not known. In addition, limited experiments intended to induce attacks have not succeeded. Although these attacks appeared to be consistent with gastrointestinal and laryngeal angioedema, and they have not been observed in wild-type littermates, because they have occurred so infrequently and have not been induced under controlled conditions, it is difficult to state with certainty that they are the result of C1INH deficiency. To test the hypothesis that the deficiency of C1INH resulted in increased vascular permeability, the mice were given intravenous injections of Evans blue dye. Both homozygous and heterozygous deficient mice displayed an obvious increase in vascular permeability in comparison with C1INHþ/þ littermates. Within a few minutes after the injection, the skin of the C1INHþ/þ mice became slightly blue while that of the deficient mice became intensely blue. The blue color was more apparent on the feet and around the nose and eyes, and was enhanced by the topical inflammatory agent, mustard oil. The increased permeability was quantitated by extracting the Evans blue dye from the feet of the mice (Fig. 6); permeability clearly was significantly greater in both the C1INH/ and C1INHþ/ mice. The response to a topical inflammatory stimulus also was exaggerated in the deficient mice in comparison with the wild-type mice. Although the amount of vascular permeability in the untreated ears of the deficient mice was not significantly increased in comparison with the C1INHþ/þ mice, the application of mustard oil in an amount that resulted in only a slight increase in C1INHþ/þ mice caused a dramatic increase in the deficient mice (Han et al., 2002). Proof that the increased vascular permeability was the result of C1INH deficiency and not some unforeseen associated defect was

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Fig 6 Increased vascular permeability in the footpads of C1INH-deficient mice. Evans blue dye was extracted from the paws of mice of the indicated genotypes and quantitated spectrophotometrically. Mice were either untreated or treated with intravenous C1INH (C1I), DX88 (DX), or Hoe140 (Hoe). Reprinted with permission from Han et al. (2002).

provided by the demonstration that the intravenous injection of human C1INH in an amount calculated to restore plasma levels to approximately normal completely reversed the increased vascular permeability (Fig. 6). The identity of the phenotypes of the C1INH/ and C1INHþ/ mice was unexpected. Because no human with complete deficiency has ever been identified, we predicted that complete deficiency would be lethal. However, the C1INH/ mice are viable, have normal fertility, and are otherwise perfectly normal, except for the increased vascular permeability, to which they are well-compensated. Although the factors responsible for the mild phenotype are not yet determined, there are several possible explanations. First, it is possible that below a critical C1INH level, activation of the proteolytic pathways regulated by C1INH is maximal and that a further decrease in the C1INH level has no additional effect. In the case of the complement system, however, based on the C4 and hemolytic complement levels, this does not seem to be the case. Reagents are being developed to determine whether contact system activation is maximal. A related possibility is that the presumed substrate, HK, is depleted in both the C1INHþ/ and C1INH/ mice.

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Therefore it would not be possible to release additional bradykinin. Lastly, other inhibitors may play a larger role in regulation of C1INH target proteases than is the case in the human. This could explain both the fact that the phenotype in the mouse is less severe than in the human and that the phenotypes of the two mice are the same. No data related to this possibility are currently available. To test the hypothesis that increased vascular permeability in C1INH deficiency is mediated by bradykinin generated by contact system activation, C1INH/ mice were treated with a highly specific plasma kallikrein inhibitor, DX88 (Dyax Corporation, Cambridge, MA), with the bradykinin type 2 receptor (Bk2R) antagonist, Hoe140 (Sigma Chemical Co., St. Louis, MO), and with the ACE inhibitor, captopril (Sigma Chemical Co.). Both DX88 and Hoe140 completely reversed the increased vascular permeability (Fig. 6). Captopril, which prevents the degradation of bradykinin, markedly increased vascular permeability. In addition, mice deficient in both C1INH and the Bk2R did not have any increase in vascular permeability and were indistinguishable from wild-type littermates (Fig. 6). Mice also were treated with a recombinant C1INH protein in which the amino terminal 97 amino acids had been deleted and that also contained the C1INH Ala-443 ! Val substitution described in Section V.A. This protein, which is relatively specific for the contact system proteases, was as effective as normal plasma-derived C1INH in reversing the increased vascular permeability (Han Lee et al., 2003). These data, taken together, provide strong support for the hypothesis that the increased vascular permeability in C1INH-deficient mice is mediated by bradykinin. These experiments also strengthen the argument that the mediator in HAE is bradykinin. In addition, the last experiment indicates that the amino-terminal heavily glycosylated nonserpin domain does not influence inactivation of contact system proteases in vivo. As pointed out by Schmaier (2002), these studies also support the idea that contact system activation is constantly taking place within the vascular system, but is tightly regulated by C1INH. This regulation not only maintains the endothelial barrier to the escape of fluid and protein, but also may influence blood pressure regulation and the balance of pro- and antithrombotic effects of the contact, the renin–angiotensin, and the fibrinolytic systems. VI. Modulation of Inflammation

A. Disease Models in Which Treatment with C1INH Has Been Beneficial Treatment with C1INH is effective at improving outcome in a variety of inflammatory disease models, including, in particular, reperfusion injury, hyperacute transplant rejection, and gram-negative endotoxemia. In addition,

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evidence suggests that C1INH might be useful in pancreatitis (Niederau et al., 1995; Schneider et al., 1999; Testoni et al., 1995; Yamaguchi et al., 1997), in traumatic and hemorrhagic shock (Horstick et al., 2001; Kochilas et al., 1997), and in the prevention of the vascular leak syndromes and inflammatory responses associated with thermal injury (Henze et al., 1997; Khorram-Sefat et al., 1998; Radke et al., 2000), interleukin (IL)-2 therapy (Hack et al., 1993, 1994; Ogilvie et al., 1994), and cardiopulmonary bypass (Tassani et al., 2001). B. Reperfusion Injury In several animal models of myocardial reperfusion injury, administration of C1INH just prior to or at the time of reperfusion resulted in decreased infarct size, decreased neutrophil accumulation in the myocardium, and decreased plasma levels of creatine kinase, C3a, and C5a (Buerke et al., 1995, 1998; Horstick et al., 1997, 2002). One study also showed that expression of P-selectin and ICAM-1 within the cardiac vasculature was abolished (Buerke et al., 1998). Similar beneficial effects have been observed in other reperfusion injury, including skeletal muscle injury and middle cerebral artery occlusion models (Akita et al., 2003; De Simoni et al., 2003; Nielsen et al., 2002). An early study treated three patients with C1INH following emergency surgical revascularization after failed percutaneous transluminal coronary angioplasty (Bauernschmitt et al., 1998). Hemodynamic stability could not be maintained following surgery due to myocardial reperfusion injury, but was rapidly achieved after initiation of therapy with C1INH. One study has evaluated C1INH use in patients with myocardial infarction and demonstrated diminished complement activation with reduction of troponin T and creatine kinase in patients who also received early thrombolytic therapy, suggesting that there may be some therapeutic efficacy (de Zwaan et al., 2002). Reperfusion injury also plays a role in solid organ transplantation. C1INH has been used in dogs and sheep in lung transplant models and was shown to improve early pulmonary dysfunction (Graeter et al., 1997; Salvatierra et al., 1997). Presumably, the effect of C1INH in reperfusion injury is largely due to inhibition of complement activation, which clearly is involved in the mediation of damage (Weisman et al., 1990). The observation that a small molecule inhibitor of C1s has effects similar to those of C1INH further strengthens this argument (Buerke et al., 2001). However, the finding of supression of selectin and ICAM-1 expression following therapy with C1INH in cardiac reperfusion injury is intriguing (Buerke et al., 1998). Data from different studies differ in whether complement activation plays a major role in the induction of adhesion molecule expression (Shandelya et al., 1993; Solvik et al., 2001). Of possible relevance is our observation that C1INH expresses the sialyl LewisX epitope via which it binds directly to both E and P selectins (Cai and Davis, 2003). This could provide a mechanism to concentrate

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C1INH at sites of intense complement or contact system activation. Furthermore, other preliminary data suggest that this binding may result in suppression of selectin expression (Cai and Davis, 2003). It is possible, therefore, that the effect of C1INH may be multifactorial and include complement inhibition, contact system inhibition, and inhibition of adhesion molecule expression. C. Hyperacute Transplant Rejection Hyperacute transplant rejection, as occurs particularly in the case of xenotransplantation, is complement mediated. Inhibition of natural antibodymediated complement activation is required for survival of xenotransplants. C1INH has been successfully used in a variety of in vitro, ex vivo, and in vivo models of xenotransplantation. In an early study, added C1INH inhibited the activation of porcine aortic endothelial cells incubated with human serum (Dalmasso and Platt, 1993). Subsequently, a surface-bound form of C1INH was used to protect Chinese hamster ovary cells and pig endothelial cells from lysis by human complement (Fukuta et al., 2003; Matsunami et al., 2000). Ex vivo perfusion of pig kidneys with human blood in the presence of added C1INH prolonged survival of the kidneys from 79 to 327 min (Fiane et al., 1999). Complement activation was reduced in comparison with controls, as were platelet and neutrophil activation, while no contact system activation was observed in either treated or control kidneys. These data suggested that platelet and neutrophil activation were largely a result of complement activation and that C1INH might prove useful in prevention of hyperacute rejection. Similar observations were made with an ex vivo pig lung with human blood model (Schelzig et al., 2001). Recently, soluble C1INH was shown to protect kidneys from hyperacute rejection in a pig to a cynomolgus monkey model (Hecker et al., 2002; Przemek et al., 2002). D. Endotoxin Shock The complement system has been implicated both in the pathogenesis of, and protection from, endotoxin shock. (Caliezi et al., 2001; Czermak et al., 1999; Fischer et al., 1997; Laudes et al., 2002; Strachan et al., 2000). In addition, the contact system may play a role in the mediation of septic shock (Colman, 1999). Based on the assumption that inhibition of complement and/ or contact system activation might be beneficial in sepsis/endotoxin shock, therapy with C1INH has been studied in a variety of animal models and has been demonstrated to improve the outcome in several respects (Caliezi et al., 2001; Fischer et al., 1997; Giebler et al., 1999; Guerrero et al., 1993; Jansen et al., 1998; Scherer et al., 1996; Schmidt et al., 1999a, 1999b; Triantaphyllopoulos and Cho, 1986). In endotoxin shock in dogs, C1INH prevented hypoxemia, the increase in intrapulmonary shunt, and the decrease in contact system factors

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induced by endotoxin (Guerrero et al., 1993). Similarly, in a rabbit model of endotoxin-induced hypercoagulability, C1INH infusion improved mean arterial pressure, increased central venous oxygen saturation, prevented the decrease in antithrombin III, and reduced fibrin deposition in the liver and lungs (Scherer et al., 1996). Additional observations in the rat and rabbit have confirmed and extended these observations (Giebler et al., 1999; Schmidt et al., 1999a, 1999b). In a lethal Escherichia coli sepsis model in baboons, C1INH supplementation prolonged survival, improved pathological changes in major target organs, blunted the decreases in plasma levels of factor XII and prekallikrein, decreased the appearance of C4 cleavage products, and reduced the production of TNF-a, IL-10, IL-6, and IL-8 (Jansen et al., 1998). C3- and C4-deficient mice were much more sensitive to endotoxin shock than were wild-type controls and were defective in the clearance of endotoxin from the circulation (Fischer et al., 1997). C3, therefore, appears to be required for normal endotoxin clearance. C1INH was depleted during endotoxemia in the C3/C4-deficient mice, which suggested that prolonged activation of the contact system secondary to inadequate endotoxin clearance resulted in enhanced consumption of C1INH. We recently have shown that both active plasma-derived C1INH and reactive center-cleaved C1INH (iC1INH) that has no protease inhibitory activity protected mice from lethal endotoxin shock (Fig. 7) (Liu et al., 2003). Both forms of C1INH and recombinant full-length C1INH also bound to Salmonella typhimurium lipopolysaccharide (LPS), as judged by ELISA and by nondenaturing polyacrylamide gel shift experiments. A recombinant C1INH variant in which the amino-terminal 97 amino acids were deleted, however, did not bind to LPS. Furthermore, this binding inhibited the binding of LPS to the mouse macrophage-like cell line, RAW 264.7, probably by preventing the interaction of endotoxin with LPS-binding protein. This inhibition prevented macrophage activation, as demonstrated by C1INHmediated suppression of TNF-a mRNA synthesis by LPS-treated RAW264.7 cells and by leukocytes in whole blood (Fig. 8). These data suggest, therefore, that C1INH contributes to protection from gram-negative endotoxin shock via three mechanisms: inhibition of excessive complement activation, which would limit the amount of C5a generated (Czermak et al., 1999; Laudes et al., 2002; Strachan et al., 2000); inhibition of contact system activation, which would limit the amount of activated plasma kallikrein, factor XIIa, and bradykinin generated (Caliezi et al., 2001; Colman, 1999); and direct inhibition of endotoxin binding to macrophages, which thereby suppresses macrophage activation (Liu et al., 2003). Experience with the use of C1INH in patients with sepsis has been limited (Caliezi et al., 2001, 2002; Fronhoffs et al., 2000; Hack et al., 1992, 1993, 1994; Marx et al., 1999; Zeerleder et al., 2003). In the only randomized, double-blind

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Fig 7 Active C1INH and reactive center loop-cleaved, inactive C1INH prolong survival of mice with lethal gram-negative endotoxemia. Mice (C57BL / 6J) were injected with either LPS intraperitoneally (ip) following treatment with C1INH ip (filled squares) or intravenously (iv) (filled circles), with iC1INH iv (open circles), or with mixtures of LPS and C1INH ip (filled triangles) or iC1INH ip (open triangles). Control mice were injected with LPS ip alone (filled diamonds) or with C1INH iv (n ¼ 4) alone (open diamonds). The indicated P values are for each treatment group in comparison with the group treated with LPS only. Reprinted with permission from Liu et al. (2003).

study, patients with sepsis secondary to a variety of organisms were treated with C1INH every 12 h for 36 h (Caliezi et al., 2002). This therapy had a beneficial effect on renal function and multiple organ dysfunction was less pronounced in treated patients, but there was no effect on overall mortality. In addition, C1INH therapy in these patients reduced neutrophil activation, as reflected by decreased levels of circulating a1-antitrypsin–elastase complexes and of lactoferrin in treated as compared with placebo-treated patients. In addition, the treated patients had less IL-8 release and less complement activation than controls. In summary, although it is clear that C1INH plays an important biological role in prevention of sepsis/septic shock via inhibition of the complement and contact system and perhaps via a direct interaction with endotoxin, its potential as a therapeutic agent in sepsis, although promising, requires further study.

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Fig 8

( continued )

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Fig 8 Inhibition of LPS-induced TNF-a mRNA expression by C1INH. (A) RAW 264.7 cells were induced with LPS (175 ng/ml) for 0, 5, 15, 30, 60, and 120 min at 37 8C in the presence of C1INH (150 mg/ml). (B) RAW 264.7 cells were induced with LPS (175 ng/ml) for 30 min at 37 8C in the presence of C1INH (150, 75, 37.5, 10, 5, 1, and 0 mg/ml). (C) RAW 264.7 cells were induced with LPS (175 ng/ml) for 30 min at 37 8C in the presence of iC1INH (150, 75, 37.5, 10, 5, 1, and 0 mg/ml). For (A–C) reverse transcriptase polymerase chain reaction (RT-PCR) was performed using mouse TNF-a and b-actin cDNA primers. (D) Total RNA from whole human blood cells was isolated after treatment with LPS (175 ng/ml) in the presence of C1INH (150, 75, 37.5, 10, 5, and 0 mg/ml). RT-PCR was performed using human TNF-a and b-actin oligonucleotide primers. Reprinted with permission from Liu et al. (2003).

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VII. Conclusions

The primary biological roles of C1INH may be described as falling into four overlapping categories: regulation of vascular permeability, modulation of inflammation, blood pressure regulation, and antifibrinolytic/procoagulant effects. Its most obvious role is in the regulation of vascular permeability via inhibition of activation of the contact system, which prevents excessive generation of bradykinin. This function is most dramatically demonstrated by the recurrent episodes of angioedema observed in patients with HAE. The antiinflammatory effects of C1INH are mediated via its inhibition of both the complement and contact systems and include its effects on vascular permeability. Regulation of contact system activation limits a number of proinflammatory effects of both bradykinin and plasma kallikrein (Table I). Regulation of activation of all three pathways of the complement system limits the generation of the primary complement-derived inflammatory mediators: C3a, C5a, and the terminal component membrane attack complex. C1INH, therefore, may act to suppress pathological change in any disease in which complement participates in mediation of damage. This role is demonstrated by the studies cited earlier in which C1INH supplementation provides benefit in inflammatory conditions such as reperfusion injury, hyperacute transplant rejection, and sepsis. In addition, by preventing excessive complement activation, C1INH maintains the levels of the second and fourth components of complement, which offer protection from the development of autoimmune disease. This is demonstrated by the fact that patients with HAE, who have secondary low levels of C2 and C4, similar to patients with genetic deficiencies of these proteins, are susceptible to the development of autoimmune disease, particularly systemic lupus erythematosus. HAE patients, even in the absence of overt symptoms, express a variety of autoimmune phenomena (Brickman et al., 1986a, 1986b). Two additional potential antiinflammatory functions of C1INH have been described recently. The direct interaction of C1INH with gram-negative endotoxin, in the mouse, participates in protection from endotoxin shock (Liu et al., 2003). Second, the ability of C1INH to bind directly to endothelial cells, either at decreased temperature or after treatment with TNF-a, may indicate other roles for C1INH (Bergamaschini et al., 2001a, 2001b; Cai and Davis, 2003). At the least, this binding may provide a mechanism to concentrate C1INH at localized sites of inflammation to allow more efficient regulation of both complement and contact system activation. This is particularly relevant because, as discussed in Section IV.B, the endothelial cell surface is a major site of contact system activation (Fig. 5). This binding to endothelial cells, via selectins, may also play a more direct role in the modulation of inflammation by inhibiting the transmigration of leukocytes to the extravascular space (Cai and Davis, 2003).

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Because bradykinin acts to decrease blood pressure, C1INH, by inhibiting kallikrein and limiting bradykinin generation, also plays an indirect role in blood pressure regulation. However, this role is complex because kallikrein also may act to convert prorenin to renin (Table I). Finally, regulation of contact system activation may have an antifibrinolytic and procoagulant effect because bradykinin releases tPA from endothelial cells and inhibits thrombininduced platelet aggregation, while kallikrein activates urokinase plasminogen activator (Table I). In addition, under some circumstances C1INH may play a direct role in the inhibition of tPA. Therefore, although C1INH is the primary inhibitor of only four complement and contact system proteases (C1r, C1s, factor XIIa, and plasma kallikrein), through the increased understanding of these systems, combined with a number of informative animal models, it has become apparent that the biological effects of C1INH are much broader than originally appreciated. References Akita, N., Nakase, H., Kaido, T., Kanemoto, Y., and Sakaki, T. (2003). Neurosurgery 52, 395. Aoki, N., Moroi, M., Matsuda, M., and Tachiya, K. (1977). J. Clin. Invest. 60, 361. Barnathan, E. S., Kuo, A., and Rosenfeld, L. (1990). J. Biol. Chem. 265, 2865. Bauernschmitt, R., Bohrer, H., and Hagl, S. (1998). Intensive Care Med. 24, 635. Bergamaschini, L., Gatti, S., Caccamo, L., Prato, P., Latham, L., Trezza, P., Maggioni, M., Gobbo, G., and Fassati, L. R. (2001a). Transpl. Proc. 33, 939. Bergamaschini, L., Gobbo, G., Gatti, S., Caccamo, L., Prato, P., Maggioni, M., Braidotti, P., Di Stefano, R., and Fassati, L. R. (2001b). Clin. Exp. Immunol. 126, 412. Berrettini, M., Lammle, B., White, T., Heeb, M. J., Schwarz, H. P., Zuraw, B., Curd, J., and Griffin, J. H. (1986). Blood 68, 455. Boackle, S. A., and Holers, V. M. (2003). Curr. Dir. Autoimmun. 6, 154. Bock, S. C., Skriver, K., Nielsen, E., Thogersen, H. C., Wiman, B., Donaldson, V. H., Eddy, R. L., Marrinan, J., Radziejewska, E., Huber, R., Shows, T. B., and Magnussen, S. (1986). Biochemistry 25, 4292. Bos, I. G. A., Hack, C. E., and Abrahams, J. P. (2002). Immunobiology 205, 518. Bos, I. G. A., Lubbers, Y. T. P., Roem, D., Abrahams, J. P., Hack, C. E., and Eldering, E. (2003). J. Biol. Chem. 278, 29463. Brickman, C. M., Tsokos, G. C., Balow, J. E., Lawley, T. J., Santaella, M., Hammer, C. H., and Frank, M. M. (1986a). J. Allergy Clin. Immunol. 77, 749. Brickman, C. M., Tsokos, G. C., Chused, T. M., Balow, J. E., Lawley, T. J., Santaella, M., Hammer, C. H., Linton, G. F., and Frank, M. M. (1986b). J. Allergy Clin. Immunol. 77, 758. Brown, N. J., Nadeau, J. H., and Vaughan, D. E. (1997). Thromb. Haemost. 77, 522. Buerke, M., Murohara, T., and Lefer, A. M. (1995). Circulation 91, 393. Buerke, M., Prufer, D., Dahm, M., Oelert, H., Meyer, J., and Darius, H. (1998). J. Pharmacol. Exp. Ther. 286, 429. Buerke, M., Schwertz, H., Seitz, W., Meyer, J., and Darius, H. (2001). J. Immunol. 167, 5375. Buhler, R., Hovinga, J. K., Aebi-Huber, I., Furlan, M., and Lammle, B. (1995). Blood Coagul. Fibrinolysis 6, 223. Cai, S., and Davis, A. E., III. (2003). J. Imm. 171, 4786. Caldwell, E. E., Andreasen, A. M., Blietz, M. A., Serrahn, J. N., VanderNoot, V., Park, Y., Yu, G., Linhardt, R. J., and Weiler, J. M. (1999). Arch. Biochem. Biophys. 361, 215.

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INDEX

A Abs. See Antibodies ACE. See Enzymes, angiotensin-converting ACF. See Auxiliary factor Activation-induced cytidine deaminase (AID), 306–311, 307f, 310f ADCC. See Cytoxicity, antibody-dependent cellular Adenocarcinomas, 259 Agonists, 27–28, 27t AIA. See Arthritis AID. See Activation-induced cytidine deaminase Alanines, 252 Algorithms, 265–266 Allergen(s). See also Sensitization, allergic classes/forms of, 108, 113–114 cross-reactive, 133 encoding DNA, 131 extract-based immunotherapy, 127–132, 128t–129t factors, 109 food, 109 grass pollen, 135, 139 modified, 135–142, 136t–137t, 141t natural, 134–135 recombinant, 132–135 respiratory, 108–110 specific immunotherapy, 115–120, 116t, 118t T cells and, 113–114 Allergy(ies). See also Sensitization, allergic in children, 107 definition of, 105 mechanisms of, 164–165 Type I, 105–114, 107t, 112f types of, 105–106 AMA1. See Antigens, apical membrane 365

Amino acid sequences, 251 substitutions, 346 Amino acid residues, 157–161, 160f between Fcc/RF, 179–180 of FcRn, 165–166, 167t of IgG, 166–169, 167t, 169t in SpA binding, 187, 188t in SpG binding, 190–192, 190t Amino terminal nonserpin domains, 333–335, 335f AML. See Leukemia, acute myeloid Anaphylatoxins, 177 Angioedema, 345–347 Animals, 222, 264–269 Antagonists, 349 Antibodies (Abs). See also Arthritis, Rheumatoid allergen-specific, 115–120, 116t–118t anti-CD20, 50, 53–54 auto, 179, 218–219 blocking, 121, 127 CamPath1, 53 cell-surface carbohydrate, 62 glycoprotein, 58 Herceptin, 62 IgG, 262 levels of, 52 monoclonal, 50–51, 62 MUC1 and, 252 responses of, 54 tumor, 58–61, 60f Antibodies, CEA monoclonal (MAbs), 61 Antibodies, immunoglobulin E (IGE), 105–106 epitopes, 109

366

INDEX

Antibodies, immunoglobulin E (IGE) (continued) mediated allergies, 109–110, 115–120, 116t–118t T cells and, 113–114 Antigen-combining sites, 198–201, 199t anaphylatoxin binding and, 177 C1g binding as, 160f, 174–176, 175t C3b/C4b and, 176–177 Cc2-Cc3 interface and, 197–198 complement, 160f, 173–177, 175t Fc receptor-binding sites as, 155–173, 156f, 158t, 159f–160f, 164f, 166f, 167t, 168f, 169t, 171f lectins and, 184–186 peptides and, 178–181 polymeric immunoglobulin receptors and, 172–173 proteins and, 178–183, 186–197, 188t, 191t RF and, 179–181 transcytosis/catabolism and, 165–172, 166f, 167t, 168f, 169t, 171f Antigens. See also Cells allergies and, 108–111 delivery of, 73 MAM-6 DF3, 250 manipulation of, 76–78 Tn, 266 transplantation, 55 tumor, 58–61, 60f viral, 52, 55 Antigens, apical membrane (AMA1), 73 Antigens, carcinoembryonic (CEA), 61 Antigens, epithelial membrane (EMA), 250 Antigens, prostate-specific (PSA), 83 Antigens, Thomsen-Friedenreich, 255 AP. See Apyrimidinic-endonuclease APCs, 72–75 antibodies and, 114 transfection of, 78 Apyrimidinic (AP)-endonuclease, 310f Aronica, M. G., 7–9 Arthritis adjuvant, 228 antigen-induced (AIA), 224–225, 234–235 collagen-induced (CIA), 223–224, 234–235 in HTLV mouse, 230–231

in IL-1ra mouse, 232 immunizations and, 232–233, 234–235 in Ipr mouse, 230 K/BxN, 228–230, 235 mutant IL-6 mouse, 232 other models for, 232–233 pristane-induced, 226–227 proteoglycan-induced (PGIA), 226, 234–235 psoriatic, 233 streptococcal cell wall (SCW), 226–227 zymosan-induced, 227–228 Arthritis, rheumatoid (RA), 235–238. See also Rheumatoid factors adjuvant arthritis and, 228 AIA and, 224–225 animal models of, 222 CIA and, 223–224 cytokines/macrophages and, 219 effector mechanisms and, 220f, 220–222 features of, 217 fibroblasts and, 219, 308 K/BxN arthritis and, 228–230 MHC and, 217–218 mouse models of, 217–237, 220f, 234t, 235f PGIA and, 226 pristane-induced arthritis and, 227 SCW and, 226 T cells and, 217–218 theories of, 217–219 zymosan-induced arthritis and, 227–228 ASC-MUC1. See MUC1purified from ascites fluid of cancer patients Asthma, 21–22, 113–114 Ataxia telangiectasia (AT), 315–316 Atopy, 114 Auto-Abs, 236 Auxiliary factor (ACF), 306 Avery, O. T., 50

B Bacillus Calmettte-Gue´ rin (BCG), 270 Bagnara, G. P., 7–9 BCG. See Bacillus Calmettte-Gue´ rin BCRs. See Receptors, B cell Bellani, P., 22 Biragyn, A., 72 Bloodstream, 25–26 Bradykinins, 344–345, 345t, 346 Brizzi, M. F., 7–9

INDEX

C Calcineurin, 23 Calreticulin (CRT), 76–77 Cancer. See also Carcinogenesis breast, 270 cervical, 255 heptocellular, 49 immune responses to, 261–264 immunity evasion/tolerance and, 55–58 incidence of, 53–54 in mice, 53 renal cell, 281 treatment of, 49–52, 50t tumor antigens and, 58–61, 60f vaccinations for, 53–54, 79–80 Captoprils, 348f, 349 Carbohydrates, 58, 64, 255 Carcinogenesis, 258–259 Carcinomas. See Cancer Catabolism FcRn and, 165–166, 167t, 169–171, 171f IgG and, 166–169, 167t, 169t, 170–171, 171f Rc receptors in, 165, 166f therapeutic implications of, 171–172 Cationic Ags, 224 CCAAT/enhancer-binding proteins (C/EBP), 30–31 CEA. See Antigens, carcinoembryonic C/EBP. See CCAAT/enhancer-binding proteins Cells antigen-presenting, 72–75 endothelial, 341–344, 342f of immune system, 221–222 Kuppfer, 222 malignant, 59, 60f mast, 221–222, 225 mononuclear, 230 moraxella catarrhalis, 194 specialized, 59 Staphylococcus aureus, 186–190, 188t suppressor, 57 tumor, 65–66, 255, 258–260, 275 Wrzburg T, 21 Cells, B, 53, 233–234 activation of, 300 DCs and, 65–66 deficiencies, 305–313, 307f, 310f, 312f immature/mature, 296 malignancies, 59–60 of patients, 301–302

367

Cells, dendritic (DCs), 221, 271–276, 281, 303 Cells, follicular dendritic (FDCs), 72 Cells, germinal (GC) founder, 307 Cells, natural killer (NK), 53 Cells, polymorphonuclear (PMN), 5 Cells, T, 50, 233–234. See also Cytoxic T lymphocytes allergies and, 113–114, 120 CD4þ, 54, 63–64, 70, 77, 230 CD8þ, 54, 63–64, 71 expansion of, 54 HIGM and, 315 IGE and, 113–114 leukemia virus type I, human (HTLV-1), 230–231 MUC1 and, 257 of newborns, 299 priming, 68 proliferation of, 63–64 protein, 182–183 provision of, 69–71 RA and, 217–218 receptors, 51–52 responses, 52, 54, 56 tumor-infiltrating, 56–57 Cellular responses, 1 Cellular therapy, 51 CFA. See Complete Freund’s adjuvant Chakraborty, A., 24 Chemokines, 72 inflammatory, 2, 13, 16 as mediators, 1–2 Chemotaxis, 344 Chemotherapy, 53–54 Chimpanzees, 264–265 CIA. See Arthritis CIS. See Protein, cytokine-inducible SH2 c-Jun N-terminal kinase (JNK), 32 Class Switch Recombination (CSR), 316 primary antibody repertoire in, 295–296 secondary repertoire in, 296–298 CLL. See Leukemia, chronic lymphocytic CML. See Leukemia, chronic myeloid CMV. See Cytomegalovirus Collagen Ab, anti, 224 induced arthritis, 223–224, 234–235 xenogenic type II, 223

368

INDEX

Complement(s) activation regulation, 337–339, 338f C3 component, 218–219 cascades, 220–221, 220f Complete Freund’s adjuvant (CFA), 62, 223 Component-resolved diagnosis (CRD), 132 Coombs, R. R. A., 105, 111 CRD. See Component-resolved diagnosis CRT. See Calreticulin CSR. See Class Switch Recombination CTL. See Cytoxic T lymphocytes Cyclophosphamides, 277 Cytokines, 263 action of, 25 blockade of, 236 CIA and, 223–224 inflammatory, 2–3, 13, 16 as mediators, 1–2 RA and, 219 Th1, 120 Th2, 113, 120, 122 Cytokines, interleukin (IL), 2, 182 blockage of, 219, 225 STATs and, 13–14 Cytokines, pleiotropic (IFN-c), 11–14, 76–78 Cytomegalovirus (CMV), 50–51 Cytoxic T lymphocytes (CTL), 52–53 activities of, 76–77 MUC1 and, 262–263, 277 responses of, 57, 61–62, 71 in vitro, 64–66 Cytoxicity, antibody-dependent cellular (ADCC), 5, 176 Cytoxicity, cellular, 156

D Davidson, D., 22 DCs. See Cells, dendritic Defensins, 72 Dermatitis, atopic, 113–114 Detergent lysis, 12 Diisopropyl fluorophosphonate (DGP), 6 DIP. See Distal interphalangeal Diptheria, 50, 276 Disease, graft-versus-host (GvH), 51 Disease, graft-versus-leukemia (GvL), 51 Diseases antibody-mediated, 220–222, 220f, 238 infectious, 49, 83

Diseases, allergic. See also Allergen(s); Allergy(ies); Sensitization, allergic immunotherapy of, 105–106 Type I, 105–114, 107t, 112f Distal interphalangeal (DIP), 217 DLI. See Donor lymphocyte infusion DNA (deoxyribonucleic acid), 82 allergen-encoding, 131 breaks, 297 naked vaccines, 66–68, 67f sequences, 62, 232 single-stranded, 310, 310f technology, 115 tumor vaccines, 68–71, 74–75, 78–79, 271 DNA, double-strand break (DSBs), 295, 308–309 DNA-dependent protein kinase (DNA-PK), 295–296 Donor lymphocyte infusion (DLI), 51 DSBs. See DNA, double-strand break Dyax Corporation, 349 Dysplasia, extodermal, 305

E EBV. See Virus, Epstein-Barr EGF. See Epidermal growth factor Ehrlich, Paul, 50 Electrophoretic mobility shift assay (EMSA), 6, 8 analyses, 9, 24 mobility, 21 Electroporation, 78 ELISA testing, 280 ELISPOT assays, 284 EMA. See Antigens, epithelial membrane EMSA. See Electrophoretic mobility shift assay Endoplasmic reticulum, 76 Endotoxin shock, 351–355, 353f, 354f–355f Enzymes, 343 Enzymes, angiotensin-converting (ACE), 343 Eosinophils, 114, 221 Epidermal growth factor (EGF), 3 Escherichia coli, 309

F Fab regions, 155 proteins and, 181–183 RF-Fc IgG interactions of, 180–181 SpA and, 188–190 SpG and, 192

369

INDEX

FACS. See Fluorescence-activated cell sorter Fc receptor-binding sites Fcc receptors as, 155–161, 156f, 158t, 159f–160f transcytosis/catabolism and, 165–173, 166f, 167t, 168f, 169t, 171f FDCs. See Cells, follicular dendritic Fibroblasts, 219, 308 Fluorescence-activated cell sorter (FACS), 14, 23 fMLP. See Peptides, N-formulated Fn. See Protein(s) France, 279 Freeze-thaw cycles, 5–6, 12

G Galactosyltransferases (GT), 186 Galectins, 185 GAS. See Interferon-activated sequences, c GC. See Cells, germinal founder G-CSF, 4–7 Gell, P. G. H., 105, 111 Gene(s) -based tumor vaccines, 66 CD40, 303–304, 304f expression, 2–3, 78 human TNF (huTNF), 231 immediate-early, 17 kB (NEMO), 304–305 PU.1, 28–30 transcription, 2, 17–28 uracil-N glycosylase (UNG), 311–314, 315f Genotypes, 303 Glutamines, 252 Glycloproteins, 264, 273–274 as tumor vaccines, 61–64 Glycolipids, 58 Glycopeptides, 274 Glycoproteins, 58 Glycoproteins, histidine-rich (HRG), 183 GM-CSF. See Granulocyte-macrophage colony-stimulating factor GMP. See Guanosine monophosphate Granulocyte-macrophage colony-stimulating factor (GM-CSF), 2 effects of, 63–64 encoding plasmids, 70 STATs and, 7–11 Granulopoiesis, 5, 30

Growth factor(s), 2 epidermal, 62 HER2, 62 receptors, 3–4 GT. See Galactosyltransferases Guanosine monophosphate (GMP), 64 GvH. See Disease, graft-versus-host GvL. See Disease, graft-versus-leukemia Gylcosylation, 254–255

H HA. See Hemagglutinin Haines, B. A., 72 Hemagglutinin (HA), 71, 73 Heparin, 335–336 Hepatitis B, 49 High-molecular-weight kininogen (HK), 340–343, 342f HIGM. See Hyper-IgM syndromes HIV. See Human immunodeficiency virus HK. See High-molecular-weight kininogen HPV. See Virus, human papilloma HRG. See Glycoproteins, histidine-rich HSPs. See Proteins, heat shock HSV-1. See Virus, herpes simplex hTERT. See Telomerase reverse transcriptase HTLV-1. See Cells, T Human immunodeficiency virus (HIV), 49, 182–183 -1, 196–197 treatment of, 50, 71 huTNF. See Gene(s) Hybridizations, 306 Hygiene hypothesis, 108 Hyper-IgM syndromes (HIGM) AT and, 315–316 -1 and, 301–303, 302f -2, 306–311, 307f, 310f -3 and, 303–304, 304f -4 and, 313–314 B cell deficiencies and, 305–313, 307f, 310f, 312f CD40/CD40-L defects and, 298–301, 299f, 300t other, 315–316 T cell deficiencies and, 315 UNG deficiences and, 311–314, 315f Hyperplasia, synovial, 230 Hypoxemia, 351–352

370

INDEX

I IC. See Immune complexes IFN. See Interferon IFN-c. See Cytokines, pleiotropic Ig. See Immunoglobulins IGE. See Antibodies, immunoglobulin E IKB kinase complex (IKK), 17 IKK. See IKB kinase complex IKKc. See Gene(s) IL. See Cytokines, interleukin Immune complexes (IC), 155, 218–219 Immune deficiencies, 298. See also Hyper-IgM syndromes Immune responses to cancer, 261–264, 268 of humans/MUC1, 260–261 humoral, 263 Immunity, 54 adaptive/innate, 53, 219 evasion/tolerance of, 55–58 induced, 78 Immunizations. See also Vaccination(s) arthritis and, 232–233, 234–235 DC-tumor cell, 275 intramuscular, 75 intrasplenic (i.spl), 75 of mice, 276 somatic transgene, 75–76 Immunobiology, MUC1 in animal models, 264–269 biosynthesis/structure of, 251–256 history of, 250–251 in human milk, 253 immune responses to, 260–264 o-linked glycosylation of, 254–255 physiology of, 256–260 studies of, 249 tumor cells and, 258–260, 281–282, 283f vaccines, 269–275, 276–281, 282–284, 283f VNTR region and, 252–253, 255, 266 Immunodeficiencies, 301 Immunogenicity, 81 Immunoglobulins (Ig), 198–201, 199t. See also Class Switch Recombination anaphylatoxin binding and, 177 C1g binding as, 160f, 174–176, 175t C3b/C4b and, 176–177 Cc2-Cc3 interface and, 197–198 classes of, 157–165, 158t, 159f–160f, 164f

complement-binding sites and, 160f, 173–177, 175t constant domains of, 155 Fc receptor-binding sites of, 155–173, 156f, 158t, 159f–160f, 164f, 166f, 167t, 168f, 169t, 171f human, 178, 181–182, 189 interactions of, 155–201 lectins and, 184–186 mouse, 189 peptides and, 178–181 polymeric receptors, 172–173 proteins and, 178–183, 186–197, 188t, 191t RF and, 179–181 structural unit of, 155, 156f transcytosis/catabolism and, 165–172, 166f, 167t, 168f, 169t, 171f Immunoglobulins, intravenous (IVIG), 171–172 Immunoglobulins, polymeric (pIgs), 172 Immunology, 2, 57, 249 Immunoprecipitation, 18 Immunostimulatory sequences (ISSs), 67 Immunosuppression, 53, 82 Immunotherapy allergen extract-based, 127–132, 128t–129t allergen-specific, 121–127, 124t–125t of allergic diseases, 105–142, 107t, 112f, 116t–118t models, 122–127, 124t–125t modified allergens for, 135–142, 136t–137t, 141t passive, 49–52 recombinant allergens and, 132–135 targets, 115–120, 116t–118t Type I allergy and, 105–114, 107t, 112f Infections in children, 54 treatment of, 49–52, 50t Infiltration, synovial, 230 Inflammation, 223 C11NH treatments and, 349–350 endotoxin shock and, 351–355, 353f, 354f–355f modulation of, 331–332 reperfusion injury and, 350–351 transplant rejection and, 351 Influenza, 71, 73 Inhibitions of alternative pathway activation, 339 of complement pathway activation, 337, 338f

371

INDEX

of lectin pathway activation, 337–339 serpine mechanisms of, 333, 334f Inhibitor, C1 (C11NH), 356 contact system activation and, 340–345, 340f, 342f, 345t factors, 331–332 function/structure of, 333–336, 334f, 335f heparin and, 335–336 inflammation and, 349–355, 353f, 354f–355f mouse analysis, 347–349, 348f role of, 332 serpin/nonserpin mechanisms and, 333–335, 334f, 335f vascular permeability and, 345–349, 348f Injury, reperfusion, 350–351 Interferon (IFN), 53, 261 Interferon-activated sequences, c (GAS), 4, 11 Interferon-regulated factors (IRF), 28–29 Interferon-stimulated response element (ISRE), 4 IRF. See Interferon-regulated factors Isohemagglutinins, 305 i.spl. See Immunizations ISRE. See Interferon-stimulated response element ISSs. See Immunostimulatory sequences ITAM. See Tyrosine-based activator ITIM. See Tyrosine-based inhibitor IVIG. See Immunoglobulins, intravenous

J Jacalins, 184 Janus kinases (JAKs), 3 -1, 11 -2, 7–8, 11, 14 STAT cascades, 4, 10, 16 Jenner, Edward, 49 JNK. See c-Jun N-terminal kinase Joints, 217 carpal/tarsal, 229 destruction of, 222, 223–224

L Lactation, 181 Lectins animal, 185–186 antigen-combining sites and, 184–186 Galectin-1/-3, 185 GT, 186 Jacalin as, 184 mannan-binding (MBL), 337–339 mannose-binding proteins as, 185–186 pathway activation, 337–339 Leukemia, 51 Leukemia, acute myeloid (AML), 279 Leukemia, chronic lymphocytic (CLL), 53 Leukemia, chronic myeloid (CML), 60, 60f Leukocytes, 1, 7, 352, 354f–355f Leukotriene B4, 1 Ligands, 198 CD40, 298–301, 299f, 301t HIGM1, 301–303, 302f LIMP-II. See Lysosomal integral membrane protein-II Lipid mediators, 1 monophosphoryl A (MPL), 127 Lipopolysaccharide (LPS), 14 activation by, 21–22, 301 inhibition of, 23 Liposomes, 127 Livers, 222 Loveless, Mary, 121 LPS. See Lipopolysaccharide Lymphocytes, 232, 263 Lymphoid organs, 295, 306–307 Lymphomas, 52 follicular, 56 murine, 306 non-Hodgkin’s, 53 Lysosomal integral membrane protein-II (LIMP-II), 78

K Kallikreins, 340–343, 342f, 344, 345t, 346, 349 Keyhole limpet hemocyanin (KLH), 56, 270 Kinase inhibitory domain (KIR), 4 KIR. See Kinase inhibitory domain KLH. See Keyhole limpet hemocyanin Klyushnenkova, E., 72 Kwak, L. W., 72

M MAbs. See Antibodies, CEA monoclonal MAC. See Membrane attack complex Macaques, 264–265 Macrophages, 232 alveolar, 222 RA and, 219, 221–222

372

INDEX

Major histocompatibility complex (MHC), 228 Class I, 64, 76 Class II-associated, 59–61, 77, 182 Class II-restricted, 114 RA and, 217–218 role of, 227 Malaria, 73 Mannose-binding proteins A (MBP-A), 185–186 Mannose-binding-lectin (MBL) pathways, 220, 220f MBL. See Lectins MBL-associated proteases (MASPs), 337–339 MBP-A. See Mannose-binding proteins A mBSA. See Methylated bovine serum albumin MCP. See Metacarpophalangeal Mediators, 263 chemokine, 1–2 cytokine, 1–2 inflammatory, 32, 114, 156 Melanocytes, 59 Melanomas, 59, 60 Membrane attack complex (MAC), 220, 220f MET, 267–268, 269 Metabolites, 27 Metacarpophalangeal (MCP), 217 Metatarsophalangeal (MTP), 217 Methylated bovine serum albumin (mBSA), 224–225 MHC. See Major histocompatibility complex Mice cancer in, 53 CIA and, 223–224 HTLV, 230–231 Ig, 189 IL-1ra, 232 immunization of, 276 Ipr, 230 models of RA, 217–237, 220f, 234t, 235f mutant IL-6, 232 RA models in, 217–237 wild-type, 349 Microorganisms, 1 Molecules, 1, 256–257 alternative adjuvant, 63 CD40, 298–301, 299f, 301t inflammatory, 219 J chain, 172–173 Morphine, 24–25 MPL. See Lipid

MTP. See Metatarsophalangeal MUC1purified from ascites fluid of cancer patients (ASC-MUC1), 273–274 Mucin, polymorphic epithelial (PEM), 250 Mucin, polymorphic urinary (PUM), 250 Mucins. See also Immunobiology, MUC1 family of, 249, 250–251 MUC1 as, 61, 250–251 Murine models, 265–269 Mutations, 297–298 Myelomas, 53, 64

N NEMO. See Gene(s) Neutropenia, 303 Neutrophil(s), 221, 341 activation of, 1, 18–22, 28–34 aggregation/degranulation of, 344–345 apoptosis, 25–28, 27t differentiation, 30–31 extracts, 19–20 future direction for, 15–17, 16t, 32–34 G-CSF and, 4–7 GM-CSF and, 7–11 IFN-c and, 11–14 IL-10 and, 13–14 morphine and, 24–25 NF-kB family and, 17–28 other agent in, 14–17 polymorphonuclear, 1 roles of, 1–3 STATS and, 4–17 NHEJ. See Nonhomologous end-joining pathway Nitric oxide (NO), 24–25 Nitrogen cavitation, 5–6, 19 NK. See Cells, natural killer NMR. See Nuclear magnetic resonance NO. See Nitric oxide Nonhomologous end-joining (NHEJ) pathways, 295, 310f Nuclear magnetic resonance (NMR), 161, 252

O Oligosaccharides, 176 Open reading frames (ORFs), 79, 306 Oppenheimer, J. J., 72 ORFs. See Open reading frames Osteoclasts, 222

INDEX

P PAF. See Platelet-activating factor PBMC. See Peripheral blood mononuclear cells PCR. See Polymerase chain reaction PDGF. See Platelet-derived growth factor PDTC. See Pyrrolidine dithiocarbamate PEG. See Polyethyleneglycol Pegoraro, L., 7–9 PEM. See Mucin, polymorphic epithelial Peptide(s), 58 antigen-combining sites and, 178–181 Fc-binding, 179 identification of, 60–61 immunologenic, 278 MHC Class I, 64, 76 MHC Class II-associated, 59–61, 77, 182 MHC Class II-restricted, 114 poly, 179 synthetic, 264 tumor vaccines and, 64–66 vaccines, 269–271 Peptides, N-formulated (fMLP), 15, 19–120 Peptostreptococcal magnus, 193–194 Peripheral blood mononuclear cells (PBMC), 12, 15, 23–24, 139 Peroxynitrites, 25 PG. See Proteoglycan PGIA. See Arthritis Phagocytes, 1, 5 Phagocytosis, 74, 156 Phenotypes, 303 Phenylmethylsulfonyl fluoride (PMSF), 9 Phosphatases, 4 PIAS. See Protein inhibitors of activated STATs pIgR. See Receptors pIgs. See Immunoglobulins, polymeric PIP. See Proximal interphalangeal Plasmids encoding, 70 Fas/EGFP, 74 Platelet-activating factor (PAF), 19 Platelet-derived growth factor (PDGF), 3 Platelets, 341, 344 PLGA. See Poly-d,l-lactic-coglycolic acid PMN. See Cells, polymorphonuclear PMSF. See Phenylmethylsulfonyl fluoride Pneumonia, 50

373

Poly-d,l-lactic-coglycolic acid (PLGA), 267 Polyethyleneglycol (PEG), 130 Polymerase chain reaction (PCR), 139, 308 Polymorphisms, 255 Polypeptides, 179 PpL. See Protein, peptostreptococcal L Prekallikreins, 340–343, 342f Prenancies, 260–261 Prl. See Protein(s) Prolines, 251, 252 Prostaglandin D2, 27 Protein(s), 58. See also Signal transducers and activators of transcription ATF/CREB, 31–32 bacterial immunoglobulin-binding, 186–190, 188t Cc2-Cc3, 197–198 clusterin, 178 corresponding, 2–3 cytokine signaling, 4 fibronectin (Fn), 178 Fos family of, 31–32 Fv, 181–182 inflammatory, 21–22 Jun family of, 31–32 prolactin (Prl), 181 rag, 295 synthesis, 2 T cell, 182–183 as tumor vaccines, 61–64 viral immunoglobulin-binding, 195–197 Protein, cytokine-inducible SH2 (CIS), 4 Protein inhibitors of activated STATs (PIAS), 4 Protein, peptostreptococcal L (PpL), 193–194 Protein, staphylococcus A (SpA), 186–190, 188t Protein, streptococcal G (SpG), 190–192, 190t Protein, streptococcal H (SpH), 192 Proteins, heat shock (HSPs), 76–77, 231 Proteins, SOCS -1, 7 -3, 7, 14 Proteoglycan (PG), 226 Proximal interphalangeal (PIP), 217 PSA. See Antigens, prostate-specific Public health, 49 PUM. See Mucin, polymorphic urinary PVXCP. See Virus, potato X Pyrrolidine dithiocarbamate (PDTC), 23–24

374

INDEX

R RA. See Arthritis, rheumatoid Reactive oxygen intermediates (ROI), 22–24 Receptors Fc cell, 155–172, 156f, 158t, 159f–160f, 160f, 164f, 166f, 167f, 168f, 169t, 171f, 221 high-affinity FceRI, 119–120 low-affinity FceRII, 119–120 NKG2A, 56–57 polymeric immunoglobulin (pIgR), 172–173 surface, 17–18 T cell, 51–52 Receptors, B cell (BCRs), 295 Receptors, TNF (TNFR), 300–301 Receptors, toll-like (TLR), 232–233 Rel homology domains, 17 Respiratory tracts, 109–110 RF. See Rheumatoid factors Rheumatoid factors (RF), 179–181, 218, 237–238 ROI. See Reactive oxygen intermediates Rosso, A., 7–9 Ruffini, P. A., 72

S SC. See Secretory component SCID. See Severe combined immunodeficient SCW. See Arthritis SDS-PAGE. See Sodium dodecyl sulfatepolyacrylamide gel electrophoresis Secretory component (SC), 172–173 Sensitization, allergic allergic immune response in, 111–114, 112f concept of, 106–111, 107t to cross-reactive allergens, 133 Sepsis, 352–353 SEREX (serological identification of antigens by recombinant expression cloning), 58 Serines, 251 Serpine mechanisms, 333, 334f non, 333–335, 335f Severe combined immunodeficient (SCID), 219, 299f SFV. See Virus, Semliki Forest SHM. See Somatic hypermutations Sialylation, 252 ‘Sigma Chemical Co., 349 Signal transducers and activators of transcription (STATs)

-1, 3–5, 7–13 -2, 3–5 -3, 3–11 -4, 3–5 -5, 3–5, 8–11, 14 -6, 3–5 -7, 3–5 family members, 10–11, 15–17, 16t G-CSF and, 4–7 genes, 3 GM-CSF and, 7–11 IFN-c and, 11–14 IL-10 and, 13–14 JAK cascades, 4, 10, 16 other agents and, 14–17 SIN. See Virus, Sindbis SLE. See Systemic lupus erthematosus Sloan Kettering Cancer Center, 277 SNAP. See S-nitroso-N-acetypencillamine S-nitroso-N-acetypencillamine (SNAP), 25 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 6 Somatic hypermutations (SHM), 295, 316. See also Hyper-IgM syndromes primary antibody repertoire in, 295–296 secondary repertoire in, 296–298 Sonication, 12 SpA. See Protein, staphylococcus A SpH. See Protein, streptococcal H Spondylitis, ankylosing, 233 STATs. See Signal transducers and activators of transcription Streptococcus pyogenes, 192, 194 Surenhu, M., 72 Systemic lupus erthematosus (SLE), 171–172

T Taiwan, 49 Technology, molecular, 57 Telomerase reverse transcriptase (hTERT), 61 tg. See Transgenic mouse models TGF. See Tumor growth factor Threonines, 251, 252 TLR. See Receptors, toll-like TLR9. See Toll-like receptor 9 TNF. See Tumor necrosis factor TNFR. See Receptors, TNF Toll-like receptor 9 (TLR9), 67 Transcription factors AP-1 family of, 31–32

375

INDEX

C/EBP family of, 30–31 Ets family of, 28–30 in neutrophil activation, 28–34 Transcription factors, NF-kB family components, 18–22 HIGM and, 304–305 regulation of, 24–25 ROI and, 22–24 role of, 25–28, 27t Transcytosis FcRn and, 165–166, 167t, 169–171, 171f IgG and, 166–169, 167t, 169t, 170–171, 171f Rc receptors in, 165, 166f therapeutic implications of, 171–172 Transgenic (tg) mouse models, 56, 266–268. See also MET Tumor(s), 49. See also Vaccines, tumor breast, 279–280 cells, 65–66, 255, 258–260 DC-tumor cell, 275 families, 60 immunology, 249 metatasis, 258 pancreatic, 268–269 resurgence of, 54 types of, 53, 56–57 vaccines, 68–69 virus-associated, 52 Tumor growth factor (TGF), 2, 53 Tumor necrosis factor (TNF), 2 -a, 219, 225 superfamily, 299–300 Tweady, D. J., 24 Tyrosine-based activator (ITAM), 156 Tyrosine-based inhibitor (ITIM), 156

U

DC-based, 271–276 DNA, 66–69, 67f, 74–75, 78–79, 271 gentle fusion, 69–71 hepatitis B, 49 MUC1, 269–275, 276–281, 282–284, 283f peptide, 269–271 prophylactic allergy, 142 recombinant vaccinia virus, 276 RNA, 83–84 VP22-E7, 74 Vaccines, tumor active vaccination and, 52–54 DNA, 68–71, 74–75, 78–79 evasion/tolerance of, 55–58 gene-based, 66 glycloproteins/proteins as, 61–64 introduction to, 49–58 passive immunotherapy and, 49–52 peptides and, 64–66 RNA vaccines and, 83–84 strategies for, 61–84 tumor antigens and, 58–61, 60f Vancurova, I., 22 Vascular permeability, 331–332 regulation of, 345–349, 348f VEE. See Venezuelan equine encephalitis Venezuelan equine encephalitis (VEE), 79 Virus, Epstein-Barr (EBV), 50–51, 57 Virus, herpes simplex (HSV-1), 195–196 Virus, human papilloma (HPV), 63 Virus, potato X (PVXCP), 70 Virus, Semliki Forest (SFV), 79 Virus, Sindbis (SIN), 79 Viruses, alpha, 79–80, 83–84 Vitronectins, 343 von Pirquet, Clemens, 105

UNG. See Gene(s)

W V

Vaccination(s) active, 52–54 anti-MUC1, 257 booster, 80–83 for cancer, 53–54 children and, 49 preventive, 49 strategies, 61–84 therapeutic, 49 Vaccines alphavirus-based cancer, 79–80

Women, 260–261

X X-ray crystallography, 166, 179, 193

Y Yang, M. F., 72 Yarden, Y., 7–9

Z Zymogens, protease, 340 Zymosan, 227–228