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Processed MHC class I alloantigen as the stimulus for CD4 ÷ T-cell dependent antibody-mediated graft rejection J. Andrew Bradley, Allan McI. Mowat and Eleanor M. Bolton The traditional view of graft rejection is one of direct recognition of allogeneic MHC molecules by effector T cells, the phenotype of which may be predicted by the nature of the MHC disparity. In this article, Andrew Bradley and colleagues discuss recent evidence that suggests this view may be an oversimplification, and argue that additional effector mechanisms, such as alloantibody, need to be reconsidered. Rejection of an organ allograft is characterized by a highly complex series of cellular and humoral interactions in which the T cell plays a central and essential role. In recent years, considerable progress has been made towards understanding how T cells initiate, regulate and effect graft rejection. Inevitably, much attention has focused on the relative roles of the phenotypically distinct CD4 ~ and CD8 + T-cell subpopulations in the rejection process. Many studies have attempted to define the requirements for these subsets in rejection, using two basic approaches: first, adoptive transfer analysis in T-cell deficient animals and, secondly, in vivo treatment of normal animals with monoclonal antibodies (mAbs) to selectively deplete or block the function of one or other of the T-cell subsets (reviewed in Ref. 1). The consensus view from such studies is that both CD4 + and CD8* T cells contribute to the rejection of fully allogeneic grafts, but overall responsibility for orchestrating the response is usually assigned to the CD4 + T-cell subset. It is widely accepted that graft rejection in recipients who have not had previous exposure to transplantation antigens, that is, first-set rejection, is mediated by effector T cells rather than by antibody-dependent effector mechanisms, and for many years there has been intense debate concerning the relative importance of specific cytotoxic T cells (CTL) - predominantly CD8 ÷ T cells - compared with cells that induce delayed type hypersensitivity (DTH) - usually CD4 + T cells - as the principal effector cell types *-3. Rejection entails a considerable degree of collaboration between these distinct cell types, a fact well illustrated by the elegant experiments of Rosenberg and colleagues 4 on skin graft rejection using allophenic mice. Allophenic mice are genetic mosaics which develop from the fused blastomeres of two different H-2 strains, such that their individual cells express one or other set (but not both) of the parental major histocompatibility (MHC) complex molecules s. Transplantation of allophenic skin to either parental strain results in the
rapid and complete destruction of the epidermis by a non-specific inflammatory response consistent with a DTH effector mechanism. However, all the melanocytes and hair follicles expressing the nonshared allogeneic MHC antigens are specifically destroyed, leaving the adjacent syngeneic cells perfectly intact 4. This implies a specific CTL effector mechanism. The balance between CD4* and CD8 + T cells as the principal effector cells mediating rejection is likely to depend on such factors as the type of graft and, in particular, the nature of the MHC disparity between donor and recipient. The availability of mutant and recombinant rodent strains that differ from each other at isolated MHC subregions has enabled these variables to be examined in greater detail, although most such studies have confined their attention to the rejection of skin grafts. The T-cell requirement for rejection of skin grafts bearing isolated MHC disparities Adoptive transfer experiments in T-cell depleted mice bearing mutant MHC class I- or class II-disparate skin grafts have shown that purified CD8 +, but not CD4*, T cells reject MHC class I-disparate grafts, whereas only CD4* effector T cells destroy MHC class II-disparate grafts 6,7. Isolated MHC class I disparities have been the most extensively studied and the exclusive role for CD8 ÷ effector T cells has been confirmed by mAb depletion of T-cell subsets in vivo s,9. Surprisingly, in certain MHC class I-disparate mouse strain combinations, selective depletion of CD8* T cells by anti-CD8 mAb treatment does not prevent skin graft rejection, suggesting that CD4 + T cells may also participate in the rejection of such grafts ~'9. The presence of an anti-class I alloantibody response in recipients of allelic class I-disparate grafts is further evidence for CD4+ T-cell activation, since the functional overlap between CD8* and CD4 + T cells does not extend to the provision of cognate T-cell help by CD8* T cells for the production of antibody. Nevertheless, the prevailing
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viewpoint view remains that CD8 + effector cells are essential for reiection of MHC class I-disparate grafts. Rosenberg and colleagues have recently attributed rejection of MHC class I-disparate skin grafts in antiCD8 treated mice to a unique population of anti-CD8 resistant, CD8 + CTL which are dependent for their generation on help from CD4 + T cells triggered by additional alloantigens co-expressed by MHC class ldisparate grafts ~.
Rejection of MHC class 1-disparate kidney allografts Organ grafts are more directly relevant to clinical transplantation than skin grafts and, since there are likely to be important differences in the nature of the rejection response between indirectly vascularized skin grafts and directly vascularized organ grafts, we chose to examine the role of CD4 + and CD8 ~ T cells in the rejection of rat kidney allografts across an isolated allelic MHC class I disparity~'J(uL In these studies, we used PVG congenic rats, which bear the RTI" haplotype and are high responders to the RT1A ~'class I antigen. These animals, which rapidly reject class I-disparate skin and organ grafts ~e';~, were treated in vivo with mAbs to specifically deplete CDS+ or CD4 + T cells. Interestingly, although anti-CD8 treatment was highly effective at depleting CD8* T cells, both phenotypically and functionally, it did not prolong the survival of RTIN' class I-incompatible kidney allografts from donors of the PVG recombinant strain, RS. Moreover, unlike anti-CD8-treated mice, there was no evidence that anti-CDS-resistant CD8 ~ CTLs were responsible for rejection in this experimental model and the grafts in anti-CDS-treated animals were destroyed in the absence of detectable CDS* effector T ceils within the graft infiltrate. In contrast to anti-CD8 treatment, administration of anti-CD4 mAb markedly delayed rejection and, in some animals, led to permanent survival of class I-disparate kidney grafts. From these experiments we concluded that, under certain circumstances, CD4 ~ T cells alone may be sufficient to initiate rejection of MHC class I-disparate organ grafts. Conversely, CD8* T cells are neither necessary nor, by themselves, sufficient.
Processed MHC class I alloantigen as the stimulus for rejection? The above studies raise intriguing questions as to the nature of the alloantigen recognized by CD4 + T cells and the effector mechanisms responsible for rejection of MHC class I-disparate kidney grafts. To answer these questions it is necessary to consider, briefly, current concepts of how T cells may recognize alloantigen. During a conventional T-cell response to a protein antigen, the c(~ T-cell receptor (TCR) molecule recognizes processed antigenic peptides presented by self MHC molecules ]4 J-. The T-cell response to alloantigens may, therefore, be exceptional because it may be directed at intact a[logeneic MHC molecules themselves ~s. To what extent the TCR recognizes the endogenous peptide in the binding groove of the allogeneic MHC molecule and to what extent it recognizes epitopes of the allogeneic MHC molecule adjacent to
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the peptide-binding groove is still unclear ~". In either event, it is difficult to reconcile the possibility that allogeneic MHC molecules are recognized directly by host T cells with the dogma that T cells only recognize antigen complexed with syngeneic MHC molecules. The expression of CD4 and CD8 by T cells normally defines their MHC-binding specificity]*: CD8 ÷ T cells recognize antigen in the context of self MHC class I molecules and respond directly to allogeneic MHC class 1, whereas CD4* T cells recognize antigen in the context of self MHC class II molecules and respond directly to allogeneic MHC class 1I molecules. How, then, might CD4 + T cells be primed against alloantigens from MHC class l-disparate grafts? There are three possibilities. First, CD4 + T cells may recognize and be activated by intact allogeneic MHC class I molecules. We think that this is unlikely, but it cannot be completely discounted because exceptional CD4 + Tcell clones have been described that are lyric towards MHC class I targets -'°'-'~. Secondly, CD4" T cells may be activated, not by MHC class I alloantigens themselves, but by other alloantigenic determinants co-expressed by the graft *. Although we cannot entirely exclude the possibility that there may be additional antigenic differences between the recombinant rat strains used in our studies, their existence and potential role in activating CD4 ÷ T cells must be largely speculative. The third, and in our view the most likely, possibility is that CD4 + T cells are stimulated by graft MHC class I alloantigens which have been processed by recipient antigen-presenting cells (APCs) and presented as antigenic class 1 peptide fragments in the antigenhinding groove of self MHC class II molecules. This is analogous to the way in which CD4" T cells recognize conventional antigens and has been called the 'indirect pathway' of allorecognition. Although this pathway of allorecognition was originally postulated in the early 1980s:: -", convincing evidence that it may be important in graft rejection has emerged only very recently z~-~. In particular, studies in the rat have clearly demonstrated that immunization with peptides from allogeneic MHC class I molecules may prime recipient (~D4+ T cells 2- and lead to accelerated reiection of skin all(> grafts-'". Although we have not directly addressed the question of how CD4 + T cells are activated to class I alloantigens, it is notable that when PV(; RTI" rats arc' immunized with purified RT1A ~' MHC class I antigen, they mount a strong T helper ceil-dependent cytotoxic alloantibody response (C. Kelly, J.A. Bradley and J. Fabre, unpublished observations). This observation clearly illustrates the potential for indirect allorecognition of processed RTIN ~class I by RT1 ~' CD4 + helper T cells, since neither intact, membrane-bound MHC class I molecules nor other poorly defined alloantigens are present to provide helper determinants in this system. It is interesting to note that when host and graft are mismatched for MHC class I but matched for MHC class II molecules, as in the studies discussed here, host CD4 + T cells could, in principle, recognize allogeneic class I peptides presented by syngeneic MHC class II
I N,,. t
viewpoint molecules on APCs originating from either the graft recipient or the donor organ. The latter route of sensitization would necessitate physiological presentation of stimulatory peptides derived from internalized cell-surface MHC class I molecules. Such a concept is not simply a theoretical possibility, since naturally occurring peptides derived from endogenously synthesized cell membrane proteins have recently been isolated from the binding cleft of MHC class II molecules32.
T cells are not necessary for rejection of such grafts in anti-CD8-treated recipients. It seems unlikely that CD4 + T cells activated through the indirect pathway would be able to recognize native allogeneic MHC class I target-bearing cells within the graft. However, it is interesting to speculate that MHC class II-restricted CD4* effector T cells might recognize endogenously synthesized allo-class I peptides presented naturally in the context of donor MHC class II by tissue cells within the graft. The vascular endothelium of MHC class l-disparate rat kidney allografts, which is usually regarded as the prime target for rejection, remains largely MHC class II-negative during rejection and would, therefore, escape recognition by MHC class If-restricted CD4 + effector T cells. However, renal tubular cells are strongly class II-positive and are, therefore, potential targets of such CD4 + effector T cells I°. Alternatively, CD4 + effector T cells could recognize MHC class I alloantigen that has been processed and presented in the context of MHC class II by host APCs within the graft itself. Such cells might then mediate a classical DTH reaction within the graft through release of cytokines, such as tumour necrosis factor ~ (TNF-~) and gamma-interferon (IFN-7) , which either damage the kidney directly or recruit nonspecific cellular effectors. Direct evidence to support a role for this effector pathway in rejection of MHC class I-disparate kidney grafts is lacking, although numerous CD4 + T cells and large numbers of activated macrophages can be identified in such grafts during rejection ~°.
CD4 ÷ T-cell-dependent effector mechanisms What might the CD4 + T-cell-dependent mechanism of rejection be for MHC class l-disparate kidney allografts? CD4 + T cells could, through CD4+-CD8 ÷ T-cell collaboration, promote the generation of CD8 + MHC class I-specific cytotoxic cells which, according to the traditional view, are the principal effectors of rejection (Fig. 1). However, as already noted, CD8 ÷ effector
Priming I
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~ ~')
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Fig. 1. Putative events underlying rejectton of an MHC class I-disparate graft highlighting the central role played by T cells and the redundancy of effector mechanisms. (a) CD8 + T cells directly recognize allogeneic MHC class I molecules expressed by donor APCs and differentiate into CD8* class I-specific CTL. This usually requires help from CD4 + T cells primed via the indirect pathway, but in certain situations CD8 + T cells are autonomous. (b) Host CD4 ÷ T cells are stimulated by an allogeneic peptide derived from donor MHC class I and presented in the context of syngeneic class II MHC, that is, via the indirect pathway of allorecognition. Because grafts are matched for MHC class II, APCs of either host or donor origin may, in principle, present class I peptide in the context of self MHC class II molecules. CD4 +effector T cells activated within the graft by this pathway effect rejection by release of cytokines, which mediate local nonspecific tissue injury. (c) Donor native class I molecules shed from the graft are recognized and processed by recipient B cells. Allogeneic class I peptides are then presented in the context of self MHC class II molecules to CD4 ÷ T cells and, as a result, B cells receive help to produce MHC class I-specific alloantibody.
1mmu,or,,gy roaay
Rejection of MHC class I-disparate grafts by alloantibody In addition to the cellular effector pathways already discussed, a further possibility is that CD4 + T cells activated by the indirect pathway mediate graft rejection via T-cell-B-cell collaboration, and the provision of help for antibody production. It is well recognized that pre-existing alloantibody is responsible for hyperacute rejection of human kidney allografts in sensitized recipients and the antibodies responsible are invariably directed against MHC class I molecules3s,34. However, the role of antibody in acute rejection by nonsensitized recipients is controversial. Rejection of RT1A '~ class I-disparate kidney grafts in both unmodified and anti-CD8 treated high-responder recipients is accompanied by a strong alloantibody response. In recent experiments, we have shown that passive transfer of immune serum obtained from such animals completely restores the ability of either cyclosporin or anti-CD4 mAb-treated recipients to reject an MHC class I-disparate kidney graft m°. The effect is allospecific, since fully allogeneic 'third party' PVG kidneys of the RT1 c haplotype display prolonged survival in such recipients, indicating that anti-RT1A ~ antibody is the critical mediator of rejection. Moreover, the histological pattern of graft rejection in recipients given immune serum is very similar to that observed in rejecting grafts from unmodified recipients. In both cases, damage to the microvasculature of the graft is an early feature, followed by ischaemia and
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viewpoint haemorrhage. Whether antibody mediates these effects through (1) antibody-dependent cellular cytotoxicity or (2) by complement fixation leading to endothelial activation and injury, followed by release of inflammatory mediators, is not known. Of course, these mechanisms are not mutually exclusive. These observations on the role of antibody in rejection of MHC class l-disparate kidney grafts in the rat challenge the widely held view that acute rejection in nonsensitized recipients is mediated by cellular, rather than antibody-dependent, effector mechanisms. Support for cellular mechanisms is derived largely from numerous studies in the 1950s and 1960s which demonstrated that the ability of mice to reject skin or tumour allografts was readily restored by transfer of immune cells, whereas attempts to cause graft rejection by passive transfer of immune serum were uniformly unsuccessful~,3L Even transfer of relatively large amounts of high titre immune serum did not have a detrimental effect on graft survival ~:. Similar results were subsequently obtained when vascularized organ grafts were examined in the rat and, in some strain combinations, transfer of immune serum led, paradoxically, to markedly improved, and sometimes permanent, graft survival (passive enhancement) 3s'~. How can a role for antibody in acute rejection of kidney allografts in the rat be reconciled with the results from these earlier animal studies? There are several possible explanations. First, the strain combinations and immunization protocols used to raise immune serum in some of the earlier studies of skin and organ graft rejection may have been biased towards the generation of anti-class II rather than anti-class I alloantibody. Whereas anticlass I antibody may produce graft damage, there is little evidence that anti-class II antibody is detrimental after transplantation, and the phenomenon of passive enhancement is largely attributable to anti-class lI alloantibodies 4°. Secondly, many of the early studies were concerned with rejection of skin grafts rather than vascularized organ grafts. Skin grafts may be less susceptible to antibody-mediated injury, since a substantial component of their vascular supply may be derived from the recipient. Interestingly, exceptional mouse strain combinations were noted where passive transfer of immune serum could cause skin graft rejection < . Third, there are important species and strain differences in the efficiency of complement. Early attempts to produce hyperacute rejection of rat kidney allografts were relatively unsuccessful, and this was attributed, in part, to species differences in complement activity42. Whether or not hyperimmune serum causes hyperacute rejection or enhancement of rat organ grafts is critically dependent on the strain combinations examined4L In our studies, passive transfer of anti-RT1A~ immune serum is unable to cause rejection of RTlA~'-disparate kidney grafts in PVG rats bearing the low responder RTI" haplotype, and we have attributed this to differences in complement activity between RTI ° and RT1' rats >. Other recent studies in the rat also point to the potential role of antibody in kidney allograft rejec-
1mm,,,otogy r,, ay
tion 44, and evidence is now emerging from clinical studies that the detrimental effect of anti-class 1 antibodies may not be confined to hyperacute rejection: Halloran et al? ~ have recently shown that the presence of circulating anti-class I antibodies following renal transplantation is associated with an increased severity of rejection episodes and histological evidence of microvascular injury4s. These results suggest there is a need to reassess the role of antibody in organ graft rejection. They also caution that the current approaches to specific immunotherapy, directed at individual T-cell subsets, may need to take into account the fact that MHC restriction may not necessarily predict the nature of the effector T-cell or the effector mechanism. This work was supported by the Western Infirmary Kidney Research Fund and the British Heart Foundation. J. Andrew Bradley and Eleanor M. Bolton are at the University Department of Surgery and Allan Mcl. Mowat is at the Department of Imnmnoh,gy, Western Infirmary, Glasgow, UK G11 6NT.
References 1 Bradley, J.A. and Bolton~E.M. (1992} Transplant. Rev. 6~ 115-129 2 Hall, B.M. (1991) Transplantation 51, 1141-1151 3 Mason, D.W. and Morris, P.J. (1986)Annu. Ret'. lmmunoL 4, 119-145 4 Rosenberg, A.S. and Singer, A. (1988) Proc. Natl Acad. Sci. USA 85, 7739-7742 5 Mintz, B. and Silvers, W.K. (1970) Transplantation 9, 497-505 6 Sprent, J., Schaefer, M., Lo, D. and Korngold, R. (1986) J. Exp. Med. 163,999-1011 7 Rosenberg, A.S., Mizuochi, T., Sharrow, S.O. and Singer, A. (1987) J. Exp. Med. 165, 1296-1315 8 Ichikawa, T., Nakayama, E., Uenaka, A., Monden, M. and Mori, T. (1987)J. Exp. Med. 166, 982-990 9 Rosenberg, A,S., Munitz, T.I., Maniero, T.G. and Singer, A, (1991)I. Exp. Med. 173, 1463-1471 10 Gracie, J.A., Bolton, E.M., Porteous, C and Bradley, J.A. (1990).l. Exp. Med. 172, 1547-1557 11 Bradley, J.A., Gracie, J.A., Porteous, C. and Bolton, E.M. (1991) Transplantation Proc. 23,266-267 12 Howard, J.C and Butcher, G.W. ( 1981 ) So,rod. I. lmmunol. 14, 687-691 13 Stewart, R., Butcher, G.W., Herbert, J. and Roser, B. (1985 ) Transplantation 40, 427-432 14 Davis, M.M. and Bjorkman, P.J. (1988) Nature ,334, 395-402 15 Townsend, A. and Bodmer, H. (1989) Atom. Rev. lmmunol. 7, 601-624 16 Monaco, J.J. (1992) lmmunol. Today 13, 173-179 17 Neefjes, 3.,1. and Ploegh, H.L. (1992)Immured. Today 13,179-184 18 Lechler, R.I., l,ombardi, G., Batchelor, J.R., Reinsmoen, N. and Bach, F.H. (1990) 1mmunol. Today 1I, 83-88 19 Swain, S.L. (1983) Immunol. Rev. 74, 129-142 20 Flomenberg, N., Naito, K., Duffy, E. et al. (1983) Eur. J. lmmunol. 13, 905-911 21 Strassman, G. and Bach, F.H. (1984)]. Immured. 133, 1705-1709
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22 Butcher, G.W. and Howard, J.C. (1982) Transplantation 34, 161-166 23 Lechler, R.I. and Batchelor, J.R. (1982) J. Exp. Med. 155, 31-41 24 Golding, H. and Singer, A. (1984)J. Immunol. 133, 597-605 25 Sherwood, R.A., Brent, L. and Rayfield, L.S. (1986) Eur. J. Immunol. 16, 569-574 26 Dalchau, R., Fangmann, J. and Fabre, J.W. (1992) Eur. J. Immunol. 22, 669-677 27 Fangmann, J., Dalchau, R., Sawyer, G.J., Priestley, C.A. and Fabre, J.W. Eur. J. Immunol. (in press) 28 Parker, K.E., Dalchau, R., Fowler, V.J. et al. (1992) Transplantation 53, 918-924 29 Fangmann, J., Dalchau, R. and Fabre, J.W. (1992) J. Exp. Med. 175, 1521-1529 30 Benichou, G., Takizawa, P.A., Olson, C.A., McMillan, M. and Sercarz, E.E. (1992) J. Exp. Med. 175, 305-308 31 Chen, B.P., Madrigal, A. and Parham, P. (1990) J. Exp. Med. 172, 779-788 32 Rudensky, A.Y., Preston-Hurlburt, P., Hong, S.C., Barlow, A. and Janeway, C.A. (1991) Nature 353, 622-627 33 Kissmeyer-Nielson, F., Olsen, S., Peteresen, V.P. and
Fjeldborg, O. (1966) Lancet ii, 662-665 34 Williams, G.M., Hume, D.M., Hudson, R.P. et al. (1968) New Engl. J. Med. 279, 611-618 35 Mitchison, N.A. (1954) Proc. Royal Soc. Lond. 142, 72-87 36 Brent, L. and Medawar, P.B. (1962) Proc. Royal Soc. Lond. B155, 392-416 37 Billingham, R.E. and Brent, L. (1956) Brit. ]. Exp. Path. 37, 566-569 38 Stuart, F.P., Siatoh, R. and Fitch, F.W. (1968) Science 160, 463 39 Fabre, J.W. and Morris, P.J. (1974) Transplantation 18,429-435 40 Morris, P.J. (1980) Immunol. Rev. 49, 93-125 41 Steinmuller, D. (1962) Ann. New York Acad. Sci. 99, 629-644 42 French, M.E. (1972) Transplantation 13,447-451 43 Oluwole, S.F., Tezuka, K., Wasfie, T. et al. (1989) Transplantation 48,751-755 44 Baldwin, W.M., Paul, L.C., Clads, F.H.J. and Deha, M.R. (1986) in Progress in Transplantation (Morris, P.J. and Tilney, N.L., eds), p. 85, Churchill Livingston 45 Halloran, P.F., Schlaut, J., Solez, K. and Srinivasa, N.S. (1992) Transplantation 53,550-555
The evolution of immune memory and germinal centers Moon H. Nahm, Frans G.M. Kroese and Joseph W. Hoffmann Antibody responses in homoiothermic and poikilothermic vertebrates are significantly different in their heterogeneity and affinity range, and in the speed of the secondary response following repeated antigenic stimulation. This article presents the hypothesis that the evolutionary development of unique lymphoid structures, the germinal centers, in combination with the development of a distinct B-cell lineage, is a determining feature of these differences. Important data relevant to the evolution of the immune response have been acquired from the study of poikilothermic vertebrates (Table 1). Both shark and fish produce low affinity antibody (Ab) with very little heterogeneity, even after repeated immunization with antigen 1-3. Even when kept at a warm temperature, the Ab response of turtles repeatedly immunized with a given antigen shows no apparent increase in Ab affinity and titer 4. Frogs and toads have immunoglobulin (Ig) genes organized like those of mammals - they have V region gene fragments (V, D and J regions) and produce IgM-like isotype and IgG-like isotype, the expression of which depends on T cells s - yet these amphibians produce a highly restricted set of Abs (Ref. 3) of low affinity 6. In a study of secondary antidinitrophenol (DNP) response in two anuran species,
Du Pasquier and Haimovich 6 found that the secondary response occurs relatively late (three weeks after secondary immunization) and is not marked by an increase in Ab affinity. Thus, it is generally accepted that the poikilothermic vertebrate Ab response is of low affinity for the antigen, has limited heterogeneity 3, and has poor anamnestic qualities. While information from birds is more limited than from mammals, both homoiothermic classes are clearly different from poikilothermic vertebrates in terms of the anamnestic Ab response. Like mammals, chickens can produce secondary Abs that are of higher affinity 7 and heterogeneity 3 than primary Abs, and peak Ab levels are attained within one week of boosting ~. Mammals also display all the features of anamnestic Ab response: for instance, the affinity and heterogeneity of
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Processed MHC class I alloantigen as the stimulus for CD4 ÷ T-cell dependent antibody-mediated graft rejection J. Andrew Bradley, Allan McI. Mowat and Eleanor M. Bolton The traditional view of graft rejection is one of direct recognition of allogeneic MHC molecules by effector T cells, the phenotype of which may be predicted by the nature of the MHC disparity. In this article, Andrew Bradley and colleagues discuss recent evidence that suggests this view may be an oversimplification, and argue that additional effector mechanisms, such as alloantibody, need to be reconsidered. Rejection of an organ allograft is characterized by a highly complex series of cellular and humoral interactions in which the T cell plays a central and essential role. In recent years, considerable progress has been made towards understanding how T cells initiate, regulate and effect graft rejection. Inevitably, much attention has focused on the relative roles of the phenotypically distinct CD4 ~ and CD8 + T-cell subpopulations in the rejection process. Many studies have attempted to define the requirements for these subsets in rejection, using two basic approaches: first, adoptive transfer analysis in T-cell deficient animals and, secondly, in vivo treatment of normal animals with monoclonal antibodies (mAbs) to selectively deplete or block the function of one or other of the T-cell subsets (reviewed in Ref. 1). The consensus view from such studies is that both CD4 + and CD8* T cells contribute to the rejection of fully allogeneic grafts, but overall responsibility for orchestrating the response is usually assigned to the CD4 + T-cell subset. It is widely accepted that graft rejection in recipients who have not had previous exposure to transplantation antigens, that is, first-set rejection, is mediated by effector T cells rather than by antibody-dependent effector mechanisms, and for many years there has been intense debate concerning the relative importance of specific cytotoxic T cells (CTL) - predominantly CD8 ÷ T cells - compared with cells that induce delayed type hypersensitivity (DTH) - usually CD4 + T cells - as the principal effector cell types *-3. Rejection entails a considerable degree of collaboration between these distinct cell types, a fact well illustrated by the elegant experiments of Rosenberg and colleagues 4 on skin graft rejection using allophenic mice. Allophenic mice are genetic mosaics which develop from the fused blastomeres of two different H-2 strains, such that their individual cells express one or other set (but not both) of the parental major histocompatibility (MHC) complex molecules s. Transplantation of allophenic skin to either parental strain results in the
rapid and complete destruction of the epidermis by a non-specific inflammatory response consistent with a DTH effector mechanism. However, all the melanocytes and hair follicles expressing the nonshared allogeneic MHC antigens are specifically destroyed, leaving the adjacent syngeneic cells perfectly intact 4. This implies a specific CTL effector mechanism. The balance between CD4* and CD8 + T cells as the principal effector cells mediating rejection is likely to depend on such factors as the type of graft and, in particular, the nature of the MHC disparity between donor and recipient. The availability of mutant and recombinant rodent strains that differ from each other at isolated MHC subregions has enabled these variables to be examined in greater detail, although most such studies have confined their attention to the rejection of skin grafts. The T-cell requirement for rejection of skin grafts bearing isolated MHC disparities Adoptive transfer experiments in T-cell depleted mice bearing mutant MHC class I- or class II-disparate skin grafts have shown that purified CD8 +, but not CD4*, T cells reject MHC class I-disparate grafts, whereas only CD4* effector T cells destroy MHC class II-disparate grafts 6,7. Isolated MHC class I disparities have been the most extensively studied and the exclusive role for CD8 ÷ effector T cells has been confirmed by mAb depletion of T-cell subsets in vivo s,9. Surprisingly, in certain MHC class I-disparate mouse strain combinations, selective depletion of CD8* T cells by anti-CD8 mAb treatment does not prevent skin graft rejection, suggesting that CD4 + T cells may also participate in the rejection of such grafts ~'9. The presence of an anti-class I alloantibody response in recipients of allelic class I-disparate grafts is further evidence for CD4+ T-cell activation, since the functional overlap between CD8* and CD4 + T cells does not extend to the provision of cognate T-cell help by CD8* T cells for the production of antibody. Nevertheless, the prevailing
© 1992, Elsevier Science Publishers Ltd, UK.
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viewpoint view remains that CD8 + effector cells are essential for reiection of MHC class I-disparate grafts. Rosenberg and colleagues have recently attributed rejection of MHC class I-disparate skin grafts in antiCD8 treated mice to a unique population of anti-CD8 resistant, CD8 + CTL which are dependent for their generation on help from CD4 + T cells triggered by additional alloantigens co-expressed by MHC class ldisparate grafts ~.
Rejection of MHC class 1-disparate kidney allografts Organ grafts are more directly relevant to clinical transplantation than skin grafts and, since there are likely to be important differences in the nature of the rejection response between indirectly vascularized skin grafts and directly vascularized organ grafts, we chose to examine the role of CD4 + and CD8 ~ T cells in the rejection of rat kidney allografts across an isolated allelic MHC class I disparity~'J(uL In these studies, we used PVG congenic rats, which bear the RTI" haplotype and are high responders to the RT1A ~'class I antigen. These animals, which rapidly reject class I-disparate skin and organ grafts ~e';~, were treated in vivo with mAbs to specifically deplete CDS+ or CD4 + T cells. Interestingly, although anti-CD8 treatment was highly effective at depleting CD8* T cells, both phenotypically and functionally, it did not prolong the survival of RTIN' class I-incompatible kidney allografts from donors of the PVG recombinant strain, RS. Moreover, unlike anti-CD8-treated mice, there was no evidence that anti-CDS-resistant CD8 ~ CTLs were responsible for rejection in this experimental model and the grafts in anti-CDS-treated animals were destroyed in the absence of detectable CDS* effector T ceils within the graft infiltrate. In contrast to anti-CD8 treatment, administration of anti-CD4 mAb markedly delayed rejection and, in some animals, led to permanent survival of class I-disparate kidney grafts. From these experiments we concluded that, under certain circumstances, CD4 ~ T cells alone may be sufficient to initiate rejection of MHC class I-disparate organ grafts. Conversely, CD8* T cells are neither necessary nor, by themselves, sufficient.
Processed MHC class I alloantigen as the stimulus for rejection? The above studies raise intriguing questions as to the nature of the alloantigen recognized by CD4 + T cells and the effector mechanisms responsible for rejection of MHC class I-disparate kidney grafts. To answer these questions it is necessary to consider, briefly, current concepts of how T cells may recognize alloantigen. During a conventional T-cell response to a protein antigen, the c(~ T-cell receptor (TCR) molecule recognizes processed antigenic peptides presented by self MHC molecules ]4 J-. The T-cell response to alloantigens may, therefore, be exceptional because it may be directed at intact a[logeneic MHC molecules themselves ~s. To what extent the TCR recognizes the endogenous peptide in the binding groove of the allogeneic MHC molecule and to what extent it recognizes epitopes of the allogeneic MHC molecule adjacent to
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the peptide-binding groove is still unclear ~". In either event, it is difficult to reconcile the possibility that allogeneic MHC molecules are recognized directly by host T cells with the dogma that T cells only recognize antigen complexed with syngeneic MHC molecules. The expression of CD4 and CD8 by T cells normally defines their MHC-binding specificity]*: CD8 ÷ T cells recognize antigen in the context of self MHC class I molecules and respond directly to allogeneic MHC class 1, whereas CD4* T cells recognize antigen in the context of self MHC class II molecules and respond directly to allogeneic MHC class 1I molecules. How, then, might CD4 + T cells be primed against alloantigens from MHC class l-disparate grafts? There are three possibilities. First, CD4 + T cells may recognize and be activated by intact allogeneic MHC class I molecules. We think that this is unlikely, but it cannot be completely discounted because exceptional CD4 + Tcell clones have been described that are lyric towards MHC class I targets -'°'-'~. Secondly, CD4" T cells may be activated, not by MHC class I alloantigens themselves, but by other alloantigenic determinants co-expressed by the graft *. Although we cannot entirely exclude the possibility that there may be additional antigenic differences between the recombinant rat strains used in our studies, their existence and potential role in activating CD4 ÷ T cells must be largely speculative. The third, and in our view the most likely, possibility is that CD4 + T cells are stimulated by graft MHC class I alloantigens which have been processed by recipient antigen-presenting cells (APCs) and presented as antigenic class 1 peptide fragments in the antigenhinding groove of self MHC class II molecules. This is analogous to the way in which CD4" T cells recognize conventional antigens and has been called the 'indirect pathway' of allorecognition. Although this pathway of allorecognition was originally postulated in the early 1980s:: -", convincing evidence that it may be important in graft rejection has emerged only very recently z~-~. In particular, studies in the rat have clearly demonstrated that immunization with peptides from allogeneic MHC class I molecules may prime recipient (~D4+ T cells 2- and lead to accelerated reiection of skin all(> grafts-'". Although we have not directly addressed the question of how CD4 + T cells are activated to class I alloantigens, it is notable that when PV(; RTI" rats arc' immunized with purified RT1A ~' MHC class I antigen, they mount a strong T helper ceil-dependent cytotoxic alloantibody response (C. Kelly, J.A. Bradley and J. Fabre, unpublished observations). This observation clearly illustrates the potential for indirect allorecognition of processed RTIN ~class I by RT1 ~' CD4 + helper T cells, since neither intact, membrane-bound MHC class I molecules nor other poorly defined alloantigens are present to provide helper determinants in this system. It is interesting to note that when host and graft are mismatched for MHC class I but matched for MHC class II molecules, as in the studies discussed here, host CD4 + T cells could, in principle, recognize allogeneic class I peptides presented by syngeneic MHC class II
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viewpoint molecules on APCs originating from either the graft recipient or the donor organ. The latter route of sensitization would necessitate physiological presentation of stimulatory peptides derived from internalized cell-surface MHC class I molecules. Such a concept is not simply a theoretical possibility, since naturally occurring peptides derived from endogenously synthesized cell membrane proteins have recently been isolated from the binding cleft of MHC class II molecules32.
T cells are not necessary for rejection of such grafts in anti-CD8-treated recipients. It seems unlikely that CD4 + T cells activated through the indirect pathway would be able to recognize native allogeneic MHC class I target-bearing cells within the graft. However, it is interesting to speculate that MHC class II-restricted CD4* effector T cells might recognize endogenously synthesized allo-class I peptides presented naturally in the context of donor MHC class II by tissue cells within the graft. The vascular endothelium of MHC class l-disparate rat kidney allografts, which is usually regarded as the prime target for rejection, remains largely MHC class II-negative during rejection and would, therefore, escape recognition by MHC class If-restricted CD4 + effector T cells. However, renal tubular cells are strongly class II-positive and are, therefore, potential targets of such CD4 + effector T cells I°. Alternatively, CD4 + effector T cells could recognize MHC class I alloantigen that has been processed and presented in the context of MHC class II by host APCs within the graft itself. Such cells might then mediate a classical DTH reaction within the graft through release of cytokines, such as tumour necrosis factor ~ (TNF-~) and gamma-interferon (IFN-7) , which either damage the kidney directly or recruit nonspecific cellular effectors. Direct evidence to support a role for this effector pathway in rejection of MHC class I-disparate kidney grafts is lacking, although numerous CD4 + T cells and large numbers of activated macrophages can be identified in such grafts during rejection ~°.
CD4 ÷ T-cell-dependent effector mechanisms What might the CD4 + T-cell-dependent mechanism of rejection be for MHC class l-disparate kidney allografts? CD4 + T cells could, through CD4+-CD8 ÷ T-cell collaboration, promote the generation of CD8 + MHC class I-specific cytotoxic cells which, according to the traditional view, are the principal effectors of rejection (Fig. 1). However, as already noted, CD8 ÷ effector
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Fig. 1. Putative events underlying rejectton of an MHC class I-disparate graft highlighting the central role played by T cells and the redundancy of effector mechanisms. (a) CD8 + T cells directly recognize allogeneic MHC class I molecules expressed by donor APCs and differentiate into CD8* class I-specific CTL. This usually requires help from CD4 + T cells primed via the indirect pathway, but in certain situations CD8 + T cells are autonomous. (b) Host CD4 ÷ T cells are stimulated by an allogeneic peptide derived from donor MHC class I and presented in the context of syngeneic class II MHC, that is, via the indirect pathway of allorecognition. Because grafts are matched for MHC class II, APCs of either host or donor origin may, in principle, present class I peptide in the context of self MHC class II molecules. CD4 +effector T cells activated within the graft by this pathway effect rejection by release of cytokines, which mediate local nonspecific tissue injury. (c) Donor native class I molecules shed from the graft are recognized and processed by recipient B cells. Allogeneic class I peptides are then presented in the context of self MHC class II molecules to CD4 ÷ T cells and, as a result, B cells receive help to produce MHC class I-specific alloantibody.
1mmu,or,,gy roaay
Rejection of MHC class I-disparate grafts by alloantibody In addition to the cellular effector pathways already discussed, a further possibility is that CD4 + T cells activated by the indirect pathway mediate graft rejection via T-cell-B-cell collaboration, and the provision of help for antibody production. It is well recognized that pre-existing alloantibody is responsible for hyperacute rejection of human kidney allografts in sensitized recipients and the antibodies responsible are invariably directed against MHC class I molecules3s,34. However, the role of antibody in acute rejection by nonsensitized recipients is controversial. Rejection of RT1A '~ class I-disparate kidney grafts in both unmodified and anti-CD8 treated high-responder recipients is accompanied by a strong alloantibody response. In recent experiments, we have shown that passive transfer of immune serum obtained from such animals completely restores the ability of either cyclosporin or anti-CD4 mAb-treated recipients to reject an MHC class I-disparate kidney graft m°. The effect is allospecific, since fully allogeneic 'third party' PVG kidneys of the RT1 c haplotype display prolonged survival in such recipients, indicating that anti-RT1A ~ antibody is the critical mediator of rejection. Moreover, the histological pattern of graft rejection in recipients given immune serum is very similar to that observed in rejecting grafts from unmodified recipients. In both cases, damage to the microvasculature of the graft is an early feature, followed by ischaemia and
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viewpoint haemorrhage. Whether antibody mediates these effects through (1) antibody-dependent cellular cytotoxicity or (2) by complement fixation leading to endothelial activation and injury, followed by release of inflammatory mediators, is not known. Of course, these mechanisms are not mutually exclusive. These observations on the role of antibody in rejection of MHC class l-disparate kidney grafts in the rat challenge the widely held view that acute rejection in nonsensitized recipients is mediated by cellular, rather than antibody-dependent, effector mechanisms. Support for cellular mechanisms is derived largely from numerous studies in the 1950s and 1960s which demonstrated that the ability of mice to reject skin or tumour allografts was readily restored by transfer of immune cells, whereas attempts to cause graft rejection by passive transfer of immune serum were uniformly unsuccessful~,3L Even transfer of relatively large amounts of high titre immune serum did not have a detrimental effect on graft survival ~:. Similar results were subsequently obtained when vascularized organ grafts were examined in the rat and, in some strain combinations, transfer of immune serum led, paradoxically, to markedly improved, and sometimes permanent, graft survival (passive enhancement) 3s'~. How can a role for antibody in acute rejection of kidney allografts in the rat be reconciled with the results from these earlier animal studies? There are several possible explanations. First, the strain combinations and immunization protocols used to raise immune serum in some of the earlier studies of skin and organ graft rejection may have been biased towards the generation of anti-class II rather than anti-class I alloantibody. Whereas anticlass I antibody may produce graft damage, there is little evidence that anti-class II antibody is detrimental after transplantation, and the phenomenon of passive enhancement is largely attributable to anti-class lI alloantibodies 4°. Secondly, many of the early studies were concerned with rejection of skin grafts rather than vascularized organ grafts. Skin grafts may be less susceptible to antibody-mediated injury, since a substantial component of their vascular supply may be derived from the recipient. Interestingly, exceptional mouse strain combinations were noted where passive transfer of immune serum could cause skin graft rejection < . Third, there are important species and strain differences in the efficiency of complement. Early attempts to produce hyperacute rejection of rat kidney allografts were relatively unsuccessful, and this was attributed, in part, to species differences in complement activity42. Whether or not hyperimmune serum causes hyperacute rejection or enhancement of rat organ grafts is critically dependent on the strain combinations examined4L In our studies, passive transfer of anti-RT1A~ immune serum is unable to cause rejection of RTlA~'-disparate kidney grafts in PVG rats bearing the low responder RTI" haplotype, and we have attributed this to differences in complement activity between RTI ° and RT1' rats >. Other recent studies in the rat also point to the potential role of antibody in kidney allograft rejec-
1mm,,,otogy r,, ay
tion 44, and evidence is now emerging from clinical studies that the detrimental effect of anti-class 1 antibodies may not be confined to hyperacute rejection: Halloran et al? ~ have recently shown that the presence of circulating anti-class I antibodies following renal transplantation is associated with an increased severity of rejection episodes and histological evidence of microvascular injury4s. These results suggest there is a need to reassess the role of antibody in organ graft rejection. They also caution that the current approaches to specific immunotherapy, directed at individual T-cell subsets, may need to take into account the fact that MHC restriction may not necessarily predict the nature of the effector T-cell or the effector mechanism. This work was supported by the Western Infirmary Kidney Research Fund and the British Heart Foundation. J. Andrew Bradley and Eleanor M. Bolton are at the University Department of Surgery and Allan Mcl. Mowat is at the Department of Imnmnoh,gy, Western Infirmary, Glasgow, UK G11 6NT.
References 1 Bradley, J.A. and Bolton~E.M. (1992} Transplant. Rev. 6~ 115-129 2 Hall, B.M. (1991) Transplantation 51, 1141-1151 3 Mason, D.W. and Morris, P.J. (1986)Annu. Ret'. lmmunoL 4, 119-145 4 Rosenberg, A.S. and Singer, A. (1988) Proc. Natl Acad. Sci. USA 85, 7739-7742 5 Mintz, B. and Silvers, W.K. (1970) Transplantation 9, 497-505 6 Sprent, J., Schaefer, M., Lo, D. and Korngold, R. (1986) J. Exp. Med. 163,999-1011 7 Rosenberg, A.S., Mizuochi, T., Sharrow, S.O. and Singer, A. (1987) J. Exp. Med. 165, 1296-1315 8 Ichikawa, T., Nakayama, E., Uenaka, A., Monden, M. and Mori, T. (1987)J. Exp. Med. 166, 982-990 9 Rosenberg, A,S., Munitz, T.I., Maniero, T.G. and Singer, A, (1991)I. Exp. Med. 173, 1463-1471 10 Gracie, J.A., Bolton, E.M., Porteous, C and Bradley, J.A. (1990).l. Exp. Med. 172, 1547-1557 11 Bradley, J.A., Gracie, J.A., Porteous, C. and Bolton, E.M. (1991) Transplantation Proc. 23,266-267 12 Howard, J.C and Butcher, G.W. ( 1981 ) So,rod. I. lmmunol. 14, 687-691 13 Stewart, R., Butcher, G.W., Herbert, J. and Roser, B. (1985 ) Transplantation 40, 427-432 14 Davis, M.M. and Bjorkman, P.J. (1988) Nature ,334, 395-402 15 Townsend, A. and Bodmer, H. (1989) Atom. Rev. lmmunol. 7, 601-624 16 Monaco, J.J. (1992) lmmunol. Today 13, 173-179 17 Neefjes, 3.,1. and Ploegh, H.L. (1992)Immured. Today 13,179-184 18 Lechler, R.I., l,ombardi, G., Batchelor, J.R., Reinsmoen, N. and Bach, F.H. (1990) 1mmunol. Today 1I, 83-88 19 Swain, S.L. (1983) Immunol. Rev. 74, 129-142 20 Flomenberg, N., Naito, K., Duffy, E. et al. (1983) Eur. J. lmmunol. 13, 905-911 21 Strassman, G. and Bach, F.H. (1984)]. Immured. 133, 1705-1709
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22 Butcher, G.W. and Howard, J.C. (1982) Transplantation 34, 161-166 23 Lechler, R.I. and Batchelor, J.R. (1982) J. Exp. Med. 155, 31-41 24 Golding, H. and Singer, A. (1984)J. Immunol. 133, 597-605 25 Sherwood, R.A., Brent, L. and Rayfield, L.S. (1986) Eur. J. Immunol. 16, 569-574 26 Dalchau, R., Fangmann, J. and Fabre, J.W. (1992) Eur. J. Immunol. 22, 669-677 27 Fangmann, J., Dalchau, R., Sawyer, G.J., Priestley, C.A. and Fabre, J.W. Eur. J. Immunol. (in press) 28 Parker, K.E., Dalchau, R., Fowler, V.J. et al. (1992) Transplantation 53, 918-924 29 Fangmann, J., Dalchau, R. and Fabre, J.W. (1992) J. Exp. Med. 175, 1521-1529 30 Benichou, G., Takizawa, P.A., Olson, C.A., McMillan, M. and Sercarz, E.E. (1992) J. Exp. Med. 175, 305-308 31 Chen, B.P., Madrigal, A. and Parham, P. (1990) J. Exp. Med. 172, 779-788 32 Rudensky, A.Y., Preston-Hurlburt, P., Hong, S.C., Barlow, A. and Janeway, C.A. (1991) Nature 353, 622-627 33 Kissmeyer-Nielson, F., Olsen, S., Peteresen, V.P. and
Fjeldborg, O. (1966) Lancet ii, 662-665 34 Williams, G.M., Hume, D.M., Hudson, R.P. et al. (1968) New Engl. J. Med. 279, 611-618 35 Mitchison, N.A. (1954) Proc. Royal Soc. Lond. 142, 72-87 36 Brent, L. and Medawar, P.B. (1962) Proc. Royal Soc. Lond. B155, 392-416 37 Billingham, R.E. and Brent, L. (1956) Brit. ]. Exp. Path. 37, 566-569 38 Stuart, F.P., Siatoh, R. and Fitch, F.W. (1968) Science 160, 463 39 Fabre, J.W. and Morris, P.J. (1974) Transplantation 18,429-435 40 Morris, P.J. (1980) Immunol. Rev. 49, 93-125 41 Steinmuller, D. (1962) Ann. New York Acad. Sci. 99, 629-644 42 French, M.E. (1972) Transplantation 13,447-451 43 Oluwole, S.F., Tezuka, K., Wasfie, T. et al. (1989) Transplantation 48,751-755 44 Baldwin, W.M., Paul, L.C., Clads, F.H.J. and Deha, M.R. (1986) in Progress in Transplantation (Morris, P.J. and Tilney, N.L., eds), p. 85, Churchill Livingston 45 Halloran, P.F., Schlaut, J., Solez, K. and Srinivasa, N.S. (1992) Transplantation 53,550-555
The evolution of immune memory and germinal centers Moon H. Nahm, Frans G.M. Kroese and Joseph W. Hoffmann Antibody responses in homoiothermic and poikilothermic vertebrates are significantly different in their heterogeneity and affinity range, and in the speed of the secondary response following repeated antigenic stimulation. This article presents the hypothesis that the evolutionary development of unique lymphoid structures, the germinal centers, in combination with the development of a distinct B-cell lineage, is a determining feature of these differences. Important data relevant to the evolution of the immune response have been acquired from the study of poikilothermic vertebrates (Table 1). Both shark and fish produce low affinity antibody (Ab) with very little heterogeneity, even after repeated immunization with antigen 1-3. Even when kept at a warm temperature, the Ab response of turtles repeatedly immunized with a given antigen shows no apparent increase in Ab affinity and titer 4. Frogs and toads have immunoglobulin (Ig) genes organized like those of mammals - they have V region gene fragments (V, D and J regions) and produce IgM-like isotype and IgG-like isotype, the expression of which depends on T cells s - yet these amphibians produce a highly restricted set of Abs (Ref. 3) of low affinity 6. In a study of secondary antidinitrophenol (DNP) response in two anuran species,
Du Pasquier and Haimovich 6 found that the secondary response occurs relatively late (three weeks after secondary immunization) and is not marked by an increase in Ab affinity. Thus, it is generally accepted that the poikilothermic vertebrate Ab response is of low affinity for the antigen, has limited heterogeneity 3, and has poor anamnestic qualities. While information from birds is more limited than from mammals, both homoiothermic classes are clearly different from poikilothermic vertebrates in terms of the anamnestic Ab response. Like mammals, chickens can produce secondary Abs that are of higher affinity 7 and heterogeneity 3 than primary Abs, and peak Ab levels are attained within one week of boosting ~. Mammals also display all the features of anamnestic Ab response: for instance, the affinity and heterogeneity of
© 1992, Elsevier Science Publishers Ltd, UK.
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