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Blocking T-cell costimulation to prevent transplant rejection
Blocking T-Cell Costimulation to Prevent Transplant Rejection X.-G. Zheng and L.A. Turka
O
VER 25 years ago, Bretscher and Cohn1 proposed a model of self-nonself discrimination. According to their model, tolerance to autoantigens was regulated in part by a requirement for antigen non-specific “second” signals in order to effect lymphocyte activation. Although their model was proposed for B cells and regulation of autoantibody production, this two-signal hypothesis was first confirmed in the T-cell system.
CD28 AS A COSTIMULATORY RECEPTOR
At our present level of understanding, T and B-cell activation is initiated by ligation of their antigen receptors. In the case of T cells, this is effected when the T-cell receptor (TCR) engages peptide bound to an appropriate major histocompatibility complex (MHC)-encoded molecule. For B cells, activation occurs when soluble antigen binds to cell surface immunoglobulin (Ig). In each case, antigen induces the lymphocyte to enter the cell cycle. However, a second “costimulatory” signal is required to complete the activation process and induce cell division. In the case of T cells, the best-characterized costimulatory signal is delivered through the CD28 molecule, a member of the Ig gene superfamily.2 CD28 is expressed on all rat and mouse T cells and on the majority of human T cells. It has two known ligands, B7-1 (CD80) and B7-2 (CD86), each of which is expressed on activated bone marrow-derived antigen-presenting cells (APCs) and, in some species, on activated endothelial cells. CD28 meets several criteria for definition as a costimulatory molecule, including the ability to transduce a signal biochemically distinct from the TCR, the ability to deliver its signal in trans (ie, on a cell other than the one presenting antigen), and the ability to prevent anergy induction (see below). CD28 stimulation has several effects on T cells. First, it dramatically lowers the number of TCRs which must be engaged for T cells to produce interleukin-2 (IL-2) and a variety of other T cell-derived cytokines such as interferon gamma (IFN-g).3,4 In the presence of T-cell costimulation, ligation of as few as 1800 TCRs can induce efficient T-cell activation. In the absence of CD28 costimulation, signaling through as many as 20,000 TCRs is needed. Thus, CD28 costimulation allows for the initial induction of T-cell activation when antigen density/availability is low. Although the downstream signaling pathways of CD28 remain controversial, critical early effects of CD28 costimulation (in
combination with TCR ligation) appear to be the induction of cytokine gene transcription and the stabilization of cytokine mRNA transcripts. The net result is greatly augmented and sustained levels of cytokine protein secretion. Second, CD28 costimulation is critical for the maintenance of an immune response.3 When signaling through CD28 is prevented by blocking antibodies or fusion proteins, T-cell proliferation and cytokine production can be initiated. However, these effector functions are lost within 48 to 72 hours. Studies in mice with a targeted deletion of the CD28 gene confirm this finding.5 These data indicate that CD28 costimulation is required to sustain an immune response, even one which was initiated by a TCR signal strong enough to bypass a need for CD28 in the inductive phase. Without CD28 signaling, T-cell responses are incomplete and transitory. Third, CD28 costimulation appears to be important to maintain T cells in a responsive state. Studies of T-cell clones in vitro indicate that TCR ligation without CD28 costimulation induces anergy in the T cells, a state in which the cells are nonresponsive to TCR engagement.6,7 In these studies, CD28 costimulation could prevent anergy induction, but it could not reverse established anergy. Anergy, however, was not permanent, requiring renewal after several weeks by TCR ligation without costimulation, and also was reversible with IL-2. This latter finding suggested a mechanism for the breakdown of self-tolerance, in which exposure of T cells to autoantigens in an inflammatory setting (eg, a viral infection) in which many cytokines were present might break anergy. Several studies of naive T cells have confirmed the need for costimulation to prevent anergy, although many reports have found that TCR ligation without costimulation leads to T-cell apoptosis.8 T-cell activation induces low and transitory expression of the cell survival gene Bcl-xL, a gene which blocks apoptotic death in T cells induced by Cytokine withdrawal. CD28 costimulation augments the level and duration of Bcl-xL expression, suggesting a mechanism by which it may prevent apoptosis.9 In addition, by inducing sustained levels of cytokine secre-
From the Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA. Address reprint requests to Laurence A. Turka, Department of Medicine, University of Pennsylvania, 901 Stellar-Chance Laboratories, 422 Curie Blvd, Philadelphia, PA 19104-6100.
0041-1345/98/$19.00 PII S0041-1345(98)00568-5
© 1998 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
2146
Transplantation Proceedings, 30, 2146–2149 (1998)
T-CELL COSTIMULATION
tion, CD28 may prevent apoptosis occurring as a result of growth factor deprivation in cycling cells.10 CTLA4, A NEGATIVE REGULATOR OF CD28
T cells also express CTLA4, a molecule homologous to CD28. CTLA4 transduces a negative signal to the T cell, specifically inhibiting CD28-mediated costimulation. Consistent with this, CTLA4-deficient mice develop widespread lymphoproliferation with lymphoid infiltration of visceral organs and die within several weeks of birth.11,12 Studies showing that inhibition of CD28-mediated signals in these mice prevent development of this phenotype indicate that the inhibitory function of CTLA4 is CD28 specific. They also show that the pathology in CTLA4-deficient mice is not due to interference with negative selection in the thymus per se.13 CTLA4 signals appear to inhibit IL-2 production and IL-2 receptor expression, leading to cell cycle arrest.14 Apoptosis may subsequently occur consequent to growth factor withdrawal. Recently, Perez et al15 have linked this pathway to the induction of anergy in vivo, showing that deliberate CTLA4 ligation was necessary to induce T-cell tolerance. In their studies in a model of antigen immunization, blocking CD28 costimulation prevented a primary T-cell response at the time of antigen stimulation but did not prevent subsequent responses to antigen. It is unknown whether or not this role of CTLA4 ligation for tolerance induction is generalizable to other immune response such as transplantation. THE CD40:CD154 SYSTEM
CD40, a member of the tumor necrosis factor receptor family, is expressed on a variety of cell types, including B cells, macrophages, dendritic cells, and endothelial cells. Its ligand, CD154 (formerly called CD40L), is expressed on activated T cells. Engagement of CD40 by CD154 has a variety of effects.16 In the case of B cells, it provides a signal which prevents B-cell apoptosis and simultaneously promotes a switch from IgM to IgG and IgE production. Consistent with this, defects in CD154 are the genetic basis for X-linked hyperIgM syndrome. In the case of macrophages and endothelial cells, CD40 stimulation is a proinflammatory stimulus, inducing IL-12 secretion and adhesion molecule expression, respectively. Most relevantly for the topic of this review, CD40 stimulation of APCs (including B cells, macrophages, and dendritic cells) is a strong inducer of B7 expression, particularly B7-1.17,18 In experimental transplant models, blockade of the CD154:CD40 pathway completely prevents the induction of B7-1 expression which is otherwise normally observed within the allograft itself and peripheral lymphoid tissues.18 Interruption of the CD154:CD40 pathway potently inhibits both humoral and cellular immune responses. Antibodies to CD154 induce transplantation tolerance in murine models of islet and cardiac transplantation.18 –20 The mechanism by which blocking this pathway inhibits T-cell re-
2147
sponses is uncertain. CD154 might directly transduce a costimulatory signal to T cells, but this issue remains controversial. The finding that CD40 engagement induced B7 molecule expression on APCs has led to the suggestion that blockade of the CD154:CD40 pathway might act by preventing B7 expression and thus indirectly inhibit CD28mediated T-cell costimulation. Indeed, in animal models such as experimental allergic encephalomyelitis or the response to adenoviral gene therapy vectors, the ability of anti-CD154 antibodies to completely suppress the immune response appears to be attributable to inhibition of B7 expression.21,22 However, in the case of transplantation where B7-1 expression is inhibited by anti-CD154 antibodies, blockade of B7-1 cannot by itself induce transplantation tolerance and therefore is not the sole mechanism of action of anti-CD154 antibodies.18,23 Recently, CD40 stimulation induce other potentially important molecules on APCs, including CD44H, which has been implicated as a true T-cell costimulatory molecule.24 It is likely that the tolerance-inducing effects of anti-CD154 antibodies in transplantation are attributable to interrupting a variety of sequences in the immune response, including cytokine production, adhesion molecule induction, and B7 and CD44H expression. BLOCKING COSTIMULATORY SIGNALS PREVENTS TRANSPLANT REJECTION
Over the past 6 years, many groups have shown that blocking CD28-mediated costimulation prevents cellular immune responses, including transplant rejection. Most studies have used CTLA4Ig, a fusion protein using the binding region of CTLA4 fused to an immunoglobulin heavy chain.25 Consistent with CTLA4’s high affinity for B7 molecules, CTLA4Ig is a potent competitive inhibitor of CD28 for binding. The Ig tail confers a half-life of 7 to 10 days to this protein, making it feasible for in vivo use. Initial studies in a xenogeneic islet model (human to mouse) demonstrated the induction of donor-specific tolerance.26 Studies in a rat vascularized cardiac allograft model showed a significant delay in rejection (median graft survival 33 days compared with a control of 9 days), but failed to show long-term allograft survival.27 It was felt that the higher precursor frequency of allogeneic versus xenogeneic cells and the immunogenicity of the endothelium present in a vascularized graft might account for the disparate results. Subsequently, many investigators have shown that blocking CD28-mediated T-cell costimulation induces transplantation tolerance.28 In general, tolerance induction has required one of two strategies: either chronic (ie, 2 to 3-week) administration of CTLA4Ig or a single dose of CTLA4Ig plus concomitant use of donor spleen or bone marrow cells (donor specific transfusion [DST]). In the absence of either of these interventions, long-term graft survival can be achieved, but severe chronic rejection is almost uniformly seen.29 It can be envisioned that both strategies are actually acting by the same mechanism, if we postulate that acute rejection occurs as a result of direct allorecognition (ie, T
2148
cells recognize foreign MHC on donor cells) and that chronic rejection occurs as a result of indirect allorecognition (ie, T cells recognize foreign peptides processed by self-APCs and presented on self-MHC). In this instance, a short course of CTLA4Ig would tolerize the host to direct allorecognition, the only form which was occurring several days after transplantation. If indirect allopresentation does not ensue until later, the resulting T-cell response would not be blocked and gradual rejection would occur. When CTLA4Ig is given for several weeks, indirect allorecognition occurs during the time frame of administration of the drug and the T-cell response becomes suppressed. DST might effect the same result, by promoting indirect allopresentation during the first few days. The large load of donor MHC class II-positive cells delivered to peripheral lymphoid tissue by DST may result in an early burst of indirect allopresentation, allowing for tolerance induction to this pathway with a short course of CTLA4Ig. The ability to prevent chronic rejection is, an important feature of costimulatory blockade. In many transplant models, long-term graft survival is seen in a heterotopic system, but histologic examination of the grafts shows severe chronic rejection. This, for example, is seen using cyclosporine (CyA).30 Adding DST and CTLA4Ig to CyA prevents the occurrence of histologic lesions in a rat model of chronic cardiac rejection.31 As chronic rejection is the primary cause of late graft loss in clinical renal, cardiac, and lung transplantation, this means that costimulatory blockade is a promising adjunctive form of therapy to prevent the development of chronic rejection. Since 1997, CTLA4Ig has been studied in primate models. This type of large animal testing is critical prior to human clinical trials. As rodents are notoriously susceptible to tolerance induction, primate studies are required to determine which agents might truly be suitable for human trials. Levisetti et al32 reported on five primate recipients of allogeneic islet transplants treated with a 14-day course of CTLA4Ig. Two of the five had long-term graft survival and the other three experienced early rejection. Interestingly, none of the five developed alloantibodies, an important consideration given the fact that sensitization occurring as a result of transplant rejection is a major problem for patients on the cadaveric kidney waiting list. Kirk et al33 have used CTLA4Ig, anti-CD154 monoclonal antibodies (MAbs), or a combination of the two in a study of primate renal transplantation using eight animals. Although only preliminary conclusions can be drawn due to the small study size, it appeared that the anti-CD154 MAb alone was effective in delaying, but not preventing, rejection. However, the combination of CTLA4Ig and anti-CD154 MAbs was able to induce long-term rejection-free graft survival. This type of synergy of CTLA4Ig and anti-CD154 MAb had previously been observed by Larsen et al34 in murine skin allografts.34 However, in that study, CyA abrogated this synergistic effect. This important issue will need to be addressed in the design and implementation of clinical trials.
ZHENG AND TURKA
CONCLUSIONS
The strategy of blocking costimulatory signals to prevent transplant rejection holds the potential to improve longterm graft survival, prevent chronic rejection, and induce tolerance. Each of these has been seen in rodent models of transplantation, although whether or not this can be translated into similar advances in large animals and humans remains to be seen. The challenge to be faced in this arena is to design appropriate clinical trials to address these questions. REFERENCES 1. Bretscher P, Cohn M: Science 169:1042, 1970 2. June CH, Bluestone JA, Nadler LM, et al: Immunol Today 15:321, 1994 3. Wells A, Gudmundsdottir H, Turka L: J Clin Invest 100:3173, 1997 4. Viola A, Lanzavecchia A: Science 273:104, 1996 5. Green J, Noel P, Sperling A, et al: Immunity 1:501, 1994 6. Harding FA, McArthur JG, Gross JA, et al: Nature 356:607, 1992 7. Jenkins MK, Schwartz RH: J Exp Med 165:302, 1987 8. Noel PJ, Boise LH, Green JM, et al: J Immunol 157:636, 1996 9. Boise LH, Minn AJ, Noel PJ, et al: Immunity 3:87, 1995 10. Mueller DL, Seiffert S, Fang W, et al: J Immunol 156:1764, 1996 11. Tivol EA, Boriello F, Schweitzer AN, et al: Immunity 3:541, 1995 12. Waterhouse P, Penninger JM, Timms E, et al: Science 270:985, 1995 13. Tivol E, Boyd S, McKeon S, et al: J Immunol 158:5091, 1997 14. Walunas TL, Bakker CY, Bluestone JA: J Exp Med 183: 2541, 1996 15. Perez V, Parijs LV, Biuckians A, et al: Immunity 6:411, 1997 16. Durie FH, Foy TM, Masters SR, et al: Immunol Today 15:406, 1994 17. Ranheim EA, Kipps TJ: J Exp Med 177:925, 1993 18. Hancock WW, Sayegh MH, Zheng X-G, et al: Proc Natl Acad Sci USA 93:12967, 1996 19. Larsen CP, Alexander DZ, Hollenbaugh D, et al: Transplantation 61:4, 1996 20. Parker DC, Greiner DL, Phillps NE, et al: Proc Natl Acad Sci USA 92:9560, 1995 21. Grewal IS, Foellmer HG, Grewal KD, et al: Science 273: 1864, 1996 22. Yang Y, Wilson JM: Science 273:1862, 1996 23. Zheng X, Sayegh M, Zheng X-G, et al: J Immunol 159:1169, 1997 24. Guo Y, Wu Y, Shinde S, et al: J Exp Med 184:955, 1996 25. Linsley PS, Brady W, Urnes M, et al: J Exp Med 174:561, 1991 26. Lenschow D, Zeng Y, Thistlethwaite R, et al: Science 257:789, 1992 27. Turka LA, Linsley PS, Lin H, et al: Proc Natl Acad Sci USA 89:11102, 1992 28. Sayegh MH, Turka LA: J Am Soc Nephrol 6:1143, 1995
T-CELL COSTIMULATION 29. Sayegh M, Zheng X-G, Magee C, et al: Transplantation 64:1646, 1998 30. Azuma H, Chandraker A, Nadeau K, et al: Proc Natl Acad Sci USA (in press) 31. Chandraker A, Russell M, Glysing-Jensen T, et al: Transplantation 63:1053, 1997
2149 32. Levisetti M, Padrid P, Szot G, et al: J Immunol 159:5187, 1997 33. Kirk A, Harlan D, Armstrong N, et al: Proc Natl Acad Sci USA 94:8789, 1997 34. Larsen CP, Elwood ET, Alexander DZ, et al: Nature 381: 434, 1996
O
VER 25 years ago, Bretscher and Cohn1 proposed a model of self-nonself discrimination. According to their model, tolerance to autoantigens was regulated in part by a requirement for antigen non-specific “second” signals in order to effect lymphocyte activation. Although their model was proposed for B cells and regulation of autoantibody production, this two-signal hypothesis was first confirmed in the T-cell system.
CD28 AS A COSTIMULATORY RECEPTOR
At our present level of understanding, T and B-cell activation is initiated by ligation of their antigen receptors. In the case of T cells, this is effected when the T-cell receptor (TCR) engages peptide bound to an appropriate major histocompatibility complex (MHC)-encoded molecule. For B cells, activation occurs when soluble antigen binds to cell surface immunoglobulin (Ig). In each case, antigen induces the lymphocyte to enter the cell cycle. However, a second “costimulatory” signal is required to complete the activation process and induce cell division. In the case of T cells, the best-characterized costimulatory signal is delivered through the CD28 molecule, a member of the Ig gene superfamily.2 CD28 is expressed on all rat and mouse T cells and on the majority of human T cells. It has two known ligands, B7-1 (CD80) and B7-2 (CD86), each of which is expressed on activated bone marrow-derived antigen-presenting cells (APCs) and, in some species, on activated endothelial cells. CD28 meets several criteria for definition as a costimulatory molecule, including the ability to transduce a signal biochemically distinct from the TCR, the ability to deliver its signal in trans (ie, on a cell other than the one presenting antigen), and the ability to prevent anergy induction (see below). CD28 stimulation has several effects on T cells. First, it dramatically lowers the number of TCRs which must be engaged for T cells to produce interleukin-2 (IL-2) and a variety of other T cell-derived cytokines such as interferon gamma (IFN-g).3,4 In the presence of T-cell costimulation, ligation of as few as 1800 TCRs can induce efficient T-cell activation. In the absence of CD28 costimulation, signaling through as many as 20,000 TCRs is needed. Thus, CD28 costimulation allows for the initial induction of T-cell activation when antigen density/availability is low. Although the downstream signaling pathways of CD28 remain controversial, critical early effects of CD28 costimulation (in
combination with TCR ligation) appear to be the induction of cytokine gene transcription and the stabilization of cytokine mRNA transcripts. The net result is greatly augmented and sustained levels of cytokine protein secretion. Second, CD28 costimulation is critical for the maintenance of an immune response.3 When signaling through CD28 is prevented by blocking antibodies or fusion proteins, T-cell proliferation and cytokine production can be initiated. However, these effector functions are lost within 48 to 72 hours. Studies in mice with a targeted deletion of the CD28 gene confirm this finding.5 These data indicate that CD28 costimulation is required to sustain an immune response, even one which was initiated by a TCR signal strong enough to bypass a need for CD28 in the inductive phase. Without CD28 signaling, T-cell responses are incomplete and transitory. Third, CD28 costimulation appears to be important to maintain T cells in a responsive state. Studies of T-cell clones in vitro indicate that TCR ligation without CD28 costimulation induces anergy in the T cells, a state in which the cells are nonresponsive to TCR engagement.6,7 In these studies, CD28 costimulation could prevent anergy induction, but it could not reverse established anergy. Anergy, however, was not permanent, requiring renewal after several weeks by TCR ligation without costimulation, and also was reversible with IL-2. This latter finding suggested a mechanism for the breakdown of self-tolerance, in which exposure of T cells to autoantigens in an inflammatory setting (eg, a viral infection) in which many cytokines were present might break anergy. Several studies of naive T cells have confirmed the need for costimulation to prevent anergy, although many reports have found that TCR ligation without costimulation leads to T-cell apoptosis.8 T-cell activation induces low and transitory expression of the cell survival gene Bcl-xL, a gene which blocks apoptotic death in T cells induced by Cytokine withdrawal. CD28 costimulation augments the level and duration of Bcl-xL expression, suggesting a mechanism by which it may prevent apoptosis.9 In addition, by inducing sustained levels of cytokine secre-
From the Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA. Address reprint requests to Laurence A. Turka, Department of Medicine, University of Pennsylvania, 901 Stellar-Chance Laboratories, 422 Curie Blvd, Philadelphia, PA 19104-6100.
0041-1345/98/$19.00 PII S0041-1345(98)00568-5
© 1998 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
2146
Transplantation Proceedings, 30, 2146–2149 (1998)
T-CELL COSTIMULATION
tion, CD28 may prevent apoptosis occurring as a result of growth factor deprivation in cycling cells.10 CTLA4, A NEGATIVE REGULATOR OF CD28
T cells also express CTLA4, a molecule homologous to CD28. CTLA4 transduces a negative signal to the T cell, specifically inhibiting CD28-mediated costimulation. Consistent with this, CTLA4-deficient mice develop widespread lymphoproliferation with lymphoid infiltration of visceral organs and die within several weeks of birth.11,12 Studies showing that inhibition of CD28-mediated signals in these mice prevent development of this phenotype indicate that the inhibitory function of CTLA4 is CD28 specific. They also show that the pathology in CTLA4-deficient mice is not due to interference with negative selection in the thymus per se.13 CTLA4 signals appear to inhibit IL-2 production and IL-2 receptor expression, leading to cell cycle arrest.14 Apoptosis may subsequently occur consequent to growth factor withdrawal. Recently, Perez et al15 have linked this pathway to the induction of anergy in vivo, showing that deliberate CTLA4 ligation was necessary to induce T-cell tolerance. In their studies in a model of antigen immunization, blocking CD28 costimulation prevented a primary T-cell response at the time of antigen stimulation but did not prevent subsequent responses to antigen. It is unknown whether or not this role of CTLA4 ligation for tolerance induction is generalizable to other immune response such as transplantation. THE CD40:CD154 SYSTEM
CD40, a member of the tumor necrosis factor receptor family, is expressed on a variety of cell types, including B cells, macrophages, dendritic cells, and endothelial cells. Its ligand, CD154 (formerly called CD40L), is expressed on activated T cells. Engagement of CD40 by CD154 has a variety of effects.16 In the case of B cells, it provides a signal which prevents B-cell apoptosis and simultaneously promotes a switch from IgM to IgG and IgE production. Consistent with this, defects in CD154 are the genetic basis for X-linked hyperIgM syndrome. In the case of macrophages and endothelial cells, CD40 stimulation is a proinflammatory stimulus, inducing IL-12 secretion and adhesion molecule expression, respectively. Most relevantly for the topic of this review, CD40 stimulation of APCs (including B cells, macrophages, and dendritic cells) is a strong inducer of B7 expression, particularly B7-1.17,18 In experimental transplant models, blockade of the CD154:CD40 pathway completely prevents the induction of B7-1 expression which is otherwise normally observed within the allograft itself and peripheral lymphoid tissues.18 Interruption of the CD154:CD40 pathway potently inhibits both humoral and cellular immune responses. Antibodies to CD154 induce transplantation tolerance in murine models of islet and cardiac transplantation.18 –20 The mechanism by which blocking this pathway inhibits T-cell re-
2147
sponses is uncertain. CD154 might directly transduce a costimulatory signal to T cells, but this issue remains controversial. The finding that CD40 engagement induced B7 molecule expression on APCs has led to the suggestion that blockade of the CD154:CD40 pathway might act by preventing B7 expression and thus indirectly inhibit CD28mediated T-cell costimulation. Indeed, in animal models such as experimental allergic encephalomyelitis or the response to adenoviral gene therapy vectors, the ability of anti-CD154 antibodies to completely suppress the immune response appears to be attributable to inhibition of B7 expression.21,22 However, in the case of transplantation where B7-1 expression is inhibited by anti-CD154 antibodies, blockade of B7-1 cannot by itself induce transplantation tolerance and therefore is not the sole mechanism of action of anti-CD154 antibodies.18,23 Recently, CD40 stimulation induce other potentially important molecules on APCs, including CD44H, which has been implicated as a true T-cell costimulatory molecule.24 It is likely that the tolerance-inducing effects of anti-CD154 antibodies in transplantation are attributable to interrupting a variety of sequences in the immune response, including cytokine production, adhesion molecule induction, and B7 and CD44H expression. BLOCKING COSTIMULATORY SIGNALS PREVENTS TRANSPLANT REJECTION
Over the past 6 years, many groups have shown that blocking CD28-mediated costimulation prevents cellular immune responses, including transplant rejection. Most studies have used CTLA4Ig, a fusion protein using the binding region of CTLA4 fused to an immunoglobulin heavy chain.25 Consistent with CTLA4’s high affinity for B7 molecules, CTLA4Ig is a potent competitive inhibitor of CD28 for binding. The Ig tail confers a half-life of 7 to 10 days to this protein, making it feasible for in vivo use. Initial studies in a xenogeneic islet model (human to mouse) demonstrated the induction of donor-specific tolerance.26 Studies in a rat vascularized cardiac allograft model showed a significant delay in rejection (median graft survival 33 days compared with a control of 9 days), but failed to show long-term allograft survival.27 It was felt that the higher precursor frequency of allogeneic versus xenogeneic cells and the immunogenicity of the endothelium present in a vascularized graft might account for the disparate results. Subsequently, many investigators have shown that blocking CD28-mediated T-cell costimulation induces transplantation tolerance.28 In general, tolerance induction has required one of two strategies: either chronic (ie, 2 to 3-week) administration of CTLA4Ig or a single dose of CTLA4Ig plus concomitant use of donor spleen or bone marrow cells (donor specific transfusion [DST]). In the absence of either of these interventions, long-term graft survival can be achieved, but severe chronic rejection is almost uniformly seen.29 It can be envisioned that both strategies are actually acting by the same mechanism, if we postulate that acute rejection occurs as a result of direct allorecognition (ie, T
2148
cells recognize foreign MHC on donor cells) and that chronic rejection occurs as a result of indirect allorecognition (ie, T cells recognize foreign peptides processed by self-APCs and presented on self-MHC). In this instance, a short course of CTLA4Ig would tolerize the host to direct allorecognition, the only form which was occurring several days after transplantation. If indirect allopresentation does not ensue until later, the resulting T-cell response would not be blocked and gradual rejection would occur. When CTLA4Ig is given for several weeks, indirect allorecognition occurs during the time frame of administration of the drug and the T-cell response becomes suppressed. DST might effect the same result, by promoting indirect allopresentation during the first few days. The large load of donor MHC class II-positive cells delivered to peripheral lymphoid tissue by DST may result in an early burst of indirect allopresentation, allowing for tolerance induction to this pathway with a short course of CTLA4Ig. The ability to prevent chronic rejection is, an important feature of costimulatory blockade. In many transplant models, long-term graft survival is seen in a heterotopic system, but histologic examination of the grafts shows severe chronic rejection. This, for example, is seen using cyclosporine (CyA).30 Adding DST and CTLA4Ig to CyA prevents the occurrence of histologic lesions in a rat model of chronic cardiac rejection.31 As chronic rejection is the primary cause of late graft loss in clinical renal, cardiac, and lung transplantation, this means that costimulatory blockade is a promising adjunctive form of therapy to prevent the development of chronic rejection. Since 1997, CTLA4Ig has been studied in primate models. This type of large animal testing is critical prior to human clinical trials. As rodents are notoriously susceptible to tolerance induction, primate studies are required to determine which agents might truly be suitable for human trials. Levisetti et al32 reported on five primate recipients of allogeneic islet transplants treated with a 14-day course of CTLA4Ig. Two of the five had long-term graft survival and the other three experienced early rejection. Interestingly, none of the five developed alloantibodies, an important consideration given the fact that sensitization occurring as a result of transplant rejection is a major problem for patients on the cadaveric kidney waiting list. Kirk et al33 have used CTLA4Ig, anti-CD154 monoclonal antibodies (MAbs), or a combination of the two in a study of primate renal transplantation using eight animals. Although only preliminary conclusions can be drawn due to the small study size, it appeared that the anti-CD154 MAb alone was effective in delaying, but not preventing, rejection. However, the combination of CTLA4Ig and anti-CD154 MAbs was able to induce long-term rejection-free graft survival. This type of synergy of CTLA4Ig and anti-CD154 MAb had previously been observed by Larsen et al34 in murine skin allografts.34 However, in that study, CyA abrogated this synergistic effect. This important issue will need to be addressed in the design and implementation of clinical trials.
ZHENG AND TURKA
CONCLUSIONS
The strategy of blocking costimulatory signals to prevent transplant rejection holds the potential to improve longterm graft survival, prevent chronic rejection, and induce tolerance. Each of these has been seen in rodent models of transplantation, although whether or not this can be translated into similar advances in large animals and humans remains to be seen. The challenge to be faced in this arena is to design appropriate clinical trials to address these questions. REFERENCES 1. Bretscher P, Cohn M: Science 169:1042, 1970 2. June CH, Bluestone JA, Nadler LM, et al: Immunol Today 15:321, 1994 3. Wells A, Gudmundsdottir H, Turka L: J Clin Invest 100:3173, 1997 4. Viola A, Lanzavecchia A: Science 273:104, 1996 5. Green J, Noel P, Sperling A, et al: Immunity 1:501, 1994 6. Harding FA, McArthur JG, Gross JA, et al: Nature 356:607, 1992 7. Jenkins MK, Schwartz RH: J Exp Med 165:302, 1987 8. Noel PJ, Boise LH, Green JM, et al: J Immunol 157:636, 1996 9. Boise LH, Minn AJ, Noel PJ, et al: Immunity 3:87, 1995 10. Mueller DL, Seiffert S, Fang W, et al: J Immunol 156:1764, 1996 11. Tivol EA, Boriello F, Schweitzer AN, et al: Immunity 3:541, 1995 12. Waterhouse P, Penninger JM, Timms E, et al: Science 270:985, 1995 13. Tivol E, Boyd S, McKeon S, et al: J Immunol 158:5091, 1997 14. Walunas TL, Bakker CY, Bluestone JA: J Exp Med 183: 2541, 1996 15. Perez V, Parijs LV, Biuckians A, et al: Immunity 6:411, 1997 16. Durie FH, Foy TM, Masters SR, et al: Immunol Today 15:406, 1994 17. Ranheim EA, Kipps TJ: J Exp Med 177:925, 1993 18. Hancock WW, Sayegh MH, Zheng X-G, et al: Proc Natl Acad Sci USA 93:12967, 1996 19. Larsen CP, Alexander DZ, Hollenbaugh D, et al: Transplantation 61:4, 1996 20. Parker DC, Greiner DL, Phillps NE, et al: Proc Natl Acad Sci USA 92:9560, 1995 21. Grewal IS, Foellmer HG, Grewal KD, et al: Science 273: 1864, 1996 22. Yang Y, Wilson JM: Science 273:1862, 1996 23. Zheng X, Sayegh M, Zheng X-G, et al: J Immunol 159:1169, 1997 24. Guo Y, Wu Y, Shinde S, et al: J Exp Med 184:955, 1996 25. Linsley PS, Brady W, Urnes M, et al: J Exp Med 174:561, 1991 26. Lenschow D, Zeng Y, Thistlethwaite R, et al: Science 257:789, 1992 27. Turka LA, Linsley PS, Lin H, et al: Proc Natl Acad Sci USA 89:11102, 1992 28. Sayegh MH, Turka LA: J Am Soc Nephrol 6:1143, 1995
T-CELL COSTIMULATION 29. Sayegh M, Zheng X-G, Magee C, et al: Transplantation 64:1646, 1998 30. Azuma H, Chandraker A, Nadeau K, et al: Proc Natl Acad Sci USA (in press) 31. Chandraker A, Russell M, Glysing-Jensen T, et al: Transplantation 63:1053, 1997
2149 32. Levisetti M, Padrid P, Szot G, et al: J Immunol 159:5187, 1997 33. Kirk A, Harlan D, Armstrong N, et al: Proc Natl Acad Sci USA 94:8789, 1997 34. Larsen CP, Elwood ET, Alexander DZ, et al: Nature 381: 434, 1996