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Agonistic and Antagonistic Interactions Between CTLA4Ig and Donor Alloantigens in Sensitized Rat Recipients of Cardiac Allografts
Agonistic and Antagonistic Interactions Between CTLA4Ig and Donor Alloantigens in Sensitized Rat Recipients of Cardiac Allografts K. Onodera, A. Chandraker, S. Korom, T.H.W. Stadlbauer, K. Kato, S. Kasai, M.H. Sayegh, and J.W. Kupiec-Weglinski
T
HE BLOCKADE of CD28-B7 costimulation pathway after administration of the fusion protein CTLA4Ig prevents acute and chronic allograft (Tx) rejection and may induce tolerance in some animal transplant models.1– 4 This study was designed to investigate the efficacy of and interactions between CTLA4Ig and donor allo-Ag in a welldefined model of accelerated rejection of cardiac Tx in presensitized rat recipients. MATERIALS AND METHODS Tx Model Lewis (RT1l) rats were sensitized with Brown Norway (RT1n) skin Tx (day 27), followed 1 week later (day 0) by transplantation of LBNF1 hearts. These cardiac Tx are rejected in less than 36 hours, compared to 7- to 8-day acute rejection in otherwise unsensitized hosts.5
Host Immunomoduration Because of known synergistic interactions with donor allo-Ag,1 engrafted Lewis hosts were treated with human or murine CTLA4Ig (0.5 mg/rat IV) at day 25 and day 12 in concert with erythrocyte-free LBNF1 spleen cells at day 27 (20 to .100 3 106/rat IV) and at day 0 (40 3 106). Based on the number of donor cells administered at day 27, the following experimental groups were under investigation: 1A: 20 3 106 cells; 1B: 40 to 60 3 106 cells; 1C: .100 3 106 cells. Animals in groups 2A, 2B, and 2C were conditioned with increasing dose of LBNF1 spleen cells alone, at day 27 (20 to .100 3 106) and at day 0 (40 3 106). Moreover, group 3A recipients were given a mutant form of murine CTLA4Ig (CTLA4IgY 100F) with adjunctive donor cells according to 1A regimen. Both in vitro and vivo studies indicate that CTLA4IgY 100F binds and effectively blocks B7-1 (CD80) with similar avidity as does CTLA4Ig, but it has at least 200-fold lower avidity for B7-2 (CD86) (R. Peach, unpublished observations, 1997.6 The half-life of CTLA4IgY 100F in mice is similar to that of CTLA4Ig. Control animals were either untreated or received an isotype-matched chimeric recombinant fusion protein L6.
Evaluation of Tx Survival, Tolerance Induction, and Allo-Ab Responses The survival of cardiac Tx was monitored by daily palpation, and rejection was taken as the day of complete cessation of a heart beat. To test for the induction of tolerance, CGLA4Ig-treated engrafted hosts were challenged more than 70 days after the primary transplant, with a secondary donor-specific (LBNF1) or third-party 0041-1345/98/$19.00 PII S0041-1345(97)01165-2 16
(Wistar Furth) test cardiac Tx. The circulating donor-specific IgM and IgG allo-Ab responses were tested serially in recipients serum. Briefly, Brown Norway lymph node target cells were first incubated with heat-inactivated sera samples, and the washed cells were then reacted with a mixture of FITC-conjugated goat Ab specific for the Fc portion of IgG and PE-conjugated goat Ab specific for the m chain of rat IgM. After staining, washed cells were fixed in 1% neutral buffered formalin, and the mode channel fluorescence was analyzed on an EPICS C cell sorter (Coulter, Hialeah, Fla) to assess IgM (PE channel) and IgG (FITC channel) allo-Ab levels.
RESULTS
As shown in Table 1, sensitized, otherwise untreated rats rejected their cardiac Tx in less than 2 days. Treatment with CTLA4Ig in conjunction with donor spleen cells (20 3 106 at day 27) resulted in long-term Tx acceptance (1A; mean survival time [MST] . 49.1 6 55.8 days). Long-term (.70 day) group 1A recipients accepted secondary LBNF1 (MST . 50 days) but rejected secondary Wistar Furth (MST , 10 days) test cardiac allografts, consistent with the efficacy of CTLA4Ig 1 allo-Ag to induce donor-specific tolerance in about 33% of the treated hosts. In contrast, infusion of donor spleen cells alone (2A; 20 3 106 at day 27) marginally extended Tx survival to 7.2 6 1.5 days. Increasing the number of transferred cells to 40 to 60 3 106 (1B) and to more than 100 3 106 (1C) abrogated in vivo synergistic effects between CTLA4Ig and donor allo-Ag in a dose-dependent manner. In fact, as shown in Table 1, cardiac Tx survival in rats given CTLA4Ig in concert with high number of donor cells (1B and 1C) was similar to that after infusion of allo-Ag alone (2B and 2C). Interestingly, administration of mutated form of CTLA4Ig (CTLA4IgY 100F) in 3A recipients according to the most effective (1A) From Harvard Medical School, The Surgical Research Laboratory, Department of Surgery, USA, and Medicine, Brigham and Women’s Hospital, Boston, Massachusetts; and Second Department of Surgery, Asahikawa Medical College, Asahikawa, Japan. This work was supported by USPHS grant AI23847 and AI34965. Address reprint requests to Dr J.W. Kupiec-Weglinski, The Dumont-UCLA Transplant Center, 10833 Le Conte Ave, Los Angeles, CA 90095, USA. © 1998 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 Transplantation Proceedings, 30, 16–18 (1998)
INTERACTION BETWEEN CTLA4IG AND ALLOANTIGENS
17
Table 1. Effects of Treatment with CTLA4Ig and Donor-Specific allo-Ag on Rejection of Cardiac Allografts in Presensitized Rat Recipients Group
No Rx Group 1A Group Group Group Group Group Group
1B 1C 2A 2B 2C 3A
Cardiac allograft survival (d)
0, 0, 0, 0, 0, 0, 1, 1, 1, 1 5, 7, 9, 13, 13, 16, 16, 17, 22, 24, 24, .27, 40, .42, .42, 44, 45, 60, .113, .117, .120, .120, .137, .138, .160, .160, .163 4, 12, 15, 19 1, 2, 2, 3, 3, 5, 6 5, 5, 6, 7, 7, 7, 8, 9, 9, 9 1, 1, 1, 2, 2, 4, 6, 7 0, 0, 0, 1, 1, 1, 1, 1, 1, 1, 3 4, 5, 7, 8
N
MST 6 SD (d)
10 27
0.4 6 0.5 .49.1 6 55.8
4 7 10 8 11 4
12.5 6 6.4 3.1 6 1.8 7.2 6 1.5 2.0 6 2.4 0.5 6 0.8 6.0 6 1.8
Group 1: CTLA4Ig (day 25 & 12) and S.C. (day 27 & 0) Group 2: S.C. (day 27 & 0) A: S.C. 20 3 106/rat IV at day 27 B: S.C. 40 – 60 3 106/rat IV at day 27 C: S.C. .100 3 106/rat IV at day 27 Gr. 3A: CTLA4IgY100F (day 25 & 12) and S.C. (day 27 & 0) S.C., donor-specific allo-Ag.
protocol resulted in Tx survival of 6.0 6 1.8 days, as if donor cells alone have been transferred (2A; 7.2 6 1.5 days). Skin grafts were rejected uniformly within 9 days irrespective of combined CTLA4Ig and spleen cell therapy. As shown in Fig 1, skin transplantation in otherwise untreated rats elicited a strong systemic IgM allo-Ab response, which peaked first around the time of cardiac Tx rejection at day 1 and then at about 3 weeks posttransplant. The IgM switched to IgG allo-Ab response 4 days after cardiac engraftment. Infusion of donor spleen cells alone
(2A) resulted in an early IgM peak at day 23 to 24 and a second peak between day 14 to 28. The switch from IgM to IgG occurred earlier, and it was even more intense as compared to that during unmodified accelerated rejection. Unlike in rats challenged with allo-Ag alone (2A), addition of CTLA4Ig to the most effective (1A) regimen significantly diminished IgM allo-Ab responses beyond day 4; these remained within the background values in long-term tolerant hosts. Moreover, IgG allo-Ab levels were markedly suppressed throughout the 150-day observation period.
Fig 1. Flow cytometric analysis of circulating IgM and IgG allo-Ab responses in rat cardiac allograft recipients. Each point represents the average mode channel staining 6 the standard deviation at a 1:4 dilution (N 5 3 to 5 for each data point).
18
Finally, substitution of CTLA4Ig by its mutated form in group 3A, resulted in depression of IgM, whereas the IgG allo-Ab pattern was comparable to that after infusion of allo-Ag alone (2A). DISCUSSION
The present study demonstrates that CTLA4Ig-mediated blockade of the CD28-B7 signaling pathway combined with transfusion of donor allo-Ag in the peritransplant period abrogated accelerated rejection and produced donor-specific tolerance in 33% of presensitized rat recipients. Importantly, this effect was strictly cell dose-dependent, and in the most effective regimen (1A), it required adjunctive transfusion of 20 3 106 splenocytes at day 27, that is, at the time of skin Tx challenge. However, once the number of transferred cells exceeded 40 3 106, this synergistic therapeutic effect between CTLA4Ig and donor allo-Ag significantly weakened (1B). Furthermore, when the number of cells exceeded 100 3 106, the beneficial effects of combined CTLA4Ig and allo-Ag therapy completely disappeared, resulting in accelerated rather than acute cardiac Tx rejection (1C). Hence, the most effective CTLA4Ig-mediated blockade of the CD28-B7 signaling pathway in sensitized hosts requires adjunctive infusion of allo-Ag in a dose, which by itself enhances Tx survival. CTLA4Ig is a potent suppressor of Ab responses in vivo, and in mice it depresses T cell-dependent Ab responses to sheep RBC or soluble protein Ag, keyhole limpet hemocyanid.7 Hence, we reasoned that blockade of the CD28-B7 costimulation pathway may prevent strong humoral responses, which otherwise, are critical in the immune cascade leading to accelerated rejection of cardiac Tx in this sensitized rat model.8 Indeed, indefinite graft survival in tolerant rats conditioned with CTLA4Ig plus donor allo-Ag (1A) was accompanied by virtually abrogated antidonor IgM and IgG allo-Ab serum levels. However, relatively high circulating IgM allo-Ab levels were detected around the time of cardiac engraftment in both control and experimen-
ONODERA, CHANDRAKER, KOROM ET AL
tal groups. Moreover, IgG allo-Ab levels were somewhat elevated around the time of cardiac engraftment after infusion of donor cells alone, but remained largely diminished after combined CTLA4Ig 1 donor cell therapy. Hence, the question arises as to whether such elevated allo-Ab levels influence the host immune response by themselves, or their presence merely reflects the pattern of sensitization. Whether these are putative “enhancing” Abs that may act in a protective manner comparably to the Th2-type subset at the cellular level3 remains to be determined. Finally, the selective blockade of CD80 by CTLA4IgY 100F in this study failed to replicate the immunosuppressive effects of CTLA4Ig and did not affect the IgM and IgG allo-Ab patterns elicited by donor cell infusion. This suggests the importance of CD86 blockade for long-term Tx acceptance, consistent with an early upregulation of CD86 in response to allo-Ag challenge, and prominent CD86 expression on endothelial cells.6,9
REFERENCES 1. Lin H, Bolling SF, Lindsey PS, et al: J Exp Med 178:1801, 1993 2. Pearson TC, Alexander DZ, Winn KJ, et al. Transplantation 57:1701, 1994 3. Sayegh MH, Akalin E, Hancock WW, et al: Exp Med 181:1869, 1995 4. Russel ME, Hancock WW, Akalin E et al: J Clin Invest 97:833, 1996 5. Onodera K, Lehmann M, Akalin E, et al: J Immunol 157: 1944, 1996 6. Hancock WW, Sayegh MH, Zheng XG, et al: Proc Natl Acad Sci USA 93:13967, 1996 7. Linsley PS, Wallace PM, Johnson J, et al: Science 257:792, 1992 8. Hancock WW, Di Stefano R, Braun P, et al: Transplantation 48:416, 1990 9. Hathcock KS, Laszio G, Pucill C, et al: J Exp Med 180:631, 1994
T
HE BLOCKADE of CD28-B7 costimulation pathway after administration of the fusion protein CTLA4Ig prevents acute and chronic allograft (Tx) rejection and may induce tolerance in some animal transplant models.1– 4 This study was designed to investigate the efficacy of and interactions between CTLA4Ig and donor allo-Ag in a welldefined model of accelerated rejection of cardiac Tx in presensitized rat recipients. MATERIALS AND METHODS Tx Model Lewis (RT1l) rats were sensitized with Brown Norway (RT1n) skin Tx (day 27), followed 1 week later (day 0) by transplantation of LBNF1 hearts. These cardiac Tx are rejected in less than 36 hours, compared to 7- to 8-day acute rejection in otherwise unsensitized hosts.5
Host Immunomoduration Because of known synergistic interactions with donor allo-Ag,1 engrafted Lewis hosts were treated with human or murine CTLA4Ig (0.5 mg/rat IV) at day 25 and day 12 in concert with erythrocyte-free LBNF1 spleen cells at day 27 (20 to .100 3 106/rat IV) and at day 0 (40 3 106). Based on the number of donor cells administered at day 27, the following experimental groups were under investigation: 1A: 20 3 106 cells; 1B: 40 to 60 3 106 cells; 1C: .100 3 106 cells. Animals in groups 2A, 2B, and 2C were conditioned with increasing dose of LBNF1 spleen cells alone, at day 27 (20 to .100 3 106) and at day 0 (40 3 106). Moreover, group 3A recipients were given a mutant form of murine CTLA4Ig (CTLA4IgY 100F) with adjunctive donor cells according to 1A regimen. Both in vitro and vivo studies indicate that CTLA4IgY 100F binds and effectively blocks B7-1 (CD80) with similar avidity as does CTLA4Ig, but it has at least 200-fold lower avidity for B7-2 (CD86) (R. Peach, unpublished observations, 1997.6 The half-life of CTLA4IgY 100F in mice is similar to that of CTLA4Ig. Control animals were either untreated or received an isotype-matched chimeric recombinant fusion protein L6.
Evaluation of Tx Survival, Tolerance Induction, and Allo-Ab Responses The survival of cardiac Tx was monitored by daily palpation, and rejection was taken as the day of complete cessation of a heart beat. To test for the induction of tolerance, CGLA4Ig-treated engrafted hosts were challenged more than 70 days after the primary transplant, with a secondary donor-specific (LBNF1) or third-party 0041-1345/98/$19.00 PII S0041-1345(97)01165-2 16
(Wistar Furth) test cardiac Tx. The circulating donor-specific IgM and IgG allo-Ab responses were tested serially in recipients serum. Briefly, Brown Norway lymph node target cells were first incubated with heat-inactivated sera samples, and the washed cells were then reacted with a mixture of FITC-conjugated goat Ab specific for the Fc portion of IgG and PE-conjugated goat Ab specific for the m chain of rat IgM. After staining, washed cells were fixed in 1% neutral buffered formalin, and the mode channel fluorescence was analyzed on an EPICS C cell sorter (Coulter, Hialeah, Fla) to assess IgM (PE channel) and IgG (FITC channel) allo-Ab levels.
RESULTS
As shown in Table 1, sensitized, otherwise untreated rats rejected their cardiac Tx in less than 2 days. Treatment with CTLA4Ig in conjunction with donor spleen cells (20 3 106 at day 27) resulted in long-term Tx acceptance (1A; mean survival time [MST] . 49.1 6 55.8 days). Long-term (.70 day) group 1A recipients accepted secondary LBNF1 (MST . 50 days) but rejected secondary Wistar Furth (MST , 10 days) test cardiac allografts, consistent with the efficacy of CTLA4Ig 1 allo-Ag to induce donor-specific tolerance in about 33% of the treated hosts. In contrast, infusion of donor spleen cells alone (2A; 20 3 106 at day 27) marginally extended Tx survival to 7.2 6 1.5 days. Increasing the number of transferred cells to 40 to 60 3 106 (1B) and to more than 100 3 106 (1C) abrogated in vivo synergistic effects between CTLA4Ig and donor allo-Ag in a dose-dependent manner. In fact, as shown in Table 1, cardiac Tx survival in rats given CTLA4Ig in concert with high number of donor cells (1B and 1C) was similar to that after infusion of allo-Ag alone (2B and 2C). Interestingly, administration of mutated form of CTLA4Ig (CTLA4IgY 100F) in 3A recipients according to the most effective (1A) From Harvard Medical School, The Surgical Research Laboratory, Department of Surgery, USA, and Medicine, Brigham and Women’s Hospital, Boston, Massachusetts; and Second Department of Surgery, Asahikawa Medical College, Asahikawa, Japan. This work was supported by USPHS grant AI23847 and AI34965. Address reprint requests to Dr J.W. Kupiec-Weglinski, The Dumont-UCLA Transplant Center, 10833 Le Conte Ave, Los Angeles, CA 90095, USA. © 1998 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 Transplantation Proceedings, 30, 16–18 (1998)
INTERACTION BETWEEN CTLA4IG AND ALLOANTIGENS
17
Table 1. Effects of Treatment with CTLA4Ig and Donor-Specific allo-Ag on Rejection of Cardiac Allografts in Presensitized Rat Recipients Group
No Rx Group 1A Group Group Group Group Group Group
1B 1C 2A 2B 2C 3A
Cardiac allograft survival (d)
0, 0, 0, 0, 0, 0, 1, 1, 1, 1 5, 7, 9, 13, 13, 16, 16, 17, 22, 24, 24, .27, 40, .42, .42, 44, 45, 60, .113, .117, .120, .120, .137, .138, .160, .160, .163 4, 12, 15, 19 1, 2, 2, 3, 3, 5, 6 5, 5, 6, 7, 7, 7, 8, 9, 9, 9 1, 1, 1, 2, 2, 4, 6, 7 0, 0, 0, 1, 1, 1, 1, 1, 1, 1, 3 4, 5, 7, 8
N
MST 6 SD (d)
10 27
0.4 6 0.5 .49.1 6 55.8
4 7 10 8 11 4
12.5 6 6.4 3.1 6 1.8 7.2 6 1.5 2.0 6 2.4 0.5 6 0.8 6.0 6 1.8
Group 1: CTLA4Ig (day 25 & 12) and S.C. (day 27 & 0) Group 2: S.C. (day 27 & 0) A: S.C. 20 3 106/rat IV at day 27 B: S.C. 40 – 60 3 106/rat IV at day 27 C: S.C. .100 3 106/rat IV at day 27 Gr. 3A: CTLA4IgY100F (day 25 & 12) and S.C. (day 27 & 0) S.C., donor-specific allo-Ag.
protocol resulted in Tx survival of 6.0 6 1.8 days, as if donor cells alone have been transferred (2A; 7.2 6 1.5 days). Skin grafts were rejected uniformly within 9 days irrespective of combined CTLA4Ig and spleen cell therapy. As shown in Fig 1, skin transplantation in otherwise untreated rats elicited a strong systemic IgM allo-Ab response, which peaked first around the time of cardiac Tx rejection at day 1 and then at about 3 weeks posttransplant. The IgM switched to IgG allo-Ab response 4 days after cardiac engraftment. Infusion of donor spleen cells alone
(2A) resulted in an early IgM peak at day 23 to 24 and a second peak between day 14 to 28. The switch from IgM to IgG occurred earlier, and it was even more intense as compared to that during unmodified accelerated rejection. Unlike in rats challenged with allo-Ag alone (2A), addition of CTLA4Ig to the most effective (1A) regimen significantly diminished IgM allo-Ab responses beyond day 4; these remained within the background values in long-term tolerant hosts. Moreover, IgG allo-Ab levels were markedly suppressed throughout the 150-day observation period.
Fig 1. Flow cytometric analysis of circulating IgM and IgG allo-Ab responses in rat cardiac allograft recipients. Each point represents the average mode channel staining 6 the standard deviation at a 1:4 dilution (N 5 3 to 5 for each data point).
18
Finally, substitution of CTLA4Ig by its mutated form in group 3A, resulted in depression of IgM, whereas the IgG allo-Ab pattern was comparable to that after infusion of allo-Ag alone (2A). DISCUSSION
The present study demonstrates that CTLA4Ig-mediated blockade of the CD28-B7 signaling pathway combined with transfusion of donor allo-Ag in the peritransplant period abrogated accelerated rejection and produced donor-specific tolerance in 33% of presensitized rat recipients. Importantly, this effect was strictly cell dose-dependent, and in the most effective regimen (1A), it required adjunctive transfusion of 20 3 106 splenocytes at day 27, that is, at the time of skin Tx challenge. However, once the number of transferred cells exceeded 40 3 106, this synergistic therapeutic effect between CTLA4Ig and donor allo-Ag significantly weakened (1B). Furthermore, when the number of cells exceeded 100 3 106, the beneficial effects of combined CTLA4Ig and allo-Ag therapy completely disappeared, resulting in accelerated rather than acute cardiac Tx rejection (1C). Hence, the most effective CTLA4Ig-mediated blockade of the CD28-B7 signaling pathway in sensitized hosts requires adjunctive infusion of allo-Ag in a dose, which by itself enhances Tx survival. CTLA4Ig is a potent suppressor of Ab responses in vivo, and in mice it depresses T cell-dependent Ab responses to sheep RBC or soluble protein Ag, keyhole limpet hemocyanid.7 Hence, we reasoned that blockade of the CD28-B7 costimulation pathway may prevent strong humoral responses, which otherwise, are critical in the immune cascade leading to accelerated rejection of cardiac Tx in this sensitized rat model.8 Indeed, indefinite graft survival in tolerant rats conditioned with CTLA4Ig plus donor allo-Ag (1A) was accompanied by virtually abrogated antidonor IgM and IgG allo-Ab serum levels. However, relatively high circulating IgM allo-Ab levels were detected around the time of cardiac engraftment in both control and experimen-
ONODERA, CHANDRAKER, KOROM ET AL
tal groups. Moreover, IgG allo-Ab levels were somewhat elevated around the time of cardiac engraftment after infusion of donor cells alone, but remained largely diminished after combined CTLA4Ig 1 donor cell therapy. Hence, the question arises as to whether such elevated allo-Ab levels influence the host immune response by themselves, or their presence merely reflects the pattern of sensitization. Whether these are putative “enhancing” Abs that may act in a protective manner comparably to the Th2-type subset at the cellular level3 remains to be determined. Finally, the selective blockade of CD80 by CTLA4IgY 100F in this study failed to replicate the immunosuppressive effects of CTLA4Ig and did not affect the IgM and IgG allo-Ab patterns elicited by donor cell infusion. This suggests the importance of CD86 blockade for long-term Tx acceptance, consistent with an early upregulation of CD86 in response to allo-Ag challenge, and prominent CD86 expression on endothelial cells.6,9
REFERENCES 1. Lin H, Bolling SF, Lindsey PS, et al: J Exp Med 178:1801, 1993 2. Pearson TC, Alexander DZ, Winn KJ, et al. Transplantation 57:1701, 1994 3. Sayegh MH, Akalin E, Hancock WW, et al: Exp Med 181:1869, 1995 4. Russel ME, Hancock WW, Akalin E et al: J Clin Invest 97:833, 1996 5. Onodera K, Lehmann M, Akalin E, et al: J Immunol 157: 1944, 1996 6. Hancock WW, Sayegh MH, Zheng XG, et al: Proc Natl Acad Sci USA 93:13967, 1996 7. Linsley PS, Wallace PM, Johnson J, et al: Science 257:792, 1992 8. Hancock WW, Di Stefano R, Braun P, et al: Transplantation 48:416, 1990 9. Hathcock KS, Laszio G, Pucill C, et al: J Exp Med 180:631, 1994