T CELL ANERGY BUT NOT DELETION IS THE PREDOMINANT MECHANISM FOR INDEFINITE ISLET ALLOGRAFT SURVIVAL IN RECIPIENTS TREATED WITH rhCTLA4-Ig AND ??-gp39 ANTIBODY

Abstract

48 We had previously reported that in mice, combined simultaneous blockade of costimulatory pathways by short perioperative treatment with rhCTLA4-Ig fusion protein and α-gp39 mAb results in indefinite islet allograft survival with reversal of streptozotocin (STZ)-induced diabetes. Furthermore, islet allograft survival was shown to be comparable in controls and in animals treated with either rhCTLA4-Ig or α-gp39 mAb alone, suggesting that combined simultaneous but not distinct blockade of CD28/B7 and CD40/gp39 costimulatory pathways is imperative for prolonging allograft survival. To ascertain the mechanism underlying these observations, we proceeded to transplant islets isolated from the pancreata of DBA/2 (H2d, Mlsd, Vβ6-CD4+) animals under the kidney capsule of STZ-treated diabetic MHC and MIs-disparate C3H (H2k, Mlsc, Vβ6+CD4+) recipients. The rationale for selecting this strain combination was triggered by the observation that in the donor but not the recipient, Vβ6+CD4+ were deleted during ontogeny thus prompting us to suggest that perhaps this could be used as a surrogate marker to determine the mechanism of T cell tolerance in these animals. In addition to MLR and complement-dependent microcytotoxicity assays, the proliferative status and the presence or absence of Vβ6+CD4+ T cells in the peripheral blood and splenocytes of the recipients was also determined by antibody-mediated TcR crosslinking and flow cytometric analysis respectively. The recipient treatment was as follows: Group I (n=4), isotype control mAb; Group II (n=6), anti-gp39 mAb (250µg/d on d0,2 and 4 post-Tx, i.m); Group III (n=5), rhCTLA4-Ig (200µg on d0,2,4 and 6 post-Tx, i.p) and Group IV (n=6), rhCTLA4-Ig + anti-gp39 mAb. All in vitro analysis were performed using samples obtained from the recipients either after islet allograft rejection (Groups I, II and III) or else at d81 post-Tx (Group IV). While donor-specific proliferative responses and cytotoxic antibody titers remained unchanged in Groups I, II and III, they were markedly reduced in animals in Group IV. These findings were corroborated by TcR crosslinking assays, in which the use of α-Vβ6 mAb resulted in marked proliferation of cells obtained from animals in Groups I, II and III; markedly reduced proliferation was witnessed in cells obtained from Group IV animals. Interestingly, flow cytometric analysis of T cell repertoire both in the PBMC and splenocytes of islet allograft recipients in all four groups revealed the presence of an equivalent number (∼8-9%) of donor-reactive Vβ6+CD4+ T cells arguing against deletion being the mechanism of the observed T cell tolerance. Take together, these preliminary data suggest that T cell anergy is perhaps the predominant mechanism for the indefinite islet allograft survival witnessed in animals subjected to combined simultaneous blockade of CD28/B7 and CD40/gp39 costimulatory pathways.