AN UNEXPECTED ROLE FOR CD28 AND LACK OF A ROLE FOR CD40L SIGNALING IN PERIPHERAL DELETION OF DONOR-REACTIVE CD4+ T CELLS AND ESTABLISHMENT OF MIXED CHIMERISM THROUGH COSTIMULATORY BLOCKADE

Abstract

453 Purpose: Bone marrow transplantation plus costimulatory blockade allows the establishment of long-term multilineage chimerism and tolerance across a full MHC barrier. Tolerance is associated with rapid peripheral deletion of recipient CD4+ T cells bearing donor-reactive Vβ. We evaluated the role of CD28 and CD40L in this regimen using CD28−/− and CD40L−/− mice. Methods: C57BL/6-CD28−/− and C57BL/6 wild-type (WT) mice (H-2b) received 3Gy WBI and 20×106 B10.A (H-2a) bone marrow cells on day 0, followed by single injections of anti-CD40L mAb (MR1; 0.5mg; day 0) with or without CTLA4Ig (0.5mg, day 0 or day +2). H-2b B6/129-CD40L−/− and B6/129 mice received 3Gy WBI followed by 15×106 B10.A BMC on day 0, and single injections of CTLA4Ig (0.5mg; day +2) with or without MR1 (0.5mg; day 0). Levels of donor chimerism in peripheral blood and frequency of TCR using certain Vβ were followed by FCM analysis. Tolerance was tested by grafting donor (B10.A) and third party (A.SW) tail skin. Results: In the CD28−/− experiment, five of six WT mice receiving MR1 and CTLA4Ig developed multilineage mixed chimerism, with peripheral deletion of donor-reactive CD4+ T cells at week 1 that progressed further by week 3. In contrast, CD28−/− mice failed to demonstrate measurable chimerism or deletion, regardless of CTLA4Ig administration. In the CD40L−/− experiment, two of five CD40L-deficient mice receiving only CTLA4Ig showed high levels of multilineage chimerism, similar to results in WT mice receiving both CTLA4Ig and MR1 (3 of 5 mice). Similar deletion of donor-reactive Vβ was evident among CD4+ T cells of chimeric CD40L−/− mice which received only CTLA4Ig and wild-type controls receiving MR1 and CTLA4Ig. Donor-specific skin graft tolerance (>100 days) was observed in all chimeric mice, with prompt rejection of third party skin. Conclusion: Surprisingly, signaling through CD28 on host T cells plays a role in peripheral donor-reactive T cell deletion and in permitting allogeneic marrow engraftment in mice receiving costimulatory blockade. In contrast, signaling through CD40L is not necessary in this regimen of tolerance induction. The deletion of donor-reactive CD4+ T cells observed in CD40L−/− mice which did not receive MR1 suggests that direct destruction of anti-CD40L-coated activated T cells is unlikely, and that the importance of anti-CD40L treatment in tolerance induction is due to its ability to prevent activation of APC's.