ANTI-CD40L MAB OVERCOMES CD4 T CELL-MEDIATED RESISTANCE TO XENOGENEIC MARROW ENGRAFTMENT

Abstract

O400 Aims: Costimulatory blockade can obviate the need for thymic irradiation (TI) or CD4 depletion in a non-myeloablative allogeneic BMT regimen that leads to mixed chimerism and tolerance. Anti-CD40L mAb completely overcomes CD4-mediated resistance to allogeneic marrow engraftment. We now analyze the ability of anti-CD40L mAb to overcome CD4 cell-mediated resistance to the induction of mixed xenogeneic chimerism in mice receiving non-myeloablative conditioning. Methods: C57BL/6 mice received various combinations of anti-CD4 (GK1.5) and/or anti-CD8 (2.43), anti-NK1.1 (PK136), and anti-Thy1.2 mAbs on day -5 and-1, anti-CD40L mAb MR1 (2mg, day 0), 3 Gy TBI (total body irradiation) and 7Gy TI (day 0), and 60x106 xenogeneic (F344) rat bone marrow cells (BMC). Skin grafting (donor rat, F344; third party mouse, B10.RIII) was performed one day after BMT to assess induction of specific tolerance. Chimerism in peripheral blood was followed by FCM analysis. The frequency of TCR Vβ usage was determined by FCM to assess deletion of donor-reactive T cells. Results: Transient multilineage chimerism and specific prolongation of donor skin graft survival (median survival time [MST] 78 days) were achieved in animals receiving anti-CD4, anti-CD8, anti-NK1.1, anti-Thy1.2 mAbs on day -5, -1 with TBI and TI on day 0, as we have previously reported. Mice receiving anti-CD8, anti-NK1.1, anti-Thy1.2 mAbs on day -5 and TBI (3Gy) on day 0 (without anti-CD4 mAb or TI) did not achieve mixed chimerism and donor skin grafts were rejected within 13 days (n=6). All animals treated with anti-CD4, anti-CD8, anti-NK1.1, and anti-Thy1.2 mAbs on day -5 with TBI (3Gy) on day 0 (n=5) (no TI) showed transient chimerism and prolongation of donor skin acceptance (MST 79 days). All animals treated with anti-CD8, anti-NK1.1, anti-Thy1.2 mAbs on day -5 and anti-CD40L mAb with TBI (3Gy) on day 0 (n=6) (no anti-CD4 mAb or TI) also showed transient chimerism and prolongation of donor skin acceptance (MST 68 days). Mice with transient mixed chimerism and prolongation of donor skin graft survival demonstrated specific deletion of donor-reactive CD4+ PBL. Conclusions: Treatment with anti-CD40L completely overcomes CD4-mediated resistance to xenogeneic marrow engraftment and, in combination with BMT, permits donor-specific tolerization of pre-existing peripheral CD4 cells.