ANTI-CD40 LIGAND ANTIBODY AND ANTI-CD40 ISOTYPE IGG2B ANTIBODY INDUCE EQUIVALENT MURINE CHIMERISM AND SKIN GRAFT TOLERANCE UNLIKE ANTI-CD40 ISOTYPE IGG1 ANTIBODY.

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O109 Aims: Therapeutic hematopoeitic chimerism has the potential to correct many genetic hematological diseases and to eliminate the need for chronic immunosuppressive therapy following organ transplantation. To date, protocols establishing hematopoeitic chimerism have required either recipient conditioning or administration of large doses of non-T cell depleted donor bone marrow to facilitate reliable engraftment of donor cells. While encouraging, there are issues that may restrict or possibly prevent the clinical application of these strategies, such as concerns of over-immunosuppression, the increased risk of malignancies from whole body irradiation, or unfeasible donor bone marrow requirements. Our lab has devised an alternative strategy utilizing a single, non-myelosuppressive dose of busulfan (a stem cell selective conditioning agent), costimulation blockade (anti-CD40L and CTLA4-Ig), and readily attainable numbers of T-cell depleted donor bone marrow (as low as 2x106 cells), and thus promoting a high-level chimerism and tolerance of allogeneic skin grafts. However, CD40L is found on platelets, and the use of anti-CD40L in human and primate was significantly associated with thromboembolic events. Consequently, we compared the efficacy of inducing chimerism and allogeneic skin graft tolerance between anti-CD40 IgG1, anti-CD40 IgG2b, and anti-CD40L. Methods: Skin grafts and bone marrow transplants were performed with Balb/c mice as donors and B6 mice as recipients. These mice were then treated with anti-CD40 IgG1, anti-CD40 IgG2b, or anti-CD40L (250 μg i.p. on days 0, 2, 4, 6) in combination with CTLA4-Ig (500 μg i.p. on days 0, 2, 4, 6). Skin graft rejection was identified as loss of viable epidermal donor tissue. Hematopoeitic chimerism was assessed on a monthly basis using flow cytometric assays. The memory B cell assay was performed by infecting B6 mice with LCMV on day 0 and then treating with anti-CD40 IgG1, anti-CD40 IgG2b, or anti-CD40L (250 μg i.p. on days 2, 4, 6). Spleen and bone marrow samples were then harvested on day 19. Memory B cell response was measured using an antibody secreting cell elispot assays. Results: We found the chimerism and skin graft tolerance was equivalent between anti-CD40 IgG2b and anti-CD40L (donor chimerism 46.6% and 35.5% at 60 days, respectively). However, the anti-CD40 IgG1 isotype failed to produce chimerism (1.03% at 60 days) or skin graft tolerance. This was significant in comparison to both anti-CD40L and anti- CD40 IgG2b, p<0.05. Furthermore, we found that the anti-CD40 IgG2b isotype and anti-CD40L inhibited or depleted memory B cell colony proliferation at day 21 using a LCMV based memory B cell model (frequency of LCMV memory B cells are less than 1 per 500,000 B cells for each antibody), while anti-CD40 IgG1 isotype did not deplete memory B cell colony proliferation (frequency of LCMV memory B cell 1 per 39,400 B cells, p<0.05). Conclusions: We conclude that the anti-CD40 IgG1 isotype is ineffective at inducing tolerance, while the anti-CD40 IgG2b isotype is as effective as anti-CD40L antibody. Furthermore, the anti-CD40 antibody should not have the adverse effects associated with binding to platelets that has been observed with anti-CD40L.