CD8+ T CELL TOLERANCE AFTER BONE MARROW TRANSPLANTATION USING NON-MYELOABLATIVE CONDITIONING WITH TBI AND ANTI-CD154 MAB: IMPORTANCE OF SIGNAL 1, CD4+ T CELLS AND CTLA4, BUT NO EVIDENCE FOR LONG-TERM REGULATION (2004)

O107* Aims: To investigate CD8+ T cell tolerance in a model for induction of mixed chimerism and donor-specific tolerance with bone marrow transplantation (BMT) after non-myeloablative conditioning involving anti-CD40 ligand and total body irradiation (TBI). Methods: Recipient mice were treated with TBI (3Gy, d-1), one dose of anti-CD154 mAb (2mg MR1, d0) and BMT from a fully MHC-mismatched donor to induce mixed lymphohematopoietic chimerism. Chimerism was followed by FACS analysis of peripheral blood. Immunologic tolerance was assessed by donor and third party skin grafts. To follow the fate of specific alloreactive cells, syngeneic chimeras expressing the transgenic alloreactive 2C T cell receptor on about 10% of CD8+ T cells were prepared. Deletion of alloreactive cells was followed by FACS analysis using an anti-clonotypic antibody. To assess the role of the calcineurin pathway, mice were treated with cyclosporine A (CyA 20mg/kg daily) for 2-4 weeks. Donor-specific tolerance was tested by mixed lymphocyte reaction (MLR), cytotoxicity assays (CML) and FACS for alloantibodies. Transgenic mice expressing the diphtheria toxin receptor on CD11c+ cells were used to deplete donor dendritic cells. The role of CD4+ T cells, CTLA4 and the Fas/Fas-ligand pathway was studied by using CD4+ T cell depletion at various time points, anti-CTLA4 mAb treatment and lpr mice, respectively. Results: Long-lasting mixed lymphohematopoietic chimerism and permanent donor-specific skin graft acceptance was achieved with BMT after conditioning with TBI and MR1. Donor-reactive peripheral blood 2C CD8+ T cells were rapidly deleted by d7. In recipients treated with CyA, initial chimerism was achieved. However, after stopping CyA, all the mice eventually rejected marrow and skin grafts, indicating that tolerance induction was blocked by calcineurin inhibition. Donor-specific MLR, CML and alloantibodies became positive only after complete loss of chimerism. Using transgenic mice to deplete donor dendritic cells peri-transplant permitted 80 to 90% stable donor chimerism, suggesting that donor dendritic cells do not play a major role in initial tolerance induction. An equal incidence of chimerism (ca 70%) was observed in Fas-mutated and wild-type mice, indicating that CD8+ T cell tolerance was not mediated by Fas/Fas-ligand interactions. In contrast, CD8+ T cell tolerance was dependent on the presence of recipient CD4+ T cells, since CD4+ T cell depletion abolished the achievement of chimerism and tolerance. The same was observed after anti-CTLA4 treatment. A kinetic study revealed that CD4+ T cells are only needed during the first 10 days (incidence of chimerism: no depletion 80%, depletion d-1/d3: 0%, d6: 35%, d10 70%). Adoptive transfer studies of wild-type CD8+ T cells together with tolerant CD4+ T cells from chimeric mice 1 week after BMT into RAG1−/− mice showed no evidence for suppression of skin graft rejection. Conclusions: CD8+ T cell tolerance was achieved by BMT after conditioning with anti-CD154 mAb and low dose TBI. Tolerance induction was dependent upon a functioning calcineurin pathway and CTLA4. CD8+ T cell tolerance involved rapid peripheral deletion and was dependent upon CD4+ T cells during the first 2 weeks. This finding, together with the adoptive transfer studies, strongly argues against the need for long-term regulation in our model.