Antithymocyte globulin (ATG) is a lymphoablative agent used in transplant patients. However, ATG treated recipients eventually reconstitute their T cell repertoire, develop anti-donor immune responses and either reject transplanted organ acutely or develop chronic graft injury. The goal of this study was to investigate the mechanisms of homeostatic and antigen driven recovery of T cells following lymphoablation in a mouse model of heterotopic cardiac transplantation.

Treatment with a murine analog of antithymocyte globulin mATG (25 mg/kg on d. 0 and 4 posttransplant) depletes >99.9% of total CD8 T cells in all lymphoid and non-lymphoid compartments. In contrast, about 50% of effector/memory CD44^hiCD4 T cells are resistant to depletion. Administration of depleting anti-CD4 mAb prior to mATG eliminates up to 99.9% of residual CD4 T cells. This additional CD4 cell depletion severely compromised the recovery of CD8 T cells in mATG treated heart allograft recipients compared to mATG treatment alone (0.2% vs 3-5% of spleen CD8 T cells recovered by d. 10 posttransplant, respectively). This inhibition of CD8 T cell recovery after mATG treatment was observed in both CD4 depleted and genetically CD4-deficient recipients of iso- and allografts as well as in non-transplanted mice. These results suggest that CD4 T cells are required for homeostatic CD8 T cell recovery regardless of antigen driven proliferation or surgery-induced inflammation.

To investigate the mechanisms underlying CD4 T cell help for CD8 T cell reconstitution we used a previously established model in which recipients containing donor-reactive memory CD4 T cells reject heart allografts despite treatment with anti-CD154 mAb MR1 (MST of 10 d.) Remarkably, combination of MR1 and mATG treatment significantly impaired CD8 T cell recovery and prolonged heart allograft survival in these recipients compared to mATG monotherapy (MST 32 vs. 11.5 d). Conversely, treatment with agonistic anti-CD40 antibody rescued expansion of CD8 T cells in mATG treated CD4 KO or CD4-depleted recipients. Thus, CD40/CD154 signaling was necessary and sufficient for CD8 T cell recovery in lymphopenic mice.

Our results demonstrate that residual CD4 T cells provide CD40-dependent helper signals for the expansion of both donor-specific and non-specific CD8 T cells following lymphoablation with mATG. Interfering with helper functions of CD4 T cells should improve the efficiency of ATG induction therapy in sensitized transplant recipients.

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