Synergistic effects of CTLA-4 blockade with tremelimumab and elimination of regulatory T lymphocytes in vitro and in vivo^†

Anti-CTLA-4 monoclonal antibodies (mAb) that block the interaction of CTLA-4 with CD80 and CD86 such as tremelimumab and ipilimumab are currently being tested in the clinic for cancer treatment exploiting their properties to de-repress tumor-specific cellular immunity. Addition of the fully human anti-CTLA-4 (tremelimumab) to cultures of human T cells with allogenic dendritic cells (DCs) did not increase proliferation. Magnetic bead-mediated elimination of CD4⁺ CD25⁺ regulatory T cells (T_reg) before setting up those alloreactive cultures also largely failed to increase primary proliferation. In contrast, predepletion of CD4⁺ CD25⁺ T_reg and culture in the presence of tremelimumab synergistically resulted in increased proliferation and DC:T-cell aggregation. These effects were much more prominent in CD4 than in CD8 T cells. The synergy mechanism can be traced to enhanced CTLA-4 expression in effector cells as a result of T_reg elimination, thereby offering more targets to the blocking antibody. Human T cells and allogenic DCs (derived both from healthy donors and advanced cancer patients) were coinjected in the peritoneum of Rag2^−/− IL-2Rγ^−/− mice. In these conditions, tremelimumab injected intravenously did not significantly enhance alloreactive proliferation unless T_reg cells had been predepleted. Synergistic effects in vivo were again largely restricted to the CD4 T-cell compartment. In addition, T_reg depletion and CTLA-4 blockade synergistically enhanced specific cytotoxicity raised in culture against autologous EBV-transformed cell lines. Taken together, these experiments indicate that tremelimumab therapy may benefit from previous or concomitant T_reg depletion.