Foxp3+ Alloantigen-Specific iTreg Highly Express CTLA4 and Preferentially Survive Following CTLA4-Ig Treatment.

Abstract

The CD40-CD154 costimulatory pathway is one of the most attractive targets for therapeutic intervention in transplantation. However, thromboembolic complications during early clinical trials with anti-CD154 antibodies, due to Fc-mediated platelet activation, prevented their translation. Recent studies by our group have suggested that Fc-silent anti-CD154 domain antibodies may be a safer and equally efficacious means of prolonging allograft survival. Using a transgenic mouse model where OT-I CD8 and OT-II CD4 T cells were adoptively transferred to B6 mice that then received skin grafts that ubiquitously express the OVA protein, we showed that Fc-silent anti-CD154 antibodies prolong graft survival and lead to the conversion of naïve donor-specific CD4 T cells into Foxp3+ iTreg. Given the potential for clinical translation of Fc-silent anti-CD154 therapy and long-standing interest in combination therapy with CD28 costimulation blockade, we sought to determine the effect of CTLA4-Ig on CD154 antagonism-induced iTreg generation. This question was especially pertinent due to the known negative impact of CTLA4-Ig on the frequency of Treg. While the addition of CTLA4-Ig treatment to an Fc-silent anti-CD154 regimen led to a drastic decrease in the percentage of endogenous CD4+CD25+Foxp3+ Treg (7.2% vs. 11.1% with anti-CD154 alone), the percentage of alloantigen-specific iTreg actually increased following the addition of CTLA4-Ig (25.0% vs. 14.4% with anti-CD154 alone), which may be an important mechanism of synergy with these two therapies. To determine the requirements for CD28 signals for survival of these cells, we treated animals with anti-CD28 domain antibodies that specifically block CD28 signaling while leaving CTLA4 signaling intact. Results indicated that in contrast to nTreg, the survival of iTreg is independent of CD28 costimulation. Furthermore, in our model, iTreg express higher amounts of CTLA4 (MFI 5187) than their endogenous counterparts (MFI 1668). Previous studies have shown that proliferation leads to higher expression of CTLA4 on Treg, and we hypothesize that exposure to cognate alloantigen following transplantation may supplant the requirement of CD28 for the survival of these iTreg. Taken together, these results suggest that a novel anti-CD154 therapeutic may synergize with an already-approved agent, opening the door for translation of more effective immunosuppression regimens. DISCLOSURE:Nadler, S.: Employee, Bristol Myers Squibb.