*Purpose: Clinical costimulation blockade of the CD28 pathway in the form of CTLA-4Ig has shown the ability to attenuate T cell dependent DSA responses. However, CTLA-4Ig’s mechanism of action of binding ligands CD80/86 indiscriminately blocks both CD28 costimulatory and CTLA-4 coinhibitory pathways, potentially depriving lymphocytes of critical coinhibitory signals. We have previously demonstrated that selective CD28 costimulatory blockade provides enhanced inhibition of T follicular helper (T_FH), germinal center (GC) B cell and DSA responses as compared to CTLA-4Ig following transplantation. T_FH cells differentially express large amounts of CTLA-4 in response to alloantigen and several studies have implicated CTLA-4 as a key regulator of their differentiation and function. Therefore, we hypothesize that the enhanced DSA inhibition observed with selective CD28 blockade is dependent on the preservation of critical CTLA-4 coinhibitory signaling.

*Methods: Thus, we utilized a full MHC mismatch BALB/c to B6 and minor antigen (OVA) mismatch TCR transgenic murine skin allograft models to determine whether improved inhibition of the Tfh cell-mediated alloresponse compared to CTLA-4-Ig is CTLA-4 dependent. Skin-grafted mice received anti-CD28 domain antibody (dAb) with or without anti-CTLA-4 mAb (9H10) treatment for the analysis of Tfh cell-mediated responses and DSA formation.

*Results: Anti-CD28 blockade in the presence of CTLA-4 blockade abrogated the inhibition of DSA formation observed with anti-CD28 blockade alone, with detectable anti-donor antibodies 28 days post-transplant (Fig.1A). Flow cytometric analysis of graft-draining lymph node T_FH, GC-T_FH, GC B, and plasma cells following skin transplantation did not reveal significant differences between treatment groups at 10 days post-transplant. Interestingly, examination of these compartments at day 24, immediately prior to the observed DSA breakthrough revealed significant development of T_FH (3.35% vs. 0.46% p=0.007), GC-T_FH (36% vs. 25% p=0.015), GC B (63% vs. 9.7% p=0.007) and plasma cell (5.3% vs 4% p=0.03) responses in anti-CTLA-4 treated mice compared to CD28 dAb monotherapy (Fig.1B).

*Conclusions: Thus, selective CD28 blockade inhibition of Tfh cell-mediated humoral alloimmunity is CTLA-4 dependent beyond the day 10 peak Tfh cell response, supporting that preserved CTLA-4 signaling may lead to improved control of donor-specific immunity through superior inhibition of T_FH and GC B cell responses following transplantation.

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