*Purpose: Donor-directed HLA antibodies are an important cause of renal allograft injury, but reliable and durable therapeutic options to prevent or eliminate donor-specific antibodies (DSA) do not exist. T follicular helper (Tfh) cells are a lineage of CD4+ T cells required for the provision of B cell help to generate mature antibody responses and can differentiate into memory to facilitate rapid humoral recall responses. We have previously shown that CD28 blockade effectively inhibits de novo Tfh cell-mediated humoral alloresponses, but little is known about the role of memory Tfh (mTfh) cells in alloimmunity. Thus understanding if mTfh cells accelerate humoral responses in transplantation and whether they are susceptible to costimulation blockade will guide strategies to combat HLA antibodies.

*Methods: To examine the mTfh cell alloresponse we utilized a full MHC mismatch BALB/c to B6 murine skin allograft model. Naïve B6 recipients were grafted BALB/c skin, allowed to reject, and then re-grafted BALB/c skin 4-6 weeks after rejection of the primary graft. Mice were then left untreated or treated with CTLA4Ig, and serial draining lymph node (DLN) and serum analyses were performed to measure cellular and humoral recall responses. To determine whether memory responses depend on endogenous memory B cells, we also adoptively transferred 3×10^6 BALB/c-sensitized CD45.2 CD4+CD44+ memory T cells into naïve CD45.1 B6 mice that were then grafted BALB/c skin and their DLNs examined post-transplant.

*Results: DLN analyses of BALB/c sensitized mice following skin graft re-challenge revealed an accelerated mTfh cell response compared to the primary Tfh cell response in skin-grafted naïve controls 5 days post-transplant (8.9% vs. 1.8%, p=0.005). The germinal center (GC) B cell response was also more rapid 7 days post-transplant (58.1% vs. 18.3%, p<0.001). Both recall Tfh and GC B cell responses peaked 5 and 7 days sooner than the primary peak responses, respectively, and DSA was also accelerated and of greater magnitude. Interestingly, CTLA4Ig inhibited the mTfh cell response, with a significant reduction in the frequency of Tfh cells relative to untreated controls (8.9% vs. 4.7%, p=0.032) 5 days after re-transplant. In the absence of endogenous memory B cells, memory CD45.2 CD4+ T cells adoptively transferred into CD45.1 naïve B6 mice exhibited rapid differentiation into Tfh cells 5 days after BALB/c skin-grafting, while naïve CD45.2 CD4+ T cell controls had not differentiated into Tfh cells by day 5 (5.81 vs. 0%, p=0.016).

*Conclusions: In sum, an accelerated mTfh cell response is present following transplantation that is independent of B cell memory and is inhibited by CD28 costimulation blockade. These findings support further elucidating the role of mTfh cells in recall humoral alloresponses, as their susceptibility to costimulation blockade and memory B cell independence could have important clinical implications.

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