Kidney transplantation is the treatment of choice for patients with end-stage renal disease. Although short-term outcomes are excellent, long-term allograft survival rates remain inadequate. It has been increasingly recognized that donor-specific antibodies (DSA) play a significant role in the development of chronic rejection and fibrosis that lead to late renal allograft loss. Thus, understanding the mechanisms responsible for DSA formation is necessary to improve long-term outcomes. T follicular helper (T_FH) cells are a distinct lineage of CD4⁺ T cells that provide T cell help to B cells for germinal center (GC) formation and antibody production. The critical role T_FH cells play in the development of protective antibody responses to vaccines and pathogens has been recently established, but little is known regarding their role in the generation of DSA following transplantation. To elucidate the role T_FH cells play in antibody-mediated allograft loss, we utilized a pre-clinical minor antigen (mOVA) mismatch T cell receptor transgenic murine transplant model to define both TCR transgenic and endogenous donor-specific T_FH responses to mOVA skin grafts. Flow cytometric analysis of graft-draining lymph nodes revealed a robust expansion in the frequency and absolute number of donor-specific TCR transgenic and endogenous CXCR5⁺PD-1^hi Bcl6⁺ T_FH cells, and endogenous CD95⁺GL7⁺ GC B cells in response to an mOVA skin graft as compared to a syngeneic graft. The frequency of CXCR5⁺Bcl6⁺Foxp3⁺ T follicular regulatory (T_FR) cells reciprocally decreased with an mOVA graft, along with the T_FR:T_FH ratio as compared to the response to a syngeneic graft. These donor-reactive GC changes correlated with the initiation of DSA (anti-OVA IgG) formation. Interestingly, the donor-specific T_FH response was characterized by a lower proportion of polarized GL7⁺ GC T_FH cells and decreased ICOS-L expression on GC B cells in response to the mOVA skin graft. All components of the donor-reactive GC response, including DSA formation were abrogated with CD28 costimulation pathway blockade, resulting in phenotypes similar to those following a syngeneic graft. These data serve as a platform to investigate the mechanisms of DSA formation for the development of therapeutic strategies to control humoral alloimmunity, while holding the potential to validate efforts aimed at biomarker identification in clinical transplantation to improve long-term outcomes.

CITATION INFORMATION: La Muraglia II G, Ford M, Badell I. T_FR:T_FH Cell Ratio Correlates with Anti-Donor Germinal Center Reactivity and Donor-Specific Antibody Formation Following Transplantation. Am J Transplant. 2017;17 (suppl 3).

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