It is increasingly well-recognized that donor-specific antibodies (DSA) play a significant role in the development of chronic rejection and fibrosis that contribute 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) and regulatory (T_FR) cells are distinct subsets of CD4⁺ T cells that are critical mediators of the humoral immune response. T_FH and T_FR cells function primarily within germinal centers (GC) of secondary lymphoid organs, rendering them elusive targets for immune monitoring in transplant recipients. Recent studies have identified circulating T_FH (cT_FH) cells that correlate with ongoing antibody responses after vaccination and in autoimmune disease, but little is known about the existence of cT_FH cells during transplantation. To determine the kinetics and magnitude of circulating T_FH and T_FR (cT_FR) cells during transplant, we utilized a major MHC mismatch BALB/c to B6 murine skin transplant model. Flow cytometric analysis of the peripheral blood revealed a 3-fold and 2-fold expansion in the frequency and absolute number of CD4⁺CXCR5⁺Foxp3^– cT_FH and Foxp3+ cT_FR cells, respectively, 10 days after BALB/c skin transplantation as compared to naïve and syngeneic controls. Both circulating subsets expressed increased levels of Ki-67, reduced amounts of CXCR5 and ICOS, and no detectable Bcl-6 or PD-1 relative to their draining lymph node (dLN) counterparts. Interestingly, the majority of cT_FH cells were CD44^hi 10 days after transplant at the peak of the T_FH response but reflected a more quiescent predominant CD44^lo composition by day 14, indicating a detectable phenotypic shift in the cT_FH cell pool that corresponded to peak anti-donor germinal center reactivity in the dLN. These graft-elicited changes also correlated with the initiation of anti-BALB/c IgG DSA generation. CD28 costimulation pathway blockade abrogated all components of the donor-reactive GC response, DSA formation, the observed expansion in cT_FH and cT_FR cells, and the increase in frequency of CD44^hi cT_FH cells at the peak of the alloimmune response. These data support continued investigations aimed to develop cT_FH cells as a biomarker for the clinical assessment of humoral alloimmunity to guide DSA management for improvement of long-term kidney transplant outcomes.

CITATION INFORMATION: La Muraglia II G., Wagener M., Ford M., Badell R. Circulating T Follicular Helper Cells Expand Following Transplantation and Exhibit a CD44^hi Phenotype That Correlates with Peak Germinal Center Alloreactivity Am J Transplant. 2017;17 (suppl 3).

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