*Purpose: Deleterious alloantibodies result from T follicular helper (Tfh) cell-driven germinal center (GC) reactivity. T follicular regulatory (Tfr) cells are a subset of regulatory T cells that have been identified as important regulators of GC responses and antibody formation. However, whether Tfr cells regulate Tfh cells in vivo is unknown and little is understood of their role in transplantation. Thus given the potentially beneficial impact Tfr cells could have in controlling pathologic donor-specific antibody (DSA) responses, it is of great interest to understand their role in transplantation.

*Methods: To examine the impact of Tfr cells in transplantation, we first utilized a full MHC mismatch murine skin allograft model to test for and define the kinetics of alloresponsive Tfr cells. We then used a conditional Tfr knockout (KO, Bcl6^fl/flFoxp3^Cre) mouse to study the function of Tfr cells in response to BALB/c and OVA antigen mismatch skin grafts. Graft-draining lymph node (dLN) Tfh and GC B cell, and DSA responses in the absence of Tfr cells were evaluated in comparison to wild type (WT, Bcl6^fl/flFoxp3^wt) controls. Tfh:B cell co-cultures, alloantibody subclass and affinity were also examined in the presence or absence of Tfr cells.

*Results: Tfr cells were detected following transplantation, but the frequency and number of Tfr cells remained stable over time relative to the dynamic expansion and contraction of their Tfh cell counterparts. Tfr KO mice were confirmed by the absence of CD4⁺CXCR5⁺PD1^hiFoxp3⁺ T cells. 10 days after primary Balb/c and OVA skin grafts, no significant differences were observed in the frequencies of Balb/c-reactive or OVA-specific Tfh cells (Figure 1) or H2K^d-specific or alloreactive GC B cells between WT and KO recipient mice. In Tfh:B cell co-culture studies there were no significant differences in class-switched B cell or IgG formation between WT and KO groups. Interestingly, 35 days following skin transplants, KO mice exhibited a greater frequency of Tfh cells (4.27 vs. 2.05, p=0.016) compared to WT mice in post-transplant dLNs (Figure 2). No differences were observed in the quantity (MFI and OD) or quality (antibody subclasses and affinity) of donor specific antibody formation.

*Conclusions: These findings implicate Tfr cells in humoral alloreactivity and suggest an in vivo role for Tfr cells after transplantation in mediating GC reactivity. While their absence doesn’t affect peak (d10) GC responses, it did significantly delay the contraction (d35) of GC Tfh cells. As such, Tfr cells may influence GC kinetics and the development of anamnestic alloimmunity that may manifest in secondary humoral responses.

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