Background: Donor-specific antibodies (DSA) are an important biomarker for acute rejection, transplant glomerulopathy and late allograft failure in kidney transplantation. Few studies have characterized the immunologic events that precede DSA formation. T follicular helper (T_FH) cells provide B cells with critical cognate help necessary for survival and differentiation into plasmablasts (PB) and antibody secretion in response to antigen stimulation. Moreover, the modulation of T_FH cell function by regulatory T cells (T_REG) during B cell responses that lead to DSA generation has not been investigated in the field of organ transplantation. Here we validate a set of experimental approaches to inquire specific circulating (c)T_FH, T_REG and memory (Mem) B cell functions using samples from healthy volunteers.

Methods: FACS-sorted cT_FH cells (CD4⁺CD45RO⁺CXCR5⁺), MemB cells (CD19⁺CD27⁺) and T_REG cells (CD4⁺CD25⁺CD127^–) were isolated from healthy volunteers and co-cultured in autologous and allogeneic conditions at a 1:1 ratio for 6 days in the presence or absence of SEB to mimic antigen-specific interactions. PB formation (CD19⁺CD27⁺CD38⁺) was monitored by flow cytometry while total IgG production was detected by ELISA. In parallel, CD3/CD28-stimulated CFSE-labeled cT_FH cells were incubated with or without T_REG at decreasing ratios for 6 days in vitro to ascertain the direct T_REG function on cT_FH cells.

Results: Co-culture of Mem B cells with cT_FH cells resulted in formation of 7867 ± 2199 PBs/2×10⁴ Mem B cells and total IgG secretion of 5003 ± 1639 ng/mL. Addition of T_REG suppressed PB formation and total IgG secretion in a dose-dependent manner up to 1:16 ratio, with mean of 90% suppression at 1:1 ratio (p < 0.05). T_REG inhibited proliferation of cT_FH cells with 50% inhibition at 1:1 ratio, 30% at 1:2, 12% at 1:4 and 2% at 1:8. In addition, allogeneic T_REG inhibit PB formation and IgG production by autologous cT_FH and MemB cells. Autologous and allogeneic cT_FH cells have comparable abilities to stimulate PB formation and IgG production in healthy subjects.

Conclusions: T_REG suppress PB formation and IgG secretion indirectly by tempering cT_FH cell function. These results provide valuable mechanistic understanding of the role of cT_FH cells and T_REG during antibody responses, underscoring the importance of cellular monitoring for early detection of patients at risk for DSA generation.

CITATION INFORMATION: Fadakar P, Macedo C, Yamada M, Hadi K, Metes D. Regulatory T Cells Inhibit Plasmablast Formation and IgG Production by Tempering T Follicular Helper Cell Function. Am J Transplant. 2017;17 (suppl 3).

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