Purpose: Regulatory T cells (Tregs) have been associated with clinical transplant tolerance and they have been shown to prolong graft survival in animal models without immunosuppression. Therefore, a number of centers have initiated clinical trials with ex vivo expanded Tregs. The purpose of the present study was to expand Tregs using activated/expanded allogeneic B cells and test their antigen (Ag)-specific function.

Methods & Results: Peripheral blood mononuclear cells (PBMC) from healthy volunteers were cultured using novel stimulating agent multimeric Ultra-CD40L, under B cell-promoting conditions in presence of cyclosporine A to prevent T cell growth (n=8). The resultant B cells expanded 100-1,000 fold by day 14-21 and displayed increased expression of CD80, CD86, and HLA-DR, the hallmark of mature antigen presenting cells. These expanded B cells, after irradiation were used to stimulate purified CD4⁺CD127^–CD25⁺ Tregs isolated from PBMC of an allogeneic volunteer for 28 days under Treg-promoting conditions with exogenous IL-2 and sirolimus (SRL). The Tregs expanded rapidly when SRL was removed (Fig. A). They maintained the CD4⁺CD127^–CD25⁺ phenotype with >60% being FOXP3⁺. More importantly, they dose-dependently inhibited MLR of autologous responders in an ag-specific manner (Fig. B). Additionally, statistically significant inhibition occurred even at 1:250 with Ag-specific Tregs vs only up to 1: 32 when non-specifically expanded polyclonal Tregs were used (0% inhibition controls being autologous PBMC). The Ag-specific Tregs also demonstrated infectious tolerance by generating new Tregs in CFSE-labeled autologous responder cells cultured with the specific allostimulator (n=6).

Conclusions: Ultra-CD40L activated/expanded B cells can be utilized to grow alloantigen-specific Tregs which are powerful Ag-specific immunomodulators of autologous responding cells. Upon further development and scale-up under GMP conditions, such Tregs could be more effectively utilized to achieve clinical transplant tolerance.

CITATION INFORMATION: Mathew J, McEwen S, Konieczna I, Huang X, He J, Kornbluth R, Miller J, Gallon L, Leventhal J. Ex Vivo Expansion and Functional Characterization of Human Alloantigen-Specific Regulatory T Cells. Am J Transplant. 2016;16 (suppl 3).

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