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Non-human primate natural and induced regulatory T cells suppress proliferative response to WT pig cells

Eefje Dons, Giorgio Raimondi, David K.C. Cooper, Angus Thomson

Thomas E. Starzl Transplantation Institute, Department of Surgery, University of Pittsburgh, Pittsburgh, PA, United States

Introduction: Natural regulatory T cells (nTreg) suppress xenogeneic responses. A hurdle is obtaining sufficient cell numbers. Although nTreg have expansive capacity, FoxP3 expression and suppressive capacity decrease significantly with prolonged culture. Testing of in vitro-induced Treg (iTreg), obtained by conversion of Teffectors (Teff) that retain suppressive function, would be advantageous. We examined the conversion and function of macaque iTreg in vitro, and compared responses to wild-type pig (xeno) stimulation.

Methods: Macaque nTreg and Teff were flow-sorted based on CD4+CD25+CD127- and CD4+CD25- expression, respectively, and expanded for 1 or 2 rounds (5-7d each) with anti-CD3/28 microbeads+IL-2. iTreg were generated from Teff in either TGF-beta, all-trans retinoic acid (ATRA), rapamycin, or combinations. As controls, Teff were expanded with beads and IL-2 only. FoxP3 expression was assessed by flow cytometry. For suppression assays, expanded nTreg, iTreg, and Teff were rested for 1d, stained with Violet Blue (to discriminate from responder cells) and added at different ratios to autologous CFSE-stained responder PBMC stimulated with irradiated pig or allo PBMC.

Results: 30-35ml blood yielded 0.15-0.6x106 nTreg, while sorting of Teff was stopped at 10x106. Cells could be expanded 10-fold in one round, and 100-fold in two rounds. Every expanded type had upregulated FoxP3, which decreased upon resting. Expanded nTreg had higher FoxP3 than expanded iTreg and Teff, but iTreg retained greater FoxP3 upregulation after resting. Proliferative response of CD4+ responder cells to pig PBMC was 1.5-2-fold higher than to allo PBMC. iTreg expanded in TGF-beta+ATRA or TGF-beta+rapamycin inhibited WT-stimulated proliferation at ratios (Treg:responder PBMC) up to 1:10, which was comparable to nTreg.

Conclusions: Expanded macaque iTreg have upregulated FoxP3 expression and potently suppress pig-stimulated responder T cell proliferation. Much larger numbers of iTreg can be generated compared to nTreg in a comparatively short period, an advantage for application of Treg in xenotransplantation.