Given the major roadblocks to development of Treg-based cell therapy, pharmacologic approaches to expand endogenous Tregs warrant attention, including approaches with more sustained effects than injection of IL-2 complexes. In vitro, CD3/CD28 stimulation of conventional T cells in the presence of IL-2 and TGFb promotes development of Foxp3+ inducible Treg (iTreg) cells, but their Foxp3 expression and suppressive activity are very labile. We sort to remedy this situation.

Culture of conventional CD4+CD25-YFP- T cells isolated from YFP+Foxp3+ mice for 4 d with CD3/CD28 plus IL-2 did not induce Foxp3, whereas addition of TGFb (2 ng/ml) led to ~45% Foxp3+ cells (flow cytometry). While use of rapamycin (RPM, 10-20 nM) instead of TGFb led to <10% Foxp3+ cells, their combination resulted in ~85% Foxp3+ cells. qPCR showed the combination increased Foxp3, TGFb, IL-10, Eos and Helios mRNA, and ChIP studies showed increased Smad2/3 binding at the Foxp3 promoter and CNS1 site, and increased H3K4me3 and Foxp3 binding at the CNS2 enhancer. Western blotting showed that TGFb activated the Smad pathway but also increased Akt phosphorylation and acetylation of FOXO1, whereas RPM significantly decreased Akt phosphorylation. Moreover, combined TGFb and RPM led to a 2-fold increase in Foxp3 protein compared to either agent alone, was accompanied by increased phospho-Stat5, and the associated iTregs were fully suppressive in vitro.

In vivo, daily injection of TGFb (1 [micro]g/kg/d, 7 d) or use of RPM (0.5 mg/kg/d, 28 d Alzet pump) in C57BL/6 recipients of BALB/c cardiac allografts did not increase Foxp3+ cells or reduce CD4 or CD8 T cell activation at 7 days post-Tx, or prolong allograft survival. However, combined TGFb/RPM led to a doubling of Foxp3+ Tregs when assessed at day 14 post-Tx, suppressed T cell activation, and resulted in permanent cardiac allograft survival (>100 days, p<0.01). The effects of combined TGFb/RPM on allograft survival were abrogated by use of a neutralizing mAb to TGFb.

Combined TGFb and RPM use has a powerful effect on iTreg development and induces permanent allograft survival in fully MHC-disparate mice. Much yet remains to be learnt regarding the pathways that suppress Foxp3 expression in conventional T cells, and how Foxp3 expression can be induced and stabilized to create functional Tregs. However, our work shows progress in this regard, and may ultimately have important consequences for clinical allograft recipients.

CITATION INFORMATION: Wang L., Samanta A., Han R., Zhou N., Hancock W. TGF-b Plus Rapamycin Induce Foxp3 and Promote iTreg Development and Suppressive Function, Leading to Long-Term Allograft Survival Am J Transplant. 2017;17 (suppl 3).

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