Rapamycin and abundant TCR stimulation are required for the generation of stable human induced regulatory T cells

Regulatory T cells (Tregs) are a vital sub-population of CD4⁺ T cells with major roles in immune tolerance and homeostasis. Given such properties, the use of regulatory T cells for immunotherapies has been extensively investigated, with a focus on adoptive transfer of ex vivo expanded natural Tregs (nTregs). For immunotherapies, induced Tregs (iTregs), generated in vitro from naïve CD4⁺ T cells, provide an attractive alternative, given the ease of generating cell numbers required for clinical dosage. While the combination of TGF-β, ATRA and rapamycin has been shown to generate highly suppressive iTregs, the challenge for therapeutic iTreg generation has been their instability. Here, we investigate the impact of rapamycin concentrations and α-CD3/CD28 bead ratios on human iTreg stability.

We assess iTregs generated with various concentrations of rapamycin and differing ratios of α-CD3/CD28 beads for their differentiation, stability, expression of Treg signature molecules and T helper effector cytokines, and Treg-specific demethylation region (TSDR) status.

iTregs generated in the presence of TGF-β, ATRA, rapamycin and a higher ratio of α-CD3/CD28 beads were highly suppressive and stable upon in vitro re-stimulation. These iTregs exhibited a similar expression profile of Treg signature molecules and T helper effector cytokines to nTregs, in the absence of TSDR demethylation.

This work establishes a method to generate human iTregs which maintain stable phenotype and function upon in vitro re-stimulation. Further validation in pre-clinical models will be needed to ensure its suitability for applications in adoptive transfer.