Human CD4+CD25+CD226- Tregs Demonstrate Increased Purity, Lineage Stability, and Suppressive Capacity Versus CD4+CD25+CD127lo/- Tregs for Adoptive Cell Therapy
Matthew E. Brown, Leeana D. Peters, Seif R. Hanbali, Juan M. Arnoletti, Lindsey K. Sachs, Kayla Q. Nguyen, Emma B. Carpenter, Howard R. Seay, Christopher A. Fuhrman, Amanda L. Posgai, Melanie R. Shapiro, Todd M. Brusko
Abstract
Regulatory T cell (Treg) adoptive cell therapy (ACT) represents an emerging strategy for restoring immune tolerance in autoimmune diseases. Tregs are commonly purified using a CD4 + CD25 + CD127 lo/- gating strategy, which yields a mixed population: 1) cells expressing the transcription factors, FOXP3 and Helios, that canonically define lineage stable thymic Tregs and 2) unstable FOXP3 + Helios - Tregs. Our prior work identified the autoimmune disease risk-associated locus and costimulatory molecule, CD226, as being highly expressed not only on effector T cells but also, interferon-γ (IFN-γ) producing peripheral Tregs (pTreg). Thus, we sought to determine whether isolating Tregs with a CD4 + CD25 + CD226 - strategy yields a population with increased purity and suppressive capacity relative to CD4 + CD25 + CD127 lo/- cells. After 14d of culture, expanded CD4 + CD25 + CD226 - cells displayed a decreased proportion of pTregs relative to CD4 + CD25 + CD127 lo/- cells, as measured by FOXP3 + Helios - expression and the epigenetic signature at the FOXP3 Treg-specific demethylated region (TSDR). Furthermore, CD226 - Tregs exhibited decreased production of the effector cytokines, IFN-γ, TNF, and IL-17A, along with increased expression of the immunoregulatory cytokine, TGF-β1. Lastly, CD226 - Tregs demonstrated increased in vitro suppressive capacity as compared to their CD127 lo/- counterparts. These data suggest that the exclusion of CD226-expressing cells during Treg sorting yields a population with increased purity, lineage stability, and suppressive capabilities, which may benefit Treg ACT for the treatment of autoimmune diseases.