Journal Article

Human CD4 + CD25 + CD226 - Tregs Demonstrate Increased Purity, Lineage Stability, and Suppressive Capacity Versus CD4 + CD25 + CD127 lo/- Tregs for Adoptive Cell Therapy

Carpenter, Emma B, Sachs, Lindsey K, Shapiro, Melanie R, Brusko, Todd M, Posgai, Amanda L, Hanbali, Seif R, Seay, Howard R, Arnoletti, Juan M, Peters, Leeana D, Nguyen, Kayla Q, Brown, Matthew E, Fuhrman, Christopher A

2022

Overview

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 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 cells. After 14d of culture, expanded CD4 CD25 CD226 cells displayed a decreased proportion of pTregs relative to CD4 CD25 CD127 cells, as measured by FOXP3 Helios expression and the epigenetic signature at the 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 suppressive capacity as compared to their CD127 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.

• Subject: Autoimmune Diseases / Cell- and Tissue-Based Therapy / Cytokines - metabolism / Forkhead Transcription Factors - metabolism / Humans / Interferon-gamma / T-Lymphocytes, Regulatory