CD137+CD154− Expression As a Regulatory T Cell (Treg)-Specific Activation Signature for Identification and Sorting of Stable Human Tregs from In Vitro Expansion Cultures

Abstract

Regulatory T cells (Tregs) are an attractive therapeutic tool for several different immune pathologies. Therapeutic Treg application often requires prolonged in vitro culture to generate sufficient Treg numbers or to optimize their functionality, e.g., via genetic engineering of their antigen receptors. However, purity of clinical Treg expansion cultures is highly variable, and currently, it is impossible to identify and separate stable Tregs from contaminating effector T cells, either ex vivo or after prior expansion. This represents a major obstacle for quality assurance of expanded Tregs and raises significant safety concerns. Here, we describe a Treg activation signature that allows identification and sorting of epigenetically imprinted Tregs even after prolonged in vitro culture. We show that short-term reactivation resulted in expression of CD137 but not CD154 on stable FoxP3+ Tregs that displayed a demethylated Treg-specific demethylated region, high suppressive potential, and lack of inflammatory cytokine expression. We also applied this Treg activation signature for rapid testing of chimeric antigen receptor functionality in human Tregs and identified major differences in the signaling requirements regarding CD137 versus CD28 costimulation. Taken together, CD137+CD154− expression emerges as a universal Treg activation signature ex vivo and upon in vitro expansion allowing the identification and isolation of epigenetically stable antigen-activated Tregs and providing a means for their rapid functional testing in vitro.

Regulatory T cells expressing abundant CTLA‐4 on the cell surface with a proliferative gene profile are key features of human head and neck cancer

“…15,17 It is possible that highly mutated cancers are enriched with surface-CTLA-4 + Treg cells. CD137 + surface-CTLA-4 + Treg cells, which represent TCR-activated T cells, 37,38,46 may proliferate in response to neoantigens in the tumor microenvironments from head and neck SCC.…”

Characterization of proinsulin‐specific regulatory T cells in type 1 diabetes at different ages of onset

“…Previous studies have used pre‐enrichment techniques including in vitro cultures, which can estimate the frequency of antigen‐specific T cells . CD137 acts as a Treg activation signature that allows the identification of antigen‐specific Tregs . CD137 is expressed exclusively on Tregs after antigenic stimulation.…”

“…26,48,49 CD137 acts as a Treg activation signature that allows the identification of antigen-specific Tregs. 24,25 CD137 is expressed exclusively on Tregs after antigenic stimulation.…”

“…While the availability of peptide MHC multimers has enabled detection of antigen‐specific T cells, the technical challenges associated with the synthesis and stability of peptide MHC multimers have limited their utility to specialized centres . The costimulatory molecules, CD137 and CD154 have emerged as markers to identify antigen‐specific T cells . In this context, we, therefore, analyzed the frequency and phenotype of PI‐specific Tregs in peripheral blood of two distinct pathophysiological subtypes of T1D, JOT1D and AOT1D, distinguished by the age at disease onset, using a combination of CD137, FOXP3 and CD45RA following in vitro stimulation with PI‐derived peptides.…”

“…23 The costimulatory molecules, CD137 and CD154 have emerged as markers to identify antigen-specific T cells. 24,25 In this context, we, therefore, analyzed the frequency and phenotype of PI-specific Tregs in peripheral blood of two distinct pathophysiological subtypes of T1D, JOT1D and AOT1D, distinguished by the age at disease onset, using a combination of CD137, FOXP3 and CD45RA following in vitro stimulation with PI-derived peptides.…”