CD45RA Distinguishes CD4+CD25+CD127−/low TSDR Demethylated Regulatory T Cell Subpopulations With Differential Stability and Susceptibility to Tacrolimus-Mediated Inhibition of Suppression

Abstract

BackgroundAdoptive transfer of forkhead box protein (FOX)3+ regulatory T (Treg) cells offers a promising strategy to reduce damage to an allograft by the recipient's immune system. Identification of cell surface markers sufficient to purify Treg cells expanded ex vivo to remove cellular contaminants requires optimization. Furthermore, the expanded Treg must be able to survive, expand, and suppress in allograft recipients exposed to immunosuppressants, such as tacrolimus (TAC). Reduced CD127 expression enhances identification of Treg in the human CD4+CD25+ population. CD45RA expression identifies naive CD4+CD25+ Treg with an enhanced stability of Treg phenotype.MethodsWe combine an analysis of CD45RA, CD25, and CD127 expression to identify subpopulations of CD4+CD127−/loCD25+ cells. Regulatory T cells were sorted according to expression of CD25 and CD45RA and expanded in the presence of a physiological relevant concentration of TAC. Regulatory T cell–specific demethylation region (TSDR) demethylation, FOXP3 expression, and suppression were analyzed.ResultsCD4+CD127−/loCD25+CD45RA+ Treg cells had a stable TSDR demethylated FOXP3+ phenotype after expansion whereas CD4+CD127lo/−CD25+CD45RA− Treg cell lost the TSDR demethylated phenotype. CD45RA− Treg had a greater capacity to suppress after expansion with TAC.ConclusionsAlthough CD45RA− Treg retained a greater suppressive capacity when expanded with TAC, the marked loss of the TSDR demethylated status highlights the potential for loss of stability of these cells in transplant recipients treated with TAC based immunosuppression. We show that a population of CD4+CD127−/loCD45RA+ Regulatory T cell may offer the best compromise between susceptibility to loss of suppression when exposed to TAC and maintenance of a TSDR demethylated phenotype following in vitro expansion.

Systematic testing and specificity mapping of alloantigen-specific chimeric antigen receptors in T regulatory cells

“…Importantly, A2-CAR Tregs did not make rejection worse in any reported studies (9)(10)(11), arguing against the possibility that after in vivo injection the cells acquired cytotoxic function via either Treg instability or outgrowth of contaminating conventional CAR T cells. To mitigate the risk of manufacturing CAR Tregs that were contaminated with low proportions of conventional T cells, for all in vivo studies we used naive CD45RA + CD25 hi Tregs as our starting population, since, in comparison to the CD45RAmemory Treg fraction, after expansion these cells maintain a more consistent Treg phenotype (57,58). Nevertheless, the long-terms effects of repeated CAR stimulation on Tregs is unknown.…”

Persistent regulatory T‐cell response 2 years after 3 years of grass tablet SLIT: Links to reduced eosinophil counts, sIgE levels, and clinical benefit

“…In the same study, the similar clinical outcomes were observed with a high‐dose intact allergen subcutaneous regime (Alutard SQ), suggesting similar mechanisms or both administration routes. Recently, Arroyo‐Hornero et al pointed out that CD 4+ CD 25 + CD127 ‐/lo population contained a subpopulation of Treg with differential maintenance of TSDR (regulatory T cell‐specific demethylation region) demethylation status, CD27 expression, cytokine production, and ability to suppress when exposed to immunosuppressants during in vitro expansion. A better understanding of T reg dynamics will be critical for the design of new specific immunotherapy intervention strategies.…”

Hair follicle immune privilege and its collapse in alopecia areata

“…Several neuropharmacological drugs 333 and other treatments are in development or currently available that hold potential for regenerating HF IP (Table 4), though these drugs are largely untested for use in treating AA. For example, the immunosuppressive drug FK506 (tacrolimus) 334,335 downregulates MHC class I expression and has been shown to protect HFs from IFNγ‐induced IP collapse in ex vivo studies 76 . Despite some success in rodent models, 336,337 clinical trials with topical FK506 have been unsuccessful, probably due to poor skin penetration 338‐340 .…”