Arroyo Hornero Rebeca, Betts Gareth J, Sawitzki Birgit, Vogt Katrin, Harden Paul N, Wood Kathryn J
1 Nuffield Department of Surgical Sciences, Oxford University, John Radcliffe Hospital, Oxford, United Kingdom. 2 Charité-Universitätsmedizin Berlin, Campus Virchow, Institut f. Medizinische Immunologie, Berlin, Germany. 3 Oxford Transplant Centre, Oxford University Hospitals NHS Trust, Oxford, United Kingdom.
Transplantation. 2017 Feb;101(2):302-309. doi: 10.1097/TP.0000000000001278.
Adoptive 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 CD4CD25 population. CD45RA expression identifies naive CD4CD25 Treg with an enhanced stability of Treg phenotype.
We combine an analysis of CD45RA, CD25, and CD127 expression to identify subpopulations of CD4CD127CD25 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.
CD4CD127CD25CD45RA Treg cells had a stable TSDR demethylated FOXP3 phenotype after expansion whereas CD4CD127CD25CD45RA Treg cell lost the TSDR demethylated phenotype. CD45RA Treg had a greater capacity to suppress after expansion with TAC.
Although 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 CD4CD127CD45RA 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.
过继性转移叉头框蛋白（FOX）3调节性T（Treg）细胞为减少受者免疫系统对同种异体移植物的损伤提供了一种很有前景的策略。确定足以纯化体外扩增的Treg细胞以去除细胞污染物的细胞表面标志物需要优化。此外，扩增后的Treg必须能够在接受免疫抑制剂（如他克莫司（TAC））的同种异体移植受者体内存活、扩增并发挥抑制作用。CD127表达降低可增强在人CD4CD25群体中对Treg的识别。CD45RA表达可识别具有增强的Treg表型稳定性的初始CD4CD25 Treg。
我们结合对CD45RA、CD25和CD127表达的分析来鉴定CD4CD127CD25细胞亚群。根据CD25和CD45RA的表达对调节性T细胞进行分选，并在生理相关浓度的TAC存在下进行扩增。分析调节性T细胞特异性去甲基化区域（TSDR）去甲基化、FOXP3表达和抑制作用。
扩增后，CD4CD127CD25CD45RA Treg细胞具有稳定的TSDR去甲基化FOXP3表型，而CD4CD127CD25CD45RA Treg细胞失去了TSDR去甲基化表型。CD45RA Treg在用TAC扩增后具有更强的抑制能力。
尽管CD45RA Treg在用TAC扩增时保留了更强的抑制能力，但TSDR去甲基化状态的显著丧失突出了这些细胞在接受基于TAC的免疫抑制治疗的移植受者中稳定性丧失的可能性。我们表明，CD4CD127CD45RA调节性T细胞群体可能在暴露于TAC时抑制作用丧失的易感性与体外扩增后TSDR去甲基化表型的维持之间提供最佳折衷方案。
