Evolution of TCR diversity and clonality of human CD4+ memory T cell subsets defined according to progressive increase in killer-like receptor expression

Memory T cells mount an accelerated response upon re-challenge but are heterogeneous in phenotype and function. Traditionally, memory T cells were classified into central memory, effector memory and terminally differentiated effector memory (TEMRA) cells based on their expression of CCR7 and CD45RA. Functional heterogeneity even within these subsets demonstrated the need for a different classification using more suitable markers. We applied bulk and single gene expression profiling of human CD4+ memory T cells and identified surface markers, KLRB1, KLRG1, GPR56 and KLRF1, allowing classification into “low”, “high” or “exhausted” cytokine producers. In contrast to common understanding, KLRG1 expression was not associated with exhaustion and highest simultaneous production of TNF-a and IFN-g was observed in KLRB1+KLRG1+GPR56+ T cells. We found, that only additional KLRF1 expression was associated with a decline of both cytokines. Indeed, superiority of KLRF1 to define exhausted cytokine producers compared to classical TEMRA identification was seen especially for intra-tissue T cells in patients with inflammatory liver diseases. Finally, TCR diversity and clonality analysis as well as in vitro differentiation experiments support our hypothesis of successive acquisition of KLR & GPR56 expression during CD4+ memory T cell differentiation.记忆T细胞（Memory T cells）在再次遭遇抗原激发时可启动加速的免疫应答，但其表型与功能均存在异质性。传统上，研究人员依据记忆T细胞表面CCR7与CD45RA的表达模式，将其分为中枢记忆T细胞（central memory T cell）、效应记忆T细胞（effector memory T cell）以及终末分化效应记忆T细胞（TEMRA）三类。即便在上述亚群内部仍存在功能异质性，这表明亟需采用更适宜的标志物建立全新的分类标准。本研究对人类CD4+记忆T细胞实施批量与单细胞基因表达谱分析，鉴定出KLRB1、KLRG1、GPR56及KLRF1四种表面标志物，可将细胞划分为"低细胞因子分泌型""高细胞因子分泌型"与"耗竭型"细胞因子分泌亚群。与学界主流认知相悖的是，KLRG1的表达与细胞耗竭并无关联；而KLRB1+KLRG1+GPR56+ T细胞可同时高水平分泌肿瘤坏死因子α（TNF-α）与干扰素γ（IFN-γ），其共分泌水平为各亚群之首。本研究发现，唯有额外表达KLRF1的亚群，其两种细胞因子的分泌水平均会出现显著下降。相较于经典的TEMRA鉴定方法，KLRF1用于标识耗竭型细胞因子分泌细胞的优势尤为突出，尤其体现在炎症性肝病患者的组织内T细胞群体中。最后，T细胞受体（TCR）多样性与克隆性分析，以及体外分化实验结果，均支持本研究提出的假说：CD4+记忆T细胞在分化进程中会依次获得KLR家族与GPR56的表面标志物表达。