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.