All memory T cells mount an accelerated response on antigen reencounter, but significant functional heterogeneity is present within the respective memory T-cell subsets as defined by CCR7 and CD45RA expression, thereby warranting further stratification. Here we show that several surface markers, including KLRB1, KLRG1, GPR56, and KLRF1, help define low, high, or exhausted cytokine producers within human peripheral and intrahepatic CD4 + memory T-cell populations. Highest simultaneous production of TNF and IFN-γ is observed in KLRB1 + KLRG1 + GPR56 + CD4 T cells. By contrast, KLRF1 expression is associated with T-cell exhaustion and reduced TNF/IFN-γ production. Lastly, TCRβ repertoire analysis and in vitro differentiation support a regulated, progressive expression for these markers during CD4 + memory T-cell differentiation. Our results thus help refine the classification of human memory T cells to provide insights on inflammatory disease progression and immunotherapy development.

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 (T EMRA ) cells based on expression of CCR7 and CD45RA. Functional heterogeneity even within these subsets demonstrated the need for 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 production of multiple cytokines was observed in KLRB1 + KLRG1 + GPR56 + T cells. Only additional KLRF1expression was associated with a decline in cytokine production. The superiority of KLRF1 to define exhausted cytokine producers compared to classical T EMRA identification was best exemplified for intrahepatic T cells in patients with inflammatory liver diseases.