High efficiency CRISPR knock-in demonstrates that TCF1 is insufficient to reverse T cell exhaustion

Abstract

CD8+ T cell exhaustion is a regulatory state triggered by chronic antigen stimulation in both cancer and persistent infection. The less differentiated stem-like sub-populations of exhausted T cells have been heavily studied given their importance to the efficacy of current immunotherapies. While the transcription factor TCF1 is both necessary and sufficient for formation and maintenance of these stem-like populations, it remains unclear whether TCF1 can actively de-differentiate more terminally exhausted subsets back into a stem-like state. To address this question, here we utilize and optimize a high efficiency CRISPR knock-in methodology, compatible with mouse in vivo exhaustion models, to engineer T cells that either constitutively over-express TCF1, or conditionally over-express TCF1 following differentiation of the cells into a CX3CR1+ intermediate-exhausted state. Strikingly, we find that only constitutive, and not conditional, TCF1 over-expression can increase the size of the stem-like T cell pool. Thus, while TCF1 can slow stem-like T cell differentiation, it is insufficient to revert more differentiated cells back into a stem-like state. Exhaustion is a functional state that hampers anti-cancer and antiviral CD8 T cell activity, and is preceded by a stem-like state, maintained by the transcription factor TCF1. Here authors develop mouse models that allow a precise understanding of the developmental trajectory between the stem-cell-like and exhausted states of CD8 T cells and find that while constitutive overexpression of TCF1 expands the stem-like T cell pool, TCF1 expression specifically in already exhausted cells is unable to promote dedifferentiation.