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.
Data availability
All data generated in this study are provided in the article itself, its supplementary information and in the Source Data file. The multi-ome (scRNAseq and scATACseq) sequencing data from Fig. 6 and Supplementary Fig. 4 have been deposited in GEO NCBI under the accession code GSE312328. Source data are provided with this paper.
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Acknowledgements
We thank the Peter MacCallum Cancer Centre Flow Cytometry (RRID: SCR_025550), Genotyping (RRID: SCR_025622), Victorian Centre for Functional Genomics (VCFG) (RRID: SCR_025582), Research Laboratory Support Services (RRID: SCR_025699) and Animal Core facilities for their access and support. We thank Sara Alaei from the Peter MacCallum Cancer Centre Molecular Genomics Core (RRID: SCR_025695) for conducting the multi-ome analysis. This work was funded by the Australian Cancer Research Foundation (for the Peter Mac Flow Cytometry facilities), Victorian Cancer Agency Mid-Career Fellowships 21019 (I.A.P.) and 20011 (P.A.B., 2021–2025), a CRI Lloyd J. Old STAR Grant CRI5578 (P.A.B.) and the CLEARbridge Foundation (I.A.P. and P.A.B.). The authors acknowledge the contributions of K. Gill, M. Rear, G. Sissing, I. Halligan and B. Wall who act as consumer representatives. Images in Figs. 1A, G, 2 A, 3A, F, 4 A and 5 A were created with BioRender.com. The authors have no conflicting financial interests.
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M.N.M.: conceptualization, investigation, methodology, formal analysis, visualization, writing—original draft, writing—review and editing, A.C.: conceptualization, methodology. N.K.: investigation. SS: investigation. N.Y.L.S.: formal analysis, visualization. K.M.Y.: methodology. I.P.N.: methodology, investigation. S.R.: methodology, investigation, writing—review and editing. C.D.T.D.: investigation. B.H.: investigation. K.M.R.: investigation. I.M.: conceptualization, methodology. P.A.B.: conceptualization, funding acquisition, methodology, project administration, resources, supervision, writing—original draft, writing—review and editing. I.A.P.: conceptualization, funding acquisition, methodology, project administration, resources, supervision, writing—original draft, writing—review and editing.
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de Menezes, M.N., Chen, A.X.Y., Kulkarni, N. et al. High efficiency CRISPR knock-in demonstrates that TCF1 is insufficient to reverse T cell exhaustion. Nat Commun (2026). https://doi.org/10.1038/s41467-026-69671-y
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DOI: https://doi.org/10.1038/s41467-026-69671-y
