Background Self-renewing T cells maintain robust immune responses against pathogens and tumors. In acute infections, T cells maintain self-renewal capabilities after pathogen clearance to generate memory. In chronic infections and tumors, self-renewing T cells replenish effector cells for persistent cytotoxic activity. In both contexts, the stem-like state is upheld by the transcription factor TCF1 (encoded by Tcf7). TCF1 is expressed in naive, memory, and self-renewing cells, where it maintains their long-term proliferative potential. Upon effector or exhaustion differentiation, cells down-regulate TCF1 to commit to a short-lived state. TCF1 silencing is thought to be irreversible, in line with evidence that the Tcf7 locus acquires repressive epigenetic states upon differentiation. However, effectors can dedifferentiate to form memory cells, raising the possibility that plasticity in TCF1 regulation allows for re-establishment of self-renewal in different settings.
Methods Following cell state transitions in single cells is critical to understanding cell lineage decisions, but in vivo models preclude single-cell tracking. We therefore developed an ex vivo system for tracking T cell differentiation under acute or chronic stimulation. Combining this system with long-term live imaging, we followed TCF1 regulation and lineage decision dynamics in clonal lineages over multiple cell generations, and complemented imaging readouts with time-resolved single-cell transcriptomics. Finally, to elucidate Tcf7 regulation mechanisms, we performed a CRISPR screen of regulatory targets including chromatin regulators and transcription factors.
Results Through combined imaging and transcriptomic readouts, we identify a remarkably flexible strategy for effector vs memory decision making, whereby T cells decide early whether to maintain or lose memory potential after antigen recognition, but following pathogen clearance may regain lost memory potential. This flexibility is implemented by a cis-epigenetic switch that reversibly silences the memory regulator TCF1 in response to TCR and inflammatory signaling. Strikingly, TCF1 silencing and memory decision making is stochastic, such that multiple lineages can arise even in the absence of heterogeneity in external signals. Upon chronic stimulation, cells progressively lose the ability to re-activate TCF1 and re-enter a self-renewing state, and become locked in an exhausted state. From CRISPR screening, we identify distinct chromatin and transcription factor sub-circuits responsible for initiation and lockdown of TCF1 silencing.
Conclusions Our findings reveal remarkable flexibility in the lineage pathways through which self-renewing T cells can emerge, enabling for adaptive immune response to be tailored to different types and severities of threats. The molecular mechanisms uncovered further inform strategies for maintaining T cell persistence for immunotherapy.
Ethics Approval All animals were used in accordance with the Institutional Animal Care and Use Committee guidelines for the University of Washington, registered protocol number 4397–01.
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