In Vivo CRISPR Screen Identifies FLI1 as Regulator of Effector T Cells

Immunotherapy aims to enhance antitumor immune responses, but the duration of patient benefit is often limited, in part due to T-cell exhaustion. To investigate pathways regulating differentiation into exhausted (T_EX) versus effector (T_EFF) CD8⁺ T cells, Chen and colleagues developed OpTICS, an in vivo CRISPR screening approach in which Cas9⁺ antigen-specific murine CD8⁺ T cells are transduced with a library of single guide RNAs targeting 120 transcription factor genes. T cells were then adoptively transferred into recipient mice, and the OpTICS platform identified candidates that inhibited optimal T-cell activation and T_EFF-cell differentiation in models of lymphocytic choriomeningitis virus (LCMV) infection. The gene encoding the ETS family transcription factor FLI1 was a top hit that repressed T_EFF cell differentiation, and further investigation revealed that CRISPR–Cas9-mediated mutagenesis of Fli1 increased CD8⁺ T-cell proliferation and T_EFF-cell populations during acutely resolved LCMV infection. Similarly, during chronic LCMV infection, FLI1 depletion in antigen-specific CD8⁺ T cells enriched for a signature of T_EFF-cell gene expression, supporting the idea that FLI1 deficiency promoted T_EFF-cell fate. Mechanistically, loss of FLI1 in CD8⁺ T cells shifted chromatin accessibility, especially in intronic or intergenic regions near known T_EFF-cell genes. ETS and RUNX motifs were highly enriched in regions with increased accessibility in the absence of FLI1. Given that the RUNX family member RUNX3 has been well established as a central driver of T_EFF-cell differentiation, RUNX3 was hypothesized to promote T_EFF-cell fate downstream of FLI1 loss. Indeed, overexpression of RUNX3 in the absence of FLI1 enhanced the CD8⁺ T-cell response to LCMV infection and skewed the T-cell population toward T_EFF-like cells. Importantly, loss of FLI1 in CD8⁺ T cells not only improved protective immunity in several models of viral or bacterial infection but also enhanced antitumor immunity, controlling tumor progression in a subcutaneous B16 melanoma model. Together, these results show that FLI1 inhibits T_EFF-cell fate by restricting RUNX3 genome access, highlighting FLI1 as a potential immunotherapy target that may enhance optimal T_EFF-cell differentiation in cancer and infection.

Chen Z, Arai E, Khan O, Zhang Z, Ngiow SF, He Y, et al. In vivo CD8⁺ T cell CRISPR screening reveals control by Fli1 in infection and cancer. Cell. 2021;184:1262–80.

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