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Background Fli-1 is a transcription factor essential for cellular homeostasis and development, with aberrant expression linked to several cancers. Despite its associations with cancers, Fli-1 role in T cell biology, especially in the context of cancer immunotherapy, remained largely underexplored. Emerging evidence links Fli-1 to T cell activation and exhaustion. Our recent study revealed that Fli-1 regulates gene transcription for CD8 + T cell activation and differentiation into effector and memory subsets post-allogeneic hematopoietic cell transplantation. Additionally, Chen et al. showed that Fli1 loss enhances T-cell immunity in infection and cancer models.1 Methods To further explore the role of Fli1 in T cell antitumor immunity, we created Fli1-deficient, antigen-specific CD8 + T cells by crossing Fli1-floxed mice with CD4-Cre mice and breeding them with OT-1 or Pmel TCR transgenic strain. To translate findings into a therapeutic setting, we used CRISPR to knock out Fli-1 in CD19-directed human CAR T cells (figure 1).Results Invitro, tumor-specific Fli1 KO CD8 + T cells showed elevated activation (CD25, IFN-γ, GZB), proliferation (Ki-67), and cytolytic function after antigen activation in vitro. The adoptive transfer of Fli1 KO T cells significantly delayed tumor growth in MC38-OVA and B16-F10 melanoma models in vivo, with increased T cell infiltration and higher production of IFN-γ, TNF-α, and GZB. scRNA-seq revealed a shift toward activated T cell subsets and GO and GSEA analysis confirmed enrichment in immune activation and cytokine signaling.Fli-1 KO CAR T cells maintained normal CAR expression and expansion but exhibited enhanced tumor killing, increased IFN-γ and CD69 levels, and reduced exhaustion markers (PD-1, TIM-3, LAG-3). Using a human B cell lymphoma (Raji) xenograft model, Fli1 KO CAR T cells improved tumor control and extended survival.Conclusions In summary, targeting Fli-1 enhances CD8 + T cell antitumor activity and may represent a promising strategy to boost adoptive cancer immunotherapy.Reference Chen Z, Arai E, Khan O, Zhang Z, Ngiow SF, He Y, Huang H, Manne S, Cao Z, Baxter AE, Cai Z, Freilich E, Ali MA, Giles JR, Wu JE, Greenplate AR, Hakeem MA, Chen Q, Kurachi M, Nzingha K, Ekshyyan V, Mathew D, Wen Z, Speck NA, Battle A, Berger SL, Wherry EJ, Shi J. In vivo CD8 + T cell CRISPR screening reveals control by Fli1 in infection and cancer. Cell 2021 Mar 4;184(5):1262-1280.e22. doi: 10.1016/j.cell.2021.02.019. Epub 2021 Feb 25. PMID: 33636129; PMCID: PMC8054351.Abstract 814 Figure 1Schematic illustration of CRISPR/Cas9-mediated knockout strategy targeting the fli1 gene in CD19 CAR T cell