Suppression of IFN-g expression in Treg cells is dependent on the TCR signaling pathway 2332935

Abstract Introduction Regulatory T cells (Tregs) suppress excessive immune responses but promote tumor immune evasion by limiting anti-tumor immunity. Unlike effector T cells, Tregs restrict expression of interferon-γ (IFN-γ), a key anti-tumor cytokine, through incompletely defined mechanisms. Identifying pathways that constrain IFN-γ in Tregs may enable functional reprogramming to enhance anti-tumor immunity. Methods We performed a genome-wide CRISPR/Cas9 loss-of-function screen in Tregs to identify genes involved in the suppression of IFN-γ expression. Candidate genes, including Zap70, were individually validated. Tregs were transduced with a retroviral vector carrying sgZap70 or non-targeting control (sgNT2) and cultured in vitro, with RNA-seq to assess temporal transcriptional dynamics. In vivo anti-tumor immune responses were evaluated using an adoptive T-cell transfer tumor model. Results The CRISPR screen identified Zap70 as a candidate regulator of IFN-γ suppression in Tregs. Genetic ablation of Zap70 and additional TCR pathway components resulted in a significant increase in IFN-γ expression while preserving Foxp3 expression. Time-course RNA-seq revealed a progressive upregulation of the IL-12 signaling axis components Il12rb2, Jak2, and Stat4 in Zap70-deficient Tregs. In adoptive transfer tumor models, mice receiving Zap70-deficient Tregs exhibited reduced tumor burden compared with controls. Conclusion Our data identify TCR—Zap70 signaling as a critical suppressor of a selective Il-12—Jak2—Stat4 transcriptional program in Tregs that constrains IFN-γ induction. Loss of Zap70 enables a delayed but coordinated activation of this axis while maintaining Treg lineage stability. Importantly, this transcriptional reprogramming translates into anti-tumor activity in vivo, as adoptive transfer of Zap70-deficient Tregs results in reduced tumor burden. Together, these findings establish TCR signaling in Tregs as a mechanistically defined and functionally actionable target for enhancing anti-tumor immune responses. Funding Source NIH Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)