Single-cell gain-of-function mapping reveals latent regulatory programs governing CD8+ T cell fate.

CD8+ T cells mediate host defense and tumor immunity through specialized differentiation states, yet the regulatory programs that guide these states may also limit their functional potential. Loss-of-function studies have defined many regulators required for T cell differentiation, but they do not readily reveal regulatory activities that emerge only when transcription factors are ectopically expressed outside their native lineage, dosage, or temporal context. Here, we developed single-cell gain-of-function (GOF) sequencing (scGOF-seq), a multiplexed platform for in vivo mapping of transcription factor overexpression in antigen-specific CD8+ T cells across immunocompetent models of infection and cancer. By enforcing expression of canonical T cell regulators, lineage-silenced developmental factors, and temporally restricted transcription factors, scGOF-seq uncovered unexpected in vivo activities. Developmental regulators normally silenced in T cells, including NANOG, SOX2, OCT4 and GATA2, reshaped T cell differentiation in context-dependent ways, with NANOG promoting stemness-associated phenotypes and accumulation during chronic infection. In parallel, sustained cMyc expression outside its native temporal window generated a stem-like, effector-featured state with enhanced metabolic fitness, reduced terminal exhaustion, and profound antigen-dependent expansion exceeding 5,000-fold. Importantly, cMyc GOF maintained cell-cycle checkpoint signatures and demonstrated a strong dependence on antigen presence for proliferation across the tested conditions. scGOF-seq further identified cooperating transcription factor modules that complemented cMyc-driven programs and improved T cell responses in solid tumors. These findings establish systematic GOF perturbation as a framework for uncovering latent and temporally constrained regulatory activities in CD8+ T cells and guiding immune-state engineering.