Single-cell tracking reveals tumor-reactive T cell plasticity during melanoma TIL therapy

Adoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TILs) can induce durable responses in metastatic melanoma, yet the clonal and transcriptional dynamics that govern tumor-reactive T cell fate during ex vivo expansion and after transfer remain poorly understood. Here, we performed longitudinal single-cell RNA and T cell receptor (TCR) sequencing across five timepoints, from baseline tumors through a two-phase ex vivo expansion to post-infusion peripheral blood and tumor biopsies, in seven melanoma patients, generating a high-resolution map of both CD8+ and CD4+ tumor-reactive T cell plasticity. During early expansion (preREP), tumor-reactive CD8+ T cells were reinvigorated from an exhausted state and acquired distinct HLA-II-high or KLF2-high transcriptional profiles. In-depth characterization of the CD4+ compartment revealed lineage-dependent reinvigoration. After transfer, both CD8+ and CD4+ tumor-reactive clones acquired stem-like profiles and tissue-homing markers before re-infiltrating tumor lesions. Mechanistically, we identify two processes linked to treatment failure in non-responders: de novo expansion of immunosuppressive regulatory T cells, and co-transfer of Type 17 T cells. These data provide a comprehensive longitudinal framework of T cell plasticity during TIL-ACT.