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

Adoptive cell therapy with tumor-infiltrating lymphocytes (TILs) induces durable responses in metastatic melanoma, yet the clonal and transcriptional dynamics governing tumor-reactive T cell fate during ex vivo expansion and after transfer remain poorly understood. Here, we perform longitudinal single-cell RNA and T cell receptor sequencing across five time points, from baseline tumors through two-phase ex vivo expansion to post-infusion blood and tumor biopsies, in seven melanoma patients, resolving both the CD8+ and CD4+ compartments. Tumor-reactive CD8+ T cells are reinvigorated from exhaustion and acquire HLA-II-high or KLF2-high profiles. We further dissect the tumor-responsive CD4+ compartment in depth, revealing lineage-dependent reinvigoration in which follicular helper T cells adopt an effector state while exhausted CD4+ T cells retain dysfunction. In non-responders, across three cohorts, co-transferred type 17 T cells and de novo regulatory T cell expansion after transfer associate with treatment failure. These data define subtype-specific signatures across both lineages to guide TIL expansion.