Tumor-infiltrated double-negative regulatory T cells predict outcome of T cell-based immunotherapy in nasopharyngeal carcinoma

Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TILs) has demonstrated clinical success in solid tumors. We analyze 47 TIL infusion products and 62 pretreatment tumor microenvironments (TMEs) from a randomized phase 2 clinical study of concurrent chemoradiotherapy plus TIL-ACT (NCT02421640). Using single-cell and bulk RNA sequencing along with flow cytometry, we identify 14 CD3⁺ T cell clusters within 26 TIL infusion products: 11 CD3⁺CD8⁺ TILs, 2 CD3⁺CD4⁺ TILs, and 1 CD3⁺CD8^-CD4^- double-negative (DN) TIL. (DN) TILs, significantly associated with poor TIL-ACT outcomes, exhibit an activated regulatory T cell-like phenotype and include two CD56⁺ and four CD56^- subsets. Among them, CD56^-KZF2⁺ (DN) TILs are predominantly suppressive. (DN) TILs inhibit CD8⁺ TIL expansion via Fas-FasL, transforming growth factor β (TGF-β), and interleukin (IL)-10 signaling. Distinct CD8⁺ T subsets differentially impact on TIL-ACT outcomes, while 9 baseline TME gene signatures and 14 intracellular T cell genes hold prognostic value. Our findings identify predictive TIL subsets and biomarkers for TIL-ACT outcomes.