Longitudinal profiling of tumor-reactive T cells in adoptive cell therapy with tumor-infiltrating lymphocytes

Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TILs) provides an effective treatment with long-term efficacy for patients with advanced metastatic melanoma. However, only a limited fraction of patients respond and the underlying reasons remain unclear. Here, us-ing single-cell RNA and T cell receptor (TCR) sequencing (scRNA-seq and scTCR-seq), we tracked the transcriptional alterations and fate decisions of tumor-reactive T cells through longi-tudinal sample obtained from patients undergoing TIL therapy, unveiling potential mechanisms leading to resistance. Our analyses revealed that exhausted CD8+ T cells (Tex), CD4+ T help-er 1 (Th1) /Tex, and CD4+ follicular helper T cells (Tfh) expanded during the initial phase of the expansion protocol but stagnated or contracted later. During expansion, tumor-reactive T cells lose their characteristic profile and become highly activated, whereas after TIL transfer, they rapidly extravasate and emerge with a stem/memory-like profile. However, in metastatic lesions excised after ACT (post-ACT), tumor-reactive T cells re-acquired high expression of exhaustion markers, which were elevated in non-responders (NRs) compared to responders (R). Additionally, we found that seven days after transfer, NRs were characterized by elevated frequencies of peripheral regulatory T cells (Tregs), which showed a highly activated tran-scriptomic profile post-ACT, thus potentially contributing to an immunosuppressive tumor mi-croenvironment (TME). Finally, resistance to TIL therapy was associated with the co-transfer of high numbers of activated CD4+ T helper 17 (Th17) and Type 17 CD8+ T cell (Tc17) sub-populations increasingly present in the TIL product of NRs.
Altogether, these findings provide insights into T cell fates during TIL therapy and will con-tribute to the development of new ACT to improve patient outcomes and induce durable re-sponses to TIL therapy.