<i>Ex Vivo</i> Expansion of Melanoma Tumor-Infiltrating Lymphocytes Leads to a Dominant Exhausted T-cell Population with a Lack of Memory Markers

Tumor-infiltrating lymphocytes (TIL) can be isolated from patient tumors, greatly expanded ex vivo, and returned to the patient for therapeutic effect. Recent clinical trials have highlighted the efficacy of TILs for a subset of patients and supported FDA approval for melanoma. How TILs evolve during the manufacturing process is still unknown and likely critical to improving the therapy for more patients. To characterize cell modification during TIL expansion, we performed single-cell RNA and T-cell receptor sequencing of TILs isolated from patient tumors and their paired ex vivo expanded cell products. We found large transcriptional differences between pre- and post-expansion TILs. Post-expansion TILs were predominantly exhausted and lacked naïve or memory cell phenotypes, including a decreased percentage of CD39/CD69 double-negative (DN) "stem-like" T cells. Coactivating receptors CD137 and CD27 decreased, whereas CD30 increased. Among coinhibitory receptors, PDCD1 (PD-1) decreased, whereas HAVCR2 (TIM3) and LAG3 showed the largest increases with expansion. Other gene families that showed large increases with ex vivo growth included cytotoxicity- and APC-associated genes. Individual clonotypes were distributed among multiple cell differentiation states, which exhibited high degrees of plasticity during expansion. Although ex vivo expanded TILs are predominantly terminally differentiated, exhausted, and transcriptionally highly distinct from the initial TILs, there is also a large progenitor exhausted CD8 T-cell population, and DN numbers increase. Future work to amplify subpopulations of TILs with memory cell phenotypes, such as the DN cells, will likely further improve this therapy.