Ex vivo expansion of melanoma tumor infiltrating lymphocytes leads to a dominant exhausted T cell population with lack of memory markers.

Background.Tumor infiltrating lymphocytes (TILs) 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.Methods.To characterize the process of TIL expansion we performed single-cell RNA- and TCR-sequencing of TILs isolated from patient tumors and their paired ex vivo expanded cell products. Pre- and post-expansion TILs were characterized by unsupervised clustering and cell state assignment using the ProjecTIL framework, differential abundance, and clonotype analysis.Results.We found that there are large transcriptional differences between pre- and post-expansion TILs. Post-expansion TILs were predominantly exhausted and lacked naive or memory cell phenotypes, including a decreased percentage of CD39/CD69 double negative (DN) cells. Co-activating receptors CD137 and CD27 decreased while CD30 increased, whereas among co-inhibitory receptors PD1 decreased while 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.Conclusions.Although ex vivo expanded TILs are predominantly terminally differentiated, exhausted and transcriptionally highly distinct from the initial TILs, there is also a large Tpex population and DN numbers increase. Future work to amplify subpopulations of TILs, such as the DN cells, will likely further improve this therapy.