The Outcome of TIL Expansion Is Highly Influenced by Spatial Heterogeneity of the Tumor T-Cell Repertoire and Differences in Intrinsic Growth Capacity between T-Cell Clones.

PURPOSE

During our efforts to develop tumor-infiltrating lymphocyte (TIL) therapy to counter the devastating recurrence rate in patients with primary resectable pancreatic ductal adenocarcinoma (PDA), we found that PDA TILs can readily be expanded and that the majority of resulting TIL cultures show reactivity against the autologous tumor. However, the fraction of tumor-reactive T cells is low. We investigated to which extent this was related to the expansion.

EXPERIMENTAL DESIGN

We compared the clonal composition of TIL preparations before and after expansion using T-cell receptor (TCR) deep sequencing. Our findings for PDA were benchmarked to experiments with melanoma TILs.

RESULTS

We found that the TIL TCR repertoire changes dramatically during expansion, leading to loss of tumor- dominant T-cell clones and overgrowth by newly emerging T-cell clones that are barely detectable in the tumor. These changes are primarily driven by differences in the intrinsic expansion capacity of T-cell clones. Single-cell experiments showed an association between poor proliferative capacity and expression of markers related to antigen experience and dysfunction. Furthermore, we found that spatial heterogeneity of the TIL repertoire resulted in TCR repertoires that are greatly divergent between TIL cultures derived from distant tumor samples of the same patient.

CONCLUSIONS

Culture-induced changes in clonal composition are likely to affect tumor reactivity of TIL preparations. TCR deep sequencing provides important insights into the factors that govern the outcome of TIL expansion and thereby a path toward optimization of the production of TIL preparations with high therapeutic efficacy..