Honigsberg, R.; Cruz, T. D.; Yoffe, L.; Tang, M. S.; Ozgenel, D. O.; Markowitz, G.; Micheal, M.; Singh, A.; Altorki, N. K.; Elemento, O.; Villena-Vargas, J.

PD-1 immune checkpoint blockade (ICB) with platinum chemotherapy (ChemoIO) has transformed treatment for resectable non-small cell lung cancer (NSCLC). PD-1 ICB was established and created to reinvigorate tumor T cells and reduce T cell exhaustion. However, the impact of PD-1 blockade on the induction and maintenance of tumor-relevant tdLN T cells and functional T cell memory in the context of surgical resection is not fully understood. We hypothesized that T cells activated in the tumor draining lymph node (tdLN) also respond to PD-I ICB. We assessed clonal dynamics of tumor-specific tdLN T cell clones by using single- cell TCR barcoding integrated with transcriptional profiling (scRNA sequencing) by using machine learning (ML) and bioinformatic approaches. We displayed that within the tumor tdLN-paired clones were less exhausted than clones not clonally linked to the tdLN. We established that there are two distinct LN-paired anti-tumor clonal populations: those that directly link from the tdLN and tumor (tdLN-tumor paired) with tumor killing capacity, and clones that circulate within the blood with cytotoxic memory surveillance potential. Furthermore, the transcriptional distinction of these two LN-paired subsets was set in the earliest memory state of expanded tumor-specific tdLN T cells and reflected in the tumor as similarly, more differentiated, effector subsets. ChemoIO was also found to directly impact tdLN-paired clonal diversity and memory phenotypic differentiation. Finally, our findings demonstrate that ChemoIO is enhancing the cytotoxic function of circulating LN-paired clones in the tdLN and subsequently also altering circulating blood LN-paired clonal phenotype. Together our results established that tumor-relevant T cells are transcriptionally altered by PD- 1 ICB locally in the tdLN and provide evidence that lymph node derived T cells are a primary source of anti-tumor immunity in surgically resectable NSCLC.