Abstract 5877: Intratumoral lymphocyte networks harbor TCF1+ PD1+ progenitor CD8 T cells in lung cancer

Cancer immunotherapies that activate cytotoxic T cell responses against tumors have been remarkably effective in some patients, but the determinants of response remain unclear. To investigate how spatial organization of T cells might influence immunotherapy response, we developed a tissue-based cyclic immunofluorescence (t-CyCIF) platform to characterize preclinical genetically engineered mouse models (GEMMs) of cancer. We optimized a 21-antibody panel delineating major myeloid and lymphoid cell subsets, that included key markers for characterizing T cell phenotypes and function. The panel was used to characterize the Kras/p53-mutant GEMM of lung adenocarcinoma, treated with anti-PD-1/anti-CTLA-4 immune checkpoint blockade (ICB) therapy or a neoantigen-targeted long-peptide vaccine. Spatial profiling by t-CyCIF identified intratumoral lymphocyte networks (“lymphonets”) as a key feature associated with a productive immune response against lung tumors. Lymphonets were predominantly composed of B cells and conventional CD4 T cells, but harbored tumor-reactive CD8 T cell populations. TCF1+ PD1+ progenitor CD8 T cells, a subset previously correlated with patient response to ICB, were almost exclusively localized to lymphonets inside tumors. Lymphonets of similar composition harboring TCF1+ PD1+ cells were also found in early-stage human lung adenocarcinomas. In response to ICB and vaccine therapies in mice, cytotoxic CD8 T cells (Granzyme B+/Perforin+) colocalized with TCF1+ PD1+ cells in lymphonets, likely having differentiated from this progenitor pool. Notably, vaccination further induced a highly proliferative (Ki67hi) and cytotoxic (GranzymeBhiPerforinhi) population of CD8 T cells localized outside of tumors that we posit enter tumors without passing through the intratumoral progenitor cell state. Upon becoming dysfunctional, marked by loss of Granzyme B/Perforin expression and upregulation of inhibitory receptors, CD8 T cells were excluded from lymphonets and localized predominantly to the tumor margin. Altogether, these results shed light on the spatial dynamics associated with a productive response to immunotherapies and open avenues for exploration into the function of lymphonets in supporting cytotoxic CD8 T cell responses in tumors. Citation Format: Megan L. Burger, Giorgio Gaglia, Claire C. Ritch, Danae Rammos, Yang Dai, Grace E. Crossland, Sara Z. Tavana, Simon Warchol, Alex M. Jaeger, Santiago Naranjo, Shannon Coy, Ajit J. Nirmal, Robert Krueger,