PROJECT SUMMARY: Checkpoint blockade has had tremendous impact on cancer therapy, but only a subset of patients respond. While non-response may be due to lack of tumor-associated antigen (TAA), our group and others have shown that ?as important as having TAA? is the immune system?s capacity to present TAA on antigen presenting cells, specifically a subset of ?cross-presenting? Batf3- expressing dendritic cells (DC). Without Batf3-DC, anti-tumor effects of PD1 blockade are lost. Therefore we hypothesize that increasing cross-priming DC will improve the efficacy of PD1 blockade. To increase cross-priming, we developed an in situ vaccination (ISV) combining (i) Flt3L, to recruit DC (ii) radiotherapy (XRT) to load DC with TAA, and (iii) a TLR agonist to activate the TAA-loaded DCs. We tested this novel ISV (Flt3L/XRT/polyIC) in advanced stage lymphoma patients and observed dramatic systemic remissions in some patients. To improve the ISV, we developed a murine ISV model which recapitulated the clinical findings and also increased cure rates of PD1 blockade from 0% to ~75%, which prompted a new trial, using ISV plus pembrolizumab, opened in 02/19. Still, the unmet clinical need is great, to understand and improve upon our ISV and PD1 blockade, we will develop methods to measure cross-priming, using sophisticated mouse models (Aim 1) and deep immune monitoring of patient samples from the two trials (Aim 2,3). In Aim 1, we will determine how ISV enhances PD1 blockade in mouse models in which we preserve only cross-priming or only direct-priming (by the tumor) or both. Specifically, we will: (a) determine if cross-priming is necessary for ISV anti-tumor effects (b) define a signature of cross-primed T cells, including their expression of checkpoint molecules, and (c) characterize resistance mechanisms. In Aim 2, we will demonstrate that ISV cross-primes patients? tumor-reactive T cells by analyzing banked samples from our ISV-treated patients. Specifically, we will: (a) use ex vivo autologous tumor:T cell co-cultures, flow-sorting, and immunoSEQ to identify anti-tumor T cells, and (b) use 5? scRNAseq/ TCRseq to quantify and characterize those T cells and then correlate them with clinical responses. In Aim 3, we will analyze banked samples from our ongoing ISV plus pembrolizumab trial which also includes administration of a surrogate Ag at the ISV site. Specifically, we will: (a) assess whether surrogate Ag cross-presentation is improved by ISV and PD1 blockade and whether it predicts clinical outcomes, and (b) determine mechanisms of tumoral suppression of cross-priming. Lymphomas kill ~21,000 patients annually in the U.S. The most common lymphomas are incurable with standard therapies and minimally responsive to checkpoint blockade. Improving our understanding of ISV and PD1 blockade will improve their efficacy, and thereby improve the lives of our patients.