Abstract 3241: Single-cell RNA and TCR sequencing of tumor infiltrating lymphocytes identifies changes in pancreatic ductal adenocarcinomas following neoadjuvant treatment with combined anti-PD-1 blocking and anti-CD137 agonist therapy

Introduction: Pancreatic ductal adenocarcinomas (PDACs) are immunogenically “cold” tumors that lack effector T cell infiltration associated with immune checkpoint inhibitor (ICI) responsiveness. Neoadjuvant immunotherapy clinical trials facilitate understanding of the complexities of combination treatment strategies on immunosuppressive mechanisms. Our recent multi-omics analysis of a neoadjuvant trial comparing the GVAX vaccine alone or with nivolumab and supporting preclinical data uncovered crosstalk between CD8+CD137+ T cells and neutrophils as an axis for therapeutic intervention. These studies have formed the foundation for the addition of the CD137 agonist, urelumab, to our combination regimen to improve T cell activation in PDACs. Methods: Patients were enrolled on NCT02451982 and initiated treatment 2 weeks prior to surgical resection. We compared specimens from 3 treatment arms for downstream analysis: Arm A: an allogeneic whole cell vaccine, GVAX (n=7); Arm B: GVAX and nivolumab (n=4); Arm C: GVAX, nivolumab, and urelumab (n=4). Samples underwent dissociation and Percoll separation to enrich for immune cells, followed by single-cell RNA- and TCR-seq using 10X Genomics. Computational analysis was performed for cell type classification with Seurat, differential expression with DESeq2 of pseudobulk data, cell-cell communication with Domino, and TCR-seq analysis with scRepertoire. Results: Single-cell RNA-seq analysis identified changes in immune cell proportions and immune cell states across treatment arms. Alignment of scTCR-seq to RNA data showed greater CD8+ T cell expansion following triple agent therapy. Gene set enrichment identified changes in CD8+ T cell, tumor associated macrophage (TAM), and B cell responses to cytokine signaling, locomotion, amino acid metabolism, and cell adhesion pathways in Arm C. Domino analysis demonstrated increases in the naïve T cell signal CCR7 in Arm A and integrin ligand signal ICAM1 in Arm C between the CD8+ T cells and other cell types present in PDACs. CCR7 expression was significantly downregulated in CD8+ T cells in Arm C whereas CCL5, a T cell agonist cytokine, and LCP2, a TCR signaling activator and T cell receptor which is also required for integrin signaling, were significantly upregulated in CD8+ T cells in Arm C. The increased integrin ligand signaling via ICAM1 and LCP2 in Arm C is also associated with changes in CD8+ T cell motility observed from the differential expression analysis. Conclusions: