T-cell localization, activation, and clonal expansion in human pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy resistant to most therapies, including immune checkpoint blockade. To elucidate mechanisms of immunotherapy resistance, we assessed immune parameters in resected human PDA. We demonstrate significant interpatient variability in T-cell number, localization, and phenotype. CD8⁺ T cells, Foxp3⁺ regulatory T cells, and PD-1⁺ and PD-L1⁺ cells were preferentially enriched in tertiary lymphoid structures that were found in most tumors compared with stroma and tumor cell nests. Tumors containing more CD8⁺ T cells also had increased granulocytes, CD163⁺ (M2 immunosuppressive phenotype) macrophages, and FOXP3⁺ regulatory T cells. PD-L1 was rare on tumor cells, but was expressed by CD163⁺ macrophages and an additional stromal cell subset commonly found clustered together adjacent to tumor epithelium. The majority of tumoral CD8⁺ T cells did not express molecules suggestive of recent T-cell receptor (TCR) signaling. However, 41BB⁺PD-1⁺ T cells were still significantly enriched in tumors compared with circulation. Tumoral CD8⁺PD-1⁺ T cells commonly expressed additional inhibitory receptors, yet were mostly T-BET^hi and EOMES^lo, consistent with a less terminally exhausted state. Analysis of gene expression and rearranged TCR genes by deep sequencing suggested most patients have a limited tumor-reactive T-cell response. Multiplex immunohistochemistry revealed variable T-cell infiltration based on abundance and location, which may result in different mechanisms of immunotherapy resistance. Overall, the data support the need for therapies that either induce endogenous, or provide engineered, tumor-specific T-cell responses, and concurrently relieve suppressive mechanisms operative at the tumor site.