Project description:Immune checkpoint blockade (ICB) has revolutionized cancer treatment, but most patients do not respond. PD-1 blockade “unleashes” CD8 T cells, including those specific for mutation-associated neoantigens (MANA), but factors in the tumor microenvironment can inhibit responses by dampening MANA-specific T cell function. Recent advances in single cell transcriptomics are revealing global T cell dysfunction programs in tumor-infiltrating lymphocytes (TIL). However, the vast majority of TIL do not recognize tumor antigens and little is known about transcriptional programs of true MANA-specific TIL. Here, we use an integrated approach to identify MANA-specific T cell clones using the MANA functional expansion of specific T cells (MANAFEST) assay in neoadjuvant anti-PD-1-treated lung cancers and use their TCR CDR3 as barcodes to track them and analyze their transcriptional programs and function in the tumor microenvironment using single cell RNA sequencing. We find both MANA-and virus-specific clones in TIL and adjacent normal lung, regardless of response status. MANA-specific, influenza (flu)-specific and EBV-specific TIL each have unique transcriptional programs. Most MANA-specific clones are tissue resident memory (TRM) cells, an incompletely activated cytolytic program, including EOMES deficiency, and higher levels of genes encoding T cell inhibitory molecules and Tox2. Notably, MANA-specific T cells express low levels of IL-7R and are functionally less responsive to IL-7 compared with tissue-resident flu-specific clones. MANA-specific clones from anti-PD-1 non-responding tumors express TCR with markedly lower ligand-dependent signaling capability, are largely confined to HOBIThi TRM subsets and coordinately up-regulate genes encoding specific checkpoints, killer inhibitory receptors, and intracellular inhibitors of T cell activation and cytotoxicity. These findings provide mechanistic and potential therapeutic insights into overcoming resistance to PD-1 blockade.

Project description:The differentiation and effector function of tumor infiltrating lymphocytes and macrophages in the tumor microenvironment is critical for productive anti-tumor immune responses, although direct evidence for this remains poorly characterized in brain cancer patients. Using mass cytometry, single-cell RNA sequencing, and quantitative multiplex immunofluorescence, we comprehensively characterized the phenotype and single-cell transcriptome of myeloid cells and T lymphocytes that infiltrated malignant gliomas, and identified how such populations changed following neoadjuvant PD-1 checkpoint blockade in recurrent glioblastoma patients. Cells of the myelo-monocytic lineage represented approximately three quarters of the immune cell infiltrate, with T lymphocytes representing the next major immune cell subset by density and percentage, but following neoadjuvant PD-1 antibody blockade, the ratio of myeloid cells to T cells significantly decreased. We then used single-cell RNA sequencing to comprehensively define the transcriptional profiles of lymphoid and myeloid populations in these tumors. Recurrent GBM patients treated with neoadjuvant PD-1 checkpoint inhibition possessed a greater fraction of CD8+ T cells with an effector T cell transcriptional program. The increased effector T cell populations were associated with the distinct upregulation of the chemokines, CXCL9 and CXCL10 by an interferon-responsive macrophage cluster. Importantly, we also identified that neoadjuvant PD-1 blockade was associated with significantly increased cellular interactions specifically between CD8+ T cells and tumor associated macrophages within the tumor microenvironment. Together, these findings suggest that effector T cell infiltration and differentiation are increased with neoadjuvant PD-1 blockade but impeded by adaptive resistance and immunosuppression from tumor associated macrophages. Future therapeutic strategies to target this dominant immunosuppressive myeloid cell population may be needed to achieve true, therapeutic interventions in this patient population.

Dataset Information

Distinct transcriptional programs characterize neoantigen-specific T cells in lung cancers treated with neoadjuvant PD-1 blockade [single-cell RNA-seq]

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