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:BackgroundMerkel cell carcinoma (MCC) often responds to PD-1 pathway blockade, regardless of tumor-viral status (~80% of cases driven by the Merkel cell polyomavirus (MCPyV)). Prior studies have characterized tumor-specific T cell responses to MCPyV, which have typically been CD8, but little is known about the T cell response to UV-induced neoantigens.MethodsA patient in her mid-50s with virus-negative (VN) MCC developed large liver metastases after a brief initial response to chemotherapy. She received anti-PD-L1 (avelumab) and had a partial response within 4 weeks. Whole exome sequencing (WES) was performed to determine potential neoantigen peptides. Characterization of peripheral blood neoantigen T cell responses was evaluated via interferon-gamma (IFNγ) ELISpot, flow cytometry and single-cell RNA sequencing. Tumor-resident T cells were characterized by multiplexed immunohistochemistry.ResultsWES identified 1027 tumor-specific somatic mutations, similar to the published average of 1121 for VN-MCCs. Peptide prediction with a binding cut-off of ≤100 nM resulted in 77 peptides that were synthesized for T cell assays. Although peptides were predicted based on class I HLAs, we identified circulating CD4 T cells targeting 5 of 77 neoantigens. In contrast, no neoantigen-specific CD8 T cell responses were detected. Neoantigen-specific CD4 T cells were undetectable in blood before anti-PD-L1 therapy but became readily detectible shortly after starting therapy. T cells produced robust IFNγ when stimulated by neoantigen (mutant) peptides but not by the normal (wild-type) peptides. Single cell RNAseq showed neoantigen-reactive T cells expressed the Th1-associated transcription factor (T-bet) and associated cytokines. These CD4 T cells did not significantly exhibit cytotoxicity or non-Th1 markers. Within the pretreatment tumor, resident CD4 T cells were also Th1-skewed and expressed T-bet.ConclusionsWe identified and characterized tumor-specific Th1-skewed CD4 T cells targeting multiple neoantigens in a patient who experienced a profound and durable partial response to anti-PD-L1 therapy. To our knowledge, this is the first report of neoantigen-specific T cell responses in MCC. Although CD4 and CD8 T cells recognizing viral tumor antigens are often detectible in virus-positive MCC, only CD4 T cells recognizing neoantigens were detected in this patient. These findings suggest that CD4 T cells can play an important role in the response to anti-PD-(L)1 therapy.

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Transcriptional programs of neoantigen-specific TIL in anti-PD-1-treated lung cancers.

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