Tumor-reactive T cells in HNSCC patients are directed against cancer-associated fibroblasts

Endogenous T-cell (TC) responses against tumors determine patient prognosis and are the basis for successful immune checkpoint-blocking cancer immunotherapy. Yet, cell subsets and antigens they target in the tumor microenvironment remain largely unknown. In contrast to tumor cells, which are a clonally diverse and unstable cell population, non-malignant cells of the tumor stroma, particularly cancer-associated fibroblasts (CAFs) represent a large subset of genetically stable cells which nonetheless essentially contribute to tumor progression in many cancers. Here, we studied in a cohort of HNSCC patients to what degree tumor-reactive TCs may target CAFs and which CAF-associated antigens are recognized. Using autologous TCs, tumor cell, and CAF cultures from HNSCC patients, we show that tumor-reactive type 1 TC responses against CAFs commonly occur in HNSCC patients to a similar extent as TC responses against the tumor cells themselves. Due to simultaneous MHC-I and -II expression, CAFs were directly recognized by CD8+ cytotoxic and CD4+ helper TCs. Then, we performed automated 2D proteome fractionation of CAF lysates together with mass spectrometry-based proteome identification, differential transcriptome analyses of CAFs and laser-capture-microdissected tumor stroma and TC function assays. We identified several common antigens including TXNDC17, NANS, DNAJB11, and THBS2 being differentially expressed on CAFs and recognized by tumor-reactive TC repertoires of multiple patients. Taken together, CAFs are a major target cell type of tumor-reactive TC responses in HNSCC patients. Due to their MHC-II expression, differential expression of common CAF-selective immunogenic antigens and their essential role during tumor progression, they represent promising targets for anti-cancer immunotherapies. Comparative transcriptomic analysis of CAF, TUC, NF cells and dissected normal and tumor stroma