{
  "abstract": "Background Methods to identify and characterize antigen-reactive T cell receptors (TCRs) represent important tools to understand and exploit T cell responses in patients with cancer and beyond. Current methods are hampered by the rarity of individual T cell clones and limited applicability to monitor both major histocompatibility complex (MHC) class I-restricted and MHC class II-restricted responses, hence insufficiently reflecting the entire antigen-reactive repertoire of a patient. To obtain broad and deep insight into polyclonal, antigen-specific TCR repertoires, we developed the ‘epitope-specific expansion culture with subsequent identification of TCRs’ (ESPEC-SUIT) assay to identify and track antigen-specific TCRs.Methods In vitro stimulation of peripheral blood mononuclear cells with (vaccine-targeted) neoantigens was verified in cytokine secretion assays and read-out by TCRβ repertoire sequencing (TCRseq). Candidate antigen-reactive clonotypes were defined by specific expansion in cultures stimulated with relevant antigen, followed by TCR cloning and validation in co-cultures of TCR transgenic effector cells and peptide-presenting targets. Using TCRseq information, candidate and validated clonotypes were traced and characterized in bulk and single-cell repertoire sequencing data of longitudinally collected blood samples and tumor tissue.Results In a cohort of 32 patients with cancer, we demonstrate that ESPEC-SUIT supports strong, robust and reproducible expansion of CD4 + and CD8+ T cells in response to various antigens. TCRseq revealed highly polyclonal neoepitope-specific T cell responses, which can be further characterized with respect to cross-reactivity, affinity or human leukocyte antigen (HLA) restriction. In a subcohort of 10 patients, we selected 341 ESPEC-SUIT-derived TCRs for cloning and in vitro functional validation from >2000 candidates and confirmed antigen-reactivity for >75%. We exemplify the usefulness of this TCR discovery method for downstream analysis in neoepitope vaccinated patients with glioma, where we found longitudinal changes in candidate TCR frequencies in blood mirroring antigen-specific ex vivo Enzyme-Linked ImmunoSpot (ELISpot) responses. Furthermore, up to 67% of candidates could be detected in on-treatment brain tumor tissue and exhibited gene expression signatures overlapping with clonotypes of confirmed specificity to the vaccine antigen.Conclusion ESPEC-SUIT provides unprecedented insight into highly polyclonal, antigen-specific T cell responses and enables discovery of large numbers of TCRs for a given antigen. It represents an efficient, cost-effective and scalable framework for the interrogation of tumor-reactive T cell responses in patients with cancer.",
  "authors": [
    {
      "affiliations": [
        "Clinical Cooperation Unit Neuroimmunology and Brain Tumor Immunology, German Cancer Research Center, Heidelberg, Germany",
        "Immune Monitoring Unit, National Center for Tumor Diseases Heidelberg, Heidelberg, Germany",
        "German Cancer Consortium (DKTK), DKFZ core center, Heidelberg, Germany",
        "Department of Neurology, Medical Faculty Mannheim, Mannheim Center for Translational Neuroscience (MCTN), Heidelberg University, Mannheim, Germany"
      ],
      "name": "Katharina Lindner"
    },
    {
      "affiliations": [
        "Clinical Cooperation Unit Neuroimmunology and Brain Tumor Immunology, German Cancer Research Center, Heidelberg, Germany",
        "Immune Monitoring Unit, National Center for Tumor Diseases Heidelberg, Heidelberg, Germany",
        "German Cancer Consortium (DKTK), DKFZ core center, Heidelberg, Germany",
        "Department of Neurology, Medical Faculty Mannheim, Mannheim Center for Translational Neuroscience (MCTN), Heidelberg University, Mannheim, Germany",
        "Faculty of Biosciences, Heidelberg University, Heidelberg, Germany"
      ],
      "name": "Saskia R Stange"
    },
    {
      "affiliations": [
        "Clinical Cooperation Unit Neuroimmunology and Brain Tumor Immunology, German Cancer Research Center, Heidelberg, Germany",
        "Immune Monitoring Unit, National Center for Tumor Diseases Heidelberg, Heidelberg, Germany",
        "German Cancer Consortium (DKTK), DKFZ core center, Heidelberg, Germany",
        "Department of Neurology, Medical Faculty Mannheim, Mannheim Center for Translational Neuroscience (MCTN), Heidelberg University, Mannheim, Germany",
        "Faculty of Medicine, Heidelberg University, Heidelberg, Germany"
      ],
      "name": "Julian Benedikt Hlawatsch"
    },
    {
      "affiliations": [
        "Clinical Cooperation Unit Neuroimmunology and Brain Tumor Immunology, German Cancer Research Center, Heidelberg, Germany",
        "German Cancer Consortium (DKTK), DKFZ core center, Heidelberg, Germany",
        "Department of Neurology, Medical Faculty Mannheim, Mannheim Center for Translational Neuroscience (MCTN), Heidelberg University, Mannheim, Germany",
        "Faculty of Biosciences, Heidelberg University, Heidelberg, Germany"
      ],
      "name": "Moritz Hamberger"
    },
    {
      "affiliations": [
        "Clinical Cooperation Unit Neuroimmunology and Brain Tumor Immunology, German Cancer Research Center, Heidelberg, Germany",
        "German Cancer Consortium (DKTK), DKFZ core center, Heidelberg, Germany",
        "Department of Neurology, Medical Faculty Mannheim, Mannheim Center for Translational Neuroscience (MCTN), Heidelberg University, Mannheim, Germany"
      ],
      "name": "Tamara Boschert"
    },
    {
      "affiliations": [
        "Clinical Cooperation Unit Neuroimmunology and Brain Tumor Immunology, German Cancer Research Center, Heidelberg, Germany",
        "German Cancer Consortium (DKTK), DKFZ core center, Heidelberg, Germany",
        "Department of Neurology, Medical Faculty Mannheim, Mannheim Center for Translational Neuroscience (MCTN), Heidelberg University, Mannheim, Germany",
        "DKFZ Hector Cancer Institute at the University Medical Center, Mannheim, Germany"
      ],
      "name": "Chin Leng Tan"
    },
    {
      "affiliations": [
        "Clinical Cooperation Unit Neuroimmunology and Brain Tumor Immunology, German Cancer Research Center, Heidelberg, Germany",
        "Immune Monitoring Unit, National Center for Tumor Diseases Heidelberg, Heidelberg, Germany",
        "German Cancer Consortium (DKTK), DKFZ core center, Heidelberg, Germany",
        "Department of Neurology, Medical Faculty Mannheim, Mannheim Center for Translational Neuroscience (MCTN), Heidelberg University, Mannheim, Germany"
      ],
      "name": "Ingrid Hülsmeyer"
    },
    {
      "affiliations": [
        "Clinical Cooperation Unit Neuroimmunology and Brain Tumor Immunology, German Cancer Research Center, Heidelberg, Germany",
        "Immune Monitoring Unit, National Center for Tumor Diseases Heidelberg, Heidelberg, Germany",
        "German Cancer Consortium (DKTK), DKFZ core center, Heidelberg, Germany",
        "Department of Neurology, Medical Faculty Mannheim, Mannheim Center for Translational Neuroscience (MCTN), Heidelberg University, Mannheim, Germany"
      ],
      "name": "Samira Moreen Sachse"
    },
    {
      "affiliations": [
        "Clinical Cooperation Unit Neuroimmunology and Brain Tumor Immunology, German Cancer Research Center, Heidelberg, Germany",
        "Immune Monitoring Unit, National Center for Tumor Diseases Heidelberg, Heidelberg, Germany",
        "German Cancer Consortium (DKTK), DKFZ core center, Heidelberg, Germany",
        "Department of Neurology, Medical Faculty Mannheim, Mannheim Center for Translational Neuroscience (MCTN), Heidelberg University, Mannheim, Germany"
      ],
      "name": "Simone Jünger"
    },
    {
      "affiliations": [
        "Clinical Cooperation Unit Neuroimmunology and Brain Tumor Immunology, German Cancer Research Center, Heidelberg, Germany",
        "Immune Monitoring Unit, National Center for Tumor Diseases Heidelberg, Heidelberg, Germany",
        "German Cancer Consortium (DKTK), DKFZ core center, Heidelberg, Germany",
        "Department of Neurology, Medical Faculty Mannheim, Mannheim Center for Translational Neuroscience (MCTN), Heidelberg University, Mannheim, Germany"
      ],
      "name": "Alexander Ernst"
    },
    {
      "affiliations": [
        "Clinical Cooperation Unit Neuroimmunology and Brain Tumor Immunology, German Cancer Research Center, Heidelberg, Germany",
        "Immune Monitoring Unit, National Center for Tumor Diseases Heidelberg, Heidelberg, Germany",
        "German Cancer Consortium (DKTK), DKFZ core center, Heidelberg, Germany",
        "Department of Neurology, Medical Faculty Mannheim, Mannheim Center for Translational Neuroscience (MCTN), Heidelberg University, Mannheim, Germany"
      ],
      "name": "Olga Głatki"
    },
    {
      "affiliations": [
        "Clinical Cooperation Unit Neuroimmunology and Brain Tumor Immunology, German Cancer Research Center, Heidelberg, Germany",
        "Immune Monitoring Unit, National Center for Tumor Diseases Heidelberg, Heidelberg, Germany",
        "German Cancer Consortium (DKTK), DKFZ core center, Heidelberg, Germany",
        "Department of Neurology, Medical Faculty Mannheim, Mannheim Center for Translational Neuroscience (MCTN), Heidelberg University, Mannheim, Germany"
      ],
      "name": "Sofiya Vinogradova"
    },
    {
      "affiliations": [
        "Clinical Cooperation Unit Neuroimmunology and Brain Tumor Immunology, German Cancer Research Center, Heidelberg, Germany",
        "Immune Monitoring Unit, National Center for Tumor Diseases Heidelberg, Heidelberg, Germany",
        "German Cancer Consortium (DKTK), DKFZ core center, Heidelberg, Germany",
        "Department of Neurology, Medical Faculty Mannheim, Mannheim Center for Translational Neuroscience (MCTN), Heidelberg University, Mannheim, Germany",
        "Faculty of Biosciences, Heidelberg University, Heidelberg, Germany"
      ],
      "name": "Lena Fleischhacker"
    },
    {
      "affiliations": [
        "Research Group GMP & T Cell Therapy, German Cancer Research Center, Heidelberg, Germany"
      ],
      "name": "Stefan B Eichmüller"
    },
    {
      "affiliations": [
        "German Cancer Consortium (DKTK), DKFZ core center, Heidelberg, Germany",
        "Division of Translational Medical Oncology, German Cancer Research Center, Heidelberg, Germany",
        "National Center for Tumor Diseases Heidelberg, Heidelberg, Germany",
        "Institute of Human Genetics, Heidelberg University, Heidelberg, Germany"
      ],
      "name": "Stefan Fröhling"
    },
    {
      "affiliations": [
        "National Center for Tumor Diseases Heidelberg, Heidelberg, Germany",
        "Department of Dermatology, University Hospital Heidelberg Dermatological Clinic Centre, Heidelberg, Germany"
      ],
      "name": "Jessica C Hassel"
    },
    {
      "affiliations": [
        "National Center for Tumor Diseases Heidelberg, Heidelberg, Germany",
        "Clinical Cooperation Unit Applied Tumor Immunity, German Cancer Research Center, Heidelberg, Germany",
        "Department of Medical Oncology, Medical Faculty Heidelberg, Heidelberg University, Heidelberg University Hospital, Heidelberg, Germany"
      ],
      "name": "Inka Zörnig"
    },
    {
      "affiliations": [
        "Clinical Cooperation Unit Applied Tumor Immunity, German Cancer Research Center, Heidelberg, Germany"
      ],
      "name": "Yanhong Lyu"
    },
    {
      "affiliations": [
        "Clinical Cooperation Unit Applied Tumor Immunity, German Cancer Research Center, Heidelberg, Germany"
      ],
      "name": "Zhiqin Huang"
    },
    {
      "affiliations": [
        "National Center for Tumor Diseases Heidelberg, Heidelberg, Germany",
        "Clinical Cooperation Unit Applied Tumor Immunity, German Cancer Research Center, Heidelberg, Germany",
        "Department of Medical Oncology, Medical Faculty Heidelberg, Heidelberg University, Heidelberg University Hospital, Heidelberg, Germany"
      ],
      "name": "Dirk Jäger"
    },
    {
      "affiliations": [
        "National Center for Tumor Diseases Heidelberg, Heidelberg, Germany",
        "Clinical Cooperation Unit Applied Tumor Immunity, German Cancer Research Center, Heidelberg, Germany",
        "Department of Medical Oncology, Medical Faculty Heidelberg, Heidelberg University, Heidelberg University Hospital, Heidelberg, Germany"
      ],
      "name": "Frank Momburg"
    },
    {
      "affiliations": [
        "National Center for Tumor Diseases Heidelberg, Heidelberg, Germany",
        "Clinical Cooperation Unit Applied Tumor Immunity, German Cancer Research Center, Heidelberg, Germany",
        "Department of Medical Oncology, Medical Faculty Heidelberg, Heidelberg University, Heidelberg University Hospital, Heidelberg, Germany"
      ],
      "name": "Marten Meyer"
    },
    {
      "affiliations": [
        "Clinical Cooperation Unit Neuroimmunology and Brain Tumor Immunology, German Cancer Research Center, Heidelberg, Germany",
        "German Cancer Consortium (DKTK), DKFZ core center, Heidelberg, Germany",
        "Department of Neurology, Medical Faculty Mannheim, Mannheim Center for Translational Neuroscience (MCTN), Heidelberg University, Mannheim, Germany"
      ],
      "name": "Lukas Bunse"
    },
    {
      "affiliations": [
        "Clinical Cooperation Unit Neuroimmunology and Brain Tumor Immunology, German Cancer Research Center, Heidelberg, Germany",
        "German Cancer Consortium (DKTK), DKFZ core center, Heidelberg, Germany",
        "Department of Neurology, Medical Faculty Mannheim, Mannheim Center for Translational Neuroscience (MCTN), Heidelberg University, Mannheim, Germany"
      ],
      "name": "Edward William Green"
    },
    {
      "affiliations": [
        "Clinical Cooperation Unit Neuroimmunology and Brain Tumor Immunology, German Cancer Research Center, Heidelberg, Germany",
        "Immune Monitoring Unit, National Center for Tumor Diseases Heidelberg, Heidelberg, Germany",
        "German Cancer Consortium (DKTK), DKFZ core center, Heidelberg, Germany",
        "Department of Neurology, Medical Faculty Mannheim, Mannheim Center for Translational Neuroscience (MCTN), Heidelberg University, Mannheim, Germany",
        "DKFZ Hector Cancer Institute at the University Medical Center, Mannheim, Germany",
        "Helmholtz Institute for Translational Oncology Mainz (HI-TRON Mainz) – a Helmholtz Institute of the DKFZ, Mainz, Germany"
      ],
      "name": "Michael Platten"
    },
    {
      "affiliations": [
        "Clinical Cooperation Unit Neuroimmunology and Brain Tumor Immunology, German Cancer Research Center, Heidelberg, Germany",
        "Immune Monitoring Unit, National Center for Tumor Diseases Heidelberg, Heidelberg, Germany",
        "German Cancer Consortium (DKTK), DKFZ core center, Heidelberg, Germany",
        "Department of Neurology, Medical Faculty Mannheim, Mannheim Center for Translational Neuroscience (MCTN), Heidelberg University, Mannheim, Germany"
      ],
      "name": "Isabel Poschke"
    }
  ],
  "title": "ESPEC-SUIT: a versatile and robust platform to identify and track antigen-specific T cell receptors in patients with cancer",
  "uid": "21835ce4-9423-5a57-9767-9d15af0251cc"
}