{
  "abstract": "Background Emerging evidence suggests a correlation between CD8 + T cell-tumor cell proximity and immunotherapy response. However, it is unknown whether these cells can be captured as functional clusters from clinical samples. In defined human co-cultures, tumor antigen-recognizing T cells outcompeted unmatched T cells in forming clusters with tumor cells, prompting us to investigate whether this feature could be used to isolate tumor-reactive T cells directly from cancer specimens.Methods We analyzed a cohort of 21 melanoma metastases from various anatomical sites. After mild digestion, these samples were analyzed by conventional and imaging flow cytometry. Fluorescence-activated cell sorting was used to isolate heterotypic T cell clusters for single-cell RNA- and T cell receptor (TCR)-sequencing and functional experiments. For latter experiments, single T cells and T cells from clusters were expanded using a rapid expansion protocol, after which they were used to treat autologous melanoma cells ex vivo and in mice.Results By conventional and imaging flow cytometry, we found that from 21/21 human melanoma metastases, we were able to isolate heterotypic clusters, comprising CD8 + T cells interacting with one or more tumor cells and/or antigen-presenting cells (APCs). Single-cell RNA-sequencing revealed that CD8+ T cells from clusters were enriched for tumor-reactive and exhausted gene signatures. Integration with TCR-sequencing showed increased clonality of clustered T cells, indicative of expansion. Tumor cells and various APCs that had conjugated with these T cells exhibited specific enriched cell states, which were linked to distinct patterns of cell-cell communication. TCR-matched T cells showed more exhaustion and co-modulation when conjugated to APCs than to tumor cells. Compared to single T cells, CD8+ T cells that were expanded from clusters ex vivo exerted on average 9-fold increased killing activity towards autologous melanomas, which was accompanied by enhanced cytokine production. Upon adoptive cell transfer (ACT) into mice, T cells from clusters showed significantly enhanced patient-derived xenograft (PDX) tumor control associated with more T cell infiltration and activation. The frequency of CD8+tumor infiltrating lymphocytes (TIL) with a high cluster signature score at baseline was predictive of TIL-therapy response.Conclusions Together, these results demonstrate that tumor-reactive CD8 + T cells are enriched in functional clusters with tumor cells and/or APCs and that they can be isolated and expanded from clinical samples. Typically excluded during single cell sorting by flow cytometry, these distinct heterotypic CD8+ T cell clusters serve as a valuable source amenable to deciphering functional tumor-immune cell interactions, while they may also be therapeutically explored.Ethics Approval The study was approved by the Medical Ethical Review Board of the NKI-AvL (under studies B16MEL, IRBm23-029) and executed in compliance with the ethical regulations. All patients provided prior informed consent to research usage of material not required for diagnostics. Animal work procedures performed in NSG mice were approved by the animal experimental committee (Instantie voor Dierenwelzijn) of the NKI according to Dutch law and performed in accordance with ethical and procedural guidelines established by the NKI and Dutch legislation. Animal experiments in (hIL2-)NOG mice were conducted in conformity with EU directive 2010/63 (regional animal ethics committee of Gothenburg approvals #4684/23).",
  "authors": [
    {
      "affiliations": [
        "Oncode Institute, Netherlands Cancer Institute, Amsterdam, Netherlands"
      ],
      "name": "Sofía Ibáñez-Molero"
    },
    {
      "affiliations": [
        "Netherlands Cancer Institute, Amsterdam, Netherlands"
      ],
      "name": "Johanna Veldman"
    },
    {
      "affiliations": [
        "Oncode Institute, Netherlands Cancer Institute, Amsterdam, Netherlands"
      ],
      "name": "Juan Simón-Nieto"
    },
    {
      "affiliations": [
        "Netherlands Cancer Institute, Amsterdam, Netherlands"
      ],
      "name": "Joleen JH Traets"
    },
    {
      "affiliations": [
        "Oncode Institute, Netherlands Cancer Institute, Amsterdam, Netherlands"
      ],
      "name": "Austin George"
    },
    {
      "affiliations": [
        "Oncode Institute, Netherlands Cancer Institute, Amsterdam, Netherlands"
      ],
      "name": "Kelly Hoefakker"
    },
    {
      "affiliations": [
        "Oncode Institute, Netherlands Cancer Institute, Amsterdam, Netherlands"
      ],
      "name": "Anita Karomi"
    },
    {
      "affiliations": [
        "Netherlands Cancer Institute, Amsterdam, Netherlands"
      ],
      "name": "Rolf Harkes"
    },
    {
      "affiliations": [
        "Netherlands Cancer Institute, Amsterdam, Netherlands"
      ],
      "name": "Bram van den Broek"
    },
    {
      "affiliations": [
        "Oncode Institute, Netherlands Cancer Institute, Amsterdam, Netherlands"
      ],
      "name": "Su Min Pack"
    },
    {
      "affiliations": [
        "Oncode Institute, Netherlands Cancer Institute, Amsterdam, Netherlands"
      ],
      "name": "Liselotte Tas"
    },
    {
      "affiliations": [
        "Oncode Institute, Netherlands Cancer Institute, Amsterdam, Netherlands"
      ],
      "name": "Nils L Visser"
    },
    {
      "affiliations": [
        "Oncode Institute, Netherlands Cancer Institute, Amsterdam, Netherlands"
      ],
      "name": "Susan E van Hal-van Veen"
    },
    {
      "affiliations": [
        "Oncode Institute, Netherlands Cancer Institute, Amsterdam, Netherlands"
      ],
      "name": "Paula Alóndiga-Mérida"
    },
    {
      "affiliations": [
        "Netherlands Cancer Institute, Amsterdam, Netherlands"
      ],
      "name": "Maartje Alkemade"
    },
    {
      "affiliations": [
        "Netherlands Cancer Institute, Amsterdam, Netherlands"
      ],
      "name": "Iris M Seignette"
    },
    {
      "affiliations": [
        "Netherlands Cancer Institute, Amsterdam, Netherlands"
      ],
      "name": "Renaud Tissier"
    },
    {
      "affiliations": [
        "Netherlands Cancer Institute, Amsterdam, Netherlands"
      ],
      "name": "Marja Nieuwland"
    },
    {
      "affiliations": [
        "Netherlands Cancer Institute, Amsterdam, Netherlands"
      ],
      "name": "Martijn van Baalen"
    },
    {
      "affiliations": [
        "VIB Center for Cancer Biology, KU Leuven, Leuven, Belgium"
      ],
      "name": "Joanna Poźniak"
    },
    {
      "affiliations": [
        "Sanquin, Amsterdam, Netherlands"
      ],
      "name": "Erik Mul"
    },
    {
      "affiliations": [
        "Sanquin, Amsterdam, Netherlands"
      ],
      "name": "Simon Tol"
    },
    {
      "affiliations": [
        "Sahlgrenska Center for Cancer Research, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden"
      ],
      "name": "Sofia Stenqvist"
    },
    {
      "affiliations": [
        "Sahlgrenska Center for Cancer Research, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden",
        "Harry Perkins Institute of Medical Research and University of Western Australia, Perth, Australia"
      ],
      "name": "Lisa M Nilsson"
    },
    {
      "affiliations": [
        "Sahlgrenska Center for Cancer Research, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden",
        "Harry Perkins Institute of Medical Research and University of Western Australia, Perth, Australia"
      ],
      "name": "Jonas A Nilsson"
    },
    {
      "affiliations": [
        "Netherlands Cancer Institute, Amsterdam, Netherlands"
      ],
      "name": "John Haanen"
    },
    {
      "affiliations": [
        "Netherlands Cancer Institute, Amsterdam, Netherlands"
      ],
      "name": "Winan J van Houdt"
    },
    {
      "affiliations": [
        "Oncode Institute, Netherlands Cancer Institute, Amsterdam, Netherlands"
      ],
      "name": "Daniel S Peeper"
    }
  ],
  "title": "745 Heterotypic CD8 T cell clusters isolated from clinical samples are biologically distinct and enriched for antitumor activity",
  "uid": "45197d1d-b773-59d1-84fa-48af542f355a"
}