{
  "abstract": "Background T cell in vitro sensitization (IVS) involving autologous dendritic cells (DCs) and T cells can induce tumor-specific T cells from peripheral blood lymphocytes (PBLs). However, the conditions of IVS generating polyclonal cytotoxic T lymphocytes (CTLs) for adoptive cell therapy against highly heterogenous solid tumors remain to be established. Alpha-type-1 polarized dendritic cells (αDC1), a type of DCs induced by mediators of innate responses against viruses, are particularly effective in converting non-cytolytic CD8+ T cell into effector CTLs. Here we tested the feasibility of generating large numbers of multi-epitope-specific T cells with potent effector functions using αDC1-assisted IVS.Methods Monocyte-derived DCs were matured with different stimuli to generate αDC1 or previously used standard DC (sDC). IVS was performed by coculturing T cells with autologous DC loaded with tumor peptides, viral peptides, or apoptotic tumor cells. 4-1BB enrichment and rapid expansion protocol (REP) were performed to enrich and expand antigen-specific T cells from bulk in vitro sensitized T cells. Flow cytometry, single-cell secretome, ELISA, IFNγ ELISPOT, and LDH cytotoxicity assay were used to determine T cell functionality. TCRvb sequencing was used to analyze TCR clonotypes. DCs’ antigen processing and presenting functions was analyzed by RNA sequencing and immunoprecipitation-mass spectrometry.Results Using MART-1 as a model of tumor-associated antigen (TAA), we observed that αDC1 induces de novo activation of TAA-specific CTLs from heathy donors with robust effector functions. Following with 4-1BB enrichment and REP, αDC1-sensitized TAA-specific CTLs were thoroughly purified and expanded, while retained potent antigen-specific cytotoxicity. The aDC1-induced T cell product included strongly enhanced poly-functionality, compared to sDC-sensitized T cells. Similar advantages of αDC1-sensitized T cells were observed when αDC1 or sDC were loaded with CMV peptide pool or apoptotic tumor cells, reflecting specialized antigen processing and presenting machinery and (co)stimulatory functions of αDC1. Accordingly, αDC-sensitized CTLs showed strongly enhanced poly-clonality, as determined by broad spectrum of their TCR variants, compared to sDC-sensitized CTLs.Conclusions αDC1-assisted IVS is a feasible tool to generate polyclonal, multi-epitope-specific CTLs with elevated poly-functionality from PBLs, as potential alternative to TIL therapy of solid tumors, especially in the particularly challenging group of patients with low numbers of TILs.Ethics Approval Peripheral blood cones (byproduct of platelets collection) were obtained from healthy volunteers under the Roswell Park Comprehensive Cancer Center (RPCCC) Institutional Review Board (IRB)-approved protocol 163222, or purchased from StemCell Technologies Inc, Vancouver, Canada. Surgically resected samples from ovarian cancer patients were collected at RPCCC following RPCCC IRB-approved protocol 130120. All procedures were performed in compliance with relevant laws and institutional guidelines. Informed consent was obtained from all donors.",
  "authors": [
    {
      "affiliations": [
        "Roswell Park Comprehensive Cancer Center, Buffalo, NY, USA"
      ],
      "name": "Bowen Dong"
    },
    {
      "affiliations": [
        "Roswell Park Comprehensive Cancer Center, Buffalo, NY, USA"
      ],
      "name": "Duna Massillon"
    },
    {
      "affiliations": [
        "University of Pittsburgh School of Medicine, Pittsburgh, PA, USA"
      ],
      "name": "Pawel Kalinski"
    }
  ],
  "title": "321 Generation of multi-epitope-specific T cells for adoptive cell therapy for solid tumors using polarized DCs",
  "uid": "e242a272-dadc-52ad-a57b-0aa4d34c657e"
}