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@ARTICLE{Ehrenfried:289913,
      author       = {A. R. Ehrenfried$^*$ and S. Zens$^*$ and L. K. Steffens$^*$
                      and H. Kehm$^*$ and A. Paul$^*$ and C. Lauenstein$^*$ and M.
                      Volkmar and I. Poschke$^*$ and Z. Meng$^*$ and R.
                      Offringa$^*$},
      title        = {{T}-{C}ell-{B}ased {P}latform for {F}unctional {S}creening
                      of {T}-{C}ell {R}eceptors {I}dentified in {S}ingle-{C}ell
                      {RNA} {S}equencing {D}ata {S}ets of {T}umor-{I}nfiltrating
                      {T}-{C}ells.},
      journal      = {Bio-protocol},
      volume       = {14},
      number       = {8},
      issn         = {2331-8325},
      address      = {Sunnyvale, CA},
      publisher    = {bio-protocol.org},
      reportid     = {DKFZ-2024-00905},
      pages        = {e4972},
      year         = {2024},
      note         = {2024 Apr 20;14(8):e4972 / HI-TRON},
      abstract     = {The advent of single-cell RNA sequencing (scRNAseq) has
                      enabled in-depth gene expression analysis of several
                      thousand cells isolated from tissues. We recently reported
                      the application of scRNAseq toward the dissection of the
                      tumor-infiltrating T-cell repertoire in human pancreatic
                      cancer samples. In this study, we demonstrated that combined
                      whole transcriptome and T-cell receptor (TCR) sequencing
                      provides an effective way to identify tumor-reactive TCR
                      clonotypes on the basis of gene expression signatures. An
                      important aspect in this respect was the experimental
                      validation of TCR-mediated anti-tumor reactivity by means of
                      an in vitro functional assay, which is the subject of the
                      present protocol. This assay involves the transient
                      transfection of mRNA gene constructs encoding TCRα/β pairs
                      into a well-defined human T-cell line, followed by
                      co-cultivation with the tumor cells of interest and
                      detection of T-cell activation by flow cytometry. Due to the
                      high transfectability and the low background reactivity of
                      the mock-transfected T-cell line to a wide variety of tumor
                      cells, this assay offers a highly robust and versatile
                      platform for the functional screening of large numbers of
                      TCR clonotypes as identified in scRNAseq data sets. Whereas
                      the assay was initially developed to test TCRs of human
                      origin, it was more recently also applied successfully for
                      the screening of TCRs of murine origin. Key features •
                      Efficient functional screening of-and discrimination
                      between-TCRs isolated from tumor-reactive vs. bystander
                      T-cell clones. • Applicable to TCRs from CD8+ and CD4+
                      tumor-infiltrating T-cells originating from patient-derived
                      tumor samples and syngeneic mouse tumor models. • Rapid
                      flow cytometric detection of T-cell activation by means of
                      TNFα and CD107a expression after a 5 h T-cell/tumor cell
                      co-cultivation.},
      keywords     = {Anti-tumor reactivity (Other) / T-cell activation (Other) /
                      T-cell receptor (Other) / Tumor-infiltrating lymphocytes
                      (Other) / mRNA transfection (Other)},
      cin          = {D200 / D170},
      ddc          = {570},
      cid          = {I:(DE-He78)D200-20160331 / I:(DE-He78)D170-20160331},
      pnm          = {314 - Immunologie und Krebs (POF4-314)},
      pid          = {G:(DE-HGF)POF4-314},
      typ          = {PUB:(DE-HGF)16},
      pubmed       = {pmid:38686347},
      pmc          = {pmc:PMC11056003},
      doi          = {10.21769/BioProtoc.4972},
      url          = {https://inrepo02.dkfz.de/record/289913},
}