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000289913 1001_ $$0P:(DE-HGF)0$$aEhrenfried, Aaron Rodriguez$$b0$$eFirst author
000289913 245__ $$aT-Cell-Based Platform for Functional Screening of T-Cell Receptors Identified in Single-Cell RNA Sequencing Data Sets of Tumor-Infiltrating T-Cells.
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000289913 520__ $$aThe 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.
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000289913 650_7 $$2Other$$aAnti-tumor reactivity
000289913 650_7 $$2Other$$aT-cell activation
000289913 650_7 $$2Other$$aT-cell receptor
000289913 650_7 $$2Other$$aTumor-infiltrating lymphocytes
000289913 650_7 $$2Other$$amRNA transfection
000289913 7001_ $$0P:(DE-He78)17b0a821061db042fe4b6eff379d1ba6$$aZens, Stefan$$b1$$udkfz
000289913 7001_ $$0P:(DE-He78)fec6ef4ffbd92f019f904ba6809ec1b3$$aSteffens, Laura Katharina$$b2$$udkfz
000289913 7001_ $$0P:(DE-He78)37e53d333a7b0896397cbaa885b046b1$$aKehm, Hannes$$b3$$udkfz
000289913 7001_ $$0P:(DE-He78)2b298456f6596fd1f3fbb23c521ccb62$$aPaul, Alina$$b4$$udkfz
000289913 7001_ $$0P:(DE-He78)67614eacdbe6fe4404a46d2f8d01ac52$$aLauenstein, Claudia$$b5$$udkfz
000289913 7001_ $$aVolkmar, Michael$$b6
000289913 7001_ $$0P:(DE-He78)e9c55f46b4b06cf835834ee7e3e00db8$$aPoschke, Isabel$$b7$$udkfz
000289913 7001_ $$0P:(DE-He78)a7ebe54d5cb6cfa15c663da9ce1f9ab2$$aMeng, Zibo$$b8$$udkfz
000289913 7001_ $$0P:(DE-He78)81ae96953d6149e4307057d71a190019$$aOffringa, Rienk$$b9$$eLast author$$udkfz
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