T-Cell-Based Platform for Functional Screening of T-Cell Receptors Identified in Single-Cell RNA Sequencing Data Sets of Tumor-Infiltrating T-Cells

Aaron Rodriguez Ehrenfried; Stefan Zens; Laura K Steffens; Hannes Kehm; Alina Paul; Claudia Lauenstein; Michael Volkmar; Isabel Poschke; Zibo Meng; Rienk Offringa

doi:10.21769/BioProtoc.4972

T-Cell-Based Platform for Functional Screening of T-Cell Receptors Identified in Single-Cell RNA Sequencing Data Sets of Tumor-Infiltrating T-Cells

Bio Protoc. 2024 Apr 20;14(8):e4972. doi: 10.21769/BioProtoc.4972.

Authors

Aaron Rodriguez Ehrenfried^{1

2

3}, Stefan Zens^{1

3}, Laura K Steffens^{1

3}, Hannes Kehm^{1

3}, Alina Paul^{1

3}, Claudia Lauenstein¹, Michael Volkmar², Isabel Poschke^{4

5}, Zibo Meng^{1

6

7}, Rienk Offringa^{1

6

7}

Affiliations

¹ Division of Molecular Oncology of Gastrointestinal Tumors, German Cancer Research Center (DKFZ), Heidelberg, Germany.
² Helmholtz-Institute for Translational Oncology by DKFZ (HI-TRON), Mainz, Germany.
³ Faculty of Biosciences, Heidelberg University, Heidelberg, Germany.
⁴ Clinical Cooperation Unit Neuroimmunology and Brain Tumor Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany.
⁵ Immune Monitoring Unit, National Center for Tumor Diseases (NCT), Heidelberg, Germany.
⁶ Department of General Visceral and Transplantation Surgery, University Hospital Heidelberg, Heidelberg, Germany.
⁷ Sino-German Laboratory of Personalized Medicine for Pancreatic Cancer, Union Hospital, Tongji Medical College, Wuhan, China.

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; T-cell activation; T-cell receptor; Tumor-infiltrating lymphocytes; mRNA transfection.