A versatile T cell-based assay to assess therapeutic antigen-specific PD-1-targeted approaches

Abstract

Blockade of programmed cell death protein 1 (PD-1) immune checkpoint receptor signaling is an established standard treatment for many types of cancer and indications are expanding. Successful clinical trials using monoclonal antibodies targeting PD-1 signaling have boosted preclinical research, encouraging development of novel therapeutics. Standardized assays to evaluate their bioactivity, however, remain restricted. The robust bioassays available all lack antigen-specificity. Here, we developed an antigen-specific, short-term and high-throughput T cell assay with versatile readout possibilities. A genetically modified T cell receptor (TCR)-deficient T cell line was stably transduced with PD-1. Transfection with messenger RNA encoding a TCR of interest and subsequent overnight stimulation with antigen-presenting cells, results in eGFP-positive and granzyme B-producing T cells for single cell or bulk analysis. Control antigen-presenting cells induced reproducible high antigen-specific eGFP and granzyme B expression. Upon PD-1 interaction, ligand-positive antigen-presenting immune or tumor cells elicited significantly lower eGFP and granzyme B expression, which could be restored by anti-PD-(L)1 blocking antibodies. This convenient cell-based assay shows a valuable tool for translational and clinical research on antigen-specific checkpoint-targeted therapy approaches.

Rapid Assessment of Functional Avidity of Tumor-Specific T Cell Receptors Using an Antigen-Presenting Tumor Cell Line Electroporated with Full-Length Tumor Antigen mRNA

“…To analyze the WT1 peptide-presenting capacity of the four model APC candidates, we used an in-house developed T-cell model assay, based on TCR-deficient CD8 + Jurkat 2D3 cells that are electroporated with TCRαβ-encoding mRNAs and express enhanced green fluorescent protein (EGFP) via nuclear factor of activated T cells (NFAT) upon antigen-specific TCR triggering [28,29]. Transgenic TCR expression for two HLA-A2-restricted TCRs directed against two epitopes of the WT1 protein, WT1 37−45 and WT1 126−134 (WT1.37 and WT1.126 TCR, respectively), was maximal for both TCRs 24 h after electroporation (92.75 ± 1.5% WT1.37 TCR + and 94.48 ± 0.67% WT1.126 TCR + 2D3 cells; Supplementary Figure S1A).…”

“…The TCRαβ-deficient, CD8αβ and NFAT-EGFP stably-transfected T cell acute leukemia 2D3 cell line [28,29] Blood samples of healthy anonymous donors were purchased from the Blood Service of the Flemish Red Cross (Mechelen, Belgium) following the approval by the Ethics Committee of the Antwerp University Hospital and the University of Antwerp (reference number 16/35/357). Peripheral blood mononuclear cells were isolated by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare, Diegem, Belgium), and CD8 T cells were selected using human CD8 magnetic microbeads for magnetic-activated cell sorting (MACS) according to the manufacturer's instructions (Miltenyi Biotec, Leiden, The Netherlands).…”

“…The cloning of WT1-specific TCR genes, generation of the pST1 DNA plasmids containing the TCR constructs and generation of WT1-specific TCR mRNA by in vitro transcription (IVT) were performed as previously described [28,29]. Clinical-grade codon-optimized Sig-DC-LAMP WT1 mRNA encoding isoform D of WT1 [21] was purchased from eTheRNA immunotherapies (Niel, Belgium).…”

Efficient and Non-genotoxic RNA-Based Engineering of Human T Cells Using Tumor-Specific T Cell Receptors With Minimal TCR Mispairing

“…Samples were measured on a FACScan flow cytometer (BD Biosciences). The human acute T cell leukemia cell lines Jurkat Clone E6-1 (ATCC, TIB-152) and 2D3 ( 46 ) were maintained in Roswell Park Memorial Institute 1640 (RPMI) culture medium (Gibco Invitrogen) supplemented with 10% fetal bovine serum (FBS; Gibco Invitrogen). 2D3 cells were generated from TCRαβ-deficient Jurkat 76 cells by transduction with human CD8 alpha-E2A-CD8 beta construct (both Jurkat 76 cells and CD8-encoding plasmid were kind gifts of Prof. Hans Stauss, Institute of Immunity and Transplantation, University College London, London, UK) and with a plasmid vector containing the enhanced green fluorescent protein ( EGFP ) gene under the control of a nuclear factor of activated T-cell (NFAT) promoter (NFAT-EGFP plasmid kindly provided by Prof. Takashi Saito, Riken Research Center for Allergy and Immunology, Yokohama, Japan).…”

The Emerging Role of In Vitro-Transcribed mRNA in Adoptive T Cell Immunotherapy

“…Several groups have used mRNA electroporation to monitor T cell response to immune-based therapy. 62,63 Transient expression of molecules has been shown to stimulate and expand T cells, 64 and future work will investigate how additional localized factors may improve T cell function both during expansion and therapeutic application. Beyond adoptive T cell therapy, mRNA is now also becoming an option for other forms of immunotherapy such as bispecific T cell engagers (BiTE).…”