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.

Keywords: PD-1/PD-L1 immune checkpoint pathway; antigen-specific; bioassay; flow cytometry; immune checkpoint inhibition.

Figure 1. Efficiency of PD-1 transduction, TCR mRNA electroporation and cryopreservation of 2D3 cells

(A) Representative flow cytometry T-cell receptor (TCRαβ) and programmed death-1 (PD-1) protein surface expression profiles and corresponding isotype controls of non-transduced PD-1⁻ 2D3 and PD-1-transduced (PD-1⁺) 2D3 cells 24 hours after TCR mRNA electroporation (fresh; 10-14 replicates) and after thawing of TCR mRNA-electroporated cells (cryo; 6 replicates). (B) Percentage viability and recovery upon TCR mRNA electroporation of PD-1⁻ and PD-1⁺ 2D3 cells. Data information: in (B), means are depicted. ^*P ≤ 0.05 (Student’s t-test). Abbreviations: cryo, transfected effector cells were cryopreserved prior to co-culture; fresh, stimulator cells were co-cultured immediately following transfection; ns, not significant; PD-1, programmed death-1 protein; TCR, T-cell receptor.

Figure 2. Validation of antigen-specific TCR function of transfected 2D3 and PD-1⁺ 2D3 cells

(A–B) Activation profiles of freshly used or thawed WT1-specific TCR mRNA-electroporated PD-1⁻ and PD-1⁺ 2D3 cells left unstimulated (-) versus 24 hours co-culture with unloaded (T2^-pept) and WT1 peptide-pulsed (T2^+pept) stimulator cells at a ratio of 2:1. Comparable results were obtained with gp100 TCR-positive PD-1⁻ and PD-1⁺ 2D3 cells. (A) Representative example of TCR activation-mediated eGFP expression within the viable CD8⁺ cell population as assessed with flow cytometry (freshly used WT1 TCR mRNA-electroporated PD-1⁺ 2D3 cells). (B) The left graph shows the mean percentage (± SEM) WT1-specific TCR activation-mediated eGFP expression from 2–8 replicate experiments. The right panel depicts the mean amount (± SEM) of secreted granzyme B determined with ELISA in cell-free 24-hour culture supernatant of 10⁵ effector cells for 2–4 replicate experiments. Data information: ^*P ≤ 0.05, ^**P < 0.01, ^***P < 0.001 (one-way ANOVA). Abbreviations: cryo, transfected effector cells were cryopreserved prior to co-culture; eGFP, enhanced green fluorescent protein; fresh, stimulator cells were co-cultured immediately following transfection; PD-1, programmed death-1 protein; SEM, standard error of mean; TCR, T-cell receptor; WT1, Wilms’ tumor 1.

Figure 3. TCR⁺PD-1⁺ 2D3 cells as a model assay for evaluation of involvement of PD-1 signaling in cell-mediated antigen-specific T-cell activation

(A–C) WT1 (A, B) and gp100 (A, C) specific T-cell activation expressed as percentage viable CD8⁺ eGFP⁺ PD-1⁻ and PD-1⁺ 2D3 cells (± SEM) after 24 hours co-culture with different PD-L1⁺ stimulator cells. Neutralizing antibody against PD-1 (αPD-1; A, B; 15 µg/mL in WT1 model (A, B), 5 µg/mL in gp100 model (A)) or PD-L1 (αPD-L1; C) was added to cells 1 hour prior to co-culture to verify PD-1-mediated signaling, where indicated. (A) PD-1-dependent stimulating capacity of two differently generated peptide-pulsed mature monocyte-derived dendritic cells (WT1 (4 DC donors tested in two independent experiments); gp100 (4 DC donors in four independent experiments)). (B)Impact of induced PD-L1 expression on peptide-pulsed THP-1 leukemic cells on WT1-specific T-cell activation (6 replicate experiments). (C) gp100-specific PD-1⁺ T cell-activating capacity of peptide-pulsed wild-type or stably transduced PD-L1⁺ MCF-7 breast carcinoma cells (4 replicate experiments). Data information: in A, the horizontal line represents the median percentage eGFP expression (n = 4). ^*P ≤ 0.05, ^**P < 0.01, ^***P < 0.001 (repeated measures one-way ANOVA with Bonferroni post-hoc test). Abbreviations: eGFP, enhanced green fluorescent protein; gp100, glycoprotein 100; PD-1, programmed death 1 protein; PD-L1, programmed death-ligand 1; TCR, T-cell receptor; WT1, Wilms’ tumor 1.