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

Abstract

The functional avidity of T-cell receptor (TCR)-engineered T cells towards their cognate epitope plays a crucial role in successfully targeting and killing tumor cells expressing the tumor-associated antigen (TAA). When evaluating in vitro functional T-cell avidity, an important aspect that is often neglected is the antigen-presenting cell (APC) used in the assay. Cell-based models for antigen-presentation, such as tumor cell lines, represent a valid alternative to autologous APCs due to their availability, off-the-shelf capabilities, and the broad range of possibilities for modification via DNA or messenger RNA (mRNA) transfection. To find a valuable model APC for in vitro validation of TAA Wilms' tumor 1 (WT1)-specific TCRs, we tested four different WT1 peptide-pulsed HLA-A2+ tumor cell lines commonly used in T-cell stimulation assays. We found the multiple myeloma cell line U266 to be a suitable model APC to evaluate differences in mean functional avidity (EC50) values of transgenic TCRs following transfection in 2D3 Jurkat T cells. Next, to assess the dose-dependent antigen-specific responsiveness of WT1 TCR-engineered 2D3 T cells to endogenously processed epitopes, we electroporated U266 cells with different amounts of full-length antigen WT1 mRNA. Finally, we analyzed the functional avidity of WT1 TCR-transfected primary CD8 T cells towards WT1 mRNA-electroporated U266 cells. In this study, we demonstrate that both the APC and the antigen loading method (peptide pulsing versus full-length mRNA transfection) to analyze T-cell functional avidity have a significant impact on the EC50 values of a given TCR. For rapid assessment of the functional avidity of a cloned TCR towards its endogenously processed MHC I-restricted epitope, we showcase that the TAA mRNA-transfected U266 cell line is a suitable and versatile model APC.

Keywords: T-cell functional avidity; Wilms’ tumor 1; cancer immunotherapy; full-length antigen mRNA electroporation; model antigen-presenting cell.

Figure 1

HLA-A2 and WT1 expression on four model antigen-presenting cell (APC) lines. Histograms (relative to mode) show the surface expression of HLA-A2 (upper panel) and the intracellular expression of WT1 (lower panel) of T2 (orange), U266 (red), Raji-A2 (green), and K562-A2 (blue) cell lines. HLA-A2 or WT1 expression (filled histograms) and isotype control (black line). The table shows HLA-A2 delta median fluorescence intensity (dMFI) values and percentage of HLA-A2 positive cells minus isotype staining (upper histograms) or percentages of WT1 positive cells minus isotype staining (lower histograms) for each cell line. HLA-A2, human leukocyte antigen A*02:01; WT1, Wilms’ tumor 1 protein.

Figure 2

Epitope-specific T-cell activation by four model APC lines. Epitope-specific TCR activation was measured by expression of EGFP after WT1_37–45 (A,C) or WT1_126–134 (B,D) peptide-specific TCR-transfected 2D3 cells were cultured for 18–22 h with model APCs T2, U266, Raji-A2 or K562-A2 cells that were pulsed with decreasing concentrations of WT1 peptide. Control depicts unstimulated 2D3 cells only. Graphs show the results of three to five independent replicates, showing (A,B) mean % (± SEM) of EGFP positive cells and (C,D) % of maximal EGFP expression (± SEM). (A,B) Data were analyzed using one-way ANOVA followed by Dunnett’s post hoc test (comparing to non-peptide pulsed cells). EC50, the concentration of WT1 peptide at which 50% of the maximal EGFP expression is reached. *, p < 0.05; **, p < 0.01; ***, p < 0.001; and ****, p < 0.0001.

Figure 3

Epitope-specific TCR-engineered 2D3 cells can recognize full-length antigen WT1 mRNA-electroporated U266 cells in a dose-dependent manner. (A,B) Intracellular expression of WT1 is shown for U266 cells 24 h after electroporation with increasing amounts of WT1 mRNA per 5 × 10⁶ U266 cells. (C,D) 2D3 cells were electroporated with WT1_37–45- or WT1_126–134-specific TCR mRNAs. Specific activation was detected by NFAT-promoted EGFP expression in 2D3 cells after 18–22 h co-culture with U266 cells electroporated with increasing amounts of WT1 mRNA. Graphs show the mean percentage of WT1⁺ U266 cells ± SEM (A), the median fluorescence intensity (MFI) of U266 for WT1 expression ± SEM (B), the percentage of maximal EGFP expression ± SEM (C) and EC50, the amount of WT1 mRNA at which 50% of the maximal EGFP expression is reached (D) of 3–4 independent replicates. (A,B) One-way ANOVA followed by Tukey’s post hoc test. (C) One-way ANOVA followed by Dunnett’s post hoc test (comparing to mock-electroporated cells). *, p < 0.05; **, p < 0.01; and ****, p < 0.0001.

Figure 4

Analysis of functional avidity of WT1 epitope-specific primary CD8 T cells using WT1 peptide-pulsed and WT1 mRNA-electroporated U266 cells. Surface expression of both CD69 and CD137 activation markers was measured on WT1.37 (triangles) and WT1.126 (squares) peptide-specific DSE-engineered primary CD8 T cells 24 h after co-culture with U266 cells that were either pulsed with decreasing (A,B) concentrations of WT1_37–45 or WT1_126–134 peptide, or WT1 mRNA-electroporated (C,D). T cells only condition was used as a control. Graphs show mean ± SEM of % CD69/CD137 double positive CD8 T cells (A,C) or % of maximal CD69/CD137 expression ± SEM (B,D) for 6–8 donors. EC50, the concentration of WT1 peptides or amounts of electroporated WT1 mRNA at which 50% of the maximal upregulation of CD69 and CD137 activation markers is reached. (A,C) One-way ANOVA followed by Dunnett’s post hoc test (comparing to non-peptide pulsed or mock-electroporated cells). *, p < 0.05.