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.
Highlights
T-cell receptor (TCR) gene therapy is a promising strategy in cancer immunotherapy, capitalizing on the use of TCR-engineered T cells targeting tumor-associated antigens (TAAs) expressed by cancer cells [1]
MRNA would be needed to reach upregulation of CD69 and CD137 in half of the maximal percentage of cells (Figure 4D), which is in line with the results using the 2D3 cell line (6.54 μg for Wilms’ tumor 1 (WT1).37 TCR+ ; Figure 3D). These findings show that evaluation of T-cell functional avidity with WT1 peptide-pulsed or WT1 messenger RNA (mRNA)-electroporated U266 cells remains constant for the TCRs analyzed regardless of the source of T cells, and that this system can help to distinguish TCRs that will respond to epitope densities of naturally processed WT1 protein
Our study demonstrates the relevance of comparing the antigen-presenting cell (APC) used in T-cell assays and the influence they may have when evaluating T-cell functional avidity
Summary
T-cell receptor (TCR) gene therapy is a promising strategy in cancer immunotherapy, capitalizing on the use of TCR-engineered T cells targeting tumor-associated antigens (TAAs) expressed by cancer cells [1]. T cell, defined as functional avidity, is a measurement of its effector response towards a particular surface density of the epitope [4]. It is evaluated in vitro by analyzing the response of T cells in peptide titration experiments. The mean functional avidity, usually described by EC50, represents the peptide dose at which half-maximal activation of the T-cell population is reached. This value depends on the affinity and avidity of the TCR for its cognate peptide-MHC (pMHC) ligand and, it varies between different T-cell clones or TCR-engineered T cells. The analysis of antigen-specific T-cell responses is vital at a clinical and research level to obtain the best TCRs for adoptive T-cell therapies [7,8]
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