T cells are a key part of the adaptive immune system, capable of recognizing and eliminating cancer cells. This ability has led to major advances in T cell-based immunotherapies. In 2023, certain T cell receptors (TCRs) from tumor-infiltrating lymphocytes were found to recognize multiple tumor antigens. Notably, the MEL8 TCR clone was associated with long-term remission in a melanoma patient.
The MEL8 TCR clone can recognize not only melanoma but also other cancers, such as breast, prostate, and pancreatic cancers. It targets three tumor antigens that share a common amino acid motif, allowing these “multipronged” T cells to detect multiple antigens at once. This broad recognition gives them an advantage over conventional T cells and makes them promising candidates for future immunotherapies.
While the multipronged nature of MEL8 TCRs has been described, their sensitivity to its tumor associated antigens is still unclear. In my project, I aimed to investigate the sensitivity of this TCR using Glass Supported Lipid Bilayer (SLB) experiments. These SLBs mimic the membrane of antigen-presenting cells, allowing us to observe TCR-pMHC interactions in real time within a living T cell. Using high-resolution, single-cell quantitative microscopy, we can study how T cells engage with antigens at the immunological synapse.
To generate the MEL8 T-cell receptor for real-time observation, we engineered this receptor using orthotopic T-cell receptor (TCR) replacement (OTR) via CRISPR/Cas9, preserving near-physiological function.
To generate the MEL8 T-cell receptor for real-time observation, we engineered this receptor using orthotopic T-cell receptor (TCR) replacement (OTR) via CRISPR/Cas9, preserving near-physiological function.
With orthotopic TCR replacement, T cells display TCR regulation patterns that closely resemble those of natural T cells during antigen stimulation, including both TCR expression levels and pMHC multimer staining patterns. By inserting the transgenic TCR directly into the endogenous TCR gene locus, orthotopic replacement significantly reduces mispairing. Its main advantage lies in enabling long-term physiological regulation and generating safer, more controlled T-cell products. Moreover, even low numbers of these T cells can be effective in clinical models, such as donor-lymphocyte infusions or tumor-infiltrating lymphocyte therapies.
For the lipid bilayer, which serves as an antigen-presenting cell, we produced pMHC complexes with the respective tumor-associated antigens to ultimately embed them into the planar glass-supported lipid bilayer. To load the cancer epitope of choice onto the MHC molecule, we used UV-mediated ligand exchange.
The conditional MHC complex is irradiated with UV light at 350 nm. The photolabile peptide ligand cleaves into two fragments that dissociate from the MHC binding groove. The resulting empty MHC class I complex has a short half-life at 37°C if not stabilized by a bound peptide. By displacing the conditional ligand, MHC complexes loaded with an epitope of choice can be generated in a high-throughput manner.
We employed cell-free differential scanning fluorimetry (DSF) to measure the thermal stability of destabilized or reassembled MHC-I complexes. The minima of the curves specify the melting temperature (Tm) of the HLA-peptide complexes. Tm values well above 37°C indicate strong peptide binding to MHC-I at physiological temperatures, whereas values around 37°C correlate only with weak and below 36°C with absent binding.
Differential Scanning Fluorimetry (DSF) is a thermal shift assay that measures protein stability as temperature increases. A fluorescent dye binds to hydrophobic regions that are normally buried inside proteins and become exposed during unfolding. As more protein unfolds with rising temperature, fluorescence increases, reaching a peak when the protein is fully denatured.