T cells are a central component of the adaptive immune system and play a critical role in recognizing and eliminating cancer cells, forming the basis of T cell–based immunotherapies. In 2023, certain T cell receptors (TCRs) derived from tumor-infiltrating lymphocytes were shown to recognize multiple tumor antigens, challenging the long-standing paradigm that a single TCR recognizes a single antigen. Notably, the MEL8 TCR clone was among these multipronged receptors and was associated with long-term remission in a melanoma patient.
The MEL8 TCR clone recognizes not only melanoma but also other cancers, including breast, prostate, and pancreatic cancers. It targets three distinct tumor antigens that share a common amino acid motif, enabling these “multipronged” T cells to detect multiple antigens simultaneously. This broad antigen recognition provides an advantage over conventional T cells and highlights their potential for future immunotherapies.
Although the multipronged nature of the MEL8 TCR has been described, its sensitivity to tumor-associated antigens remains unclear. In my project, I aimed to investigate the antigen sensitivity of the MEL8 TCR using glass-supported lipid bilayer (SLB)–based single-cell, quantitative microscopy experiments.
T cells can recognize antigens presented as peptide–MHC complexes on antigen-presenting cells with remarkable precision. To better understand the molecular and cellular factors underlying this sensitivity in a physiological context, imaging-based approaches allow the examination of TCR–pMHC interactions at the immunological synapse. In my project, I aimed to use functionalized glass-supported lipid bilayers (SLBs) that mimic antigen-presenting cell membranes as a platform for monitoring TCR engagement in real time within living T cells.
To enable real-time observation, we engineered the MEL8 T cell receptor using orthotopic TCR replacement (OTR) via CRISPR/Cas9, preserving near-physiological function.
T cells can recognize antigens presented as peptide–MHC complexes on antigen-presenting cells with remarkable precision. To better understand the molecular and cellular factors underlying this sensitivity in a physiological context, imaging-based approaches allow the examination of TCR–pMHC interactions at the immunological synapse. In my project, I aimed to use functionalized glass-supported lipid bilayers (SLBs) that mimic antigen-presenting cell membranes as a platform for monitoring TCR engagement in real time within living T cells.
To enable real-time observation, we engineered the MEL8 T cell receptor using orthotopic 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.
For the construction of the lipid bilayer, which functions as an artificial antigen-presenting cell, peptide–MHC complexes bearing tumor-associated antigens are generated and incorporated into planar glass-supported lipid bilayers. Loading of the selected cancer epitope onto the MHC molecule is achieved through UV-mediated ligand exchange.
The conditional MHC complex is exposed to UV light at 350 nm, causing the photolabile peptide to break into two fragments that then dissociate from the MHC binding groove. Without a stabilizing peptide, the resulting empty MHC class I complex is short-lived at 37°C. This approach allows high-throughput generation of MHC complexes loaded with a chosen epitope by replacing the conditional ligand.
The thermal stability of destabilized or reconstituted MHC class I complexes assessed using cell-free differential scanning fluorimetry (DSF). The melting temperature (Tm) of HLA–peptide complexes is determined from the minima of the DSF curves. Tm values well above 37 °C indicate strong peptide binding at physiological temperatures, whereas values around 37 °C correspond to weak binding and values below 36 °C suggest 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.
Although my project had some limitations, it provides important insights. The multipronged T-cell receptor and its beneficial cross-reactivity were previously unknown. By examining rare patients with advanced solid cancers who achieved complete and durable remission, we gain the opportunity to identify unique T cells with superior therapeutic potential. Working with these exceptional samples allows us to explore how such T cells recognize and eliminate cancer cells.
Applying advanced engineering and microscopy techniques can reveal how these unique cells recognize tumor antigens, as well as their sensitivity and specificity. Understanding these mechanisms is crucial for guiding the development of more effective next generation immunotherapies.
Applying advanced engineering and microscopy techniques can reveal how these unique cells recognize tumor antigens, as well as their sensitivity and specificity. Understanding these mechanisms is crucial for guiding the development of more effective next generation immunotherapies.