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2026BIOMED003.pdf
Doctoral thesis

Toward functional TCR-pMHC dataset generation : microfluidic T-cell receptor repertoire grafting and primary-cell antigen-specificity screening workflows

  • 2026

PhD: Università della Svizzera italiana

T-cell receptor (TCR)
Peptide–MHC complexes (pMHC)
TCR antigen specificity
TCR–pMHC dataset generation
TCR repertoire grafting
Paired TCRαβ sequencing
Droplet microfluidics
Primary-cell antigen-specificity screening
Cell–cell interaction assays
Functional TCR validation
English A major challenge in T-cell immunology is that T-cell receptor (TCR) sequences can now be obtained at high throughput, whereas their cognate peptide-MHC (pMHC) specificities remain much harder to define experimentally. This gap limits both mechanistic studies of adaptive immunity and the generation of high-quality datasets for TCR-specificity prediction. The aim of this thesis was therefore to develop experimental strategies to preserve, recover, and functionally test antigen-reactive human TCR repertoires, with the broader goal of contributing to future TCR-pMHC dataset generation. The work followed two complementary routes. The first focused on Microfludic Grafting of T cell Receptors (MIGOT), a droplet-microfluidic workflow designed to capture natively paired TCRαβ sequences from single primary T cells by in-droplet reverse transcription and overlap-extension PCR, clone them into molecular libraries, and re-express them in TCR-knockout Jurkat NFAT reporter cells. Several key steps of this workflow were successfully established, including single-cell encapsulation, paired TCRαβ recovery, plasmid and lentiviral library construction, and receptor expression in Jurkat cells. However, end-to-end functional validation remained limited. Although grafted TCR receptors could support signaling after polyclonal stimulation, antigen-specific reactivation at library level was weak or absent across multiple validation systems. Long-read sequencing of defined libraries further revealed substantial repertoire distortion, including strong clonal skewing and likely loss of pairing fidelity, indicating that the current MIGOT workflow does not yet preserve repertoire structure robustly enough for reliable library-scale antigen discovery. The second route developed complementary primary-cell-based workflows using peptide- pulsed antigen-presenting cells to directly enrich antigen-reactive healthy donor CD4+ and CD8+ T cells. These experiments showed that peptide-reactive populations could be generated, sorted, expanded, and rechallenged, but also highlighted major limitations, including donor scarcity, background proliferation, and unstable preservation of weakly enriched repertoires. Overall, this thesis defines both the promise and the current bottlenecks of two experimental strategies for linking TCR sequence to antigen specificity and provides a methodological foundation for future improvements toward scalable, functionally grounded TCR-pMHC mapping.
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  • English
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Medicine
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green
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https://n2t.net/ark:/12658/srd1336317
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