Scalable TCR synthesis and screening enables antigen reactivity mapping in vitiligo

Gagliione, S. A.; Mukkamala, R. S.; Krishna, C.; Smith, B. E.; Wadsworth, M. H.; Jelinsky, S. A.; Perez, C. R.; Schmidt-Hong, L.; Katz, E. L.; Gellatly, K. J.; Ali, L. R.; Shen, J.; Holec, P. V.; Zhao, Q. H.; Chan, A. O.; Xu, E. J.; Kravarik, K. M.; Guzova, J. A.; Dobson, C. S.; Singh, H.; Garber, M.; Dougan, M.; Dougan, S. K.; Harris, J. E.; Winkler, A.; Birnbaum, M. E.

doi:10.1101/2025.04.24.649726

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bioRxiv · 10.1101/2025.04.24.649726

Scalable TCR synthesis and screening enables antigen reactivity mapping in vitiligo

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Abstract

T cell receptors (TCRs) mediate antigen recognition in adaptive immunity, yet large-scale mapping of TCR-antigen interactions remains a major challenge. Current approaches to synthesize and functionally screen TCRs remain technically complex and limited in throughput. We introduce a modular strategy, TCRAFT, to rapidly construct tens of thousands of TCRs for <$1 each while maintaining TCR-{beta} pairing with >99% accuracy. We integrate this approach with a high-throughput antigen discovery platform to enable library-on-library TCR-antigen screening. We reconstruct and screen 3,808 TCRs from vitiligo lesions, linking TCR specificity to transcriptional phenotypes for antigen-reactive T cells. To demonstrate scalability, we synthesize and screen 30,810 TCRs from donors with pancreatic ductal adenocarcinoma to capture antigen-specific TCRs. This workflow reduces the cost and complexity of large-scale TCR screening, enabling the expansion of the known landscape of antigen-specific TCRs in vitiligo with a method that can be readily extended to other immunological applications.

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Gagliione, S. A., Mukkamala, R. S., Krishna, C., Smith, B. E., Wadsworth, M. H., Jelinsky, S. A., Perez, C. R., Schmidt-Hong, L., Katz, E. L., Gellatly, K. J., Ali, L. R., Shen, J., Holec, P. V., Zhao, Q. H., Chan, A. O., Xu, E. J., Kravarik, K. M., Guzova, J. A., Dobson, C. S., Singh, H., Garber, M., Dougan, M., Dougan, S. K., Harris, J. E., Winkler, A., Birnbaum, M. E.. 2025-04-26. Scalable TCR synthesis and screening enables antigen reactivity mapping in vitiligo. https://doi.org/10.1101/2025.04.24.649726

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