Barcoding intracellular reverse transcription enables high-throughput phenotype-coupled T cell receptor analyses.

Assays linking cellular phenotypes with T cell or B cell antigen receptor sequences are crucial for characterizing adaptive immune responses. Existing methodologies are limited by low sample throughput and high cost. Here, we present INtraCEllular Reverse Transcription with Sorting and sequencing (INCERTS), an approach that combines molecular indexing of receptor repertoires within intact cells and fluorescence-activated cell sorting (FACS). We demonstrate that INCERTS enables efficient processing of millions of cells from pooled human peripheral blood mononuclear cell (PBMC) samples while retaining robust association between T cell receptor (TCR) sequences and cellular phenotypes. We used INCERTS to discover antigen-specific TCRs from patients with cancer immunized with a novel mutant KRAS peptide vaccine. After ex vivo stimulation, 28 uniquely barcoded samples were pooled prior to FACS into peptide-reactive and non-reactive CD4

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Déclaration de conflit d'intérêts

Declaration of interests H.B.L. is a founder of Infinity Bio, Portal Bioscience, Alchemab, and ImmuneID. H.B.L., S.J., and J.M.M. are listed as inventors on a patent application related to this work. E.J.F. is on the scientific advisory board of Resistance Bio and is a consultant for Mestag Therapeutics and Merck. E.M.J. reports other support from Abmeta, other support from Adventris, personal fees from Achilles, personal fees from DragonFly, personal fees from Parker Institute and CPRIT, personal fees from Surge and HDTbio, grants from Lustgarten, grants from Genentech, personal fees from Mestag, personal fees from Medical Home Group, grants from BMS, and grants from Break Through Cancer outside the submitted work. E.M.J. is the Dana and Albert “Cubby” Broccoli Professor of Oncology. N.Z. receives research support from BMS. N.Z. is consultant for Genentech. N.Z. is a consultant for and receives other support from Adventris Pharmaceuticals. N.Z. is a scientific advisor for RyboDyn, Inc. N.Z. is co-inventor on filed patent applications related to cancer vaccines. N.A. is a paid consultant for Tempus, Mirati, and QED. N.A. receives institutional funding from Agios, Inc., Array, Atlas, Bayer HealthCare, BMS, Celgene, Debio, Eli Lilly and Company, EMD Serono, Incyte Corporation, Intensity, Merck & Co., Inc., and Taiho Pharmaceuticals Co., Ltd. N.A. participates on advisory boards for Incyte, QED, and Glaxo Smith Kline.