Figure 1

Schematic representation of the Rapid TCR: Epitope Ranker (RAPTER) assay. RAPTER uses hashing reagents and T cell activation-induced markers (AIMs) to select and characterize antigen-specific T cells. (A) In this schematic, PBMC are distributed evenly across assay wells. Desired T cell stimuli (protein, peptides, RNA, etc.) are added to individual wells in an arrayed format and cultured with cells for the desired assay time. (B) Following stimulation, T cells from each well are labeled with unique hash tag oligonucleotide (HTO)-labeled reagents, e.g., anti-CD2 antibodies, to zip-code the assay well location of all T cells and, hence, their corresponding antigen exposure. (C) HTO-labeled samples are pooled and stained with desired flow cytometry and scRNA-SEQ reagents, including fluorescently tagged antibodies and CITE-seq antibodies. (D) Antigen-specific T cells are isolated by fluorescence activated cell sorting (FACS) using T cell AIMs for functional enrichment. (E) Sorted cells are loaded into the 10X Genomics single cell Chromium partitioner and 5′ sequencing libraries for the transcriptome, HTO, TCR-seq, and, if desired, CITE-seq, and oligo-multimers are generated for HT sequencing. (F) Individual HTO assignments are bioinformatically demultiplexed to link individual T cell TCR and phenotype with specific antigen reactivity. (G) Antigen-specific TCR sequences are further resolved by evaluating frequency, clone size, and level of CD137/4-1BB expression.