Single-cell immune repertoire sequencing can provide invaluable information about the response of the adaptive immune system to disease and therapy. However, existing approaches for pairing T-cell or B-cell receptor (TCR/BCR) sequences at the single-cell level are still relatively low throughput and costly. These limitations are particularly acute for methods that aim to combine receptor sequences with complementary cell-type information, as defined by cell-surface protein expression. Here, we propose to develop and validate an affordable high- throughput technology for simultaneous pairing of TCRs and determination of cell state based on cell surface protein expression. The proposed approach builds on Split Pool Ligation-based Transcriptome sequencing (SPLiT-seq), our recently developed single-cell sequencing method that is based on combinatorial indexing. The combinatorial indexing approach uses intact fixed cells or nuclei as ‘reaction vessels’ to physically partition nucleic acids of interest and bypass the need for microfluidic cell isolation. Cells undergo multiple rounds of splitting, barcoding, and re-pooling, generating cell-specific barcode combinations for each tagged molecule. Here, we will extend the SPLiT-seq workflow for use with DNA-barcoded antibodies and for the specific detection of TCR transcripts. We will demonstrate scaling up of the technology to sequence millions of cells in a single experiment – at least an order of magnitude greater throughput than currently available approaches. By combining combinatorial indexing with targeted detection of cell surface markers, which can provide high resolution cellular profiles with limited sequencing reads, and similarly targeted detection of adaptive immune repertoires, which we will also optimize for sequencing efficiency, we will overcome practical limitations imposed by sequencing cost. Throughput, accuracy and costs of this approach will be quantitatively compared with flow cytometry, mass cytometry, bulk TCR sequencing and the 10x Genomics single-cell sequencing platform. An ability to assess TCR sequences and cellular profiles on a large scale will permit tracking of clonal relationships and corresponding cellular profiles of T or B cells infiltrating tumors and in peripheral blood. This analysis will provide mechanistic insights into the roles of T and B cells in tumor-specific responses and allow for identification of therapeutically relevant T and B cell receptors. To demonstrate utility for the study of cancer, we will apply this approach to study paired human tumor and blood sample T cells from cancer patients undergoing tumor- resection surgeries.

This research seeks to develop an affordable and high-throughput method for multi-modal single-cell immunophenotyping. The technology will be applied for studying T cell responses in human cancer.