FLAIRR-Seq: A Method for Single-Molecule Resolution of Near Full-Length Antibody H Chain Repertoires.

Current Adaptive Immune Receptor Repertoire sequencing (AIRR-seq) using short-read sequencing strategies resolve expressed Ab transcripts with limited resolution of the C region. In this article, we present the near-full-length AIRR-seq (FLAIRR-seq) method that uses targeted amplification by 5' RACE, combined with single-molecule, real-time sequencing to generate highly accurate (99.99%) human Ab H chain transcripts. FLAIRR-seq was benchmarked by comparing H chain V (IGHV), D (IGHD), and J (IGHJ) gene usage, complementarity-determining region 3 length, and somatic hypermutation to matched datasets generated with standard 5' RACE AIRR-seq using short-read sequencing and full-length isoform sequencing. Together, these data demonstrate robust FLAIRR-seq performance using RNA samples derived from PBMCs, purified B cells, and whole blood, which recapitulated results generated by commonly used methods, while additionally resolving H chain gene features not documented in IMGT at the time of submission. FLAIRR-seq data provide, for the first time, to our knowledge, simultaneous single-molecule characterization of IGHV, IGHD, IGHJ, and IGHC region genes and alleles, allele-resolved subisotype definition, and high-resolution identification of class switch recombination within a clonal lineage. In conjunction with genomic sequencing and genotyping of IGHC genes, FLAIRR-seq of the IgM and IgG repertoires from 10 individuals resulted in the identification of 32 unique IGHC alleles, 28 (87%) of which were previously uncharacterized. Together, these data demonstrate the capabilities of FLAIRR-seq to characterize IGHV, IGHD, IGHJ, and IGHC gene diversity for the most comprehensive view of bulk-expressed Ab repertoires to date.

An Unexpected Role for Cell Density Rather than IgM in Cell-Surface Display of the Fc Receptor for IgM on Human Lymphocytes.

The Fc receptor for IgM, FcMR, is unusual in that it is preferentially expressed by cells of the adaptive immune system. It is, moreover, the only constitutively expressed Fc receptor on human T cells. Efforts to decipher the normal functions of FcMR have been complicated by species-specific expression patterns in lymphocytes from mice (B cells) versus humans (B, NK, and T cells). In human cells, FcMR cell-surface expression has been reported to be low at baseline ex vivo, with one suggested contribution being ligand-induced internalization by serum IgM. Indeed, preincubation overnight in IgM-free culture medium is recommended for studies of FcMR because surface display is increased under these conditions. We investigated FcMR display on human lymphocytes in PBMCs and found that, surprisingly, cell-surface FcMR was unaffected by IgM abundance and was instead downregulated in high-cell density cultures by a yet undefined mechanism. We further found that ex vivo processing of whole blood decreased surface FcMR, supporting the idea that FcMR expression is likely to be greater on circulating lymphocytes than previously appreciated. Collectively, these findings prompt new predictions of where and when FcMR might be available for functional interactions in vivo.

Reference long-read isoform-aware transcriptomes of 4 human peripheral blood lymphocyte subsets.

Long-read sequencing technologies such as isoform sequencing can generate highly accurate sequences of full-length mRNA transcript isoforms. Such long-read transcriptomics may be especially useful in investigations of lymphocyte functional plasticity as it relates to human health and disease. However, no long-read isoform-aware reference transcriptomes of human circulating lymphocytes are readily available despite being valuable as benchmarks in a variety of transcriptomic studies. To begin to fill this gap, we purified 4 lymphocyte populations (CD4+ T, CD8+ T, NK, and Pan B cells) from the peripheral blood of a healthy male donor and obtained high-quality RNA (RIN > 8) for isoform sequencing and parallel RNA-Seq analyses. Many novel polyadenylated transcript isoforms, supported by both isoform sequencing and RNA-Seq data, were identified within each sample. The datasets met several metrics of high quality and have been deposited to the Gene Expression Omnibus database (GSE202327, GSE202328, GSE202329) as both raw and processed files to serve as long-read reference transcriptomes for future studies of human circulating lymphocytes.