Myasthenia gravis (MG) is an autoantibody-mediated autoimmune disorder of the neuromuscular junction. A small subset of patients (<10%) with MG, have autoantibodies targeting muscle-specific tyrosine kinase (MuSK). MuSK MG patients respond well to CD20-mediated B cell depletion therapy (BCDT); most achieve complete stable remission. However, relapse often occurs. To further understand the immunomechanisms underlying relapse, we studied autoantibody-producing B cells over the course of BCDT. We developed a fluorescently labeled antigen to enrich for MuSK-specific B cells, which was validated with a novel Nalm6 cell line engineered to express a human MuSK-specific B cell receptor. B cells (≅ 2.6 million) from 12 different samples collected from nine MuSK MG patients were screened for MuSK specificity. We successfully isolated two MuSK-specific IgG4 subclass-expressing plasmablasts from two of these patients, who were experiencing a relapse after a BCDT-induced remission. Human recombinant MuSK mAbs were then generated to validate binding specificity and characterize their molecular properties. Both mAbs were strong MuSK binders, they recognized the Ig1-like domain of MuSK, and showed pathogenic capacity when tested in an acetylcholine receptor (AChR) clustering assay. The presence of persistent clonal relatives of these MuSK-specific B cell clones was investigated through B cell receptor repertoire tracing of 63,977 unique clones derived from longitudinal samples collected from these two patients. Clonal variants were detected at multiple timepoints spanning more than five years and reemerged after BCDT-mediated remission, predating disease relapse by several months. These findings demonstrate that a reservoir of rare pathogenic MuSK autoantibody-expressing B cell clones survive BCDT and reemerge into circulation prior to manifestation of clinical relapse. Overall, this study provides both a mechanistic understanding of MuSK MG relapse and a valuable candidate biomarker for relapse prediction.
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.
Long-read sequencing technologies such as isoform sequencing (Iso-Seq) 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 four 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 Iso-Seq and parallel RNA-Seq analyses. Many novel polyadenylated transcript isoforms, supported by both Iso-Seq 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 (GEO) database (GSE202327, GSE202328, GSE202329) as both raw and processed files to serve as long-read reference transcriptomes for future studies of human circulating lymphocytes.
Current Adaptive Immune Receptor Repertoire Sequencing (AIRR-seq) strategies resolve immunoglobulin (IG) variable region transcripts with limited resolution of the constant region. Here we present a novel near full-length AIRR-seq (FLAIRR-Seq) method that utilizes targeted heavy chain amplification by rapid amplification of cDNA ends (RACE), combined with single molecule, real-time (SMRT) sequencing to generate highly accurate (>Q40, 99.99%), immunoglobulin heavy chain transcripts. FLAIRR-seq was benchmarked by comparing V, D, and J gene usage, complementarity-determining region 3 (CDR3) length, and the extent of total variable gene somatic hypermutation to matched datasets generated with standard RACE AIRR-seq and full-length isoform sequencing methods. Together these data demonstrate robust, unbiased FLAIRR-seq performance using RNA samples derived from peripheral blood mononuclear cells, purified B cells, and whole blood, which recapitulated results generated by commonly used methods, while resolving novel constant region features. FLAIRR-seq data provides, for the first time, simultaneous, single-molecule characterization of variable and constant region genes and alleles, allele-resolved subisotype definition, and identification of class-switch recombination within a clonal lineage. FLAIRR-seq, in conjunction with IGHC genotyping, of the IgM- and IgG repertoires from 10 individuals resulted in the identification of 32 unique IGHC alleles; 28/32 (87%) that were not represented by sequences curated in the IMmunoGeneTics Information System (IMGT) database. Together, these data demonstrate the capabilities of FLAIRR-seq to characterize IGHV, D, J and C gene diversity for the most comprehensive view of B cell repertoires to date.
Myasthenia gravis (MG) is an autoantibody-mediated autoimmune disorder of the neuromuscular junction. In a subset of patients with MG, muscle-specific tyrosine kinase (MuSK) autoantibodies are present. MuSK MG patients respond well to CD20-mediated B cell depletion therapy (BCDT); most achieve complete stable remission. However, relapse often occurs. To further understand the immunomechanisms underlying relapse, we sought to study autoantibody-producing B cells over the course of BCDT. We developed a fluorescently labeled antigen to enrich for MuSK-specific B cells, which was validated with a novel Nalm6 cell line engineered to express a human MuSK-specific B cell receptor. B cells (≅ 2.6 million) from 12 different samples collected from nine MuSK MG patients were screened for MuSK specificity. We successfully isolated two MuSK-specific IgG4 subclass-expressing plasmablasts from two of these patients, who were experiencing a relapse after a BCDT-induced remission. Human recombinant MuSK mAbs were then generated to validate binding specificity and characterize their molecular properties. Both mAbs were strong MuSK binders, they recognized the Ig1-like domain of MuSK, and showed pathogenic capacity when tested in an AChR clustering assay. The presence of persistent clonal relatives of these MuSK-specific B cell clones was investigated through B cell receptor repertoire tracing of 63,977 unique clones derived from longitudinal samples collected from these two patients. Clonal variants were detected at multiple timepoints spanning more than five years and reemerged after BCDT-mediated remission, predating disease relapse by several months. These results indicate that a reservoir of rare pathogenic MuSK autoantibody-expressing B cell clones survive BCDT and reemerge into circulation prior to manifestation of clinical relapse. This study provides both a mechanistic understanding of MuSK MG relapse and a valuable candidate biomarker for relapse prediction.
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