Analysis of IGH allele content in a sample group of rheumatoid arthritis patients demonstrates unrevealed population heterogeneity.

Immunoglobulin heavy chain (IGH) germline gene variations influence the B cell receptor repertoire, with resulting biological consequences such as shaping our response to infections and altering disease susceptibilities. However, the lack of information on polymorphism frequencies in the IGH loci at the population level makes association studies challenging. Here, we genotyped a pilot group of 30 individuals with rheumatoid arthritis (RA) to examine IGH allele content and frequencies in this group. Eight novel IGHV alleles and one novel IGHJ allele were identified in the study. 15 cases were haplotypable using heterozygous IGHJ6 or IGHD anchors. One variant, IGHV4-34*01_S0742, was found in three out of 30 cases and included a single nucleotide change resulting in a non-canonical recombination signal sequence (RSS) heptamer. This variant allele, shown by haplotype analysis to be non-expressed, was also found in three out of 30 healthy controls and matched a single nucleotide polymorphism (SNP) described in the 1000 Genomes Project (1KGP) collection with frequencies that varied between population groups. Our finding of previously unreported alleles in a relatively small group of individuals with RA illustrates the need for baseline information about IG allelic frequencies in targeted study groups in preparation for future analysis of these genes in disease association studies.

Integrated single cell and spatial transcriptomics reveal autoreactive differentiated B cells in joints of early rheumatoid arthritis.

B cells play a significant role in established Rheumatoid Arthritis (RA). However, it is unclear to what extent differentiated B cells are present in joint tissue already at the onset of disease. Here, we studied synovial biopsies (n = 8) captured from untreated patients at time of diagnosis. 3414 index-sorted B cells underwent RNA sequencing and paired tissue pieces were subjected to spatial transcriptomics (n = 4). We performed extensive bioinformatics analyses to dissect the local B cell composition. Select plasma cell immunoglobulin sequences were expressed as monoclonal antibodies and tested by ELISA. Memory and plasma cells were found irrespective of autoantibody status of the patients. Double negative memory B cells were prominent, but did not display a distinct transcriptional profile. The tissue architecture implicate both local B cell maturation via T cell help and plasma cell survival niches with a strong CXCL12-CXCR4 axis. The immunoglobulin sequence analyses revealed clonality between the memory B and plasma cell pools further supporting local maturation. One of the plasma cell-derived antibodies displayed citrulline autoreactivity, demonstrating local autoreactive plasma cell differentiation in joint biopsies captured from untreated early RA. Hence, plasma cell niches are not a consequence of chronic inflammation, but are already present at the time of diagnosis.

High-Quality Library Preparation for NGS-Based Immunoglobulin Germline Gene Inference and Repertoire Expression Analysis.

Next generation sequencing (NGS) of immunoglobulin (Ig) repertoires (Rep-seq) enables examination of the adaptive immune system at an unprecedented level. Applications include studies of expressed repertoires, gene usage, somatic hypermutation levels, Ig lineage tracing and identification of genetic variation within the Ig loci through inference methods. All these applications require starting libraries that allow the generation of sequence data with low error rate and optimal representation of the expressed repertoire. Here, we provide detailed protocols for the production of libraries suitable for human Ig germline gene inference and Ig repertoire studies. Various parameters used in the process were tested in order to demonstrate factors that are critical to obtain high quality libraries. We demonstrate an improved 5'RACE technique that reduces the length constraints of Illumina MiSeq based Rep-seq analysis but allows for the acquisition of sequences upstream of Ig V genes, useful for primer design. We then describe a 5' multiplex method for library preparation, which yields full length V(D)J sequences suitable for genotype identification and novel gene inference. We provide comprehensive sets of primers targeting IGHV, IGKV, and IGLV genes. Using the optimized protocol, we produced IgM, IgG, IgK, and IgL libraries and analyzed them using the germline inference tool IgDiscover to identify expressed germline V alleles. This process additionally uncovered three IGHV, one IGKV, and six IGLV novel alleles in a single individual, which are absent from the IMGT reference database, highlighting the need for further study of Ig genetic variation. The library generation protocols presented here enable a robust means of analyzing expressed Ig repertoires, identifying novel alleles and producing individualized germline gene databases from humans.

Autoimmune reactivity to malondialdehyde adducts in systemic lupus erythematosus is associated with disease activity and nephritis.

BACKGROUND: Immunoglobulin M (IgM) autoreactivity to malondialdehyde (MDA) protein modifications is part of the natural antibody repertoire in health and may have beneficial functions. In contrast, IgG anti-MDA are increased in chronic inflammation and autoimmunity and may instead have pathogenic properties. METHODS: Herein, we investigated serum IgG anti-MDA levels by enzyme-linked immunosorbent assay (ELISA) in 398 systemic lupus erythematosus (SLE) patients in the Swedish Karolinska SLE cohort and compared these to findings in 225 US SLE patients from New York University and Johns Hopkins University. RESULTS: In two independent cohorts, IgG anti-MDA levels correlated positively with disease activity by the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI; p < 0.0001, Spearman R = 0.3). Meta-analysis found an odds ratio of 2.7 (confidence interval (CI) 1.9-3.9; p < 0.0001) for high anti-MDA IgG levels with active disease (SLEDAI ≥ 6). Furthermore, IgG anti-MDA correlated directly with erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), soluble tumor necrosis factor receptors (sTNFR-1, sTNFR-2), and vascular cell adhesion molecule 1 (VCAM-1) measurements, and inversely with complement factors (C1q, C2, C3, C4). Importantly, IgG anti-MDA levels were significantly elevated in SLE patients with active nephritis (p = 0.0005) and correlated with cystatin C estimated glomerular filtration rate and albuminuria. CONCLUSIONS: Elevated IgG anti-MDA in SLE patients was associated with high disease activity, with active lupus nephritis, and with biomarkers of systemic inflammation. This natural antibody reactivity may have potential prognostic utility, and may also actively contribute to pathogenesis.

Depressed serum IgM levels in SLE are restricted to defined subgroups.

Natural IgM autoantibodies have been proposed to convey protection from autoimmune pathogenesis. Herein, we investigated the IgM responses in 396 systemic lupus erythematosus (SLE) patients, divided into subgroups based on distinct autoantibody profiles. Depressed IgM levels were more common in SLE than in matched population controls. Strikingly, an autoreactivity profile defined by IgG anti-Ro/La was associated with reduced levels of specific natural IgM targeting phosphorylcholine (PC) antigens and malondialdehyde (MDA) modified-protein, as well as total IgM, while no differences were detected in SLE patients with an autoreactivity profile defined by anti-cardiolipin/ß2glycoprotein-I. We also observed an association of reduced IgM levels with the HLA-DRB1*03 allelic variant among SLE patients and controls. Associations of low IgM anti-PC with cardiovascular disease were primarily found in patients without antiphospholipid antibodies. These studies further highlight the clinical relevance of depressed IgM. Our results suggest that low IgM levels in SLE patients reflect immunological and genetic differences between SLE subgroups.

Autoreactivity to malondialdehyde-modifications in rheumatoid arthritis is linked to disease activity and synovial pathogenesis.

Oxidation-associated malondialdehyde (MDA) modification of proteins can generate immunogenic neo-epitopes that are recognized by autoantibodies. In health, IgM antibodies to MDA-adducts are part of the natural antibody pool, while elevated levels of IgG anti-MDA antibodies are associated with inflammatory and autoimmune conditions. Yet, in human autoimmune disease IgG anti-MDA responses have not been well characterized and their potential contribution to disease pathogenesis is not known. Here, we investigate MDA-modifications and anti-MDA-modified protein autoreactivity in rheumatoid arthritis (RA). While RA is primarily associated with autoreactivity to citrullinated antigens, we also observed increases in serum IgG anti-MDA in RA patients compared to controls. IgG anti-MDA levels significantly correlated with disease activity by DAS28-ESR and serum TNF-alpha, IL-6, and CRP. Mass spectrometry analysis of RA synovial tissue identified MDA-modified proteins and revealed shared peptides between MDA-modified and citrullinated actin and vimentin. Furthermore, anti-MDA autoreactivity among synovial B cells was discovered when investigating recombinant monoclonal antibodies (mAbs) cloned from single B cells, and 3.5% of memory B cells and 2.3% of plasma cells were found to be anti-MDA positive. Several clones were highly specific for MDA-modification with no cross-reactivity to other antigen modifications such as citrullination, carbamylation or 4-HNE-carbonylation. The mAbs recognized MDA-adducts in a variety of proteins including albumin, histone 2B, fibrinogen and vimentin. Interestingly, the most reactive clone, originated from an IgG1-bearing memory B cell, was encoded by near germline variable genes, and showed similarity to previously reported natural IgM. Other anti-MDA clones display somatic hypermutations and lower reactivity. Importantly, these anti-MDA antibodies had significant in vitro functional properties and induced enhanced osteoclastogenesis, while the natural antibody related high-reactivity clone did not. We postulate that these may represent distinctly different facets of anti-MDA autoreactive responses.