B cell transcriptomics and immunoglobulin genetics in rheumatoid arthritis

Rheumatoid Arthritis (RA) is a complex autoimmune disease characterized by inflammation of the peripheral joints. The exact disease pathology remains to be elucidated, but it has been hypothesized that autoreactive B cells and autoantibodies play a major role in the etiology. In this thesis, I studied synovial B cells at the time of RA diagnosis using single cell RNA sequencing, spatial transcriptomics and isolation of monoclonal antibodies. In a separate track, I optimized Next Generation Sequencing library preparation methods to study antibody repertoires, infer germline antibody alleles and I applied these methods to a local disease study group. Using single cell RNA sequencing and spatial transcriptomics from patient paired tissue pieces, we found mostly memory B cells and naïve B cells in lymphoid rich biopsies. CD27++ plasma cells were found to have a strong identity signal as shown by both methods. Similarily, both methods showed a strong signature for T-B cell crosstalk that we discuss to drive differentiation to memory and plasma cells. Furthermore, we found the CXCL12-CXCR4 axis to be an active component of a plasma cell niche in synovial tissue. Studies of the B cell receptor from the single cell RNA sequencing data imply a clonal recall response from memory cells differentiating into plasma cells due to recurrent antigen exposure. We also identified an anticitrullinated protein reactivity towards modified vimentin in the tissue biopsies. Moreover, we compared 5’ rapid amplification of cDNA ends (5’RACE) antibody library preparation methods to a 5’ multiplex (5’MTPX) approach. We detected a 5’RACE amplicon length limitation that occurs when the sequences of the antibody heavy chain repertoire have long 5’ untranslated regions (UTRs) or long complementary determining regions 3 (CDR3s). Furthermore, we tested a single lambda constant chain primer versus a mix of single lambda constant chain specific primers and found that there was good detection of independent variable-joining segment (V-J) recombination that allows identification of lambda chain germline alleles. Finally, we used the 5’MTPX approach to study the antibody heavy chain alleles of thirty individuals from the local Epidemiological Investigation of Rheumatoid Arthritis (EIRA) study. We found common structural variations as well as few novel alleles. In haplotype analysis, we observed that the IGHV4-34 gene was seemingly hemizygous. This could be linked to a polymorphism in the recombination signal sequence of one chromosome leading to the absence of the allele in the expressed repertoire. Notably, this variation was also found in thirty control subjects from the EIRA study group and in the SNP data from the Finnish population group in the 1000 Genomes Project. Hence, we stress the importance of population stratification when performing disease association studies. In summary, I discuss RA therapy with respect to T-B cell interaction and a plasma cell survival niche, suitable antibody library generation methods for deep repertoire studies and germline gene inference studies, and the relevance of this for larger disease association studies

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

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 are associated with inflammatory 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. Several clones were highly specific for MDA-modification with no cross-reactivity to other antigen modifications. The mAbs recognized MDA-adducts in a variety of proteins. Interestingly, the most reactive clone, originated from an IgG1-bearing memory B cell, was encoded by germline variable genes, and showed similarity to previously reported natural IgM. Other anti-MDA clones display somatic hypermutations and lower reactivity. 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

Development of Hepatocellular Carcinoma Associated with Anabolic Androgenic Steroid Abuse in a Young Bodybuilder: A Case Report

Introduction. Many different etiological factors are involved in the development of hepatocellular carcinoma (HCC). We report the case of HCC in a 37-year-old male professional bodybuilder with extensive anabolic androgenic (AAS) steroid abuse. Case Presentation. Because of increasing epigastric and abdominal pain, abdominal ultrasound was performed in a 37-year-old male professional bodybuilder. A hyperechoic lesion in the liver was detected in segment VI. The magnetic resonance imaging showed hepatomegaly and confirmed the lesion, which showed features of a hepatocellular adenoma (HCA). Laboratory values were inconspicuous. After laparoscopic segmentectomy the histological examination revealed HCC. Conclusion. While the development of HCA in the liver by chronic intake of AAS is well known, little is known about the association with HCC. The presented case may indicate aetiological association of chronic intake of AAS and the development of HCC

Table_2_Analysis of IGH allele content in a sample group of rheumatoid arthritis patients demonstrates unrevealed population heterogeneity.docx

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.</p

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 &lt; 0.0001, Spearman R = 0.3). Meta-analysis found an odds ratio of 2.7 (confidence interval (CI) 1.9-3.9; p &lt; 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

Image_1_Analysis of IGH allele content in a sample group of rheumatoid arthritis patients demonstrates unrevealed population heterogeneity.jpeg

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.</p

Table_3_Analysis of IGH allele content in a sample group of rheumatoid arthritis patients demonstrates unrevealed population heterogeneity.docx

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.</p

Image_2_Analysis of IGH allele content in a sample group of rheumatoid arthritis patients demonstrates unrevealed population heterogeneity.jpeg

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.</p

Pro12Ala polymorphism of the peroxisome proliferator-activated receptor γ2 in patients with fatty liver diseases

AIM: To test the occurrence of the Pro12Ala mutation of the peroxisome proliferator-activated receptor-γ (PPARγ)2-gene in patients with non-alcoholic fatty liver disease (NAFLD) or alcoholic fatty liver disease (AFLD)