| 1. |
- Bernet, Néstor Vazquez, et al.
(författare)
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High-Quality Library Preparation for NGS-Based Immunoglobulin Germline Gene Inference and Repertoire Expression Analysis
- 2019
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Ingår i: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Carlberg, Konstantin, et al.
(författare)
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Integrated Single Cell and Spatial Transcriptomics Reveal Autoreactive Differentiated B Cells in Joints of Early Rheumatoid Arthritis
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Rheumatoid Arthritis (RA) is a prevalent autoimmune disease characterized by inflammation of peripheral joints. Patients can be subdivided by the presence or absence of Rheumatoid Factor and anti-citrullinated protein antibodies (ACPA) in their circulation. Inflammation of the joint tissue is associated with infiltration of leukocytes from the blood, which can result in generation of lymphoid structures composed of B and T cells. Previous studies have shown that both memory B cells and antibody-secreting plasma cells populate the rheumatic joint tissue when captured from established and often long-standing disease. However, it has remained unclear, whether these cells are autoreactive and whether the associated lymphoid structures are present at the site of inflammation already at the time of diagnosis. Here, we used an integrated single cell and spatial transcriptomic approach to study B and plasma cells in synovial tissue of ACPA- and ACPA+ RA patients at this early time point. We found evidence for T cell help to B cells and presence of memory B and plasma cell pools in ACPA- as well as in ACPA+ RA. Our results demonstrated common supportive microenvironments in both patient subgroups, clonal relationships between the memory B and plasma cell pools and autoreactivity within the plasma cell compartment. These findings challenge our understanding of the dynamics of local adaptive immune responses in the RA joint of ACPA- and ACPA+ patients at the time of diagnosis, with direct implications for B and T cell targeting therapies for both patient subgroups.
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| 3. |
- Grönwall, Caroline, et al.
(författare)
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Depressed serum IgM levels in SLE are restricted to defined subgroups
- 2017
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Ingår i: Clinical Immunology. - : Elsevier BV. - 1521-6616 .- 1521-7035. ; 183, s. 304-315
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Tidskriftsartikel (refereegranskat)abstract
- 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 phosphoiylcholine (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/beta(2)glycoprotein-I. We also observed an association of reduced IgM levels with the HEA-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.
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| 4. |
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| 5. |
- Hardt, Uta, et al.
(författare)
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Autoimmune reactivity to malondialdehyde adducts in systemic lupus erythematosus is associated with disease activity and nephritis
- 2018
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Ingår i: Arthritis Research & Therapy. - : Springer Science and Business Media LLC. - 1478-6362. ; 20
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 6. |
- Hardt, Uta
(författare)
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B cell transcriptomics and immunoglobulin genetics in rheumatoid arthritis
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 7. |
- Hardt, Uta, et al.
(författare)
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Integrated single cell and spatial transcriptomics reveal autoreactive differentiated B cells in joints of early rheumatoid arthritis
- 2022
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Ingår i: Scientific Reports. - : Springer Nature. - 2045-2322. ; 12:1
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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