Autoreactive B cells against malondialdehyde-induced protein cross-links are present in the joint, lung, and bone marrow of rheumatoid arthritis patients.

Autoantibodies to malondialdehyde (MDA) proteins constitute a subset of anti-modified protein autoantibodies in rheumatoid arthritis (RA), which is distinct from citrulline reactivity. Serum anti-MDA IgG levels are commonly elevated in RA and correlate with disease activity, CRP, IL6, and TNF-α. MDA is an oxidation-associated reactive aldehyde that together with acetaldehyde mediates formation of various immunogenic amino acid adducts including linear MDA-lysine, fluorescent malondialdehyde acetaldehyde (MAA)-lysine, and intramolecular cross-linking. We used single-cell cloning, generation of recombinant antibodies (n = 356 from 25 donors), and antigen-screening to investigate the presence of class-switched MDA/MAA+ B cells in RA synovium, bone marrow, and bronchoalveolar lavage. Anti-MDA/MAA+ B cells were found in bone marrow plasma cells of late disease and in the lung of both early disease and risk-individuals and in different B cell subsets (memory, double negative B cells). These were compared with previously identified anti-MDA/MAA from synovial memory and plasma cells. Seven out of eight clones carried somatic hypermutations and all bound MDA/MAA-lysine independently of protein backbone. However, clones with somatic hypermutations targeted MAA cross-linked structures rather than MDA- or MAA-hapten, while the germline-encoded synovial clone instead bound linear MDA-lysine in proteins and peptides. Binding patterns were maintained in germline converted clones. Affinity purification of polyclonal anti-MDA/MAA from patient serum revealed higher proportion of anti-MAA versus anti-MDA compared to healthy controls. In conclusion, IgG anti-MDA/MAA show distinct targeting of different molecular structures. Anti-MAA IgG has been shown to promote bone loss and osteoclastogenesis in vivo and may contribute to RA pathogenesis.

Occurrence and localization of FOXP3 + cells in kidney biopsies in lupus nephritis and ANCA-associated vasculitis.

The study aims to increase the understanding regarding the role of regulatory T cells (Tregs) in lupus nephritis (LN) and ANCA-associated vasculitis (AAV) by comparing their localization in renal tissue and changes following immunosuppressive therapy. Kidney biopsies from 12 patients with LN and 7 patients with AAV were examined. Kidney biopsies had been performed both at active disease and following immunosuppressive treatment. Clinical data was collected at both biopsy occasions. Expression of Forkhead Box P 3 (Foxp3) in renal tissue was assessed by immunohistochemistry. An arbitrary scale was used to estimate the number of Foxp3+ cells. In LN, 8/12 (67%) had positive tissue staining for Foxp3 at baseline, most pronounced in inflammatory infiltrates, but also interstitially and in a peri-glomerular pattern. At second biopsies, after immunosuppressive treatment, 4/12 (33%) still had detectable Foxp3+ cells, found in persisting inflammatory infiltrates and some in the interstitium. Patients with a good clinical response to treatment had high grade of Foxp3+ cells in first biopsies. In AAV, only 2/7 (29%) had positive staining for Foxp3 at baseline, in inflammatory infiltrates and to a lesser extent in the interstitium, despite large areas of inflammatory infiltrates in all patients. At follow-up, 2/7 (29%) biopsies were positive for Foxp3. Our data show a higher presence of Foxp3+ cells in renal tissue from LN patients compared to AAV, suggesting that Tregs may be differently involved in the control of inflammatory mechanisms in these diseases. These findings could have further implication for therapeutic approaches aiming at restoring the immunological tolerance. Key Points • Foxp3+-cells are present in larger amount in renal tissue in lupus nephritis vs. ANCA-associated vasculitis. • Our data suggest that Foxp3+ regulatory T cells are involved in the control of inflammatory processes in lupus nephritis.

Anti-Citrullinated Protein Antibody Reactivity towards Neutrophil-Derived Antigens: Clonal Diversity and Inter-Individual Variation.

BACKGROUND: Why the adaptive immune system turns against citrullinated antigens in rheumatoid arthritis (RA) and whether anti-citrullinated protein antibodies (ACPAs) contribute to pathogenesis are questions that have triggered intense research, but still are not fully answered. Neutrophils may be crucial in this context, both as sources of citrullinated antigens and also as targets of ACPAs. To better understand how ACPAs and neutrophils contribute to RA, we studied the reactivity of a broad spectrum of RA patient-derived ACPA clones to activated or resting neutrophils, and we also compared neutrophil binding using polyclonal ACPAs from different patients. METHODS: Neutrophils were activated by Ca2+ ionophore, PMA, nigericin, zymosan or IL-8, and ACPA binding was studied using flow cytometry and confocal microscopy. The roles of PAD2 and PAD4 were studied using PAD-deficient mice or the PAD4 inhibitor BMS-P5. RESULTS: ACPAs broadly targeted NET-like structures, but did not bind to intact cells or influence NETosis. We observed high clonal diversity in ACPA binding to neutrophil-derived antigens. PAD2 was dispensable, but most ACPA clones required PAD4 for neutrophil binding. Using ACPA preparations from different patients, we observed high patient-to-patient variability in targeting neutrophil-derived antigens and similarly in another cellular effect of ACPAs, the stimulation of osteoclast differentiation. CONCLUSIONS: Neutrophils can be important sources of citrullinated antigens under conditions that lead to PAD4 activation, NETosis and the extrusion of intracellular material. A substantial clonal diversity in targeting neutrophils and a high variability among individuals in neutrophil binding and osteoclast stimulation suggest that ACPAs may influence RA-related symptoms with high patient-to-patient variability.

Rheumatoid Arthritis-Specific Autoimmunity in the Lung Before and at the Onset of Disease.

OBJECTIVE: The lung is implicated as a site for breach of tolerance prior to onset of seropositive rheumatoid arthritis (RA). To substantiate this, we investigated lung-resident B cells in bronchoalveolar lavage (BAL) samples from untreated early RA patients and anti-citrullinated protein antibody (ACPA)-positive individuals at risk for developing RA. METHODS: Single B cells (n = 7,680) were phenotyped and isolated from BAL samples from individuals at risk of RA (n = 3) and at RA diagnosis (n = 9). The immunoglobulin variable region transcripts were sequenced and selected for expression as monoclonal antibodies (n = 141). Monoclonal ACPAs were tested for reactivity patterns and binding to neutrophils. RESULTS: Using our single-cell approach, we found significantly increased proportions of B lymphocytes in ACPA+ compared to ACPA- individuals. Memory and double-negative B cells were prominent in all subgroups. Upon antibody re-expression, 7 highly mutated citrulline-autoreactive clones originating from different memory B cell subsets were identified, both in individuals at risk of RA and early RA patients. Lung IgG variable gene transcripts from ACPA+ individuals carried frequent mutation-induced N-linked Fab glycosylation sites (P < 0.001), often in the framework 3 of the variable region. Two of the lung ACPAs bound to activated neutrophils, 1 from an individual at risk of RA and 1 from an early RA patient. CONCLUSION: T cell-driven B cell differentiation resulting in local class switching and somatic hypermutation are evident in lungs before as well as in early stages of ACPA+ RA. Our findings add to the notion of lung mucosa being a site for initiation of citrulline autoimmunity preceding seropositive RA.

The human bone marrow plasma cell compartment in rheumatoid arthritis - Clonal relationships and anti-citrulline autoantibody producing cells.

A majority of circulating IgG is produced by plasma cells residing in the bone marrow (BM). Long-lived BM plasma cells constitute our humoral immune memory and are essential for infection-specific immunity. They may also provide a reservoir of potentially pathogenic autoantibodies, including rheumatoid arthritis (RA)-associated anti-citrullinated protein autoantibodies (ACPA). Here we investigated paired human BM plasma cell and peripheral blood (PB) B-cell repertoires in seropositive RA, four ACPA+ RA patients and one ACPA- using two different single-cell approaches, flow cytometry sorting, and transcriptomics, followed by recombinant antibody generation. Immunoglobulin (Ig) analysis of >900 paired heavy-light chains from BM plasma cells identified by either surface CD138 expression or transcriptome profiles (including gene expression of MZB1, JCHAIN and XBP1) demonstrated differences in IgG/A repertoires and N-linked glycosylation between patients. For three patients, we identified clonotypes shared between BM plasma cells and PB memory B cells. Notably, four individuals displayed plasma cells with identical heavy chains but different light chains, which may indicate receptor revision or clonal convergence. ACPA-producing BM plasma cells were identified in two ACPA+ patients. Three of 44 recombinantly expressed monoclonal antibodies from ACPA+ RA BM plasma cells were CCP2+, specifically binding to citrullinated peptides. Out of these, two clones reacted with citrullinated histone-4 and activated neutrophils. In conclusion, single-cell investigation of B-cell repertoires in RA bone marrow provided new understanding of human plasma cells clonal relationships and demonstrated pathogenically relevant disease-associated autoantibody expression in long-lived plasma cells.

Identification of proteinase 3 autoreactive CD4+T cells and their T-cell receptor repertoires in antineutrophil cytoplasmic antibody-associated vasculitis.

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is an autoimmune disease involving autoreactivity to proteinase 3 (PR3) as demonstrated by presence of ANCAs. While autoantibodies are screened for diagnosis, autoreactive T cells and their features are less well-studied. Here, we investigated PR3-specific CD4+T cell responses and features of autoreactive T cells in patients with PR3-AAV, using a cohort of 72 patients with either active or inactive disease. Autoreactive PR3-specific CD4+T cells producing interferon γ in response to protein stimulation were found to express the G-protein coupled receptor 56 (GPR56), a cell surface marker that distinguishes T cells with cytotoxic capacity. GPR56+CD4+T cells were significantly more prominent in the blood of patients with inactive as compared to active disease, suggesting that these cells were affected by immunosuppression and/or that they migrated from the circulation to sites of organ involvement. Indeed, GPR56+CD4+T cells were identified in T-cell infiltrates of affected kidneys and an association with immunosuppressive therapy was found. Moreover, distinct TCR gene segment usage and shared (public) T cell clones were found for the PR3-reactive TCRs. Shared T cell clones were found in different patients with AAV carrying the disease-associated HLA-DP allele, demonstrating convergence of the autoreactive T cell repertoire. Thus, we identified a CD4+T cell signature in blood and in affected kidneys that display PR3 autoreactivity and associates with T cell cytotoxicity. Our data provide a basis for novel rationales for both immune monitoring and future therapeutic intervention in PR3-AAV.

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.

A subset of antibodies targeting citrullinated proteins confers protection from rheumatoid arthritis.

Although elevated levels of anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis (RA), the in vivo functions of these antibodies remain unclear. Here, we have expressed monoclonal ACPAs derived from patients with RA, and analyzed their functions in mice, as well as their specificities. None of the ACPAs showed arthritogenicity nor induced pain-associated behavior in mice. However, one of the antibodies, clone E4, protected mice from antibody-induced arthritis. E4 showed a binding pattern restricted to skin, macrophages and dendritic cells in lymphoid tissue, and cartilage derived from mouse and human arthritic joints. Proteomic analysis confirmed that E4 strongly binds to macrophages and certain RA synovial fluid proteins such as α-enolase. The protective effect of E4 was epitope-specific and dependent on the interaction between E4-citrullinated α-enolase immune complexes with FCGR2B on macrophages, resulting in increased IL-10 secretion and reduced osteoclastogenesis. These findings suggest that a subset of ACPAs have therapeutic potential in RA.

Diversity and Clonality of T Cell Receptor Repertoire and Antigen Specificities in Small Joints of Early Rheumatoid Arthritis.

OBJECTIVE: CD4+ T cells are implicated in rheumatoid arthritis (RA) pathology from the strong association between RA and certain HLA class II gene variants. This study was undertaken to examine the synovial T cell receptor (TCR) repertoire, T cell phenotypes, and T cell specificities in small joints of RA patients at time of diagnosis before therapeutic intervention. METHODS: Sixteen patients, of whom 11 patients were anti-citrullinated protein antibody (ACPA)-positive and 5 patients were ACPA-, underwent ultrasound-guided synovial biopsy of a small joint (n = 13) or arthroscopic synovial biopsy of a large joint (n = 3), followed by direct sorting of single T cells for paired sequencing of the αß TCR together with flow cytometry analysis. TCRs from expanded CD4+ T cell clones of 4 patients carrying an HLA-DRB1*04:01 allele were artificially reexpressed to study antigen specificity. RESULTS: T cell analysis demonstrated CD4+ dominance and the presence of peripheral helper T-like cells in both patient groups. We identified >4,000 unique TCR sequences, as well as 225 clonal expansions. Additionally, T cells with double α-chains were a recurring feature. We identified a biased gene usage of the Vß chain segment TRBV20-1 in CD4+ cells from ACPA+ patients. In vitro stimulation of T cell lines expressing selected TCRs with an extensive panel of citrullinated and viral peptides identified several different virus-specific TCRs (e.g., human cytomegalovirus and human herpesvirus 2). Still, the majority of clones remained orphans with unknown specificity. CONCLUSION: Minimally invasive biopsies of the RA synovium allow for single-cell TCR sequencing and phenotyping. Clonally expanded, viral-reactive T cells account for part of the diverse CD4+ T cell repertoire. TRBV20-1 bias in ACPA+ patients suggests recognition of common antigens.

Combination of Two Monoclonal Anti-Citrullinated Protein Antibodies Induced Tenosynovitis, Pain, and Bone Loss in Mice in a Peptidyl Arginine Deiminase-4-Dependent Manner.

OBJECTIVE: The appearance of anti-citrullinated protein antibodies (ACPAs) in the circulation represents a major risk factor for developing rheumatoid arthritis (RA). Patient-derived ACPAs have been shown to induce pain and bone erosion in mice, suggesting an active role in the pathogenicity of RA. We undertook this study to investigate whether ACPAs can induce tenosynovitis, an early sign of RA, in addition to pain and bone loss and whether these symptoms are dependent on peptidyl arginine deiminase 4 (PAD4). METHODS: Monoclonal ACPAs generated from plasma cells of RA patients were transferred to wild-type and PAD4-deficient mice. Pain-like behavior and macroscopic inflammation were monitored for a period of 4 weeks, followed by the analyses of tenosynovitis in the ankle joints using magnetic resonance imaging (MRI) and bone microarchitecture in the tibia using an X-ray microscope. Microscopic changes in the tendon sheath were analyzed in decalcified ankle joint sections. RESULTS: The combination of 2 monoclonal ACPAs (1325:04C03 and 1325:01B09) induced long-lasting pain-like behavior and trabecular bone loss in mice. Although no synovitis was observed macroscopically, we detected tenosynovitis in the ACPA-injected mice by MRI. Microscopic analyses of the joints revealed a cellular hyperplasia and a consequent enlargement of the tendon sheath in the ACPA-treated group. In PAD4-/- mice, the effects of ACPAs on pain-like behavior, tenosynovitis, and bone loss were significantly reduced. CONCLUSION: Monoclonal ACPAs can induce tenosynovitis in addition to pain and bone loss via mechanisms dependent on PAD4-mediated citrullination.

Five commercially-available antibodies react differentially with allelic forms of human HLA-DR beta chain.

Allelic variants of HLA-DRB1 have been associated with a variety of autoimmune and infectious diseases. Although the precise molecular mechanisms by which HLA-DRB1 alleles predispose to a particular disease are currently unclear, it has been shown that mRNA expression levels of HLA-DRB1 are dependent on the different alleles. We aimed to measure HLA-DR beta chain levels in peripheral blood mononuclear cells of individuals carrying HLA-DRB1*03:01/*04:01 and HLA-DRB1*03:01/*15:01 alleles by western blotting, using five commercially-available HLA-DRB antibodies. We observed highly heterogeneous binding of the tested antibodies to the different allelic forms of the HLA-DR beta chain. Overall, we show that current immunological research that employs available antibodies to detect HLA-DR beta chains is biased towards detection of specific variants of the protein; this may cause significant discrepancy in quantification of protein expression in a heterogeneous human population.

Modulating T-cell activation with antisense oligonucleotides targeting lymphocyte cytosolic protein 2.

Dysregulated T-cell activation is a hallmark of several autoimmune diseases such as rheumatoid arthritis (RA) and multiple sclerosis (MS). The lymphocyte cytosolic protein 2 (LCP2), also known as SLP-76, is essential for the development and activation of T cells. Despite the critical role of LCP2 in T-cell activation and the need for developing drugs that modify T-cell activation, no LCP2 inhibitors have been developed. This can be explained by the "undruggable" nature of LCP2, lacking a structure permissive to standard small molecule inhibitor modalities. Here, we explored an alternative drug modality, developing antisense oligonucleotides (ASOs) targeting LCP2 mRNAs, and evaluated its activity in modulating T-cell activation. We identified a set of 3' UTR targeting LCP2 ASOs, which knocked down LCP2 in a human T-cell line and primary human T cells and found that these suppressed T-cell receptor mediated activation. We also found that the ASOs suppressed FcεR1-mediated mast cell activation, in line with the role of LCP2 in mast cells. Taken together, our data provide examples of how immunomodulatory ASOs that interfere with undruggable targets can be developed and propose that such drug modalities can be used to treat autoimmune diseases.

Single cell sequencing identifies clonally expanded synovial CD4+ TPH cells expressing GPR56 in rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease affecting synovial joints where different CD4+ T cell subsets may contribute to pathology. Here, we perform single cell sequencing on synovial CD4+ T cells from anti-citrullinated protein antibodies (ACPA)+ and ACPA- RA patients and identify two peripheral helper T cell (TPH) states and a cytotoxic CD4+ T cell subset. We show that the adhesion G-protein coupled receptor 56 (GPR56) delineates synovial CXCL13high TPH CD4+ T cells expressing LAG-3 and the tissue-resident memory receptors CXCR6 and CD69. In ACPA- SF, TPH cells display lower levels of GPR56 and LAG-3. Further, most expanded T cell clones in the joint are within CXCL13high TPH CD4+ T cells. Finally, RNA-velocity analyses suggest a common differentiation pathway between the two TPH clusters and effector CD4+ T cells. Our study provides comprehensive immunoprofiling of the synovial CD4+ T cell subsets in ACPA+ and ACPA- RA.

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.

Antibodies to a Citrullinated Porphyromonas gingivalis Epitope Are Increased in Early Rheumatoid Arthritis, and Can Be Produced by Gingival Tissue B Cells: Implications for a Bacterial Origin in RA Etiology.

Based on the epidemiological link between periodontitis and rheumatoid arthritis (RA), and the unique feature of the periodontal bacterium Porphyromonas gingivalis to citrullinate proteins, it has been suggested that production of anti-citrullinated protein antibodies (ACPA), which are present in a majority of RA patients, may be triggered in the gum mucosa. To address this hypothesis, we investigated the antibody response to a citrullinated P. gingivalis peptide in relation to the autoimmune ACPA response in early RA, and examined citrulline-reactivity in monoclonal antibodies derived from human gingival B cells. Antibodies to a citrullinated peptide derived from P. gingivalis (denoted CPP3) and human citrullinated peptides were analyzed by multiplex array in 2,807 RA patients and 372 controls; associations with RA risk factors and clinical features were examined. B cells from inflamed gingival tissue were single-cell sorted, and immunoglobulin (Ig) genes were amplified, sequenced, cloned and expressed (n=63) as recombinant monoclonal antibodies, and assayed for citrulline-reactivities by enzyme-linked immunosorbent assay. Additionally, affinity-purified polyclonal anti-cyclic-citrullinated peptide (CCP2) IgG, and monoclonal antibodies derived from RA blood and synovial fluid B cells (n=175), were screened for CPP3-reactivity. Elevated anti-CPP3 antibody levels were detected in RA (11%), mainly CCP2+ RA, compared to controls (2%), p<0.0001, with a significant association to HLA-DRB1 shared epitope alleles, smoking and baseline pain, but with low correlation to autoimmune ACPA fine-specificities. Monoclonal antibodies derived from gingival B cells showed cross-reactivity between P. gingivalis CPP3 and human citrullinated peptides, and a CPP3+/CCP2+ clone, derived from an RA blood memory B cell, was identified. Our data support the possibility that immunity to P. gingivalis derived citrullinated antigens, triggered in the inflamed gum mucosa, may contribute to the presence of ACPA in RA patients, through mechanisms of molecular mimicry.

Rituximab in Systemic Lupus Erythematosus: Transient Effects on Autoimmunity Associated Lymphocyte Phenotypes and Implications for Immunogenicity.

B cell abnormalities are common in systemic lupus erythematosus (SLE), and include expansion of double negative (DN) and age-associated-like B cells (ABC-like). We aimed to investigate rituximab (RTX) effects on DN and ABC-like B-cell subsets and, when possible, also secondary effects on T cells. Fifteen SLE patients, fulfilling the ACR 1982 criteria, starting RTX and followed longitudinally up to two years, were analyzed for B- and T- lymphocyte subsets using multicolor flow cytometry. DN were defined as IgD-CD27- and ABC-like as CD11c+CD21- within the DN gate. Additional phenotyping was performed adding CXCR5 in the B-cell panel. Cellular changes were further analyzed in the context of the generation of anti-drug antibodies (ADA) against RTX and clinical information. The SLE patients were mainly females (86.6%), of median age 36.7 (29.8-49.4) years and disease duration of 6.1 (1.6-11.8) years. Within the DN subset, ABC-like (IgD-CD27-CD11c+CD21-) B cell frequency reduced from baseline median level of 20.4% to 11.3% (p=0.03), at early follow-up. The DN B cells were further subdivided based on CXCR5 expression. Significant shifts were observed at the early follow-up in the DN2 sub-cluster (CD11c+CXCR5-), which reduced significantly (-15.4 percentage points, p=0.02) and in the recently described DN3 (CD11c-CXCR5-) which increased (+13 percentage points, p=0.03). SLE patients treated with RTX are at high risk of developing ADA. In our cohort, the presence of ADA at 6 months was associated with lower frequencies of DN cells and to a more pronounced expansion of plasmablasts at early follow-up. The frequency of follicular helper T cells (TFH, CD4+PD-1+CXCR5+) and of peripheral helper T cells (TPH, CD4+PD-1+CXCR5-) did not change after RTX. A sub-cluster of PD-1highCD4+ T cells showed a significant decrease at later follow-up compared to early follow-up (p=0.0039). It is well appreciated that RTX transiently influences B cells. Here, we extend these observations to cell phenotypes which are believed to directly contribute to autoimmunity in SLE. We show early transient effects of RTX on ABC-like memory B cells, later effects on PD-1high CD4+ cells, and possible implications for RTX immunogenicity. Further insight in such effects and their monitoring may be of clinical relevance.

Three-dimensional spatial transcriptomics uncovers cell type localizations in the human rheumatoid arthritis synovium.

The inflamed rheumatic joint is a highly heterogeneous and complex tissue with dynamic recruitment and expansion of multiple cell types that interact in multifaceted ways within a localized area. Rheumatoid arthritis synovium has primarily been studied either by immunostaining or by molecular profiling after tissue homogenization. Here, we use Spatial Transcriptomics, where tissue-resident RNA is spatially labeled in situ with barcodes in a transcriptome-wide fashion, to study local tissue interactions at the site of chronic synovial inflammation. We report comprehensive spatial RNA-Seq data coupled to cell type-specific localization patterns at and around organized structures of infiltrating leukocyte cells in the synovium. Combining morphological features and high-throughput spatially resolved transcriptomics may be able to provide higher statistical power and more insights into monitoring disease severity and treatment-specific responses in seropositive and seronegative rheumatoid arthritis.