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.

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.

Early prediction of clinical response to anti-TNF treatment using multi-omics and machine learning in rheumatoid arthritis.

OBJECTIVES: Advances in immunotherapy by blocking TNF have remarkably improved treatment outcomes for Rheumatoid arthritis (RA) patients. Although treatment specifically targets TNF, the downstream mechanisms of immune suppression are not completely understood. The aim of this study was to detect biomarkers and expression signatures of treatment response to TNF inhibition. METHODS: Peripheral blood mononuclear cells (PBMCs) from 39 female patients were collected before anti-TNF treatment initiation (day 0) and after 3 months. The study cohort included patients previously treated with MTX who failed to respond adequately. Response to treatment was defined based on the EULAR criteria and classified 23 patients as responders and 16 as non-responders. We investigated differences in gene expression in PBMCs, the proportion of cell types and cell phenotypes in peripheral blood using flow cytometry and the level of proteins in plasma. Finally, we used machine learning models to predict non-response to anti-TNF treatment. RESULTS: The gene expression analysis in baseline samples revealed notably higher expression of the gene EPPK1 in future responders. We detected the suppression of genes and proteins following treatment, including suppressed expression of the T cell inhibitor gene CHI3L1 and its protein YKL-40. The gene expression results were replicated in an independent cohort. Finally, machine learning models mainly based on transcriptomic data showed high predictive utility in classifying non-response to anti-TNF treatment in RA. CONCLUSIONS: Our integrative multi-omics analyses identified new biomarkers for the prediction of response, found pathways influenced by treatment and suggested new predictive models of anti-TNF treatment in RA patients.

Contact-dependent interference with invariant NKT cell activation by herpes simplex virus-infected cells.

Invariant CD1d-restricted NKT (iNKT) cells play important roles in generating protective immune responses against infections. In this study, we have investigated the role of human iNKT cells in HSV-1 infection and their interaction with epidermal keratinocytes. These cells express CD1d and are the primary target of the virus. Keratinocytes loaded with α-galactosyl ceramide (α-GalCer) could stimulate IFN-γ production and CD25 upregulation by iNKT cells. However, both α-GalCer-dependent and cytokine-dependent activation of iNKT cells was impaired after coculture with HSV-1-infected cells. Notably, CD1d downregulation was not observed on infected keratinocytes, which were also found to inhibit TCR-independent iNKT cell activation. Further examination of the cytokine profile of iNKT-keratinocyte cocultures showed inhibition of IFN-γ, IL-5, IL-10, IL-13, and IL-17 secretion but upregulation of IL-4 and TNF-α after the infection. Moreover, cell-to-cell contact between infected keratinocytes and iNKT cells was required for the inhibition of activation, as the cell-free supernatants containing virus did not affect activation. Productive infection of iNKT cells was however not required for the inhibitory effect. After coculture with infected cells, iNKT cells were no longer responsive to further stimulation with α-GalCer-loaded CD1d-expressing cells. We found that exposure to HSV-1-infected cells resulted in impaired TCR signaling downstream of ZAP70. Additionally, infected cells upregulated the expression of the negative T cell regulator, galectin-9; however, blocking experiments indicated that the impairment of iNKT cell responses was independent of galectin-9. Thus, interference with activation of human iNKT cells by HSV-1 may represent a novel immunoevasive strategy used by the virus to avoid immune clearance.

Molecular signature of methotrexate response among rheumatoid arthritis patients.

Background: Methotrexate (MTX) is the first line treatment for rheumatoid arthritis (RA), but failure of satisfying treatment response occurs in a significant proportion of patients. Here we present a longitudinal multi-omics study aimed at detecting molecular and cellular processes in peripheral blood associated with a successful methotrexate treatment of rheumatoid arthritis. Methods: Eighty newly diagnosed patients with RA underwent clinical assessment and donated blood before initiation of MTX, and 3 months into treatment. Flow cytometry was used to describe cell types and presence of activation markers in peripheral blood, the expression of 51 proteins was measured in serum or plasma, and RNA sequencing was performed in peripheral blood mononuclear cells (PBMC). Response to treatment after 3 months was determined using the EULAR response criteria. We assessed the changes in biological phenotypes during treatment, and whether these changes differed between responders and non-responders with regression analysis. By using measurements from baseline, we also tried to find biomarkers of future MTX response or, alternatively, to predict MTX response. Results: Among the MTX responders, (Good or Moderate according to EULAR treatment response classification, n = 60, 75%), we observed changes in 29 partly overlapping cell types proportions, levels of 13 proteins and expression of 38 genes during treatment. These changes were in most cases suppressions that were stronger among responders compared to non-responders. Within responders to treatment, we observed a suppression of FOXP3 gene expression, reduction of immunoglobulin gene expression and suppression of genes involved in cell proliferation. The proportion of many HLA-DR expressing T-cell populations were suppressed in all patients irrespective of clinical response, and the proportion of many IL21R+ T-cells were reduced exclusively in non-responders. Using only the baseline measurements we could not detect any biomarkers or prediction models that could predict response to MTX. Conclusion: We conclude that a deep molecular and cellular phenotyping of peripheral blood cells in RA patients treated with methotrexate can reveal previously not recognized differences between responders and non-responders during 3 months of treatment with MTX. This may contribute to the understanding of MTX mode of action and explain non-responsiveness to MTX therapy.

NKG2D performs two functions in invariant NKT cells: direct TCR-independent activation of NK-like cytolysis and co-stimulation of activation by CD1d.

Invariant NKT cells are important in the activation and regulation of immune responses. They can also function as CD1d-restricted killer cells. However, the role of activating innate NK-cell receptors expressed on NKT cells in triggering cytolytic function is poorly characterized. Here, we initially confirmed that the cellular stress-ligand receptor NKG2D is expressed on CD4- NKT cells, whereas most CD4+ NKT cells lack this receptor. Interestingly, NKG2D+ NKT cells frequently expressed perforin, and both NKG2D and perforin localized at the site of contact with NKG2D ligand-expressing target cells. CD4- NKT cells degranulated in response to NKG2D engagement in a redirected activation assay independent of stimulation via their invariant TCR. NKT cells killed P815 cells coated with anti-NKG2D mAb and CD1d-negative K562 tumor target cells in an NKG2D-dependent manner. Furthermore, NKG2D engagement co-stimulated TCR-mediated NKT-cell activation in response to endogenous CD1d-presented ligands or suboptimal levels of anti-CD3 triggering. These data indicate that the CD4- subset of human NKT cells can mediate direct lysis of target cells via NKG2D engagement independent of CD1d, and that NKG2D also functions as a co-stimulatory receptor in these cells. NKG2D thus plays both a direct and a co-stimulatory role in the activation of NKT cells.

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.

Different Hierarchies of Anti-Modified Protein Autoantibody Reactivities in Rheumatoid Arthritis.

OBJECTIVE: Anti-citrullinated protein antibodies (ACPAs) are a hallmark of seropositive rheumatoid arthritis (RA). Yet, the precise disease-relevant autoantigens that are targeted by ACPAs remains a matter of debate. This study utilized patient-derived monoclonal ACPAs, rather than serum autoantibody analysis, to characterize the multireactivity to different protein modifications and to reveal autoantibody subsets in patients with RA. METHODS: Twelve human monoclonal ACPAs (positive by the second-generation cyclic citrullinated peptide test) were generated from 6 RA patients, and a head-to-head comparison of their reactivities was performed. For profiling, we used a complementary DNA-based protein array (Engine GmbH) and 3 peptide-screening platforms with RA autoantigens (Thermo Fisher Scientific), citrullinated and carbamylated peptides (NimbleGen/Roche), or histone-derived peptides with different posttranslational modifications (JPT Histone Code), covering >207,000 peptides (>7,800 gene products). RESULTS: The fine-specificity profiles of the investigated ACPAs varied, but all of the monoclonal ACPAs displayed multireactivity to a large number of citrullinated peptides/proteins, each characterized by specific binding properties. ACPA subsets could be defined by clone-distinct consensus binding motifs (e.g., Cit-Gly, Gly-Cit, or Arg-Cit-Asp), with the most common ACPA recognition being that of a Gly in the +1 flanking position, but with additional amino acid preferences. For ACPA protein recognition, we observed a preference for citrullinated RNA-binding proteins with high Arg/Gly content. Six of the 12 ACPA clones also bound acetylated lysine (KAc) or homocitrulline peptide motifs, displaying a similar affinity or higher apparent affinity than that for citrullinated peptides. CONCLUSION: ACPAs and anti-modified protein autoantibodies represent overlapping facets of RA autoimmunity and bind to a wide variety of modified proteins, extending well beyond the historically recognized set of RA autoantigens. So far, KAc reactivity has been detected only in the context of anti-carbamylated and anti-citrullinated peptide autoantibody responses, postulating the existence of hierarchies of autoreactivity in RA. Future investigations of ACPA fine specificities and functionality should take into consideration the presence of consensus Cit/Carb/KAc motifs and the multireactivity of these autoantibodies in patients with RA.

A Refined Protocol for Identifying Citrulline-specific Monoclonal Antibodies from Single Human B Cells from Rheumatoid Arthritis Patient Material.

We describe here a detailed, refined protocol for the generation of citrulline-specific monoclonal antibodies from single human B cells from rheumatoid arthritis (RA) patients. This protocol provides a detailed guide of the procedure starting from single B cells of your choice and followed by amplification of the variable region of immunoglobulin genes by RT-PCR, subsequent immunoglobulin gene cloning, recombinant IgG1 monoclonal antibody (mAb) production and quality controls. The produced mAbs can be used for further studies including reactivity towards candidate antigens and functionality both in vitro and in vivo. This protocol can be used to generate antigen-specific mAbs from B cells derived from different tissues and compartments, including peripheral blood, synovial fluid, digested biopsies, bone marrow aspirations, and bronchoalveolar lavage fluid. Notably, although examples are given on how to identify citrulline-specific autoantibodies the general methods can also be applied to other reactivities.

Recognition of Amino Acid Motifs, Rather Than Specific Proteins, by Human Plasma Cell-Derived Monoclonal Antibodies to Posttranslationally Modified Proteins in Rheumatoid Arthritis.

OBJECTIVE: Antibodies against posttranslationally modified proteins are a hallmark of rheumatoid arthritis (RA), but the emergence and pathogenicity of these autoantibodies are still incompletely understood. The aim of this study was to analyze the antigen specificities and mutation patterns of monoclonal antibodies (mAb) derived from RA synovial plasma cells and address the question of antigen cross-reactivity. METHODS: IgG-secreting cells were isolated from RA synovial fluid, and the variable regions of the immunoglobulins were sequenced (n = 182) and expressed in full-length mAb (n = 93) and also as germline-reverted versions. The patterns of reactivity with 53,019 citrullinated peptides and 49,211 carbamylated peptides and the potential of the mAb to promote osteoclastogenesis were investigated. RESULTS: Four unrelated anti-citrullinated protein autoantibodies (ACPAs), of which one was clonally expanded, were identified and found to be highly somatically mutated in the synovial fluid of a patient with RA. The ACPAs recognized >3,000 unique peptides modified by either citrullination or carbamylation. This highly multireactive autoantibody feature was replicated for Ig sequences derived from B cells from the peripheral blood of other RA patients. The plasma cell-derived mAb were found to target distinct amino acid motifs and partially overlapping protein targets. They also conveyed different effector functions as revealed in an osteoclast activation assay. CONCLUSION: These findings suggest that the high level of cross-reactivity among RA autoreactive B cells is the result of different antigen encounters, possibly at different sites and at different time points. This is consistent with the notion that RA is initiated in one context, such as in the mucosal organs, and thereafter targets other sites, such as the joints.

Differential ACPA Binding to Nuclear Antigens Reveals a PAD-Independent Pathway and a Distinct Subset of Acetylation Cross-Reactive Autoantibodies in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) associated anti-citrullinated protein autoantibodies (ACPA) target a wide range of modified proteins. Citrullination occurs during physiological processes such as apoptosis, yet little is known about the interaction of ACPA with nuclear antigens or apoptotic cells. Since uncleared apoptotic cells and neutrophil extracellular trap (NET) products have been postulated to be central sources of autoantigen and immunostimulation in autoimmune disease, we sought to characterize the anti-nuclear and anti-neutrophil reactivities of ACPA. Serology showed that a subset of anti-CCP2 seropositive RA patients had high reactivity to full-length citrullinated histones. In contrast, seronegative RA patients displayed elevated IgG reactivity to native histone compared to controls, but no citrulline-specific reactivity. Screening of 10 single B-cell derived monoclonal ACPA from RA patients revealed that four ACPA exhibited strong binding to apoptotic cells and three of these had anti-nuclear (ANA) autoantibody reactivity. Modified histones were confirmed to be the primary targets of this anti-nuclear ACPA subset following immunoprecipitation from apoptotic cell lysates. Monoclonal ACPA were also screened for reactivities against stimulated murine and human neutrophils, and all the nuclear-reactive monoclonal ACPA bound to NETs. Intriguingly, one ACPA mAb displayed a contrasting cytoplasmic perinuclear neutrophil binding and may represent a different NET-reactive ACPA subset. Notably, studies of CRISPR-Cas9 PAD4 KO cells and cells from PAD KO mice showed that the cytoplasmic NET-binding was fully dependent on PAD4, whilst nuclear- and histone-mediated NET reactivity was largely PAD-independent. Our further analysis revealed that the nuclear binding could be explained by consensus-motif driven ACPA cross-reactivity to acetylated histones. Specific acetylated histone peptides targeted by the monoclonal antibodies were identified and the anti-modified protein autoantibody (AMPA) profile of the ACPA was found to correlate with the functional activity of the antibodies. In conclusion, when investigating monoclonal ACPA, we could group ACPA into distinct subsets based on their nuclear binding-patterns and acetylation-mediated binding to apoptotic cells, neutrophils, and NETs. Differential anti-modified protein reactivities of RA-autoantibody subsets could have an important functional impact and provide insights in RA pathogenesis.

Pathogenic Citrulline-Multispecific B Cell Receptor Clades in Rheumatoid Arthritis.

OBJECTIVE: Anti-citrullinated protein antibodies (ACPAs) have proven highly useful as biomarkers for rheumatoid arthritis (RA). However, composition and functionality of the associated autoreactive B cell repertoire have not been directly assessed. We aimed to selectively investigate citrullinated autoantigen-specific B cell receptors (BCRs) involved in RA and initiate studies on their pathogenicity. METHODS: Blood samples were obtained from patients in a University of Minnesota cohort with ACPA-positive RA (n = 89). Tetramer sets bearing citrullinated filaggrin peptide cfc1 or citrullinated α-enolase peptide were constructed to specifically capture autoreactive B cells from the unaltered, polyclonal repertoire in RA patients. Citrullinated peptide tetramer-bound B cells were subjected to flow cytometric cell sorting and single-cell IGH, IGK, and IGL gene sequencing for B cell lineage determinations. BCR gene sequences were also expressed as recombinant monoclonal antibodies (mAb) for direct evaluation of citrullinated autoantigen binding and effector functionality. RESULTS: Using citrullinated peptide tetramer enrichment to investigate single autoreactive blood B cells, we identified biased V-region gene usage and conserved junction arrangements in BCRs from RA patients. Parsimonious clustering of related immunoglobulin gene nucleotide sequences revealed clonal expansions of rare individual B cell clades, in parallel with divergent sequence mutations. Correspondingly, recombinant mAb generated from such BCR lineages demonstrated citrulline-dependent cross-reactivity extending beyond the citrullinated peptides used for B cell capture. A pair of citrullinated autoantigen-specific mAb with cross-reactive binding profiles also promoted arthritis in mice. CONCLUSION: Our findings suggest that broad ACPA specificities in RA arise from a restricted repertoire of evolving citrulline-multispecific B cell clades with pathogenic potential.