Epigenetic programming underpins B cell dysfunction in human SLE.

Systemic lupus erythematosus (SLE) is characterized by the expansion of extrafollicular pathogenic B cells derived from newly activated naive cells. Although these cells express distinct markers, their epigenetic architecture and how it contributes to SLE remain poorly understood. To address this, we determined the DNA methylomes, chromatin accessibility profiles and transcriptomes from five human B cell subsets, including a newly defined effector B cell subset, from subjects with SLE and healthy controls. Our data define a differentiation hierarchy for the subsets and elucidate the epigenetic and transcriptional differences between effector and memory B cells. Importantly, an SLE molecular signature was already established in resting naive cells and was dominated by enrichment of accessible chromatin in motifs for AP-1 and EGR transcription factors. Together, these factors acted in synergy with T-BET to shape the epigenome of expanded SLE effector B cell subsets. Thus, our data define the molecular foundation of pathogenic B cell dysfunction in SLE.

Diversity, cellular origin and autoreactivity of antibody-secreting cell population expansions in acute systemic lupus erythematosus.

Acute systemic lupus erythematosus (SLE) courses with surges of antibody-secreting cells (ASCs) whose origin, diversity and contribution to serum autoantibodies remain unknown. Here, deep sequencing, proteomic profiling of autoantibodies and single-cell analysis demonstrated highly diversified ASCs punctuated by clones expressing the variable heavy-chain region VH4-34 that produced dominant serum autoantibodies. A fraction of ASC clones contained autoantibodies without mutation, a finding consistent with differentiation outside the germinal centers. A substantial ASC segment was derived from a distinct subset of newly activated naive cells of considerable clonality that persisted in the circulation for several months. Thus, selection of SLE autoreactivities occurred during polyclonal activation, with prolonged recruitment of recently activated naive B cells. Our findings shed light on the pathogenesis of SLE, help explain the benefit of agents that target B cells and should facilitate the design of future therapies.

Distinct Effector B Cells Induced by Unregulated Toll-like Receptor 7 Contribute to Pathogenic Responses in Systemic Lupus Erythematosus.

Systemic Lupus Erythematosus (SLE) is characterized by B cells lacking IgD and CD27 (double negative; DN). We show that DN cell expansions reflected a subset of CXCR5- CD11c+ cells (DN2) representing pre-plasma cells (PC). DN2 cells predominated in African-American patients with active disease and nephritis, anti-Smith and anti-RNA autoantibodies. They expressed a T-bet transcriptional network; increased Toll-like receptor-7 (TLR7); lacked the negative TLR regulator TRAF5; and were hyper-responsive to TLR7. DN2 cells shared with activated naive cells (aNAV), phenotypic and functional features, and similar transcriptomes. Their PC differentiation and autoantibody production was driven by TLR7 in an interleukin-21 (IL-21)-mediated fashion. An in vivo developmental link between aNAV, DN2 cells, and PC was demonstrated by clonal sharing. This study defines a distinct differentiation fate of autoreactive naive B cells into PC precursors with hyper-responsiveness to innate stimuli, as well as establishes prominence of extra-follicular B cell activation in SLE, and identifies therapeutic targets.

B cell subset composition segments clinically and serologically distinct groups in chronic cutaneous lupus erythematosus.

OBJECTIVE: While the contribution of B-cells to SLE is well established, its role in chronic cutaneous lupus erythematosus (CCLE) remains unclear. Here, we compare B-cell and serum auto-antibody profiles between patients with systemic lupus erythematosus (SLE), CCLE, and overlap conditions. METHODS: B-cells were compared by flow cytometry amongst healthy controls, CCLE without systemic lupus (CCLE+/SLE-) and SLE patients with (SLE+/CCLE+) or without CCLE (SLE+/CCLE-). Serum was analyed for autoreactive 9G4+, anti-double-stranded DNA, anti-chromatin and anti-RNA antibodies by ELISA and for anti-RNA binding proteins (RBP) by luciferase immunoprecipitation. RESULTS: Patients with CCLE+/SLE- share B-cell abnormalities with SLE including decreased unswitched memory and increased effector B-cells albeit at a lower level than SLE patients. Similarly, both SLE and CCLE+/SLE- patients have elevated 9G4+ IgG autoantibodies despite lower levels of anti-nucleic acid and anti-RBP antibodies in CCLE+/SLE-. CCLE+/SLE- patients could be stratified into those with SLE-like B-cell profiles and a separate group with normal B-cell profiles. The former group was more serologically active and more likely to have disseminated skin lesions. CONCLUSION: CCLE displays perturbations in B-cell homeostasis and partial B-cell tolerance breakdown. Our study demonstrates that this entity is immunologically heterogeneous and includes a disease segment whose B-cell compartment resembles SLE and is clinically associated with enhanced serological activity and more extensive skin disease. This picture suggests that SLE-like B-cell changes in primary CCLE may help identify patients at risk for subsequent development of SLE. B-cell profiling in CCLE might also indentify candidates who would benefit from B-cell targeted therapies.

The Regulation of Inherently Autoreactive VH4-34-Expressing B Cells in Individuals Living in a Malaria-Endemic Area of West Africa.

Plasmodium falciparum malaria is a deadly infectious disease in which Abs play a critical role in naturally acquired immunity. However, the specificity and nature of Abs elicited in response to malaria are only partially understood. Autoreactivity and polyreactivity are common features of Ab responses in several infections and were suggested to contribute to effective pathogen-specific Ab responses. In this article, we report on the regulation of B cells expressing the inherently autoreactive VH4-34 H chain (identified by the 9G4 mAb) and 9G4+ plasma IgG in adults and children living in a P. falciparum malaria-endemic area in West Africa. The frequency of 9G4+ peripheral blood CD19+ B cells was similar in United States adults and African adults and children; however, more 9G4+ B cells appeared in classical and atypical memory B cell compartments in African children and adults compared with United States adults. The levels of 9G4+ IgG increased following acute febrile malaria but did not increase with age as humoral immunity is acquired or correlate with protection from acute disease. This was the case, even though a portion of 9G4+ B cells acquired phenotypes of atypical and classical memory B cells and 9G4+ IgG contained equivalent numbers of somatic hypermutations compared with all other VHs, a characteristic of secondary Ab repertoire diversification in response to Ag stimulation. Determining the origin and function of 9G4+ B cells and 9G4+ IgG in malaria may contribute to a better understanding of the varied roles of autoreactivity in infectious diseases.

Antibody-Array-Based Proteomic Screening of Serum Markers in Systemic Lupus Erythematosus: A Discovery Study.

A discovery study was carried out where serum samples from 22 systemic lupus erythematosus (SLE) patients and matched healthy controls were hybridized to antibody-coated glass slide arrays that interrogated the level of 274 human proteins. On the basis of these screens, 48 proteins were selected for ELISA-based validation in an independent cohort of 28 SLE patients. Whereas AXL, ferritin, and sTNFRII were significantly elevated in patients with active lupus nephritis (LN) relative to SLE patients who were quiescent, other molecules such as OPN, sTNFRI, sTNFRII, IGFBP2, SIGLEC5, FAS, and MMP10 exhibited the capacity to distinguish SLE from healthy controls with ROC AUC exceeding 90%, all with p < 0.001 significance. These serum markers were next tested in a cohort of 45 LN patients, where serum was obtained at the time of renal biopsy. In these patients, sTNFRII exhibited the strongest correlation with eGFR (r = -0.50, p = 0.0014) and serum creatinine (r = 0.57, p = 0.0001), although AXL, FAS, and IGFBP2 also correlated with these clinical measures of renal function. When concurrent renal biopsies from these patients were examined, serum FAS, IGFBP2, and TNFRII showed significant positive correlations with renal pathology activity index, while sTNFRII displayed the highest correlation with concurrently scored renal pathology chronicity index (r = 0.57, p = 0.001). Finally, in a longitudinal cohort of seven SLE patients examined at ∼3 month intervals, AXL, ICAM-1, IGFBP2, SIGLEC5, sTNFRII, and VCAM-1 demonstrated the ability to track with concurrent disease flare, with significant subject to subject variation. In summary, serum proteins have the capacity to identify patients with active nephritis, flares, and renal pathology activity or chronicity changes, although larger longitudinal cohort studies are warranted.

Neutrophil-mediated IFN activation in the bone marrow alters B cell development in human and murine systemic lupus erythematosus.

Inappropriate activation of type I IFN plays a key role in the pathogenesis of autoimmune disease, including systemic lupus erythematosus (SLE). In this study, we report the presence of IFN activation in SLE bone marrow (BM), as measured by an IFN gene signature, increased IFN regulated chemokines, and direct production of IFN by BM-resident cells, associated with profound changes in B cell development. The majority of SLE patients had an IFN signature in the BM that was more pronounced than the paired peripheral blood and correlated with both higher autoantibodies and disease activity. Pronounced alterations in B cell development were noted in SLE in the presence of an IFN signature with a reduction in the fraction of pro/pre-B cells, suggesting an inhibition in early B cell development and an expansion of B cells at the transitional stage. These B cell changes strongly correlated with an increase in BAFF and APRIL expression in the IFN-high BM. Furthermore, we found that BM neutrophils in SLE were prime producers of IFN-α and B cell factors. In NZM lupus-prone mice, similar changes in B cell development were observed and mediated by IFN, given abrogation in NZM mice lacking type-I IFNR. BM neutrophils were abundant, responsive to, and producers of IFN, in close proximity to B cells. These results indicate that the BM is an important but previously unrecognized target organ in SLE with neutrophil-mediated IFN activation and alterations in B cell ontogeny and selection.

AID expression in peripheral blood of children living in a malaria holoendemic region is associated with changes in B cell subsets and Epstein-Barr virus.

The development of endemic Burkitt's lymphoma (eBL) is closely associated with Epstein-Barr virus (EBV) infection and holoendemic malaria infections. The role of EBV in the development of malignancy has been studied in depth, but there is still little known about the mechanisms by which malaria affects Burkitt's lymphomagenesis. Activation induced cytidine deaminase (AID) expression is necessary for the introduction of c-myc translocations that are characteristic of BL, but a link between AID and EBV or malaria is unclear. To determine whether frequency of malaria exposure leads to increased AID expression in peripheral blood mononuclear cells (PBMC) we examined two cohorts of children in western Kenya with endemic and sporadic malaria transmission dynamics. High frequency of malaria exposure led to increased expression of AID, which coincided with decreases in the IgM(+) memory B cells. In the children from the malaria endemic region, the presence of a detectible EBV viral load was associated with higher AID expression compared to children with undetectable EBV, but this effect was not seen in children with sporadic exposure to malaria. This study demonstrates that intensity of malaria transmission correlates with AID expression levels in the presence of EBV suggesting that malaria and EBV infection have a synergistic effect on the development of c-myc translocations and BL.

High-throughput flow cytometry data normalization for clinical trials.

Flow cytometry datasets from clinical trials generate very large datasets and are usually highly standardized, focusing on endpoints that are well defined apriori. Staining variability of individual makers is not uncommon and complicates manual gating, requiring the analyst to adapt gates for each sample, which is unwieldy for large datasets. It can lead to unreliable measurements, especially if a template-gating approach is used without further correction to the gates. In this article, a computational framework is presented for normalizing the fluorescence intensity of multiple markers in specific cell populations across samples that is suitable for high-throughput processing of large clinical trial datasets. Previous approaches to normalization have been global and applied to all cells or data with debris removed. They provided no mechanism to handle specific cell subsets. This approach integrates tightly with the gating process so that normalization is performed during gating and is local to the specific cell subsets exhibiting variability. This improves peak alignment and the performance of the algorithm. The performance of this algorithm is demonstrated on two clinical trial datasets from the HIV Vaccine Trials Network (HVTN) and the Immune Tolerance Network (ITN). In the ITN data set we show that local normalization combined with template gating can account for sample-to-sample variability as effectively as manual gating. In the HVTN dataset, it is shown that local normalization mitigates false-positive vaccine response calls in an intracellular cytokine staining assay. In both datasets, local normalization performs better than global normalization. The normalization framework allows the use of template gates even in the presence of sample-to-sample staining variability, mitigates the subjectivity and bias of manual gating, and decreases the time necessary to analyze large datasets.

Increased IgD and CD27 Double Negative (DN) B cell population in pediatric onset autoimmune hepatitis.

Autoimmune hepatitis (AIH) is a chronic, inflammatory liver disease of unknown aetiology which requires lifelong immunosuppression. Most therapeutic and outcome studies of AIH have been conducted predominantly in Caucasian (European Ancestry, EA) cohorts, with the exclusion of African American (AA) patients due to inadequate sample size. It is known that AA patients have a severe phenotype of autoimmune diseases and demonstrate a poor response to conventional medical therapy. Understanding cellular and molecular pathways which determine AIH severity and progression in AA patients is likely to lead to the discovery of novel, personalised and better tolerated therapies. The aim of the study is to determine the distinct effector B cell phenotypes which contribute to disease severity and progression of AIH in AA children as compared to their EA cohorts. PBMCs were isolated from blood samples collected from patients visiting Children's Healthcare of Atlanta (CHOA) and were grouped into AA, (n = 12), EA, (n = 11) and controls (n = 12) and were processed for flow cytometry. Markers of B cell development, maturation and activation were assessed namely CD19, CD21, IgD, CD27, CD38, CD11c, CD24, CD138. AA children with AIH demonstrated an expansion of CD19 + ve, Activated Naïve (aN), (CD19+ IgD-/CD27- Double Negative (DN2) ([CD19+/IgD-/CD27++CD38++) cells. Plasmablasts were significantly higher along with Signalling Lymphocytic activation molecule F7 (SLAMF7). Unswitched memory [CD19+] IgD+CD27+ (USM) B cells were significantly contracted in AA patients with AIH. B cell phenotyping reveals a distinct profile in AA AIH patients with a major skewing towards the expansion of effector pathways which have been previously characterised in severe SLE in AA patients. These results suggest that the quantification and therapeutic target of B cell pathway could contribute substantially to the clinical approach to AIH especially in the AA population.

Distinct transcriptomes and autocrine cytokines underpin maturation and survival of antibody-secreting cells in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by multiple autoantibody types, some of which are produced by long-lived plasma cells (LLPC). Active SLE generates increased circulating antibody-secreting cells (ASC). Here, we examine the phenotypic, molecular, structural, and functional features of ASC in SLE. Relative to post-vaccination ASC in healthy controls, circulating blood ASC from patients with active SLE are enriched with newly generated mature CD19-CD138+ ASC, similar to bone marrow LLPC. ASC from patients with SLE displayed morphological features of premature maturation and a transcriptome epigenetically initiated in SLE B cells. ASC from patients with SLE exhibited elevated protein levels of CXCR4, CXCR3 and CD138, along with molecular programs that promote survival. Furthermore, they demonstrate autocrine production of APRIL and IL-10, which contributed to their prolonged in vitro survival. Our work provides insight into the mechanisms of generation, expansion, maturation and survival of SLE ASC.

Anergic responses characterize a large fraction of human autoreactive naive B cells expressing low levels of surface IgM.

B cell anergy represents an important mechanism of peripheral immunological tolerance for mature autoreactive B cells that escape central tolerance enforced by receptor editing and clonal deletion. Although well documented in mice, the extent of its participation in human B cell tolerance remains to be fully established. In this study, we characterize the functional behavior of strictly defined human naive B cells separated on the basis of their surface IgM (sIgM) expression levels. We demonstrate that cells with lower sIgM levels (IgM(lo)) are impaired in their ability to flux calcium in response to either anti-IgM or anti-IgD cross-linking and contain a significantly increased frequency of autoreactive cells compared with naive B cells with higher levels of sIgM. Phenotypically, in healthy subjects, IgM(lo) cells are characterized by the absence of activation markers, reduction of costimulatory molecules (CD19 and CD21), and increased levels of inhibitory CD22. Functionally, IgM(lo) cells display significantly weaker proliferation, impaired differentiation, and poor Ab production. In aggregate, the data indicate that hyporesponsiveness to BCR cross-linking associated with sIgM downregulation is present in a much larger fraction of all human naive B cells than previously reported and is likely to reflect a state of anergy induced by chronic autoantigen stimulation. Finally, our results indicate that in systemic lupus erythematosus patients, naive IgM(lo) cells display increased levels of CD95 and decreased levels of CD22, a phenotype consistent with enhanced activation of autoreactive naive B cells in this autoimmune disease.

SLE Antibody-Secreting Cells Are Characterized by Enhanced Peripheral Maturation and Survival Programs.

Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by multiple autoantibodies, some of which are present in high titers in a sustained, B cell-independent fashion consistent with their generation from long-lived plasma cells (LLPC). Active SLE displays high numbers of circulating antibody-secreting cells (ASC). Understanding the mechanisms of generation and survival of SLE ASC would contribute important insight into disease pathogenesis and novel targeted therapies. We studied the properties of SLE ASC through a systematic analysis of their phenotypic, molecular, structural, and functional features. Our results indicate that in active SLE, relative to healthy post-immunization responses, blood ASC contain a much larger fraction of newly generated mature CD19- CD138+ ASC similar to bone marrow (BM) LLPC. SLE ASC were characterized by morphological and structural features of premature maturation. Additionally, SLE ASC express high levels of CXCR4 and CD138, and molecular programs consistent with increased longevity based on pro-survival and attenuated pro-apoptotic pathways. Notably, SLE ASC demonstrate autocrine production of APRIL and IL-10 and experience prolonged in vitro survival. Combined, our findings indicate that SLE ASC are endowed with enhanced peripheral maturation, survival and BM homing potential suggesting that these features likely underlie BM expansion of autoreactive PC.

Syk inhibition with fostamatinib leads to transitional B lymphocyte depletion.

Cell signaling initiated by the B cell receptor is critical to normal development of B lymphocytes, most notably at the transitional B cell stage. Inhibition of this signaling pathway with the syk inhibitor, fostamatinib, has produced significant efficacy in lymphoid malignancies and autoimmune conditions. Here, we demonstrate that short-term use of fostamatinib impairs B lymphocyte development at the transitional stage without affecting mature B cell populations. Additionally, IL-10 producing B cells remained relatively constant throughout the treatment period. These findings provide insight into the mechanism of action of B cell receptor inhibition in autoimmune disease. As the development of agents targeting B cell receptor signaling proceeds, monitoring for long-term consequences as well as functional evaluation of B cell subsets may further improve our understanding of this rapidly growing class of novel agents.

Safety, immunological effects and clinical response in a phase I trial of umbilical cord mesenchymal stromal cells in patients with treatment refractory SLE.

BACKGROUND: Reports of clinical improvement following mesenchymal stromal cell (MSC) infusions in refractory lupus patients at a single centre in China led us to perform an explorative phase I trial of umbilical cord derived MSCs in patients refractory to 6 months of immunosuppressive therapy. METHODS: Six women with a SLEDAI >6, having failed standard of care therapy, received one intravenous infusion of 1×106 MSCs/kg of body weight. They maintained their current immunosuppressives, but their physician was allowed to adjust corticosteroids initially for symptom management. The clinical endpoint was an SRI of 4 with no new British Isles Lupus Activity Guide (BILAG) As and no increase in Physician Global Assessment score of >0.3 with tapering of prednisone to 10 mg or less by 20 weeks. RESULTS: Of six patients, five (83.3%; 95% CI 35.9% to 99.6%) achieved the clinical endpoint of an SRI of 4. Adverse events were minimal. Mechanistic studies revealed significant reductions in CD27IgD double negative B cells, switched memory B cells and activated naïve B cells, with increased transitional B cells in the five patients who met the endpoint. There was a trend towards decreased autoantibody levels in specific patients. Two patients had increases in their Helios+Treg cells, but no other significant T cell changes were noted. GARP-TGFß complexes were significantly increased following the MSC infusions. The B cell changes and the GARP-TGFß increases significantly correlated with changes in SLEDAI scores. CONCLUSION: This phase 1 trial suggests that umbilical cord (UC) MSC infusions are very safe and may have efficacy in lupus. The B cell and GARP-TGFß changes provide novel insight into mechanisms by which MSCs may impact disease. TRIAL REGISTRATION NUMBER: NCT03171194.

Novel human transitional B cell populations revealed by B cell depletion therapy.

Transitional cells represent a crucial step in the differentiation and selection of the mature B cell compartment. Human transitional B cells have previously been variably identified based on the high level of expression of CD10, CD24, and CD38 relative to mature B cell populations and are expanded in the peripheral blood following rituximab-induced B cell-depletion at reconstitution. In this study, we take advantage of the gradual acquisition of the ABCB1 transporter during B cell maturation to delineate refined subsets of transitional B cells, including a late transitional B cell subset with a phenotype intermediate between T2 and mature naive. This late transitional subset appears temporally following the T1 and T2 populations in the peripheral compartment after rituximab-induced B cell reconstitution (and is thus termed T3) and is more abundant in normal peripheral blood than T1 and T2 cells. The identity of this subset as a developmental intermediate between early transitional and mature naive B cells was further supported by its ability to differentiate to naive during in vitro culture. Later transitional B cells, including T2 and T3, are found at comparatively increased frequencies in cord blood and spleen but were relatively rare in bone marrow. Additional studies demonstrate that transitional B cells mature across a developmental continuum with gradual up-regulation of mature markers, concomitant loss of immature markers, and increased responsiveness to BCR cross-linking in terms of proliferation, calcium flux, and survival. The characterization of multiple transitional B cell subpopulations provides important insights into human B cell development.

Challenges and Opportunities for Consistent Classification of Human B Cell and Plasma Cell Populations.

The increasingly recognized role of different types of B cells and plasma cells in protective and pathogenic immune responses combined with technological advances have generated a plethora of information regarding the heterogeneity of this human immune compartment. Unfortunately, the lack of a consistent classification of human B cells also creates significant imprecision on the adjudication of different phenotypes to well-defined populations. Additional confusion in the field stems from: the use of non-discriminatory, overlapping markers to define some populations, the extrapolation of mouse concepts to humans, and the assignation of functional significance to populations often defined by insufficient surface markers. In this review, we shall discuss the current understanding of human B cell heterogeneity and define major parental populations and associated subsets while discussing their functional significance. We shall also identify current challenges and opportunities. It stands to reason that a unified approach will not only permit comparison of separate studies but also improve our ability to define deviations from normative values and to create a clean framework for the identification, functional significance, and disease association with new populations.

Clinical Efficacy and Safety of Baminercept, a Lymphotoxin ß Receptor Fusion Protein, in Primary Sjögren's Syndrome: Results From a Phase II Randomized, Double-Blind, Placebo-Controlled Trial.

OBJECTIVE: To evaluate the clinical efficacy and safety of baminercept, a lymphotoxin ß receptor IgG fusion protein (LTßR-Ig), for the treatment of primary Sjögren's syndrome (SS), and to explore the possible mechanisms of action of this treatment. METHODS: In this multicenter trial, 52 patients with primary SS were randomized in a 2:1 ratio to receive subcutaneous injections of 100 mg of baminercept every week for 24 weeks or matching placebo. The primary end point was the change between screening and week 24 in the stimulated whole salivary flow (SWSF) rate. Secondary end points included the European League Against Rheumatism Sjögren's Syndrome Disease Activity Index (ESSDAI), as well as measurements of select chemokines and cytokines and enumeration of peripheral blood B and T cell subsets. RESULTS: The change from baseline to week 24 in the SWSF rate was not significantly different between the baminercept and placebo treatment groups (baseline-adjusted mean change -0.01 versus 0.07 ml/minute; P = 0.332). The change in the ESSDAI during treatment was also not significantly different between the treatment groups (baseline-adjusted mean change -1.23 versus -0.15; P = 0.104). Although the incidence of adverse events was similar between the treatment groups, baminercept therapy was associated with a higher incidence of liver toxicity, including 2 serious adverse events. Baminercept also produced a significant decrease in plasma levels of CXCL13 and significant changes in the number of circulating B and T cells, consistent with its known inhibitory effects on LTßR signaling. CONCLUSION: In this trial, treatment with baminercept failed to significantly improve glandular and extraglandular disease in patients with primary SS, despite evidence from mechanistic studies showing that it blocks LTßR signaling.