Peripheral B cell abnormalities in patients with systemic lupus erythematosus in quiescent phase: Decreased memory B cells and membrane CD19 expression

Background:Belimumab (BLM), a recombinant human monoclonal antibody directed against B-cell activating factor (BAFF), is the first approved biological agent for patients with active severe systemic lupus erythematosus (SLE) and lupus nephritis (LN). There is clinical evidence that combining BLM with B cell depleting therapy can ameliorate disease activity in severe, refractory SLE patients1. Although BLM is a B cell directed therapy and has been shown to significantly decrease total B cells, flow cytometry observations suggest a rapid increase of circulating memory B cells (MBC)2.Objectives:To investigate dynamics of B-cell subsets in SLE patients treated with or without BLM, with a focus on assessing MBC characteristics.Methods:Extensive B cell subset phenotyping was performed by high-sensitivity (HS) flow cytometry (acquisition of 107 leukocytes; per EuroFlow protocols3) on samples from active LN or SLE patients with major organ involvement treated with standard of care (SOC) consisting of high dose steroids and mycophenolate mofetil combined with or without the addition of BLM. MBC gene expression profiles were characterized with single-cell RNA and V(D)J sequencing (ScRNA-SEQ).Results:By employing HS flowcytometry, we established that the absolute increase in circulating MBC in SLE and LN patients was significant for patients who initiated BLM (Figure 1). The increase was observed in a broad range of MBC subsets (Unswitched, IgG1+, IgG2+, IgA1+, IgA2+) at 2 and 4 weeks following initiation of BLM treatment. This rise in MBC could hypothetically be attributed to either proliferation of blood MBC, BLM induced migration of tissue-resident MBCs or BLM related retention of tissue-destined MBC in the blood. ScRNA-SEQ analysis of cell cycle gene-expression was performed and established in both groups a non-proliferating phenotype [in approximately ~94%] of MBC post-treatment, including absence of MKI67 as active proliferation marker. Clonal diversity analysis comparing week 2 with baseline revealed an unexpected decrease of the largest MBC clones in BLM, whereas no change in clonality was observed with SOC alone. Together these data indicate that proliferation is unlikely to be responsible for the observed increase in MBC by BLM. Furthermore, a clear difference was found in gene-expression levels between both treatment groups: BLM was responsible for the upregulation of 72 vs 10 genes in SOC, likewise 162 vs 32 genes were downregulated. Most importantly, a significant downregulation of the migration genes SELL (CD62L), CCR7, ITGB1, RAC2 and ICAM2, were specifically seen in BLM treated patients. This may reflect disrupted lymphocyte trafficking, preventing MBCs from transmigrating from the blood into tissue owing to reduced migration molecules, or preventing MBCs from being retained at the tissue level owing to reduction in tissue adhesion proteins.Conclusion:The addition of BLM to SOC significantly increases MBCs in patients with SLE independently of proliferation, accompanied by a strong modulation of gene expression, including reduced expression of migration markers pointing towards disrupted lymphocyte trafficking. These data may have important implications for improving treatment strategies in patients with LN or severe SLE, as a deeper depletion of autoreactive MBCs could be established by adding B-cell-depleting therapy after the initiation of BLM.Figure 1.Change in pre-germinal center and memory B cell counts from baseline to week 4 of patients with SLE or LN treated with SOC (n=8) or SOC+BLM (n=11).References:[1]Arends EJ et al. Long-term effects of combined B cell immunomodulation with rituximab and belimumab in severe, refractory systemic lupus erythematosus: 2-year results. Nephrol Dial Transplant. 2020 Jun 27 gfaa117.[2]Stohl W et al. Belimumab reduces autoantibodies, normalizes low complement levels, and reduces select B cell populations in patients with SLE. Arthritis Rheum. 2012;64(7):2328-2337.[3]Blanco et al, Age-associated distribution of B and plasma cell subsets in peripheral blood - J Allergy Clin Immunol 2018 141 2208-2219.Disclosure of Interests:Eline J. Arends: None declared, Mihaela Zlei: None declared, Christopher M. Tipton: None declared, Zgjim Osmani: None declared, Sylvia Kamerling: None declared, Ton Rabelink: None declared, Ignacio Sanz: None declared, Jacques J.M. van Dongen Paid instructor for: BD Biosciences: Educational Services (fees for LUMC), Consultant of: BD Biosciences and Cytognos (fees for LUMC), Grant/research support from: GSK (flow cytometry studies for GSK BLISS-BELIEVE study NCT03312907), Cees van Kooten: None declared, Y.K. Onno Teng Consultant of: Aurinia provided financial compensation for consultancy, Grant/research support from: GSK provided belimumab for free for the Synbiose-2 clinical trial and provided an unrestricted grant to conduct the study.

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OP0005 ELEVATED STAT1 EXPRESSION BUT NOT PHOSPHORYLATION IN LUPUS B CELLS CORRELATES WITH DISEASE ACTIVITY AND INCREASED PLASMABLAST SUSCEPTIBILITY

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2020-eular.2955 ◽

2020 ◽

Vol 79 (Suppl 1) ◽

pp. 4-5

Author(s):

A. Aue ◽

F. Szelinski ◽

S. Weißenberg ◽

A. Wiedemann ◽

T. Rose ◽

...

Keyword(s):

Systemic Lupus Erythematosus ◽

B Cells ◽

Autoimmune Diseases ◽

Disease Activity ◽

B Cell ◽

Lupus Erythematosus ◽

Type I ◽

Type I Ifn ◽

Systemic Lupus ◽

B Cell Subsets

Background:Systemic lupus erythematosus (SLE) is characterized by two pathogenic key signatures, type I interferon (IFN) (1.) and B-cell abnormalities (2.). How these signatures are interrelated is not known. Type I-II IFN trigger activation of Janus kinase (JAK) – signal transducer and activator of transcription (STAT).Objectives:JAK-STAT inhibition is an attractive therapeutic possibility for SLE (3.). We assess STAT1 and STAT3 expression and phosphorylation at baseline and after IFN type I and II stimulation in B-cell subpopulations of SLE patients compared to other autoimmune diseases and healthy controls (HD) and related it to disease activity.Methods:Expression of STAT1, pSTAT1, STAT3 and pSTAT3 in B and T-cells of 21 HD, 10 rheumatoid arthritis (RA), 7 primary Sjögren’s (pSS) and 22 SLE patients was analyzed by flow cytometry. STAT1 and STAT3 expression and phosphorylation in PBMCs of SLE patients and HD after IFNα and IFNγ incubation were further investigated.Results:SLE patients showed substantially higher STAT1 but not pSTAT1 in B and T-cell subsets. Increased STAT1 expression in B cell subsets correlated significantly with SLEDAI and Siglec-1 on monocytes, a type I IFN marker (4.). STAT1 activation in plasmablasts was IFNα dependent while monocytes exhibited dependence on IFNγ.Figure 1.Significantly increased expression of STAT1 by SLE B cells(A) Representative histograms of baseline expression of STAT1, pSTAT1, STAT3 and pSTAT3 in CD19+ B cells of SLE patients (orange), HD (black) and isotype controls (grey). (B) Baseline expression of STAT1 and pSTAT1 or (C) STAT3 and pSTAT3 in CD20+CD27-, CD20+CD27+ and CD20lowCD27high B-lineage cells from SLE (orange) patients compared to those from HD (black). Mann Whitney test; ****p≤0.0001.Figure 2.Correlation of STAT1 expression by SLE B cells correlates with type I IFN signature (Siglec-1, CD169) and clinical activity (SLEDAI).Correlation of STAT1 expression in CD20+CD27- näive (p<0.0001, r=0.8766), CD20+CD27+ memory (p<0.0001, r=0.8556) and CD20lowCD27high (p<0.0001, r=0.9396) B cells from SLE patients with (A) Siglec-1 (CD169) expression on CD14+ cells as parameter of type I IFN signature and (B) lupus disease activity (SLEDAI score). Spearman rank coefficient (r) was calculated to identify correlations between these parameters. *p≤0.05, **p≤0.01. (C) STAT1 expression in B cell subsets of a previously undiagnosed, active SLE patient who was subsequently treated with two dosages of prednisolone and reanalyzed.Conclusion:Enhanced expression of STAT1 by B-cells candidates as key node of two immunopathogenic signatures (type I IFN and B-cells) related to important immunopathogenic pathways and lupus activity. We show that STAT1 is activated upon IFNα exposure in SLE plasmablasts. Thus, Jak inhibitors, targeting JAK-STAT pathways, hold promise to block STAT1 expression and control plasmablast induction in SLE.References:[1]Baechler EC, Batliwalla FM, Karypis G, Gaffney PM, Ortmann WA, Espe KJ, et al. Interferon-inducible gene expression signature in peripheral blood cells of patients with severe lupus. Proc Natl Acad Sci U S A. 2003;100(5):2610-5.[2]Lino AC, Dorner T, Bar-Or A, Fillatreau S. Cytokine-producing B cells: a translational view on their roles in human and mouse autoimmune diseases. Immunol Rev. 2016;269(1):130-44.[3]Dorner T, Lipsky PE. Beyond pan-B-cell-directed therapy - new avenues and insights into the pathogenesis of SLE. Nat Rev Rheumatol. 2016;12(11):645-57.[4]Biesen R, Demir C, Barkhudarova F, Grun JR, Steinbrich-Zollner M, Backhaus M, et al. Sialic acid-binding Ig-like lectin 1 expression in inflammatory and resident monocytes is a potential biomarker for monitoring disease activity and success of therapy in systemic lupus erythematosus. Arthritis Rheum. 2008;58(4):1136-45.Disclosure of Interests:Arman Aue: None declared, Franziska Szelinski: None declared, Sarah Weißenberg: None declared, Annika Wiedemann: None declared, Thomas Rose: None declared, Andreia Lino: None declared, Thomas Dörner Grant/research support from: Janssen, Novartis, Roche, UCB, Consultant of: Abbvie, Celgene, Eli Lilly, Roche, Janssen, EMD, Speakers bureau: Eli Lilly, Roche, Samsung, Janssen

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THU0035 OX40L EXPRESSING NEUTROPHILS INDUCE CD4 T FOLLICULAR AND PERIPHERAL HELPER CELL DIFFERENTIATION IN SYSTEMIC LUPUS ERYTHEMATOSUS

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2020-eular.2739 ◽

2020 ◽

Vol 79 (Suppl 1) ◽

pp. 230.2-231

Author(s):

A. Pappalardo ◽

E. Wojciechowski ◽

I. Odriozola ◽

I. Douchet ◽

N. Merillon ◽

...

Keyword(s):

Systemic Lupus Erythematosus ◽

T Cells ◽

B Cells ◽

Lupus Erythematosus ◽

Cd4 T Cells ◽

Close Contact ◽

Memory B Cells ◽

Systemic Lupus ◽

Research Support

Background:Neutrophils have been described as potent antigen-presenting cells able to activate T cells by MHC/TCR interaction and costimulatory molecules in tumor immunity. However, little is known about the direct interaction between neutrophils and CD4 T cells with respect to systemic lupus erythematosus (SLE). We have previously shown that OX40L expressed by monocytes from SLE patients promote the differentiation of naïve and memory cells into IL21 secreting T cells that are able to help B cells1,2.Objectives:In this study, we investigate OX40L expression on neutrophils from SLE patients and contribution of these OX40L+neutrophils in SLE pathogenesis to modulation of the B cell helper role of CD4 T cells.Methods:Surface expression of co-stimulatory molecules (OX40L, ICOSL, GITRL, 4-1BBL) on neutrophils from SLE patients and healthy donors (HD) was measured by flow cytometry (FC). Neutrophils from HD were stimulated with TLR7 or TLR8 agonists and IFNα after 5 hours of culture, OX40L expression was measured by FC and Western Blotting. CD4 T cells were cultured with the stimulated neutrophils for 3 days. At the end of the co-culture, percentages of IL21-expressing T follicular (Tfh) and peripheral helper (Tph) cells measured by FC. These generated T cells were also cultured in the presence of memory B cells. After 5 days of co-culture, plasmablast generation and Ig levels were assessed by FC and ELISA, respectively. Inhibition of OX40-OX40L interaction in vitro was achieved using ISB 830, a novel anti-OX40 mAb currently used in clinical trials.Results:Among the co-stimulatory molecules tested, percentages of OX40L+neutrophils in SLE (n=54) were increased compared to HD (n=25)(mean + SD: HD = 1,34%±1.62 vs SLE = 4,53%±8.1; p=0.29). OX40L expression positively correlated with SLE disease activity score (SLEDAI) (p = 0,04; r = 0,31) and with anti-DNA antibodies (p= 0,04, r = 0,33). Of note, the percentage of OX40L+neutrophils was higher in anti-sm-RNP+patients (n=16, mean= 9%±9.8), compared to anti-sm-RNP-patients (n=27, mean = 1,4%±2.5; p = 0,02). The percentage of OX40L+neutrophils was higher in patients with class III or IV lupus nephritis, and inflammatory infiltrate within the kidney biopsy disclosed OX40L+neutrophils, in close contact with T cells. Neutrophils from HD express OX40L with TLR8 agonist, or IFNα priming followed by TLR7 agonist. When memory CD4 T cells were cultured in the presence of TLR8-stimulated neutrophils, the proportion of IL21-expressing Tfh (CXCR5+PD1+) and Tph (CXCR5-PD1hi) were increased, compared to culture with unstimulated neutrophils. This process was dependent on OX40-OX40L interactions, since in vitro treatment with the anti-OX40 blocking antibody ISB 830, inhibited the differentiation of memory T cells into Tfh and Tph. Both generated Tfh and Tph were able to promote the differentiation of memory B cells into Ig-secreting plasmablasts.Conclusion:Our results disclose an unprecedented phenomenon where cross-talk between TLR7/8-activated neutrophils and CD4 lymphocytes operates through OX40L-OX40 costimulation, and neutrophils promote the differentiation of pro-inflammatory Tfh and Tph, as well as IL21 production. Therefore, OX40L/OX40 should be considered as a potentially therapeutic axis in SLE patients.References:[1]Jacquemin et al. Immunity 2015;[2]Jacquemin et al. JCI Insight 2018Disclosure of Interests:Angela Pappalardo Grant/research support from: Ichnos Sciences, Elodie Wojciechowski: None declared, Itsaso Odriozola: None declared, Isabelle Douchet: None declared, Nathalie Merillon: None declared, Andrea Boizard-Moracchini: None declared, Pierre Duffau: None declared, Estibaliz Lazaro: None declared, Marie-Agnes Doucey Employee of: Ichnos Sciences, Lamine Mbow Employee of: Ichnos Sciences, Christophe Richez Consultant of: Abbvie, Amgen, Mylan, Pfizer, Sandoz and UCB., Patrick Blanco Grant/research support from: Ichnos Sciences

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The expansion of activated naive DNA autoreactive B cells and its association with disease activity in systemic lupus erythematosus patients

Arthritis Research & Therapy ◽

10.1186/s13075-021-02557-0 ◽

2021 ◽

Vol 23 (1) ◽

Author(s):

Kittikorn Wangriatisak ◽

Chokchai Thanadetsuntorn ◽

Thamonwan Krittayapoositpot ◽

Chaniya Leepiyasakulchai ◽

Thanitta Suangtamai ◽

...

Keyword(s):

Systemic Lupus Erythematosus ◽

B Cells ◽

Disease Activity ◽

B Cell ◽

Lupus Erythematosus ◽

Systemic Lupus ◽

Autoreactive B Cells ◽

Dsdna Antibody ◽

Cell Subpopulations ◽

B Cell Subsets

Abstract Background Autoreactive B cells are well recognized as key participants in the pathogenesis of systemic lupus erythematosus (SLE). However, elucidating the particular subset of B cells in producing anti-dsDNA antibodies is limited due to their B cell heterogeneity. This study aimed to identify peripheral B cell subpopulations that display autoreactivity to DNA and contribute to lupus pathogenesis. Methods Flow cytometry was used to detect total B cell subsets (n = 20) and DNA autoreactive B cells (n = 15) in SLE patients’ peripheral blood. Clinical disease activities were assessed in SLE patients using modified SLEDAI-2 K and used for correlation analyses with expanded B cell subsets and DNA autoreactive B cells. Results The increases of circulating double negative 2 (DN2) and activated naïve (aNAV) B cells were significantly observed in SLE patients. Expanded B cell subsets and DNA autoreactive B cells represented a high proportion of aNAV B cells with overexpression of CD69 and CD86. The frequencies of aNAV B cells in total B cell populations were significantly correlated with modified SLEDAI-2 K scores. Further analysis showed that expansion of aNAV DNA autoreactive B cells was more related to disease activity and serum anti-dsDNA antibody levels than to total aNAV B cells. Conclusion Our study demonstrated an expansion of aNAV B cells in SLE patients. The association between the frequency of aNAV B cells and disease activity patients suggested that these expanded B cells may play a role in SLE pathogenesis.

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Multiple Functions of B Cells in the Pathogenesis of Systemic Lupus Erythematosus

International Journal of Molecular Sciences ◽

10.3390/ijms20236021 ◽

2019 ◽

Vol 20 (23) ◽

pp. 6021 ◽

Cited By ~ 3

Author(s):

Kongyang Ma ◽

Wenhan Du ◽

Xiaohui Wang ◽

Shiwen Yuan ◽

Xiaoyan Cai ◽

...

Keyword(s):

Systemic Lupus Erythematosus ◽

B Cells ◽

B Cell ◽

Lupus Erythematosus ◽

Therapeutic Interventions ◽

Systemic Lupus ◽

Autoantibody Production ◽

Regulatory B Cell ◽

Functional Features ◽

B Cell Subsets

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by excessive autoantibody production and multi-organ involvement. Although the etiology of SLE still remains unclear, recent studies have characterized several pathogenic B cell subsets and regulatory B cell subsets involved in the pathogenesis of SLE. Among pathogenic B cell subsets, age-associated B cells (ABCs) are a newly identified subset of autoreactive B cells with T-bet-dependent transcriptional programs and unique functional features in SLE. Accumulation of T-bet+ CD11c+ ABCs has been observed in SLE patients and lupus mouse models. In addition, innate-like B cells with the autoreactive B cell receptor (BCR) expression and long-lived plasma cells with persistent autoantibody production contribute to the development of SLE. Moreover, several regulatory B cell subsets with immune suppressive functions have been identified, while the impaired inhibitory effects of regulatory B cells have been indicated in SLE. Thus, further elucidation on the functional features of B cell subsets will provide new insights in understanding lupus pathogenesis and lead to novel therapeutic interventions in the treatment of SLE.

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Glucocorticoid-induced leucine zipper (GILZ) inhibits B cell activation in systemic lupus erythematosus

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2015-207744 ◽

2015 ◽

Vol 75 (4) ◽

pp. 739-747 ◽

Cited By ~ 21

Author(s):

Sarah A Jones ◽

Andrew E J Toh ◽

Dragana Odobasic ◽

Marie-Anne Virginie Oudin ◽

Qiang Cheng ◽

...

Keyword(s):

Systemic Lupus Erythematosus ◽

B Cells ◽

B Cell ◽

Lupus Erythematosus ◽

Leucine Zipper ◽

Germinal Centre ◽

Cell Phenotype ◽

Systemic Lupus ◽

Human B Cells

ObjectivesSystemic lupus erythematosus (SLE) is a serious multisystem autoimmune disease, mediated by disrupted B cell quiescence and typically treated with glucocorticoids. We studied whether B cells in SLE are regulated by the glucocorticoid-induced leucine zipper (GILZ) protein, an endogenous mediator of anti-inflammatory effects of glucocorticoids.MethodsWe conducted a study of GILZ expression in blood mononuclear cells of patients with SLE, performed in vitro analyses of GILZ function in mouse and human B cells, assessed the contributions of GILZ to autoimmunity in mice, and used the nitrophenol coupled to keyhole limpet haemocyanin model of immunisation in mice.ResultsReduced B cell GILZ was observed in patients with SLE and lupus-prone mice, and impaired induction of GILZ in patients with SLE receiving glucocorticoids was associated with increased disease activity. GILZ was downregulated in naïve B cells upon stimulation in vitro and in germinal centre B cells, which contained less enrichment of H3K4me3 at the GILZ promoter compared with naïve and memory B cells. Mice lacking GILZ spontaneously developed lupus-like autoimmunity, and GILZ deficiency resulted in excessive B cell responses to T-dependent stimulation. Accordingly, loss of GILZ in naïve B cells allowed upregulation of multiple genes that promote the germinal centre B cell phenotype, including lupus susceptibility genes and genes involved in cell survival and proliferation. Finally, treatment of human B cells with a cell-permeable GILZ fusion protein potently suppressed their responsiveness to T-dependent stimuli.ConclusionsOur findings demonstrated that GILZ is a non-redundant regulator of B cell activity, with important potential clinical implications in SLE.

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Belimumab alters transitional B-cell subset proportions in patients with stable systemic lupus erythematosus

Lupus ◽

10.1177/0961203319869468 ◽

2019 ◽

Vol 28 (11) ◽

pp. 1337-1343 ◽

Cited By ~ 1

Author(s):

A Benitez ◽

K Torralba ◽

M Ngo ◽

L M Salto ◽

K S Choi ◽

...

Keyword(s):

Systemic Lupus Erythematosus ◽

B Cells ◽

B Cell ◽

Lupus Erythematosus ◽

Mononuclear Cells ◽

Cell Subset ◽

Systemic Lupus ◽

Peripheral Blood Mononuclear ◽

Healthy Donors ◽

Immature B Cells

Objective We evaluated the effects of the B-cell activating factor (BAFF)-targeting antibody Belimumab on human nonmemory B-cell pools. Human B-cell pools were identified using surface markers adapted from mouse studies that specifically assessed reductions in immature B cells due to BAFF depletion. Patients with systemic lupus erythematosus (SLE) have high levels of both BAFF and immature B cells. Mechanistic mouse studies provide a framework for understanding human responses to therapies that target B cells. Methods Peripheral blood mononuclear cells were isolated from healthy donors and SLE patients on Belimumab or standard-of-care therapy (SCT). Cells were stained for flow cytometry to identify B-cell subsets based on CD21/CD24. Differences in subset proportions were determined by one-way ANOVA and Tukey’s post hoc test. Results Patients treated with Belimumab show alterations in the nonmemory B-cell pool characterized by a decrease in the Transitional 2 (T2) subset ( p = 0.002), and an increase in the proportion of Transitional 1 (T1) cells ( p = 0.005) as compared with healthy donors and SCT patients. The naïve B-cell compartment showed no significant differences between the groups ( p = 0.293). Conclusion Using a translational approach, we show that Belimumab-mediated BAFF depletion reduces the T2 subset in patients, similar to observations in mouse models with BAFF depletion.

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