FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W3089246427> ?p ?o ?g. }

• W3089246427 endingPage "82" @default.
• W3089246427 startingPage "69" @default.
• W3089246427 abstract "B-cell secretion of autoantibodies drives autoimmune diseases, including systemic lupus erythematosus and idiopathic inflammatory myositis. Few therapies are presently available for treatment of these patients, often resulting in unsatisfactory effects and helping only some of the patients. We developed a screening assay for evaluation of novel targets suspending B-cell maturation into antibody secreting cells, which could contribute to future drug development. The assay was employed for testing 43 high quality chemical probes and compounds inhibiting under-explored protein targets, using primary cells from patients with autoimmune disease. Probes inhibiting bromodomain family proteins and histone methyl transferases demonstrated abrogation of B-cell functions to a degree comparable to a positive control, the JAK inhibitor tofacitinib. Inhibition of each target rendered a specific functional cell and potential disease modifying effect, indicating specific epigenetic protein targets as potential new intervention points for future drug discovery and development efforts. B-cell secretion of autoantibodies drives autoimmune diseases, including systemic lupus erythematosus and idiopathic inflammatory myositis. Few therapies are presently available for treatment of these patients, often resulting in unsatisfactory effects and helping only some of the patients. We developed a screening assay for evaluation of novel targets suspending B-cell maturation into antibody secreting cells, which could contribute to future drug development. The assay was employed for testing 43 high quality chemical probes and compounds inhibiting under-explored protein targets, using primary cells from patients with autoimmune disease. Probes inhibiting bromodomain family proteins and histone methyl transferases demonstrated abrogation of B-cell functions to a degree comparable to a positive control, the JAK inhibitor tofacitinib. Inhibition of each target rendered a specific functional cell and potential disease modifying effect, indicating specific epigenetic protein targets as potential new intervention points for future drug discovery and development efforts. AT A GLANCE COMMENTARYSundström Y, et al.BackgroundTo meet the unmet medical need for patients suffering from B-cell driven chronic autoimmune diseases, we have developed a phenotypic screening method based on patient derived cells. This method is probing the role of novel targets on end-stage B-cell maturation processes, with inhibition of antibody production as primary endpoint.Translational SignificanceThis phenotypic screening method has the potential to be used in future personalized medicine strategies for immune mediated diseases, using peripheral blood mononuclear cells from patients or from healthy donors. We here screen 43 chemical probes and compounds, and identify potential novel targets for development of future B-cell therapy. B cells and autoantibodies are important drivers of autoimmune inflammatory diseases. Autoantibodies are central in the pathology of systemic lupus erythematosus (SLE) and are postulated to drive inflammation also in idiopathic inflammatory myositis (IIM).1Mammen N. Narasimhan S. de Gironcoli S. Tuning the morphology of gold clusters by substrate doping.J Am Chem Soc. 2011; 133: 2801-2803Crossref PubMed Scopus (50) Google Scholar Yet, B cells are also essential in regulating immune reactions by presenting antigens to T cells and by secreting immune modulatory cytokines. SLE patients can display a range of autoantibodies to nuclear antigens including DNA, ribonucleoprotein, and chromatin proteins. IgG-autoantigen immune complexes have been hypothesized to be immune-stimulatory by activation via Fc2Nagelkerke S.Q. Schmidt D.E. de Haas M. et al.Genetic Variation in Low-To-Medium-Affinity Fcgamma Receptors: Functional Consequences, Disease Associations, and Opportunities for Personalized Medicine.Front Immunol. 2019; 10: 2237Crossref PubMed Scopus (39) Google Scholar and toll-like receptor (TLR)3Devarapu S.K. Anders H.J. Toll-like receptors in lupus nephritis.J Biomed Sci. 2018; 25: 35Crossref PubMed Scopus (39) Google Scholar pathways, and are deposited in tissues causing activation of complement. Peripheral blood B cells in SLE have been reported to be phenotypically and functionally abnormal in several ways4Jenks S.A. Cashman K.S. Zumaquero E. et al.Distinct Effector B Cells Induced by Unregulated Toll-like Receptor 7 Contribute to Pathogenic Responses in Systemic Lupus Erythematosus.Immunity. 2018; 49 (725-39 e6)Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar,5Jenks S.A. Cashman K.S. Woodruff M.C. et al.Extrafollicular responses in humans and SLE.Immunol Rev. 2019; 288: 136-148Crossref PubMed Scopus (93) Google Scholar; for example, there is an increased frequency of double negative memory B cells lacking CD27 in circulation.6Jacobi A.M. Reiter K. Mackay M. et al.Activated memory B cell subsets correlate with disease activity in systemic lupus erythematosus: delineation by expression of CD27, IgD, and CD95.Arthritis Rheum. 2008; 58: 1762-1773Crossref PubMed Scopus (211) Google Scholar,7Wei C. Anolik J. Cappione A. et al.A new population of cells lacking expression of CD27 represents a notable component of the B cell memory compartment in systemic lupus erythematosus.J Immunol. 2007; 178: 6624-6633Crossref PubMed Scopus (380) Google Scholar These IgD−CD27− double negative memory B cells are associated with disease activity as well as with renal disease in SLE.7Wei C. Anolik J. Cappione A. et al.A new population of cells lacking expression of CD27 represents a notable component of the B cell memory compartment in systemic lupus erythematosus.J Immunol. 2007; 178: 6624-6633Crossref PubMed Scopus (380) Google Scholar. In addition, extrafollicular maturation processes of B cells have, in mouse models, been shown to have pathogenic relevance in different phases of autoimmune disease.5Jenks S.A. Cashman K.S. Woodruff M.C. et al.Extrafollicular responses in humans and SLE.Immunol Rev. 2019; 288: 136-148Crossref PubMed Scopus (93) Google Scholar These phenotypic and functional alterations strongly suggest B cells as main drivers of autoimmune pathology in SLE. Consequently, several B-cell targeting therapies have been evaluated and some have moved forward to clinical development.8Hofmann K. Clauder A.K. Manz R.A. Targeting B Cells and Plasma Cells in Autoimmune Diseases.Front Immunol. 2018; 9: 835Crossref PubMed Scopus (147) Google Scholar B-cell depletion by the anti-CD20 targeting monoclonal rituximab is approved for treatment of patients with rheumatoid arthritis and antineutrophil cytoplasmic antibody-vasculitis and is commonly used off-lable in several autoimmune conditions, but failed to meet the efficacy end-point in SLE. In contrast, blocking B-cell activating factor (BAFF, Belimumab), reducing B-cell survival, represents the only biological drug that is currently approved for treatment of SLE. Blocking BAFF mainly targets mature B cells, but other inhibitors are under clinical development for A proliferation-inducing ligand (APRIL) and its receptor that could also target long-lived plasma cells. Other strategies include monoclonal antibodies and chimeric antigen receptor-T-cells targeting several B-cell subtypes (CD19, CD20, and CD22 expressing cells) while sparing mature long-lived antibody-secreting plasma cells. Recently, bortezomib, a small molecule proteasome inhibitor, was shown to deplete plasma cells and ameliorate refractory SLE,9Alexander T. Sarfert R. Klotsche J. et al.The proteasome inhibitior bortezomib depletes plasma cells and ameliorates clinical manifestations of refractory systemic lupus erythematosus.Ann Rheum Dis. 2015; 74: 1474-1478Crossref PubMed Scopus (231) Google Scholar although clinical development is hampered by association with adverse events.10Ishii T. Tanaka Y. Kawakami A. et al.Multicenter double-blind randomized controlled trial to evaluate the effectiveness and safety of bortezomib as a treatment for refractory systemic lupus erythematosus.Mod Rheumatol. 2018; 28: 986-992Crossref PubMed Scopus (29) Google Scholar Other approaches, such as CD40 and CD40L blocking therapies, may also prove useful in regulating B-cell functions in autoimmune diseases. While these efforts are promising, there is presently only a few therapies available for the treatment of patients with SLE and IIM, often resulting in partial responses and only helping a fraction of the patients. Hence, there is an unmet medical need in B-cell driven autoimmune diseases, including SLE and IIM. Patient derived primary cells (eg, xenografts, organoids, tissue slice cultures, short term cell lines) have been explored in vitro for new exploratory treatment strategies for cancer, however such approaches are not commonly used in drug discovery efforts for chronic inflammatory conditions. Instead, drug development mainly in SLE but also to some degree in IIM (inclusion body myositis and polymyositis) depends primarily on studies of animal models. The clinical relevance of animal models can be questioned,11Seok J. Warren H.S. Cuenca A.G. et al.Genomic responses in mouse models poorly mimic human inflammatory diseases.Proc Natl Acad Sci U S A. 2013; 110: 3507-3512Crossref PubMed Scopus (2031) Google Scholar and we believe that phenotypic screens using primary cells from patients are better suited for translational research from bed to benchside and then back to bed, for translational target evaluation. The Structural Genomics Consortium (SGC, www.thesgc.org), a non–profit public-private organization that aim to accelerate discovery of new medicine through open research, has during the last decade developed chemical probes in collaboration with partners from the pharmaceutical industry. These small inhibitory molecules are selective, potent, and are made available to the scientific communitywithout restrictions on research use.12Arrowsmith C.H. Audia J.E. Austin C. et al.The promise and peril of chemical probes.Nat Chem Biol. 2015; 11: 536-541Crossref PubMed Scopus (529) Google Scholar The SGC probes target mainly epigenetic regulators,13Scheer S. Ackloo S. Medina T.S. et al.A chemical biology toolbox to study protein methyltransferases and epigenetic signaling.Nat Commun. 2019; 10: 19Crossref PubMed Scopus (62) Google Scholar,14Wu Q. Heidenreich D. Zhou S. et al.A chemical toolbox for the study of bromodomains and epigenetic signaling.Nat Commun. 2019; 10: 1915Crossref PubMed Scopus (60) Google Scholar but also other proteins with critical immune functionality are contained within this collection (https://www.thesgc.org/chemical-probes). In this study, our aim was to screen these chemical probes in a patient-derived primary B-cell assay for functions relevant in autoimmune diseases (SLE and IIM). Thus, we developed an assay using peripheral blood mononuclear cells (PBMCs) from patients and healthy donors and investigated the effect of 43 probes and compounds on induced B-cell maturation and antibody secretion. Our results show that 9 of these novel probes, which inhibit bromodomain family proteins and histone methyl transferases, demonstrate abrogation of B-cell functions to a degree comparable to a positive control, the JAK inhibitor tofacitinib. We also discuss small-molecule targeting of epigenetic regulators as a potiential B-cell modulating therapeutic strategy in systemic autoimmune disease. Peripheral blood was collected from 20 blood donors including 5 patients diagnosed with SLE and fulfilling the 1982 revised American College of Rheumatology (ACR) classification criteria for SLE15Tan E.M. Cohen A.S. Fries J.F. et al.The 1982 revised criteria for the classification of systemic lupus erythematosus.Arthritis Rheum. 1982; 25: 1271-1277Crossref PubMed Scopus (12455) Google Scholar and/or the SLICC-2012 criteria16Petri M. Orbai A.M. Alarcon G.S. et al.Derivation and validation of the Systemic Lupus International Collaborating Clinics classification criteria for systemic lupus erythematosus.Arthritis Rheum. 2012; 64: 2677-2686Crossref PubMed Scopus (1) Google Scholar (SLE, age 36–73 years, all females, disease duration 4–36 years), 8 with IIM (7 with polymyositis and 1 with dermatomyositis, age 36–74 years, 6 females, disease duration 5–19 years) and 7 healthy donors (anonymized blood donors, age and gender unknown). All patients provided informed consent and the study was approved by the Stockholm ethical review board, in line with the Code of Ethics of the World Medical Association (Declaration of Helsinki). Two of the IIM and one of the SLE patients were untreated. Ongoing treatment of others included corticosteroids (4 IIM and 3 SLE patients), methotrexate (2 SLE), hydroxychloroquine (2 SLE), mycophenolic acid (2 IIM), azathioprine (1 IIM), belimumab (1 SLE) and rituximab (1 IIM). All patients attended the Rheumatology clinic at the Karolinska University Hospital, Stockholm, Sweden. A set of 43 chemical probes inhibiting epigenetic regulators, protein kinases and other proteins were tested in this study. The chemical probes are small molecules and met the following criteria: In vitro potency of <100 nM, >30-fold selectivity vs other subfamilies, demonstration of on-target effect in cells at 1 µM (detailed information is available at https://www.thesgc.org/chemical-probes). To validate probe effects, negative control compunds were used when available. Negative (inactive) controls have a similar chemical structure as active probes (Fig 6, B) but do not bind to the primary targets. PBMCs were isolated from heparinized blood or citrated buffy coat preparations by gradient centrifugation using Ficoll-Paque PLUS density gradient media (GE Healthcare Bioscience). After washing the PBMCs with the 1x Dulbecco's Phosphate Buffered Saline (DPBS) without calcium and magnesium (Sigma Aldrich), cells were suspended in Roswell Park Memorial Institute (RPMI) media supplemented with 10% heat-inactivated foetal calf serum (Sigma Aldrich), 1% L-glutamine (2 mM; Sigma Aldrich) and Penicillin-Streptomycin (10 mL/L; Sigma Aldrich) to a final concentration of 106 cells/mL. A final volume of 200 μl/well were seeded in 96 well round-bottom plates (Sarstedt). Probes and compounds diluted to 1μM (1:10,000) or 0,1μM (1:100,000) in dimethyl sulphoxide (DMSO), or only DMSO (0,01%, v/v) were added and plates were incubated 30 minutes at 37°C. Soluble rhCD40L (final concentration 500 ng/mL, R&D Systems), rhIL-21 (final concentration 50 ng/mL, R&D Systems), rhIL-10 (final concentration 200 ng/mL, R&D Systems), rhIL-4 (final concentration 200 ng/mL, R&D Systems) and CpG ODN2006 (final concentration 1 μg/mL, Miltenyi Biotec) were added, and plates were further incubated at 37°C for 6 days. Cells and supernatant were collected for subsequent analysis by flow cytometry and enzyme-linked immunosorbent assay (ELISA, IgG and IgM), respectively. In a separate set of experiments, PBMCs were cultured for 6 days in presence or absence of stimulation cocktail as above. On day 6 of culture, cells were washed with DPBS twice, a new stimulatory cocktail, as well as chemical probes, were added to stimulated cells and plates incubated for a further 4 days. Cell culture supernatants were collected, and IgG content was determined by ELISA. Cells from triplicate wells were collected and pooled, washed twice with DPBS, stained with LIVE and/or DEAD fixable near-IR dead cell marker (ThermoFisher), and stained for surface markers. Cells were resuspended in Stabilizing Fixative buffer (BD Biosciences) before analysis by flow cytometry. Additionally, B-cell proliferation was monitored by intracellular Ki67 AF488 (BD Biosciences) using buffers in the Fix & Perm Cell Permeabilization Kit (BD Biosciences). Phenotypes of B cells before and after 6 days of culture were determined by flow cytometric analysis using mouse monoclonal antibody clones: CD19 PECy7 HIB19 (Biolegend), CD27 BV421 O323 (Biolegend), IgD FITC IA6-2 (BD Biosciences), Ki67 AF488 B56 (BD Biosciences), CD38 PerCPCy5.5 HIT2 (BD Biosciences), CD45 BV510 HI30 (BD Biosciences), and IgG APC IS11-3B2.2.3 (Miltenyi Biotec). Data of labelled cells were acquired using a Beckman Coulter Gallios instrument and FlowJo software (BD) was used for analyzing cell populations. Immunoglobulins (Ig) in culture supernatants were measured using a human IgG ELISA development kit (Mabtech) according to manufacturer's instructions, or F(ab)2 polyclonal goat anti-human IgM (Jackson Immuno Research) as capture antibody and detection with horseradish peroxidase-conjugated mouse μ-specific anti-human IgM (Southern Biotech, clone SA-DA4). IL-2, IL-6, IL-8, IL-12p40, IL-12p70, IL-13, IL-17, GM-CSF, IFN-α, IFN-γ, TNF-α from Bio-Plex Pro Human Cytokine Screening assay and IL-27, IL-35, APRIL, BAFF from Bio-plex Pro Human inflammation assay were measured in supernatant collected at day 3 and day 6 on Bio-Plex 200 following the standard protocol recommended by the manufacturer. Data was analysed using Bio-Plex Manager 6.1 with standard curve fitted using 5-parameter regression. Statistical analysis was performed using the paired t-test, Mann-Whitney U test, the Kruskal-Wallis test or Pearson's correlation. Differences were considered statistically significant if P < 0.05. We stimulated PBMC with a combination of rhIL-4, rhIL-10, rhIL21, rhsCD40L, and CpG (ODN2006) and analyzed the secretion of antibodies over time. IgG production was detected above baseline from day 4 of culture (Fig 1, A), while IgM induction started already at day 2 (Fig 1, B). Flow cytometry analysis of PBMCs with or without stimuli on day 0, 3 and 6 of the culture was used to investigate the effect of stimulation on B-cell phenotype. CD19-expressing B cells were identified as naïve (CD27−IgD+), memory (CD27+IgD−), IgG switched (surface IgG+) and plasmablasts (CD27+IgD−CD38+). The stimulation protocol increased the proportion of memory B cells and plasmablasts from day 3 of cell culture (Fig 1, C), but did not significantly affect the proportion of live B cells at day 6 of cell culture (average ± SD 70 ± 17% without stimulation, 50 ± 21% with stimulation, n = 16). Cytokine secretion was evaluated using a panel of 15 cytokines (IL-2, IL-6, IL-8, IL-12p40, IL-13, IL-17, IL-27, IL-35, TNF-α, APRIL, BAFF, IFN-α2, INF-γ, IL-12p70, GM-CSF) on day 3 and 6 of cell culture (Supplemantry Fig 1). While IL-6 secretion was induced by stimulation as detected on day 3 of culture, most other cytokines were detected at lower concentrations in stimulated compared to unstimulated conditions on day 6. GM-CSF was undetectable, and IFN-α2, INF-γ, and IL-12p70 were detected at very low levels (generally <5 pg/mL, not shown). Expression of the proliferation marker Ki67 indicate that B cells were induced to proliferate by the stimulation. A large proportion of CD19+ B cells express Ki67 upon stimulation, including both memory B cells and naïve B cells (Fig 1, E). B cells were rarely expressing Ki67 immediately upon PBMCs isolation as evidenced in both naive and memory B cells, (% Ki67+ cells n = 4 median [range]: 0.05% [0.03–0.3] and 0.2% [0.05–1.7], respectively). Interestingly, a substantial fraction of plasmablasts express Ki67 before culturing the cells (6.6% [0.45–18]) and the fraction of plasmablasts expressing Ki67 increases to the same level in both medium control and stimulated condition after 6 days of culture (17% [4.4–35] and 19% [7.7–22], respectively). Although the stimulation mainly induced proliferation of B cells, we noted a slight stimulation-induced Ki67 expression in non-B cells (CD45+CD19−) (2.8% [1.7–4.5] in medium control and 9.5% [6.7–14] in stimulated cells). The assay was thereafter used to analyze the B-cell maturation stage and response to stimulation in PBMCs isolated from 5 patients with SLE, 6 patients with IIM and7 healthy donors. Patients with SLE showed a lower total frequency of B cells among lymphocytes in freshly isolated PBMCs as compared to healthy controls (Fig 2, A), and a higher proportion of memory B cells (Fig 2, B). Despite these basal differences in B-cell phenotype, stimulation induced an accumulation and maturation of B cells in both patients and healthy donors (Fig 2, A-C), and IgG as well as IgM secretion was induced to a similarlevel (Fig 2, D). Of note, stimulation did not substantially increase the % of B cells in 3 of the 4 SLE donors (Fig 2, A). This may be due to previously described diminished responses to stimulation of B cells obtained from SLE patients.17Odendahl M. Jacobi A. Hansen A. et al.Disturbed peripheral B lymphocyte homeostasis in systemic lupus erythematosus.J Immunol. 2000; 165: 5970-5979Crossref PubMed Scopus (506) Google Scholar,18Gies V. Schickel J.N. Jung S. et al.Impaired TLR9 responses in B cells from patients with systemic lupus erythematosus.JCI Insight. 2018; 3: e96795Crossref PubMed Scopus (33) Google Scholar Normalization of secreted IgG levels to the B-cell fraction of PBMCs showed similarly that the ability of the B cells to mature and produce IgG did not differ between healthy controls and patients with SLE or IMM (data not shown). Similarly, there was no statistically significant correlation between IgG concentrations induced by stimulation and the numbers of B cells or their maturation stages in the initial PBMCs isolated on day 0 (Supplemantry Fig 2). IgG levels were low without added stimulation (average ±SD 446 ± 575 ng/mL) and was induced by addition of the stimulation cocktail (median [range] 35 [2.5–187] times the levels detected without added stimulation). Next, we sought to investigate whether our defined set of chemical probes could affect B-cell phenotype and function in healthy controls and patients. We used a panel of 43 different probes and compounds at 1 concentration (1 μM or 0.1 μM) in the above described B-cell stimulation assay. The concentration used in our studies followed recommendations by the provider, based on experiences and results from applying these probes to various cellular assays (demonstrated on-target effect and not associated with toxicity). PBMCs from the 20 blood donors presented in Fig 2, including healthy donors (n = 7) and patients with SLE (n = 5) and IIM (n = 8), were preincubated with probes before addition of stimuli and cultured for 6 days. The JAK protein kinase inhibitor tofacitinib, which has previously been shown to affect induced B cell maturation in vitro,19Rizzi M. Lorenzetti R. Fischer K. et al.Impact of tofacitinib treatment on human B-cells in vitro and in vivo.J Autoimmun. 2017; 77: 55-66Crossref PubMed Scopus (42) Google Scholar was used as a positive control. After 6 days of incubation, the secretion of IgG and IgM was measured by ELISA. In addition, cells were analysed by flow cytometry for fraction of B cells, their maturation stage, and the cell viability at day 6 of culture. The data is depicted as average fold change compared to DMSO vehicle control for each patient group (Fig 3, Supplemantry Fig 3), and absolute values in presence and absence of stimulation is shown in Fig 2. An arbitrary cutoff for probe effect was set at (average for n = 20 donors) >50% reduction of IgG secretion, without overt cytotoxic effects as analyzed by flow cytometry. Nine of the 43 chemical probes met these criteria (Fig 3) including probes inhibiting methyltransferases EZH2 (UNC1999), G9a/GLP (UNC0642 and UNC0638), WDR5 (OICR-9429), PRMT Type 1 (MS023) and PRMT5 (GSK591), as well as inhibitors of the bromodomains and extraterminal domain (BET) (PFI-1, JQ1, and Bromosporine). The addition of DMSO alone at 0,01% did not significantly affect any of the parameters measured (data not shown). None of the chemical probes showed a statistically significant increase in IgG secretion (Supplemantry Fig 3). The results from the selected 9 inhibitors and the positive control are shown for individual blood donors in Fig 3, B. There was no statistically significant difference of probe effects comparing healthy donors to patient groups, and we saw no statistically significant correlation between the percentage of B cells, the fraction of memory B cell or the fraction of plasmablasts at the time of blood sampling and the effect of the probes after 6 days of cell culture (data not shown). However, we observed an interesting tendency in that inhibitors of methyltransferases reduced the accumulation of B cells, whereas BET inhibitors tended to reduce their maturation into memory cells (Fig 3, B). All 9 probes reduced, to different degrees, the proportion of plasmablasts and IgG secretion, with similar effect as tofacitinib (Fig 3, B).Fig 3Testing chemical probes and compounds in the B-cell assay using cells from patients with SLE, IIM and healthy controls PBMCs from healthy donors (n = 7), SLE patients (n = 5), and IIM patients (n = 8) were cultured in presence of the stimulation cocktail, with the addition of indicated compounds for 6 days. The percentage of subpopulations of B cells and viablity of cells as analysed by flow cytometry and the secretion of IgG and IgM analysed by ELISA in presence of probes is shown as fold change compared to DMSO vehicle (averages of the respective blood donor group). The effect of the compounds are shown by color, where blue indicates increase compared to vehicle control and red decrease of the parameter analysed.The heat map key is set separately for ELISA (IgG and IgM) and flow cytometry (B cells, memory, IgG+ B cells, plasmablasts, viability), as the ranges differ considerably between these 2 sets of data (median (min-max) for ELISA 0.092 (0.03–5.30) and for flow cytometry 1.00 (0.12–2.30). Yellow indicates no effect of the probe compared to vehicle. The compounds were added at 1μM (0.1μM for compounds marked with *), and target proteins are listed. In (B), individual data points of a selection of the data shown in (A) are given. Each dot shows the results from 1 blood donor (blue healthy controls (HC), red SLE and green IIM), as fold changed in presence of probe compared to vehicle control for the same blood donor. Results from patients previously treated with B cell targeted therapy is marked with +. The median value for all donors is given by a line for each probe. For some probes, the efficient reduction of the number of CD19+ B cells or CD27+IgD− memory B cells resulted in missing data in the downstreams figures. Tofacitinib was included in 14 of the 20 experiments. Dotted lines indicate no effect compared to vehicle control. PBMCs, peripheral blood mononuclear cells; SLE, systemic lupus erythematosus; IIM, idiopathic inflammatory myositis.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Futhermore, since the stimulation cocktail induced proliferation of B cells (Fig 1, E) we also investigated the effect on Ki67 expression in different B-cell subtypes. Inhibition of G9a/GLP (UNC0642 and UNC0638) and PRMT5 (GSK591) reduced the proliferation of naïve B cells (Fig 4, A, P < 0.01), in line with the reduced proportion of total B cells found in presence of these probes (Fig 3, B). Reflecting the lower frequency of memory B cells observed following exposure to the probes (Figure 3b), BET inhibitors tended to affect the proliferation of memory B cells (Fig 4, B, not significant). GSK591 clearly inhibited proliferation of total B cells compared to vehicle control (median (range) Ki67-expressing cells: 17% (1.3–32) and 73% (35–87), respectively, P < 0.05), including both naïve (Fig 4, A) and memory B cells (Fig 4, B). None of the probes affected proliferation of CD19− non-B cells significantly, while GSK591 showed a strong such tendency (Fig 4, C, not significant). Next we sought to investigate whether the probes affect the functionality of already matured B cells. Hence, we added probes to PBMCs after 6 days of stimulation, allowing B cells to mature into IgG secreting plasmablasts before inhibiting the epigenetic regulators (Fig 5). IgG secretion was induced by cytokines to a level of 4232 ± 1711 ng/mL (Mean ± SD n = 6) after 6 + 4 days of stimulation (77 ± 80 ng/mL without stimulation). Bromodomain targeting probes JQ1 and PFI-1 had a clear suppressive effect on IgG secretion in already matured B cells (Fig 5), suppressing by more than 60% on average. UNC1999 showed a variable suppressive effect, while tofacitinib suppressed IgG secretion by 37%. We then investigated whether the effects by the probes on IgG secretion in the B-cell assay were due to on-target mediated effects by including negative control compounds available for JQ1, UNC1999, UNC0638, GSK591, and OICR-9429. These compounds are similar in structure to the active compounds (Fig 6, B), but do not bind and inhibit the function of the primary target protein. We were able to confirm on-target effects of GSK591, OICT-9429, and JQ1 (Fig 6, A), while UNC0638 and UNC1999 displayed off-target or scaffold effects. Targeting B cells and their differentiation into antibody-producing plasmablasts could constitute a promising treatment strategy for autoimmune diseases.8Hofmann K. Clauder A.K. Manz R.A. Targeting B Cells and Plasma Cells in Autoimmune Diseases.Front Immunol. 2018; 9: 835Crossref PubMed Scopus (147) Google Scholar In this study, we intestigated the potential of novel chemical probes targeting epigenetic regulators and thereby identified novel druggable targets with the potential to modulate B-cell functionality also in B cells from autoimmune patients. The differentiation of mature B cells into antibody-secreting cells occurs through genetic reprogramming20Boothby M.R. Hodges E. Thomas J.W. Molecular regulation of peripheral B cells and their progeny in immunity.Gene Dev. 2019; 33: 26-48Crossref PubMed Scopus (30) Google Scholar invo" @default.
• W3089246427 created "2020-10-01" @default.
• W3089246427 creator A5006790660 @default.
• W3089246427 creator A5010007630 @default.
• W3089246427 creator A5011002229 @default.
• W3089246427 creator A5015790535 @default.
• W3089246427 creator A5020622451 @default.
• W3089246427 creator A5021773969 @default.
• W3089246427 creator A5025148001 @default.
• W3089246427 creator A5037276492 @default.
• W3089246427 creator A5060818623 @default.
• W3089246427 creator A5067910913 @default.
• W3089246427 date "2021-03-01" @default.
• W3089246427 modified "2023-10-16" @default.
• W3089246427 title "Identifying novel B-cell targets for chronic inflammatory autoimmune disease by screening of chemical probes in a patient-derived cell assay" @default.
• W3089246427 cites W1485900506 @default.
• W3089246427 cites W1516646538 @default.
• W3089246427 cites W1642392901 @default.
• W3089246427 cites W1951116999 @default.
• W3089246427 cites W1967380173 @default.
• W3089246427 cites W1970854602 @default.
• W3089246427 cites W1975420246 @default.
• W3089246427 cites W1980268334 @default.
• W3089246427 cites W1984035854 @default.
• W3089246427 cites W2009754477 @default.
• W3089246427 cites W2036072927 @default.
• W3089246427 cites W2043332368 @default.
• W3089246427 cites W2053644097 @default.
• W3089246427 cites W2090510808 @default.
• W3089246427 cites W2122057816 @default.
• W3089246427 cites W2146203649 @default.
• W3089246427 cites W2146853551 @default.
• W3089246427 cites W2156049613 @default.
• W3089246427 cites W2159860161 @default.
• W3089246427 cites W2161771297 @default.
• W3089246427 cites W2266921405 @default.
• W3089246427 cites W2289400000 @default.
• W3089246427 cites W2318554958 @default.
• W3089246427 cites W2541126885 @default.
• W3089246427 cites W2547567393 @default.
• W3089246427 cites W2562869729 @default.
• W3089246427 cites W2775911035 @default.
• W3089246427 cites W2786704362 @default.
• W3089246427 cites W2792413515 @default.
• W3089246427 cites W2793218229 @default.
• W3089246427 cites W2799828163 @default.
• W3089246427 cites W2800778103 @default.
• W3089246427 cites W2801097217 @default.
• W3089246427 cites W2805529518 @default.
• W3089246427 cites W2809080525 @default.
• W3089246427 cites W2897114391 @default.
• W3089246427 cites W2899707256 @default.
• W3089246427 cites W2907480849 @default.
• W3089246427 cites W2907539128 @default.
• W3089246427 cites W2913497115 @default.
• W3089246427 cites W2922125755 @default.
• W3089246427 cites W2949150764 @default.
• W3089246427 cites W2949416082 @default.
• W3089246427 cites W2953288458 @default.
• W3089246427 cites W2954940431 @default.
• W3089246427 cites W2969794251 @default.
• W3089246427 cites W2971917030 @default.
• W3089246427 doi "https://doi.org/10.1016/j.trsl.2020.09.003" @default.
• W3089246427 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/32977027" @default.
• W3089246427 hasPublicationYear "2021" @default.
• W3089246427 type Work @default.
• W3089246427 sameAs 3089246427 @default.
• W3089246427 citedByCount "1" @default.
• W3089246427 countsByYear W30892464272022 @default.
• W3089246427 crossrefType "journal-article" @default.
• W3089246427 hasAuthorship W3089246427A5006790660 @default.
• W3089246427 hasAuthorship W3089246427A5010007630 @default.
• W3089246427 hasAuthorship W3089246427A5011002229 @default.
• W3089246427 hasAuthorship W3089246427A5015790535 @default.
• W3089246427 hasAuthorship W3089246427A5020622451 @default.
• W3089246427 hasAuthorship W3089246427A5021773969 @default.
• W3089246427 hasAuthorship W3089246427A5025148001 @default.
• W3089246427 hasAuthorship W3089246427A5037276492 @default.
• W3089246427 hasAuthorship W3089246427A5060818623 @default.
• W3089246427 hasAuthorship W3089246427A5067910913 @default.
• W3089246427 hasBestOaLocation W30892464271 @default.
• W3089246427 hasConcept C1491633281 @default.
• W3089246427 hasConcept C159654299 @default.
• W3089246427 hasConcept C203014093 @default.
• W3089246427 hasConcept C2778453870 @default.
• W3089246427 hasConcept C2779075594 @default.
• W3089246427 hasConcept C54355233 @default.
• W3089246427 hasConcept C71924100 @default.
• W3089246427 hasConcept C86803240 @default.
• W3089246427 hasConceptScore W3089246427C1491633281 @default.
• W3089246427 hasConceptScore W3089246427C159654299 @default.
• W3089246427 hasConceptScore W3089246427C203014093 @default.
• W3089246427 hasConceptScore W3089246427C2778453870 @default.
• W3089246427 hasConceptScore W3089246427C2779075594 @default.
• W3089246427 hasConceptScore W3089246427C54355233 @default.
• W3089246427 hasConceptScore W3089246427C71924100 @default.
• W3089246427 hasConceptScore W3089246427C86803240 @default.
• W3089246427 hasFunder F4320326631 @default.

• next