Publications by authors named "Rudi W Hendriks"

154 Publications

Aberrant B Cell Receptor Signaling in Naïve B Cells from Patients with Idiopathic Pulmonary Fibrosis.

Cells 2021 May 26;10(6). Epub 2021 May 26.

Department of Pulmonary Medicine, Erasmus MC, University Medical Center, 3015 GD Rotterdam, The Netherlands.

Idiopathic pulmonary fibrosis (IPF) is a chronic and ultimately fatal disease in which an impaired healing response to recurrent micro-injuries is thought to lead to fibrosis. Recent findings hint at a role for B cells and autoimmunity in IPF pathogenesis. We previously reported that circulating B cells from a fraction of patients, compared with healthy controls, express increased levels of the signaling molecule Bruton's tyrosine kinase (BTK). However, it remains unclear whether B cell receptor (BCR) signaling is altered in IPF. Here, we show that the response to BCR stimulation is enhanced in peripheral blood B cells from treatment-naïve IPF patients. We observed increased anti-immunoglobulin-induced phosphorylation of BTK and its substrate phospholipase Cγ2 (PLCγ2) in naïve but not in memory B cells of patients with IPF. In naïve B cells of IPF patients enhanced BCR signaling correlated with surface expression of transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI) but not B cell activating factor receptor (BAFFR), both of which provide pro-survival signals. Interestingly, treatment of IPF patients with nintedanib, a tyrosine kinase inhibitor with anti-fibrotic and anti-inflammatory activity, induced substantial changes in BCR signaling. These findings support the involvement of B cells in IPF pathogenesis and suggest that targeting BCR signaling has potential value as a treatment option.
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http://dx.doi.org/10.3390/cells10061321DOI Listing
May 2021

Involvement of Dendritic Cells and Th17 Cells in Induced Tertiary Lymphoid Structures in a Chronic Beryllium Disease Mouse Model.

Mediators Inflamm 2021 6;2021:8845966. Epub 2021 May 6.

Department of Pulmonary Medicine, Erasmus University Medical Center (Erasmus MC), Dr. Molewaterplein 50, 3015 GE Rotterdam, Netherlands.

Objective: To study airway pathophysiology and the role of dendritic cells (DCs) and IL-17 receptor (IL-17R) signals in a mouse model for CBD.

Methods: Here, we present a CBD mouse model in which mice were exposed to beryllium during three weeks. We also exposed IL-17R-deficient mice and mice in which DCs were depleted.

Results: Eight weeks after the initial beryllium exposure, an inflammatory response was detected in the lungs. Mice displayed inflammation of the lower airways that included focal dense infiltrates, granuloma-like foci, and tertiary lymphoid structure (TLS) containing T cells, B cells, and germinal centers. Alveolar cell analysis showed significantly increased numbers of CD4 T cells expressing IFN, IL-17, or both cytokines. The pathogenic role of IL-17R signals was demonstrated in IL-17R-deficient mice, which had strongly reduced lung inflammation and TLS development following beryllium exposure. In CBD mice, pulmonary DC subsets including CD103 conventional DCs (cDCs), CD11b cDCs, and monocyte-derived DCs (moDCs) were also prominently increased. We used diphtheria toxin receptor-mediated targeted cell ablation to conditionally deplete DCs and found that DCs are essential for the maintenance of TLS in CBD. Furthermore, the presence of antinuclear autoantibodies in the serum of CBD mice showed that CBD had characteristics of autoimmune disease.

Conclusions: We generated a translational model of sarcoidosis driven by beryllium and show that DCs and IL-17R signals play a pathophysiological role in CBD development as well as in established CBD in vivo.
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http://dx.doi.org/10.1155/2021/8845966DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8123089PMC
May 2021

Loss of immune homeostasis in patients with idiopathic pulmonary arterial hypertension.

Thorax 2021 May 7. Epub 2021 May 7.

Department of Pulmonary Medicine, Erasmus Universiteit Rotterdam, Rotterdam, The Netherlands

Introduction: Autoreactivity against pulmonary vascular structures is thought to be involved in idiopathic pulmonary arterial hypertension (IPAH), but the underlying mechanisms remain poorly understood. We hypothesised that aberrant B-cell activation contributes to IPAH aetiology.

Methods: Mice with enhanced B-cell activation due to B-cell-specific overexpression of the B-cell receptor (BCR) signalling molecule Bruton's tyrosine kinase (BTK) were subjected to lung injury and examined for several pulmonary hypertension (PH) indices. Peripheral blood lymphocytes from patients with IPAH (n=13), connective tissue disease-associated PAH (CTD-PAH, n=9), congenital heart disease PAH (n=7), interstitial lung disease associated PH (n=17) and healthy controls (n=19) were characterised by 14-colour flow cytometry.

Results: Following pulmonary injury, BTK-overexpressing mice showed prolonged activation of B cells and CXCR5 follicular T-helper (Tfh) cells, as well as features of PH development. Patients with CTD-PAH and CHD-PAH displayed reduced proportions of circulating non-switched-memory B cells (p=0.03, p=0.02, respectively). Interestingly, we observed increased BTK protein expression in naive (p=0.007) and memory B-cell subsets of patients with IPAH and CTD-PAH. BTK was particularly high in patients with IPAH with circulating autoantibodies (p=0.045). IPAH patients had low frequencies of circulating CXCR5 Tfh cells (p=0.005). Hereby, the increased BTK protein expression in B cells was associated with high proportions of Tfh17 (p=0.018) and Tfh17.1 (p=0.007) cells within the circulating Tfh population.

Conclusions: Our study shows that pulmonary injury in combination with enhanced B-cell activation is sufficient to induce PH symptoms in mice. In parallel, immune homeostasis in patients with IPAH is compromised, as evidenced by increased BCR signalling and cTfh17 polarisation, indicating that adaptive immune activation contributes to IPAH disease induction or progression.
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http://dx.doi.org/10.1136/thoraxjnl-2020-215460DOI Listing
May 2021

A Versatile Protocol to Quantify BCR-mediated Phosphorylation in Human and Murine B Cell Subpopulations.

Bio Protoc 2021 Feb 5;11(3):e3902. Epub 2021 Feb 5.

Department of Pulmonary Medicine, Erasmus MC Rotterdam, The Netherlands.

Signal transduction is the process by which molecular signals are transmitted from the cell surface to its interior, resulting in functional changes inside the cell. B cell receptor (BCR) signaling is of crucial importance for B cells, as it regulates their differentiation, selection, survival, cellular activation and proliferation. Upon BCR engagement by antigen several protein kinases, lipases and linker molecules become phosphorylated. Phosphoflow cytometry (phosphoflow) is a flow cytometry-based method allowing for analysis of protein phosphorylation in single cells. Due to recent advances in methodology and antibody availability - together with the relatively easy quantification of phosphorylation - phosphoflow is increasingly and more commonly used, compared to classical western blot analysis. It can however be challenging to set-up a method that works for all targets of interest. Here, we present a step-by-step phosphoflow protocol allowing the evaluation of the phosphorylation status of signaling molecules in conjunction with extensive staining to identify various human and murine B cell subpopulations, as was previously published in the original paper by Rip (2020). Next to a description of phosphoflow targets from the original paper, we provide directions on additional targets that play a pivotal role in BCR signaling. The step-by-step phosphoflow protocol is user-friendly and provides sensitive detection of phosphorylation of various BCR signaling molecules in human and murine B cell subpopulations.
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http://dx.doi.org/10.21769/BioProtoc.3902DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7952956PMC
February 2021

Central Role of Dendritic Cells in Pulmonary Arterial Hypertension in Human and Mice.

Int J Mol Sci 2021 Feb 10;22(4). Epub 2021 Feb 10.

Department of Pulmonary Medicine, Erasmus MC, University Medical Center Rotterdam, 3015 GD Rotterdam, The Netherlands.

The pathogenesis of idiopathic pulmonary arterial hypertension (IPAH) is not fully understood, but evidence is accumulating that immune dysfunction plays a significant role. We previously reported that 31-week-old mice develop pulmonary hypertension (PH) symptoms. These mice harbor a targeted deletion of the TNFα-induced protein-3 () gene, encoding the NF-κB regulatory protein A20, specifically in type I conventional dendritic cells (cDC1s). Here, we studied the involvement of dendritic cells (DCs) in PH in more detail. We found various immune cells, including DCs, in the hearts of mice, particularly in the right ventricle (RV). Secondly, in young mice, innate immune activation through airway exposure to toll-like receptor ligands essentially did not result in elevated RV pressures, although we did observe significant RV hypertrophy. Thirdly, PH symptoms in mice were not enhanced by concomitant mutation of bone morphogenetic protein receptor type 2 (), which is the most affected gene in PAH patients. Finally, in human IPAH lung tissue we found co-localization of DCs and CD8+ T cells, representing the main cell type activated by cDC1s. Taken together, these findings support a unique role of cDC1s in PAH pathogenesis, independent of general immune activation or a mutation in the gene.
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http://dx.doi.org/10.3390/ijms22041756DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7916474PMC
February 2021

Steroid-resistant human inflammatory ILC2s are marked by CD45RO and elevated in type 2 respiratory diseases.

Sci Immunol 2021 Jan;6(55)

Department of Pulmonary Medicine, Erasmus MC, Rotterdam, Netherlands.

Group 2 innate lymphoid cells (ILC2s) orchestrate protective type 2 immunity and have been implicated in various immune disorders. In the mouse, circulatory inflammatory ILC2s (iILC2s) were identified as a major source of type 2 cytokines. The human equivalent of the iILC2 subset remains unknown. Here, we identify a human inflammatory ILC2 population that resides in inflamed mucosal tissue and is specifically marked by surface CD45RO expression. CD45RO ILC2s are derived from resting CD45RA ILC2s upon activation by epithelial alarmins such as IL-33 and TSLP, which is tightly linked to STAT5 activation and up-regulation of the IRF4/BATF transcription factors. Transcriptome analysis reveals marked similarities between human CD45RO ILC2s and mouse iILC2s. Frequencies of CD45RO inflammatory ILC2 are increased in inflamed mucosal tissue and in the circulation of patients with chronic rhinosinusitis or asthma, correlating with disease severity and resistance to corticosteroid therapy. CD45RA-to-CD45RO ILC2 conversion is suppressed by corticosteroids via induction of differentiation toward an immunomodulatory ILC2 phenotype characterized by low type 2 cytokine and high amphiregulin expression. Once converted, however, CD45RO ILC2s are resistant to corticosteroids, which is associated with metabolic reprogramming resulting in the activation of detoxification pathways. Our combined data identify CD45RO inflammatory ILC2s as a human analog of mouse iILC2s linked to severe type 2 inflammatory disease and therapy resistance.
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http://dx.doi.org/10.1126/sciimmunol.abd3489DOI Listing
January 2021

Bacterial lysate therapy for the prevention of wheezing episodes and asthma exacerbations: a systematic review and meta-analysis.

Eur Respir Rev 2020 Dec 27;29(158). Epub 2020 Nov 27.

Dept of Paediatric Medicine, Franciscus Gasthuis & Vlietland, Rotterdam, The Netherlands.

Wheezing and asthma are a growing cause of morbidity in children and adults. Treatment is aimed at prevention of disease exacerbations and preservation of lung function. Respiratory viruses are involved in ∼40-60% of exacerbations. Bacterial lysates prevent recurrent respiratory tract infections and might reduce exacerbations. Moreover, immunomodulatory effects have been observed in human and animal studies. Here we aimed to assess the effects of bacterial lysate therapy on preschool wheezing episodes and asthma exacerbation frequency. We performed a systematic literature review based on the Preferred Reporting Items for Systematic reviews and Meta-Analysis (PRISMA) statement and a meta-analysis using Cochrane Review Manager. Out of 2016 retrieved articles, 22 studies were included, of which five provided sufficient data for a meta-analysis.The use of bacterial lysates showed a decrease of both wheezing episodes (mean difference -2.35 (-3.03- -1.67), p<0.001) and asthma exacerbations in children (mean difference -0.90 (-1.23- -0.57), p<0.001). Additionally, antibiotic use was reduced, and the duration of wheezing episodes was also decreased. No data for adults with asthma are currently available. The immunomodulatory effect seems to be dependent on increased T-helper (Th)1-cell activation and Th2-cell suppression.These favourable effects of bacterial lysates indicate that they show promise as add-on therapy in preschool wheezing and childhood asthma.
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http://dx.doi.org/10.1183/16000617.0175-2019DOI Listing
December 2020

SIRPα on Mouse B1 Cells Restricts Lymphoid Tissue Migration and Natural Antibody Production.

Front Immunol 2020 9;11:570963. Epub 2020 Oct 9.

Sanquin Research and Landsteiner Laboratory, Department of Blood Cell Research, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands.

The inhibitory immunoreceptor SIRPα is expressed on myeloid and neuronal cells and interacts with the broadly expressed CD47. CD47-SIRPα interactions form an innate immune checkpoint and its targeting has shown promising results in cancer patients. Here, we report expression of SIRPα on B1 lymphocytes, a subpopulation of murine B cells responsible for the production of natural antibodies. Mice defective in SIRPα signaling (SIRPα mice) displayed an enhanced CD11b/CD18 integrin-dependent B1 cell migration from the peritoneal cavity to the spleen, local B1 cell accumulation, and enhanced circulating natural antibody levels, which was further amplified upon immunization with T-independent type 2 antigen. As natural antibodies are atheroprotective, we investigated the involvement of SIRPα signaling in atherosclerosis development. Bone marrow (SIRPα>LDLR) chimaeric mice developed reduced atherosclerosis accompanied by increased natural antibody production. Collectively, our data identify SIRPα as a unique B1 cell inhibitory receptor acting to control B1 cell migration, and imply SIRPα as a potential therapeutic target in atherosclerosis.
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http://dx.doi.org/10.3389/fimmu.2020.570963DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7581795PMC
May 2021

The PD-1/PD-L1-Checkpoint Restrains T cell Immunity in Tumor-Draining Lymph Nodes.

Cancer Cell 2020 11 1;38(5):685-700.e8. Epub 2020 Oct 1.

Department of Pulmonary Medicine, Erasmus Medical Center, Rotterdam, the Netherlands; Erasmus MC Cancer Institute, Erasmus Medical Center, Rotterdam, the Netherlands. Electronic address:

PD-1/PD-L1-checkpoint blockade therapy is generally thought to relieve tumor cell-mediated suppression in the tumor microenvironment but PD-L1 is also expressed on non-tumor macrophages and conventional dendritic cells (cDCs). Here we show in mouse tumor models that tumor-draining lymph nodes (TDLNs) are enriched for tumor-specific PD-1 T cells which closely associate with PD-L1 cDCs. TDLN-targeted PD-L1-blockade induces enhanced anti-tumor T cell immunity by seeding the tumor site with progenitor-exhausted T cells, resulting in improved tumor control. Moreover, we show that abundant PD-1/PD-L1-interactions in TDLNs of nonmetastatic melanoma patients, but not those in corresponding tumors, associate with early distant disease recurrence. These findings point at a critical role for PD-L1 expression in TDLNs in governing systemic anti-tumor immunity, identifying high-risk patient groups amendable to adjuvant PD-1/PD-L1-blockade therapy.
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http://dx.doi.org/10.1016/j.ccell.2020.09.001DOI Listing
November 2020

DNGR1-Cre-mediated Deletion of /A20 in Conventional Dendritic Cells Induces Pulmonary Hypertension in Mice.

Am J Respir Cell Mol Biol 2020 11;63(5):665-680

Department of Pulmonary Medicine.

Chronic perivascular inflammation is a prominent feature in the lungs of idiopathic pulmonary arterial hypertension. Although the proportions of conventional dendritic cells (cDCs) and plasmacytoid DCs are increased in idiopathic pulmonary arterial hypertension lungs, it remains unknown whether activated cDCs play a pathogenic role. The gene encodes the ubiquitin-binding protein A20, which is a negative regulator of NF-κB, critically involved in DC activation. Targeting of Tnfaip3/A20 in cDCs was achieved by (DNGR1)-Cre-mediated excision of the gene in mice. Mice were evaluated for signs of pulmonary hypertension (PH) using right heart catheterization, echocardiography, and measurement of the Fulton index. Inflammation was assessed by immunohistochemistry and flow cytometry. Pulmonary cDCs and monocyte-derived DCs from 31-week-old mice showed modulated expression of cell surface activation markers compared with mice. mice developed elevated right ventricular systolic pressure and right ventricular hypertrophy. The lungs of these mice displayed increased vascular remodeling and perivascular and peribronchial immune cell infiltration resembling tertiary lymphoid organs. Proportions of activated T cells and expression of IL-1β, IL-6, and IL-10 were enhanced in the lungs of mice. Autoreactive IgA and IgG1 was detected in BAL and autoreactive IgA recognizing pulmonary endothelial antigens was present in the serum of mice. All signs of PH were ameliorated in mice by antiIL-6 antibody treatment. These results indicate that activation of the NF-κB pathway in DCs, through deletion of A20/, leads to experimental PH with accompanied pulmonary inflammation in an IL-6-dependent fashion.
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http://dx.doi.org/10.1165/rcmb.2019-0443OCDOI Listing
November 2020

A soluble allergen sensor sounds the alarm.

Authors:
Rudi W Hendriks

Nat Immunol 2020 07;21(7):724-726

Department of Pulmonary Medicine, Erasmus MC Rotterdam, Rotterdam, the Netherlands.

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http://dx.doi.org/10.1038/s41590-020-0709-2DOI Listing
July 2020

PDE3 Inhibition Reduces Epithelial Mast Cell Numbers in Allergic Airway Inflammation and Attenuates Degranulation of Basophils and Mast Cells.

Front Pharmacol 2020 1;11:470. Epub 2020 May 1.

Department of Pulmonary Medicine, Erasmus MC, Rotterdam, Netherlands.

Epithelial mast cells are generally present in the airways of patients with allergic asthma that are inadequately controlled. Airway mast cells (MCs) are critically involved in allergic airway inflammation and contribute directly to the main symptoms of allergic patients. Phosphodiesterase 3 (PDE3) tailors signaling of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), which are critical intracellular second messenger molecules in various signaling pathways. This paper investigates the pathophysiological role and disease-modifying effects of PDE3 in mouse bone marrow-derived MCs (bmMCs), human LAD2- and HMC1 mast cell lines, human blood basophils, and peripheral blood-derived primary human MCs (HuMCs). In a chronic house dust mite (HDM)-driven allergic airway inflammation mouse model, we observed that PDE3 deficiency or PDE3 inhibition (PDE3i) therapy reduced the numbers of epithelial MCs, when compared to control mice. Mouse bone marrow-derived MCs (bmMCs) and the human HMC1 and LAD2 cell lines predominantly expressed PDE3B and PDE4A. BmMCs from mice showed reduced loss of the degranulation marker CD107b compared with wild-type BmMCs, when stimulated in an immunoglobulin E (IgE)-dependent manner. Following both IgE-mediated and substance P-mediated activation, PDE3i-pretreated basophils, LAD2 cells, and HuMCs, showed less degranulation than diluent controls, as measured by surface CD63 expression. MCs lacking PDE3 or treated with the PDE3i enoximone exhibited a lower calcium flux upon stimulation with ionomycine. In conclusion PDE3 plays a critical role in basophil and mast cell degranulation and therefore its inhibition may be a treatment option in allergic disease.
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http://dx.doi.org/10.3389/fphar.2020.00470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7206980PMC
May 2020

Tnfaip3 expression in pulmonary conventional type 1 Langerin-expressing dendritic cells regulates T helper 2-mediated airway inflammation in mice.

Allergy 2020 10 14;75(10):2587-2598. Epub 2020 Jun 14.

Department of Pulmonary Medicine, Erasmus MC, Rotterdam, The Netherlands.

Background: Conventional type 1 dendritic cells (cDC1s) control anti-viral and anti-tumor immunity by inducing antigen-specific cytotoxic CD8 T-cell responses. Controversy exists whether cDC1s also control CD4 T helper 2 (Th2) cell responses, since suppressive and activating roles have been reported. DC activation status, controlled by the transcription factor NF-κB, might determine the precise outcome of Th-cell differentiation upon encounter with cDC1s. To investigate the role of activated cDC1s in Th2-driven immune responses, pulmonary cDC1s were activated by targeted deletion of A20/Tnfaip3, a negative regulator of NF-κB signaling.

Methods: To target pulmonary cDC1s, Cd207 (Langerin)-mediated excision of A20/Tnfaip3 was used, generating Tnfaip3 xCd207 (Tnfaip3 ) mice. Mice were exposed to house dust mite (HDM) to provoke Th2-mediated immune responses.

Results: Mice harboring Tnfaip3-deficient cDC1s did not develop Th2-driven eosinophilic airway inflammation upon HDM exposure, but rather showed elevated numbers of IFNγ-expressing CD8 T cells. In addition, Tnfaip3 mice harbored increased numbers of IL-12-expressing cDC1s and elevated PD-L1 expression in all pulmonary DC subsets. Blocking either IL-12 or IFNγ in Tnfaip3 mice restored Th2 responses, whereas administration of recombinant IFNγ during HDM sensitization in C57Bl/6 mice blocked Th2 development.

Conclusions: These findings indicate that the activation status of cDC1s, shown by their specific expression of co-inhibitory molecules and cytokines, critically contributes to the development of Th2 cell-mediated disorders, most likely by influencing IFNγ production in CD8 T cells.
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http://dx.doi.org/10.1111/all.14334DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7687104PMC
October 2020

Notch signaling licenses allergic airway inflammation by promoting Th2 cell lymph node egress.

J Clin Invest 2020 07;130(7):3576-3591

Department of Pulmonary Medicine.

Allergic asthma is mediated by Th2 responses to inhaled allergens. Although previous experiments indicated that Notch signaling activates expression of the key Th2 transcription factor Gata3, it remains controversial how Notch promotes allergic airway inflammation. Here we show that T cell-specific Notch deficiency in mice prevented house dust mite-driven eosinophilic airway inflammation and significantly reduced Th2 cytokine production, serum IgE levels, and airway hyperreactivity. However, transgenic Gata3 overexpression in Notch-deficient T cells only partially rescued this phenotype. We found that Notch signaling was not required for T cell proliferation or Th2 polarization. Instead, Notch-deficient in vitro-polarized Th2 cells showed reduced accumulation in the lungs upon in vivo transfer and allergen challenge, as Notch-deficient Th2 cells were retained in the lung-draining lymph nodes. Transcriptome analyses and sequential adoptive transfer experiments revealed that while Notch-deficient lymph node Th2 cells established competence for lung migration, they failed to upregulate sphingosine-1-phosphate receptor 1 (S1PR1) and its critical upstream transcriptional activator Krüppel-like factor 2 (KLF2). As this KLF2/S1PR1 axis represents the essential cell-intrinsic regulator of T cell lymph node egress, we conclude that the druggable Notch signaling pathway licenses the Th2 response in allergic airway inflammation via promoting lymph node egress.
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http://dx.doi.org/10.1172/JCI128310DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7324208PMC
July 2020

Phosphoflow Protocol for Signaling Studies in Human and Murine B Cell Subpopulations.

J Immunol 2020 05 6;204(10):2852-2863. Epub 2020 Apr 6.

Department of Pulmonary Medicine, Erasmus MC Rotterdam, NL 3000 CA Rotterdam, the Netherlands; and

BCR signaling, involving phosphorylation of various downstream molecules, including kinases, lipases, and linkers, is crucial for B cell selection, survival, proliferation, and differentiation. Phosphoflow cytometry (phosphoflow) is a single-cell-based technique to measure phosphorylated intracellular proteins, providing a more quantitative read-out than Western blotting. Recent advances in phosphoflow basically allow simultaneous analysis of protein phosphorylation in B cell (sub)populations, without prior cell sorting. However, fixation and permeabilization procedures required for phosphoflow often affect cell surface epitopes or mAb conjugates, precluding the evaluation of the phosphorylation status of signaling proteins across different B cell subpopulations present in a single sample. In this study, we report a versatile phosphoflow protocol allowing extensive staining of B cell subpopulations in human peripheral blood or various anatomical compartments in the mouse, starting from freshly isolated or frozen cell suspensions. Both human and mouse B cell subpopulations showed different basal and BCR stimulation-induced phosphorylation levels of downstream signaling proteins. For example, peritoneal B-1 cells and splenic marginal zone B cells exhibited significantly increased basal (ex vivo) signaling and increased responsiveness to in vitro BCR stimulation compared with peritoneal B-2 cells and splenic follicular B cells, respectively. In addition, whereas stimulation with anti-IgM or anti-Igκ L chain Abs resulted in strong pCD79a and pPLCγ2 signals, IgD stimulation only induced CD79a but not pPLCγ2 phosphorylation. In summary, the protocol is user friendly and quantifies BCR-mediated phosphorylation with high sensitivity at the single-cell level, in combination with extensive staining to identify individual B cell development and differentiation stages.
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http://dx.doi.org/10.4049/jimmunol.1901117DOI Listing
May 2020

T cell receptor repertoire characteristics both before and following immunotherapy correlate with clinical response in mesothelioma.

J Immunother Cancer 2020 03;8(1)

Pulmonary Medicine, Erasmus Medical Center, Rotterdam, The Netherlands

Background: Malignant pleural mesothelioma (MPM) is a highly lethal malignancy in need for new treatment options. Although immunotherapies have been shown to boost a tumor-specific immune response, not all patients respond and prognostic biomarkers are scarce. In this study, we determined the peripheral blood T cell receptor β (TCRβ) chain repertoire of nine MPM patients before and 5 weeks after the start of dendritic cell (DC)-based immunotherapy.

Materials And Methods: We separately profiled PD1 and PD1CD4 and CD8 T cells, as well as Tregs and analyzed 70 000 TCRβ sequences per patient.

Results: Strikingly, limited TCRβ repertoire diversity and high average clone sizes in total CD3 T cells before the start of immunotherapy were associated with a better clinical response. To explore the differences in TCRβ repertoire prior-DC-therapy and post-DC-therapy, for each patient the TCRβ clones present in the total CD3 T cell fractions were classified into five categories, based on therapy-associated frequency changes: expanding, decreasing, stable, newly appearing and disappearing clones. Subsequently, the presence of these five groups of clones was analyzed in the individual sorted T cell fractions. DC-therapy primarily induced TCRβ repertoire changes in the PD1CD4 and PD1CD8 T cell fractions. In particular, in the PD1CD8 T cell subpopulation we found high frequencies of expanding, decreasing and newly appearing clones. Conversion from a PD1 to a PD1 phenotype was significantly more frequent in CD8 T cells than in CD4 T cells. Hereby, the number of expanding PD1CD8 T cell clones-and not expanding PD1CD4 T cell clones following immunotherapy positively correlated with overall survival, progression-free survival and reduction of tumor volume.

Conclusion: We conclude that the clinical response to DC-mediated immunotherapy is dependent on both the pre-existing TCRβ repertoire of total CD3 T cells and on therapy-induced changes, in particular expanding PD1CD8 T cell clones. Therefore, TCRβ repertoire profiling in sorted T cell subsets could serve as predictive biomarker for the selection of MPM patients that benefit from immunotherapy.

Trial Registration Number: NCT02395679.
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http://dx.doi.org/10.1136/jitc-2019-000251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7174074PMC
March 2020

Novel, Non-Gene-Destructive Knock-In Reporter Mice Refute the Concept of Monoallelic Expression.

J Immunol 2020 05 25;204(9):2600-2611. Epub 2020 Mar 25.

Institute of Immunology, University Hospital, D-89081 Ulm, Germany; and

Accurately tuned expression levels of the transcription factor GATA-3 are crucial at several stages of T cell and innate lymphoid cell development and differentiation. Moreover, several lines of evidence suggest that expression might provide a reliable molecular marker for the identification of elusive progenitor cell subsets at the earliest stages of T lineage commitment. To be able to faithfully monitor expression noninvasively at the single-cell level, we have generated a novel strain of knock-in reporter mice, termed GATIR, by inserting an expression cassette encoding a bright fluorescent marker into the 3'-untranslated region of the endogenous locus. Importantly, in contrast to three previously published strains of reporter mice, GATIR mice preserve physiological expression on the targeted allele. In this study, we show that GATIR mice faithfully reflect endogenous expression without disturbing the development of GATA-3-dependent lymphoid cell populations. We further show that GATIR mice provide an ideal tool for noninvasive monitoring of Th2 polarization and straightforward identification of innate lymphoid cell 2 progenitor populations. Finally, as our reporter is non-gene-destructive, GATIR mice can be bred to homozygosity, not feasible with previously published strains of reporter mice harboring disrupted alleles. The availability of hetero- and homozygous reporter mice with an exceptionally bright fluorescent marker, allowed us to visualize allelic expression in individual cells simply by flow cytometry. The unambiguous results obtained provide compelling evidence against previously postulated monoallelic expression in early T lineage and hematopoietic stem cell subsets.
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http://dx.doi.org/10.4049/jimmunol.2000025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7167461PMC
May 2020

Butyrate inhibits human mast cell activation via epigenetic regulation of FcεRI-mediated signaling.

Allergy 2020 08 24;75(8):1966-1978. Epub 2020 Apr 24.

Dermatological Allergology, Dermatology and Allergy, Charité - Universitätsmedizin Berlin, Berlin, Germany.

Background: Short-chain fatty acids (SCFAs) are fermented dietary components that regulate immune responses, promote colonic health, and suppress mast cell-mediated diseases. However, the effects of SCFAs on human mast cell function, including the underlying mechanisms, remain unclear. Here, we investigated the effects of the SCFAs (acetate, propionate, and butyrate) on mast cell-mediated pathology and human mast cell activation, including the molecular mechanisms involved.

Method: Precision-cut lung slices (PCLS) of allergen-exposed guinea pigs were used to assess the effects of butyrate on allergic airway contraction. Human and mouse mast cells were co-cultured with SCFAs and assessed for degranulation after IgE- or non-IgE-mediated stimulation. The underlying mechanisms involved were investigated using knockout mice, small molecule inhibitors/agonists, and genomics assays.

Results: Butyrate treatment inhibited allergen-induced histamine release and airway contraction in guinea pig PCLS. Propionate and butyrate, but not acetate, inhibited IgE- and non-IgE-mediated human or mouse mast cell degranulation in a concentration-dependent manner. Notably, these effects were independent of the stimulation of SCFA receptors GPR41, GPR43, or PPAR, but instead were associated with inhibition of histone deacetylases. Transcriptome analyses revealed butyrate-induced downregulation of the tyrosine kinases BTK, SYK, and LAT, critical transducers of FcεRI-mediated signals that are essential for mast cell activation. Epigenome analyses indicated that butyrate redistributed global histone acetylation in human mast cells, including significantly decreased acetylation at the BTK, SYK, and LAT promoter regions.

Conclusion: Known health benefits of SCFAs in allergic disease can, at least in part, be explained by epigenetic suppression of human mast cell activation.
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http://dx.doi.org/10.1111/all.14254DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7703657PMC
August 2020

Rapid identification of human mast cell degranulation regulators using functional genomics coupled to high-resolution confocal microscopy.

Nat Protoc 2020 03 14;15(3):1285-1310. Epub 2020 Feb 14.

Department of Pathology, Stanford University School of Medicine, Stanford, California, USA.

Targeted functional genomics represents a powerful approach for studying gene function in vivo and in vitro. However, its application to gene expression studies in human mast cells has been hampered by low yields of human mast cell cultures and their poor transfection efficiency. We developed an imaging system in which mast cell degranulation can be visualized in single cells subjected to shRNA knockdown or CRISPR-Cas9 gene editing. By using high-resolution confocal microscopy and a fluorochrome-labeled avidin probe, one can directly assess the alteration of functional responses, i.e., degranulation, in single human mast cells (10-12 weeks old). The elimination of a drug or marker selection step avoids the use of potentially toxic treatment procedures, and the brief hands-on time of the functional analysis step enables high-throughput screening of shRNA or CRISPR-Cas9 constructs to identify genes that regulate human mast cell degranulation. The ability to analyze single cells substantially reduces the total number of cells required and enables the parallel visualization of the degranulation profiles of both edited and non-edited mast cells, offering a consistent internal control not found in other protocols. Moreover, our protocol offers a flexible choice between RNA interference (RNAi) and CRISPR-Cas9 genome editing for perturbation of gene expression using our human mast cell single-cell imaging system. Perturbation of gene expression, acquisition of microscopy data and image analysis can be completed within 5 d, requiring only standard laboratory equipment and expertise.
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http://dx.doi.org/10.1038/s41596-019-0288-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7197894PMC
March 2020

Overexpression of SH2-Containing Inositol Phosphatase Contributes to Chronic Lymphocytic Leukemia Survival.

J Immunol 2020 01 13;204(2):360-374. Epub 2019 Dec 13.

Department of Pulmonary Medicine, Erasmus MC, NL 3000 CA Rotterdam, the Netherlands;

Balanced activity of kinases and phosphatases downstream of the BCR is essential for B cell differentiation and function and is disturbed in chronic lymphocytic leukemia (CLL). In this study, we employed mice, which spontaneously develop CLL, and stable EMC CLL cell lines derived from these mice to explore the role of phosphatases in CLL. Genome-wide expression profiling comparing CLL cells with wild-type splenic B cells identified 96 differentially expressed phosphatase genes, including SH2-containing inositol phosphatase (). We found that B cell-specific deletion of , but not of its close homolog , significantly reduced CLL formation in mice. Treatment of EMC cell lines with Ship1/2 small molecule inhibitors resulted in the induction of caspase-dependent apoptosis. Using flow cytometry and Western blot analysis, we observed that blocking Ship1/2 abrogated EMC cell survival by exerting dual effects on the BCR signaling cascade. On one hand, specific Ship1 inhibition enhanced calcium signaling and thereby abrogated an anergic response to BCR stimulation in CLL cells. On the other hand, concomitant Ship1/Ship2 inhibition or specific Ship2 inhibition reduced constitutive activation of the mTORC1/ribosomal protein S6 pathway and downregulated constitutive expression of the antiapoptotic protein Mcl-1, in both EMC cell lines and primary CLL cells. Importantly, also in human CLL, we found overexpression of many phosphatases including SHIP2. Inhibition of SHIP1/SHIP2 reduced cellular survival and S6 phosphorylation and enhanced basal calcium levels in human CLL cells. Taken together, we provide evidence that SHIP2 contributes to CLL pathogenesis in mouse and human CLL.
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http://dx.doi.org/10.4049/jimmunol.1900153DOI Listing
January 2020

3D genome organization during lymphocyte development and activation.

Brief Funct Genomics 2020 03;19(2):71-82

Department of Pulmonary Medicine, Erasmus MC, Rotterdam, the Netherlands.

Chromosomes have a complex three-dimensional (3D) architecture comprising A/B compartments, topologically associating domains and promoter-enhancer interactions. At all these levels, the 3D genome has functional consequences for gene transcription and therefore for cellular identity. The development and activation of lymphocytes involves strict control of gene expression by transcription factors (TFs) operating in a three-dimensionally organized chromatin landscape. As lymphocytes are indispensable for tissue homeostasis and pathogen defense, and aberrant lymphocyte activity is involved in a wide range of human morbidities, acquiring an in-depth understanding of the molecular mechanisms that control lymphocyte identity is highly relevant. Here we review current knowledge of the interplay between 3D genome organization and transcriptional control during B and T lymphocyte development and antigen-dependent activation, placing special emphasis on the role of TFs.
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http://dx.doi.org/10.1093/bfgp/elz030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7115705PMC
March 2020

Enhanced Bruton's tyrosine kinase in B-cells and autoreactive IgA in patients with idiopathic pulmonary fibrosis.

Respir Res 2019 Oct 24;20(1):232. Epub 2019 Oct 24.

Department of Pulmonary Medicine, Erasmus Medical Center, 's-Gravendijkwal 230, 3015, CE, Rotterdam, The Netherlands.

Rationale: Idiopathic Pulmonary Fibrosis (IPF) is thought to be triggered by repeated alveolar epithelial cell injury. Current evidence suggests that aberrant immune activation may contribute. However, the role of B-cell activation remains unclear. We determined the phenotype and activation status of B-cell subsets and evaluated the contribution of activated B-cells to the development of lung fibrosis both in humans and in mice.

Methods: B-cells in blood, mediastinal lymph node, and lung single-cell suspensions of IPF patients and healthy controls (HC) were characterized using 14-color flow cytometry. Mice were exposed to bleomycin to provoke pulmonary fibrosis.

Results: More IgA memory B-cells and plasmablasts were found in blood (n = 27) and lungs (n = 11) of IPF patients compared to HC (n = 21) and control lungs (n = 9). IPF patients had higher levels of autoreactive IgA in plasma, which correlated with an enhanced decline of forced vital capacity (p = 0.002, r = - 0.50). Bruton's tyrosine kinase expression was higher in circulating IPF B-cells compared to HC, indicating enhanced B-cell activation. Bleomycin-exposed mice had increased pulmonary IgA germinal center and plasma cell proportions compared to control mice. The degree of lung fibrosis correlated with pulmonary germinal center B-cell proportions (p = 0.010, r = 0.88).

Conclusion: Our study demonstrates that IPF patients have more circulating activated B-cells and autoreactive IgA, which correlate with disease progression. These B-cell alterations were also observed in the widely used mouse model of experimental pulmonary fibrosis. Autoreactive IgA could be useful as a biomarker for disease progression in IPF.
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http://dx.doi.org/10.1186/s12931-019-1195-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6814043PMC
October 2019

The presence of CLL-associated stereotypic B cell receptors in the normal BCR repertoire from healthy individuals increases with age.

Immun Ageing 2019 28;16:22. Epub 2019 Aug 28.

1Department Immunology, Laboratory Medical Immunology, Erasmus MC, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands.

Background: Aging is known to induce immunosenescence, resulting in alterations in both the innate and adaptive immune system. Here we evaluated the effects of aging on B cell subsets in peripheral blood of 155 immunologically healthy individuals in four age categories (range 20-95y) via multi-parameter flow cytometry. Furthermore, we studied the naive and antigen-experienced B cell receptor (BCR) repertoire of different age groups and compared it to the clonal BCR repertoire of chronic lymphocytic leukemia (CLL), a disease typically presenting in elderly individuals.

Results: Total numbers and relative frequencies of B cells were found to decline upon aging, with reductions in transitional B cells, memory cell types, and plasma blasts in the 70 + y group. The BCR repertoire of naive mature B cells and antigen-experienced B cells did not clearly alter until age 70y. Clear changes in IGHV gene usage were observed in naive mature B cells of 70 + y individuals, with a transitional pattern in the 50-70y group. IGHV gene usage of naive mature B cells of the 50-70y, but not the 70 + y, age group resembled that of both younger (50-70y) and older (70 + y) CLL patients. Additionally, CLL-associated stereotypic BCR were found as part of the healthy control BCR repertoire, with an age-associated increase in frequency of several stereotypic BCR (particularly subsets #2 and #5).

Conclusion: Composition of the peripheral B cell compartment changes with ageing, with clear reductions in non-switched and CD27 + IgG+ switched memory B cells and plasma blasts in especially the 70 + y group. The BCR repertoire is relatively stable until 70y, whereafter differences in IGHV gene usage are seen. Upon ageing, an increasing trend in the occurrence of particular CLL-associated stereotypic BCR is observed.
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http://dx.doi.org/10.1186/s12979-019-0163-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6714092PMC
August 2019

The Effects of an IL-21 Receptor Antagonist on the Alloimmune Response in a Humanized Mouse Skin Transplant Model.

Transplantation 2019 10;103(10):2065-2074

The Rotterdam Transplant Group, Erasmus MC, Department of Internal Medicine, University Medical Center Rotterdam, Rotterdam, The Netherlands.

Background: Interleukin 21 (IL-21) is involved in regulating the expansion and effector function of a broad range of leukocytes, including T cells and B cells. In transplantation, the exact role of IL-21 in the process of allograft rejection is unknown. To further explore this, the aim of this study is to test the effect of an IL-21 receptor (IL-21R) blocking antibody on the early phase of allograft rejection in a humanized skin transplantation model in mice reconstituted with human T and B cells.

Methods: Immunodeficient Balb/c IL2rγRag2 mice were transplanted with human skin followed by adoptive transfer of human allogeneic splenocytes. Control animals were treated with a phosphate buffered saline vehicle while the other group was treated with a humanized anti-IL-21R antibody (αIL-21R).

Results: In the phosphate buffered saline-treated animals, human skin allografts were infiltrated with lymphocytes and developed a thickened epidermis with increased expression of the inflammatory markers Keratin 17 (Ker17) and Ki67. In mice treated with αIL-21R, these signs of allograft reactivity were significantly reduced. Concordantly, STAT3 phosphorylation was inhibited in this group. Of note, treatment with αIL-21R attenuated the process of T and B cell reconstitution after adoptive cellular transfer.

Conclusions: These findings demonstrate that blockade of IL-21 signaling can delay allograft rejection in a humanized skin transplantation model.
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http://dx.doi.org/10.1097/TP.0000000000002773DOI Listing
October 2019

Evidence for enhanced Bruton's tyrosine kinase activity in transitional and naïve B cells of patients with granulomatosis with polyangiitis.

Rheumatology (Oxford) 2019 12;58(12):2230-2239

Department of Pulmonary Medicine, Erasmus University Medical Center, Rotterdam, The Netherlands.

Objectives: To determine Bruton's tyrosine kinase (BTK) protein and phosphorylation levels in B cell subsets of granulomatosis with polyangiitis (GPA) patients and to investigate the effect of BTK blockade on in vitro B cell cytokine production, subset distribution and (auto)antibody production.

Methods: BTK protein and phosphorylation levels were determined by flow cytometry in peripheral blood B cells of 29 untreated GPA patients [9 active and 20 remission GPA patients (10 ANCA- and 10 ANCA+)], 9 age- and sex-matched healthy controls (HCs) and 9 untreated active RA patients. The effect of BTK blockade on in vitro B cell cytokine production, subset distribution and (auto)antibody production was determined in the same donors in peripheral blood mononuclear cell cultures.

Results: BTK protein levels were significantly increased in transitional and naïve B cells of active GPA and RA patients compared with remission GPA patients and HCs. Both B cell subsets of active patients were more sensitive to B cell receptor stimulation, as BTK and phospholipase Cγ2 phosphorylation were increased in these patients. In vitro BTK blockade had profound effects on B cell cytokine production, plasma cell formation and (auto)antibody production in both GPA patients and HCs. Interestingly, the effect of BTK blockade was less pronounced in active GPA patients, possibly due to increased activation of B cells.

Conclusion: We show that BTK protein and phosphorylation levels are most profoundly increased in newly emerging B cells of active GPA patients compared with remission patients. BTK blockade greatly inhibits in vitro B cell effector functions in GPA patients and HCs. These promising data identify BTK as an interesting novel therapeutic target in the treatment of GPA.
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http://dx.doi.org/10.1093/rheumatology/kez205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6880857PMC
December 2019

KLRG1 and NKp46 discriminate subpopulations of human CD117CRTH2 ILCs biased toward ILC2 or ILC3.

J Exp Med 2019 08 14;216(8):1762-1776. Epub 2019 Jun 14.

Department of Experimental Immunology, Amsterdam UMC, Location AMC, University of Amsterdam, Amsterdam, The Netherlands

Recently, human ILCs that express CD117 and CD127 but lack CRTH2 and NKp44 have been shown to contain precursors of ILC1, ILC2, and ILC3. However, these ILCs have not been extensively characterized. We performed an unbiased hierarchical stochastic neighbor embedding (HSNE) analysis of the phenotype of peripheral blood CD117 ILCs, which revealed the presence of three major subsets: the first expressed NKp46, the second expressed both NKp46 and CD56, and the third expressed KLRG1, but not NKp46 or CD56. Analysis of their cytokine production profiles and transcriptome revealed that NKp46 ILCs predominantly develop into ILC3s; some of them can differentiate into ILC1/NK-like cells, but they are unable to develop into ILC2s. In contrast, KLRG1 ILCs predominantly differentiate into ILC2s. Single-cell cultures demonstrate that KLRG1 ILCs can also differentiate into other ILC subsets depending on the signals they receive. Epigenetic profiling of KLRG1 ILCs is consistent with the broad differentiation potential of these cells.
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http://dx.doi.org/10.1084/jem.20190490DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6683990PMC
August 2019

DNGR1-mediated deletion of A20/Tnfaip3 in dendritic cells alters T and B-cell homeostasis and promotes autoimmune liver pathology.

J Autoimmun 2019 08 9;102:167-178. Epub 2019 Jul 9.

Department of Pulmonary Medicine, Erasmus MC, PO Box 2040, 3000 CA, Rotterdam, the Netherlands. Electronic address:

Dendritic cells (DCs) are central regulators of tolerance versus immunity. The outcome depends amongst others on DC subset and activation status. Whereas CD11b type 2 conventional DCs (cDC2s) initiate proinflammatory helper T (Th)-cell responses, CD103 cDC1s are crucial for regulatory T-cell (Treg) induction and CD8 T-cell activation. DC activation is controlled by the transcription factor NF-κB. Ablation of A20/Tnfaip3, a critical regulator of NF-κB activation, in DCs leads to constitutive DC activation and development of systemic autoimmunity. We hypothesized that the activation status of cDCs controls the development of autoimmunity. To target cDCs, DNGR1(Clec9a)-cre-mediated excision of A20/Tnfaip3 was used through generation of Tnfaip3xClec9a (Tnfaip3) mice. Immune cell activation was evaluated at 31-weeks of age. We found that DNGR1-cre-mediated deletion of A20/Tnfaip3 resulted in liver pathology characterized by inflammatory infiltrates adjacent to the portal triads. Both cDC subsets as well as monocyte-derived DCs (moDCs) in Tnfaip3 livers harbored an activated phenotype. Specifically, the costimulatory molecule CD40 in liver cDCs and moDCs was regulated by A20/Tnfaip3 expression. Livers from Tnfaip3 mice had augmented proportions of Th1, Th17, Treg, and follicular Th (Tfh)-cells compared to control mice, accompanied by an increase in IgA-producing plasma cells. Serum IgA from Tnfaip3 mice recognized self-proteins, specifically cytoplasmic proteins in liver periportal regions. These data show that enhanced activation of cDCs and moDCs, due to A20/Tnfaip3 ablation, promotes the development of organ-specific autoimmunity but not systemic autoimmunity. This model could be useful to examine the pathobiological processes contributing to autoimmune liver diseases.
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http://dx.doi.org/10.1016/j.jaut.2019.05.007DOI Listing
August 2019

Responsiveness of chronic lymphocytic leukemia cells to B-cell receptor stimulation is associated with low expression of regulatory molecules of the nuclear factor-κB pathway.

Haematologica 2020 01 16;105(1):182-192. Epub 2019 May 16.

Laboratory Medical Immunology, Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam

Chronic lymphocytic leukemia (CLL) is a disease with heterogeneous clinical and biological characteristics. Differences in Ca levels among cases, both basal and upon B-cell receptor (BCR) stimulation, may reflect heterogeneity in the pathogenesis due to cell-intrinsic factors. Our aim was to elucidate cell-intrinsic differences between BCR-responsive and -unresponsive cases. We therefore determined BCR responsiveness based on Ca influx upon α-IgM stimulation of purified CLL cell fractions from 52 patients. Phosphorylation levels of various BCR signaling molecules, and expression of activation markers were assessed by flow cytometry. Transcription profiling of responsive (n=6) and unresponsive cases (n=6) was performed by RNA sequencing. Real-time quantitative polymerase chain reaction analysis was used to validate transcript level differences in a larger cohort. In 24 cases an α-IgM response was visible by Ca influx which was accompanied by higher phosphorylation of PLCγ2 and Akt after α-IgM stimulation in combination with higher surface expression of IgM, IgD, CD19, CD38 and CD43 compared to the unresponsive cases (n=28). Based on RNA sequencing analysis several components of the canonical nuclear factor (NF)-κB pathway, especially those related to NF-κB inhibition, were expressed more highly in unresponsive cases. Moreover, upon α-IgM stimulation, the expression of these NF-κB pathway genes (especially genes coding for NF-κB pathway inhibitors but also NF-κB subunit ) was upregulated in BCR-responsive cases while the level did not change, compared to basal level, in the unresponsive cases. These findings suggest that cells from CLL cases with enhanced NF-κB signaling have a lesser capacity to respond to BCR stimulation.
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http://dx.doi.org/10.3324/haematol.2018.215566DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6939541PMC
January 2020