scholarly journals Immunogenic effects of recombinant interferon-beta therapy disrupt the JAK/STAT pathway in primary immune cells from patients with multiple sclerosis

2012 ◽  
Vol 18 (8) ◽  
pp. 1116-1124 ◽  
Author(s):  
S Gavasso ◽  
BT Gjertsen ◽  
E Anderssen ◽  
KM Myhr ◽  
C Vedeler
Keyword(s):  
Multiple Sclerosis ◽  
Flow Cytometry ◽  
Neutralizing Antibodies ◽  
Mononuclear Cells ◽  
Specific Cell ◽  
Peripheral Blood Mononuclear ◽  
Specific Flow ◽  
Recombinant Interferon ◽  
The Impact

Background: Immunogenicity of recombinant interferon-β (IFN-β) is a known complication in the therapy of relapsing–remitting multiple sclerosis (RRMS). Neutralizing antibodies (NAbs) that can interfere with efficacy are quantified using in vitro bioassays; however, these assays do not reveal the immunogenic state of the patient and are not predictive of treatment outcome. Objective: Assessment of the impact of NAbs on IFN-β responsive cells and signalling pathways in peripheral blood mononuclear cells (PBMCs) with phospho-specific flow cytometry. Method: PBMCs from 10 IFN-β-treated patients with RRMS, two untreated patients, and two healthy controls were re-stimulated in autologous sera and media with a serial dilution of IFN-β (0–8000 U/ml) and levels of phosphorylation of STAT1/3/4/5/6 transcription factors were quantified in PBMC subtypes (NAb titres 0 to > 6000 neutralizing units). Data was subjected to principal component analysis, Hotelling’s T2, and partial least squares analysis. Results: Three significantly distinct clusters of individuals were revealed in autologous sera: therapy-naïve and healthy, treated NAb-negative, and treated NAb-positive. Compared with controls STATs signalling patterns were modulated in treated NAb-negative patients and inhibited in all treated NAb-positive patients independently of NAb titres. In media no clustering of patients could be found. The predictability of NAb titres based on the phospho-flow data was 74%. Conclusion: Phospho-specific flow cytometry can delineate subset-specific cell responses that can act as surrogates for NAb exposure in blood. Immunogenic effects alter the response in primary cells even at low NAb levels. Cell line-based immunogenicity testing is not readily transferable to the immunogenic response in patients.

scholarly journals Tetracyclines Diminish In Vitro IFN-γ and IL-17-Producing Adaptive and Innate Immune Cells in Multiple Sclerosis

2021 ◽  
Vol 12 ◽  
Author(s):  
Despoina T. Florou ◽  
Athanasios Mavropoulos ◽  
Efthymios Dardiotis ◽  
Vana Tsimourtou ◽  
Vasileios Siokas ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
Flow Cytometry ◽  
Mononuclear Cells ◽  
Nkt Cells ◽  
Dependent Manner ◽  
Peripheral Blood Mononuclear ◽  
Th2 Immune Response ◽  
Ifn Γ

IntroductionLimited data from clinical trials in multiple sclerosis (MS) reported that minocycline, a widely used antibiotic belonging to the family of tetracyclines (TCs), exerts a beneficial short-lived clinical effect A similar anti-inflammatory effect of minocycline attributed to a deviation from Th1 to Th2 immune response has been reported in experimental models of MS. Whether such an immunomodulatory mechanism is operated in the human disease remains largely unknown.AimTo assess the in vitro immunomodulatory effect of tetracyclines, and in particular minocycline and doxycycline, in naïve and treated patients with MS.Material and MethodsPeripheral blood mononuclear cells from 45 individuals (35 MS patients, amongst which 15 naïve patients and 10 healthy controls, HCs) were cultured with minocycline or doxycycline and conventional stimulants (PMA/Ionomycin or IL-12/IL-18). IFN-γ and IL-17 producing T-, NK- and NKT cells were assessed by flow cytometry. The effect of TCs on cell viability and apoptosis was further assessed by flow cytometry with Annexin V staining.ResultsBoth tetracyclines significantly decreased, in a dose dependent manner, IFN-γ production in NKT and CD4+ T lymphocytes from MS patients (naïve or treated) stimulated with IL-12/IL-18 but did not decrease IFN-γ producing CD8+ T cells from naive MS or treated RRMS patients. They also decreased IL-17+ T and NKT cells following PMA and Ionomycin-stimulation. Tetracyclines did not affect the viability of cell subsets.ConclusionTetracyclines can in vitro suppress IFN-γ and IL-17- producing cells from MS patients, and this may explain their potential therapeutic effect in vivo.

scholarly journals Regulation of CTLA-4 and PD-L1 Expression in Relapsing-Remitting Multiple Sclerosis Patients after Treatment with Fingolimod, IFNβ-1α, Glatiramer Acetate, and Dimethyl Fumarate Drugs

2021 ◽  
Vol 11 (8) ◽  
pp. 721
Author(s):  
Afshin Derakhshani ◽  
Zahra Asadzadeh ◽  
Hossein Safarpour ◽  
Patrizia Leone ◽  
Mahdi Abdoli Shadbad ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
Mononuclear Cells ◽  
Demyelinating Disease ◽  
Immune Checkpoints ◽  
Peripheral Blood Mononuclear ◽  
Relapsing Remitting ◽  
Remitting Multiple Sclerosis ◽  
Multiple Sclerosis Patients ◽  
Relapsing Remitting Multiple Sclerosis ◽  
The Impact

Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system (CNS) that is characterized by inflammation which typically results in significant impairment in most patients. Immune checkpoints act as co-stimulatory and co-inhibitory molecules and play a fundamental role in keeping the equilibrium of the immune system. Cytotoxic T-lymphocyte antigen-4 (CTLA-4) and Programmed death-ligand 1 (PD-L1), as inhibitory immune checkpoints, participate in terminating the development of numerous autoimmune diseases, including MS. We assessed the CTLA-4 and PD-L1 gene expression in the different cell types of peripheral blood mononuclear cells of MS patients using single-cell RNA-seq data. Additionally, this study outlines how CTLA-4 and PD-L1 expression was altered in the PBMC samples of relapsing-remitting multiple sclerosis (RRMS) patients compared to the healthy group. Finally, it investigates the impact of various MS-related treatments in the CTLA-4 and PD-L1 expression to restrain autoreactive T cells and stop the development of MS autoimmunity.

scholarly journals Viral Protein VP4 Is a Target of Human Antibodies Enhancing Coxsackievirus B4- and B3-Induced Synthesis of Alpha Interferon

2005 ◽  
Vol 79 (22) ◽  
pp. 13882-13891 ◽  
Author(s):  
Wassim Chehadeh ◽  
Pierre-Emmanuel Lobert ◽  
Pierre Sauter ◽  
Anne Goffard ◽  
Bernadette Lucas ◽  
...  
Keyword(s):  
Healthy Subjects ◽  
Neutralizing Antibodies ◽  
Mononuclear Cells ◽  
Insulin Dependent Diabetes Mellitus ◽  
Alpha Interferon ◽  
Dependent Diabetes Mellitus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Peripheral Blood Mononuclear ◽  
Coxsackievirus B4

ABSTRACT Coxsackievirus B4 (CVB4)-induced production of alpha interferon (IFN-α) by peripheral blood mononuclear cells (PBMC) is enhanced in vitro by nonneutralizing anti-CVB4 antibodies from healthy subjects and, to a higher extent, from patients with insulin-dependent diabetes mellitus. In this study, we focused on identification of the viral target of these antibodies in CVB systems. High levels of IFN-α were obtained in supernatants of PBMC incubated with CVB4E2 or CVB3 and plasma from healthy subjects and, to a higher extent, from patients. The VP4 capsid proteins dissociated by heating at 56°C from CVB4E2 (VP4CVB4) and CVB3 (VP4CVB3) but not H antigen preincubated with plasma from healthy subjects or patients inhibited the plasma-dependent enhancement of CVB4E2- and CVB3-induced IFN-α synthesis. There was no cross-reaction between VP4CVB4 and VP4CVB3 in the inhibiting effect. IFN-α levels in culture supernatants showed dose-dependent correlation with anti-VP4 antibodies eluted from plasma specimens using VP4-coated plates. There were higher index values for anti-VP4 antibodies detected by enzyme-linked immunosorbent assay (ELISA) and higher proportions of positive detection in 40 patients than in 40 healthy subjects (80% versus 15% for anti-VP4CVB4). There was no relationship between the levels of anti-CVB neutralizing antibodies and the detection of anti-VP4 antibodies by ELISA. The CVB plasma-induced IFN-α levels obtained in PBMC cultures in the anti-VP4 antibody-positive groups were significantly higher than those obtained in the anti-VP4 antibody-negative groups regardless of the titers of anti-CVB neutralizing antibodies. These results show that VP4 is the target of antibodies involved in the plasma-dependent enhancement of CVB4E2- and CVB3-induced IFN-α synthesis by PBMC.

scholarly journals In Vitro Effects of Olive Vegetation Water on IFN-γ and IL-10 Secretion in Multiple Sclerosis Patients

2021 ◽  
Vol 1 (1) ◽  
Author(s):  
Mahboubeh Baheri ◽  
Mohammadreza Dayer ◽  
Narges Baharifar ◽  
Abdolkarim Sheikhi ◽  
Abolfazl Sheikh
Keyword(s):  
Multiple Sclerosis ◽  
Mononuclear Cells ◽  
Inhibitory Effect ◽  
Inflammatory Disorder ◽  
Peripheral Blood Mononuclear ◽  
Inflammatory Reactions ◽  
Ifn Γ ◽  
Vegetation Water ◽  
Multiple Sclerosis Patients

Background: Multiple Sclerosis (MS) is an autoimmune and inflammatory disorder of the central nervous system (CNS), which is associated with the imbalance of pro- and anti-inflammatory cytokines. Evidence indicates that nutritional interventions have some immunomodulatory impacts. Objectives: In this study, we investigated the effect of olive vegetation water (OVW) on IFN-γ and IL-10 secretion by peripheral blood mononuclear cells (PBMCs) of MS patients. Methods: In this study, PBMCs of MS patients were separated by Ficoll-Hypaque centrifugation. The cytotoxicity of OVW was assessed by the MTT assay. The treatments were performed for 48 and 72 hours, and IFN-γ and IL-10 were measured by ELISA. Results: No cytotoxicity was observed for OVW. Besides, OVW showed a significant inhibitory effect on IFN-γ secretion but augmenting effect on IL-10 secretion by PBMCs dose-dependently. Conclusions: This study indicated that OVW could have immunoregulatory effects on inflammatory reactions in MS patients.

scholarly journals JIA patient T cells differentiate into Th1, Th17 and Th1.17 effector cells under Th1 polarizing conditions

2021 ◽  
Author(s):  
Anna E Patrick ◽  
Tashawna Esmond ◽  
Kayla Shoaff ◽  
David M Patrick ◽  
David K Flaherty ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Cell Cultures ◽  
Th17 Cells ◽  
Mononuclear Cells ◽  
Healthy Children ◽  
Th1 Cell ◽  
Peripheral Blood Mononuclear ◽  
Ifn Gamma

Objective. T helper cells develop into discrete Th1, Th2 or Th17 lineages that selectively express IFN-gamma, IL-4/IL-5/IL-13, or IL-17, respectively and actively silence signature cytokines expressed by opposing lineages. Our objective was to compare Th1, Th2 and Th17 polarization in cell culture models using JIA patient samples. Methods. Peripheral blood mononuclear cells were isolated from JIA or healthy prepubescent children. T cell naive and memory phenotypes were assessed by flow cytometry. T cell proliferation was measured using a fluorescence-based assay. Th cell cultures were generated in vitro and IFN-gamma, IL-17, and TNF-alpha measured by ELISA and flow cytometry. Results. JIA Th1 cells produced increased IFN-gamma and inappropriately produced IL-17. JIA Th17 cells produced increased IL-17. JIA Th1 cell cultures develop dual producers of IFN-gamma and IL-17, which are Th1.17 cells. JIA Th1 cultures expressed elevated levels of both T-bet and ROR-gamma-T. RNA sequencing confirmed activation of immune responses and inappropriate activation of IL-17 signaling pathways in Th1 cultures. A subset of JIA patient samples was disproportionally responsible for the enhanced IFN-gamma and IL-17 phenotype and Th1.17 phenotype. Conclusions. This study reveals that JIA patient uncommitted T cell precursors, but not healthy children, inappropriately develop into inflammatory effector Th1.17 and Th17 cells under Th1 polarizing conditions.

scholarly journals P035 NUDT15 variance increases DNA-incorporated thiopurine metabolites and lymphocyte apoptosis in patients with inflammatory bowel disease

2020 ◽  
Vol 14 (Supplement_1) ◽  
pp. S149-S150
Author(s):  
H Kiyohara ◽  
T Toyonaga ◽  
S Kuronuma ◽  
A Ueno ◽  
S Okabayashi ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Japanese Patients ◽  
Lymphocyte Apoptosis ◽  
Drug Induced ◽  
Peripheral Blood Mononuclear ◽  
Inflammatory Bowel ◽  
The Impact ◽  
Cd4 Lymphocytes

Abstract Background A novel thiopurine metabolizing enzyme, nucleotide diphosphate-linked moiety X-type motif 15 (NUDT15) was associated with drug-induced leukopenia in patients with non-synonymous genetic polymorphisms. Thiopurine-induced leukopenia in Japanese patients with genetic variance in NUDT15 (c.415C>T) appears to be independent of the 6-thioguanine nucleotide concentration in red blood cells. However, detailed molecular mechanism how NUDT15 variance causes thiopurine-induced leukopenia remains unclear and NUDT15-associated subcellular thiopurine metabolism has not been investigated in patients with inflammatory bowel diseases (IBD). Methods DNA-incorporated deoxythioguanosine (dTG) was measured in the peripheral blood mononuclear cells (PBMCs) of Japanese patients with IBD under thiopurine treatment. Association of a single-nucleotide polymorphism for NUDT15 (c.415C>T) with dTG in PBMCs (dTGPBMC) was examined. Peripheral blood T lymphocytes were cultured in vitro with 6-thioguanine (6-TG) to examine the Impact of NUDT15 genotypes on incorporation into DNA, cell proliferation and apoptosis. Results NUDT15 variants had significantly higher dTGPBMC per thiopurine dosage than non-variants (homozygous variants (TT) vs. heterozygous variants (CT) vs. non-variants (CC), 4418.0 vs. 663.0 vs. 295.3 dTG mol/106 moles dA per mg/kg/day of 6-MP (Figure A)). dTGPBMC and peripheral lymphocyte counts showed a negative correlation (r = −0.30, p = 0.015) (Figure B). Peripheral blood lymphocytes from patients with NUDT15 variance showed a higher DNA-incorporated dTG associated with increased apoptosis (increase of Annexin V+ PI+ CD4+ lymphocytes; TT vs. CT vs. CC, 158.5 % vs. 80.1 % vs. 57.9 % (p = 0.0427)) (Figure C) and decreased proliferation (decrease of proliferative CD4+ lymphocytes, TT vs. CT vs. CC, 49.0 % vs. 25.0 % vs. 19.1 % (p = 0.0098)) (Figure D) when cultured with 6-thioguanine in vitro. Conclusion DNA-incorporated dTG affected by NUDT15 genotypes induces T lymphocyte apoptosis in patients with IBD.

scholarly journals MAPK pathway and B cells overactivation in multiple sclerosis revealed by phosphoproteomics and genomic analysis

2019 ◽  
Vol 116 (19) ◽  
pp. 9671-9676 ◽  
Author(s):  
Ekaterina Kotelnikova ◽  
Narsis A. Kiani ◽  
Dimitris Messinis ◽  
Inna Pertsovskaya ◽  
Vicky Pliaka ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
B Cells ◽  
Kinase Inhibitors ◽  
Mononuclear Cells ◽  
Mapk Pathway ◽  
Genomic Analysis ◽  
Nucleotide Polymorphisms ◽  
Peripheral Blood Mononuclear ◽  
Disease Modifying Drugs

Dysregulation of signaling pathways in multiple sclerosis (MS) can be analyzed by phosphoproteomics in peripheral blood mononuclear cells (PBMCs). We performed in vitro kinetic assays on PBMCs in 195 MS patients and 60 matched controls and quantified the phosphorylation of 17 kinases using xMAP assays. Phosphoprotein levels were tested for association with genetic susceptibility by typing 112 single-nucleotide polymorphisms (SNPs) associated with MS susceptibility. We found increased phosphorylation of MP2K1 in MS patients relative to the controls. Moreover, we identified one SNP located in the PHDGH gene and another on IRF8 gene that were associated with MP2K1 phosphorylation levels, providing a first clue on how this MS risk gene may act. The analyses in patients treated with disease-modifying drugs identified the phosphorylation of each receptor’s downstream kinases. Finally, using flow cytometry, we detected in MS patients increased STAT1, STAT3, TF65, and HSPB1 phosphorylation in CD19+ cells. These findings indicate the activation of cell survival and proliferation (MAPK), and proinflammatory (STAT) pathways in the immune cells of MS patients, primarily in B cells. The changes in the activation of these kinases suggest that these pathways may represent therapeutic targets for modulation by kinase inhibitors.

Genetically engineered natural killer (NK) cell immunotherapy for children poor risk b-cell (CD20+) leukemia and lymphoma (L/L).

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e13022-e13022
Author(s):  
Yaya Chu ◽  
Janet Ayello ◽  
Jessica Hochberg ◽  
Carmella Van de ven ◽  
James Murphy ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Cell Number ◽  
Genetically Engineered ◽  
Peripheral Blood Mononuclear ◽  
Poor Risk ◽  
Anti Cd20

e13022 Background: A majority of children with CD20+ L/L at relapse have a chemotherapy resistant phenotype (Cairo et al Blood, 2007; JCO, 2012). Novel, non-chemotherapy-based therapies are desperately needed for this poor risk population. NK cells play an important role in tumor surveillance post allogeneic stem cell transplantation (Beziat V et al, Leukemia, 2009) but cell number and tumor recognition limit adoptive NK cell therapy (Shereck/Cairo, PBC 2007). PBNK cells expanded with genetically engineered K562-mbIL15-41BBL cells (geK562) have been previously reported (Imai C et al, Blood. 2005). Objective: We investigated the functional activities and cytolytic effect of anti-CD20 chimeric antigen receptor (CAR+) engineered PBNK cells expanded with mK562 against CD20+ L/L both in vitro and in vivo. Methods: Peripheral blood mononuclear cells (PBMC) were expanded with mitomycin C treated geK562 cells in culture medium with 10 IU/ml IL-2 for 7 or 14 days. CD56 and CD3 expression were evaluated by flow cytometry. Retrovirus preps that express CAR+ or CAR- were generated independently. The CAR+ was constructed in a MSCV-anti-CD20BB-CD3-zeta-GFP plasmid (generously supplied by Dario Campana, MD, PhD). Expanded PBMC were transduced with retroviruses as described (Imai C et al, Blood. 2005). NK cytotoxicity was assessed by europium release assay at 2:1 E:T ratio against CD20+ Ramos. Results: CD56+CD3- PBNK cells were significantly increased compared to media alone at day7 (60.94+ 3.63% vs 8.05+0.49%, n=6, p<0.001). CD56-CD3+ PBT cells were significantly reduced compared to media alone at day 7 (22.08+2.22% vs 75.73+0.75%, n=6, p<0.001). CAR+ and CAR- retrovirus supernants infected expanded PBMC at 1%-10% range. The anti-CD20 CAR expression was further confirmed by flow cytometry and western blot. We also observe that cytotoxicity was enhanced with CAR+ PBNK compared to CAR- PBNK (41+ 1.1% vs 24.5+ 3.7%) against Ramos at E:T ratio 2:1. Conclusions: PBNK can be expanded with geK562. Anti-CD20 CAR enhances PBNK anti-tumor activity against CD20+ Ramos. Future directions include characterizing the cytotoxicity activity of engineered PBNK against L/L in vitro and survival in xenogafted mice.

Natalizumab inhibits the expression of human endogenous retroviruses of the W family in multiple sclerosis patients: a longitudinal cohort study

2013 ◽  
Vol 20 (2) ◽  
pp. 174-182 ◽  
Author(s):  
Giannina Arru ◽  
Stefania Leoni ◽  
Maura Pugliatti ◽  
Alessandra Mei ◽  
Caterina Serra ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
Flow Cytometry ◽  
Mononuclear Cells ◽  
Relative Increase ◽  
Endogenous Retroviruses ◽  
Peripheral Blood Mononuclear ◽  
Human Endogenous Retroviruses ◽  
Cell Subpopulations ◽  
Env Protein ◽  
Multiple Sclerosis Patients

Background: Several viruses were reported as co-factors triggering the pathogenesis of multiple sclerosis (MS), including the endogenous retroviruses of the HERV-W family, that were also proposed as biomarkers of disease progression and therapy outcome. Objective: The objective of this article is to clarify whether in MS patients treatment with natalizumab has effects on MSRV/syncytin-1/HERV-W expression and the possible relationship with disease outcome. Methods: Peripheral blood mononuclear cells were collected from 22 patients with relapsing–remitting disease, at entry and after three, six and 12 months of treatment with natalizumab. The cell subpopulations and the expression of MSRV env/syncytin-1/HERV-W env were analyzed by flow cytometry and by discriminatory env-specific RT-PCR assays. Results: By flow cytometry the relative amounts of T, NK and monocyte subpopulations were shown to remain fairly constant. A relative increase of B lymphocytes was observed at three to six months ( p = 0.033). The MSRV env and syncitin-1 transcripts were reduced at six to 12 months of therapy ( p = 0.0001). Accordingly, at month 12, the plasma-membrane levels of the HERV-W env protein were reduced ( p = 0.0001). B cells, NK and monocytes but not T cells expressed the HERV-W env protein. None of the patients relapsed during therapy. Conclusion: Effective therapy with natalizumab downregulates MSRV/syncytin-1/HERV-W expression.

MO251HIGHLY SENSITIVE FLOW CYTOMETRIC DETECTION OF RESIDUAL B-CELLS AFTER RITUXIMAB IN ANTI-NEUTROPHIL CYTOPLASMIC ANTIBODY-ASSOCIATED VASCULITIS PATIENTS*

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Y K O Teng ◽  
L Van Dam ◽  
Jelle Oskam ◽  
S W A Kamerling ◽  
E J Arends ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Mononuclear Cells ◽  
Plasma Cells ◽  
Memory B Cells ◽  
Cell Depletion ◽  
B Cell Depletion ◽  
Peripheral Blood Mononuclear

Abstract Background and Aims B-cell depletion with rituximab (RTX) is an effective treatment for anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) patients. Nevertheless, relapses are frequent after RTX, often preceded by B-cell repopulation suggesting that residual autoreactive B-cells persist despite therapy. Therefore, this study aimed to identify minimal residual autoimmunity (MRA) in the B-cell compartment of AAV patients treated with RTX. Method EuroFlow-based highly-sensitive flow cytometry (HSFC) was employed to study B-cell and plasma cell (PC) subsets in-depth in AAV patients before and after RTX treatment. Additionally, peripheral blood mononuclear cells (PBMCs) of these RTX-treated AAV patients were cultured and in vitro stimulated with CpG, IL-2, and IL-21 to induce antibody-secreting cells (ASC). (ANCA)-IgG was measured in these supernatants by ELISA. Results By employing EuroFlow-based HSFC, we detected circulating CD19+ B-cells at all timepoints after RTX treatment, in contrast to conventional low-sensitive flow cytometry. Pre-germinal center (Pre-GC) B-cells, memory B-cells and CD20+CD138− plasmablasts (PBs) were rapidly and strongly reduced, while CD20−CD138− PrePC and CD20-CD138+ mature (m)PCs were reduced slower and remained detectable. Both memory B-cells and CD20− PCs remained detectable after RTX. Serum ANCA-IgG decreased significantly upon RTX. Changes in ANCA levels strongly correlated with changes in naive, switched CD27+ and CD27− (double-negative) memory B-cells, but not with plasma cells. Lastly, we demonstrated in vitro ANCA production by AAV PBMCs, 24 and 48 weeks after RTX treatment reflecting MRA in the memory compartment of AAV patients. Conclusion We demonstrated that RTX induced strong reductions in circulating B-cells, but never resulted in complete B-cell depletion. Despite strongly reduced B-cell numbers after RTX, ANCA-specific memory B-cells were still detectable in AAV patients. Thus, MRA is identifiable in AAV and can provide a potential novel approach in personalizing RTX treatment in AAV patients.

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