scholarly journals OP0186 CHANGES IN CIRCULATING B CELL LEVELS AND IMMUNOPHENOTYPE ARE ASSOCIATED WITH DEVELOPMENT OF ARTHRITIS FOLLOWING TREATMENT WITH CHECKPOINT INHIBITORS

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 112.2-113
Author(s):  
M. Gatto ◽  
S. Bjursten ◽  
C. Jonell ◽  
C. Jonsson ◽  
S. Mcgrath ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
Adverse Events ◽  
B Cell ◽  
Mononuclear Cells ◽  
Checkpoint Inhibitors ◽  
Therapy Monitoring ◽  
Cell Subset ◽  
Peripheral Blood Mononuclear ◽  
Transitional B Cells

Background:Inflammatory arthritis (IA) is frequent among rheumatic side effects induced by checkpoint inhibitor (CPI) therapy for metastatic malignancies1. While T cells are likely to sustain the inflammatory process2, fewer data are available concerning the role of B cells3.Objectives:To investigate the phenotype of circulating B cells in patients who develop CPI-induced IA (CPI-IA) and to compare it with features of B cells in patients not developing immune-related adverse events (irAE) upon CPI treatment.Methods:B cell subsets at baseline (before CPI initiation) and during CPI treatment were analyzed in CPI-IA patients and in patients receiving CPI but who did not develop irAE (non-irAE). Peripheral blood mononuclear cells (PBMC) were analyzed by flow cytometry and B cells were identified as CD19+ and divided into naïve (CD27-IgD+), memory (CD27+IgD+/-), double negative (CD27-IgD-) and transitional (CD10+CD24+CD38+/hi) B cells. Levels of CD21, an activation marker on transitional B cells, were also analyzed. Non-parametric tests were used for analysis of differences between groups.Results:Six CPI-IA and 7 non-irAE patients matched for age, gender and CPI treatment were included, who had received CPI treatment due to metastatic melanoma. Flow cytometry revealed a significant increase of circulating B cells (p=0.002) (Figure 1A) and especially of transitional B cells in CPI-IA patients vs. non-irAE (median %, range: 7.8 (4.5-11.4) vs. 3.2 (1.6-4.3),p=0.007) (Figure 1B), while no remarkable changes were seen across other subsets. Transitional B cell levels significantly decreased from active to quiescent CPI-IA in all patients (p=0.008). In two CPI-IA patients for whom baseline sampling was available, the increase of transitional levels occurred early after CPI treatment and before CPI-IA onset. Levels of expression of CD21 on transitional B cells were increased in CPI-IA vs. non-irAE (p=0.01).Conclusion:Transitional B cells are expanded in CPI-IA patients and seem to increase early after start of CPI therapy. Monitoring this B cell subset might lead to closer follow-up and earlier diagnosis of CPI-IA.References:[1]Ramos-Casals M, Brahmer JR, Callahan MK, et al. Immune-related adverse events of checkpoint inhibitors. Nat Rev Dis Primers 2020;6:38[2]Murray-Brown W, Wilsdon TD, Weedon H, et al. Nivolumab-induced synovitis is characterized by florid T cell infiltration and rapid resolution with synovial biopsy-guided therapy. J Immunother Cancer 2020;8:e000281[3]Das R, Bar N, Ferreira M, et al. Early B cell changes predict autoimmunity following combination immune checkpoint blockade. J Clin Invest. 2018;128:715-2Disclosure of Interests:None declared

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

Lupus ◽  
2019 ◽  
Vol 28 (11) ◽  
pp. 1337-1343 ◽  
Author(s):  
A Benitez ◽  
K Torralba ◽  
M Ngo ◽  
L M Salto ◽  
K S Choi ◽  
...  
Keyword(s):  
Systemic Lupus Erythematosus ◽  
B Cells ◽  
B Cell ◽  
Lupus Erythematosus ◽  
Mononuclear Cells ◽  
Cell Subset ◽  
Systemic Lupus ◽  
Peripheral Blood Mononuclear ◽  
Healthy Donors ◽  
Immature B Cells

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

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.

scholarly journals Highly Sensitive Flow Cytometric Detection of Residual B-Cells After Rituximab in Anti-Neutrophil Cytoplasmic Antibodies-Associated Vasculitis Patients

2020 ◽  
Vol 11 ◽  
Author(s):  
Laura S. van Dam ◽  
Jelle M. Oskam ◽  
Sylvia W. A. Kamerling ◽  
Eline J. Arends ◽  
O. W. Bredewold ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Mononuclear Cells ◽  
Memory B Cells ◽  
Cell Depletion ◽  
B Cell Depletion ◽  
Peripheral Blood Mononuclear ◽  
Highly Sensitive

BackgroundB-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.MethodsEuroFlow-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.ResultsBy 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.ConclusionWe 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.

scholarly journals Altered B cells homeostasis in child-onset immunoglobulin A vasculitis

2020 ◽  
Author(s):  
Deying Liu ◽  
Yanfang Jiang ◽  
Jinghua Wang ◽  
Jinxiang Liu ◽  
Meng Xu ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Mononuclear Cells ◽  
Immunoglobulin A ◽  
Cell Subset ◽  
Memory B Cells ◽  
Peripheral Blood Mononuclear ◽  
Cell Numbers ◽  
Cell Counts ◽  
Immunoglobulin A Vasculitis

AbstractBackgroundImmunoglobulin A vasculitis (IgAV), also called Henoch–Schönlein purpura, is a systemic small vessels vasculitis with immunoglobulin A1-dominant immune deposits. B-cells are a heterogeneous population with unique subsets distinguished by their phenotypes and cytokine production. Here, we explored the status of B cell subsets in patients with IgAV.MethodsThirty IgAV patients and fifteen age- and sex-matched healthy individuals were enrolled in this study. Fresh blood samples were collected from both healthy and IgAV patients. Upon the distinct expressions of CD3, CD19, CD20, CD38, CD27 and IgD, peripheral blood mononuclear cells (PBMCs) were initially categorized into plasmablasts and memory B cells. Subsequently, using surface markers including CD138 and IgM, and intracellular markers containing IgM and IgG, plasmablasts and memory B cells were further divided into distinct subgroups. A total of eleven populations were detected using multiple flow cytometry.ResultsCD3-CD19+IgD+CD27-, CD3-CD19+CD20-CD38+, CD3-CD19+CD20-CD38+IgM+, and CD3-CD19+CD20-CD38+CD138+ B cells were larger in patients with IgAV than in the HCs. Only CD3-CD19+IgD-CD27+IgM+ B cell counts were reduced in IgAV. The elevated B cell numbers returned to normal after treatment. Plasma and plasmablast B cell numbers correlated with plasma IgA levels. On the contrary, CD3-CD19+IgD-CD27+IgM+ B cell numbers were negatively proportional to the plasma IgA levels while naïve B cell numbers correlated with plasma and plasmablast B cell counts.ConclusionsWe hypothesized that immunoglobulin production was abnormally elevated in IgAV and could be explained by altered B-cell subset homeostasis.

scholarly journals Aberrant Frequency of IL-10-Producing B Cells and Its Association with Treg/Th17 in Adult Primary Immune Thrombocytopenia Patients

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Fanli Hua ◽  
Lili Ji ◽  
Yanxia Zhan ◽  
Feng Li ◽  
Shanhua Zou ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Immune Thrombocytopenia ◽  
Mononuclear Cells ◽  
Cell Subset ◽  
Brefeldin A ◽  
Interleukin 10 ◽  
T Cell Subsets ◽  
Peripheral Blood Mononuclear ◽  
B10 Cells

Background. Regulatory B cells (Breg) are a distinct B cell subset with immunoregulatory properties. Pivotal to Breg function is interleukin-10. This study was to investigate the role of IL-10-producing B cell (B10) and its association with Treg and Th17 subsets in immune thrombocytopenia (ITP) patients.Methods. Peripheral blood mononuclear cells from ITP patients and controls were stimulated with PMA, ionomycin, and Brefeldin A. The frequencies of CD19+IL-10+B cells, CD3+CD4+IL-17+Th17 cells, and CD4+CD25hiFoxp3+Treg cells were analyzed by flow cytometry. The mRNA expression of Foxp3 and RORγt was detected by real-time quantitative PCR.Results. The number of B10 cells was elevated in ITP patients. After first-line therapies, it remained at high level in patients who achieved complete or partial response but decreased in those who acquired no response. There was a positive correlation between B10 cells and Tregs in ITP both before and after therapies. The ratio of Treg/Th17 decreased in ITP, and it strongly correlated with B10 cells.Conclusions. The frequency of B10 cells is elevated in ITP and it correlates with both the Tregs counts and the Treg/Th17 ratio. B10 cells to regulate functional T cell subsets might be impaired in patients with ITP.

scholarly journals Elevated STAT1 expression but not phosphorylation in lupus B cells correlates with disease activity and increased plasmablast susceptibility

Rheumatology ◽  
2020 ◽  
Vol 59 (11) ◽  
pp. 3435-3442 ◽  
Author(s):  
Arman Aue ◽  
Franziska Szelinski ◽  
Sarah Y Weißenberg ◽  
Annika Wiedemann ◽  
Thomas Rose ◽  
...  
Keyword(s):  
B Cells ◽  
Disease Activity ◽  
B Cell ◽  
Mononuclear Cells ◽  
Janus Kinase ◽  
T Cell Subsets ◽  
Type I ◽  
Type I Ifn ◽  
Peripheral Blood Mononuclear ◽  
B Cell Subsets

Abstract Objectives SLE is characterized by two pathogenic key signatures, type I IFN and B-cell abnormalities. How these signatures are interrelated is not known. Type I-II IFN trigger activation of Janus kinase (JAK) – signal transducer and activator of transcription (STAT). JAK-STAT inhibition is an attractive therapeutic possibility for SLE. We assess STAT1 and STAT3 expression and phosphorylation at baseline and after IFN type I and II stimulation in B-cell subpopulations of SLE patients compared with other autoimmune diseases and healthy controls (HD) and related it to disease activity. Methods Expression of STAT1, pSTAT1, STAT3 and pSTAT3 in B and T cells of 21 HD, 10 rheumatoid arthritis (RA), seven primary Sjögren’s (pSS) and 22 SLE patients was analysed by flow cytometry. STAT1 and STAT3 expression and phosphorylation in PBMCs (peripheral blood mononuclear cells) of SLE patients and HD after IFNα and IFNγ incubation were further investigated. Results SLE patients showed substantially higher STAT1 but not pSTAT1 in B- and T-cell subsets. Increased STAT1 expression in B-cell subsets correlated significantly with SLEDAI and Siglec-1 on monocytes, a type I IFN marker. STAT1 activation in plasmablasts was IFNα dependent while monocytes exhibited dependence on IFNγ. Conclusion Enhanced expression of STAT1 by B-cell candidates as a key node of two immunopathogenic signatures (type I IFN and B-cells) related to important immunopathogenic pathways and lupus activity. We show that STAT1 is activated upon IFNα exposure in SLE plasmablasts. Thus, Jak inhibitors, targeting JAK-STAT pathways, hold a promise to block STAT1 expression and control plasmablast induction in SLE.

scholarly journals Evidence for progenitors of chronic lymphocytic leukemia B cells that undergo intraclonal differentiation and diversification

Blood ◽  
1996 ◽  
Vol 87 (4) ◽  
pp. 1586-1594 ◽  
Author(s):  
M Dono ◽  
S Hashimoto ◽  
F Fais ◽  
V Trejo ◽  
SL Allen ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Dna Sequences ◽  
Mononuclear Cells ◽  
Lymphocytic Leukemia ◽  
Specific Primers ◽  
Region Gene ◽  
Peripheral Blood Mononuclear ◽  
Ancestral Gene

Peripheral blood mononuclear cells from five patients with IgG+ B-type chronic lymphocytic leukemia (B-CLL) were analyzed for the presence of clone-specific Ig H chain variable region gene mRNA transcripts linked to C mu and/or C alpha. This was assessed by (1) comparing the lengths of portions of the VHDJH of the IgG+ CLL clones with those of the mu and alpha isotype-expressing B cells, (2) performing clone-specific endonuclease digestion studies, and (3) determining the DNA sequences of the mu and alpha isotype-expressing cDNA. Thus, when B-cell mRNA from these five patients were reverse transcribed with C gamma-specific primers and then amplified by polymerase chain reaction, dominant cDNA were found with lengths corresponding to those of the IgG+ CLL B cell. In addition, in four cases, cDNA of lengths identical to those of the CLL B cell were detected when mRNA was reverse transcribed and amplified using c mu- and/or C alpha-specific primers, strongly suggesting clonal relatedness. These CLL-related mu- and alpha- expressing cDNA were present in greater amounts that unrelated (non- CLL) mu- and alpha-expressing cDNA from normal B cells that used genes of the same VH family. When the sequences of these CLL-related C mu- and C alpha-expressing cDNA were compared with those of the IgG+ CLL clones, it was clear that they were derived from the same ancestral gene as the IgG-expressing CLL B cell, thus documenting their common origin. Finally, nucleotide point mutations were observed in the mu- and alpha-expressing cDNA of certain patients, indicating divergence with the CLL. These data suggest that IgM+ B cells, which are precursors of the leukemic B cells, exist in increased numbers in the blood of most patients with IgG+ B-CELL and that these cells may differentiate, accumulate V genes mutations, and undergo isotype switching in vivo. In addition, the data are consistent with a sequential-hit model for the evolution of CLL.

Gastrointestinal defects and immunodeficiency syndrome with normal in vitro IgG production

2018 ◽  
Vol 5 (3) ◽  
pp. 91-99
Author(s):  
Alexandra Langlois ◽  
Bahar Torabi ◽  
Marieme Dembele ◽  
Marylin Desjardins ◽  
Reza Alizadehfar ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Mononuclear Cells ◽  
Cell Function ◽  
Current Knowledge ◽  
Genetic Defect ◽  
Peripheral Blood Mononuclear ◽  
Cardiac Malformations ◽  
Immunodeficiency Syndrome

Background: Gastrointestinal defects and immunodeficiency syndrome (GIDID) is a severe neonatal disorder usually fatal within the first months of life. We report a case presenting with intestinal atresia, combined immunodeficiency, and a novel association with hypothyroidism and cardiac malformations. The immune phenotype was remarkable for agammaglobulinemia, lymphopenia, and mildly decreased lymphocyte proliferation. We present here the unique phenotype as well as studies to determine if the agammaglobulinemia was due to an intrinsic B lymphocyte defect. Methods: Peripheral blood mononuclear cells from the patient and a healthy control were isolated by Ficoll-Hypaque centrifugation and stimulated with anti-CD40, IL-4 and IL-21 for 7 days. Total IgG production was measured by ELISA in the supernatant of the stimulated sample on day 7. Cells were stained for CD19, CD27, IgM, CD11b, CD11c, and CD14. Results: At day 7, supernatant from the patient stimulated cells contained levels of total IgG comparable to the control (755 ng/mL vs. 658 ng/mL, respectively). B cell maturation appeared impaired, as morphologically the patient sample demonstrated fewer B cell clones and cells with dendritic projections. Conclusions: Despite this typical severe clinical picture of GIDID with agammaglobulinemia, IgG production was detected under optimal stimulation for induction of plasma cells. This suggests that there may not be an inherent defect in class switching and antibody production in B cells in this disorder. It is possible that the in vivo physical or cytokine milieu may be defective for optimal B cell function. Further studies assessing the function of the immune cells as well as possible gastrointestinal loss of immunoglobulins are needed in this disease. Statement of novelty: Despite much improvement in understanding the effects of TTC7A mutations in GIDID, the root cause of hypogammaglobulinemia in these patients is still unclear. The work portrayed in this study furthers the current knowledge. It suggests that when appropriately stimulated in vitro, this patient’s B cells were capable of adequate immunoglobulin production. Moreover, to the best of our knowledge, this patient is the first with this genetic defect to be reported with hypothyroidism and cardiac malformations.

scholarly journals Generation of Monoclonal Antibodies against Immunoglobulin Proteins of the Domestic Ferret (Mustela putorius furo)

2017 ◽  
Vol 2017 ◽  
pp. 1-13 ◽  
Author(s):  
Greg A. Kirchenbaum ◽  
Ted M. Ross
Keyword(s):  
Animal Model ◽  
Monoclonal Antibodies ◽  
B Cells ◽  
B Cell ◽  
Mononuclear Cells ◽  
Unmet Need ◽  
Mustela Putorius ◽  
Peripheral Blood Mononuclear ◽  
Antibody Secreting Cells ◽  
Mustela Putorius Furo

The domestic ferret (Mustela putorius furo) serves as an animal model for the study of several viruses that cause human disease, most notably influenza. Despite the importance of this animal model, characterization of the immune response by flow cytometry (FCM) is severely hampered due to the limited number of commercially available reagents. To begin to address this unmet need and to facilitate more in-depth study of ferret B cells including the identification of antibody-secreting cells, eight unique murine monoclonal antibodies (mAb) with specificity for ferret immunoglobulin (Ig) were generated using conventional B cell hybridoma technology. These mAb were screened for reactivity against ferret peripheral blood mononuclear cells by FCM and demonstrate specificity for CD79β+B cells. Several of these mAb are specific for the light chain of surface B cell receptor (BCR) and enable segregation of kappa and lambda B cells. Additionally, a mAb that yielded surface staining of nearly all surface BCR positive cells (i.e., pan ferret Ig) was generated. Collectively, these MαF-Ig mAb offer advancement compared to the existing portfolio of polyclonal anti-ferret Ig detection reagents and should be applicable to a wide array of immunologic assays including the identification of antibody-secreting cells by FCM.

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