scholarly journals Jo-1 autoantigen-specific B cells are skewed towards distinct functional B cell subsets in anti-synthetase syndrome patients

2021 ◽  
Vol 23 (1) ◽  
Author(s):  
Jennifer Young-Glazer ◽  
Alberto Cisneros ◽  
Erin M. Wilfong ◽  
Scott A. Smith ◽  
Leslie J. Crofford ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Biology ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Cell Subset ◽  
Trna Synthetase ◽  
B Cell Subsets ◽  
Antibody Secreting Cells

Abstract Background Anti-Jo-1 autoantibodies which recognize histidyl-tRNA synthetase identify patients with the rare rheumatologic disease, anti-histidyl-tRNA synthetase syndrome (Jo-1 ARS), a phenotypically distinct subset of idiopathic inflammatory myopathies (IIM). Jo-1-binding B cells (JBCs) are implicated in disease pathogenesis, yet they have not been studied directly. We therefore aimed to characterize JBCs to better understand how they expand and function in Jo-1 ARS. Methods We enrolled 10 IIM patients diagnosed with Jo-1 ARS, 4 patients with non-Jo-1 IIM, and 8 age- and sex-matched healthy controls. We phenotypically characterized peripheral blood mononuclear cells (PBMCs) ex vivo using flow cytometry to define the B cell subsets in which JBCs reside. We further tested their ability to differentiate into antibody-secreting cells following stimulation in vitro. Results The majority of JBCs were IgM+ (not class-switched). Compared to non-JBCs in the same donors, JBCs contained a higher percentage of autoimmune-prone CD21lo cells and were increased in the CD21lo IgM+ IgD− CD27+ memory subset relative to healthy donor B cells. Whereas non-JBCs were present in the anergic BND B cell subset, JBCs were nearly absent from this compartment. JBCs were detected among plasmablasts in some donors, but a reduced frequency of JBCs differentiated into CD38hi24− plasmablasts compared to non-JBCs present in the same wells following in vitro stimulation. Conclusions JBCs are enriched for autoimmune-prone CD21lo B cells, some of which exhibit a memory phenotype in the peripheral repertoire of Jo-1 ARS patients. JBCs undergo limited class switch and show reduced capacity to differentiate into antibody-secreting cells. This suggests complex B cell biology exists beyond class-switched cells that differentiate to secrete anti-Jo-1 autoantibody (i.e., what is captured through serum autoantibody studies). New Jo-1 ARS therapies should thus ideally target non-class-switched JBCs in addition to those that have undergone IgG class-switching to most effectively block cross-talk with autoreactive T cells.

scholarly journals High-Throughput Detection of Autoantigen-Specific B Cells Among Distinct Functional Subsets in Autoimmune Donors

2021 ◽  
Vol 12 ◽  
Author(s):  
Bryan A. Joosse ◽  
James H. Jackson ◽  
Alberto Cisneros ◽  
Austin B. Santhin ◽  
Scott A. Smith ◽  
...  
Keyword(s):  
Type 1 Diabetes ◽  
B Cells ◽  
Autoimmune Diseases ◽  
B Cell ◽  
High Throughput ◽  
Cell Subset ◽  
Trna Synthetase ◽  
Antibody Secreting Cells

Antigen-specific B cells (ASBCs) can drive autoimmune disease by presenting autoantigen to cognate T cells to drive their activation, proliferation, and effector cell differentiation and/or by differentiating into autoantibody-secreting cells. Autoantibodies are frequently used to predict risk and diagnose several autoimmune diseases. ASBCs can drive type 1 diabetes even when immune tolerance mechanisms block their differentiation into antibody-secreting cells. Furthermore, anti-histidyl tRNA synthetase syndrome patients have expanded IgM+ Jo-1-binding B cells, which clinically diagnostic IgG Jo-1 autoantibodies may not fully reflect. Given the potential disconnect between the pathologic function of ASBCs and autoantibody secretion, direct study of ASBCs is a necessary step towards developing better therapies for autoimmune diseases, which often have no available cure. We therefore developed a high-throughput screening pipeline to 1) phenotypically identify specific B cell subsets, 2) expand them in vitro, 3) drive them to secrete BCRs as antibody, and 4) identify wells enriched for ASBCs through ELISA detection of antibody. We tested the capacity of several B cell subset(s) to differentiate into antibody-secreting cells following this robust stimulation. IgM+ and/or IgD+, CD27- memory, memory, switched memory, and BND B cells secreted B cell receptor (BCR) as antibody following in vitro stimulation, whereas few plasmablasts responded. Bimodal responses were observed across autoimmune donors for IgM+ CD21lo and IgM- CD21lo B cells, consistent with documented heterogeneity within the CD21lo subset. Using this approach, we detected insulin-binding B cell bias towards CD27- memory and CD27+ memory subsets in pre-symptomatic type 1 diabetes donors. We took advantage of routine detection of Jo-1-binding B cells in Jo-1+ anti-histidyl tRNA synthetase syndrome patients to show that Jo-1-binding B cells and total B cells expanded 20-30-fold using this culture system. Overall, these studies highlight technology that is amenable to small numbers of cryopreserved peripheral blood mononuclear cells that enables interrogation of phenotypic and repertoire attributes of ASBCs derived from autoimmune patients.

scholarly journals Optimisation of ex vivo memory B cell expansion/differentiation for interrogation of rare peripheral memory B cell subset responses

2018 ◽  
Vol 2 ◽  
pp. 97 ◽  
Author(s):  
Luke Muir ◽  
Paul F. McKay ◽  
Velislava N. Petrova ◽  
Oleksiy V. Klymenko ◽  
Sven Kratochvil ◽  
...  
Keyword(s):  
Cell Culture ◽  
B Cells ◽  
B Cell ◽  
Cell Expansion ◽  
Ex Vivo ◽  
Cell Subset ◽  
Human Memory ◽  
Memory B Cells ◽  
Memory B Cell ◽  
B Cell Subsets

Background:Human memory B cells play a vital role in the long-term protection of the host from pathogenic re-challenge. In recent years the importance of a number of different memory B cell subsets that can be formed in response to vaccination or infection has started to become clear. To study memory B cell responses, cells can be culturedex vivo,allowing for an increase in cell number and activation of these quiescent cells, providing sufficient quantities of each memory subset to enable full investigation of functionality. However, despite numerous papers being published demonstrating bulk memory B cell culture, we could find no literature on optimised conditions for the study of memory B cell subsets, such as IgM+memory B cells.Methods:Following a literature review, we carried out a large screen of memory B cell expansion conditions to identify the combination that induced the highest levels of memory B cell expansion. We subsequently used a novel Design of Experiments approach to finely tune the optimal memory B cell expansion and differentiation conditions for human memory B cell subsets. Finally, we characterised the resultant memory B cell subpopulations by IgH sequencing and flow cytometry.Results:The application of specific optimised conditions induce multiple rounds of memory B cell proliferation equally across Ig isotypes, differentiation of memory B cells to antibody secreting cells, and importantly do not alter the Ig genotype of the stimulated cells. Conclusions:Overall, our data identify a memory B cell culture system that offers a robust platform for investigating the functionality of rare memory B cell subsets to infection and/or vaccination.

scholarly journals Optimisation of ex vivo memory B cell expansion/differentiation for interrogation of rare peripheral memory B cell subset responses

2017 ◽  
Vol 2 ◽  
pp. 97 ◽  
Author(s):  
Luke Muir ◽  
Paul F. McKay ◽  
Velislava N. Petrova ◽  
Oleksiy V. Klymenko ◽  
Sven Kratochvil ◽  
...  
Keyword(s):  
Cell Culture ◽  
B Cells ◽  
B Cell ◽  
Cell Expansion ◽  
Ex Vivo ◽  
Cell Subset ◽  
Human Memory ◽  
Memory B Cells ◽  
Memory B Cell ◽  
B Cell Subsets

Background:Human memory B cells play a vital role in the long-term protection of the host from pathogenic re-challenge. In recent years the importance of a number of different memory B cell subsets that can be formed in response to vaccination or infection has started to become clear. To study memory B cell responses, cells can be culturedex vivo,allowing for an increase in cell number and activation of these quiescent cells, providing sufficient quantities of each memory subset to enable full investigation of functionality. However, despite numerous papers being published demonstrating bulk memory B cell culture, we could find no literature on optimised conditions for the study of memory B cell subsets, such as IgM+memory B cells.Methods:Following a literature review, we carried out a large screen of memory B cell expansion conditions to identify the combination that induced the highest levels of memory B cell expansion. We subsequently used a novel Design of Experiments approach to finely tune the optimal memory B cell expansion and differentiation conditions for human memory B cell subsets. Finally, we characterised the resultant memory B cell subpopulations by IgH sequencing and flow cytometry.Results:The application of specific optimised conditions induce multiple rounds of memory B cell proliferation equally across Ig isotypes, differentiation of memory B cells to antibody secreting cells, and importantly do not alter the Ig genotype of the stimulated cells. Conclusions:Overall, our data identify a memory B cell culture system that offers a robust platform for investigating the functionality of rare memory B cell subsets to infection and/or vaccination.

scholarly journals Carrier-directed anti-hapten responses by b-cell subsets

1977 ◽  
Vol 146 (1) ◽  
pp. 1-10 ◽  
Author(s):  
GK Lewis ◽  
JW Goodman
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Activation ◽  
Burst Size ◽  
Cell Subset ◽  
B Cell Activation ◽  
Cell Subpopulations ◽  
Activation Pathways ◽  
B Cell Subsets

The capacity of the trinitrophenyl (TNP) haptenic group, coupled to a series of chemically dissimilar carriers, to cross-stimulate putative T- dependent and T-independent murine B-cell subpepulations was determined by using an in vitro limiting dilution technique to generate primary IgM responses. It was found that TNP-Ficoll and TNP-dextran, two T- independent antigens with little or no polyclonal mitogenicity, stimulate the same population of anti-TNP precursors, which is distinct from the precursor population activated by TNP-bacterial lipopolysaccharide (LPS), a T-independent polyclonal mitogen, or TNP-horse erythrocytes (HRBC), a T-dependent antigen. On the other hand, TNP-LPS and TNP-HRBC activate the same precursor population, indicating that LPS can substitute for the T- cell signal in T-dependent B-cell responses, whereas nonmitogenic T- independent antigens cannot. However, the cumulative evidence from this and other laboratories strongly indicates that LPS and T-dependent antigens activate B cells by different mechanisms. Of particular interest, LPS is incapable of activating B cells responsive to weakly- or nonmitogenic T-independent antigens. Based on clonal burst size, T-dependent antigens are capable of inducing greater antigen-specific B-cell proliferation than T-independent antigens. However, TNP conjugates of Ficoll and dextran, which are relatively poor inducers of polyclonal B-cell activation, induced larger anti-TNP clones than did TNP-LPS, a strong polyclonal mitogen. The findings reinforce the evidence favoring existence of multiple B- cell subpopulations with distinctive activation pathways. They also strengthen the proposition that a given B-cell subset can be activated by more than one mechanism.

scholarly journals Distinct Ex Vivo Susceptibility of B-Cell Subsets to Epstein-Barr Virus Infection According to Differentiation Status and Tissue Origin

2008 ◽  
Vol 82 (9) ◽  
pp. 4400-4412 ◽  
Author(s):  
Marcus Dorner ◽  
Franziska Zucol ◽  
Christoph Berger ◽  
Rahel Byland ◽  
Gregory T. Melroe ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Ex Vivo ◽  
Memory B Cells ◽  
Barr Virus ◽  
Ebv Infection ◽  
Epstein Barr ◽  
Differentiation Status ◽  
B Cell Subsets

ABSTRACT Epstein-Barr virus (EBV) uses tonsils as the portal of entry to establish persistent infection. EBV is found in various B-cell subsets in tonsils but exclusively in memory B cells in peripheral blood. The in vitro susceptibilities of B-cell subsets to EBV infection have been studied solely qualitatively. In this work, we examined quantitatively the in vitro susceptibilities of various B-cell subsets from different tissue origins to EBV infection. First, we established a centrifugation-based inoculation protocol (spinoculation) that resulted in a significantly increased proportion of infected cells compared to that obtained by conventional inoculation, enabling a detailed susceptibility analysis. Importantly, B-cell infection occurred via the known EBV receptors and infected cells showed EBV mRNA expression patterns similar to those observed after conventional inoculation, validating our approach. Tonsillar naïve and memory B cells were infected ex vivo at similar frequencies. In contrast, memory B cells from blood, which represent B cells from various lymphoid tissues, were infected at lower frequencies than their naïve counterparts. Immunoglobulin A (IgA)-positive or IgG-positive tonsillar memory B cells were significantly more susceptible to EBV infection than IgM-positive counterparts. Memory B cells were transformed with lower efficiency than naïve B cells. This result was paralleled by lower proliferation rates. In summary, these data suggest that EBV exploits the B-cell differentiation status and tissue origin to establish persistent infection.

scholarly journals Characterization of a rare IL-10–competent B-cell subset in humans that parallels mouse regulatory B10 cells

Blood ◽  
2011 ◽  
Vol 117 (2) ◽  
pp. 530-541 ◽  
Author(s):  
Yohei Iwata ◽  
Takashi Matsushita ◽  
Mayuka Horikawa ◽  
David J. DiLillo ◽  
Koichi Yanaba ◽  
...  
Keyword(s):  
B Cells ◽  
Autoimmune Disease ◽  
B Cell ◽  
Lupus Erythematosus ◽  
Ex Vivo ◽  
Cell Subset ◽  
Cell Subpopulation ◽  
B10 Cells

Abstract Regulatory B cells control inflammation and autoimmunity in mice, including the recently identified IL-10–competent B10 cell subset that represents 1% to 3% of spleen B cells. In this study, a comparable IL-10–competent B10 cell subset was characterized in human blood. B10 cells were functionally identified by their ability to express cytoplasmic IL-10 after 5 hours of ex vivo stimulation, whereas progenitor B10 (B10pro) cells required 48 hours of in vitro stimulation before they acquired the ability to express IL-10. B10 and B10pro cells represented 0.6% and approximately 5% of blood B cells, respectively. Ex vivo B10 and B10pro cells were predominantly found within the CD24hiCD27+ B-cell subpopulation that was able to negatively regulate monocyte cytokine production through IL-10–dependent pathways during in vitro functional assays. Blood B10 cells were present in 91 patients with rheumatoid arthritis, systemic lupus erythematosus, primary Sjögren syndrome, autoimmune vesiculobullous skin disease, or multiple sclerosis, and were expanded in some cases as occurs in mice with autoimmune disease. Mean B10 + B10pro-cell frequencies were also significantly higher in patients with autoimmune disease compared with healthy controls. The characterization of human B10 cells will facilitate their identification and the study of their regulatory activities during human disease.

scholarly journals Recovery of Memory B-cell Subsets and Persistence of Antibodies in Convalescent COVID-19 Patients

2021 ◽  
Author(s):  
Anuradha Rajamanickam ◽  
Nathella Pavan Kumar ◽  
Arul Nancy P ◽  
Nandhini Selvaraj ◽  
Saravanan Munisankar ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Neutralizing Antibodies ◽  
Plasma Cells ◽  
Ex Vivo ◽  
Cell Subset ◽  
Memory B Cells ◽  
Memory B Cell ◽  
Immature B Cells ◽  
B Cell Subsets

It is essential to examine the longevity of the defensive immune response engendered by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. We examined the SARS-CoV-2-specific antibody responses and ex vivo memory B-cell subsets in seven groups of individuals with COVID-19 classified based on days since reverse-transcription polymerase chain reaction confirmation of SARS-CoV-2 infection. Our data showed that the levels of IgG and neutralizing antibodies started increasing from days 15 to 30 to days 61 to 90, and plateaued thereafter. The frequencies of naive B cells and atypical memory B cells decreased from days 15 to 30 to days 61 to 90, and plateaued thereafter. In contrast, the frequencies of immature B cells, classical memory B cells, activated memory B cells, and plasma cells increased from days 15 to 30 to days 61 to 90, and plateaued thereafter. Patients with severe COVID-19 exhibited increased frequencies of naive cells, atypical memory B cells, and activated memory B cells, and lower frequencies of immature B cells, central memory B cells, and plasma cells when compared with patients with mild COVID-19. Therefore, our data suggest modifications in memory B-cell subset frequencies and persistence of humoral immunity in convalescent individuals with COVID-19.

scholarly journals Hyper‐metabolic B cells in the spleens of old mice make antibodies with autoimmune specificities

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Daniela Frasca ◽  
Maria Romero ◽  
Denisse Garcia ◽  
Alain Diaz ◽  
Bonnie B. Blomberg
Keyword(s):  
B Cells ◽  
B Cell ◽  
Inflammatory Markers ◽  
Cell Subset ◽  
Antibody Responses ◽  
Metabolic Phenotype ◽  
Cellular Mechanisms ◽  
Autoimmune Antibodies ◽  
B Cell Subsets ◽  
Old Mice

Abstract Background Aging is associated with increased intrinsic B cell inflammation, decreased protective antibody responses and increased autoimmune antibody responses. The effects of aging on the metabolic phenotype of B cells and on the metabolic programs that lead to the secretion of protective versus autoimmune antibodies are not known. Methods Splenic B cells and the major splenic B cell subsets, Follicular (FO) and Age-associated B cells (ABCs), were isolated from the spleens of young and old mice and left unstimulated. The RNA was collected to measure the expression of markers associated with intrinsic inflammation and autoimmune antibody production by qPCR. B cells and B cell subsets were also stimulated with CpG and supernatants collected after 7 days to measure autoimmune IgG secretion by ELISA. Metabolic measures (oxygen consumption rate, extracellular acidification rate and glucose uptake) were performed using a Seahorse XFp extracellular flux analyzer. Results Results have identified the subset of ABCs, whose frequencies and numbers increase with age and represent the most pro-inflammatory B cell subset, as the cell type mainly if not exclusively responsible for the expression of inflammatory markers and for the secretion of autoimmune antibodies in the spleen of old mice. Hyper-inflammatory ABCs from old mice are also hyper-metabolic, as compared to those from young mice and to the subset of FO B cells, a feature needed not only to support their higher expression of RNA for inflammatory markers but also their higher autoimmune antibody secretion. Conclusions These results identify a relationship between intrinsic inflammation, metabolism and autoimmune B cells and suggest possible ways to understand cellular mechanisms that lead to the generation of pathogenic B cells, that are hyper-inflammatory and hyper-metabolic, and secrete IgG antibodies with autoimmune specificities.

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 Dual T cell– and B cell–intrinsic deficiency in humans with biallelic RLTPR mutations

2016 ◽  
Vol 213 (11) ◽  
pp. 2413-2435 ◽  
Author(s):  
Yi Wang ◽  
Cindy S. Ma ◽  
Yun Ling ◽  
Aziz Bousfiha ◽  
Yildiz Camcioglu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cd4 T Cells ◽  
Ex Vivo ◽  
Cell Phenotype ◽  
Loss Of Function ◽  
Inborn Errors

Combined immunodeficiency (CID) refers to inborn errors of human T cells that also affect B cells because of the T cell deficit or an additional B cell–intrinsic deficit. In this study, we report six patients from three unrelated families with biallelic loss-of-function mutations in RLTPR, the mouse orthologue of which is essential for CD28 signaling. The patients have cutaneous and pulmonary allergy, as well as a variety of bacterial and fungal infectious diseases, including invasive tuberculosis and mucocutaneous candidiasis. Proportions of circulating regulatory T cells and memory CD4+ T cells are reduced. Their CD4+ T cells do not respond to CD28 stimulation. Their CD4+ T cells exhibit a "Th2" cell bias ex vivo and when cultured in vitro, contrasting with the paucity of "Th1," "Th17," and T follicular helper cells. The patients also display few memory B cells and poor antibody responses. This B cell phenotype does not result solely from the T cell deficiency, as the patients’ B cells fail to activate NF-κB upon B cell receptor (BCR) stimulation. Human RLTPR deficiency is a CID affecting at least the CD28-responsive pathway in T cells and the BCR-responsive pathway in B cells.

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