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

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.

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

Arthritis Research & Therapy ◽

10.1186/s13075-020-02412-8 ◽

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.

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CD1c but neither CD1a nor CD1b molecules are expressed on normal, activated, and malignant human B cells: identification of a new B-cell subset

Blood ◽

10.1182/blood.v72.1.241.241 ◽

1988 ◽

Vol 72 (1) ◽

pp. 241-247 ◽

Cited By ~ 49

Author(s):

D Delia ◽

G Cattoretti ◽

N Polli ◽

E Fontanella ◽

A Aiello ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

B Cell ◽

De Novo ◽

Cell Subset ◽

Ultrastructural Level ◽

Cytoplasmic Staining ◽

Clear Cut ◽

Hodgkin's Lymphomas

Abstract The CD1 cluster of monoclonal antibodies (MoAbs) CD1a, CD1b, and CD1c, identifies molecules that are differentially expressed on hematopoietic and nonhematopoietic tissues. Our earlier finding that the mantle zone (MZ) but not the germinal center (GC) of normal lymph nodes (LN) is CD1c+, CD1a-, and CD1b- prompted us to further investigate the expression of these molecules on normal, activated, and malignant B cells. We report that blood and spleen contain CD1c+ B cells that account for 49% +/- 20.4% (mean +/- SD) and 50.9% +/- 4.4% of the total B cell population, respectively. CD1a- and CD1b-specific MoAbs are unreactive with both B and T cells; these latter are CD1c- as well. When CD1c+ and CD1c- B cells are activated in vitro, the CD1c molecule is upregulated in the former subset and induced de novo in the latter. Conversely, activated blood T cells remain CD1c-. Neither CD1a nor CD1b molecules are detected on activated T and B lymphocytes. At ultrastructural level, the CD1c+ B cells exhibit distinctive features, namely, condensed chromatin with or without a nucleolus and a unique cluster of cytoplasmic vesicles and organelles; the number of nucleolated cells is higher in the spleen (95%) than in the tonsil (40%) or blood (5%). These findings further confirm the similarity between blood and MZ B cells. The CD1c expression assessed on 27 B-cell chronic lymphocytic leukemias (B-CLL) and 46 B non-Hodgkin's lymphomas (B-NHL) was detected on 41% and 32% of cases, respectively; the latter comprised four follicular and 11 diffuse histotypes. The Burkitt's lymphomas were CD1c-negative. The B-cell neoplasms were all CD1a- and, except for four with a weak cytoplasmic staining, all CD1b- as well. The clear-cut CD1c distribution in normal LN (MZ+, GC-) contrasted with the evidence that some B-NHL cells of GC origin (eg, follicular with predominantly small cleaved cells) were CD1c+. Overall, the finding that CD1c expression is restricted to a fraction of B cells present in lymphoid organs and in peripheral blood indicates that CD1c is a powerful marker for the identification and dissection of B-cell subsets whose functional properties can now be evaluated.

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Antibody Production and In Vitro Behavior of CD27-Defined B-Cell Subsets: Persistent Hepatitis C Virus Infection Changes the Rules

Journal of Virology ◽

10.1128/jvi.80.8.3923-3934.2006 ◽

2006 ◽

Vol 80 (8) ◽

pp. 3923-3934 ◽

Cited By ~ 56

Author(s):

Vito Racanelli ◽

Maria Antonia Frassanito ◽

Patrizia Leone ◽

Maria Galiano ◽

Valli De Re ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

T Cell ◽

B Cells ◽

B Cell ◽

Feedback Inhibition ◽

Cell Subset ◽

Functional Program ◽

Cell Functions

ABSTRACT There is growing interest in the tendency of B cells to change their functional program in response to overwhelming antigen loading, perhaps by regulating specific parameters, such as efficiency of activation, proliferation rate, differentiation to antibody-secreting cells (ASC), and rate of cell death in culture. We show that individuals persistently infected with hepatitis C virus (HCV) carry high levels of circulating immunoglobulin G (IgG) and IgG-secreting cells (IgG-ASC). Thus, generalized polyclonal activation of B-cell functions may be supposed. While IgGs include virus-related and unrelated antibodies, IgG-ASC do not include HCV-specific plasma cells. Despite signs of widespread activation, B cells do not accumulate and memory B cells seem to be reduced in the blood of HCV-infected individuals. This apparent discrepancy may reflect the unconventional activation kinetics and functional responsiveness of the CD27+ B-cell subset in vitro. Following stimulation with T-cell-derived signals in the absence of B-cell receptor (BCR) engagement, CD27+ B cells do not expand but rapidly differentiate to secrete Ig and then undergo apoptosis. We propose that their enhanced sensitivity to BCR-independent noncognate T-cell help maintains a constant level of nonspecific serum antibodies and ASC and serves as a backup mechanism of feedback inhibition to prevent exaggerated B-cell responses that could be the cause of significant immunopathology.

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TLR9-deficiency in B cells Promotes Immune Tolerance via IL-10 in a Type 1 Diabetes Mouse Model

10.2337/figshare.13177016 ◽

2020 ◽

Author(s):

Ada Admin ◽

Sha Sha ◽

James A Pearson ◽

Jian Peng ◽

Youjia Hu ◽

...

Keyword(s):

Type 1 Diabetes ◽

B Cells ◽

B Cell ◽

Nod Mice ◽

Adaptive Immune ◽

Immune Molecule ◽

And Function ◽

Intrinsic Effect ◽

Specific Deletion

Toll-like receptor 9 (TLR9) is highly expressed in B cells and B cells are important in the pathogenesis of type 1 diabetes (T1D) development. However, the intrinsic effect of TLR9 in B cells on beta cell autoimmunity is not known. To fill this knowledge gap, we generated non-obese diabetic (NOD) mice with a B cell-specific deficiency of TLR9 (TLR9<sup>fl/fl</sup>/CD19-Cre+ NOD). The B cell-specific deletion of TLR9 resulted in near complete protection from T1D development. Diabetes protection was accompanied by an increased proportion of IL-10-producing B cells. We also found that TLR9-deficient B cells were hyporesponsive to both innate and adaptive immune-stimuli. This suggested that TLR9 in B cells modulates T1D susceptibility in NOD mice by changing the frequency and function of IL-10-producing B cells. Molecular analysis revealed a network of TLR9 with MMPs, TIMP1 and CD40, all of which are inter-connected with IL-10. Our study has highlighted an important connection of an innate immune molecule in B cells to the immuno-pathogenesis of T1D. Thus, targeting the TLR9 pathway, specifically in B cells, may provide a novel therapeutic strategy for T1D treatment.

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A High-Throughput Electrochemiluminescence 7-Plex Assay Simultaneously Screening for Type 1 Diabetes and Multiple Autoimmune Diseases

Journal of Visualized Experiments ◽

10.3791/61160 ◽

2020 ◽

Author(s):

Xiaofan Jia ◽

Ling He ◽

Yong Gu ◽

Hilary High ◽

Liping Yu

Keyword(s):

Type 1 Diabetes ◽

Autoimmune Diseases ◽

High Throughput

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TLR9-deficiency in B cells Promotes Immune Tolerance via IL-10 in a Type 1 Diabetes Mouse Model

10.2337/figshare.13177016.v1 ◽

2020 ◽

Author(s):

Ada Admin ◽

Sha Sha ◽

James A Pearson ◽

Jian Peng ◽

Youjia Hu ◽

...

Keyword(s):

Type 1 Diabetes ◽

B Cells ◽

B Cell ◽

Nod Mice ◽

Adaptive Immune ◽

Immune Molecule ◽

And Function ◽

Intrinsic Effect ◽

Specific Deletion

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Natural killer-like B cells promote Th17 cell differentiation and exacerbate rheumatoid arthritis

10.1101/2021.10.27.466096 ◽

2021 ◽

Author(s):

Ping Wang ◽

Jing Song ◽

Mingxin Bai ◽

Xi Zheng ◽

Yang Xie ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Cell Differentiation ◽

B Cells ◽

B Cell ◽

Disease Progression ◽

Natural Killer ◽

Th17 Cell ◽

Cell Subset ◽

Th17 Cell Differentiation

B cells are important participants in the pathogenesis of rheumatoid arthritis (RA). Besides classical B cells, novel B cell subsets are continually to be identified in recent years. Natural killer-like B (NKB) cells, a newly recognized B cell subset, are proved to be actively involved in the anti-infection immunity. However, their role in RA and the potential mechanism remain elusive. Here, we showed that NKB cells were expanded dramatically in collagen-induced arthritis (CIA) mice, demonstrating dynamic changes during the disease progression. These cells promoted CD4+ effector T cell proliferation and Th17 cell differentiation in vitro, while adoptive transfer of these cells exacerbated the arthritis severity of CIA mice. RNA Sequencing revealed that NKB cells displayed distinct differential gene expression profile under RA circumstance, potential perpetuating the disease progression. Moreover, the frequencies of NKB cells were significantly increased in RA patients, positively correlated with the clinical and immunological features. After effective therapy, these cells could be recovered to normal levels. Taken together, our results preliminarily revealed the pathogenic role of NKB cells in RA by promoting Th17 proinflammatory responses. Targeting these cells might provide potential therapeutic strategies for this persistent disease.

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Establishment of an inbred line of mice that express a synergistic immune defect precluding in vitro responses to type 1 and type 2 antigens, B cell mitogens, and a number of T cell-derived helper factors.

Journal of Experimental Medicine ◽

10.1084/jem.158.5.1401 ◽

1983 ◽

Vol 158 (5) ◽

pp. 1401-1414 ◽

Cited By ~ 3

Author(s):

J J Mond ◽

G Norton ◽

W E Paul ◽

I Scher ◽

F D Finkelman ◽

...

Keyword(s):

T Cell ◽

B Cells ◽

B Cell ◽

C3h Mice ◽

Immune Defect ◽

T Cell Help

Introduction of the CBA/N X-linked gene into C3H mice has resulted in the establishment of a new strain of mice that has profound immunologic defects. B cells from these mice show significantly impaired in vitro immune responses to the T cell-independent type 1 antigen trinitrophenyl-Brucella abortus (TNP-BA) as well as markedly reduced proliferative responses to a number of B cell mitogens when compared with the responses of the parental control mice. The in vivo response of such mice to TNP-BA is, however, comparable to that of CBA/N mice. Furthermore, B cells from C3.CBA/N mice are unresponsive to the plaque-forming cell enhancing effects induced by EL4-derived supernatant in the presence of TNP-BA, unlike B cells obtained from CBA/N or C3H/Hen mice whose responsiveness to TNP-BA can be significantly enhanced in the presence of EL4-derived supernatant. The model we have presented to best explain these results suggests that B cells from C3.CBA/N mice can be stimulated only under conditions in which they can interact with carrier-specific T cell help and not under conditions where factor-dependent responses are dominant.

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High-Throughput Ig Sequencing of Paired Blood and Spleen Samples Allows a Redefinition of Memory IgM Subsets in Humans

Blood ◽

10.1182/blood.v124.21.565.565 ◽

2014 ◽

Vol 124 (21) ◽

pp. 565-565

Author(s):

Davide Bagnara ◽

Margherita Squillario ◽

David Kipling ◽

Thierry Mora ◽

Aleksandra Walczak ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

High Throughput ◽

Germinal Center ◽

Marginal Zone ◽

Cell Subset ◽

Memory B Cells ◽

Double Negative ◽

Marginal Zone B Cells ◽

B Cell Subsets

Abstract In humans, whether B cells with the IgM+IgD+CD27+ phenotype represent an independent lineage involved in T-independent responses, similar to mouse marginal zone B cells, or whether they are part of the germinal center-derived memory B-cell pool generated during responses to T-dependent antigens, is still a debated issue. To address this question, we performed high-throughput Ig sequencing of B-cell subsets from paired blood and spleen samples and analyzed the clonal relationships between them. We isolated and analyzed 3 different B cell subsets based on CD27 and IgD staining from both blood and spleen: IgD+CD27+ (MZ) - amplified with Cmu primers IgD-CD27+ (switched and IgM-only) with Cmu, Cgamma and Calpha primers IgD-CD27- (CD27- memory or double-negative DN) with the same three primers We obtained 95729 unique sequences that clustered in 49199 different clones: 1125 clones were shared between blood and spleen of the same B-cell subset, and 1681 clones were shared between different subsets, allowing us to trace their relationships. We analyzed these clones that share sequences from different subsets/tissues for their mutation frequency distribution, CDR3-length, and VH/JH family usage, and compared these different characteristics with the bulk of sequences from their respective subset of origin. The analysis of clones shared between blood and spleen for switched IgG/IgA and for MZ subsets suggests different recirculation dynamics. For switched cells, the blood appears to be a mixture of splenic and other lymphoid tissues B cells. For MZ B cells in contrast, the blood appear to be only composed of a subgroup of the splenic repertoire, in agreement with the observation that marginal zone B cells recirculate and are mainly generated in the spleen. Clonal relationships between the IgM clones (originating from the MZ, IgM-only and double negative compartments) show that the clones involved display the characteristics of IgM-only B cells whatever their subset of origin, even in the case of the paired MZ/double-negative sequences that were not supposed to include IgM-only sequences. We therefore conclude that the clones shared between the various IgM subsets do not represent b between them, but rather correspond to a heterogeneous phenotype of the IgM-only population that concerns both IgD and CD27 expression, leading to a partial overlap with the MZ and double-negative gates. Clones shared between the MZ and the switched IgG and IgA compartment also show, for their IgM part, the mutation and repertoire characteristics of IgM-only cells and not of MZ B cells, reinforcing the conclusion that IgM-only are true memory B cells, and constitute the only subset showing clonal relationships with switched memory B cells. In summary, we report that MZ B cells have different recirculation characteristics and do not show real clonal relationships with IgM-only and switched memory B cells, in agreement with the notion that they represent a distinct differentiation pathway. In contrast, the only precursor-product relationship between IgM memory and switched B cells appear to concern a B cell subset that has been described as "IgM-only", but appears to have a more heterogeneous expression of IgD than previously reported and therefore contribute to 3-15% of the MZ compartment. Searching for markers that would permit to discriminate between marginal zone and germinal center-derived IgM memory B cells is obviously required to further delineate their respective function. Disclosures No relevant conflicts of interest to declare.

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