Class-switzched B cells display response to therapeutic B-cell depletion in rheumatoid arthritis

Introduction Reconstitution of peripheral blood (PB) B cells after therapeutic depletion with the chimeric anti-CD20 antibody rituximab (RTX) mimics lymphatic ontogeny. In this situation, the repletion kinetics and migratory properties of distinct developmental B-cell stages and their correlation to disease activity might facilitate our understanding of innate and adaptive B-cell functions in rheumatoid arthritis (RA). Methods Thirty-five 'RTX-naïve' RA patients with active arthritis were treated after failure of tumour necrosis factor blockade in an open-label study with two infusions of 1,000 mg RTX. Prednisone dose was tapered according to clinical improvement from a median of 10 mg at baseline to 5 mg at 9 and 12 months. Conventional disease-modifying antirheumatic drugs were kept stable. Subsets of CD19+ B cells were assessed by flow cytometry according to their IgD and CD27 surface expression. Their absolute number and relative frequency in PB were followed every 3 months and were determined in parallel in synovial tissue (n = 3) or synovial fluid (n = 3) in the case of florid arthritis. Results Six of 35 patients fulfilled the European League Against Rheumatism criteria for moderate clinical response, and 19 others for good clinical response. All PB B-cell fractions decreased significantly in number (P < 0.001) after the first infusion. Disease activity developed independently of the total B-cell number. B-cell repopulation was dominated in quantity by CD27-IgD+ 'naïve' B cells. The low number of CD27+IgD- class-switched memory B cells (MemB) in the blood, together with sustained reduction of rheumatoid factor serum concentrations, correlated with good clinical response. Class-switched MemB were found accumulated in flaring joints. Conclusions The present data support the hypothesis that control of adaptive immune processes involving germinal centre-derived, antigen, and T-cell-dependently matured B cells is essential for successful RTX treatment.

IgE+ B cells are scarce, but allergen-specific B cells with a memory phenotype circulate in patients with allergic rhinitis

Background Despite the critical role of immunoglobulin E (IgE) in allergy, circulating IgE+ B cells are scarce. Here, we describe in patients with allergic rhinitis B cells with a memory phenotype responding to a prototypic aeroallergen. Methods Fifteen allergic rhinitis patients with grass pollen allergy and 13 control subjects were examined. Blood mononuclear cells stained with carboxyfluorescein diacetate succinimidyl ester (CFSE) were cultured with Bahia grass pollen. Proliferation and phenotype were assessed by multicolour flow cytometry. Results In blood of allergic rhinitis patients with high serum IgE to grass pollen, most IgEhi cells were CD123+ HLA-DR- basophils, with IgE for the major pollen allergen (Pas n 1). Both B and T cells from pollen-allergic donors showed higher proliferation to grass pollen than nonallergic donors (P = 0.002, and 0.010, respectively), whereas responses to vaccine antigens and mitogen did not differ between groups. Allergen-driven B cells that divided rapidly (CD19mid CD3- CFSElo) showed higher CD27 (P = 0.008) and lower CD19 (P = 0.004) and CD20 (P = 0.004) expression than B cells that were slow to respond to allergen (CD19hi CD3- CFSEmid). Moreover, rapidly dividing allergen-driven B cells (CD19mid CFSElo CD27hi) showed higher expression of the plasmablast marker CD38 compared with B cells (CD19hi CFSEmid CD27lo) that were slow to divide. Conclusion Patients with pollen allergy but not control donors have a population of circulating allergen-specific B cells with the phenotype and functional properties of adaptive memory B-cell responses. These cells could provide precursors for allergen-specific IgE production upon allergen re-exposure. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Interleukin 7 independent development of human B cells.

Mammalian hematopoietic stem cell (HSC) commitment and differentiation into lymphoid lineage cells proceed through a series of developmentally restricted progenitor compartments. A complete understanding of this process, and how it differs from HSC commitment and differentiation into cells of the myeloid/erythroid lineages, requires the development of model systems that support HSC commitment to the lymphoid lineages. We now describe a human bone marrow stromal cell culture that preferentially supports commitment and differentiation of human HSC to CD19+ B-lineage cells. Fluorescence activated cell sorterpurified CD34++/lineage-cells were isolated from fetal bone marrow and cultured on human fetal bone marrow stromal cells in serum-free conditions containing no exogenous cytokines. Over a period of 3 weeks, CD34++/lineage- cells underwent commitment, differentiation, and expansion into the B lineage. Progressive changes included: loss of CD34, acquisition of and graded increases in the level of cell surface CD19, and appearance of immature B cells expressing mu/kappa or mu/lambda cell surface Ig receptors. The tempo and phenotype of B-cell development was not influenced by the addition of IL-7 (10 ng/ml), or by the addition of goat anti-IL-7 neutralizing antibody. These results indicate a profound difference between mouse and human in the requirement for IL-7 in normal B-cell development, and provide an experimental system to identify and characterize human bone marrow stromal cell-derived molecules crucial for human B lymphopoiesis.

HIV-1 envelope gp41 antibodies can originate from terminal ileum B cells that share cross-reactivity with commensal bacteria.

Monoclonal antibodies derived from blood plasma cells of acute HIV-1-infected individuals are predominantly targeted to the HIV Env gp41 and cross-reactive with commensal bacteria. To understand this phenomenon, we examined anti-HIV responses in ileum B cells using recombinant antibody technology and probed their relationship to commensal bacteria. The dominant ileum B cell response was to Env gp41. Remarkably, a majority (82%) of the ileum anti-gp41 antibodies cross-reacted with commensal bacteria, and of those, 43% showed non-HIV-1 antigen polyreactivity. Pyrosequencing revealed shared HIV-1 antibody clonal lineages between ileum and blood. Mutated immunoglobulin G antibodies cross-reactive with both Env gp41 and microbiota could also be isolated from the ileum of HIV-1 uninfected individuals. Thus, the gp41 commensal bacterial antigen cross-reactive antibodies originate in the intestine, and the gp41 Env response in HIV-1 infection can be derived from a preinfection memory B cell pool triggered by commensal bacteria that cross-react with Env.

Patterns of de novo allo B cells and antibody formation in chronic cardiac allograft rejection after alemtuzumab treatment.

Even though the etiology of chronic rejection (CR) is multifactorial, donor specific antibody (DSA) is considered to have a causal effect on CR development. Currently the antibody-mediated mechanisms during CR are poorly understood due to lack of proper animal models and tools. In a clinical setting, we previously demonstrated that induction therapy by lymphocyte depletion, using alemtuzumab (anti-human CD52), is associated with an increased incidence of serum alloantibody, C4d deposition and antibody-mediated rejection in human patients. In this study, the effects of T cell depletion in the development of antibody-mediated rejection were examined using human CD52 transgenic (CD52Tg) mice treated with alemtuzumab. Fully mismatched cardiac allografts were transplanted into alemtuzumab treated CD52Tg mice and showed no acute rejection while untreated recipients acutely rejected their grafts. However, approximately half of long-term recipients showed increased degree of vasculopathy, fibrosis and perivascular C3d depositions at posttransplant day 100. The development of CR correlated with DSA and C3d deposition in the graft. Using novel tracking tools to monitor donor-specific B cells, alloreactive B cells were shown to increase in accordance with DSA detection. The current animal model could provide a means of testing strategies to understand mechanisms and developing therapeutic approaches to prevent chronic rejection.

The role of B cells in solid organ transplantation.

The role of antibodies in chronic injury to organ transplants has been suggested for many years, but recently emphasized by new data. We have observed that when immunosuppressive potency decreases either by intentional weaning of maintenance agents or due to homeostatic repopulation after immune cell depletion, the threshold of B cell activation may be lowered. In human transplant recipients the result may be donor-specific antibody, C4d+ injury, and chronic rejection. This scenario has precise parallels in a rhesus monkey renal allograft model in which T cells are depleted with CD3 immunotoxin, or in a CD52-T cell transgenic mouse model using alemtuzumab to deplete T cells. Such animal models may be useful for the testing of therapeutic strategies to prevent DSA. We agree with others who suggest that weaning of immunosuppression may place transplant recipients at risk of chronic antibody-mediated rejection, and that strategies to prevent this scenario are needed if we are to improve long-term graft and patient outcomes in transplantation. We believe that animal models will play a crucial role in defining the pathophysiology of antibody-mediated rejection and in developing effective therapies to prevent graft injury. Two such animal models are described herein.

Characterization of B cells in muscle-specific kinase antibody myasthenia gravis.

OBJECTIVE: To characterize B-cell subsets in patients with muscle-specific tyrosine kinase (MuSK) myasthenia gravis (MG). METHODS: In accordance with Human Immunology Project Consortium guidelines, we performed polychromatic flow cytometry and ELISA assays in peripheral blood samples from 18 patients with MuSK MG and 9 healthy controls. To complement a B-cell phenotype assay that evaluated maturational subsets, we measured B10 cell percentages, plasma B cell-activating factor (BAFF) levels, and MuSK antibody titers. Immunologic variables were compared with healthy controls and clinical outcome measures. RESULTS: As expected, patients treated with rituximab had high percentages of transitional B cells and plasmablasts and thus were excluded from subsequent analysis. The remaining patients with MuSK MG and controls had similar percentages of total B cells and naïve, memory, isotype-switched, plasmablast, and transitional B-cell subsets. However, patients with MuSK MG had higher BAFF levels and lower percentages of B10 cells. In addition, we observed an increase in MuSK antibody levels with more severe disease. CONCLUSIONS: We found prominent B-cell pathology in the distinct form of MG with MuSK autoantibodies. Increased BAFF levels have been described in other autoimmune diseases, including acetylcholine receptor antibody-positive MG. This finding suggests a role for BAFF in the survival of B cells in MuSK MG, which has important therapeutic implications. B10 cells, a recently described rare regulatory B-cell subset that potently blocks Th1 and Th17 responses, were reduced, which suggests a potential mechanism for the breakdown in immune tolerance in patients with MuSK MG.

B cells in rheumatoid synovitis.

In rheumatoid arthritis, T cells, B cells, macrophages, and dendritic cells invade the synovial membranes, establishing complex microstructures that promote inflammatory/tissue destructive lesions. B cell involvement has been considered to be limited to autoantibody production. However, recent studies suggest that B cells support rheumatoid disease through other mechanisms. A critical element of rheumatoid synovitis is the process of ectopic lymphoid neogenesis, with highly efficient lymphoid architectures established in a nonlymphoid tissue site. Rheumatoid synovitis recapitulates the pathways of lymph node formation, and B cells play a key role in this process. Furthermore, studies of rheumatoid lesions implanted in immunodeficient mice suggest that T cell activation in synovitis is B cell dependent, indicating the role played by B cells in presenting antigens and providing survival signals.

Human B cell colony growth from pre-B cells in vitro

We describe a simple one-step technique for the growth of human B cell colonies in semi-solid agar in vitro. This method used conditioned medium from the human plasmacytoma cell line LICR-LON-H My 2 as a source of stimulating activity. A linear relationship exists between the number of B cells seeded and the number of colonies formed (r = 0.95). Most colony forming cells, approximately 1 in 500 of B cells seeded, lack surface immunoglobulin, possess Fc receptors and mark with the Leu 12 monoclonal antibody. Cells within developing colonies are found to have cytoplasmic IgM, IgA and IgG depending on the length of time in culture.