A reservoir of rituximab-resistant splenic memory B cells contributes to relapses after B-cell depletion therapy

AbstractImmune thrombocytopenia (ITP) is an autoimmune disease mediated by pathogenic antibodies directed against platelet antigens, including GPIIbIIIa. Taking advantage of spleen samples obtained from ITP patients, we characterized by multiples approaches the onset of disease relapses occurring after an initial complete response to rituximab. Analysis of splenic B cell immunoglobulin heavy chain gene repertoire at bulk level and from single anti-GPIIbIIIa B cells revealed that germinal centers were fueled by B cells originating from the ongoing lymphopoiesis, but also by rituximab-resistant memory B cells, both giving rise to anti-GPIIbIIIa plasma cells. We identified a population of splenic memory B cells that resisted rituximab through acquisition of a unique phenotype and contributed to relapses, providing a new target in B cell mediated autoimmune diseases.

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Rituximab-resistant splenic memory B cells and newly engaged naive B cells fuel relapses in patients with immune thrombocytopenia

Science Translational Medicine ◽

10.1126/scitranslmed.abc3961 ◽

2021 ◽

Vol 13 (589) ◽

pp. eabc3961

Author(s):

Etienne Crickx ◽

Pascal Chappert ◽

Aurélien Sokal ◽

Sandra Weller ◽

Imane Azzaoui ◽

...

Keyword(s):

B Cells ◽

Autoimmune Diseases ◽

B Cell ◽

Single Cell ◽

Immune Thrombocytopenia ◽

Plasma Cells ◽

Surface Expression ◽

Memory B Cells ◽

Gene Repertoire ◽

Immunoglobulin Gene

Rituximab (RTX), an antibody targeting CD20, is widely used as a first-line therapeutic strategy in B cell–mediated autoimmune diseases. However, a large proportion of patients either do not respond to the treatment or relapse during B cell reconstitution. Here, we characterize the cellular basis responsible for disease relapse in secondary lymphoid organs in humans, taking advantage of the opportunity offered by therapeutic splenectomy in patients with relapsing immune thrombocytopenia. By analyzing the B and plasma cell immunoglobulin gene repertoire at bulk and antigen-specific single-cell level, we demonstrate that relapses are associated with two responses coexisting in germinal centers and involving preexisting mutated memory B cells that survived RTX treatment and naive B cells generated upon reconstitution of the B cell compartment. To identify distinctive characteristics of the memory B cells that escaped RTX-mediated depletion, we analyzed RTX refractory patients who did not respond to treatment at the time of B cell depletion. We identified, by single-cell RNA sequencing (scRNA-seq) analysis, a population of quiescent splenic memory B cells that present a unique, yet reversible, RTX-shaped phenotype characterized by down-modulation of B cell–specific factors and expression of prosurvival genes. Our results clearly demonstrate that these RTX-resistant autoreactive memory B cells reactivate as RTX is cleared and give rise to plasma cells and further germinal center reactions. Their continued surface expression of CD19 makes them efficient targets for current anti-CD19 therapies. This study thus identifies a pathogenic contributor to autoimmune diseases that can be targeted by available therapeutic agents.

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VH1–46 Is the Dominant Immunoglobulin Heavy Chain Gene Segment in Rotavirus-Specific Memory B Cells Expressing the Intestinal Homing Receptor α4β7

The Journal of Immunology ◽

10.4049/jimmunol.174.6.3454 ◽

2005 ◽

Vol 174 (6) ◽

pp. 3454-3460 ◽

Cited By ~ 45

Author(s):

Jörn-Hendrik Weitkamp ◽

Nicole L. Kallewaard ◽

Amber L. Bowen ◽

Bonnie J. LaFleur ◽

Harry B. Greenberg ◽

...

Keyword(s):

B Cells ◽

Heavy Chain ◽

Gene Segment ◽

Immunoglobulin Heavy Chain ◽

Memory B Cells ◽

Chain Gene ◽

Heavy Chain Gene ◽

Immunoglobulin Heavy Chain Gene ◽

Homing Receptor ◽

Specific Memory

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Clustering of extensive somatic mutations in the variable region of an immunoglobulin heavy chain gene from a human B cell lymphoma

Cell ◽

10.1016/0092-8674(86)90488-5 ◽

1986 ◽

Vol 44 (1) ◽

pp. 97-106 ◽

Cited By ~ 173

Author(s):

Michael L. Cleary ◽

Timothy C. Meeker ◽

Shoshana Levy ◽

Elizabeth Lee ◽

Martha Trela ◽

...

Keyword(s):

B Cell ◽

Heavy Chain ◽

Somatic Mutations ◽

Cell Lymphoma ◽

Variable Region ◽

B Cell Lymphoma ◽

Chain Gene ◽

Heavy Chain Gene ◽

Immunoglobulin Heavy Chain Gene ◽

Human B Cell

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Potential anti-EBV effects associated with elevated interleukin-21 levels: a case report

BMC Infectious Diseases ◽

10.1186/s12879-020-05609-z ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Kristian Assing ◽

Christian Nielsen ◽

Marianne Jakobsen ◽

Charlotte B. Andersen ◽

Kristin Skogstrand ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

B Cell ◽

Plasma Cell ◽

Germinal Center ◽

Plasma Cells ◽

Cell Formation ◽

Memory B Cells ◽

Memory B Cell

Abstract Background Germinal center derived memory B cells and plasma cells constitute, in health and during EBV reactivation, the largest functional EBV reservoir. Hence, by reducing germinal center derived formation of memory B cells and plasma cells, EBV loads may be reduced. Animal and in-vitro models have shown that IL-21 can support memory B and plasma cell formation and thereby potentially contribute to EBV persistence. However, IL-21 also displays anti-viral effects, as mice models have shown that CD4+ T cell produced IL-21 is critical for the differentiation, function and survival of anti-viral CD8+ T cells able to contain chronic virus infections. Case presentation We present immunological work-up (flow-cytometry, ELISA and genetics) related to a patient suffering from a condition resembling B cell chronic active EBV infection, albeit with moderately elevated EBV copy numbers. No mutations in genes associated with EBV disease, common variable immunodeficiency or pertaining to the IL-21 signaling pathway (including hypermorphic IL-21 mutations) were found. Increased (> 5-fold increase 7 days post-vaccination) CD4+ T cell produced (p < 0.01) and extracellular IL-21 levels characterized our patient and coexisted with: CD8+ lymphopenia, B lymphopenia, hypogammaglobulinemia, compromised memory B cell differentiation, absent induction of B-cell lymphoma 6 protein (Bcl-6) dependent peripheral follicular helper T cells (pTFH, p = 0.01), reduced frequencies of peripheral CD4+ Bcl-6+ T cells (p = 0.05), compromised plasmablast differentiation (reduced protein vaccine responses (p < 0.001) as well as reduced Treg frequencies. Supporting IL-21 mediated suppression of pTFH formation, pTFH and CD4+ IL-21+ frequencies were strongly inversely correlated, prior to and after vaccination, in the patient and in controls, Spearman’s rho: − 0.86, p < 0.001. Conclusions To the best of our knowledge, this is the first report of elevated CD4+ IL-21+ T cell frequencies in human EBV disease. IL-21 overproduction may, apart from driving T cell mediated anti-EBV responses, disrupt germinal center derived memory B cell and plasma cell formation, and thereby contribute to EBV disease control.

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Hyperproliferation of B Cells Specific for a Weakly Immunogenic PorA in a Meningococcal Vaccine Model

Clinical and Vaccine Immunology ◽

10.1128/cvi.00192-08 ◽

2008 ◽

Vol 15 (10) ◽

pp. 1598-1605 ◽

Cited By ~ 6

Author(s):

Thomas A. Luijkx ◽

Jacqueline A. M. van Gaans-van den Brink ◽

Harry H. van Dijken ◽

Germie P. J. M. van den Dobbelsteen ◽

Cécile A. C. M. van Els

Keyword(s):

B Cells ◽

B Cell ◽

Bactericidal Activity ◽

Plasma Cells ◽

Affinity Maturation ◽

Memory B Cells ◽

Vaccine Antigen ◽

Cell Subpopulation ◽

Cell Subpopulations ◽

Group B

ABSTRACT Highly homologous meningococcal porin A (PorA) proteins induce protective humoral immunity against Neisseria meningitidis group B infection but with large and consistent differences in the levels of serum bactericidal activity achieved. We investigated whether a poor PorA-specific serological outcome is associated with a limited size of the specific B-cell subpopulation involved. The numbers of PorA-specific splenic plasma cells, bone marrow (BM) plasma cells, and splenic memory B cells were compared between mice that received priming and boosting with the weakly immunogenic PorA (P1.7-2,4) protein and those that received priming and boosting with the highly immunogenic PorA (P1.5-1,2-2) protein. Immunoglobulin G (IgG) titers (except at day 42), bactericidal activity, and the avidity of IgG produced against P1.7-2,4 were significantly lower at all time points after priming and boosting than against P1.5-1,2-2. These differences, however, were not associated with a lack of P1.7-2,4-specific plasma cells. Instead, priming with both of the PorAs resulted in the initial expansion of comparable numbers of splenic and BM plasma cells. Moreover, P1.7-2,4-specific BM plasma cells, but not P1.5-1,2-2-specific plasma cells, expanded significantly further after boosting. Likewise, after a relative delay during the priming phase, the splenic P1.7-2,4-specific memory B cells largely outnumbered those specific for P1.5-1,2-2, upon boosting. These trends were observed with different vaccine formulations of the porins. Our results show for the first time that B-cell subpopulations involved in a successfully maturated antibody response against a clinically relevant vaccine antigen are maintained at smaller population sizes than those associated with poor affinity maturation. This bears consequences for the interpretation of immunological memory data in clinical vaccine trials.

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High Prevalence of B-Cell Clonality in Patients with Gaucher’s Disease.

Blood ◽

10.1182/blood.v104.11.2391.2391 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2391-2391 ◽

Cited By ~ 3

Author(s):

Derralynn A. Hughes ◽

Veronique M. Duke ◽

Robert J. Baker ◽

Faith Wright ◽

Letizia Foroni ◽

...

Keyword(s):

B Cell ◽

Gaucher Disease ◽

Haematological Malignancy ◽

Chain Gene ◽

Heavy Chain Gene ◽

Immunoglobulin Heavy Chain Gene ◽

Bulk Storage ◽

Clonal Population ◽

Polyclonal Gammopathy ◽

Cell Clonality

Abstract Haematological malignancy occurs in patients with Gaucher Disease at an incidence 15 fold greater than in the non-Gaucher population. Little is understood regarding the role of glucosyl ceramide acculmulation in immune system pathology or in the development of cancer in Gaucher Disease. Furthermore whilst enzyme replacement therapy (ERT) with recombinant glucocerebrosidase has been successful in reversing symptoms relating to bulk storage e.g. hepatosplenomegaly and bone marrow infiltration, its effects on the pathogenesis of malignancy are unknown. We have measured the levels of immunlglobulin in 63 patients with Gaucher disease receiving ERT for up to 10 years and have investigated the incidence of clonal B cell populations by flow cytometry, immunoglobulin heavy chain gene rearrangement analysis, and measurement of paraproteins by electropheresis and immunofixation. Evidence of high level of immune stimulation was found by morphological and immunoglobin analysis. Examination of peripheral blood revealed atypical lymphocyte morphology in 53% Gaucher patients whilst 50.7% patients were found to have levels of immunoglobulins significantly higher than those found in healthy adults. There were no sex or age related differences between those patients with elevated immunoglobulins (18 male; median age 40yrs, 14 female; median age 48 years) and those with normal levels (13 male median age 41 years, 18 female; median age 47.5 years). Elevation of one, two or three immunoglobulin subclasses was detected in 36%, 13.1% and 1.6% of patients respectively. In patients with IgM polyclonal gammopathy levels of immunoglobulin normalised with ERT. This was not the case with IgG and IgA gammopathies which were unaffected by ERT even after 10 years of treatment. No patient with normal immunoglobulin levels prior to treatment developed a gammopathy after initiation of ERT Evidence of B-cell clonality was found in 23% of patients. 13.4% had measurable paraproteins, 9.3% exhibited a IgM+, IgD+, FMC7+ monoclonal population by flow cytometry and 26.9% had clonal immunoglobulin heavy chain gene rearrangements of which 43% were in frame and therefore functional. There was no relationship between genotype, absolute lymphocyte count (median 1.5 x109 in all groups) and age (median 45.5 years with clonal population and 44 years without) or duration of ERT (median 5.25 years with clonal population and 6.5 years without) and the existence of clonality. Our data confirms the existence of high levels of immune stimulation and B cell clonality in patients with Gaucher disease. In contrast to features of bulk storage of glucosyl ceramide, the polyclonal gammopathy and B cell clonality is not reversed by ERT and may therefore occur at low levels of storage. The existence of multiple markers of clonality will allow the response of individual patients to different modalities of therapy to be followed, and the pathophysiology of haematological malignancy to be further assessed.

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miRNA Expression Profile of B-SLL Consistent with Normal Memory B Cells:BIC/miR–155 Specific Location in Proliferation Center.

Blood ◽

10.1182/blood.v110.11.2081.2081 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2081-2081

Author(s):

Miao Wang ◽

Jan-Lukas Robertus ◽

Lu-ping Tan ◽

Geert Harms ◽

Tjasso Blokzijl ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Expression Profile ◽

Mirna Expression ◽

Direct Sequencing ◽

Memory B Cells ◽

Lymphocytic Leukemia ◽

Chain Gene ◽

Mutation Status ◽

Igh Genes

Abstract Introduction: B cell Chronic Lymphocytic Leukemia (B-CLL) is characterized by the accumulation of nonproliferating mature-appearing lymphocytes in the blood, marrow, lymph nodes, and spleen. B-CLL behaves like leukemia and is found mostly in the blood. The expression of ZAP-70 and mutation status of the immunoglobulin heavy-chain gene (IgH) can serve as prognostic markers in B-CLL. ZAP-70 positive cases usually present with unmutated IgH genes and have a bad prognosis, whereas ZAP-70 negative cases mostly present with mutated IgH genes and have good prognosis. Gene expression studies of B-CLL indicated that the profile of IgH mutated and unmutated cases are similar to normal memory B cells. Several studies showed that miRNAs play important roles in pathogenesis of B-CLL and some miRNAs correlate with the prognosis in B-CLL. B-SLL is considered to be the same disease entity as CLL, but this variant is found mostly in bone marrow and the lymphatic system. The most aggressive type of B-SLL is characterized by neoplastic cells that are more responsive to B-cell receptor signaling and are characterized by proliferation centers (PCs), a potentially important site of neoplastic cell stimulation. Until now, only a few reports have been published about the ZAP-70 expression and IgH mutation status and no data are available about the microRNA expression profile. Methods: 33 B-SLL cases were retrieved from the pathology files. ZAP-70 expression was analyzed by using immunohistochemistry. IgH mutation status was determined using PCR followed by direct sequencing. Cases with homology of ≥98% with germline sequences were considered as unmutated and cases with homologies <98% as mutated. Levels of 15 miRNAs were determined by qRT-PCR using U6 as a housekeeping gene in 28 B-SLL cases with good quality RNA. As a control we also analyzed miRNA levels in normal naïve, GC and memory B cell subsets. RNA in-situ hybridization (ISH) was used to localize the most abundantly expressed miRNAs in B-SLL tissues. Results: 16 B-CLL cases were ZAP70+ with the vast majority of tumor cells staining positive and 17 were negative. Of the ZAP70+ cases 10 carried unmutated and 3 mutated IgH genes and 3 cases failed due to bad quality DNA. 14/17 ZAP-70- cases carried mutated IgH genes and 3 cases failed. miR-150, miR-21, miR-16, miR-92 and miR-155 were expressed at high levels in all B-SLL cases independent of the ZAP70 and IgH mutation status. The miRNA expression pattern in B-SLL was very similar to normal memory B cells. RNA-ISH for BIC, the primary transcript of miR-155, demonstrated the most abundant expression in the proliferation centers of B-SLL cases. Conclusion: In B-SLL there is significant correlation between ZAP-70 expression and IgH mutation status similar to B-CLL cases. miRNA expression levels in B-SLL did not correlate with ZAP-70 or IgH status. The overall expression profile is very similar to normal memory B cells. BIC/miR-155 expression is observed specifically in the proliferation center of B-SLL tissues.

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