Multiple Regulatory Mechanisms Control B-1 B Cell Activation

B-1 cells constitute a unique subset of B cells identified in several species including mice and humans. B-1 cells are further subdivided into B-1a and B-1b subsets as the former but not the later express CD5.The B-1a subset contributes to innate type of immune responses while the B-1b B cell subset contributes to adaptive responses. B-1 cell responses to B cell receptor (BCR) as well as Toll-like receptor (TLR) ligation are tightly regulated due to the cross-reactivity of antigen specific receptors on B-1 cells to self-antigens. B-1 cells are elevated in several autoimmune diseases. CD5 plays a major role in down regulation of BCR responses in the B-1a cell subset. Reduced amplification of BCR induced signals via CD19 and autoregulation of BCR and TLR responses by B-1 cell produced IL-10 appear to have a role in regulation of both B-1a and B-1b B cell responses. Siglec G receptors and Lyn kinase also regulate B-1 cell responses but their differential role in the two B-1 cell subsets is unknown

Interleukin-10 Mediated Autoregulation of Murine B-1 B-Cells and Its Role in \u3cem\u3eBorrelia hermsii\u3c/em\u3e Infection

B cells are typically characterized as positive regulators of the immune response, primarily by producing antibodies. However, recent studies indicate that various subsets of B cells can perform regulatory functions mainly through IL-10 secretion. Here we discovered that peritoneal B-1 (B-1P) cells produce high levels of IL-10 upon stimulation with several Toll-like receptor (TLR) ligands. High levels of IL-10 suppressed B-1P cell proliferation and differentiation response to all TLR ligands studied in an autocrine manner in vitro and in vivo. IL-10 that accumulated in cultures inhibited B-1P cells at second and subsequent cell divisions mainly at the G1/S interphase. IL-10 inhibits TLR induced B-1P cell activation by blocking the classical NF-kappaB pathway. Co-stimulation with CD40 or BAFF abrogated the IL-10 inhibitory effect on B-1P cells during TLR stimulation. Finally, B-1P cells adoptively transferred from the peritoneal cavity of IL-10-/- mice showed better clearance of Borrelia hermsii than wild-type B-1P cells. This study described a novel autoregulatory property of B-1P cells mediated by B-1P cell derived IL-10, which may affect the function of B-1P cells in infection and autoimmunity

CD19 Signaling Is Impaired in Murine Peritoneal and Splenic B-1 B Lymphocytes

B-1 cells reside predominantly within the coelomic cavities, tonsils, Peyer\u27s patches, spleen (a minor fraction – ∼5%) and are absent in the lymph nodes. They are the primary sources of natural IgM in the body. B-1 cells express polyreactive B cell receptors (BCRs) that cross react with self-antigens and are thus implicated in auto-immune disorders. Previously, we reported that peritoneal B-1 cells are deficient in CD19-mediated intracellular signals leading to Ca2+ mobilization. Here, we find that splenic B-1 cells, like peritoneal B-1 cells, are defective in Ca2+ release upon B cell activation by co-cross-linking BCR and CD19. In the absence of extracellular sources of Ca2+, intracellular Ca2+ flux is similar between B-1 and B-2 cells. Moreover, the intracellular component of Ca2+ release in both subsets of B cells is mostly PI3K dependent. BCR and CD19 co-cross-linking activates Akt, a key mediator of survival and proliferation signals downstream of PI3K in splenic B-2 cells. Splenic B-1 cells, on the other hand, do not phosphorylate Akt (S473) upon similar treatment. Furthermore, BCR + CD19 cross-linking induced phosphorylation of JNK is much reduced in splenic B-1 cells. In contrast, B-1 cells exhibited increased levels of constitutively active pLyn which appears to have an inhibitory role. The CD19 induced Ca2+ response and BCR induced proliferation response were restored by a partial inhibition of pLyn with Src kinase specific inhibitors. These findings suggest a defect in CD19-mediated signals in both peritoneal and splenic B-1 B lymphocytes, which is in part, due to higher levels of constitutively active Lyn

YY1 Is Required for Germinal Center B Cell Development.

YY1 has been implicated as a master regulator of germinal center B cell development as YY1 binding sites are frequently present in promoters of germinal center-expressed genes. YY1 is known to be important for other stages of B cell development including the pro-B and pre-B cells stages. To determine if YY1 plays a critical role in germinal center development, we evaluated YY1 expression during B cell development, and used a YY1 conditional knock-out approach for deletion of YY1 in germinal center B cells (CRE driven by the immunoglobulin heavy chain γ1 switch region promoter; γ1-CRE). We found that YY1 is most highly expressed in germinal center B cells and is increased 3 fold in splenic B cells activated by treatment with anti-IgM and anti-CD40. In addition, deletion of the yy1 gene by action of γ1-CRE recombinase resulted in significant loss of GC cells in both un-immunized and immunized contexts with corresponding loss of serum IgG1. Our results show a crucial role for YY1 in the germinal center reaction

Anomalous Constitutive Src Kinase Activity Promotes B Lymphoma Survival and Growth

Background: Previously we have shown that B cell receptor (BCR) expression and B cell receptor signaling pathways are important for the basal growth of B lymphoma cells. In particular we have shown that the activation of Syk, a non-src family protein tyrosine kinase and the mitogen activated protein kinases (MAPK), ERK and JNK that mediate BCR signals are required for the constitutive growth of B lymphoma cells. Since src family protein tyrosine kinases (SFKs) like Lyn are known to be needed for the phosphorylation of BCR co-receptors, Ig-alpha and Ig-beta, we hypothesized that one or more SFKs will be constitutively activated in B lymphoma cells and may be necessary for B lymphoma growth. Results: Src kinase activity was found to be constitutively high in many murine and human B lymphoma cell lines and primary lymphoma samples. The specific pharmacological inhibitors of SFKs, PP1 and PP2 inhibited the proliferation of a number of both murine and human B lymphomas in a dose-dependent manner. Importantly, dasatinib (BMS-354825), an oral dual BCR-ABL and SFK specific inhibitor inhibited the growth of B lymphomas in the nanomolar range in vitro and strongly inhibited a mouse lymphoma growth in vivo. Among the SFKs, Lyn is predominantly phosphorylated and Lyn-specific small interfering RNA inhibited the growth of B lymphomas, supporting an important role for Lyn in B lymphoma growth. Suppression of SFK activity blocks BCR mediated signaling pathways. PMA or CpG can partially reverse the growth inhibition induced by SFK inhibition. Although blocking SFK activity inhibited the growth of a number of B lymphomas, some lymphomas such as SudHL-4, SudHL-6, OCI-Ly3 and OCI-Ly10 are more resistant due to an increased expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL. Conclusions: These studies further support our concept that BCR signaling pathways are important for the continued growth of established B lymphoma cells. Some of the intermediates in this BCR pathway are potential immunotherapeutic targets. In particular, inhibition of SFK activity alone or in synergy with inhibition of the prosurvival Bcl-2 proteins holds promise in developing more effective treatments for B lymphoma patients

Diversity of Epigenetic Features of the Inactive X-Chromosome in NK Cells, Dendritic Cells, and Macrophages

In females, the long non-coding RNA Xist drives X-chromosome Inactivation (XCI) to equalize X-linked gene dosage between sexes. Unlike other somatic cells, dynamic regulation of Xist RNA and heterochromatin marks on the inactive X (Xi) in female lymphocytes results in biallelic expression of some X-linked genes, including Tlr7, Cxcr3, and Cd40l, implicated in sex-biased autoimmune diseases. We now find that while Xist RNA is dispersed across the nucleus in NK cells and dendritic cells (DCs) and partially co-localizes with H3K27me3 in bone marrow-derived macrophages, it is virtually absent in plasmacytoid DCs (p-DCs). Moreover, H3K27me3 foci are present in only 10–20% of cells and we observed biallelic expression of Tlr7 in p-DCs from wildtype mice and NZB/W F1 mice. Unlike in humans, mouse p-DCs do not exhibit sex differences with interferon alpha production, and interferon signature gene expression in p-DCs is similar between males and females. Despite the absence of Xist RNA from the Xi, female p-DCs maintain dosage compensation of six immunity-related X-linked genes. Thus, immune cells use diverse mechanisms to maintain XCI which could contribute to sex-linked autoimmune diseases

Interleukin-10 Mediated Autoregulation of Murine B-1 B-Cells and Its Role in Borrelia hermsii Infection

B cells are typically characterized as positive regulators of the immune response, primarily by producing antibodies. However, recent studies indicate that various subsets of B cells can perform regulatory functions mainly through IL-10 secretion. Here we discovered that peritoneal B-1 (B-1P) cells produce high levels of IL-10 upon stimulation with several Toll-like receptor (TLR) ligands. High levels of IL-10 suppressed B-1P cell proliferation and differentiation response to all TLR ligands studied in an autocrine manner in vitro and in vivo. IL-10 that accumulated in cultures inhibited B-1P cells at second and subsequent cell divisions mainly at the G1/S interphase. IL-10 inhibits TLR induced B-1P cell activation by blocking the classical NF-κB pathway. Co-stimulation with CD40 or BAFF abrogated the IL-10 inhibitory effect on B-1P cells during TLR stimulation. Finally, B-1P cells adoptively transferred from the peritoneal cavity of IL-10−/− mice showed better clearance of Borrelia hermsii than wild-type B-1P cells. This study described a novel autoregulatory property of B-1P cells mediated by B-1P cell derived IL-10, which may affect the function of B-1P cells in infection and autoimmunity

B Cell Tolerance in Health and Disease

B lymphocyte receptors are generated randomly during the bone marrow developmental phase of B cells. Hence, the B cell repertoire consists of both self and foreign antigen specificities necessitating specific tolerance mechanisms to eliminate self-reactive B cells. This review summarizes the major mechanisms of B cell tolerance, which include clonal deletion, anergy and receptor editing. In the bone marrow presentation of antigen in membrane bound form is more effective than soluble form and the role of dendritic cells in this process is discussed. Toll like receptor derived signals affect activation of B cells by certain ligands such as nucleic acids and have been shown to play crucial roles in the development of autoimmunity in several animal models. In the periphery availability of BAFF, a B cell survival factor plays a critical role in the survival of self-reactive B cells. Antibodies against BAFF have been found to be effective therapeutic agents in lupus like autoimmune diseases. Recent developments are targeting anergy to control the growth of chronic lymphocytic leukemia cells

YY1 is required for germinal center B cell development and immunoglobulin class switching.

<p><b>(A)</b> Spleen cells from non-immunized <i>YY1</i>^<i>f/</i>, <i>γ1CRE</i> and <i>YY1</i>^<i>f/</i>f <i>γ1CRE</i> mice were stained with various antibodies to identify total B cells (CD19⁺AA4.1⁺, upper panel) and germinal center B cells (GC-B, DUMP^-IgD^-GL7^hiCD95^hi, lower panel). Percentages and number of <b>(B)</b> total B cells, and <b>(C)</b> GC-B cells per spleen of <i>YY1</i>^<i>f/</i>, <i>γ1CRE</i> and <i>YY1</i>^<i>f/</i>f <i>γ1CRE</i> mice. Fig A-C are from three independent experiments (<i>n</i> = 3 mice for each genotype). <b>(D)</b> We used ELISA to detect various isotypes of serum immunoglobulins from <i>YY1</i>^<i>f/</i>, <i>γ1CRE</i> and <i>YY1</i>^<i>f/</i>f <i>γ1CRE</i> mice. The concentration of IgM, IgA, total IgG, as well as IgG subclasses, IgG1, IgG2 and IgG3 were measured from sera samples that were obtained from four experiments (<i>n</i> ≥ 4 mice for each genotype). Asterisks indicate p<0.001.</p

YY1 is required for antigen-specific germinal center development and for generation of antigen-specific IgG1.

<p><b>(A)</b> Splenocytes from NP-CGG immunized <i>γ1CRE</i> and <i>YY1</i>^<i>f/</i>f <i>γ1CRE</i> mice were harvested at 14 days after immunization and stained with various antibodies, as well as PNA to detect GC B cells. We gated on CD4⁻CD8⁻F4/80⁻Gr1⁻(DUMP⁻) IgD^- cells that were subdivided into PNA⁺B220⁺ GC-B cells. GC-B cells were gated and further subsetted into NP-specific (NP⁺B220⁺) GC-B cells. Representative results are from three independent experiments. <b>(B)</b> Numbers of NP-specific (NP⁺B220⁺) GC-B cells per spleen of immunized mice (<i>n</i> = 3). <b>(C)</b> <i>γ1CRE</i> and <i>YY1</i>^<i>f/</i>f <i>γ1CRE</i> mice were immunized with NP-CGG, and 14 days later spleen sections were stained with anti-GL7, anti-IgD and anti-TCRβ antibody. GL7-rich regions demarcate germinal center B cells. <b>(D, E)</b> Serum from NP-CGG immunized <i>γ1CRE</i> and <i>YY1</i>^<i>f/</i>f <i>γ1CRE</i> mice were collected at 14 days after immunization and NP-specific serum Igs were analyzed using ELISA. <i>D</i>. The concentration of low affinity (NP26, left panel) and high affinity (NP4, right panel) IgG1 in the serum. <i>E</i>. Titer of NP-specific total IgM in the sera of immunized mice. Data are derived from sera samples that were obtained from three experiments. Asterisks indicate p<0.001.</p