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

CD5 Plays an Inhibitory Role in the Suppressive Function of Murine CD4\u3csup\u3e+\u3c/sup\u3e CD25\u3csup\u3e+\u3c/sup\u3e T\u3csub\u3ereg\u3c/sub\u3e Cells

A subset of CD4+ T cells, the CD4+ CD25+ regulatory T (Treg) cells in the lymphoid organs and peripheral blood are known to possess suppressive function. Previous in vitro and in vivo studies have indicated that T cell receptor (TCR) signal is required for development of such ‘natural regulatory (Treg) cells’ and for activation of the effector function of CD4+ CD25+ regulatory T cells. CD5 is a cell surface molecule present on all T cells and a subtype of B lymphocytes, the B-1 cells, primarily localized to coelomic cavities, Peyer\u27s patches, tonsils and spleen. CD5 acts as a negative regulator of T cell and B cell signaling via recruitment of SHP-1. Here, we demonstrate that Treg cells obtained from CD5−/− mice are more potent than those from wild type mice in suppressing the in vitro cell proliferation of anti-CD3 stimulated CD4+ CD25− responder T cells. This phenomenon was cell contact and GITR dependent. Lack of CD5 expression on Treg cells (from spleen, lymph node and thymus) did not affect the intracellular levels of Foxp3. However, CD5−/− Tregthymocytes were able to elicit a higher Ca2+ response to TCR + co-stimulatory signals than the wild type cells. CD5−/− mice expressed more Foxp3 mRNA in the colon than wild type mice, and additionally, the severity of the dextran sulfate sodium (DSS)-induced colitis in CD5−/− mice was less than the wild type strain. We suggest that manipulation of CD5 expression or the downstream signaling components of CD4+ CD25+ Treg cells as a potential strategy for therapeutic intervention in cases of auto-immune disorders

Expression of High-Affinity IgE Receptor on Human Peripheral Blood Dendritic Cells in Children

BACKGROUND: In a mouse model of viral induced atopic disease, expression of FcεRI on dendritic cells is critical. While adult human conventional (cDC) and plasmacytoid (pDC) dendritic cells have been shown to express FcεRI, it is not known if this receptor is expressed in childhood and how its expression is governed by IgE. METHODS: Following informed consent of subjects (n = 27, aged 12-188 months), peripheral blood was stained for surface expression of CD19, ILT7, CD1c, IgE, FcεRI and analyzed by flow cytometry (cDC: CD19(-) ILT7(-) CD1c(+); pDC: CD19(-) ILT7(+) CD1c(-)). Total and specific serum IgE levels to food and inhalant allergens were determined by ImmunoCAP, and the relationship between FcεRI expression on dendritic cells and sensitization, free IgE, cell bound IgE, and age was determined. RESULTS: Independent of sensitization status, FcεRI expression was noted on cDC and pDC as early as 12 months of age. Serum IgE level correlated with expression of FcεRI on cDC, but not pDC. Based on the concentration of IgE, a complex relationship was found between surface bound IgE and expression of FcεRI on cDC. pDC exhibited a linear relationship of FcεRI expression and bound IgE that was consistent through all IgE concentrations. CONCLUSIONS: In children, FcεRI expression on cDC and pDC is modulated differently by serum and cell bound IgE. IgE governance of FcεRI expression on cDC depends upon a complex relationship. Further studies are needed to determine the functional roles of FcεRI on cDC and pDC

Early Growth Response Genes Regulate B Cell Development, Proliferation, and Immune Response

Egr-1 (early growth response gene-1) is an immediate early gene encoding a zinc finger motif-containing transcription factor. Upon cross-linking of BCR, mature B cells undergo proliferation with an increase in Egr-1 message. Immature B lymphoma cells that express Egr-1 message and protein constitutively are growth inhibited when Egr-1 is down-regulated by negative signals from BCR or by antisense oligonucleotides. To test the hypothesis that Egr-1 is important for B cell development, we examined B cells from primary and secondary lymphoid organs in Egr-1−/− mice. Marginal zone B cell development was arrested in these mice, whereas the B cells in all other compartments were increased. To test the hypothesis that Egr-1 function may be partially compensated by other Egr family members, we developed transgenic mice expressing a dominant negative form of Egr-1, which lacks the trans activation domain but retains the DNA-binding domain, in a B cell-specific manner. There was a decrease in B lymphopoiesis in the bone marrow accompanied by a reduction in splenic immature and mature B cells as well as marginal zone B cells in the transgenic mice. Moreover, transgenic mice respond poorly to BCR cross-linking in vitro and T-independent and T-dependent Ags in vivo

Correlation of cDC expression of FcεRI with IgE.

<p>Expression of FcεRI and IgE on the surface of cDC was determined by flow cytometry. (A) Data are presented as fold MFI FcεRI versus fold MFI IgE expression, with non-sensitized subjects (n = 15) in red circles and sensitized subjects (n = 12) in black x's. Note the difference in the slope of the fitted lines (non-sensitized subjects in red dotted line; sensitized subjects in black solid line) above and below a fold MFI IgE of 12, suggesting that there is differential regulation of FcεRI on cDC around this level of cell bound IgE. For all subjects the data was fit with the following cubic equation (blue line): Ln(cDC FcεRI) = 2.26+0.72*ln(cDC IgE)-0.43*ln(cDC IgE)²+0.082*ln(cDC IgE)³); r = 0.86; p = 0.045. For equations based on sensitization status, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032556#pone-0032556-t003" target="_blank">Table 3</a>. (B) Expression of cell bound IgE on cDC compared with serum (free) IgE levels. Data are presented as in (A) with fold MFI IgE expression versus serum IgE (kU/L). Note that a cell bound IgE fold MFI of 12 correlates with a serum IgE of around 38 (based on statistical fitting of the curve, this value could range between 34 and 42 kU/L).</p

Expression of FcεRI on pDC does not correlate with serum IgE or age of subject.

<p>Expression of FcεRI on peripheral blood pDC was determined by flow cytometry and compared to subject's age (panel A) and serum IgE level (panel B). Data are presented as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032556#pone-0032556-g001" target="_blank">Figure 1</a>.</p

Expression of FcεRI on cDC correlates with serum IgE but not with subject's age.

<p>(A) Gating strategy used to identify expression of FcεRI and IgE on cDC and pDC. Cells were gated on scatter (R1) and lack of expression of CD19 (R2, PerCP negative). Those events that satisfied both of these criteria were examined for expression of ILT7 (R3, PE positive) versus CD1c (R4, APC positive). Cells that were CD19⁻ILT7⁺CD1c⁻ were considered pDC and CD19⁻ILT7⁻CD1c⁺ were considered cDC. The expression of FcεRI (left histograms, FITC positive) or IgE (right histograms, FITC positive) on these cells was then determined and compared to an appropriate isotype control. (B) Expression of FcεRI on peripheral blood cDC was determined by flow cytometry and compared to subject's age or (C) serum IgE level. Data are presented as fold MFI FcεRI versus age in months (B) or serum IgE in kU/L (C), with non-sensitized subjects (n = 15) in circles and sensitized subjects (n = 12) in x's. Sensitization was defined as having at least one positive allergen specific IgE by ImmunoCAP.</p