Geraldine L. Shu

Polypeptides on human B lymphocytes associated with cell activation

Two monoclonal antibodies (MoAbs), designated BB-1 and LB-2, react with distinct polypeptides expressed on activated human B cells. The BB-1 MoAb reacted with a 37,000-dalton polypeptide (Bp37) restricted to pre-B and B-cell blasts and B-cell malignancies. The LB-2 MoAb reacted with a 76,000-dalton polypeptide (p76) found on resting B cells but at higher levels on activated B cells and T cells. Buoyant tonsillar lymphoid cells with a germinal center phenotype express higher levels of Bp37 and p76 than do dense B cells of the mantle zone. Furthermore, the expression of Bp37 and p76 on tonsillar B-cell subsets was distinct from other B-cell antigens such as Bp39, Bp95, Bp135, the C3d receptor and surface IgM. Based on biochemical, cross-blocking, and tissue distribution analyses, these antigens appear to be distinct from previously described B cell and B-cell-blast markers.

Caspase Activity Is Required for Stimulated B Lymphocytes to Enter the Cell Cycle

Following activation with proliferative stimuli, including ligation of CD40, dense human tonsillar B cells (>98% cells in G0) have increased cleavage and activation of caspase-8 and -6 accompanied by decreased caspase-3 activation and apoptosis. Proliferation was blocked by either a broad specificity caspase inhibitor or inhibitors selective for caspase-6 or caspase-8. In contrast, an inhibitor selective for caspase-3 was without effect. Furthermore, induction of cyclin D and cyclin-dependent kinase 4 mRNA and protein was blocked upon inhibition of caspase-6, but not caspase-3. Thus, caspase-6-like activity is required for quiescent B cells to increase the expression of genes required for entry into G1. In support of this model, the transcriptional suppressor special AT-rich sequence-binding protein 1, a preferred caspase-6 substrate, was cleaved upon B cell stimulation. Caspase activity was not required for all signaling events, as caspase inhibitors did not affect the phosphorylation of p42/44 mitogen-activated protein kinase, the expression of the survival factor cellular inhibitor of apoptosis 2, or the production of IL-6 by stimulated G0 B cells. These findings suggest a mechanism by which caspase-6 may selectively allow entry of quiescent B cells into the cell cycle.

Caspase-6 Regulates B Cell Activation and Differentiation into Plasma Cells

Caspase (Casp) family proteases regulate not only lymphocyte apoptosis but also lymphocyte activation and development. In this study, we show that Casp6 regulates B cell activation and differentiation into plasma cells by modifying cell cycle entry. B cells from Casp6 knockout (Casp6 KO) mice examined ex vivo have more cells in G1 than wild-type B cells, and mitogen-induced G1 entry of Casp6 KO B cells is much faster than that of wild-type B cells. Even so, S phase entry and proliferation are not increased in Casp6 KO B cells. Rather than proliferating, activated Casp6 KO B cells preferentially differentiate into syndecan-1+ plasma cells and produce Abs. In Casp6 KO mice compared with WT mice, serum levels of IgG1, IgG2a, and IgG2b are increased and Ag-specific Ab responses are also enhanced along with increased percentages of syndecan-1+ plasma cells. Casp6 may regulate both B cell activation and differentiation by modifying requirements for G0 B cells to enter G1.

Targeting CD22 with the monoclonal antibody epratuzumab modulates human B-cell maturation and cytokine production in response to Toll-like receptor 7 (TLR7) and B-cell receptor (BCR) signaling

BackgroundAbnormal B-cell activation is implicated in the pathogenesis of autoimmune diseases, including systemic lupus erythematosus (SLE). The B-cell surface molecule CD22, which regulates activation through the B-cell receptor (BCR), is a potential target for inhibiting pathogenic B cells; however, the regulatory functions of CD22 remain poorly understood. In this study, we determined how targeting of CD22 with epratuzumab (Emab), a humanized anti-CD22 IgG1 monoclonal antibody, affects the activation of human B-cell subsets in response to Toll-like receptor 7 (TLR7) and BCR engagement.MethodsB-cell subsets were isolated from human tonsils and stimulated with F(ab′)2 anti-human IgM and/or the TLR7 agonist R848 in the presence of Emab or a human IgG1 isotype control. Changes in mRNA levels of genes associated with B-cell activation and differentiation were analyzed by quantitative PCR. Cytokine production was measured by ELISA. Cell proliferation, survival, and differentiation were assessed by flow cytometry.ResultsPretreatment of phenotypically naïve CD19+CD10–CD27– cells with Emab led to a significant increase in IL-10 expression, and in some but not all patient samples to a reduction of IL-6 production in response to TLR7 stimulation alone or in combination with anti-IgM. Emab selectively inhibited the expression of PRDM1, the gene encoding B-lymphocyte-induced maturation protein 1 (Blimp-1) in activated CD10–CD27– B cells. CD10–CD27–IgD– cells were highly responsive to stimulation through TLR7 as evidenced by the appearance of blasting CD27hiCD38hi cells. Emab significantly inhibited the activation and differentiation of CD10–CD27–IgD– B cells into plasma cells.ConclusionsEmab can both regulate cytokine expression and block Blimp1-dependent B-cell differentiation, although the effects of Emab may depend on the stage of B-cell development or activation. In addition, Emab inhibits the activation of CD27–IgD– tonsillar cells, which correspond to so-called double-negative memory B cells, known to be increased in SLE patients with more active disease. These data may be relevant to the therapeutic effect of Emab in vivo via modulation of the production of pro-inflammatory and anti-inflammatory cytokines by B cells. Because Blimp-1 is required by B cells to mature into antibody-producing cells, inhibition of Blimp1 may reduce autoantibody production.

Activation of Human B Cells with Monoclonal Antibody to the Bp35 Cell Surface Polypeptide

Resting human B lymphocytes can be triggered to proliferate with high concentrations of antisera to surface immunoglobulin (Ig) or with low doses of anti-Ig in the presence of T cell-derived B cell-stimulating factors (BSF) (1–3). It is thought that after an initial activation signal, e.g., by antigen or anti-Ig, receptors for BSF and other growth and differentiation factors are expressed, and that in the presence of these factors B cells divide and mature into antibody-forming cells (4–6). The receptor for the T cell growth factor interleukin-2 (IL-2) has been defined (7), but analogous structures on B cells have not yet been described. Nor is it clear how binding of the Ig receptor, which has such a small intracytoplasmic tail (8), leads to B cell activation.

Regulation of B-lineage cells by caspase 6

The caspase (Casp) family of proteases regulate both lymphocyte apoptosis and activation. Here, we show that Casp6 regulates early B‐cell development. One‐week‐old Casp6 knockout (Casp6 KO) mice have significantly more splenic B‐cell subsets than wild‐type (WT) mice. Adult Casp6 KO mice have normal levels of total splenic B cells but have increased numbers of B1a B cells and CD43+ “transitional” or splenic red pulp (RP) B cells. These results suggested that Casp6 may function to control B‐cell numbers under nonhomeostatic conditions and during B‐cell development. Consistent with this model, reconstitution of B cells was dysregulated in Casp6 KO mice after sublethal irradiation. Furthermore, bone marrow pro‐B, pre‐B and immature B‐cell numbers were significantly higher in 1‐week‐old Casp6 KO mice than in 1‐week‐old WT mice. Casp6 KO pro‐B cells proliferated more in response to IL‐7 than WT pro‐B cells, suggesting that Casp6 regulates early B‐cell responses to IL‐7. Indeed, adult and aged Casp6 KO mice had elevated numbers of IL‐7αR+Sca1+ precursors of common lymphoid progenitors, suggesting Casp6 may help regulate progenitors of B cells and early B‐lineage cells. Casp6 regulates B‐cell programs both during early development and after antigen stimulation in the periphery.