Bruton's tyrosine kinase (BTK) is pivotal in B cell activation and development through its participation in the signaling pathways of multiple hematopoietic receptors. The mechanisms controlling BTK activation were studied here by examination of the biochemical consequences of an interaction between BTK and SRC family kinases. This interaction of BTK with SRC kinases transphosphorylated BTK on tyrosine at residue 551, which led to BTK activation. BTK then autophosphorylated at a second site. The same two sites were phosphorylated upon B cell antigen receptor cross-linking. The activated BTK was predominantly membrane-associated, which suggests that BTK integrates distinct receptor signals resulting in SRC kinase activation and BTK membrane targeting.

Chronic hepatitis C virus (HCV) infection, with its cohort of life-threatening complications, affects more than 200 million persons worldwide and has a prevalence of more than 10% in certain countries. Preventive and therapeutic vaccines against HCV are thus much needed. Neutralizing antibodies (NAbs) are the foundation for successful disease prevention for most established vaccines. However, for viruses that cause chronic infection such as HIV or HCV, induction of broad NAbs from recombinant vaccines has remained elusive. We developed a vaccine platform specifically aimed at inducing NAbs based on pseudotyped virus-like particles (VLPs) made with retroviral Gag. We report that VLPs pseudotyped with E2 and/or E1 HCV envelope glycoproteins induced high-titer anti-E2 and/or anti-E1 antibodies, as well as NAbs, in both mouse and macaque. The NAbs, which were raised against HCV 1a, cross-neutralized the five other genotypes tested (1b, 2a, 2b, 4, and 5). Thus, the described VLP platform, which can be pseudotyped with a vast array of virus envelope glycoproteins, represents a new approach to viral vaccine development.

Mutation of Bruton's tyrosine kinase (Btk) impairs B cell maturation and function and results in a clinical phenotype of X-linked agammaglobulinemia. Activation of Btk correlates with an increase in the phosphorylation of two regulatory Btk tyrosine residues. Y551 (site 1) within the Src homology type 1 (SH1) domain is transphosphorylated by the Src family tyrosine kinases. Y223 (site 2) is an autophosphorylation site within the Btk SH3 domain. Polyclonal, phosphopeptide-specific antibodies were developed to evaluate the phosphorylation of Btk sites 1 and 2. Crosslinking of the B cell antigen receptor (BCR) or the mast cell Fc receptor, or interleukin 5 receptor stimulation each induced rapid phosphorylation at Btk sites 1 and 2 in a tightly coupled manner. Btk molecules were singly and doubly tyrosinephosphorylated. Phosphorylated Btk comprised only a small fraction (<5%) of the total pool of Btk molecules in the BCR-activated B cells. Increased dosage of Lyn in B cells augmented BCR-induced phosphorylation at both sites. Kinetic analysis supports a sequential activation mechanism in which individual Btk molecules undergo serial transphosphorylation (site 1) then autophosphorylation (site 2), followed by successive dephosphorylation of site 1 then site 2. The phosphorylation of conserved tyrosine residues within structurally related Tec family kinases is likely to regulate their activation.Mutation of the Bruton's tyrosine kinase (Btk) gene produces X-linked (or Bruton's) agammaglobulinemia in humans and X-linked immunodeficiency in mice (1-4). At the cellular level, Btk mutation is manifested by abnormal B cell responses to multiple critical factors, such as interleukin 5 (IL-5) (5-7), IL-6 (8), IL-10 (9), anti-CD38 (10, 11), and the B cell antigen receptor (BCR) (12-17). A mechanism for activation of Btk has been derived from study of endogenous receptor signaling pathways as well as through heterologous expression of Btk in fibroblasts. Src family tyrosine kinases are rapidly activated after stimulation of the BCR (18, 19), then they phosphorylate Btk at Y551 (site 1) (17, 20), a consensus Src family phosphorylation site in the Src homology type 1 (SH1) domain. This phosphorylation event dramatically increases Btk protein tyrosine kinase activity and is required for promotion of fibroblast growth in soft agar by the activated Btk allele, Btk* (17, 20-22). A second major phosphorylated tyrosine residue (Y223) is located within the Btk SH3 domain (23). Phosphorylation of Y223 (site 2) occurs by a Btk kinase-dependent mechanism, i.e., autophosphorylation (17). In contrast to site 1, site 2 phosphorylation has little discernible influence on Btk catalytic activity in vitro or in vivo. The role of the SH3 domain, however, in protein-protein interactions is well established, and site 2 corresponds to a conserved residue important for binding to proline-rich peptide sequences (24-30). Y223 phosphorylation may be a mechanism to modify such interactions.A critical, but unresolved, feature of Btk activation is...

SummaryInterleukin 10 (IL-10) has recently been shown to induce normal human B lymphocytes to proliferate and differentiate into immunoglobulin (Ig)-secreting cells. Herein, we show that IL-10 also promotes DNA synthesis and IgM production by anti-CD40 activated B cell chronic lymphocytic leukemia (B-CLL). Most strikingly, IL-2 and IL-10 were found to synergize to induce the proliferation and differentiation of B-CLL cells. This synergy between IL-2 and IL-10 was also observed with normal B cells which proliferated strongly and secreted large amounts of IgM, IgG, and IgA. The observed synergy is likely to be due to the IL-10-induced increase of high affinity IL-2 receptors on both normal and leukemic B cells. This increase of high affinity receptor is associated to an increase of Tac/CD25 expression that can be detected by flow cytometric analysis. Taken together, these results indicate that IL-10 permits anti-CD40 activated B cells to respond to IL-2 through an induction of high affinity IL-2 receptors. This effect of IL-IO may partly explain how T cells, which activate B cells in a CD40-dependent fashion, induce B cell proliferation and differentiation mostly through IL-2.