Mutations in the Bruton's tyrosine kinase (Btk) gene have been linked to severe early B cell developmental blocks in human X-linked agammaglobulinemia (XLA), and to milder B cell activation deficiencies in murine X-linked immune deficiency (Xid). To elucidate unequivocally potential Btk functions in mice, we generated mutations in embryonic stem cells, which eliminated the ability to encode Btk pleckstrin homology or kinase domains, and assayed their effects by RAG2-deficient blastocyst complementation or introduction into the germline. Both mutations block expression of Btk protein and lead to reduced numbers of mature conventional B cells, severe B1 cell deficiency, serum IgM and IgG3 deficiency, and defective responses in vitro to various B cell activators and in vivo to immunization with thymus-independent type II antigens. These results prove that lack of Btk function results in an Xid phenotype and further suggest a differential requirement for Btk during the early stages of murine versus human B lymphocyte development.

We compared the efficacy of intravenous gamma globulin plus aspirin with that of aspirin alone in reducing the frequency of coronary-artery abnormalities in children with acute Kawasaki syndrome in a multicenter, randomized trial. Children randomly assigned to the gamma globulin group received intravenous gamma globulin, 400 mg per kilogram of body weight per day, for four consecutive days; both treatment groups received aspirin, 100 mg per kilogram per day, through the 14th day of illness, then 3 to 5 mg per kilogram per day. Two-dimensional echocardiograms were interpreted blindly and independently by two or more readers. Two weeks after enrollment, coronary-artery abnormalities were present in 18 of 78 children (23 percent) in the aspirin group, as compared with 6 of 75 (8 percent) in the gamma globulin group (P = 0.01). Seven weeks after enrollment, abnormalities were present in 14 of 79 children (18 percent) in the aspirin group and in 3 of 79 (4 percent) in the gamma globulin group (P = 0.005). No child had serious adverse effects from receiving gamma globulin. We conclude that high-dose intravenous gamma globulin is safe and effective in reducing the prevalence of coronary-artery abnormalities when administered early in the course of Kawasaki syndrome.

The Wiskott-Aldrich syndrome (WAS) is a human X-linked immunodeficiency resulting from mutations in a gene (WASP) encoding a cytoplasmic protein implicated in regulating the actin cytoskeleton. To elucidate WASP function, we disrupted the WASP gene in mice by gene-targeted mutation. WASP-deficient mice showed apparently normal lymphocyte development, normal serum immunoglobulin levels, and the capacity to respond to both T-dependent and T-independent type II antigens. However, these mice did have decreased peripheral blood lymphocyte and platelet numbers and developed chronic colitis. Moreover, purified WASP-deficient T cells showed markedly impaired proliferation and antigen receptor cap formation in response to anti-CD3epsilon stimulation. Yet, purified WASP-deficient B cells showed normal responses to anti-Ig stimulation. We discuss the implications of our findings regarding WASP function in receptor signaling and cytoskeletal reorganization in T and B cells and compare the effects of WASP deficiency in mice and humans.

Framework monoclonal antibodies have identified a population of human lymphocytes that express the T3 glycoprotein but not the T-cell receptor (TCR) alpha- and beta-subunits. Chemical crosslinking experiments reveal that these lymphocytes express novel T3-associated polypeptides, one of which appears to be the product of the T gamma gene. The other polypeptide may represent a fourth TCR subunit, designated T delta.

The Wiskott-Aldrich syndrome protein (WASP) family of molecules integrates upstream signalling events with changes in the actin cytoskeleton. N-WASP has been implicated both in the formation of cell-surface projections (filopodia) required for cell movement and in the actin-based motility of intracellular pathogens. To examine N-WASP function we have used homologous recombination to inactivate the gene encoding murine N-WASP. Whereas N-WASP-deficient embryos survive beyond gastrulation and initiate organogenesis, they have marked developmental delay and die before embryonic day 12. N-WASP is not required for the actin-based movement of the intracellular pathogen Listeria but is absolutely required for the motility of Shigella and vaccinia virus. Despite these distinct defects in bacterial and viral motility, N-WASP-deficient fibroblasts spread by using lamellipodia and can protrude filopodia. These results imply a crucial and non-redundant role for N-WASP in murine embryogenesis and in the actin-based motility of certain pathogens but not in the general formation of actin-containing structures.

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The Vav family of Rho guanine nucleotide exchange factors is thought to orchestrate signaling events downstream of lymphocyte antigen receptors. Elucidation of Vav function has been obscured thus far by the expression of three highly related family members. We generated mice lacking all Vav family proteins and show that Vav-null mice produce no functional T or B cells and completely fail to mount both T-dependent and T-independent humoral responses. Whereas T cell development is blocked at an early stage in the thymus, immature B lineage cells accumulate in the periphery but arrest at a late “transitional” stage. Mechanistically, we show that the Vav family is crucial for both TCR and B cell receptor (BCR)–induced Ca2+ signaling and, surprisingly, is only required for mitogen-activated protein kinase (MAPK) activation in developing and mature T cells but not in B cells. Thus, the abundance of immature B cells generated in Vav-null mice may be due to intact Ras/MAPK signaling in this lineage. Although the expression of Vav1 alone is sufficient for normal lymphocyte development, our data also reveal lineage-specific roles for Vav2 and Vav3, with the first demonstration that Vav3 plays a critical compensatory function in T cells. Together, we define an essential role for the entire Vav protein family in lymphocyte development and activation and establish the limits of functional redundancy both within this family and between Vav and other Rho–guanine nucleotide exchange factors.

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