ALBERT

Description: The tetraspanin CD81 is required for numerous biological functions including fertilization, infection, cell migration and cellular interactions in the nervous and immune systems. In B cells CD81 is a component of the CD19/CD21 signaling complex. CD81 was shown to facilitate the redistribution of the B cell receptor (BCR) complex and CD21 into lipid rafts in response to co-engagement, and to modulate BCR signaling. In addition, CD81-deficient mice express low levels of cell surface CD19, thereby potentially altering signaling by the CD19/CD21 co-receptor complex. Interestingly, the onset of CD81 expression coincides with the onset of CD19 expression during B cell development. The foregoing observations suggest that CD81 might reduce the in vivo response of B cells to antigenic stimulation. To test this hypothesis we compared the response of CD81-deficient and wild type mice to T-independent (TNP-LPS) and T-dependent (TNP-KLH) antigens. Surprisingly, CD81-deficient mice mounted significantly higher IgM responses against both types of antigens. Moreover, the IgG response of CD81-deficient mice was stronger and persistent in response to T-independent antigen. We further found that CD81-deficient mice have an increase in bone marrow perisinusoidal B cells (IgM+IgD+). These cells are primarily responsible for mounting T-independent immune responses against blood-borne pathogens. In addition, CD81-deficient spleenic B cells have an intrinsic ability to produce higher amounts of IgM. These surprising results suggest that CD81 is involved in modulating B cell activation, particularly in response to infection.

Description: Wiskott-Aldrich Syndrome (WAS) is an X-linked recessive immunodeficiency disorder with eczema, thrombocytopenia and high susceptibility to opportunistic and pyogenic infections. The gene product of the WAS locus, WAS protein (WASP), is expressed in a hematopoietic-specific fashion and regulates cytoskeletal actin reorganization via Cdc42 and Arp2/3 interactions. Non-random inactivation of the X chromosome carrying the defective WASP gene in all peripheral blood cells from obligate female carriers of WAS suggests a selective advantage of hematopoietic stem cells or immature progenitor cells expressing the intact WASP gene. Because such progenitor cells are dependent on the Kit receptor/Kit ligand (KL) pathway, we tested whether or not WASP plays a role in signaling responses through Kit. WASP and interacting proteins WIP and Arp2/3 were strongly phosphorylated in response to KL stimulation of Mo7e cells. Time kinetics revealed onset of tyrosine phosphorylation of WASP as early as 1 min and a maximum at 5 min after KL stimulation. Although real-time KL-induced actin reorganization and KL-mediated spreading of bone marrow-derived mast cells (BMMC) on fibronectin-coated surfaces were grossly normal, KL-induced formation of filopodia was significantly decreased in number and size in the absence of WASP. In addition, KL-induced calcium-flux in BMMCs was aberrant in the absence of WASP suggesting that KL-dependent calcium signals and cytoskeletal rearrangement are linked through WASP. When BMMC cultures were established from WASP heterozygous female mice using KL as a growth factor, the cultures initially contained a mixture of WASP positive and negative populations. KL-driven differentiation into mature BMMCs eventually resulted in homogenous WASP positive cultures derived from the WASP positive progenitors. Thus, WASP expression conferred a selective advantage to the development of Kit-dependent hematopoiesis consistent with the selective advantage of WASP positive blood cells observed in WAS heterozygous female humans. Finally, KL-mediated gene expression in BMMCs derived from WASP negative mice or WT controls was compared and revealed in summary that at least 30% of all changes are WASP-dependent. The results indicate that WASP is downstream of Kit signaling and necessary for Kit-mediated filopodia formation, cell survival and gene expression.

Description: CD81 is a component of the CD19/CD21 coreceptor complex in B cells. This tetraspanin molecule was previously shown to enable membrane reorganization in B cells responding to complement-bound antigens. Here we stimulated B cells via their B cell receptor (BCR) and demonstrate that Cd81−/− B cells fluxed higher intracellular free calcium ion along with increased phosphorylation of PLCγ2 and Syk. The stimulated Cd81−/− B cells also proliferated faster and secreted higher amounts of antibodies. Moreover, activation of the TLR4 pathway in Cd81−/− B cells induced increased proliferation and antibody secretion. Furthermore, Cd81−/− mice mounted a significantly higher immune response to T-cell independent antigens than their wildtype counterparts. Finally, analysis of Cd81−/− B cells that were generated by bone marrow transplantation into Rag1−/− mice confirmed a cell intrinsic hyperactive phenotype. Taken together, these results indicate that CD81 plays a negative role in B cell activation in vitro and in vivo.

Description: CD81 is a component of the CD19/CD21 signaling complex in B cells. CD81 was originally discovered as target of an anti-proliferative antibody in a human B cell lymphoma. However, the exact role of CD81 in B cell function is not known. Here we studied B cells from CD81 knockout mice. We demonstrate that upon BCR induction these B cells flux higher intracellular free calcium ion; increase the phosphorylation of BCR-related proximal and distal substrates and increase their proliferation. Similarly, polyclonal activation of CD81-deficient B cells with LPS induced increased proliferation and antibody secretion. Consistent with these intrinsic B cell capabilities, CD81-deficient mice mounted significantly higher immune response upon antigenic stimulation. In addition, bone marrow perisinusoidal B cells (IgM+IgD+) capable of mounting T-independent immune responses against blood-borne pathogens were over represented in CD81-deficient mice. These cells also displayed increased calcium influx kinetics as splenic B cells and produced higher amounts of antibody after polyclonal stimulation. Taken together, these results suggest that CD81 is involved in suppressing B cell activation.

Description: Pbx1, a homeodomain transcription factor that was originally identified as the product of a proto-oncogene in acute pre-B–cell leukemia, is a global regulator of embryonic development. However, embryonic lethality in its absence has prevented an assessment of its role in B-cell development. Here, using Rag1-deficient blastocyst complementation assays, we demonstrate that Pbx1 null embryonic stem (ES) cells fail to generate common lymphoid progenitors (CLPs) resulting in a complete lack of B and NK cells, and a partial impairment of T-cell development in chimeric mice. A critical role for Pbx1 was confirmed by rescue of B-cell development from CLPs following restoration of its expression in Pbx1-deficient ES cells. In adoptive transfer experiments, B-cell development from Pbx1-deficient fetal liver cells was also severely compromised, but not erased, since transient B lymphopoiesis was detected in Rag-deficient recipients. Conditional inactivation of Pbx1 in pro-B (CD19+) cells and thereafter revealed that Pbx1 is not necessary for B-cell development to proceed from the pro-B–cell stage. Thus, Pbx1 critically functions at a stage between hematopoietic stem cell development and B-cell commitment and, therefore, is one of the earliest-acting transcription factors that regulate de novo B-lineage lymphopoiesis.

Description: The pleiotropic receptor tyrosine kinase Kit can provide cytoskeletal signals that define cell shape, positioning, and migration, but the underlying mechanisms are less well understood. In this study, we provide evidence that Kit signals through Wiskott-Aldrich syndrome protein (WASP), the central hematopoietic actin nucleation-promoting factor and regulator of the cytoskeleton. Kit ligand (KL) stimulation resulted in transient tyrosine phosphorylation of WASP, as well as interacting proteins WASP-interacting protein and Arp2/3. KL-induced filopodia in bone marrow–derived mast cells (BMMCs) were significantly decreased in number and size in the absence of WASP. KL-dependent regulation of intracellular Ca2+ levels was aberrant in WASP-deficient BMMCs. When BMMCs were derived from WASP-heterozygous female mice using KL as a growth factor, the cultures eventually developed from a mixture of WASP-positive and -negative populations into a homogenous WASP-positive culture derived from the WASP-positive progenitors. Thus, WASP expression conferred a selective advantage to the development of Kit-dependent hematopoiesis consistent with the selective advantage of WASP-positive hematopoietic cells observed in WAS-heterozygous female humans. Finally, KL-mediated gene expression in wild-type and WASP-deficient BMMCs was compared and revealed that approximately 30% of all Kit-induced changes were WASP dependent. The results indicate that Kit signaling through WASP is necessary for normal Kit-mediated filopodia formation, cell survival, and gene expression, and provide new insight into the mechanism in which WASP exerts a strong selective pressure in hematopoiesis.