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Induction of T cell antigen receptor (TCR) endocytosis has a significant impact on TCR signaling and T cell behavior, but the molecular interactions coordinating internalization of the activated TCR are poorly understood. Previously we have shown that TCR endocytosis is regulated by the Wiskott Aldrich Syndrome protein (WASp), a cytosolic effector which, upon interaction with the cdc42 Rho GTPase, couples TCR engagement to Arp 2/3 complex-mediated actin polymerization. Here we report that WASp associates in T cells with intersectin 2, an endocytic adaptor containing multiple domains including a Dbl homology (DH) domain with the potential to activate Rho GTPases. Intersectin 2 association with WASp increases after TCR engagement, and its overexpression in Cos-7 cells induces WASp translocation to endocytic vesicles within which intersectin 2 colocalizes with both WASp and cdc42. Intersectin 2, but not a DH domain-deleted (ΔDH) form of intersectin 2, and stimulation via the TCR also trigger the activation of cdc42. Induction of TCR internalization is also augmented by intersectin 2 and severely impaired by latrunculin B treatment. Thus, intersection 2 appears to function cooperatively with WASp and cdc42 to link the clathrin endocytic machinery to WASp-mediated actin polymerization and ultimately to occupancy-induced TCR endocytosis.

Macrolide resistance has been demonstrated in group B streptococcus (GBS), but there is limited information regarding mechanisms of resistance and their prevalence. We determined these in GBS obtained from neonatal blood cultures and vaginal swabs from pregnant women. Of 178 isolates from cases of neonatal GBS sepsis collected from 1995 to 1998, 8 and 4.5% were resistant to erythromycin and clindamycin, respectively, and one isolate showed intermediate penicillin resistance (MIC, 0.25 μg/ml). Of 101 consecutive vaginal or rectal/vaginal isolates collected in 1999, 18 and 8% were resistant to erythromycin and clindamycin, respectively. Tetracycline resistance was high (>80%) among both groups of isolates. Of 32 erythromycin-resistant isolates, 28 possessed the erm methylase gene (7 ermB and 21 ermTR/ermA ) and 4 harbored the mefA gene; one isolate harbored both genes. One isolate which was susceptible to erythromycin but resistant to clindamycin (MIC, 4 μg/ml) was found to have the linB gene, previously identified only in Enterococcus faecium . The mreA gene was found in all the erythromycin-resistant strains as well as in 10 erythromycin-susceptible strains. The rate of erythromycin resistance increased from 5% in 1995–96 to 13% in 1998–99, which coincided with an increase in macrolide usage during that time.

The Wiskott-Aldrich syndrome protein (WASp) has been implicated in modulation of lymphocyte activation and cytoskeletal reorganization. To address the mechanisms whereby WASp subserves such functions, we have examined WASp roles in lymphocyte development and activation using mice carrying a WAS null allele ( WAS − / − ). Enumeration of hemopoietic cells in these animals revealed total numbers of thymocytes, peripheral B and T lymphocytes, and platelets to be significantly diminished relative to wild-type mice. In the thymus, this abnormality was associated with impaired progression from the CD44 − CD25 + to the CD44 − CD25 − stage of differentiation. WASp-deficient thymocytes and T cells also exhibited impaired proliferation and interleukin (IL)-2 production in response to T cell antigen receptor (TCR) stimulation, but proliferated normally in response to phorbol ester/ionomycin. This defect in TCR signaling was associated with a reduction in TCR-evoked upregulation of the early activation marker CD69 and in TCR-triggered apoptosis. While induction of TCR-ζ, ZAP70, and total protein tyrosine phosphorylation as well as mitogen-activated protein kinase (MAPK) and stress-activated protein/c-Jun NH 2 -terminal kinase (SAPK/JNK) activation appeared normal in TCR-stimulated WAS − / − cells, TCR-evoked increases in intracellular calcium concentration were decreased in WASp-deficient relative to wild-type cells. WAS − / − lymphocytes also manifested a marked reduction in actin polymerization and both antigen receptor capping and endocytosis after TCR stimulation, whereas WAS − / − neutrophils exhibited reduced phagocytic activity. Together, these results provide evidence of roles for WASp in driving lymphocyte development, as well as in the translation of antigen receptor stimulation to proliferative or apoptotic responses, cytokine production, and cytoskeletal rearrangement. The data also reveal a role for WASp in modulating endocytosis and phagocytosis and, accordingly, suggest that the immune deficit conferred by WASp deficiency reflects the disruption of a broad range of cellular behaviors.

The Wiskott–Aldrich syndrome protein (WASp) plays a major role in coupling T cell antigen receptor (TCR) stimulation to induction of actin cytoskeletal changes required for T cell activation. Here, we report that WASp inducibly binds the sorting nexin 9 (SNX9) in T cells and that WASp, SNX9, p85, and CD28 colocalize within clathrin-containing endocytic vesicles after TCR/CD28 costimulation. SNX9, implicated in clathrin-mediated endocytosis, binds WASp via its SH3 domain and uses its PX domain to interact with the phosphoinositol 3-kinase regulatory subunit p85 and product, phosphoinositol (3,4,5)P 3 . The data reveal ligation-induced CD28 endocytosis to be clathrin- and phosphoinositol 3-kinase-dependent and TCR/CD28-evoked CD28 internalization and NFAT activation to be markedly enhanced by SNX9 overexpression, but severely impaired by expression of an SNX9 mutant (SNX9ΔPX) lacking p85-binding capacity. CD28 endocytosis and CD28-evoked actin polymerization also are impaired in WASp-deficient T cells. These findings suggest that SNX9 couples WASp to p85 and CD28 so as to link CD28 engagement to its internalization and to WASp-mediated actin remodeling required for CD28 cosignaling. Thus, the WASp/SNX9/p85/CD28 complex enables a unique interface of endocytic, actin polymerizing, and signal transduction pathways required for CD28-mediated T cell costimulation.

The Wiskott-Aldrich syndrome protein (WASp) couples actin cytoskeletal rearrangement to T cell activation, but the mechanisms involved are unknown. Here, we show that antigen-induced formation of T cell:APC conjugates and synapses is abrogated in WASp-deficient T cells and that CD2 engagement evokes interactions between the proline-rich region required for WASp translocation to the synapse and the PSTPIP1 adaptor SH3 domain and between the PSTPIp1 coiled-coil domain and both CD2 and another CD2-binding adaptor, CD2AP. The induced colocalization of these proteins at the synapse is disrupted by expression of coiled-coil domain-deleted PSTPIP1. These data, together with the impairment in CD2-induced actin polymerization observed in WASp-deficient cells, suggest that PSTPIP1 acts downstream of CD2/CD2AP to link CD2 engagement to the WASp-evoked actin polymerization required for synapse formation and T cell activation.

All members of the Wiskott–Aldrich syndrome protein (WASp) family contain a carboxyl-terminal verprolin homology, cofilin homology, and acidic region (VCA) domain that binds and activates the Arp2/3 complex, thereby linking these proteins to the induction of actin polymerization. Although the VCA domain imbues WASp and other WASp family members with the capacity to modulate cytoskeletal organization, little is known about the impact of this domain activity on lymphoid cell function. Here we demonstrate that T cell-restricted expression of VCA domain-deleted WASp (WASpΔVCA) in WAS−/−mice engenders a severe early block in T lymphopoiesis associated with impaired T cell antigen receptor αβ expression and a consequent failure to generate single-positive CD4+and CD8+T cells. These latter defects, which are not observed in WAS−/−mice, are associated with impaired induction of cellular actin polymerization and a failure in the terminal differentiation of double-negative thymocytes. These findings indicate that WASp family proteins play an essential role in modulating the signaling events required for early thymocyte development and reveal their capacity to subserve this role to depend on VCA domain-mediated actin polymerization.

Engagement of antigen receptors on lymphocytes induces a complex cascade of intracellular molecular interactions, which provides the circuitry for coupling stimulatory signals to alterations in gene expression and ultimately cell behavior. This circuitry has been intensively investigated and the results of such studies have elucidated not only the specific components of antigen-receptor-evoked signaling cascades, but also many mechanistic paradigms relevant to the signaling field as a whole. Thus, for example, the pivotal role for tyrosine phosphorylation-based signaling interactions in initiating intracellular transmission of activation signals was highlighted by the characterization of immunoreceptor tyrosine-based activation motifs (ITAMs) within antigen-receptor components and the demonstration that Src-family-mediated phosphorylation of these motifs allows for binding and activation of ZAP-70/Syk kinases, the consequent recruitment/activation of phosphatidylinositol-3 kinase and phospholipase C (PLC)-γ, and the induction of a myriad of secondary signaling cascades (Birkeland and Monroe 1997; DeFranco 1997; Qian and Weiss 1997). Similarly, the concept of signaling thresholds for expression of given cellular responses emanated from the demonstration that signaling effectors, such as accessory receptors and cytosolic tyrosine phosphatases, can modify the outcome of antigen-receptor engagement by quantitatively modulating antigen-receptor signaling (Goodnow 1996). From these and the myriad of other insights culled from studies of antigen-receptor signaling has emerged a wealth of information on the biochemical networks that enable antigen stimulation to be translated to such diverse outcomes as proliferation, cytokine/antibody production, differentiation, death or transformation.