Hebin Liu

Dynamics of subsynaptic vesicles and surface microclusters at the immunological synapse.

Vesicles dynamically deliver the T cell adaptor protein LAT to sites of T cell signalosomes. Submariner Adaptor Engagement of the T cell antigen receptor (TCR) on the surface of a T cell with peptide-loaded major histocompatibility complex on the surface of an antigen-presenting cell occurs at a specialized contact point known as the immunological synapse (IS). Stimulation of the TCR triggers the activation of the proximal kinases Lck and ZAP-70, which leads to the formation at the IS of microclusters of kinases and adaptor molecules that are required for T cell signaling. Two of these adaptor molecules are the cytosolic protein SLP-76 and the plasma membrane–associated protein LAT. SLP-76 and LAT form distinct microclusters, but how they interact to propagate T cell signals is unclear (see the Perspective by Billadeau). Purbhoo et al. used imaging techniques to show that a fraction of LAT was enriched in intracellular vesicles that resided below the IS and that these vesicles moved to the IS, where they interacted with SLP76-containing microclusters. LAT-containing vesicles were corralled by TCR–ZAP-70 microclusters in the IS and were more mobile than the SLP-76 microclusters, but they slowed when they interacted with SLP-76. Cells that contained a mutant LAT that could not interact with SLP-76 (through the associated protein GADS) contained fewer SLP-76 microclusters, and LAT-containing vesicles interacted less with them. Together, these data suggest that vesicular LAT interacts with protein microclusters at the IS and contributes to the propagation of T cell signaling. Imaging studies have identified clusters of kinases and adaptor proteins that serve as centers of signaling at the contact points between T cells and antigen-presenting cells (APCs). Here, we report that the kinase ZAP-70 and the adaptor proteins LAT and SLP-76 accumulated in separate clusters at the interface between T cells and coverslips coated with a stimulatory antibody against CD3, a component of the T cell antigen receptor complex. A fraction of LAT was detected in motile vesicles that repeatedly moved to surface microclusters of SLP-76 and the adaptor protein GADS (growth factor receptor–bound protein–related adaptor downstream of Shc), where they exhibited decreased motility. LAT molecules in which the residues tyrosine 171 and tyrosine 191 (which are required for the binding of LAT to GADS) were mutated to phenylalanine did not dwell at clusters of SLP-76. At immunological synapses, LAT-containing vesicles also colocalized with microclusters of SLP-76, as detected in experiments in which laser tweezers were used to position T cell–APC conjugates vertically for high-resolution imaging. Phosphorylation of LAT was most prominent when vesicular LAT colocalized with SLP-76. Indeed, the abundance of phosphorylated LAT within a microcluster of SLP-76 was greatest in those clusters that had more recent interactions with LAT-containing vesicles. Finally, negative signals by the inhibitory receptor ILT2 disrupted the assembly of SLP-76–containing microclusters. Together, these data show that the movement of LAT-containing vesicles is linked to the organization of protein microclusters and suggest an important role for vesicular LAT in the SLP-76 signalosome.

CTLA-4 disrupts ZAP70 microcluster formation with reduced T cell/APC dwell times and calcium mobilization

CTLA‐4 is a co‐receptor that modulates the threshold of T cell activation and autoimmunity. We previously showed that CTLA‐4 reverses the TCR‐mediated stop signal needed for T cell/APC interactions [Schneider et al., Science 2006, 313: 1972]. In this study, using a different T cell system, we show that CTLA‐4 expression changed the behavior of T8.1 T cells by reducing the contact time between T cell and APC, preventing re‐inforced contacts, and reducing the contact area at the immunological synapse. This led to a major reduction in Ca2+ influx/mobilization and interleukin‐2 production. Further, anti‐CD3/CTLA‐4 increased T cell motility on antibody‐coated glass slides, concurrent with an abrogation of ZAP70 microcluster formation. Our findings further support a role for CTLA‐4 in limiting the interaction between T cell and APC that is needed for optimal activation.

Functional defects of SKAP-55-deficient T cells identify a regulatory role for the adaptor in LFA-1 adhesion.

ABSTRACT The ADAP-SKAP-55 module regulates T-cell receptor (TCR)-induced integrin clustering and adhesion in T cells. However, it has been unclear whether ADAP and/or SKAP-55 is an effector of the response. ADAP controls SKAP-55 expression such that ADAP−/− T cells are also deficient in SKAP-55 expression. In this study, we report the phenotype of the SKAP-55-deficient mouse. SKAP-55−/− T cells retain ADAP expression yet show defects in β1 and β2 integrin adhesion, leukocyte function-associated antigen 1 (LFA-1) clustering, production of the cytokines interleukin-2 and gamma interferon, and proliferation. This dependency was also reflected in more-transient conjugation times in response to the superantigen staphylococcal enterotoxin A on dendritic cells and a reduced number of cells with TCR/CD3 microcluster localization at the immunological synapse. SKAP-55−/− T cells showed the same general impairment of function as ADAP−/− T cells, indicating that SKAP-55 is an effector of the ADAP-SKAP-55 module. At the same time, the requirement for ADAP and SKAP-55 was not absolute, since a subset of peripheral T cells adhered with loss of expression of either adaptor. Further, dependency on SKAP-55 or ADAP differed with the strength of the TCR signal. As with the ADAP−/− mouse, SKAP-55-deficient mice showed no major effects on lymphoid development or the appearance of peripheral T cells, B cells, and NK cells. Our findings identify a clear effector role for SKAP-55 in LFA-1 adhesion in peripheral T cells and demonstrate that dependency on SKAP-55 and ADAP differs among T cells and differs with the strength of the TCR signal.

SH2 domain containing leukocyte phosphoprotein of 76-kDa (SLP-76) feedback regulation of ZAP-70 microclustering

T cell receptor (TCR) signaling involves CD4/CD8-p56lck recruitment of ZAP-70 to the TCR receptor, ZAP-70 phosphorylation of LAT that is followed by LAT recruitment of the GADS-SLP-76 complex. Back regulation of ZAP-70 by SLP-76 has not been documented. In this paper, we show that anti-CD3 induced ZAP-70 cluster formation is significantly reduced in the absence of SLP-76 (i.e., J14 cells) and in the presence of a mutant of SLP-76 (4KE) in Jurkat and primary T cells. Both the number of cells with clusters and the number of clusters per cell were reduced. This effect was not mediated by SLP-76 SH2 domain binding to ZAP-70 because SLP-76 failed to precipitate ZAP-70 and an inactivating SH2 domain mutation (i.e., R448L) on SLP-76 4KE did not reverse the inhibition of ZAP-70 clustering. Mutation of R448 on WT SLP-76 still supported ZAP-70 clustering. Intriguingly, by contrast, LAT clustering occurred normally in the absence of SLP-76, or the presence of 4KE SLP-76 indicating that this transmembrane adaptor can operate independently of ZAP-70-GADS-SLP-76. Our findings reconfigure the TCR signaling pathway by showing SLP-76 back-regulation of ZAP-70, an event that could ensure that signaling components are in balance for optimal T cell activation.

The chemokine CXCL12 generates costimulatory signals in T cells to enhance phosphorylation and clustering of the adaptor protein SLP-76.

Chemokine and antigen signals converge on an adaptor protein to enhance T cell activation. Chemokine Enhancement of TCR Signals Stimulation of the T cell receptor (TCR) activates multiple receptor-proximal signals, including the phosphorylation and clustering of the adaptor protein SLP-76. In addition to stimulating the chemotaxis of T cells through its G protein–coupled receptor CXCR4, the chemokine CXCL12 acts as a costimulatory signal for T cells. Smith et al. performed live confocal imaging of TCR-stimulated human T cells and found that simultaneous stimulation with CXCL12 enhanced the number, stability, and phosphorylation of SLP-76 microclusters and the expression of TCR target genes. Mutation of either of two critical tyrosine residues in SLP-76 blocked this effect of CXCL12, as did inhibition of the coupling of CXCR4 to Gi-family G proteins. These data suggest that T cells simultaneously exposed to CXCL12 and antigen are enhanced in activation, thereby boosting the immune response. The CXC chemokine CXCL12 mediates the chemoattraction of T cells and enhances the stimulation of T cells through the T cell receptor (TCR). The adaptor SLP-76 [Src homology 2 (SH2) domain–containing leukocyte protein of 76 kD] has two key tyrosine residues, Tyr113 and Tyr128, that mediate signaling downstream of the TCR. We investigated the effect of CXCL12 on SLP-76 phosphorylation and the TCR-dependent formation of SLP-76 microclusters. Although CXCL12 alone failed to induce SLP-76 cluster formation, it enhanced the number, stability, and phosphorylation of SLP-76 microclusters formed in response to stimulation of the TCR by an activating antibody against CD3, a component of the TCR complex. Addition of CXCL12 to anti-CD3–stimulated cells resulted in F-actin polymerization that stabilized SLP-76 microclusters in the cells’ periphery at the interface with antibody-coated coverslips and increased the interaction between SLP-76 clusters and those containing ZAP-70, the TCR-associated kinase that phosphorylates SLP-76, as well as increased TCR-dependent gene expression. Costimulation with CXCL12 and anti-CD3 increased the extent of phosphorylation of SLP-76 at Tyr113 and Tyr128, but not that of other TCR-proximal components, and mutation of either one of these residues impaired the CXCL12-dependent effect on SLP-76 microcluster formation, F-actin polymerization, and TCR-dependent gene expression. The effects of CXCL12 on SLP-76 microcluster formation were dependent on the coupling of its receptor CXCR4 to Gi-family G proteins (heterotrimeric guanine nucleotide–binding proteins). Thus, we identified a costimulatory mechanism by which CXCL12 and antigen converge at SLP-76 microcluster formation to enhance T cell responses.

Identification of an N-terminal Transactivation Domain of Runx1 That Separates Molecular Function from Global Differentiation Function

RUNX1, or AML1, is a transcription factor that is the most frequent target for chromosomal gene translocations in acute leukemias. RUNX1 is essential for definitive hematopoiesis in embryos and profoundly influences adult steady-state hematopoiesis both positively and negatively. To investigate this wide range of normal activities and the pathological role of RUNX1, it is important to define the functions of different domains of the protein. RUNX1, RUNX2, and RUNX3 are highly conserved in their DNA binding runt homology domain and contain divergent sequences of unknown function N-terminal to this domain. Here we analyzed the role of the N-terminal sequence and the α-helix of the runt homology domain of Runx1 in DNA binding, transactivation, and megakaryocytopoiesis. Both the N terminus and the α-helix were found to reduce DNA binding of Runx1 and be essential for transactivation of the granulocyte-macrophage colony-stimulating factor and Iα1 promoters by Runx1. The N terminus of Runx1, including the α-helix, was also required for transactivation of a Gal4 reporter when expressed as fusion proteins with a Gal4 DNA binding domain, and the N terminus alone was capable of stimulating transcription when fused to the Gal4 DNA binding domain. The N terminus and the α-helix, however, were not required for megakaryocyte development from embryonic stem cells differentiated in vitro. Thus, our findings define a second transactivation domain of Runx1 that is differentially required for activation of transcription of some Runx1-dependent promoters and megakaryocytopoiesis.

The Immune Adaptor SLP-76 Binds to SUMO-RANGAP1 at Nuclear Pore Complex Filaments to Regulate Nuclear Import of Transcription Factors in T Cells

Summary While immune cell adaptors regulate proximal T cell signaling, direct regulation of the nuclear pore complex (NPC) has not been reported. NPC has cytoplasmic filaments composed of RanGAP1 and RanBP2 with the potential to interact with cytoplasmic mediators. Here, we show that the immune cell adaptor SLP-76 binds directly to SUMO-RanGAP1 of cytoplasmic fibrils of the NPC, and that this interaction is needed for optimal NFATc1 and NF-κB p65 nuclear entry in T cells. Transmission electron microscopy showed anti-SLP-76 cytoplasmic labeling of the majority of NPCs in anti-CD3 activated T cells. Further, SUMO-RanGAP1 bound to the N-terminal lysine 56 of SLP-76 where the interaction was needed for optimal RanGAP1-NPC localization and GAP exchange activity. While the SLP-76-RanGAP1 (K56E) mutant had no effect on proximal signaling, it impaired NF-ATc1 and p65/RelA nuclear entry and in vivo responses to OVA peptide. Overall, we have identified SLP-76 as a direct regulator of nuclear pore function in T cells.