End-binding protein 1 controls signal propagation from the T cell receptor

The role of microtubules (MTs) in the control and dynamics of the immune synapse (IS) remains unresolved. Here, we show that T cell activation requires the growth of MTs mediated by the plus-end specific protein end-binding 1 (EB1). A direct interaction of the T cell receptor (TCR) complex with EB1 provides the molecular basis for EB1 activity promoting TCR encounter with signalling vesicles at the IS. EB1 knockdown alters TCR dynamics at the IS and prevents propagation of the TCR activation signal to LAT, thus inhibiting activation of PLCγ1 and its localization to the IS. These results identify a role for EB1 interaction with the TCR in controlling TCR sorting and its connection with the LAT/PLCγ1 signalosome.     

Dynamics of T cell activation threshold tuning

T lymphocytes are believed to alter their sensitivity to TCR stimulation by means of a tunable cellular activation threshold. We present two modelling examples which show that the concept of a tunable threshold can be made mechanistically plausible. The tunable threshold is treated as an emergent property of the dynamics of the T cell's signalling machinery. In addition, we discuss how the dynamic properties of activation threshold tuning can be determined experimentally with the aid of these two models. We propose a novel 'avidity selection' mechanism for the initial stages of the immune response, based on the properties of the T cell activation threshold tuning mechanism we propose for the commitment to differentiation. Our main finding is that activation threshold tuning allows T cells to respond to relevant ligands with a detection threshold that is (i) uniform across both the T cell repertoire and the secondary lymphoid tissues, while (ii) retaining tolerance to autostimulation. Our analysis indicates that central tolerance enhances the efficiency of peripheral tolerance, casting new light on the role of negative selection in the thymus.     

Cooperative interactions at the SLP‐76 complex are critical for actin polymerization

T‐cell antigen receptor (TCR) engagement induces formation of multi‐protein signalling complexes essential for regulating T‐cell functions. Generation of a complex of SLP‐76, Nck and VAV1 is crucial for regulation of the actin machinery. We define the composition, stoichiometry and specificity of interactions in the SLP‐76, Nck and VAV1 complex. Our data reveal that this complex can contain one SLP‐76 molecule, two Nck and two VAV1 molecules. A direct interaction between Nck and VAV1 is mediated by binding between the C‐terminal SH3 domain of Nck and the VAV1 N‐terminal SH3 domain. Disruption of the VAV1:Nck interaction deleteriously affected actin polymerization. These novel findings shed new light on the mechanism of actin polymerization after T‐cell activation.     

COP9 signalosome controls the Carma1–Bcl10–Malt1 complex upon T‐cell stimulation

The Carma1–Bcl10–Malt1 (CBM) complex connects T‐cell receptor (TCR) signalling to the canonical IκB kinase (IKK)/NF (nuclear factor)‐κB pathway. Earlier studies have indicated that the COP9 signalosome (CSN), a pleiotropic regulator of the ubiquitin/26S proteasome system, controls antigen responses in T cells. The CSN is required for the degradation of the NF‐κB inhibitor IκBα, but other molecular targets involved in T‐cell signalling remained elusive. Here, we identify the CSN subunit 5 (CSN5) as a new interactor of Malt1 and Carma1. T‐cell activation triggers the recruitment of the CSN to the CBM complex, and CSN downregulation impairs TCR‐induced IKK activation. Furthermore, the CSN is required for maintaining the stability of Bcl10 in response to T‐cell activation. Taken together, our data provide evidence for a functional link between the evolutionarily conserved CSN and the adaptive immunoregulatory CBM complex in T cells.     

Extensive MHC class I-restricted CD8 T lymphocyte responses against various yeast genera in humans

The human cellular immune response against 14 distantly related yeast species was analyzed by intracellular cytokine staining of lymphocytes after ex vivo stimulation of whole blood. While the CD4 T cell response was marginal, extensive MHC class I-restricted CD8 T cell responses were detected against a number of species including spoiling, environmental and human pathogenic yeasts. The yeast-specific CD8 T cells expressed interferon-gamma but lacked expression of CD27 and CCR7, indicating that they were end-differentiated effector memory cells. Mainly intact yeast cells rather than spheroplasts were able to induce cytokine expression in T cells demonstrating that the dominant immunogens were located in the yeast cell wall. Together these data underline the importance of the cellular immune response in protecting humans against yeast and fungal infections. And, from another perspective, recombinant yeast suggests itself as a potential vaccine candidate to efficiently induce antigen-specific CD8 T cell responses.     

The Spectrin cytoskeleton regulates the Hippo signalling pathway

The Spectrin cytoskeleton is known to be polarised in epithelial cells, yet its role remains poorly understood. Here, we show that the Spectrin cytoskeleton controls Hippo signalling. In the developing Drosophila wing and eye, loss of apical Spectrins (alpha/beta‐heavy dimers) produces tissue overgrowth and mis‐regulation of Hippo target genes, similar to loss of Crumbs (Crb) or the FERM‐domain protein Expanded (Ex). Apical beta‐heavy Spectrin binds to Ex and co‐localises with it at the apical membrane to antagonise Yki activity. Interestingly, in both the ovarian follicular epithelium and intestinal epithelium of Drosophila, apical Spectrins and Crb are dispensable for repression of Yki, while basolateral Spectrins (alpha/beta dimers) are essential. Finally, the Spectrin cytoskeleton is required to regulate the localisation of the Hippo pathway effector YAP in response to cell density human epithelial cells. Our findings identify both apical and basolateral Spectrins as regulators of Hippo signalling and suggest Spectrins as potential mechanosensors.     

热休克与ConA激活对T淋巴细胞的双重作用

为模拟天然免疫条件下,病毒等抗原对T淋巴细胞激活同时伴有体温升高的环境,我们建立了人外周血T淋巴细胞体外激活和热休克的模型。热休克对ConA激活细胞中多肽的合成既有协同也有抑制作用。ConA激活细胞受热休克的影响较静止细胞小,而其热休克蛋白(HSP)的诱导合成则较强,为了解HSP在淋巴细胞中的作用提供了线索。     

Expression of chemokine-like factor 1 is upregulated during T lymphocyte activation

Chemokine-like factor 1 (CKLF1) is a cytokine with chemotactic effects on leukocytes and a functional ligand of CCR4. This cytokine is widely expressed and the level of expression is reported to be upregulated in asthma and rheumatoid arthritis (RA), disease conditions in which T lymphocytes are over-activated. In order to determine the expression profile of CKLF1 in activated T lymphocytes, we first employed a PCR-based method on human blood fractions cDNA panels and found that CKLF1 was upregulated in activated CD4+ and CD8+ cells, with no obvious changes in CD19+ cells. We further performed kinetic analyses of CKLF1 expression in phytohemagglutinin (PHA)-stimulated human peripheral blood lymphocytes (PBL) at both the mRNA and protein levels. In resting PBL, the constitutive expression of CKLF1 was low at mRNA level and barely detectable at the protein level; however, both were remarkably upregulated by PHA, appearing at 8h after PHA-stimulation and persisting up to 72h. These results suggest that CKLF1 may be involved in T lymphocyte activation and further study of CKLF1 function will prove valuable.     

Divalent Cations Crosslink Vimentin Intermediate Filament Tail Domains to Regulate Network Mechanics

Intermediate filament networks in the cytoplasm and nucleus are critical for the mechanical integrity of metazoan cells. However, the mechanism of crosslinking in these networks and the origins of their mechanical properties are not understood. Here, we study the elastic behavior of in vitro networks of the intermediate filament protein vimentin. Rheological experiments reveal that vimentin networks stiffen with increasing concentrations of Ca²⁺ and Mg²⁺, showing that divalent cations act as crosslinkers. We quantitatively describe the elastic response of vimentin networks over five decades of applied stress using a theory that treats the divalent cations as crosslinkers: at low stress, the behavior is entropic in origin, and increasing stress pulls out thermal fluctuations from single filaments, giving rise to a nonlinear response; at high stress, enthalpic stretching of individual filaments significantly modifies the nonlinearity. We investigate the elastic properties of networks formed by a series of protein variants with stepwise tail truncations and find that the last 11 amino acids of the C-terminal tail domain mediate crosslinking by divalent ions. We determined the single-filament persistence length, l_P ≈ 0.5 μm, and Young's modulus, Y ≈ 9 MPa; both are consistent with literature values. Our results provide insight into a crosslinking mechanism for vimentin networks and suggest that divalent ions may help regulate the cytoskeletal structure and mechanical properties of cells.     

Integrin α4β7 Co-Stimulation of Human Peripheral Blood T Cell Proliferation

The Integrin α4β7 mediates lymphocyte adhesion to VCAM-1 on activated endothelium, fibronectin in the extracellular matrix, and the mucosal vascular addressin MAdCAM-1. It is unclear whether α4β7 performs any function beyond directing specific adhesion reactions. We addressed the possibility that triggering of α4β7 with a specific monoclonal antibody was capable of delivering an accessory stimulus that would coactivate T cells and lead to proliferation. At submitogenic levels of anti-CD3 stimulation, triggering of α4β7 by immobilized mAb ACT-1 resulted in T cell blastogenesis, IL-2 production, expression of the IL-2 receptor α chain CD25, and ultimately DNA synthesis. These results indicate that the integrin α4β7 is involved in more than lymphocyte adhesion and homing but also plays a role in cell signaling.     

Identification of amino acid residues lining the pore of a gap junction channel

Gap junctions represent a ubiquitous and integral part of multicellular organisms, providing the only conduit for direct exchange of nutrients, messengers and ions between neighboring cells. However, at the molecular level we have limited knowledge of their endogenous permeants and selectivity features. By probing the accessibility of systematically substituted cysteine residues to thiol blockers (a technique called SCAM), we have identified the pore-lining residues of a gap junction channel composed of Cx32. Analysis of 45 sites in perfused Xenopus oocyte pairs defined M3 as the major pore-lining helix, with M2 (open state) or M1 (closed state) also contributing to the wider cytoplasmic opening of the channel. Additional mapping of a close association between M3 and M4 allowed the helices of the low resolution map (Unger et al., 1999. Science. 283:1176-1180) to be tentatively assigned to the connexin transmembrane domains. Contrary to previous conceptions of the gap junction channel, the residues lining the pore are largely hydrophobic. This indicates that the selective permeabilities of this unique channel class may result from novel mechanisms, including complex van der Waals interactions of permeants with the pore wall, rather than mechanisms involving fixed charges or chelation chemistry as reported for other ion channels.