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Bernard Malissen

Publications and source records attributed to Bernard Malissen.

At least 19 recordsLinked to original sources
PubMed2005

The H-2Kk MHC peptide-binding groove anchors the backbone of an octameric antigenic peptide in an unprecedented mode.

A wealth of data has accumulated on the structure of mouse MHC class I (MHCI) molecules encoded by the H-2(b) and H-2(d) haplotypes. In contrast, there is a dearth of structural data regarding H-2(k)-encoded molecules. Therefore, the structures of H-2K(k) complexed to an octameric peptide from influenza A virus (HA(259-266)) and to a nonameric peptide from SV40 (SV40(560-568)) have been determined by x-ray crystallography at 2.5 and 3.0 A resolutions, respectively. The structure of the H-2K(k)-HA(259-266) complex reveals that residues located on the floor of the peptide-binding groove contact directly the backbone of the octameric peptide and force it to lie deep within the H-2K(k) groove. This unprecedented mode of peptide binding occurs despite the presence of bulky residues in the middle of the floor of the H-2K(k) peptide-binding groove. As a result, the Calpha atoms of peptide residues P5 and P6 are more buried than the corresponding residues of H-2K(b)-bound octapeptides, making them even less accessible to TCR contact. When bound to H-2K(k), the backbone of the SV40(560-568) nonapeptide bulges out of the peptide-binding groove and adopts a conformation reminiscent of that observed for peptides bound to H-2L(d). This structural convergence occurs despite the totally different architectures of the H-2L(d) and H-2K(k) peptide-binding grooves. Therefore, these two H-2K(k)-peptide complexes provide insights into the mechanisms through which MHC polymorphism outside primary peptide pockets influences the conformation of the bound peptides and have implications for TCR recognition and vaccine design.

Animals↗
PubMed2005

Selective defect in antigen-induced TCR internalization at the immune synapse of CD8 T cells bearing the ZAP-70(Y292F) mutation.

Cbl proteins have been implicated in ligand-induced TCR/CD3 down-modulation, but underlying mechanisms are unclear. We analyzed the effect of mutation of a cbl-binding site on ZAP-70 (ZAP-Y292F) on dynamics, internalization, and degradation of the TCR/CD3 complex in response to distinct stimuli. Naive CD8 T cells expressing the P14 transgenic TCR from ZAP-Y292F mice were selectively affected in TCR/CD3 down-modulation in response to antigenic stimulation, whereas neither anti-CD3 Ab-, and PMA-induced TCR down-modulation, nor constitutive receptor endocytosis/cycling were impaired. We further established that the defect in TCR/CD3 down-modulation in response to Ag was paralleled by an impaired TCR/CD3 internalization and CD3zeta degradation. Analysis of T/APC conjugates revealed that delayed redistribution of TCR at the T/APC contact zone was paralleled by a delay in TCR internalization in the synaptic zone in ZAP-Y292F compared with ZAP-wild-type T cells. Cbl recruitment to the synapse was also retarded in ZAP-Y292F T cells, although F-actin and LFA-1 redistribution was similar for both cell types. This study identifies a step involving ZAP-70/cbl interaction that is critical for rapid internalization of the TCR/CD3 complex at the CD8 T cell/APC synapse.

Animals↗
PubMed2005

The type 1 cysteinyl leukotriene receptor triggers calcium influx and chemotaxis in mouse alpha beta- and gamma delta effector T cells.

Linker for activation of T cells (LAT) is essential for T cell activation. Mice with mutations of distinct LAT tyrosine residues (LatY136F and Lat3YF) develop lymphoproliferative disorders involving TCR alphabeta or gammadelta T cells that trigger symptoms resembling allergic inflammation. We analyzed whether these T cells share a pattern of gene expression that may account for their pathogenic properties. Both LatY136F alphabeta and Lat3YF gammadelta T cells expressed high levels of the type 1 cysteinyl leukotriene receptor (CysLT(1)). Upon binding to the 5(S)-hydroxy-6(R)-S-cysteinylglycyl-7,9-trans-11,14-cis-eicosatetraenoic acid (LTD(4)) cysteinyl leukotriene, CysLT(1) induced Ca(2+) flux and caused chemotaxis in both LatY136F alphabeta and Lat3YF gammadelta T cells. Wild-type in vitro-activated T cells, but not resting T cells, also migrated toward LTD(4) however with a lower magnitude than T cells freshly isolated from LatY136F and Lat3YF mice. These results suggest that CysLT(1) is likely involved in the recruitment of activated alphabeta and gammadelta T cells to inflamed tissues.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Autistic effector T cells in mice with a point mutation in the LAT adaptor fail to respond to Listeria monocytogenes infection.

The adaptor protein linker for activation of T cells (LAT) is an important transducer of extracellular T cell stimuli. In mice with a point mutation in LAT (LatY136F), TCR signaling is substantially compromised and LatY136F T cells are unresponsive to CD3 cross-linking in vitro. Nevertheless, LatY136F mice develop a polyclonal lymphoproliferation of CD4(+) T cells, which display a T(h)2-polarized effector phenotype. In this study, LatY136F mice were infected with the intracellular bacterium Listeria monocytogenes and the antigen-specific responses of T cells were determined. Both CD4(+) and CD8(+) LatY136F T cells were unresponsive to L. monocytogenes infection. In contrast, when CD4(+) T cells from wild-type mice were adoptively transferred into LatY136F hosts, they responded normally to L. monocytogenes, indicating that the LatY136F milieu permits T(h)1 responses. Furthermore, we analyzed whether the infection would influence the capacity of LatY136F CD4(+) T cells to produce IL-4 and IFN-gamma. While L. monocytogenes infection results in T(h)1-type T cell responses in wild-type animals, we found that it did not shift the strong T(h)2 polarization of LatY136F T cells towards a T(h)1 pattern. In conclusion, our results suggest that the activation and T(h)2 polarization of the LatY136F CD4(+) T cells is not influenced by infection with an intracellular pathogen known to induce robust T(h)1 responses, and is thus likely driven by T cell intrinsic mechanisms.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Chemical inhibitors when timing is critical: a pharmacological concept for the maturation of T cell contacts.

Cellular signal transduction proceeds through a complex network of molecular interactions and enzymatic activities. The timing of these molecular events is critical for the propagation of a signal and the generation of a specific cellular response. To define the timing of signalling events, we introduce the combination of high-resolution confocal microscopy with the application of small-molecule inhibitors at various stages of signal transduction in T cells. Inhibitors of Src-family tyrosine kinases and actin dynamics were employed to dissect the role of the lymphocyte-specific tyrosine kinase Lck in the formation and maintenance of T cell receptor/CD3-dependent contacts. Anti-CD3epsilon-coated coverslips served as a highly defined stimulus. The kinetics of the recruitment of the yellow fluorescent protein-tagged signalling protein ZAP-70 were detected by high-resolution confocal microscopy. The analysis revealed that at 5 min after receptor engagement, Lck activity was required for maintenance of contacts. In contrast, after 20 min of receptor engagement, the contacts were Lck-independent. The relevance of the timing of inhibitor application provides a pharmacological concept for the maturation of T cell-substrate contacts.

Animals↗
PubMed2005

Rapid in vivo analysis of mutant forms of the LAT adaptor using Pax5-Lat double-deficient pro-B cells.

Following injection into recombinase-activating gene-deficient (Rag1(-/-)) mice, pro-B cells lacking the Pax5 transcription factor (Pax5(-/-)) develop into most major hematopoietic lineages, with the notable exception of B cells. We assessed whether Pax5(-/-) pro-B cells that were also rendered deficient for the linker for activation of T cells (LAT), an adaptor essential for T cell receptor signaling, can be used for the rapid in vivo analysis of mutant forms of LAT. We showed that Pax5(-/-) Lat(-/-) pro-B cell lines can be infected with recombinant retroviruses expressing a LAT cDNA and sorted for the expression of LAT. When injected into Rag1(-/-) mice, they restore normal intrathymic T cell development and give rise to functional peripheral T cells. Considering that the handling of Pax5(-/-) pro-B cell lines is easier than that of bone marrow hematopoietic precursors, we used them for the rapid functional analysis of a novel Lat allelic series. When compared to knock-in and transgenic approaches, a major advantage of our Pax5(-/-) pro-B cell-based experimental approach consists in the production of mice bearing a given mutation within 2-3 months. Therefore, it constitutes a powerful first-line screen for mutations worth fastidious knock-in approaches.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Dynamics and function of Langerhans cells in vivo: dermal dendritic cells colonize lymph node areas distinct from slower migrating Langerhans cells.

Langerhans cells (LCs) are prominent dendritic cells (DCs) in epithelia, but their role in immunity is poorly defined. To track and discriminate LCs from dermal DCs in vivo, we developed knockin mice expressing enhanced green fluorescent protein (EGFP) under the control of the langerin (CD207) gene. By using vital imaging, we showed that most EGFP(+) LCs were sessile under steady-state conditions, whereas skin inflammation induced LC motility and emigration to lymph nodes (LNs). After skin immunization, dermal DCs arrived in LNs first and colonized areas distinct from slower migrating LCs. LCs reaching LNs under steady-state or inflammatory conditions expressed similar levels of costimulatory molecules. Langerin and EGFP were also expressed on thymic DCs and on blood-derived, CD8alpha(+) DCs from all secondary lymphoid organs. By using a similar knockin strategy involving a diphtheria toxin receptor (DTR) fused to EGFP, we demonstrated that LCs were dispensable for triggering hapten-specific T cell effectors through skin immunization.

Animals↗
PubMed2005

Role of the LAT adaptor in T-cell development and Th2 differentiation.

LAT (linker for activation of T cells) is an integral membrane adaptor protein that constitutes in T cells a major substrate of the ZAP-70 protein tyrosine kinase. LAT coordinates the assembly of a multiprotein signaling complex through phosphotyrosine-based motifs present within its intracytoplasmic segment. The resulting "LAT signalosome" links the TCR to the main intracellular signalling pathways that regulate T-cell development and T-cell function. Early studies using transformed T-cell lines suggested that LAT acts primarily as a positive regulator of T-cell receptor (TCR) signalling. The partial or complete inhibition of T-cell development observed in several mouse lines harboring mutant forms of LAT was congruent with that view. More recently, LAT "knock-ins" harboring point mutations in the four COOH-terminal tyrosine residues, were found to develop lymphoproliferative disorders involving polyclonal T cells that produced high amounts of T helper-type 2 (Th2) cytokines. This unexpected finding revealed that LAT also constitutes a negative regulator of TCR signalling and T-cell homeostasis. Although LAT is also expressed in mast cells, natural killer cells, megakaryocytes, platelets, and early B cells, the present review specifically illustrates the role LAT plays in the development and function of mouse T cells. As discussed, the available data underscore that a novel immunopathology proper to defective LAT signalosome is taking shape.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Innate and adaptive immunity: specificities and signaling hierarchies revisited.

The conventional classification of known immune responses by specificity may need re-evaluation. The immune system can be classified into two subsystems: the innate and adaptive immune systems. In general, innate immunity is considered a nonspecific response, whereas the adaptive immune system is thought of as being very specific. In addition, the antigen receptors of the adaptive immune response are commonly viewed as 'master sensors' whose engagement dictates lymphocyte function. Here we propose that these ideas do not genuinely reflect the organization of immune responses and that they bias our view of immunity as well as our teaching of immunology. Indeed, the level of specificity and mode of signaling integration used by the main cellular participants in the adaptive and innate immune systems are more similar than previously appreciated.

Animals↗
PubMed2005

Disruption of the langerin/CD207 gene abolishes Birbeck granules without a marked loss of Langerhans cell function.

Langerin is a C-type lectin expressed by a subset of dendritic leukocytes, the Langerhans cells (LC). Langerin is a cell surface receptor that induces the formation of an LC-specific organelle, the Birbeck granule (BG). We generated a langerin(-/-) mouse on a C57BL/6 background which did not display any macroscopic aberrant development. In the absence of langerin, LC were detected in normal numbers in the epidermis but the cells lacked BG. LC of langerin(-/-) mice did not present other phenotypic alterations compared to wild-type littermates. Functionally, the langerin(-/-) LC were able to capture antigen, to migrate towards skin draining lymph nodes, and to undergo phenotypic maturation. In addition, langerin(-/-) mice were not impaired in their capacity to process native OVA protein for I-A(b)-restricted presentation to CD4(+) T lymphocytes or for H-2K(b)-restricted cross-presentation to CD8(+) T lymphocytes. langerin(-/-) mice inoculated with mannosylated or skin-tropic microorganisms did not display an altered pathogen susceptibility. Finally, chemical mutagenesis resulted in a similar rate of skin tumor development in langerin(-/-) and wild-type mice. Overall, our data indicate that langerin and BG are dispensable for a number of LC functions. The langerin(-/-) C57BL/6 mouse should be a valuable model for further functional exploration of langerin and the role of BG.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2005

Single and combined deletions of the NTAL/LAB and LAT adaptors minimally affect B-cell development and function.

NTAL (non-T-cell activation linker, also called LAB) and LAT (linker for activation of T cells) are evolutionarily related transmembrane adaptor proteins that are phosphorylated upon immunoreceptor engagement. Using quantitative reverse transcription-PCR, both NTAL and LAT were found to be expressed in B cells. However, LAT expression was limited to early B cells, whereas NTAL expression typified mature B cells. To delineate their roles in B-cell development and function, Ntal-deficient mice were generated and crossed with Lat-deficient mice. B cells developed in Lat(-/-) Ntal(-/-) double-deficient mice and in mice lacking either of the two adaptors with the same efficiency as in wild-type mice. Upon B-cell antigen receptor cross-linking, Ntal(-/-) B cells exhibited slightly increased Ca(2+) mobilization and proliferation. In addition, Ntal-deficient mice had increased levels of natural antibodies and slightly increased humoral response to a T-dependent antigen. Normal titers of serum-specific immunoglobulins were produced in response to a T-cell-independent antigen. Although NTAL is also expressed in plasma cells, its absence did not affect the hypergammaglobulinemia E and G1 that developed in mice with a mutation in tyrosine 136 of LAT. Therefore, NTAL does not play a role in B cells symmetric to the role played by LAT in T cells.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Linker for activation of T cells integrates positive and negative signaling in mast cells.

The transmembrane adapter linker for activation of T cells (LAT) is thought to couple immunoreceptors to intracellular signaling pathways. In mice, its intracytoplasmic domain contains nine tyrosines which, when phosphorylated upon receptor aggregation, recruit Src-homology 2 domain-containing cytosolic enzymes and adapters. The four distal tyrosines are critical for both TCR and FcepsilonRI signaling. Unexpectedly, knock-in mice expressing LAT with a point mutation of the first or of the last three of these tyrosines exhibited an abnormal T cell development characterized by a massive expansion of TH2-like alphabeta or gammadelta T cells, respectively. This phenotype suggests that, besides positive signals, LAT might support negative signals that normally regulate terminal T cell differentiation and proliferation. We investigated here whether LAT might similarly regulate mast cell activation, by generating not only positive but also negative signals, following FcR engagement. To this end, we examined IgE- and/or IgG-induced secretory and intracellular responses of mast cells derived from knock-in mice expressing LAT with combinations of tyrosine mutations (Y136F, Y(175, 195, 235)F, or Y(136, 175, 195, 235)F). A systematic comparison of pairs of mutants enabled us to dissect the respective roles played by the five proximal and the four distal tyrosines. We found that LAT tyrosines differentially contribute to exocytosis and cytokine secretion and differentially regulate biological responses of mucosal- and serosal-type mast cells. We also found that, indeed, both positive and negative signals may emanate from distinct tyrosines in LAT, whose integration modulates mast cell secretory responses.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Negative regulation of mast cell signaling and function by the adaptor LAB/NTAL.

Engagement of the Fcepsilon receptor I (FcepsilonRI) on mast cells and basophils initiates signaling pathways leading to degranulation. Early activation events include tyrosine phosphorylation of two transmembrane adaptor proteins, linker for activation of T cells (LAT) and non-T cell activation linker (NTAL; also called LAB; a product of Wbscr5 gene). Previous studies showed that the secretory response was partially inhibited in bone marrow-derived mast cells (BMMCs) from LAT-deficient mice. To clarify the role of NTAL in mast cell degranulation, we compared FcepsilonRI-mediated signaling events in BMMCs from NTAL-deficient and wild-type mice. Although NTAL is structurally similar to LAT, antigen-mediated degranulation responses were unexpectedly increased in NTAL-deficient mast cells. The earliest event affected was enhanced tyrosine phosphorylation of LAT in antigen-activated cells. This was accompanied by enhanced tyrosine phosphorylation and enzymatic activity of phospholipase C gamma1 and phospholipase C gamma2, resulting in elevated levels of inositol 1,4,5-trisphosphate and free intracellular Ca2+. NTAL-deficient BMMCs also exhibited an enhanced activity of phosphatidylinositol 3-OH kinase and Src homology 2 domain-containing protein tyrosine phosphatase-2. Although both LAT and NTAL are considered to be localized in membrane rafts, immunogold electron microscopy on isolated membrane sheets demonstrated their independent clustering. The combined data show that NTAL is functionally and topographically different from LAT.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Chemokine receptor CCR9 contributes to the localization of plasma cells to the small intestine.

Humoral immunity in the gut-associated lymphoid tissue is characterized by the production of immunoglobulin A (IgA) by antibody-secreting plasma cells (PCs) in the lamina propria. The chemokine CCL25 is expressed by intestinal epithelial cells and is capable of inducing chemotaxis of IgA+ PCs in vitro. Using a newly generated monoclonal antibody against murine CCR9, we show that IgA+ PCs express high levels of CCR9 in the mesenteric lymph node (MLN) and Peyer's patches (PPs), but down-regulate CCR9 once they are located in the small intestine. In CCR9-deficient mice, IgA+ PCs are substantially reduced in number in the lamina propria of the small intestine. In adoptive transfer experiments, CCR9-deficient IgA+ PCs show reduced migration into the small intestine compared with wild-type controls. Furthermore, CCR9 mutants fail to mount a regular IgA response to an orally administered antigen, although the architecture and cell type composition of PPs and MLN are unaffected and are functional for the generation of IgA PCs. These findings provide profound in vivo evidence that CCL25/CCR9 guides PCs into the small intestine.

Animals↗
PubMed2003

LAT regulates gammadelta T cell homeostasis and differentiation.

LAT (linker for activation of T cells) is essential for T cell receptor signaling. Mice homozygous for a mutation of the three C-terminal LAT tyrosine residues showed a block in alphabeta T cell development and a partially impaired gammadelta T cell development. Without intentional immunization, they accumulated gammadelta T cells in the spleen and lymph nodes that chronically produced T helper type 2 cytokines in large amounts, and caused the maturation of plasma cells secreting immunoglobulin E (IgE) and IgG1. These effects are very similar to that triggered in the alphabeta lineage by a mutation involving a distinct LAT tyrosine. Thus, LAT is an essential regulator of T cell homeostasis and terminal differentiation.

Adaptor Proteins, Signal Transducing↗