André Veillette

The CD4 and CD8 T cell surface antigens are associated with the internal membrane tyrosine-protein kinase p56lck

The CD4 and CD8 T cell antigens are thought to transduce an independent signal during the process of T cell activation. We report our evaluation of the possible involvement of the lymphocyte-specific tyrosine kinase p56lck in these transduction pathways. Our data demonstrate that p56lck is specifically modulated with either CD4 or CD8 following antibody-mediated cross-linking of these molecules and that a large fraction of the total cellular lck protein can be coimmunoprecipitated with these surface glycoproteins. These results suggest that p56lck is functionally and physically associated with CD4/CD8 in normal murine T lymphocytes and support the concept that an independent signal is transduced by the interaction of these surface molecules with major histocompatibility complex determinants.

The Syk Protein Tyrosine Kinase Is Essential for Fcγ Receptor Signaling in Macrophages and Neutrophils

ABSTRACT The cytoplasmic protein tyrosine kinase Syk has two amino-terminal SH2 domains that engage phosphorylated immunoreceptor tyrosine-based activation motifs in the signaling subunits of immunoreceptors. Syk, in conjunction with Src family kinases, has been implicated in immunoreceptor signaling in both lymphoid and myeloid cells. We have investigated the role of Syk in Fcγ receptor (FcγR)-dependent and -independent responses in bone marrow-derived macrophages and neutrophils by using mouse radiation chimeras reconstituted with fetal liver cells from Syk−/− embryos. Chimeric mice developed an abdominal hemorrhage starting 2 to 3 months after transplantation that was ultimately lethal. Syk-deficient neutrophils derived from the bone marrow were incapable of generating reactive oxygen intermediates in response to FcγR engagement but responded normally to tetradecanoyl phorbol acetate stimulation. Syk-deficient macrophages were defective in phagocytosis induced by FcγR but showed normal phagocytosis in response to complement. The tyrosine phosphorylation of multiple cellular polypeptides, including the FcγR γ chain, as well as Erk2 activation, was compromised inSyk −/− macrophages after FcγR stimulation. In contrast, the induction of nitric oxide synthase in macrophages stimulated with lipopolysaccharide and gamma interferon was not dependent on Syk. Surprisingly, Syk-deficient macrophages were impaired in the ability to survive or proliferate on plastic petri dishes. Taken together, these results suggest that Syk has specific physiological roles in signaling from FcγRs in neutrophils and macrophages and raise the possibility that in vivo, Syk is involved in signaling events other than those mediated by immunoreceptors.

Circulating Precursor CCR7loPD-1hi CXCR5+ CD4+ T Cells Indicate Tfh Cell Activity and Promote Antibody Responses upon Antigen Reexposure

Follicular B helper T (Tfh) cells support high affinity and long-term antibody responses. Here we found that within circulating CXCR5⁺ CD4⁺ T cells in humans and mice, the CCR7(lo)PD-1(hi) subset has a partial Tfh effector phenotype, whereas CCR7(hi)PD-1(lo) cells have a resting phenotype. The circulating CCR7(lo)PD-1(hi) subset was indicative of active Tfh differentiation in lymphoid organs and correlated with clinical indices in autoimmune diseases. Thus the CCR7(lo)PD-1(hi) subset provides a biomarker to monitor protective antibody responses during infection or vaccination and pathogenic antibody responses in autoimmune diseases. Differentiation of both CCR7(hi)PD-1(lo) and CCR7(lo)PD-1(hi) subsets required ICOS and BCL6, but not SAP, suggesting that circulating CXCR5⁺ helper T cells are primarily generated before germinal centers. Upon antigen reencounter, CCR7(lo)PD-1(hi) CXCR5⁺ precursors rapidly differentiate into mature Tfh cells to promote antibody responses. Therefore, circulating CCR7(lo)PD-1(hi) CXCR5⁺ CD4⁺ T cells are generated during active Tfh differentiation and represent a new mechanism of immunological early memory.

Binding of SAP SH2 domain to FynT SH3 domain reveals a novel mechanism of receptor signalling in immune regulation

SAP (or SH2D1A), an adaptor-like molecule expressed in immune cells, is composed almost exclusively of a Src homology 2 (SH2) domain. In humans, SAP is mutated and either absent or non-functional in X-linked lymphoproliferative (XLP) syndrome, a disease characterized by an inappropriate response to Epstein-Barr virus (EBV) infection. Through its SH2 domain, SAP associates with tyrosines in the cytoplasmic domain of the SLAM family of immune cell receptors, and is absolutely required for the function of these receptors. This property results from the ability of SAP to promote the selective recruitment and activation of FynT, a cytoplasmic Src-related protein tyrosine kinase (PTK). Here, we demonstrate that SAP operates in this pathway by binding to the SH3 domain of FynT, through a second region in the SAP SH2 domain distinct from the phosphotyrosine-binding motif. We demonstrate that this interaction is essential for SAP-mediated signalling in T cells, and for the capacity of SAP to modulate immune cell function. These observations characterize a biologically important signalling mechanism in which an adaptor molecule composed only of an SH2 domain links a receptor devoid of intrinsic catalytic activity to the kinase required for its function.

High Expression of Inhibitory Receptor SHPS-1 and Its Association with Protein-tyrosine Phosphatase SHP-1 in Macrophages

SHPS-1 (or SIRP) is a member of the immunoglobulin (Ig) superfamily abundantly expressed in neurons and other cell types. Within its cytoplasmic domain, it possesses at least two immunoreceptor tyrosine-based inhibitory motifs, which are targets for tyrosine phosphorylation and mediate the recruitment of SHP-2, an Src homology 2 (SH2) domain-containing protein-tyrosine phosphatase. Since other immunoreceptor tyrosine-based inhibitory motifs-containing receptors have critical roles in the negative regulation of hemopoietic cell functions, we wanted to examine the expression of SHPS-1 in cells of hematological lineages. By analyzing a panel of hemopoietic cell lines, evidence was provided that SHPS-1 is abundantly expressed in macrophages and, to a lesser extent, in myeloid cells. No expression was detected in T-cell or B-cell lines. Expression of SHPS-1 could also be documented in normal ex vivo peritoneal macrophages. Further studies showed that SHPS-1 was an efficient tyrosine phosphorylation substrate in macrophages. However, unlike in non-hemopoietic cells, tyrosine-phosphorylated SHPS-1 in macrophages associated primarily with SHP-1 and not SHP-2. Finally, our analyses allowed us to identify several isoforms of SHPS-1 in mouse cells. In part, this heterogeneity was due to differential glycosylation of SHPS-1. Additionally, it was caused by the production of at least two distinct shps-1 transcripts, coding for SHPS-1 polypeptides having different numbers of Ig-like domains in the extracellular region. Taken together, these findings indicate that SHPS-1 is likely to play a significant role in macrophages, at least partially as a consequence of its capacity to recruit SHP-1.

Proximal protein tyrosine kinases in immunoreceptor signaling

Immunoreceptor engagement results in the sequential activation of several classes of protein tyrosine kinases, including the Src and Syk/Zap-70 families. Recent progress has been made in our understanding of the regulation and function of these molecules. First, it was revealed that membrane compartmentation of protein tyrosine kinases may be essential for their proper biological function. Second, Src family kinases were found to act not only as positive regulators, but also as inhibitors of cell activation. Third, it was appreciated that Csk, a potent inhibitor of Src kinases, is regulated by an assortment of protein-protein interactions. Fourth, differences in the regulation of Syk and Zap-70 were observed, suggesting significant distinctions in the purpose of these two kinases in immunoreceptor signaling. And fifth, it was suggested that proximal kinases implicated in immunoreceptor-mediated signal transduction may be regulated by protein degradation via binding to c-Cbl, a ubiquitin ligase.

Immune regulation by SLAM family receptors and SAP-related adaptors

The signalling lymphocytic activation molecule (SLAM) family of receptors is expressed by a wide range of immune cells. Through their cytoplasmic domain, SLAM family receptors associate with SLAM-associated protein (SAP)-related molecules, a group of cytoplasmic adaptors composed almost exclusively of an SRC homology 2 domain. SAP, the prototype of the SAP family, is mutated in a human immunodeficiency named X-linked lymphoproliferative (XLP) disease. Recent observations indicate that SLAM family receptors, in association with SAP family adaptors, have crucial roles during normal immune reactions in innate and adaptive immune cells. The latest progress in this field is reviewed here.

Phosphorylation-Dependent Regulation of T-Cell Activation by PAG/Cbp, a Lipid Raft-Associated Transmembrane Adaptor

ABSTRACT PAG/Cbp (hereafter named PAG) is a transmembrane adaptor molecule found in lipid rafts. In resting human T cells, PAG is tyrosine phosphorylated and associated with Csk, an inhibitor of Src-related protein tyrosine kinases. These modifications are rapidly lost in response to T-cell receptor (TCR) stimulation. Overexpression of PAG was reported to inhibit TCR-mediated responses in Jurkat T cells. Herein, we have examined the physiological relevance and the mechanism of PAG-mediated inhibition in T cells. Our studies showed that PAG tyrosine phosphorylation and association with Csk are suppressed in response to activation of normal mouse T cells. By expressing wild-type and phosphorylation-defective (dominant-negative) PAG polypeptides in these cells, we found that the inhibitory effect of PAG is dependent on its capacity to be tyrosine phosphorylated and to associate with Csk. PAG-mediated inhibition was accompanied by a repression of proximal TCR signaling and was rescued by expression of a constitutively activated Src-related kinase, implying that it is due to an inactivation of Src kinases by PAG-associated Csk. We also attempted to identify the protein tyrosine phosphatases (PTPs) responsible for dephosphorylating PAG in T cells. Through cell fractionation studies and analyses of genetically modified mice, we established that PTPs such as PEP and SHP-1 are unlikely to be involved in the dephosphorylation of PAG in T cells. However, the transmembrane PTP CD45 seems to play an important role in this process. Taken together, these data provide firm evidence that PAG is a bona fide negative regulator of T-cell activation as a result of its capacity to recruit Csk. They also suggest that the inhibitory function of PAG in T cells is suppressed by CD45. Lastly, they support the idea that dephosphorylation of proteins on tyrosine residues is critical for the initiation of T-cell activation.

Dok-3, a Novel Adapter Molecule Involved in the Negative Regulation of Immunoreceptor Signaling

ABSTRACT Adapters are typically viewed as molecules coordinating the recruitment of positive effectors of cell signaling. Herein, we report the identification of Dok-3, a novel adapter molecule belonging to the Dok family. Our studies show that Dok-3 is highly expressed in several hemopoietic cell types, including B cells and macrophages. It undergoes rapid tyrosine phosphorylation in response to immunoreceptor-mediated cellular activation, seemingly as a result of the action of Src family kinases. This phosphorylation induces the binding of Dok-3 to at least two inhibitory molecules, the 5′ inositol phosphatase SHIP and the protein tyrosine kinase Csk. We also demonstrate that augmented expression of wild-type Dok-3 in a B-cell line results in an inhibition of immunoreceptor-mediated nuclear factor of activated T-cells (NFAT) activation and cytokine release, while introduction of a Dok-3 mutant with impaired ability to associate with SHIP and Csk enhances B-cell responsiveness. Taken together, these results indicate that Dok-3 is an adapter involved in the recruitment of inhibitory molecules and that it may play a significant role in the negative regulation of immunoreceptor signaling in hemopoietic cells such as B cells and macrophages.

The Carboxyl-terminal Region of Biliary Glycoprotein Controls Its Tyrosine Phosphorylation and Association with Protein-tyrosine Phosphatases SHP-1 and SHP-2 in Epithelial Cells

Biliary glycoprotein (Bgp, C-CAM, or CD66a) is an immunoglobulin-like cell adhesion molecule and functions as a tumor suppressor protein. We have previously shown that the Bgp1 isoform responsible for inhibition of colonic, liver, prostate, and breast tumor cell growth contains within its cytoplasmic domain two tyrosine residues positioned in immunoreceptortyrosine-based inhibition motif (ITIM) consensus sequences. Moreover, we determined that these residues, upon phosphorylation, associate with the protein-tyrosine phosphatase SHP-1. In this report, we have further evaluated the structural bases of the association of Bgp1 with Tyr phosphatases. First, we demonstrate that Bgp1 also associates with the SHP-2 Tyr phosphatase, but not with an unrelated Tyr phosphatase, PTP-PEST. Association of Bgp1 and SHP-2 involves the Tyr residues within the Bgp1 ITIM sequences, Val at position +3 relative to the second Tyr (Tyr-515), and the SHP-2 N-terminal SH2 domain. In addition, our results indicate that residues +4, +5, and +6 relative to Tyr-515 in the Bgp1 cytoplasmic domain play a significant role in these interactions, as their deletion reduced Bgp1 Tyr phosphorylation and association with SHP-1 and SHP-2 by as much as 80%. Together, these results indicate that both SHP-1 and SHP-2 interact with the Bgp1 cytoplasmic domain via ITIM-like sequences. Furthermore, they reveal that the C-terminal amino acids of Bgp1 are critical for these interactions.