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1.
T cell-specific adapter (TSAd) protein and adapter protein in lymphocytes of unknown function (ALX) are two related Src homology 2 (SH2) domain-containing signaling adapter molecules that have both been shown to regulate TCR signal transduction in T cells. TSAd is required for normal TCR-induced synthesis of IL-2 and other cytokines in T cells and acts at least in part by promoting activation of the LCK protein tyrosine kinase at the outset of the TCR signaling cascade. By contrast, ALX functions as a negative-regulator of TCR-induced IL-2 synthesis through as yet undetermined mechanisms. In this study, we report a novel T cell-expressed adapter protein named SH2D4A that contains an SH2 domain that is highly homologous to the TSAd protein and ALX SH2 domains and that shares other structural features with these adapters. To examine the function of SH2D4A in T cells we produced SH2D4A-deficient mice by homologous recombination in embryonic stem cells. T cell development, homeostasis, proliferation, and function were all found to be normal in these mice. Furthermore, knockdown of SH2D4A expression in human T cells did not impact upon their function. We conclude that in contrast to TSAd and ALX proteins, SH2D4A is dispensable for TCR signal transduction in T cells.  相似文献   

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T cell-specific adapter protein (TSAd), encoded by the SH2D2A gene, interacts with Lck through its C terminus and thus modulates Lck activity. Here we mapped Lck phosphorylation and interaction sites on TSAd and evaluated their functional importance. The three C-terminal TSAd tyrosines Tyr(280), Tyr(290), and Tyr(305) were phosphorylated by Lck and functioned as docking sites for the Lck Src homology 2 (SH2) domain. Binding affinities of the TSAd Tyr(P)(280) and Tyr(P)(290) phosphopeptides to the isolated Lck SH2 domain were similar to that observed for the Lck Tyr(P)(505) phosphopeptide, whereas the TSAd Tyr(P)(305) peptide displayed a 10-fold higher affinity. The proline-rich Lck SH3-binding site on TSAd as well as the Lck SH2 domain were required for efficient tyrosine phosphorylation of TSAd by Lck. Interaction sites on TSAd for both Lck SH2 and Lck SH3 were necessary for TSAd-mediated modulation of proximal TCR signaling events. We found that 20-30% of TSAd molecules are phosphorylated in activated T cells and that the proportion of TSAd to Lck molecules in such cells is approximately 1:1. Therefore, in activated T cells, a considerable number of Lck molecules may potentially be engaged by TSAd. In conclusion, Lck binds to TSAd prolines and phosphorylates and interacts with the three C-terminal TSAd tyrosines. We propose that through multivalent interactions with Lck, TSAd diverts Lck from phosphorylating other substrates, thus modulating its functional activity through substrate competition.  相似文献   

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TSAd/Lad is a T cell adaptor molecule involved in p56 lck -mediated T cell activation. To investigate the functions of TSAd in T cells, we generated transgenic (TG) mice expressing the SH2 domain of TSAd (TSAd-SH2) under the control of the p56 lck proximal promoter. In T cells from TSAd-SH2 TG mice, T cell receptor (TCR)-mediated early signaling events, such as Ca2+ flux and ERK activation, were normal; however, late activation events, such as IL-2 production and proliferation, were significantly reduced. Moreover, TCR-induced cell adhesion to extracellular matrix (ECM) proteins and migration through ECM proteins were defective in T cells from TSAd-SH2 TG mice. Furthermore, the contact hypersensitivity (CHS) reaction, an inflammatory response mainly mediated by T helper 1 (Th1) cells, was inhibited in TSAd-SH2 TG mice. Taken together, these results show that TSAd, particularly the SH2 domain of TSAd, is essential for the effector functions of T cells.  相似文献   

6.
Natural killer (NK) cells trigger cytotoxicity and interferon (IFN)‐γ secretion on engagement of the natural‐killer group (NKG)2D receptor or members of the natural cytotoxicity receptor (NCR) family, such as NKp46, by ligands expressed on tumour cells. However, it remains unknown whether T cells can regulate NK cell‐mediated anti‐tumour responses. Here, we investigated the early events occurring during T cell–tumour cell interactions, and their impact on NK cell functions. We observed that on co‐culture with some melanomas, activated CD4+ T cells promoted degranulation, and NKG2D‐ and NKp46‐dependent IFN‐γ secretion by NK cells, probably owing to the capture of NKG2D and NKp46 ligands from the tumour‐cell surface (trogocytosis). This effect was observed in CD4+, CD8+ and resting T cells, which showed substantial amounts of cell surface major histocompatibility complex class I chain‐related protein A on co‐culture with tumour cells. Our findings identify a new, so far, unrecognized mechanism by which effector T cells support NK cell function through the capture of specific tumour ligands with profound implications at the crossroad of innate and adaptive immunity.  相似文献   

7.
X-linked lymphoproliferative syndrome (XLP) is an immunodeficiency characterized by life-threatening infectious mononucleosis and EBV-induced B cell lymphoma. The gene mutated in XLP encodes SLAM (signaling lymphocytic activation molecule-associated protein)-associated protein (SAP), a small SH2 domain-containing protein. SAP associates with 2B4 and SLAM, activating receptors expressed by NK and T cells, and prevents recruitment of SH2 domain-containing protein tyrosine phosphatase-2 SHP-2) to the cytoplasmic domains of these receptors. The phenotype of XLP may therefore result from perturbed signaling through SAP-associating receptors. We have addressed the functional consequence of SAP deficiency on 2B4-mediated NK cell activation. Ligating 2B4 on normal human NK cells with anti-2B4 mAb or interaction with transfectants bearing the 2B4 ligand CD48 induced NK cell cytotoxicity. In contrast, ligation of 2B4 on NK cells from a SAP-deficient XLP patient failed to initiate cytotoxicity. Despite this, CD2 or CD16-induced cytotoxicity of SAP-deficient NK cells was similar to that of normal NK cells. Thus, selective impairment of 2B4-mediated NK cell activation may contribute to the immunopathology of XLP.  相似文献   

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We previously reported the isolation of a cDNA encoding a T cell-specific adapter protein (TSAd). Its amino acid sequence contains an SH2 domain, tyrosines in protein binding motifs, and proline-rich regions. In this report we show that expression of TSAd is induced in normal peripheral blood T cells stimulated with anti-CD3 mAbs or anti-CD3 plus anti-CD28 mAbs. Overexpression of TSAd in Jurkat T cells interfered with TCR-mediated signaling by down-modulating anti-CD3/PMA-induced IL-2 promoter activity and anti-CD3 induced Ca2+ mobilization. The TCR-induced tyrosine phosphorylation of phospholipase C-gamma1, SH2-domain-containing leukocyte-specific phosphoprotein of 76kDa, and linker for activation of T cells was also reduced. Furthermore, TSAd inhibited Zap-70 recruitment to the CD3zeta-chains in a dose-dependent manner. Consistent with this, Lck kinase activity was reduced 3- to 4-fold in COS-7 cells transfected with both TSAd and Lck, indicating a regulatory effect of TSAd on Lck. In conclusion, our data strongly suggest an inhibitory role for TSAd in proximal T cell activation.  相似文献   

10.
We demonstrate that IL-2-activated NK cells or lymphokine-activated killer cells recognize and kill syngeneic CD4(+) and CD8(+) T cells that have been activated by APCs. Induction with APC required TCR-specific Ag, and lysis was perforin mediated. Brefeldin A, which disrupts protein transport, inhibited the sensitivity induced by activation. In BALB/c, expression of NKG2D ligands correlated with lysis and could be inhibited by brefeldin A. As well, addition of anti-NKG2D mAb to a killing assay completely abrogated lysis. Transduction of mouse NKG2D into a human NK cell line, YTSeco, conferred upon it the ability to kill activated BALB/c T cells, indicating that NKG2D is necessary for recognition. Our data provide a basis for studying a role for NK cells in T cell regulation.  相似文献   

11.

Background

The chemokine CXCL12/SDF-1α interacts with its G-protein coupled receptor CXCR4 to induce migration of lymphoid and endothelial cells. T cell specific adapter protein (TSAd) has been found to promote migration of Jurkat T cells through interaction with the G protein β subunit. However, the molecular mechanisms for how TSAd influences cellular migration have not been characterized in detail.

Principal Findings

We show that TSAd is required for tyrosine phosphorylation of the Lck substrate IL2-inducible T cell kinase (Itk). Presence of Itk Y511 was necessary to boost TSAd''s effect on CXCL12 induced migration of Jurkat T cells. In addition, TSAd''s ability to promote CXCL12-induced actin polymerization and migration of Jurkat T lymphocytes was dependent on the Itk-interaction site in the proline-rich region of TSAd. Furthermore, TSAd-deficient murine thymocytes failed to respond to CXCL12 with increased Itk phosphorylation, and displayed reduced actin polymerization and cell migration responses.

Conclusion

We propose that TSAd, through its interaction with both Itk and Lck, primes Itk for Lck mediated phosphorylation and thereby regulates CXCL12 induced T cell migration and actin cytoskeleton rearrangements.  相似文献   

12.
NKG2D is an activating immunoreceptor, first recognized on NK cells but subsequently found on γδ T cells, CD8+ αβ T cells and macrophages. In NK cells, inhibitory signals are generally dominate over activating signals. However, activating signals mediated through engagement of NKG2D by its ligands on target cells can bypass signals transmitted through inhibitory NK receptors, allowing NKG2D to function as a “master-switch” in determining the activation status of NK cells. NKG2D is important for T cell and NK cell-mediated immunity to viruses and tumours, and has roles in autoimmune disease, allogeneic transplantation, and xenotransplantation. Depending upon the situation, development of strategies to either block or to enhance the interactions between NKG2D and its ligands may have important implications for human health and disease.  相似文献   

13.
Nielsen N  Ødum N  Ursø B  Lanier LL  Spee P 《PloS one》2012,7(2):e31959
In mouse models of chronic inflammatory diseases, Natural Killer (NK) cells can play an immunoregulatory role by eliminating chronically activated leukocytes. Indirect evidence suggests that NK cells may also be immunoregulatory in humans. Two subsets of human NK cells can be phenotypically distinguished as CD16(+)CD56(dim) and CD16(dim/-)CD56(bright). An expansion in the CD56(bright) NK cell subset has been associated with clinical responses to therapy in various autoimmune diseases, suggesting an immunoregulatory role for this subset in vivo. Here we compared the regulation of activated human CD4(+) T cells by CD56(dim) and CD56(bright) autologous NK cells in vitro. Both subsets efficiently killed activated, but not resting, CD4(+) T cells. The activating receptor NKG2D, as well as the integrin LFA-1 and the TRAIL pathway, played important roles in this process. Degranulation by NK cells towards activated CD4(+) T cells was enhanced by IL-2, IL-15, IL-12+IL-18 and IFN-α. Interestingly, IL-7 and IL-21 stimulated degranulation by CD56(bright) NK cells but not by CD56(dim) NK cells. NK cell killing of activated CD4(+) T cells was suppressed by HLA-E on CD4(+) T cells, as blocking the interaction between HLA-E and the inhibitory CD94/NKG2A NK cell receptor enhanced NK cell degranulation. This study provides new insight into CD56(dim) and CD56(bright) NK cell-mediated elimination of activated autologous CD4(+) T cells, which potentially may provide an opportunity for therapeutic treatment of chronic inflammation.  相似文献   

14.
Both human and mouse cytomegaloviruses (CMVs) encode proteins that inhibit the activation of NK cells by down-regulating cellular ligands for the activating NK cell receptor NKG2D. Up to now, three ligands for the NKG2D receptor, named RAE-1, H60, and MULT-1, have been identified in mice. The resistance of mouse strains to murine CMV (MCMV) infection is determined by their ability to generate an effective NK cell response. The MCMV gene m152, a member of the m145 gene family, down-regulates the expression of RAE-1 in order to avoid NK cell control in vivo. Here we report that the m155 gene, another member of the m145 gene family, encodes a protein that interferes with the expression of H60 on the surfaces of infected cells. Deletion of the m155 gene leads to an only partial restoration of H60 expression on the cell surface, suggesting the involvement of another, so far unknown, viral inhibitor. In spite of this, an m155 deletion mutant virus shows NK cell-dependent attenuation in vivo. The acquisition of endo-beta-N-acetylglucosaminidase H resistance and the preserved half-life of H60 in MCMV-infected cells indicate that the m155-mediated effect must take place in a compartment after H60 exits from the ERGIC-cis-Golgi compartment.  相似文献   

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Natural killer (NK) cells express an activating receptor, 2B4, that enhances cellular cytotoxicity. Upon NK cell activation by ligation of 2B4, the intracellular domain of 2B4 associates with the X-linked lymphoproliferative disease (XLP) gene product, signaling lymphocytic activation molecule-associated protein/SH2D1A (SAP/SH2D1A). Defective intracellular association of 2B4 with mutated SAP/SH2D1A is likely to underlie the defects in cytotoxicity observed in NK cells from patients with XLP. We report here a role for phosphoinositide 3-kinase (PI3K) in the recruitment and association of SAP/SH2D1A to 2B4 in human NK cells. The activation of normal NK cells by ligation of 2B4 leads to the phosphorylation of 2B4, recruitment of SAP/SH2D1A, and association of the p85 regulatory subunit of PI3K. The inhibition of PI3K enzymatic activity with either wortmannin or LY294002 prior to 2B4 ligation does not alter the association of 2B4 with the p85 subunit but prevents the recruitment of SAP/SH2D1A to 2B4. In addition, PI3K inhibitors significantly diminish the cytotoxic function of primary NK cells. This observed inhibition of cytotoxicity, present in normal NK cells, was less apparent or absent in NK cells derived from a patient with XLP. These data indicate that the cytotoxicity of activated NK cells is mediated by the association of 2B4 and SAP/SH2D1A, and that this association is dependent upon the activity of PI3K.  相似文献   

17.

Background

T cell specific adapter protein (TSAd), encoded by the SH2D2A gene, modulates signaling downstream of the T cell receptor (TCR). Young, unchallenged SH2D2A-deficient C57BL/6 mice exhibit a relatively normal immune phenotype. To address whether SH2D2A regulates physiologic immune responses, SH2D2A-deficient TCR-transgenic BALB/c mice were generated. The transgenic TCR recognizes a myeloma-derived idiotypic (Id) peptide in the context of the major histocompatibility complex (MHC) class II molecule I-Ed, and confers T cell mediated resistance to transplanted multiple myeloma development in vivo.

Principal Findings

The immune phenotype of SH2D2A-deficient C57BL/6 and BALB/c mice did not reveal major differences compared to the corresponding wild type mice. When challenged with myeloma cells, Id-specific TCR-transgenic BALB/c mice lacking SH2D2A displayed increased resistance towards tumor development. Tumor free TCR-transgenic SH2D2A-deficient mice had higher numbers of Id-specific single positive CD4+ thymocytes compared to TCR-transgenic wild-type mice.

Conclusion

Our results suggest a modulatory role for SH2D2A in T cell mediated immune surveillance of cancer. However, it remains to be established whether its effect is T-cell intrinsic. Further studies are required to determine whether targeting SH2D2A function in T cells may be a potential adjuvant in cancer immunotherapy.  相似文献   

18.
Activation of V gamma 9V delta 2 T cells by NKG2D   总被引:5,自引:0,他引:5  
Human Vgamma9 Vdelta2 T cells recognize phosphorylated nonpeptide Ags (so called phosphoantigens), certain tumor cells, and cells treated with aminobisphosphonates. NKG2D, an activating receptor for NK cells, has been described as a potent costimulatory receptor in the Ag-specific activation of gammadelta and CD8 T cells. This study provides evidence that Vgamma9 Vdelta2 T cells may also be directly activated by NKG2D. Culture of PBMC with immobilized NKG2D-specific mAb or NKG2D ligand MHC class I related protein A (MICA) induces the up-regulation of CD69 and CD25 in NK and Vgamma9 Vdelta2 but not in CD8 T cells. Furthermore, NKG2D triggers the production of TNF-alpha but not of IFN-gamma, as well as the release of cytolytic granules by Vgamma9 Vdelta2 T cells. Purified Vgamma9 Vdelta2 T cells kill MICA-transfected RMA mouse cells but not control cells. Finally, DAP10, which mediates NKG2D signaling in human NK cells, was detected in resting and activated Vgamma9 Vdelta2 T cells. These remarkable similarities in NKG2D function in NK and Vgamma9 Vdelta2 T cells may open new perspectives for Vgamma9 Vdelta2 T cell-based immunotherapy, e.g., by Ag-independent killing of NKG2D ligand-expressing tumors.  相似文献   

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Regulatory NK cell receptors can contribute to antigen-specific adaptive immune responses by modulating T cell receptor (TCR)-induced T cell activation. We investigated the potential of the NK cell receptor 2B4 (CD244) to enhance tumor antigen-induced activation of human T cells. 2B4 is a member of the CD2 receptor subfamily with both activating and inhibitory functions in NK cells. In T cells, its expression is positively associated with the acquisition of a cytolytic effector memory phenotype. Recombinant chimeric receptors that link extracellular single-chain Fv fragments specific for the tumor-associated surface antigens CD19 and GD2 to the signaling domains of human 2B4 and/or TCRζ were expressed in non-specifically activated peripheral blood T cells by retroviral gene transfer. While 2B4 signaling alone failed to induce T cell effector functions or proliferation, it significantly augmented the antigen-specific activation responses induced by TCRζ. 2B4 costimulation did not affect the predominant effector memory phenotype of expanding T cells, nor did it increase the proportion of T cells with regulatory phenotype (CD4+CD25hiFoxP3+). These data support a costimulatory role for 2B4 in human T cell subpopulations. As an amplifier of TCR-mediated signals, 2B4 may provide a powerful new tool for immunotherapy of cancer, promoting sustained activation and proliferation of gene-modified antitumor T cells.  相似文献   

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