Exclusion of lipid rafts and decreased mobility of CD94/NKG2A receptors at the inhibitory NK cell synapse

CD94/NKG2A is an inhibitory receptor expressed by most human natural killer (NK) cells and a subset of T cells that recognizes human leukocyte antigen E (HLA-E) on potential target cells. To elucidate the cell surface dynamics of CD94/NKG2A receptors, we have expressed CD94/NKG2A-EGFP receptors in the rat basophilic leukemia (RBL) cell line. Photobleaching experiments revealed that CD94/NKG2A-EGFP receptors move freely within the plasma membrane and accumulate at the site of contact with ligand. The enriched CD94/NKG2A-EGFP is markedly less mobile than the nonligated receptor. We observed that not only are lipid rafts not required for receptor polarization, they are excluded from the site of receptor contact with the ligand. Furthermore, the lipid raft patches normally observed at the sites where FcepsilonR1 activation receptors are cross-linked were not observed when CD94/NKG2A was coengaged along with the activation receptor. These results suggest that immobilization of the CD94/NKG2A receptors at ligation sites not only promote sustenance of the inhibitory signal, but by lipid rafts exclusion prevent formation of activation signaling complexes.     

CD94/NKG2-A inhibitory complex blocks CD16-triggered Syk and extracellular regulated kinase activation, leading to cytotoxic function of human NK cells.

The CD94/NKG2-A complex is the inhibitory receptor for the nonclassical MHC class I molecule HLA-E on human NK cells. Here we studied the molecular mechanisms underlying the inhibitory activity of CD94/NKG2-A on NK cell functions by analyzing its interference on CD16-initiated signaling pathways involved in the control of cytolytic activity. Both tyrosine phosphorylation and activation of Syk kinase together with tyrosine phosphorylation of CD16 receptor zeta subunit are markedly inhibited by the coengagement of CD94/NKG2-A complex. As a downstream consequence, CD94/NKG2-A cross-linking impairs the CD16-induced activation of extracellular regulated kinases (ERKs), a pathway involved in NK cytotoxic function. The block of ERK activation is exerted at an early, PTK-dependent stage in the events leading to p21ras activation, as the CD16-induced tyrosine phosphorylation of Shc adaptor protein and the formation of Shc/Grb-2 complex are abrogated by CD94/NKG2-A simultaneous engagement. Our observations indicate that CD94/NKG2-A inhibits the CD16-triggered activation of two signaling pathways involved in the cytotoxic activity of NK cells. They thus provide molecular evidence to explain the inhibitory function of CD94/NKG2-A receptor on NK effector functions.     

Vav1 controls DAP10-mediated natural cytotoxicity by regulating actin and microtubule dynamics

The NK cell-activating receptor NKG2D recognizes several MHC class I-related molecules expressed on virally infected and tumor cells. Human NKG2D transduces activation signals exclusively via an associated DAP10 adaptor containing a YxNM motif, whereas murine NKG2D can signal through either DAP10 or the DAP12 adaptor, which contains an ITAM sequence. DAP10 signaling is thought to be mediated, at least in part, by PI3K and is independent of Syk/Zap-70 kinases; however, the exact mechanism by which DAP10 induces natural cytotoxicity is incompletely understood. Herein, we identify Vav1, a Rho GTPase guanine nucleotide exchange factor, as a critical signaling mediator downstream of DAP10 in NK cells. Specifically, using mice deficient in Vav1 and DAP12, we demonstrate an essential role for Vav1 in DAP10-induced NK cell cytoskeletal polarization involving both actin and microtubule networks, maturation of the cytolytic synapse, and target cell lysis. Mechanistically, we show that Vav1 interacts with DAP10 YxNM motifs through the adaptor protein Grb2 and is required for activation of PI3K-dependent Akt signaling. Based on these findings, we propose a novel model of ITAM-independent signaling by Vav downstream of DAP10 in NK cells.     

Inhibitory receptor signals suppress ligation-induced recruitment of NKG2D to GM1-rich membrane domains at the human NK cell immune synapse

NKG2D is an activating receptor expressed on all human NK cells and a subset of T cells. In cytolytic conjugates between NK cells and target cells expressing its ligand MHC class I chain-related gene A, NKG2D accumulates at the immunological synapse with GM1-rich microdomains. Furthermore, NKG2D is specifically recruited to detergent-resistant membrane fractions upon ligation. However, in the presence of a strong inhibitory stimulus, NKG2D-mediated cytotoxicity can be intercepted, and recruitment of NKG2D to the immunological synapse and detergent-resistant membrane fractions is blocked. Also, downstream phosphorylation of Vav-1 triggered by NKG2D ligation is circumvented by coengaging inhibitory receptors. Thus, we propose that one way in which inhibitory signaling can control NKG2D-mediated activation is by blocking its recruitment to GM1-rich membrane domains. The accumulation of activating NK cell receptors in GM1-rich microdomains may provide the necessary platform from which stimulatory signals can proceed.     

B cell lipid rafts regulate both peptide-dependent and peptide-independent APC-T cell interaction

Formation of an immunological synapse (IS) between APCs and T CD4(+) lymphocytes is a key event in the initiation and the termination of the cognate immune response. We have analyzed the contribution of the APC to IS formation and report the implication of the actin cytoskeleton, the signaling proteins and the lipid rafts of B lymphocytes. Recruitment of MHC class II molecules to the IS is concomitant with actin cytoskeleton-dependent B cell raft recruitment. B cell actin cytoskeleton disruption abrogates both IS formation and T cell activation, whereas protein kinase C inhibition only impairs T cell activation. Pharmacological B cell lipid raft disruption inhibited peptide-dependent T lymphocyte activation and induced peptide-independent but HLA-DR-restricted APC-T cell conjugate formation. Such peptide-independent conjugates did not retain the ability to activate T cells. Thus, B cell lipid rafts are bifunctional by regulating T cell activation and imposing peptide stringency.     

Vav1/Rac-dependent actin cytoskeleton reorganization is required for lipid raft clustering in T cells.

Formation of the immunological synapse (IS) in T cells involves large scale molecular movements that are mediated, at least in part, by reorganization of the actin cytoskeleton. Various signaling proteins accumulate at the IS and are localized in specialized membrane microdomains, known as lipid rafts. We have shown previously that lipid rafts cluster and localize at the IS in antigen-stimulated T cells. Here, we provide evidence that lipid raft polarization to the IS depends on an intracellular pathway that involves Vav1, Rac, and actin cytoskeleton reorganization. Thus, lipid rafts did not translocate to the IS in Vav1-deficient (Vav1-/-) T cells upon antigen stimulation. Similarly, T cell receptor transgenic Jurkat T cells also failed to translocate lipid rafts to the IS when transfected with dominant negative Vav1 mutants. Raft polarization induced by membrane-bound cholera toxin cross-linking was also abolished in Jurkat T cells expressing dominant negative Vav1 or Rac mutants and in cells treated with inhibitors of actin polymerization. However, Vav overexpression that induced F-actin polymerization failed to induce lipid rafts clustering. Therefore, Vav is necessary, but not sufficient, to regulate lipid rafts clustering and polarization at the IS, suggesting that additional signals are required.     

Remodelling of cortical actin where lytic granules dock at natural killer cell immune synapses revealed by super-resolution microscopy

Natural Killer (NK) cells are innate immune cells that secrete lytic granules to directly kill virus-infected or transformed cells across an immune synapse. However, a major gap in understanding this process is in establishing how lytic granules pass through the mesh of cortical actin known to underlie the NK cell membrane. Research has been hampered by the resolution of conventional light microscopy, which is too low to resolve cortical actin during lytic granule secretion. Here we use two high-resolution imaging techniques to probe the synaptic organisation of NK cell receptors and filamentous (F)-actin. A combination of optical tweezers and live cell confocal microscopy reveals that microclusters of NKG2D assemble into a ring-shaped structure at the centre of intercellular synapses, where Vav1 and Grb2 also accumulate. Within this ring-shaped organisation of NK cell proteins, lytic granules accumulate for secretion. Using 3D-structured illumination microscopy (3D-SIM) to gain super-resolution of ~100 nm, cortical actin was detected in a central region of the NK cell synapse irrespective of whether activating or inhibitory signals dominate. Strikingly, the periodicity of the cortical actin mesh increased in specific domains at the synapse when the NK cell was activated. Two-colour super-resolution imaging revealed that lytic granules docked precisely in these domains which were also proximal to where the microtubule-organising centre (MTOC) polarised. Together, these data demonstrate that remodelling of the cortical actin mesh occurs at the central region of the cytolytic NK cell immune synapse. This is likely to occur for other types of cell secretion and also emphasises the importance of emerging super-resolution imaging technology for revealing new biology.     

CD28 and lipid rafts coordinate recruitment of Lck to the immunological synapse of human T lymphocytes

In T lymphocytes, the Src family kinase Lck associates lipid rafts and accumulates at the immunological synapse (IS) during T cell stimulation by APCs. Using CD4- or CD28-deficient murine T cells, it was suggested that recruitment of Lck to the IS depends on CD4, whereas CD28 sustains Lck activation. However, in human resting T cells, CD28 is responsible for promoting recruitment of lipid rafts to the IS by an unknown mechanism. Thus, we performed a series of experiments to determine 1) whether Lck is recruited to the IS through lipid rafts; and 2) whether Lck recruitment to the IS of human resting T cells depends on CD4 or on CD28 engagement. We found that CD28, but not CD4, stimulation induced recruitment of Lck into detergent-resistant domains as well as its accumulation at the IS. We also found that Lck recruitment to the IS depends on the CD28 COOH-terminal PxxPP motif. Thus, the CD28-3A mutant, generated by substituting the prolines in positions 208, 211, and 212 with alanines, failed to induce Lck and lipid raft accumulation at the synapse. These results indicate that CD28 signaling orchestrates both Lck and lipid raft recruitment to the IS to amplify T cell activation.     

NKG2D and its ligands

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.     

HLA-E and HLA-G expression on porcine endothelial cells inhibit xenoreactive human NK cells through CD94/NKG2-dependent and -independent pathways

Human NK cells contribute a significant role to host defense as well as xenogeneic cytotoxicity. Previous studies using human 721.221 cell line have shown that peptides derived from the leader sequence of the HLA-G binds and up-regulates the surface expression of HLA-E molecules, which was considered to consequently provide negative signals to human NK cells. However, the direct role of HLA-G in inhibiting human NK cells remains controversial. In this study, we showed that the expression of HLA-G or HLA-E in porcine endothelial cells directly protected sensitive porcine cells from human NK cell-mediated xenogeneic cytotoxicity. Ab blocking assays using F(ab')2 of the HLA class I-specific mAb PA2.6 indicated that the protection was directly mediated by the expression of HLA-G and HLA-E on the porcine cells. The HLA-E-mediated protection was blocked by anti-human CD94 Ab. In addition, the engagement of HLA-E lead to the phosphorylation of the CD94/NKG2 complex and the recruitment of SH2 domain-containing protein phosphatase 1 (SHP-1) to the complex. Therefore, HLA-E protected porcine cells from xenoreactive human NK cells through a CD94/NKG2-dependent pathway. In contrast, HLA-G inhibited human NK cells in the absence of CD94/NKG2 phosphorylation or SHP-1 recruitment, and the inhibition was not blocked by anti-CD94 Ab. Therefore, HLA-G protected porcine cells from human NK cells through a CD94/NKG2-independent pathway. These results demonstrated that both HLA-E and HLA-G could directly inhibit human NK cells in the absence of other endogenous HLA class I molecules. These results also have practical implications in preventing xenograft rejection mediated by human NK cells.     

Kinetics and peptide dependency of the binding of the inhibitory NK receptor CD94/NKG2-A and the activating receptor CD94/NKG2-C to HLA-E.

The lytic function of human natural killer (NK) cells is markedly influenced by recognition of class I major histocompatibility complex (MHC) molecules, a process mediated by several types of activating and inhibitory receptors expressed on the NK cell. One of the most important of these mechanisms of regulation is the recognition of the non-classical class I MHC molecule HLA-E, in complex with nonamer peptides derived from the signal sequences of certain class I MHC molecules, by heterodimers of the C-type lectin-like proteins CD94 and NKG2. Using soluble, recombinant HLA-E molecules assembled with peptides derived from different leader sequences and soluble CD94/NKG2-A and CD94/NKG2-C proteins, the binding of these receptor-ligand pairs has been analysed. We show first that these interactions have very fast association and dissociation rate constants, secondly, that the inhibitory CD94/NKG2-A receptor has a higher binding affinity for HLA-E than the activating CD94/NKG2-C receptor and, finally, that recognition of HLA-E by both CD94/NKG2-A and CD94/NKG2-C is peptide dependent. There appears to be a strong, direct correlation between the binding affinity of the peptide-HLA-E complexes for the CD94/NKG2 receptors and the triggering of a response by the NK cell. These data may help to understand the balance of signals that control cytotoxicity by NK cells.     

Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa and phospholipase C gamma 1 are required for NF-kappa B activation and lipid raft recruitment of protein kinase C theta induced by T cell costimulation

The NF-kappaB activation pathway induced by T cell costimulation uses various molecules including Vav1 and protein kinase C (PKC)theta. Because Vav1 inducibly associates with further proteins including phospholipase C (PLC)gamma1 and Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76), we investigated their role for NF-kappaB activation in Jurkat leukemia T cell lines deficient for expression of these two proteins. Cells lacking SLP-76 or PLCgamma1 failed to activate NF-kappaB in response to T cell costimulation. In contrast, replenishment of SLP-76 or PLCgamma1 expression restored CD3/CD28-induced IkappaB kinase (IKK) activity as well as NF-kappaB DNA binding and transactivation. PKCtheta activated NF-kappaB in SLP-76- and PLCgamma1-deficient cells, showing that PKCtheta is acting further downstream. In contrast, Vav1-induced NF-kappaB activation was normal in SLP-76(-) cells, but absent in PLCgamma1(-) cells. CD3/CD28-stimulated recruitment of PKCtheta and IKKgamma to lipid rafts was lost in SLP-76- or PLCgamma1-negative cells, while translocation of Vav1 remained unaffected. Accordingly, recruitment of PKCtheta to the immunological synapse strictly relied on the presence of SLP-76 and PLCgamma1, but synapse translocation of Vav1 identified in this study was independent from both proteins. These results show the importance of SLP-76 and PLCgamma1 for NF-kappaB activation and raft translocation of PKCtheta and IKKgamma.     

Phosphatidylinositol 3-kinase activation is required to form the NKG2D immunological synapse

The receptor NKG2D allows natural killer (NK) cells to detect virally infected, stressed, and tumor cells. In human cells, NKG2D signaling is mediated through the associated DAP10 adapter. Here we show that engagement of NKG2D by itself is sufficient to stimulate the formation of the NK immunological synapse (NKIS), with recruitment of NKG2D to the center synapse. Mutagenesis studies of DAP10 revealed that the phosphatidylinositol 3-kinase binding site, but not the Grb2 binding site, was required and sufficient for recruitment of DAP10 to the NKIS. Surprisingly, we found that in the absence of the Grb2 binding site, Grb2 was still recruited to the NKIS. Since the recruitment of Grb2 was dependent on phosphatidylinositol-(3,4,5)-trisphosphate (PIP₃), we explored the possibility that recruitment to the NKIS is mediated by a pleckstrin homology (PH) domain-containing binding partner for Grb2. We found that the PH domain of SOS1, but not that of Vav1, was able to be recruited by PIP₃. These results provide new insights into the mechanism of immunological synapse formation and also demonstrate how multiple mechanisms can be used to recruit the same signaling proteins to the plasma membrane.     

Vav1 dephosphorylation by the tyrosine phosphatase SHP-1 as a mechanism for inhibition of cellular cytotoxicity

Here, we present data suggesting a novel mechanism for regulation of natural killer (NK) cell cytotoxicity through inhibitory receptors. Interaction of activation receptors with their ligands on target cells induces cytotoxicity by NK cells. This activation is under negative control by inhibitory receptors that recruit tyrosine phosphatase SHP-1 upon binding major histocompatibility class I on target cells. How SHP-1 blocks the activation pathway is not known. To identify SHP-1 substrates, an HLA-C-specific inhibitory receptor fused to a substrate-trapping mutant of SHP-1 was expressed in NK cells. Phosphorylated Vav1, a regulator of actin cytoskeleton, was the only protein detectably associated with the catalytic site of SHP-1 during NK cell contact with target cells expressing HLA-C. Vav1 trapping was independent of actin polymerization, suggesting that inhibition of cellular cytotoxicity occurs through an early dephosphorylation of Vav1 by SHP-1, which blocks actin-dependent activation signals. Such a mechanism explains how inhibitory receptors can block activating signals induced by different receptors.     

Ligand binding to inhibitory killer cell Ig-like receptors induce colocalization with Src homology domain 2-containing protein tyrosine phosphatase 1 and interruption of ongoing activation signals

Interaction of NK cells with target cells leads to formation of an immunological synapse (IS) at the contact site. NK cells form two distinctly different IS, the inhibitory NK cell IS (NKIS) and the cytolytic NKIS. Cognate ligand binding is sufficient to induce clustering of inhibitory killer cell Ig-like receptors (KIR) and phosphorylation of both the receptor and the phosphatase Src homology domain 2-containing protein tyrosine phosphatase 1 (SHP-1). Recruitment and activation of SHP-1 by a signaling competent inhibitory receptor are essential early events for NK cell inhibition. We have in the present study used three-dimensional immunofluorescence microscopy to analyze distribution of inhibitory KIR, SHP-1, LFA-1, and lipid rafts within the NKIS during cytolytic and noncytolytic interactions. NK clones retrovirally transduced with the inhibitory KIR2DL3 gene fused to GFP demonstrate colocalization of KIR2DL3 with SHP-1 in the center of early inhibitory NKIS. Ligand binding translocates the receptor to the center of the IS where activation signals are accumulating and provides a docking site for SHP-1. SHP-1 and rafts cluster in the center of early inhibitory NKIS and late cytolytic NKIS, and whereas rafts continue to increase in size in cytolytic conjugates, they are rapidly dissolved in inhibitory conjugates. Furthermore, rafts are essential only for cytolytic, not for inhibitory, outcome. These results indicate that the outcome of NK cell-target cell interactions is dictated by early quantitative differences in cumulative activating and inhibitory signals.     

Qa-1(b)-dependent modulation of dendritic cell and NK cell cross-talk in vivo

Dendritic cells (DC) trigger activation and IFN-gamma release by NK cells in lymphoid tissues, a process important for the polarization of Th1 responses. Little is known about the molecular signals that regulate DC-induced NK cell IFN-gamma synthesis. In this study, we analyzed whether the interaction between Qa-1(b) expressed on DC and its CD94/NKG2A receptor on NK cells affects this process. Activation of DC using CpG-oligodeoxynucleotides in Qa-1(b)-deficient mice, or transfer of CpG-oligodeoxynucleotide-activated Qa-1(b)-deficient DC into wild-type mice, resulted in dramatically increased IFN-gamma production by NK cells, as compared with that induced by Qa-1(b)-expressing DC. Masking the CD94/NKG2A inhibitory receptor on NK cells in wild-type mice similarly enhanced the IFN-gamma response of these cells to Qa-1(b)-expressing DC. Furthermore, NK cells from CD94/NKG2A-deficient mice displayed higher IFN-gamma production upon DC stimulation. These results demonstrate that Qa-1(b) is critically involved in regulating IFN-gamma synthesis by NK cells in vivo through its interaction with CD94/NKG2A inhibitory receptors. This receptor-ligand interaction may be essential to prevent unabated cytokine production by NK cells during an inflammatory response.     

Molecular determinants regulating the pairing of NKG2 molecules with CD94 for cell surface heterodimer expression

The lytic capacity of a NK cell is regulated, in part, by the balance in cell surface expression between inhibitory CD94/NKG2A and activating CD94/NKG2C heterodimers. We demonstrate that, in the absence of DAP12, rhesus monkey NKG2A is preferentially expressed at the cell surface with CD94 due to a single amino acid difference in the transmembrane of NKG2A and NKG2C. Furthermore, in the context of an NKG2A transmembrane, the stalk domain of NKG2C was found to enhance heterodimer formation with CD94 compared with the stalk domain of NKG2A. In the presence of DAP12, the ability of NKG2C to compete for cell surface CD94 heterodimerization is enhanced and approaches that of NKG2A. Finally, allelic differences that affect the ability of rhesus NKG2A to reach the cell surface with CD94 could also be mapped to the transmembrane. These differences in the ability of inhibitory and activating NKG2 molecules to reach the cell surface provide a mechanism for the regulation of NK cell activity.     

The CD43 coreceptor molecule recruits the zeta-chain as part of its signaling pathway

CD43 is an abundant cell surface sialoglycoprotein implicated in hemopoietic cell adhesion and activation. Cell stimulation through CD43 results in recruitment of different signaling proteins, including members of the Src family kinases, Syk, phospholipase Cgamma2, the adapter protein Shc, the guanine nucleotide exchange factor Vav, and activation of protein kinase C. In this study, we report that in human T lymphocytes, the zeta-chain is part of the CD43 signaling pathway. Upon CD43 engagement, the zeta-chain was tyrosine-phosphorylated, generating docking sites for tyrosine-phosphorylated zeta-associated protein of 70 kDa and Vav. In vitro kinase assays suggested that zeta-associated protein of 70 kDa could account for the kinase activity associated with the zeta-chain following CD43 engagement. Cross-linking CD43 on the surface of the Lck-deficient JCaM.1 cells failed to phosphorylate the zeta-chain and associated proteins, suggesting that Lck is a key element in the CD43 signaling pathway leading to zeta phosphorylation. CD43 engagement with beads coated with anti-CD43 mAb resulted in concentration of the zeta-chain toward the bead attachment site, but interestingly, the distribution of the T cell Ag receptor complex remained unaffected. Recruitment of the zeta-chain through CD43-mediated signals was not restricted to T lymphocytes because phosphorylation and redistribution of the zeta-chain was also observed in NK cells. Our results provide evidence that the zeta-chain functions as a scaffold molecule in the CD43 signaling pathway, favoring the recruitment and formation of downstream signaling complexes involved in the CD43-mediated cell activation of T lymphocytes and other leukocytes such as NK cells.     

Cutting edge: quantitative imaging of raft accumulation in the immunological synapse

Although the accumulation of lipid rafts at the immunological synapse is now well accepted, the degree of the accumulation, the localization within the fine structure of the immunological synapse, and the region from which lipid rafts are recruited have not been defined. In this work we show that lipid rafts preferentially accumulate in the central zone of the immunological synapse, the central supramolecular activation complex (C-SMAC). However, quantitative analyses indicate that the level of recruitment of lipid rafts to the C-SMAC is relatively small and suggests that rearrangement of lipid rafts from the peripheral zone of the synapse into the C-SMAC can account for this accumulation. We also assessed the effects of CD28 deficiency on lipid raft recruitment to the immunological synapse. The accumulation of lipid occurred independently of the CD28/B7 system and was not measurably altered by CD28.     

Development and function of CD94-deficient natural killer cells

The CD94 transmembrane-anchored glycoprotein forms disulfide-bonded heterodimers with the NKG2A subunit to form an inhibitory receptor or with the NKG2C or NKG2E subunits to assemble a receptor complex with activating DAP12 signaling proteins. CD94 receptors expressed on human and mouse NK cells and T cells have been proposed to be important in NK cell tolerance to self, play an important role in NK cell development, and contribute to NK cell-mediated immunity to certain infections including human cytomegalovirus. We generated a gene-targeted CD94-deficient mouse to understand the role of CD94 receptors in NK cell biology. CD94-deficient NK cells develop normally and efficiently kill NK cell-susceptible targets. Lack of these CD94 receptors does not alter control of mouse cytomegalovirus, lymphocytic choriomeningitis virus, vaccinia virus, or Listeria monocytogenes. Thus, the expression of CD94 and its associated NKG2A, NKG2C, and NKG2E subunits is dispensable for NK cell development, education, and many NK cell functions.