Signaling at the inhibitory natural killer cell immune synapse regulates lipid raft polarization but not class I MHC clustering.

Natural killer (NK) cell cytotoxicity is determined by a balance of positive and negative signals. Negative signals are transmitted by NK inhibitory receptors (killer immunoglobulin-like receptors, KIR) at the site of membrane apposition between an NK cell and a target cell, where inhibitory receptors become clustered with class I MHC ligands in an organized structure known as an inhibitory NK immune synapse. Immune synapse formation in NK cells is poorly understood. Because signaling by NK inhibitory receptors could be involved in this process, the human NK tumor line YTS was transfected with signal-competent and signal-incompetent KIR2DL1. The latter were generated by truncating the KIR2DL1 cytoplasmic tail or by introducing mutations in the immunoreceptor tyrosine-based inhibition motifs. The KIR2DL1 mutants retained their ability to cluster class I MHC ligands on NK cell interaction with appropriate target cells. Therefore, receptor-ligand clustering at the inhibitory NK immune synapse occurs independently of KIR2DL1 signal transduction. However, parallel examination of NK cell membrane lipid rafts revealed that KIR2DL1 signaling is critical for blocking lipid raft polarization and NK cell cytotoxicity. Moreover, raft polarization was inhibited by reagents that disrupt microtubules and actin filaments, whereas synapse formation was not. Thus, NK lipid raft polarization and inhibitory NK immune synapse formation occur by fundamentally distinct mechanisms.

Vpr is preferentially targeted by CTL during HIV-1 infection.

The HIV-1 accessory proteins Vpr, Vpu, and Vif are essential for viral replication, and their cytoplasmic production suggests that they should be processed for recognition by CTLs. However, the extent to which these proteins are targeted in natural infection, as well as precise CTL epitopes within them, remains to be defined. In this study, CTL responses against HIV-1 Vpr, Vpu, and Vif were analyzed in 60 HIV-1-infected individuals and 10 HIV-1-negative controls using overlapping peptides spanning the entire proteins. Peptide-specific IFN-gamma production was measured by ELISPOT assay and flow-based intracellular cytokine quantification. HLA class I restriction and cytotoxic activity were confirmed after isolation of peptide-specific CD8(+) T cell lines. CD8(+) T cell responses against Vpr, Vpu, and Vif were found in 45%, 2%, and 33% of HIV-1-infected individuals, respectively. Multiple CTL epitopes were identified in functionally important regions of HIV-1 Vpr and Vif. Moreover, in infected individuals in whom the breadth of HIV-1-specific responses was assessed comprehensively, Vpr and p17 were the most preferentially targeted proteins per unit length by CD8(+) T cells. These data indicate that despite the small size of these proteins Vif and Vpr are frequently targeted by CTL in natural HIV-1 infection and contribute importantly to the total HIV-1-specific CD8(+) T cell responses. These findings will be important in evaluating the specificity and breadth of immune responses during acute and chronic infection, and in the design and testing of candidate HIV vaccines.

Inhibition of natural killer cell-mediated cytotoxicity by Kaposi's sarcoma-associated herpesvirus K5 protein.

Kaposi's sarcoma-associated herpesvirus (KSHV) K3 and K5 proteins dramatically downregulate MHC class I molecules. However, although MHC class I downregulation may protect KSHV-infected cells from cytotoxic T lymphocyte recognition, these cells become potential targets for natural killer (NK) cell-mediated lysis. We now show that K5 also downregulates ICAM-1 and B7-2, which are ligands for NK cell-mediated cytotoxicity receptors. As a consequence, K5 expression drastically inhibits NK cell-mediated cytotoxicity. Conversely, de novo expression of B7-2 and ICAM-1 resensitizes the K5-expressing cells to NK cell-mediated cytotoxicity. This is a novel viral immune evasion strategy where KSHV K5 achieves immune avoidance by downregulation of cellular ligands for NK cell-mediated cytotoxicity receptors.

Downregulation of major histocompatibility complex class I molecules by Kaposi's sarcoma-associated herpesvirus K3 and K5 proteins.

The T-cell-mediated immune response plays a central role in the defense against intracellular pathogens. To avoid this immune response, viruses have evolved elaborate mechanisms that target and modulate many different aspects of the host's immune system. A target common to many of these viruses is the major histocompatibility complex (MHC) class I molecules. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes K3 and K5 zinc finger membrane proteins which remove MHC class I molecules from the cell surface. K3 and K5 exhibit 40% amino acid identity to each other and localize primarily near the plasma membrane. While K3 and K5 dramatically downregulated class I molecules, they displayed different specificities in downregulation of HLA allotypes. K5 significantly downregulated HLA-A and -B and downregulated HLA-C only weakly, but not HLA-E, whereas K3 downregulated all four HLA allotypes. This selective downregulation of HLA allotypes by K5 was partly due to differences in amino acid sequences in their transmembrane regions. Biochemical analyses demonstrated that while K3 and K5 did not affect expression and intracellular transport of class I molecules, their expression induced rapid endocytosis of the molecules. These results demonstrate that KSHV has evolved a novel immune evasion mechanism by harboring similar but distinct genes, K3 and K5, which target MHC class I molecules in different ways.

The human thioesterase II protein binds to a site on HIV-1 Nef critical for CD4 down-regulation.

A HIV-1 Nef affinity column was used to purify a 35-kDa Nef-interacting protein from T-cell lysates. The 35-kDa protein was identified by peptide microsequence analysis as the human thioesterase II (hTE) enzyme, an enzyme previously identified in a yeast two-hybrid screen as a potential Nef-interacting protein. Immunofluorescence studies showed that hTE localizes to peroxisomes and that coexpression of Nef and hTE leads to relocalization of Nef to peroxisomes. Interaction of Nef and hTE was abolished by point mutations in Nef at residues Asp(108), Leu(112), Phe(121), Pro(122), and Asp(123). All of these mutations also abrogated the ability of Nef to down-regulate CD4 from the surface of HIV-infected cells. Based on the x-ray and NMR structures of Nef, these residues define a surface on Nef critical for CD4 down-regulation. A subset of these mutations also affected the ability of Nef to down-regulate major histocompatibility complex class I. These results, taken together with previous studies, identify a region on Nef critical for most of its known functions. However, not all Nef alleles bind to hTE with high affinity, so the role of hTE during HIV infection remains uncertain.

N-linked carbohydrate on human leukocyte antigen-C and recognition by natural killer cell inhibitory receptors.

The possible role of carbohydrate in the interaction of HLA-C with a human inhibitory natural Killer cell Immunoglobulin-like Receptor with two Ig domains, KIR2DL1, was investigated. Transfectants of 721.221 (a class I MHC-negative human B cell line) expressing only HLA-Cw4 or -Cw6 or their respective non-glycosylated mutants (N86Q, S88A) were made. The binding of a KIR2DL1-Ig fusion protein to the non-glycosylated mutant HLA-Cw4- or -Cw6-expressing cells was markedly decreased compared to the wild type-expressing cells. The ability to induce an inhibitory signal in the NK tumor line YTS transfected with KIR2DL1 was also impaired in the nonglycosylated mutant expressing cells. Furthermore, in a second functional assay, mutant HLA-Cw4 and -Cw6 molecules had impaired ability to induce signal transduction in BW cells expressing a KIR2DL1-CD3 zeta chain chimeric protein. Thus, the deletion of the N-linked glycosylation signal in HLA-Cw4 and -Cw6 greatly reduced recognition by KIR2DL1. Alternative interpretations of the data are discussed.

The human natural killer cell immune synapse.

Inhibitory killer Ig-like receptors (KIR) at the surface of natural killer (NK) cells induced clustering of HLA-C at the contacting surface of target cells. In this manner, inhibitory immune synapses were formed as human NK cells surveyed target cells. At target/NK cell synapses, HLA-C/KIR distributed into rings around central patches of intercellular adhesion molecule-1/lymphocyte function-associated antigen-1, the opposite orientation to mature murine T cell-activating synapses. This organization of protein was stable for at least 20 min. Cells could support multiple synapses simultaneously, and clusters of HLA-C moved as NK cells crawled over target cells. Clustering required a divalent metal cation, explaining how metal chelators inhibit KIR function. Surprisingly, however, formation of inhibitory synapses was unaffected by ATP depletion and the cytoskeletal inhibitors, colchicine and cytochalsins B and D. Clearly, supramolecular organization within plasma membranes is critical for NK cell immunosurveillance.

The selective downregulation of class I major histocompatibility complex proteins by HIV-1 protects HIV-infected cells from NK cells.

To avoid detection by CTL, HIV encodes mechanisms for removal of class I MHC proteins from the surface of infected cells. However, class I downregulation potentially exposes the virus-infected cell to attack by NK cells. Human lymphoid cells are protected from NK cell cytotoxicity primarily by HLA-C and HLA-E. We present evidence that HIV-1 selectively downregulates HLA-A and HLA-B but does not significantly affect HLA-C or HLA-E. We then identify the residues in HLA-C and HLA-E that protect them from HIV down-regulation. This selective downregulation allows HIV-infected cells to avoid NK cell-mediated lysis and may represent for HIV a balance between escape from CTL and maintenance of protection from NK cells. These results suggest that subpopulations of CTL and NK cells may be uniquely suited for combating HIV.

Spatial light modulator based on a deformed-helix ferroelectric liquid crystal and a thin a-Si:H amorphous photoconductor.

We have developed an optically addressed spatial light modulator (OASLM) based on a deformed-helix ferroelectric liquid crystal (DHFLC) and a thin (0.5-mum) amorphous a-Si:H photoconductor. The thin photoconductor permits operation in the transmission mode of a read beam. The OASLM, operated in the integrated mode, exhibits gray levels, a high contrast (>1:40), and a high spatial resolution (exceeding 57 lines/mm). This is a significant improvement over previously reported OASLM's based on DHFLC's.

Rhodopsin mutation G90D and a molecular mechanism for congenital night blindness.

Mutations in the gene for the visual pigment rhodopsin cause retinitis pigmentosa (RP) and congenital night blindness. Inheritance of the diseases is generally autosomal dominant and about 40 different rhodopsin mutations have been documented. Although the cell death and retinal degeneration associated with RP have been suggested to result from improper folding and accumulation of the mutant proteins in rod photoreceptor cells, this may not account for the disease in all cases. For example, RP mutations at Lys 296, site of Schiff base linkage to the retinal chromophore, result in constitutive activation of the protein in vitro; that is, the mutants can catalytically activate the G protein transducin in the absence of chromophore and in the absence of light. Similarly, mutation of Ala 292-->Glu activates opsin in vitro and causes night blindness. We show here that the mutation Gly 90-->Asp (G90D) in the second transmembrane segment of rhodopsin, which causes congenital night blindness, also constitutively activates opsin. Furthermore, we show that Asp 90 can substitute for the Schiff base counterion, Glu 113, which is located in the third transmembrane segment of the protein. This demonstrates the proximity of Asp 90 and Lys 296 in the three-dimensional structure of rhodopsin and suggests that the constitutively activating mutations operate by a common molecular mechanism, disrupting a salt bridge between Lys 296 and the Schiff base counterion, Glu 113.

Constitutive activation of opsin: influence of charge at position 134 and size at position 296.

In previous studies, mutation of Lys296 or Glu113 in opsin has been shown to result in constitutive activation of the protein--that is, these mutants can activate the G protein transducin in the absence of chromophore and in the absence of light. These and other data have led to the suggestion that a salt bridge between Lys296 and Glu113 helps to constrain opsin to an inactive conformation. It is shown here that of 12 different amino acids substituted at position 296, all, except Arg and the wild-type Lys, are constitutively active at neutral pH, lending further support to this suggestion. However, activation of opsin appears also to be influenced significantly by the size of amino acid side chain at position 296. Thus, there are multiple effects of the mutations. Wild-type opsin is also shown to be weakly active at pH 6.1. Five other charged amino acids in the membrane-embedded region of the protein (Asp83, Glu122, Glu134, Arg135, and Glu201) were mutated to see if they affect constitutive activity. Of these amino acids, only mutation of Glu134 results in an increase in the activity of opsin. Changing Glu134 to Gln increases the activity of opsin, while changing Glu134 to Asp inhibits activity. These results suggest that a negative charge on Glu134 is important in stabilizing the inactive state of opsin. Glu134 is highly conserved in all visual pigments and most of the other G protein-linked receptors.

Mechanism of activation and inactivation of opsin: role of Glu113 and Lys296.

In previous studies, mutation of either Lys296 or Glu113 in bovine rhodopsin has been shown to result in constitutive activation of the apoprotein form, opsin [Robinson et al. (1992) Neuron 9, 719-725]. In this report, pH-rate profiles for the rhodopsin-catalyzed exchange of GTPgS for GDP on transducin are established for the constitutively active opsin mutants. All of the mutants, including the double-mutant E113Q,K296G, show a bell-shaped pH-rate profile. Therefore, it is evident that at least two ionizable groups in addition to Lys296 and Glu113 control the formation of the active opsin state. The sole effect of mutation at position 113 or 296 is to alter the ionization constant of the group with the higher pKa, called pka2. pKa2 decreases in the following order: rhodopsin/light (9.0) > K296E = K296G = E113Q,K296G (8.0) > E113Q (6.8) > K296H (6.6) >> wild-type opsin (< 5.0). These results are consistent with a model where activation of opsin involves (i) breaking of the salt bridge between Lys296 and Glu113, (ii) deprotonation of Lys296, and (iii) the net uptake of a proton from the solvent. Furthermore, exogenous addition of the chromophore all-trans-retinal shifts the wild-type and E113Q opsin equilibrium to favor the active state. In all these respects, the light-independent activation of the opsin mutants appears to proceed by a mechanism similar to that of light-activated rhodopsin.

Constitutively active mutants of rhodopsin.

Two critical amino acids in the visual pigment rhodopsin are Lys-296, the site of attachment of retinal to the protein through a protonated Schiff base linkage, and Glu-113, the Schiff base counterion. Mutation of Lys-296 or Glu-113 results in constitutive activation of opsin, as assayed by its ability to activate transducin in the absence of added chromophore. We conclude that opsin is constrained to an inactive conformation by a salt bridge between Lys-296 and Glu-113. Recently, one of the mutants, K296E, was found in a family with retinitis pigmentosa, suggesting that degeneration of the photoreceptor cells in individuals with this mutation may result from persistent stimulation of the phototransduction pathway.

The effect of thymosin on patients with disseminated malignancies. A phase I study.

A Phase I clinical trial of thymosin administered in doses of 10 to 250 mg/M2 intramuscularly for seven days was undertaken in ten patients with disseminated malignancies and evidence of immunoincompetence. Toxicity was minimal; one patient experienced a mild urticarial rash which cleared spontaneously, two patients developed low grade fever and one patient experienced pain at the injection site. There was no evidence of systemic toxicity or parenchymal organ dysfunction. Thymosin administration was associated with an increase in the E-rosette forming capacity of the patient's lymphocytes and the development of new skin test reactivity to recall antigens in some of these patients. One objective tumor response was noted. These findings are preliminary but are encouraging for further utilization of thymosin as an immunostimulant in cancer patients with immunoincompetence.