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Nonobese diabetic (NOD) mice, a model of insulin-dependent diabetes mellitus, have a defect in natural killer (NK) cell-mediated functions. Here we show impairment in an activating receptor, NKG2D, in NOD NK cells. While resting NK cells from C57BL/6 and NOD mice expressed equivalent levels of NKG2D, upon activation NOD NK cells but not C57BL/6 NK cells expressed NKG2D ligands, which resulted in downmodulation of the receptor. NKG2D-dependent cytotoxicity and cytokine production were decreased because of receptor modulation, accounting for the dysfunction. Modulation of NKG2D was mostly dependent on the YxxM motif of DAP10, the NKG2D-associated adaptor that activates phosphoinositide 3 kinase. These results suggest that NK cells may be desensitized by exposure to NKG2D ligands.

CEACAM1 functions as a novel heterophilic ligand for TIM-3 and is necessary for TIM-3-mediated tolerance, which has marked consequences for inflammation, infection and cancer. Interaction of CEACAM1 and TIM-3 T cell regulation is mediated by a number of inhibitory cell surface molecules such as T-cell immunoglobulin domain and mucin domain-3 (TIM-3), a possible target for cancer immunotherapy that has been reported to act as an immune checkpoint in tumour-induced immune suppression. However, the tolerance inducing ligand for TIM-3 has yet to be uncovered, and TIM-3 may also possess activating functions under some circumstances. This study identifies carcinoembryonic antigen cell adhesion molecule 1 (CAECAM1) as the heterophilic ligand for murine TIM-3 that is required for TIM-3 to mediate tolerance and ensure its optimal maturation, cell surface expression and regulatory function. Co-blockade of CEACAM1 and TIM-3 leads to enhancement of anti-tumour immune responses, with improved elimination of tumours in mouse colorectal cancer models. T-cell immunoglobulin domain and mucin domain-3 (TIM-3, also known as HAVCR2) is an activation-induced inhibitory molecule involved in tolerance and shown to induce T-cell exhaustion in chronic viral infection and cancers.sup.1,2,3,4,5. Under some conditions, TIM-3 expression has also been shown to be stimulatory. Considering that TIM-3, like cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death 1 (PD-1), is being targeted for cancer immunotherapy, it is important to identify the circumstances under which TIM-3 can inhibit and activate T-cell responses. Here we show that TIM-3 is co-expressed and forms a heterodimer with carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1), another well-known molecule expressed on activated T cells and involved in T-cell inhibition.sup.6,7,8,9,10. Biochemical, biophysical and X-ray crystallography studies show that the membrane-distal immunoglobulin-variable (IgV)-like amino-terminal domain of each is crucial to these interactions. The presence of CEACAM1 endows TIM-3 with inhibitory function. CEACAM1 facilitates the maturation and cell surface expression of TIM-3 by forming a heterodimeric interaction in cis through the highly related membrane-distal N-terminal domains of each molecule. CEACAM1 and TIM-3 also bind in trans through their N-terminal domains. Both cis and trans interactions between CEACAM1 and TIM-3 determine the tolerance-inducing function of TIM-3. In a mouse adoptive transfer colitis model, CEACAM1-deficient T cells are hyper-inflammatory with reduced cell surface expression of TIM-3 and regulatory cytokines, and this is restored by T-cell-specific CEACAM1 expression. During chronic viral infection and in a tumour environment, CEACAM1 and TIM-3 mark exhausted T cells. Co-blockade of CEACAM1 and TIM-3 leads to enhancement of anti-tumour immune responses with improved elimination of tumours in mouse colorectal cancer models. Thus, CEACAM1 serves as a heterophilic ligand for TIM-3 that is required for its ability to mediate T-cell inhibition, and this interaction has a crucial role in regulating autoimmunity and anti-tumour immunity., Author(s): Yu-Hwa Huang [sup.1] , Chen Zhu [sup.2] , Yasuyuki Kondo [sup.1] , Ana C. Anderson [sup.2] , Amit Gandhi [sup.1] , Andrew Russell [sup.3] , Stephanie K. Dougan [sup.4] [...]

Author(s): Aaron R. Everitt [sup.1] , Simon Clare [sup.1] , Thomas Pertel [sup.2] , Sinu P. John [sup.2] , Rachael S. Wash [sup.1] , Sarah E. Smith [sup.1] , Christopher [...], Interferon-inducible transmembrane (IFITM) protein 3 is shown to be an innate defence mechanism against viral infection invivo; furthermore, a subset of the patients hospitalized during the H1N1 2009 pandemic carried a variant form of the IFITM3 gene. Immune defence against flu virus Interferon-inducible transmembrane (IFITM) proteins restrict the replication of certain pathogenic viruses, but no in vivo role for these proteins has been known until now. Paul Kellam and colleagues now report that IFITM3 is essential for protecting mice infected with influenza viruses from developing fulminant viral pneumonia. The authors further find that a small subset of humans hospitalized for infection with pandemic H1N1/09 swine flu or seasonal influenza virus carried a variant of IFITM3 with reduced antiviral activity. These results suggest that IFITM3 has a pivotal role in defence against influenza infection. The 2009 H1N1 influenza pandemic showed the speed with which a novel respiratory virus can spread and the ability of a generally mild infection to induce severe morbidity and mortality in a subset of the population. Recent in vitro studies show that the interferon-inducible transmembrane (IFITM) protein family members potently restrict the replication of multiple pathogenic viruses.sup.1,2,3,4,5,6,7. Both the magnitude and breadth of the IFITM proteins' in vitro effects suggest that they are critical for intrinsic resistance to such viruses, including influenza viruses. Using a knockout mouse model.sup.8, we now test this hypothesis directly and find that IFITM3 is essential for defending the host against influenza A virus in vivo. Mice lacking Ifitm3 display fulminant viral pneumonia when challenged with a normally low-pathogenicity influenza virus, mirroring the destruction inflicted by the highly pathogenic 1918 'Spanish' influenza.sup.9,10. Similar increased viral replication is seen in vitro, with protection rescued by the re-introduction of Ifitm3. To test the role of IFITM3 in human influenza virus infection, we assessed the IFITM3 alleles of individuals hospitalized with seasonal or pandemic influenza H1N1/09 viruses. We find that a statistically significant number of hospitalized subjects show enrichment for a minor IFITM3 allele (SNP rs12252-C) that alters a splice acceptor site, and functional assays show the minor CC genotype IFITM3 has reduced influenza virus restriction in vitro. Together these data reveal that the action of a single intrinsic immune effector, IFITM3, profoundly alters the course of influenza virus infection in mouse and humans.

Multi-layered antiviral activity of TRIM5 TRIM5 is an E3 ubiquitin ligase with known antiretroviral restriction factor activity, although the mechanisms involved are poorly understood. Luban and colleagues now demonstrate that TRIM5 activates innate immune signalling pathways and acts as a pattern recognition receptor specific for the retrovirus capsid lattice. TRIM5 is a RING domain-E3 ubiquitin ligase that restricts infection by human immunodeficiency virus (HIV)-1 and other retroviruses immediately following virus invasion of the target cell cytoplasm.sup.1,2. Antiviral potency correlates with TRIM5 avidity for the retrovirion capsid lattice.sup.3,4 and several reports indicate that TRIM5 has a role in signal transduction.sup.5,6,7, but the precise mechanism of restriction is unknown.sup.8. Here we demonstrate that TRIM5 promotes innate immune signalling and that this activity is amplified by retroviral infection and interaction with the capsid lattice. Acting with the heterodimeric, ubiquitin-conjugating enzyme UBC13-UEV1A (also known as UBE2N-UBE2V1), TRIM5 catalyses the synthesis of unattached K63-linked ubiquitin chains that activate the TAK1 (also known as MAP3K7) kinase complex and stimulate AP-1 and NF[kappa]B signalling. Interaction with the HIV-1 capsid lattice greatly enhances the UBC13-UEV1A-dependent E3 activity of TRIM5 and challenge with retroviruses induces the transcription of AP-1 and NF-[kappa]B-dependent factors with a magnitude that tracks with TRIM5 avidity for the invading capsid. Finally, TAK1 and UBC13-UEV1A contribute to capsid-specific restriction by TRIM5. Thus, the retroviral restriction factor TRIM5 has two additional activities that are linked to restriction: it constitutively promotes innate immune signalling and it acts as a pattern recognition receptor specific for the retrovirus capsid lattice., Author(s): Thomas Pertel [sup.1] , Stéphane Hausmann [sup.1] , Damien Morger [sup.2] , Sara Züger [sup.2] , Jessica Guerra [sup.1] , Josefina Lascano [sup.1] , Christian Reinhard [sup.1] , Federico [...]

Plasma cells daily secrete their own mass in antibodies, which fold and assemble in the endoplasmic reticulum (ER). To reach these levels, cells require pERp1, a novel lymphocyte-specific small ER-resident protein, which attains expression levels as high as BiP when B cells differentiate into plasma cells. Although pERp1 has no homology with known ER proteins, it does contain a CXXC motif typical for oxidoreductases. In steady state, the CXXC cysteines are locked by two parallel disulfide bonds with a downstream C[(X).sub.6]C motif, and pERp1 displays only modest oxidoreductase activity, pERpl emerged as a dedicated folding factor for IgM, associating with both heavy and light chains and promoting assembly and secretion of mature IgM.

New World monkeys of the genus Aotus synthesize a fusion protein (AoT5Cyp) containing tripartite motif-containing 5 (TRIM5) and cyclophilin A (CypA) that potently blocks HIV-1 infection. We attempted to generate a human HIV-1 inhibitor modeled after AoT5Cyp, by fusing human CypA to human TRIM5 (hT5Cyp). Of 13 constructs, 3 showed substantial HIV-1-inhibitory activity when expressed in human cell lines. This activity required capsid binding by CypA and correlated with CypA linkage to the TRIM5a capsid-specificity determinant and the ability to form cytoplasmic bodies. CXCR4-and CCR5-tropic HIV-1 clones and primary isolates were inhibited from infecting multiple human macrophage and T cell lines and primary cells by hT5Cyp, as were [HIV-2.sub.ROD], [SIV.sub.AGM]tan, [FIV.sub.PET], and a circulating HIV-1 isolate previously reported to be AoT5Cyp resistant. The anti-HIV-1 activity of hT5Cyp was surprisingly more effective than that of the well-characterized rhesus TRIM5α, especially in T cells. hT5Cyp also blocked HIV-1 infection of primary [CD4.sup.+] T cells and macrophages and conferred a survival advantage to these cells without disrupting their function. Extensive attempts to elicit HIV-1 resistance to hT5Cyp were unsuccessful. Finally, [Rag2.sup.[-/-]]γ[c.sup.[-/-]] mice were engrafted with human [CD4.sup.+] T cells that had been transduced by optimized lentiviral vectors bearing hT5Cyp. Upon challenge with HIV-1, these mice showed decreased viremia and productive infection in lymphoid organs and preserved numbers of human [CD4.sup.+] T cells. We conclude that hT5Cyp is an extraordinarily robust inhibitor of HIV-1 replication and a promising anti-HIV-1 gene therapy candidate., Introduction Over 60 million people have been infected with HIV-1, and nearly half of these people have died as a consequence of this infection (1). Despite the discovery 25 years [...]

Src family tyrosine kinases are signaling intermediates in a diverse array of cellular events including cell differentiation, motility, proliferation, and survival. In nonairway smooth muscle cells, muscarinic receptors directly interact with Src family tyrosine kinases. As little is known about the expression and signaling of these Src family tyrosine kinases in human airway smooth muscle cells, we determined the expression of Src family members and characterized the muscarinic receptor-mediated activation of Lyn kinase in these cells. RT-PCR revealed mRNA transcripts for FYN, c-SRC, YES, FRK, and LYN. Fyn, c-Src, Yes, and Lyn were identified in cultured airway smooth muscle cells by immunoblot analysis. In both nontransformed human cultured airway smooth muscle cells and cells transduced with wild-type human Lyn kinase, carbachol increased Lyn kinase activity. Pertussis toxin pretreatment failed to block carbachol activation of Lyn kinase but did attenuate the carbachol-induced increase in ERK/MAPK phosphorylation. Moreover, carbachol inhibited adenylyl cyclase but failed to increase total inositol phosphate synthesis in these cells. The present study shows that Lyn kinase is expressed in human cultured airway smooth muscle cells at both the mRNA and protein levels and that carbachol, an [M.sub.2] muscarinic receptor agonist in these cells, activates Lyn kinase by a pertussis toxin-insensitive signaling pathway. Src kinase; lentivirus; carbachol; immunoblot; cell culture

In a previous study, we showed that isoproterenol induced actin depolymerization in human airway smooth muscle cells by both protein kinase A (PKA)-dependent and -independent signaling pathways. We now investigate the signaling pathway of PKA-independent actin depolymerization induced by isoproterenol in these cells. Cells were briefly exposed to isoproterenol or PG[E.sub.1] in the presence and absence of specific inhibitors of Src-family tyrosine kinases, phosphatidylinositol-3-kinase (PI3 kinase), or MAP kinase, and actin depolymerization was measured by concomitant staining of filamentous actin with FITC-phalloidin and globular actin with Texas red DNase I. Isoproterenol, cholera toxin, and PG[E.sub.1] induced actin depolymerization, indicated by a decrease in the intensity of filamentous/globular fluorescent staining. Pretreatment with the Src kinase inhibitors 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyriimidine(PP2) or geldanamycin or the PKA inhibitor Rp-cAMPS only partly inhibited isoproterenol- or PG[E.sub.1]-induced actin depolymerization. In contrast. PP2 and geldanamycin did not inhibit forskolin-induced actin depolymerization, and AG-213 (an EGF receptor tyrosine kinase inhibitor) did not inhibit isoproterenol- or PG[E.sub.1]-induced actin depolymerization. PI3 kinase or MAP kinase inhibition did not inhibit isoproterenol-induced actin depolymerization. Moreover, isoproterenol but not forskolin induced tyrosine phosphorylation of an Src family member at position 416. These results further confirm that both PKA-dependent and PKA-independent pathways mediate actin depolymerization in human airway smooth muscle cells and that the PKA-independent pathway by which isoproterenol induces actin depolymerization in human airway smooth muscle cells involves Src protein tyrosine kinases and the [G.sub.s] protein. protein kinase A; tyrosine 416; phosphatidylinositol-3-kinase; 4-amino-5-(4-chlorophenyl)-7 -(t-butyl)pyrazolo[3,4-d]pyriimidine; geldanamycin

Author(s): Yu-Hwa Huang; Chen Zhu; Yasuyuki Kondo; Ana C. Anderson; Amit Gandhi; Andrew Russell; Stephanie K. Dougan; Britt-Sabina Petersen; Espen Melum; Thomas Pertel; Kiera L. Clayton; Monika Raab; Qiang Chen; [...]

Background Viruses can evade immune surveillance, but the underlying mechanisms are insufficiently understood. Here, we sought to understand the mechanisms by which natural killer (NK) cells recognize HIV-1-infected cells and how this virus can evade NK-cell-mediated immune pressure. Methods and Findings Two sequence mutations in p24 Gag associated with the presence of specific KIR/HLA combined genotypes were identified in HIV-1 clade C viruses from a large cohort of infected, untreated individuals in South Africa (n = 392), suggesting viral escape from KIR+ NK cells through sequence variations within HLA class I--presented epitopes. One sequence polymorphism at position 303 of p24 Gag ([T.sub.[Gag.sub.303]V]), selected for in infected individuals with both KIR2DL3 and [HLA-C.sup.*] 03:04, enabled significantly better binding of the inhibitory KIR2DL3 receptor to [HLA-C.sup.*] 03:04-expressing cells presenting this variant epitope compared to the wild-type epitope (wild-type mean 18.01 ± 10.45 standard deviation [SD] and variant mean 44.67 ± 14.42 SD, p = 0.002). Furthermore, activation of primary KIR2DL3+ NK cells from healthy donors in response to [HLA-C.sup.*] 03:04+ target cells presenting the variant epitope was significantly reduced in comparison to cells presenting the wild-type sequence (wild-type mean 0.78 [+ or -] 0.07 standard error of the mean [SEM] and variant mean 0.63 ± 0.07 SEM, p = 0.012). Structural modeling and surface plasmon resonance of KIR/ peptide/HLA interactions in the context of the different viral sequence variants studied supported these results. Future studies will be needed to assess processing and antigen presentation of the investigated HIV-1 epitope in natural infection, and the consequences for viral control. Conclusions These data provide novel insights into how viruses can evade NK cell immunity through the selection of mutations in HLA-presented epitopes that enhance binding to inhibitory NKcell receptors. Better understanding of the mechanisms by which HIV-1 evades NK-cell-mediated immune pressure and the functional validation of a structural modeling approach will facilitate the development of novel targeted immune interventions to harness the antiviral activities of NK cells., Introduction Natural killer (NK) cells are an important component of the antiviral innate immune response. They have the ability to lyse target cells without prior antigen sensitization and to regulate [...]

Abstract Background Vpx is a virion-associated protein encoded by SIVSM, a lentivirus endemic to the West African sooty mangabey (Cercocebus atys). HIV-2 and SIVMAC, zoonoses resulting from SIVSM transmission to humans or Asian rhesus macaques (Macaca mulatta), also encode Vpx. In myeloid cells, Vpx promotes reverse transcription and transduction by these viruses. This activity correlates with Vpx binding to DCAF1 (VPRBP) and association with the DDB1/RBX1/CUL4A E3 ubiquitin ligase complex. When delivered experimentally to myeloid cells using VSV G-pseudotyped virus-like particles (VLPs), Vpx promotes reverse transcription of retroviruses that do not normally encode Vpx. Results Here we show that Vpx has the extraordinary ability to completely rescue HIV-1 transduction of human monocyte-derived dendritic cells (MDDCs) from the potent antiviral state established by prior treatment with exogenous type 1 interferon (IFN). The magnitude of rescue was up to 1,000-fold, depending on the blood donor, and was also observed after induction of endogenous IFN and IFN-stimulated genes (ISGs) by LPS, poly(I:C), or poly(dA:dT). The effect was relatively specific in that Vpx-associated suppression of soluble IFN-β production, of mRNA levels for ISGs, or of cell surface markers for MDDC differentiation, was not detected. Vpx did not rescue HIV-2 or SIVMAC transduction from the antiviral state, even in the presence of SIVMAC or HIV-2 VLPs bearing additional Vpx, or in the presence of HIV-1 VLPs bearing all accessory genes. In contrast to the effect of Vpx on transduction of untreated MDDCs, HIV-1 rescue from the antiviral state was not dependent upon Vpx interaction with DCAF1 or on the presence of DCAF1 within the MDDC target cells. Additionally, although Vpx increased the level of HIV-1 reverse transcripts in MDDCs to the same extent whether or not MDDCs were treated with IFN or LPS, Vpx rescued a block specific to the antiviral state that occurred after HIV-1 cDNA penetrated the nucleus. Conclusion Vpx provides a tool for the characterization of a potent, new HIV-1 restriction activity, which acts in the nucleus of type 1 IFN-treated dendritic cells.