Distinct requirements for IFNs and STAT1 in NK cell function

NK cell functions were examined in mice with a targeted mutation of the STAT1 gene, an essential mediator of IFN signaling. Mice deficient in STAT1 displayed impaired basal NK cytolytic activity in vitro and were unable to reject transplanted tumors in vivo, despite the presence of normal numbers of NK cells. IL-12 enhanced NK-mediated cytolysis, but poly(I:C) did not, and a similar phenotype occurred in mice lacking IFNalpha receptors. Molecules involved in activation and lytic function of NK cells (granzyme A, granzyme B, perforin, DAP10, and DAP12) were expressed at comparable levels in both wild-type and STAT1(-/-) mice, and serine esterase activity necessary for CTL function was normal, showing that the lytic machinery was intact. NK cells with normal cytolytic activity could be derived from STAT1(-/-) bone marrow progenitors in response to IL-15 in vitro, and enhanced NK lytic activity and normal levels of IFN-gamma were produced in response to IL-12 treatment in vivo. Despite these normal responses to cytokines, STAT1(-/-) mice could not reject the NK-sensitive tumor RMA-S, even following IL-12 treatment in vivo. Whereas in vitro NK cytolysis was also reduced in mice lacking both type I and type II IFN receptors, these mice resisted tumor challenge. These results demonstrate that both IFN-alpha and IFN-gamma are required to maintain NK cell function and define a STAT1-dependent but partially IFN-independent pathway required for NK-mediated antitumor activity.     

Cutting Edge: Selective role of ubiquitin in MHC class I antigen presentation

The importance of ubiquitination in MHC class I-restricted Ag processing remains unclear. To address this issue, we overexpressed wild-type and dominant-negative lysineless forms of ubiquitin (Ub) in mammalian cells using an inducible vaccinia virus system. Overexpression of the lysineless Ub nearly abrogated polyubiquitination and potently inhibited epitope presentation from a cytosolic N-end rule substrate as well as endoplasmic reticulum (ER)-targeted model Ags. In contrast, there was little impact on Ag presentation from cytosolic proteins. These trends were location dependent; redirecting cytosolic Ag to the ER rendered presentation lysineless Ub-sensitive, whereas retargeting exocytic Ag to the cytosol had the inverse effect. This dichotomy was further underscored by small interfering RNA knockdown of the ER-associated Ub ligase Hrd1. Thus, Ub-dependent degradation appears to play a major role in the MHC class I-restricted processing of ER-targeted proteins and a more restricted role in the processing of cytosolic proteins.     

Group I metabotropic glutamate receptors mediate a dual role of glutamate in T cell activation

Metabotropic glutamate receptors (mGluR) are present in cells of the nervous system, where they are activated by one of the main neurotransmitters, glutamate. They are also expressed in cells outside the nervous system. We identified and characterized two receptors belonging to group I mGluR, mGlu1R and mGlu5R, in human cell lines of lymphoid origin and in resting and activated lymphocytes from human peripheral blood. Both are highly expressed in the human Jurkat T cell line, whereas mGlu5R is expressed only in the human B cell line SKW6.4. In blood lymphocytes, mGlu5R is expressed constitutively, whereas mGlu1R is expressed only upon activation via the T cell receptor-CD3 complex. Group I receptors in the central nervous system are coupled to phospholipase C, whereas in blood lymphocytes, activation of mGlu5R does not trigger this signaling pathway, but instead activates adenylate cyclase. On the other hand, mGlu5R does not mediate ERK1/2 activation, whereas mGlu1R, which is coupled neither to phospholipase C nor to calcium channels and whose activation does not increase cAMP, activates the mitogen-activated protein kinase cascade. The differential expression of mGluR in resting and activated lymphocytes and the different signaling pathways that are triggered when mGlu1Rs or mGlu5Rs are activated point to a key role of glutamate in the regulation of T cell physiological function. The study of the signaling pathways (cAMP production and ERK1/2 phosphorylation) and the proliferative response obtained in the presence of glutamate analogs suggests that mGlu1R and mGlu5R have distinct functions. mGlu5R mediates the reported inhibition of cell proliferation evoked by glutamate, which is reverted by the activation of inducible mGlu1R. This is a novel non-inhibitory action mechanism for glutamate in lymphocyte activation. mGlu1R and mGlu5R thus mediate opposite glutamate effects in human lymphocytes.     

Cutting Edge: NK cells mediate IgG1-dependent hyperacute rejection of xenografts

Classic hyperacute rejection is dependent on the activation of the terminal components of complement. Recently, xenoantibodies with limited abilities to activate the classical pathway of complement in vitro have been implicated in the acute vascular rejection of xenografts. It is unclear how these Abs affect their pathogenic activities in vivo. In this study, we demonstrate the ability of an anti-Gal-alpha1,3Gal (Gal) IgG1, with modest complement-activating abilities in vitro, to induce xenograft rejection. This rejection was dependent on the activation of complement, on FcgammaR-mediated interactions, and on the presence of NK cells. Inhibition of any one of these factors resulted in the abrogation of IgG1-mediated rejection. In contrast, an anti-Gal IgG3 mAb induced classic, hyperacute rejection that was solely dependent on complement activation. Our observations implicate two types of IgG-mediated rejection; one that is dependent on complement activation, and a second that is uniquely dependent on complement, FcgammaR, and NK cells.     

Cutting edge: inhibitory functions of the killer cell lectin-like receptor G1 molecule during the activation of mouse NK cells

The killer cell lectin-like receptor G1 (KLRG1) is the mouse homolog of the rat mast cell function-associated Ag and contains an immunoreceptor tyrosine-based inhibitory motif in its cytoplasmic domain. In this study we demonstrate that both pathogenic and nonpathogenic in vivo activation of NK cells induces the expression of KLRG1 on their cell surface. Upon infection with murine CMV, this induction peaks between days 5 and 7 with about 90% of the NK cells expressing KLRG1. On day 1.5 post-murine CMV infection of C57BL/6 mice, the main producers of IFN-gamma are the KLRG1-negative NK cells. This effect has been recapitulated in vitro as we show that engagement of KLRG1 on a transfected NK cell line inhibits both cytokine production and NK cell-mediated cytotoxicity. Taken together, these data illustrate the crucial role played by KLRG1 during the termination of mouse NK cell activation.     

A novel role for Gab2 in bFGF-mediated cell survival during retinoic acid-induced neuronal differentiation

Gab proteins amplify and integrate signals stimulated by many growth factors. In culture and animals, retinoic acid (RA) induces neuronal differentiation. We show that Gab2 expression is detected in neurons in three models of neuronal differentiation: embryonic carcinoma (EC) stem cells, embryonic stem cells, and primary neural stem cells (NSCs). RA treatment induces apoptosis, countered by basic FGF (bFGF). In EC cells, Gab2 silencing results in hypersensitivity to RA-induced apoptosis and abrogates the protection by bFGF. Gab2 suppression reduces bFGF-dependent activation of AKT but not ERK, and constitutively active AKT, but not constitutively active MEK1, reverses the hypersensitization. Thus, Gab2-mediated AKT activation is required for bFGF's protection. Moreover, Gab2 silencing impairs the differentiation of EC cells to neurons. Similarly, in NSCs, Gab2 suppression reduces bFGF-dependent proliferation as well as neuronal survival and production upon differentiation. Our findings provide the first evidence that Gab2 is an important player in neural differentiation, partly by acting downstream of bFGF to mediate survival through phosphoinositide 3 kinase-AKT.     

Plasmacytoid dendritic cells regulate Th cell responses through OX40 ligand and type I IFNs

Dendritic cells (DCs) show a functional plasticity in determining Th responses depending on their maturational stage or on maturational signals delivered to the DCs. Human plasmacytoid DCs (PDCs) can induce either Th1- or Th2-type immune responses upon exposure to viruses or IL-3, respectively. In this study we have investigated the Th-polarizing capacity of PDCs after short (24-h) or long (72-h) culture with stimuli and have assessed the expression and function of OX40 ligand (OX40L) in PDC-mediated Th polarization in addition to type I IFN-dependent responses. IL-3-treated PDCs expressed OX40L, but produced almost no IFN-alpha in response to T cell stimulation (CD40 ligand or T cell interaction), resulting in the preferential priming of Th2 cells through OX40L-dependent mechanisms. Meanwhile, PDCs were rapidly endowed by viral infection (Sendai virus) with a high potency to develop IFN-gamma-producing Th cells depending on their capacity to residually produce IFN-alpha. Although Sendai virus-stimulated PDCs simultaneously expressed OX40L in their maturational process, the Th1-inducing effect of endogenous type I IFNs may overcome and thus conceal the OX40L-dependent Th2 responses. However, during maturation in response to Sendai virus over the longer 72-h period, the expression level of OX40L was up-regulated, whereas the residual IFN-alpha-producing ability was down-regulated, and consequently, the PDCs with prolonged Sendai virus stimulation induced Th2 responses to some extent. Thus, PDCs have the distinct means to dictate an appropriate response to environmental stimuli.     

Cutting Edge: CD28 controls dominant regulatory T cell activity during active immunization

Ligation of CD28 during Ag recognition plays an important role in the generation of effective T cell responses. However, its peripheral control of regulatory T cell function remains obscure. In this study, we show that naive wild-type or CD28(-/-) CD4(+)CD25(-) T cells exposed to peptide in vivo develop regulatory activity that suppresses the response of adoptively transferred naive T cells to a subsequent immunogenic challenge. We find that although CD28 is engaged during the initial peptide-priming event and is essential to sustain T cell survival, it is not sufficient to prevent the dominance of regulatory T cell function. Immunization with adjuvant abrogates regulatory dominance, reducing overall Foxp3 expression in a CD28-dependent manner. We conclude that CD28 licenses active immunization by regulating Ag-induced immunoregulation.     

Cutting Edge: KIR3DS1, a gene implicated in resistance to progression to AIDS, encodes a DAP12-associated receptor expressed on NK cells that triggers NK cell activation

The killer cell Ig-like receptor (KIR) gene, KIR3DS1, has been implicated in slowing disease progression in HIV infection; however, little is known about its expression, function, or ligand specificity. Using retrovirally transduced NKL cells and peripheral blood NK cells from KIR3DS1-positive donors we assessed expression of this gene by flow cytometry and its function by in vitro assays measuring KIR3DS1-induced cell-mediated cytotoxicity and cytokine production. In the present study, we demonstrate that KIR3DS1 is expressed on peripheral blood NK cells and triggers both cytotoxicity and IFN-gamma production. Using cotransfection and coimmunoprecipitation, we found that KIR3DS1 associates with the ITAM-bearing adaptor, DAP12. Soluble KIR3DS1-Ig fusion proteins did not bind to EBV-transformed B lymphoid cell lines transfected with HLA-Bw4 80I or 80T allotypes, suggesting that if KIR3DS1 does recognize HLA-Bw4 ligands, this may be peptide dependent.     

Prevention of bone marrow cell apoptosis and regulation of hematopoiesis by type I IFNs during systemic responses to pneumocystis lung infection

We recently demonstrated that lack of type I IFN signaling (IFNAR knockout) in lymphocyte-deficient mice (IFrag(-/-)) results in bone marrow (BM) failure after Pneumocystis lung infection, whereas lymphocyte-deficient mice with intact IFNAR (RAG(-/-)) had normal hematopoiesis. In the current work, we performed studies to define further the mechanisms involved in the induction of BM failure in this system. BM chimera experiments revealed that IFNAR expression was required on BM-derived but not stroma-derived cells to prevent BM failure. Signals elicited after day 7 postinfection appeared critical in determining BM cell fate. We observed caspase-8- and caspase-9-mediated apoptotic cell death, beginning with neutrophils. Death of myeloid precursors was associated with secondary oxidative stress, and decreasing colony-forming activity in BM cell cultures. Treatment with N-acetylcysteine could slow the progression of, but not prevent, BM failure. Type I IFN signaling has previously been shown to expand the neutrophil life span and regulate the expression of some antiapoptotic factors. Quantitative RT-PCR demonstrated reduced mRNA abundance for the antiapoptotic factors BCL-2, IAP2, MCL-1, and others in BM cells from IFrag(-/-) compared with that in BM cells from RAG(-/-) mice at day 7. mRNA and protein for the proapoptotic cytokine TNF-α was increased, whereas mRNA for the growth factors G-CSF and GM-CSF was reduced. In vivo anti-TNF-α treatment improved precursor cell survival and activity in culture. Thus, we propose that lack of type I IFN signaling results in decreased resistance to inflammation-induced proapoptotic stressors and impaired replenishment by precursors after systemic responses to Pneumocystis lung infection. Our finding may have implications in understanding mechanisms underlying regenerative BM depression/failure during complex immune deficiencies such as AIDS.     

Cutting Edge: Localization of linker for activation of T cells to lipid rafts is not essential in T cell activation and development

It has been proposed that upon T cell activation, linker for activation of T cells (LAT), a transmembrane adaptor protein localized to lipid rafts, orchestrates formation of multiprotein complexes and activates signaling cascades in lipid rafts. However, whether lipid rafts really exist or function remains controversial. To address the importance of lipid rafts in LAT function, we generated a fusion protein to target LAT to nonraft fractions using the transmembrane domain from a nonraft protein, linker for activation of X cells (LAX). Surprisingly, this fusion protein functioned well in TCR signaling. It restored MAPK activation, calcium flux, and NFAT activation in LAT-deficient cells. To further study the function of this fusion protein in vivo, we generated transgenic mice that express this protein. Analysis of these mice indicated that it was fully capable of replacing LAT in thymocyte development and T cell function. Our results demonstrate that LAT localization to lipid rafts is not essential during normal T cell activation and development.     

A functional role of I kappa B-epsilon in endothelial cell activation

The NF-kappa B inhibitor I kappa B-epsilon is a new member of the I kappa B protein family, but its functional role in regulating NF-kappa B-mediated induction of adhesion molecule expression is unknown. In vascular endothelial cells, I kappa B-epsilon associates predominantly with the NF-kappa B subunit Rel A and to a lesser extent with c-Rel, whereas I kappa B-alpha and I kappa B-beta associate with Rel A only. Following stimulation with TNF-alpha, pyrrolidine dithiocarbamate (PDTC), N-acetylcysteine, and dexamethasone prevented I kappa B kinase-induced I kappa B-alpha, but not I kappa B-beta or I kappa B-epsilon phosphorylation and degradation. Since the activation of NF-kappa B is required for the induction of adhesion molecule expression, we examined the role of I kappa B-epsilon in the transactivation of promoters from VCAM-1, ICAM-1, and E-selectin. Using reporter gene constructs of adhesion molecule promoters, PDTC inhibited VCAM-1 and E-selectin, but to a lesser extent, ICAM-1 promoter activity. Subcloning of kappa B cis-acting elements of VCAM-1, E-selectin, and ICAM-1 into a heterologous promoter construct revealed that PDTC inhibited VCAM-1 and E-selectin, but to a lesser extent, ICAM-1 kappa B promoter activity. By electrophoretic mobility shift assay, NF-kappa B heterodimers containing c-Rel specifically bind to the kappa B motif in the ICAM-1, but not VCAM-1 or E-selectin promoter. Indeed, overexpression of c-Rel induced ICAM-1 kappa B promoter activity to a greater extent than that of E-selectin and overexpression of I kappa B-epsilon inhibited ICAM-1 and VCAM-1 promoter activity in endothelial cells. These findings indicate that c-Rel-associated I kappa B-epsilon is involved in the induction of ICAM-1 expression.     

Cutting edge: an NK cell-independent role for Slamf4 in controlling humoral autoimmunity

Several genes within a syntenic region of human and mouse chromosome 1 are associated with predisposition to systemic lupus erythematosus. Analyses of lupus-prone congenic mice have pointed to an important role for the signaling lymphocyte activation molecule family (slamf)6 surface receptor in lupus pathogenesis. In this article, we demonstrate that a second member of the Slamf gene family, Slamf4 (Cd244), contributes to lupus-related autoimmunity. B6.Slamf4(-/-) mice spontaneously develop activated CD4 T cells and B cells and increased numbers of T follicular helper cells and a proportion develop autoantibodies to nuclear Ags. B6.Slamf4(-/-) mice also exhibit markedly increased autoantibody production in the B6.C-H-2bm12/KhEg → B6 transfer model of lupus. Although slamf4 function is best characterized in NK cells, the enhanced humoral autoimmunity of B6.Slamf4(-/-) mice is NK cell independent, as judged by depletion studies. Taken together, our findings reveal that slamf4 has an NK cell-independent negative regulatory role in the pathogenesis of lupus a normally non-autoimmune prone genetic background.     

Stat-1 is not essential for inhibition of B lymphopoiesis by type I IFNs

Type I IFNs, IFN-alpha, -beta, and -omega, are cytokine family members with multiple immune response roles, including the promotion of cell growth and differentiation. Conversely, the type I IFNs are potent inhibitors of IL-7-dependent growth of early B lineage progenitors, effectively aborting further B lineage differentiation at the pro-B cell stage. Type I IFNs alpha and beta function via receptor-mediated activation of a Jak/Stat signaling pathway in which Stat-1 is functionally important, because many IFN-induced responses are abrogated in Stat-1-deficient mice. To the contrary, we show here that the inhibition of IL-7-dependent B lymphopoiesis by IFN-alphabeta is unaffected in Stat-1-deficient mice. The present data indicate that the type I IFNs can activate an alternative signaling pathway in which neither Stat-1 nor phosphatidylinositol 3'-kinase are essential components.     

Cutting Edge: Distinct NK receptor profiles are imprinted on CD8 T cells in the mucosa and periphery during the same antigen challenge: role of tissue-specific factors

NK cell receptors (NKRs) modulate T lymphocyte responses by modifying the Ag activation threshold. However, what governs their expression on T cells remains unclear. In this study we show that different NKRs are imprinted on CD8 T cells in the gut mucosa and periphery during the same Ag challenge. After a viral, bacterial, and tumor challenge, most CD8 peritoneal exudate lymphocytes expressed NKG2A but not 2B4. In contrast, most CD8 intraepithelial lymphocytes exhibited 2B4 but not NKG2A. Our data suggest that tissue-specific factors may determine the pattern of NKR expression. In the gut, CD70 licensing appears to promote 2B4 induction on mucosal CD8 T cells. Conversely, retinoic acid produced by the intestinal dendritic cells may suppress NKG2A expression. Thus, tissue-specific factors regulate NKR expression and may confer T cells with differing effector functions in a tissue and site-specific manner.