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Background and aimsSecond-generation direct-acting antivirals (2G DAA) to cure HCV have led to dramatic clinical improvements. HCV-associated hepatocellular carcinoma (HCC), however, remains common. Impaired immune tumor surveillance may play a role in HCC development. Our cohort evaluated the effects of innate immune types and clinical variables on outcomes including HCC.MethodsParticipants underwent full HLA class I/KIR typing and long-term HCV follow-up.ResultsA total of 353 HCV+ participants were followed for a mean of 7 years. Cirrhosis: 25% at baseline, developed in 12% during follow-up. 158 participants received 2G DAA therapy. HCC developed without HCV therapy in 20 subjects, 24 HCC after HCV therapy, and 10 of these after 2G DAA. Two predictors of HCC among 2G DAA-treated patients: cirrhosis (OR, 10.0, p = 0.002) and HLA/KIR profiles predicting weak natural killer (NK) cell-mediated immunity (NK cell complementation groups 6, 9, 11, 12, OR of 5.1, p = 0.02). Without 2G DAA therapy: cirrhosis was the main clinical predictor of HCC (OR, 30.8, p < 0.0001), and weak NK-cell-mediated immunity did not predict HCC.ConclusionCirrhosis is the main risk state predisposing to HCC, but weak NK-cell-mediated immunity may predispose to post-2G DAA HCC more than intermediate or strong NK-cell-mediated immunity.

The activating Ly-49D receptor and the inhibitory Ly-49A receptor mediate opposing effects on natural killer (NK) cell cytotoxicity after interaction with the same major histocompatibility complex ligand, H2-D(d). To compare Ly-49D and Ly-49A interactions with H2-D(d), we created mutations in H2-D(d) and examined the functional ability of these mutants to activate lysis through Ly-49D or to inhibit lysis through Ly-49A. Specific single amino acid changes in either the H2-D(d) alpha(1) helix or the alpha(2) helix abrogated Ly-49D-mediated cytotoxicity, but these changes had no significant effect on Ly-49A-dependent inhibition. Each of three alpha(2) domain mutations in the floor of the peptide binding groove reduced functional recognition by either Ly-49D or Ly-49A, but all three were required to fully abrogate inhibition by Ly-49A. Our studies indicate that Ly-49D/H2-D(d) interactions require distinct determinants compared with Ly-49A/H2-D(d) interactions. These differences have important implications for the integration of activating and inhibitory signals in NK cells.

Inhibitory receptors expressed on natural killer (NK) cells abrogate positive signals upon binding corresponding major histocompatibility complex (MHC) class I molecules on various target cells. By directly micromanipulating the effector-target cell encounter using an optical tweezers system which allowed temporal and spatial control, we demonstrate that Ly49-MHC class I interactions prevent characteristic cellular responses in NK cells upon binding to target cells. Furthermore, using this system, we directly demonstrate that an NK cell already bound to a resistant target cell may simultaneously bind and kill a susceptible target cell. Thus, although Ly49-mediated inhibitory signals can prevent many types of effector responses, they do not globally inhibit cellular function, but rather the inhibitory signal is spatially restricted towards resistant targets.

Although activation of natural killer (NK) cytotoxicity is generally inhibited by target major histocompatibility complex (MHC) class I expression, subtle features of NK allorecognition suggest that NK cells possess receptors that are activated by target MHC I. The mouse Ly-49D receptor has been shown to activate NK cytotoxicity, although recognition of MHC class I has not been demonstrated previously. To define Ly-49D-ligand interactions, we transfected the mouse Ly-49D receptor into the rat NK line, RNK-16 (RNK.mLy-49D). As expected, anti- Ly-49D monoclonal antibody 12A8 specifically stimulated redirected lysis of the Fc receptor- bearing rat target YB2/0 by RNK.mLy-49D transfectants. RNK.mLy-49D effectors were tested against YB2/0 targets transfected with the mouse MHC I alleles H-2Dd, Db, Kk, or Kb. RNK.mLy-49D cells lysed YB2/0.Dd targets more efficiently than untransfected YB2/0 or YB2/0 transfected with Db, Kk, or Kb. This augmented lysis of H-2Dd targets was specifically inhibited by F(ab')2 anti-Ly-49D (12A8) and F(ab')2 anti-H-2Dd (34-5-8S). RNK.mLy-49D effectors were also able to specifically lyse Concanavalin A blasts isolated from H-2(d) mice (BALB/c, B10.D2, and DBA/2) but not from H-2(b) or H-2(k) mice. These experiments show that the activating receptor Ly-49D specifically interacts with the MHC I antigen, H-2Dd, demonstrating the existence of alloactivating receptors on murine NK cells.

Interferon gamma (IFN-gamma) has been implicated in T helper type 1 (Th1) cell development through its ability to optimize interleukin 12 (IL-12) production from macrophages and IL-12 receptor expression on activated T cells. Various systems have suggested a role for IFN-gamma derived from the innate immune system, particularly natural killer (NK) cells, in mediating Th1 differentiation in vivo. We tested this requirement by reconstituting T cell and IFN-gamma doubly deficient mice with wild-type CD4(+) T cells and challenging the mice with pathogens that elicited either minimal or robust IL-12 in vivo (Leishmania major or Listeria monocytogenes, respectively). Th1 cells developed under both conditions, and this was unaffected by the presence or absence of IFN-gamma in non-T cells. Reconstitution with IFN-gamma-deficient CD4(+) T cells could not reestablish control over L. major, even in the presence of IFN-gamma from the NK compartment. These data demonstrate that activated T cells can maintain responsiveness to IL-12 through elaboration of endogenous IFN-gamma without requirement for an exogenous source of this cytokine.

Activation of T and natural killer (NK) cells leads to the tyrosine phosphorylation of pp36 and to its association with several signaling molecules, including phospholipase Cgamma-1 and Grb2. Microsequencing of peptides derived from purified rat pp36 protein led to the cloning, in rat and man, of cDNA encoding a T- and NK cell-specific protein with several putative Src homology 2 domain-binding motifs. A rabbit antiserum directed against a peptide sequence from the cloned rat molecule recognized tyrosine phosphorylated pp36 from pervanadate-treated rat thymocytes. When expressed in 293T human fibroblast cells and tyrosine-phosphorylated, pp36 associated with phospholipase Cgamma-1 and Grb2. Studies with GST-Grb2 fusion proteins demonstrated that the association was specific for the Src homology 2 domain of Grb-2. Molecular cloning of the gene encoding pp36 should facilitate studies examining the role of this adaptor protein in proximal signaling events during T and NK cell activation.

The lytic activity of natural killer (NK) cells is inhibited by the expression of class I major histocompatibility complex (MHC) antigens on target cells. In murine NK cells, Ly-49A mediates inhibition of cytotoxicity in response to the class I MHC antigen H-2Dd. In this report, we studied the function of mouse Ly-49A in both the rat NK cell tumor line, RNK-16, transfected with Ly-49A cDNA, and in primary NK cells. We show that ligation of Ly-49A by H-2Dd inhibits early signaling events during target cell stimulation, including polyphosphoinositide turnover and tyrosine phosphorylation. We also show that Ly-49A directly associates with the cytoplasmic tyrosine phosphatase SHP-1, and that Ly-49A function is impaired in NK cells from SHP-1 mutant viable motheaten mice and from SHP-1-deficient motheaten mice. Finally, we demonstrate that mutational substitution of the tyrosine within the proposed SHP-1 binding motif in Ly-49A completely abrogates inhibition of NK cell cytotoxicity through this receptor. These results demonstrate that Ly-49A interrupts early activating signals in NK cells, and that SHP-1 is an important mediator of Ly-49A function.

The small GTPase accelerators regulator of G protein signalling (RGS) proteins are important regulators of proximal signalling from G protein coupled receptors. Although natural killer (NK) cells express a number of G-protein coupled receptors, expression of RGS proteins has not been investigated. We analysed the expression of RGS proteins in rat NK cells, and detected mRNA for RGS1, RGS2, RGS5, RGS8, RGS16, and RGS18. Interestingly, when we included a panel of different leucocyte subsets, we found that RGS8 was selectively expressed by NK cells. NK cells are under control of both activating and inhibitory receptors and, utilizing a xenogeneic system where the mouse activating Ly49D or inhibitory Ly49A receptors were transfected into the rat RNK-16 cell line, the potential regulation of RGS proteins by single NK cell receptors was studied. We found that ligation of Ly49D led to a rapid and transient increase in message for RGS2, while Ly49A ligation up-regulated RGS2, RGS16, and RGS18 mRNA. Both receptors also induced a prolonged increase in RGS2 endogenous protein levels. These findings suggest that RGS proteins may be influenced by or involved in NK cell receptor events, suggesting a crosstalk between G-protein coupled receptors and NK cell receptors. [ABSTRACT FROM AUTHOR]

We here report the molecular cloning of a novel family of killer-cell lectin-like (KLR) receptors in the rat and the mouse, termed KLRI. In both species, there are two members, KLRI1 and KLRI2. While the extracellular lectin-like domains of KLRI1 and KLRI2 are similar [74% (rat) and 83% (mouse) amino acid identity], they differ intracellularly. KLRI1 has two immunoreceptor tyrosine-based inhibition motifs (ITIMs) in the cytoplasmic domain, suggesting an inhibitory function. KLRI2 has no ITIM, but a positively charged lysine residue in the transmembrane region, suggesting association with activating adapter molecules.Klri1andKlri2are localized within the natural killer (NK) cell gene complex on rat chromosome 4 and mouse chromosome 6. By RT-PCR and Northern blot analysis KLRI1 and KLRI2 were selectively expressed by NK cells in both rat and mouse. Epitope-tagged expression constructs of rat KLRI1 and rat KLRI2 induced surface expression of a nondisulphide-linked protein ofMr 36,000/39,000 andMr 34,000, respectively. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells express a superfamily of surface proteins that share common structural features: dimeric type II integral membrane proteins with extracellular domains resembling C-type lectins. These receptors are encoded by a single genetic region called the NK complex (NKC). The NKC encompasses several families of genes including NKR-PI, Ly-49, CD94/NKG2, and NKG2D. Different NKC-encoded receptors have been shown to activate or to inhibit NK-cell function, and different receptors within the same family can have opposing functions. Within an individual NK cell, inhibitory receptors typically predominate over stimulatory receptors, calling into question the teleologic requirement or physiologic significance of lectin-like activating receptors in NK cells. Despite the widespread expression of inhibitory receptors, however, subtle features of activating receptor biology enable them to stimulate effector functions in vivo and in vitro. Activating receptors and inhibitory receptors differ in their subset expression, in their structural constraints for binding to common ligands, in their ligand repertoires, and in that divergent families of activating receptors utilize different signaling pathways. These subset, binding, repertoire, and signaling diversities may allow activating receptors to manifest their effect in spite of inhibitory receptor functions during pathologic conditions in vivo. In this review, we will present a detailed analysis of the data supporting this hypothesis with particular relevance toward physiologic NK-cell functions. [ABSTRACT FROM AUTHOR]

Rat natural killer (NK) cells recognize MHC-I molecules encoded by both the classical (RTI-A) and non-classical (KTI-C/E/M) MHC class I (MHC-I) regions. We have identified a receptor, the STOK2 antigen, which belongs to the Ly-49 family of killer cell lectin-like receptors, and we have localized the gene encoding it to the rat natural killer cell gene complex. We have also shown that it inhibits NK cytotoxicity when recognizing its cognate MHC-I ligand RTI-AI on a target cell. This is the first inhibitory Ly-49-MHC-I interaction identified in the rat and highlights the great similarity between rat and mouse Ly-49 receptors and their MHC ligands. However, the mode of rat NK-cell recognition of target cells indicates that positive recognition of allo-MHC determinants, especially those encoded by the RT1-C/E/M region, is a prevalent feature. NK cells recruited to the peritoneum as a consequence of alloimmunization display positive recognition of allodeterminants. In one case, NK cells activated in this way have been shown to be specific for the immunizing, non-classical class I molecule RT1-E[sup11]. These findings show that allospecific NK cells sometimes show features reminiscent of the adaptive immune response. [ABSTRACT FROM AUTHOR]

Early studies indicate that rats may have a repertoire of MHC class Ib-reactive Ly49 stimulatory receptors capable of mounting memory-like NK cell alloresponses. In this article, we provide molecular and functional evidence for this assumption. Pairs of Ly49 receptors with sequence similarities in the lectin-like domains, but with opposing signaling functions, showed specificity for ligands with class Ia-like structural features encoded from the first telomeric MHC class Ib gene cluster, RT1-CE, which is syntenic with the H2-D/H2-L/H2-Q cluster in mice. The activating Ly49s4 receptor and its inhibitory counterparts, Ly49i4 and Ly49i3, reacted with all allelic variants of RT1-U, whereas Ly49s5 and Ly49i5 were specific for RT1-Eu. NK cell cytolytic responses were predictably activated and inhibited, and potent in vivo NK alloresponses were induced by repeated MHC class Ib alloimmunizations. Additional Ly49-class Ib interactions, including RT1-Cl with the Ly49s4/Ly49i4/Ly49i3 group of receptors, were characterized using overexpressed receptor/ligand pairs, in vitro functional assays, and limited mutational analyses. Obvious, as well as subtle, Ly49-class Ib interactions led to ligand-induced receptor calibration and NK subset expansions in vivo. Together, these studies suggest that in vivo NK alloresponses are controlled by pleomorphic Ly49-class Ib interactions, some of which may not be easily detectable in vitro. [ABSTRACT FROM AUTHOR]