A Genome-Wide CRISPR/Cas9-Based Screen Identifies Heparan Sulfate Proteoglycans as Ligands of Killer-Cell Immunoglobulin-Like Receptors.

While human leukocyte antigen (HLA) and HLA-like proteins comprise an overwhelming majority of known ligands for NK-cell receptors, the interactions of NK-cell receptors with non-conventional ligands, particularly carbohydrate antigens, is less well described. We previously found through a bead-based HLA screen that KIR3DS1, a formerly orphan member of the killer-cell immunoglobulin-like receptor (KIR) family, binds to HLA-F. In this study, we assessed the ligand binding profile of KIR3DS1 to cell lines using Fc fusion constructs, and discovered that KIR3DS1-Fc exhibited binding to several human cell lines including ones devoid of HLA. To identify these non-HLA ligands, we developed a magnetic enrichment-based genome-wide CRISPR/Cas9 knock-out screen approach, and identified enzymes involved in the biosynthesis of heparan sulfate as crucial for the binding of KIR3DS1-Fc to K562 cells. This interaction between KIR3DS1 and heparan sulfate was confirmed via surface plasmon resonance, and removal of heparan sulfate proteoglycans from cell surfaces abolished KIR3DS1-Fc binding. Testing of additional KIR-Fc constructs demonstrated that KIR family members containing a D0 domain (KIR3DS1, KIR3DL1, KIR3DL2, KIR2DL4, and KIR2DL5) bound to heparan sulfate, while those without a D0 domain (KIR2DL1, KIR2DL2, KIR2DL3, and KIR2DS4) did not. Overall, this study demonstrates the use of a genome-wide CRISPR/Cas9 knock-out strategy to unbiasedly identify unconventional ligands of NK-cell receptors. Furthermore, we uncover a previously underrecognized binding of various activating and inhibitory KIRs to heparan sulfate proteoglycans that may play a role in NK-cell receptor signaling and target-cell recognition.

Association between the killer cell immunoglobulin-like receptor a haplotype and childhood acute lymphoblastic leukemia.

Killer immunoglobulin-like receptors (KIRs) have the ability to regulate natural killer (NK) cell function through inhibition/activation mechanisms. Healthy human cells express HLA class I ligands on their surface, which are recognized by NK cells to avoid spontaneous cell destruction. The associations of KIRs and/or HLA class 1 ligands in leukemic patients have been studied in some populations, with some of these studies demonstrating an association of specific types with leukemia. KIRs and their corresponding HLA class 1 ligands were investigated in Saudi patients with ALL and AML and compared to healthy controls. The homozygous A haplotype was found significantly more often in ALL patients ≤18years-old than in control individuals. No significant association was observed in KIRs and their corresponding HLA ligands in this study.

Activating Killer Immunoglobulin Receptors and HLA-C: a successful combination providing HIV-1 control.

Several studies demonstrated a relevant role of polymorphisms located within the HLA-B and -C loci and the Killer Immunoglobulin Receptors (KIRs) 3DL1 and 3DS1 in controlling HIV-1 replication. KIRs are regulatory receptors expressed at the surface of NK and CD8+ T-cells that specifically bind HLA-A and -B alleles belonging to the Bw4 supratype and all the -C alleles expressing the C1 or C2 supratype. We here disclose a novel signature associated with the Elite Controller but not with the long-term nonprogressor status concerning 2DS activating KIRs and HLA-C2 alleles insensitive to miRNA148a regulation. Overall, our findings support a crucial role of NK cells in the control of HIV-1 viremia.

Impairment of neurovascular coupling in Type 1 Diabetes Mellitus in rats is prevented by pancreatic islet transplantation and reversed by a semi-selective PKC inhibitor.

Streptozotocin (STZ)-induced chronic hyperglycemia has a detrimental effect on neurovascular coupling, linked to increased PKC-mediated phosphorylation and PKC isoform expression changes. Here, we sought to determine whether: 1) selective PKC-α/ß/γ inhibitor, GF109203X, could reverse the effects of chronic hyperglycemia on cerebrovascular reactivity; 2) pancreatic islet transplantation could prevent the development of cerebrovascular impairment seen in a rat model of Type 1 Diabetes. We studied the effect of GF109203X in diabetic (DM), non-diabetic (ND), and transplanted (TR) Lewis rats during either sciatic nerve stimulation (SNS) or the topical applications of the large-conductance Ca2+-operated K+(BKCa) channel opener, NS1619, or the K+ inward rectifier (Kir) channel agonist, KCl. Pial arteriole diameter changes were monitored using a closed cranial window in vivo microscopy technique. The pial arteriole dilatory response associated with SNS was decreased by ~45%, when comparing DM vs either ND or TR rats. Also, pial arteriolar dilations to topical KCl and NS1619 were largely attenuated in DM rats, but not in ND or TR animals. These responses were completely restored by the acute application of GF109203X to the brain surface. The PKC inhibitor had no effect on vascular responses in normoglycemic and TR animals. In conclusion, DM-associated chronic impairment of neurovascular coupling may be readily reversed by a PKC-α/ß/γ inhibitor or prevented via pancreatic islet transplantation. We believe that specific PCK isoforms (α/ß/γ) are mechanistically linked to the neurovascular uncoupling seen with hyperglycemia.

Influenza Virus Targets Class I MHC-Educated NK Cells for Immunoevasion.

The immune response to influenza virus infection comprises both innate and adaptive defenses. NK cells play an early role in the destruction of tumors and virally-infected cells. NK cells express a variety of inhibitory receptors, including those of the Ly49 family, which are functional homologs of human killer-cell immunoglobulin-like receptors (KIR). Like human KIR, Ly49 receptors inhibit NK cell-mediated lysis by binding to major histocompatibility complex class I (MHC-I) molecules that are expressed on normal cells. During NK cell maturation, the interaction of NK cell inhibitory Ly49 receptors with their MHC-I ligands results in two types of NK cells: licensed ("functional"), or unlicensed ("hypofunctional"). Despite being completely dysfunctional with regard to rejecting MHC-I-deficient cells, unlicensed NK cells represent up to half of the mature NK cell pool in rodents and humans, suggesting an alternative role for these cells in host defense. Here, we demonstrate that after influenza infection, MHC-I expression on lung epithelial cells is upregulated, and mice bearing unlicensed NK cells (Ly49-deficient NKCKD and MHC-I-deficient B2m-/- mice) survive the infection better than WT mice. Importantly, transgenic expression of an inhibitory self-MHC-I-specific Ly49 receptor in NKCKD mice restores WT influenza susceptibility, confirming a direct role for Ly49. Conversely, F(ab')2-mediated blockade of self-MHC-I-specific Ly49 inhibitory receptors protects WT mice from influenza virus infection. Mechanistically, perforin-deficient NKCKD mice succumb to influenza infection rapidly, indicating that direct cytotoxicity is necessary for unlicensed NK cell-mediated protection. Our findings demonstrate that Ly49:MHC-I interactions play a critical role in influenza virus pathogenesis. We suggest a similar role may be conserved in human KIR, and their blockade may be protective in humans.

The role genes encoding of killer cell immunoglobulin-like receptors (KIRs) and their ligands in susceptibility to and progression of HIV infection.

NK cells are a part of the innate antiviral response. Their activity is regulated by signals from the surface receptors. Some of them, known as killer cell immunoglobulin-like receptors (KIRs), determine the quality and intensity of the immunological response, together with their ligands (HLA class I). KIR genes are very polymorphic, and this is reflected in the NK activity modulation. The stimulation of NK cells, especially in the early stages of the infection, can reduce the transmission of HIV or slow down the progression of infection. The varied KIR/HLA repertoire is a limiting factor for the risk of HIV infection and disease progression. Such diversity enables optimal regulation of NK cells and maintenance of the balance between activation to eliminate infected cells and inhibition. The control of NK cell activity via KIR3DL1/3DS1 and HLA-Bw4 (especially Bw4-80I) seems to be very important in the HIV context. With a few exceptions, it leads to a reduction of susceptibility to HIV infection and better viremia control, and slows down depletion of CD4+ T cells. Incompatibility of sexual partners for KIRs and HLA may oblige NK cells from the exposed partner to reject incoming cells from the HIV-positive partner. The presence of the inhibitory KIR, in the absence of its ligand, results in a lower threshold of NK cell activation, which reduces the chance of infection. The presence of an inhibitory receptor with a low affinity to the ligand (KIR2DL3+HLA-C1) is associated with lower susceptibility, and the effective NK cell inhibition (KIR2DL2+HLA-C1) results in increased susceptibility to HIV infection. The advantage of activating KIRs, especially in the presence of their ligands, is associated with higher cytolytic abilities, and thus reduced risk of HIV infection. If the virus is not eliminated in an early stage of infection, massive activation of NK is unfavorable due to the excessive stimulation of the immune system.

Killer immunoglobulin receptor genes and their HLA-C ligand are associated with Type 1 diabetes in an Eastern Indian population.

AIM: Killer immunoglobulin-like receptors (KIRs) and their interaction with HLA class I ligands have been shown to be associated with Type 1 diabetes mellitus. The aim of our study was to investigate the influence of KIR genes and their HLA-C ligands for susceptibility to Type 1 diabetes in patients from Eastern India. METHODS: A total of 135 patients with Type 1 diabetes and 98 healthy subjects from Eastern India were typed for KIR genes and HLA-C ligands using PCR-based genotyping. The frequencies of these genes were compared between patients and controls. RESULTS: Comparison of KIR genes between Type 1 diabetes patients and healthy subjects revealed significantly different frequencies of KIRs 2DL2 and 2DS4. The presence of HLA-C1 was negatively associated with disease. The presence of both HLA-C1 and -C2 showed a negative association with Type 1 diabetes, whereas the absence of C1 and presence of C2 was positively associated with disease. Stratification analysis of HLA-C ligands and KIRs showed significant associations between Type 1 diabetes and 2DL2+/C1-, 2DL2-/C1+, 2DL3+/C1+, 2DL3+/C1- and 2DS2+/C1-. CONCLUSIONS: Our results suggest that the interaction of KIRs with HLA-C ligands are significant and certain combinations contribute to susceptibility to and protection against Type 1 diabetes.

KIR genes and their human leukocyte antigen ligands in the progression to cirrhosis in patients with chronic hepatitis C.

Natural killer (NK) cells play pivotal roles in immune responses against infection with viruses, such as hepatitis C virus (HCV), and killer cell immunoglobulin-like receptors (KIRs) are related to the activation and inhibition of NK cells. The aim of this study was to investigate the possibility that KIR genes and their human leukocyte antigen (HLA) ligands influence progression to cirrhosis in patients infected with genotype 1 of HCV. A total of 145 Brazilian patients with confirmed chronic hepatitis C grouped from F0 to F4 according to fibrosis progression to cirrhosis were evaluated. Genotyping of KIR and HLA genes was performed by polymerase chain reaction with sequence-specific oligonucleotide probes. The HLA-C2 KIR ligand was more frequent in patients than in healthy controls (74.5% vs 64.3%, p = 0.04, odds ratio (OR) = 1.6, 95% confidence interval (CI) = 1.03-2.52). Moreover, the HLA-C1C2 genotype was more frequent in patients with advanced fibrosis or cirrhosis (F3-F4 group) than in patients in the F0-F2 group (61.6% vs 44.7%, p = 0.06) and in the F4 group compared with the F0-F3 group (65.7% vs 46.7%, p = 0.05, OR = 2.19, 95% CI = 1.01-4.73). NK and KIR ligands may contribute to the development of liver damage in patients chronically infected by HCV.

The beneficial impact of missing KIR ligands and absence of donor KIR2DS3 gene on outcome following unrelated hematopoietic SCT for myeloid leukemia in the Chinese population.

The effect of natural killer (NK) cell alloreactivity on the outcome of unrelated hematopoietic SCT (HSCT) remains a topic of debate. NK cell alloreactivity after allogeneic HSCT is regulated by killer-cell Ig-like receptors (KIRs). To investigate the influence of KIRs on outcome after unrelated HSCT, we retrospectively analyzed the HLA and KIR genotypes of 116 donor-recipient pairs. We found that missing KIR ligands in recipients were significantly associated with a decreased leukemic relapse risk (P=0.019, HR=0.329), mainly in myeloid disease (P=0.003, HR=0.193). This beneficial effect was seen in AML/myelodysplastic syndrome and also in chronic myeloid leukemia. In myeloid disease, missing KIR ligands also improved 5-year OS (P=0.034, HR=0.430) and disease-free survival (DFS) (P=0.024, HR=0.445). Meanwhile, the presence of donor-activating KIR2DS3 gene was associated with increased relapse risk (P=0.003, HR=5.046), decreased OS (P=0.004, HR=3.181) and DFS (P=0.003, HR=2.919) in myeloid disease. No effect was seen in patients with lymphoid disease. Our study indicated that, in unrelated HSCT for myeloid leukemia, missing KIR ligands in recipients offered a lower relapse risk and a long-term survival advantage. The presence of KIR2DS3 in the donor was an important risk factor for myeloid leukemia.

The role of missing killer cell immunoglobulin-like receptor ligands in T cell replete peripheral blood stem cell transplantation from HLA-identical siblings.

The contribution of natural killer (NK) cells to graft-versus-malignancy (GVM) effects following hematopoietic stem cell transplantation (HSCT) remains uncertain, particularly in the HLA-identical setting. A model considering missing HLA ligands to the donor's inhibitory killer cell immunoglobulin-like receptor (KIR), termed the missing KIR ligand model, has been established in T cell depleted bone marrow transplantation (BMT), but lacks validity in other cohorts with different treatment characteristics. We hypothesized that the impact of missing KIR ligands on relapse-free survival (RFS) and overall survival (OS) in T cell replete peripheral blood SCT (PBSCT) differs from that in the T cell depleted BMT setting, and retrospectively evaluated 100 consecutive, HLA-identical sibling transplantations for hematologic malignancies. In addition to KIR ligand status, we considered the donors' activating KIRs and grafted NK, T, and CD34(+) cell doses. Our findings demonstrate noninferiority for OS (P = .005) and RFS (P = .002) for the heterozygous HLA-C group KIR ligand status (C1/2; n = 47) compared with patients missing either C1 or C2 (n = 53). Similarly, OS (P = .031) and RFS (P = .034) of Bw4-positive patients was noninferior to that of patients missing a Bw4 ligand to KIR3DL1. By multivariate analysis, C1/2 heterozygous patients had a favorable risk ratio (RR) for relapse (RR = 0.28; P = .003), RFS (RR = 0.56; P = .046), and acute graft-versus-host disease grade II-IV (RR = 0.36; P = .05). Following reduced-intensity conditioning (RIC), but not standard-intensity conditioning, myeloablative (MA) transplantation, a grafted NK cell dose above the median (3.4 x 10(7)/kg) was associated with a lower risk of relapse (RR = 0.57; P = .003) and improved survival (RR = 0.78; P = .03). Overall, our findings support a role for NK alloreactivity in HLA-identical HSCT, but argue against a favorable impact of missing KIR ligands in the given setting. We conclude that the mechanism favoring the missing KIR ligand constellation in T cell depleted BMT may not operate in T cell replete PBSCT. The reasons for this differential effect remain unresolved.

Killer cell immunoglobulin-like receptors influence the innate and adaptive immune responses.

Natural killer (NK) cells are a subset of lymphocytes which play a crucial role in early innate immune response against infection and tumor transformation. Furthermore, they secrete interferon- (IFN-) and tumor necrosis factor (TNF) prompting adaptive immunity. NK cells distinguish the unhealthy cells from the healthy ones through an array of cell-surface receptors. Human NK cells use inhibitory and activating killer cell Ig-like receptors (KIR) as primary probe to discriminate between healthy and unhealthy cells. The inhibitory KIRs recognize HLA class I molecules and trigger signals that stop NK killing. The activating KIRs are believed to recognize the determinants associated with infections and tumors, and trigger signals that activate NK killing. Therefore, the effector function of a given NK cell depends upon the receptors that it expresses and ligands that it recognizes on the targets. Genes encoding KIRs and HLA ligands are located on different chromosomes, and vary in number and type. The independent segregation of KIR and HLA genes results in variable KIR-HLA combinations in individuals, which may determine the individual's immunity and susceptibility to disease.