Inhibitory Killer Ig-like Receptors Research Articles

The Effect of Natural Killer Cell Killer Ig-Like Receptor Alloreactivity on the Outcome of Bone Marrow Stem Cell Transplantation for Severe Combined Immunodeficiency (SCID)

Natural killer (NK) cell alloreactions against recipient cells in the setting of bone marrow transplantation have been associated with decreased rates of graft-versus-host disease (GVHD) and improved survival in transplant recipients with myeloid leukemia. These alloreactions are predicted by the absence of recipient HLA class I ligands for donor inhibitory killer Ig-like receptors (KIR). We hypothesized that donor NK cell alloreactions against recipient cells may affect the development of T and B-cell functions and incidence of GVHD in infants with severe combined immunodeficiency (SCID). Of the 156 patients with SCID who had received related bone marrow transplants without pretransplant chemotherapy or posttransplant GVHD prophylaxis, 137 patient-donor pairs were evaluated for the absence of recipient HLA class I ligands for donor inhibitory KIR. Analysis showed that the absence of a KIR ligand had no effect on the incidence or severity of GVHD (RR [corrected] = 0.95, p = 0.84), development of T-cell function (RR [corrected] = 1.05, p = 0.69), production of IgA (p = 0.46) or IgM (p = 0.33), or on 5-year survival (RR [corrected] = 1.21, p = 0.10). Further, in patients possessing native NK cells, the absence of KIR ligands in donors for recipient-inhibitory KIR did not alter transplantation outcomes. This study suggests that inhibitory KIR/HLA interactions do not play a significant role in bone marrow transplantation for SCID.

Human Natural Killer Cells Achieve Tolerance by Preferentially Endowing Functional Competence to Inhibitory Killer Ig-Like Receptors Specific for Self-HLA Class I.

Natural killer (NK) cells are capable of cytotoxic targeting of virally infected cells and some tumor cells. It has been well-demonstrated that NK cells recognize target cells that have down-regulated MHC class I antigen expression (i.e. “missing self recognition”), and that it is the lack of class I engagement of inhibitory receptors such as the killer Ig-like receptors (KIR) that thereby allows NK activation and effector function. How these same inhibitory receptors achieve self-tolerance and simultaneously avoid autoimmunity in humans has not been clear, as more than 60% of individuals have inhibitory KIR for which they lack the HLA ligand. We demonstrate that mature NK cells achieve self-tolerance by preferentially endowing functional competence to the inhibitory KIRs for which they exhibit the cognate HLA ligands. To allow evaluation of inhibitory KIR and avoid interference from potentially class-I recognizing activating KIR, we analyzed NK cells from 10 individuals with various HLA backgrounds, but who were all homozygous for KIR haplotype-A. KIR haplotype-A contains the inhibitory KIR receptors 2DL3, 2DL1, and 3DL1, specific for HLA-Cw3, -Cw4, and -Bw4 ligands respectively, in addition to at most one other activating KIR whose ligand is unknown. Using 6-color staining and flow cytometric analysis of intracellular IFN-γ production, we evaluated the responsiveness of 30 inhibitory KIR-expressing NK subsets following activation with 721.221, a target cell line deficient in class I expression, with 721.221 transfectants expressing HLA-Cw3, -Cw4, or -Bw4 ligands, and with B-lymphocyte cell lines with diverse HLA phenotypes. NK cells exclusively expressing an inhibitory KIR for self-HLA demonstrated increased IFN-γ when coincubated with target cells lacking the cognate HLA ligand, whereas NK cells exclusively expressing an inhibitory KIR for non-self HLA were hyporesponsive to all targets. In all individuals, NK cells expressing inhibitory KIR specific for self-HLA were significantly more responsive than NK cells expressing inhibitory KIR for non-self HLA (p<0.001). All 3 inhibitory KIR (KIR2DL3, 2DL1, and 3DL1) demonstrated capacity for tolerance, with predictable response patterns based on the HLA background of the individual. NK cells lacking all inhibitory NK receptors (KIR−CD94/NKG2A−ILT2−) were identifiable and were markedly hyporesponsive. KIR−CD94/NKG2A+ILT2+ NK cells were also minimally responsive, comparable to the NK subset expressing inhibitory KIR for non-self HLA. These results demonstrate NK tolerance in humans, consistent with the licensing model proposed in mice: NK cells expressing inhibitory KIR to self-HLA are significantly more responsive than NK cells expressing inhibitory KIR for non-self HLA, and are rendered tolerant to self through inhibition upon binding to self-HLA ligands. NK cells expressing inhibitory KIR for non-self HLA are hyporesponsive and therefore rendered tolerant and incapable of autoreactivity when ligand is not engaged.

Lack of Predictive Value of KIR-HLA Genotypes for Autologous Transplant Outcome for Multiple Myeloma.

In allogeneic hematopoietic cell transplantation (HCT), donor alloreactive natural killer (NK) cells can prevent leukemia relapse and prolong survival. Donor NK alloreactivity has been shown to be influenced by the killer Ig-like receptor (KIR) repertoire of the donor and the HLA class I KIR ligand phenotype of the recipient. In T-cell depleted HLA-identical sibling HCT, it has been shown that lack of recipient class I ligand for donor inhibitory KIR is associated with improved outcome in acute myelogenous leukemia. In the normal population, more than 60% of individuals lack class I ligand for one or more of their inhibitory KIR. Because autoreactive NK cells are known to occur in normal individuals with low frequency, we sought to determine if these same NK clones driven by autologous KIR-HLA interactions might expand and play a role following treatment involving autologous stem cell infusion for hematologic malignancy. 233 patients with multiple myeloma who underwent high-dose chemotherapy with autologous stem cell infusion were evaluated with HLA and KIR genotyping. KIR-HLA genotype combinations were then correlated with event-free survival (EFS) and overall survival (OS) following autologous HCT. Cox regression models were fit to examine the association of KIR-HLA combinations with the hazards of mortality and relapse. We found that there was no association between lack of KIR ligand for inhibitory KIR and improved EFS (p=0.69) or OS (p=0.36), nor was there any association between presence of specific activating KIRs and transplant outcome. Furthermore, the cumulative effect of more than one activating KIR did not affect outcome, including the specific combination of KIR2DS1 and KIR2DS2 (EFS p=0.86 and OS p=0.67), which have been previously reported to be associated with lower leukemia relapse in allogeneic HLA-identical HCT. The combination of activating KIR with their known or putative HLA ligands was also not associated with improvement in transplant outcome. Patients were also evaluated for KIR haplotype (AA versus AB and BB). There was no association between KIR haplotype and DFS (p=0.59) or OS (p=0.96). These data suggest that KIR genotypes and KIR-HLA genotype combinations are not associated with improved outcome in multiple myeloma patients undergoing autologous HCT. This implies that NK cells in autologous HCT do not significantly impact outcome, either because they are not expanded in the post-HCT period or because they have no activity against autologous multiple myeloma cells.

Cutting Edge: Resistance to HIV-1 Infection among African Female Sex Workers Is Associated with Inhibitory KIR in the Absence of Their HLA Ligands

NK cells are regulated in part by killer Ig-like receptors (KIR) that interact with HLA molecules on potential target cells. KIR and HLA loci are highly polymorphic and certain KIR/HLA combinations were found to protect against HIV disease progression. We show in this study that KIR/HLA interactions also influence resistance to HIV transmission. HIV-exposed but seronegative female sex workers in Abidjan, Côte d'Ivoire, frequently possessed inhibitory KIR genes in the absence of their cognate HLA genes: KIR2DL2/KIR2DL3 heterozygosity in the absence of HLA-C1 and KIR3DL1 homozygosity in the absence of HLA-Bw4. HIV-seropositive female sex workers were characterized by corresponding inhibitory KIR/HLA pairings: KIR2DL3 homozygosity together with HLA-C1 and a trend toward KIR3DL1/HLA-Bw4 homozygosity. Absence of ligands for inhibitory KIR could lower the threshold for NK cell activation. In addition, exposed seronegatives more frequently possessed AB KIR genotypes, which contain more activating KIR. The data support an important role for NK cells and KIR/HLA interactions in antiviral immunity.

Impact of donor KIR genotype and recipient HLA ligand incompatibility on relapse related mortality and acute graft versus host disease in high-risk patients undergoing haploidentical non-myeloablative peripheral blood stem cell transplants: Differences between lymphoid and myeloid malignancies

Failure to recognize the appropriate HLA class I ligands on the target cell triggers the inhibitory killer Ig-like receptors (KIR) to initiate cytotoxicity and may contribute to graft-vs.-leukemia (GVL) effect. Past studies have shown GVL advantage of the absence (incompatibility) of recipient HLA ligands to donor KIR genes in select patients (pts) undergoing myeloablative transplantation. Here we examine if similar advantage is seen in non-myeloablative transplant (NMT). The KIR-HLA ligand combinations studied were: KIR2DL1-Cw4 (Asn77Lys80); KIR2DL2/3-Cw3 (Ser77Asn80); KIR3DL1-Bw4; and KIR3DL2-A3/A11.

Association of Killer Cell Ig-like Receptor (KIR) with an Adaptor Protein Shc

Cytotoxic function of killer cells is inhibited by specific recognition of class I MHC molecules on target cells by inhibitory killer Ig-like receptors (KIR) expressed on NK cells and some cytotoxic T cells. The inhibitory effect of KIR is accomplished by recruitment of SH2-containing protein tyrosine phosphatase (SHP) to the phosphotyrosine residues in the cytoplasmic tail. Methods: By in vitro coprecipitation experiments and transfection analysis, we investigated the association of KIR with an adaptor protein Shc in Jurkat T cells. Results: The cytoplasmic tail of KIR appeared to associate with an adaptor protein Shc in Jurkat T celilysates. Similar in vitro experiments showed that phosphorylated KIR cytoplasmic tail bound SHP-1 and Shc in Jurkat T cell lysates. The association of KIR with Shc was further confirmed by transfection analysis in 293T cells. Interestingly, however, Shc appeared to be replaced by SHP-2 upon engagement of KIR in 293T cells. Conclusion: Our data indicate that KIR associate with an adaptor protein Shc in Jurkat T cells, and suggest that KIR might have an additional role which is mediated by this adaptor protein.

Integrated Genotyping and mRNA Expression Profiling of Killer Immunoglobulin-Like Receptors.

Natural killer (NK) cells belong to a subgroup of lymphocytes (CD3-CD56+) which play an important role in the cellular immune response against virus-infected cells and tumors. The activity of NK cells is regulated by a balance of triggering and inhibitory receptors, including Killer Ig-like Receptor (KIR) molecules which interact with specific HLA class I molecules, predominantly HLA-C, on target cells. The 17 known KIR genes are divided into two classes: activating KIRs and inhibitory KIRs. There is strong evidence that inhibitory KIR mismatch between donor and recipient improves the outcome of haploidentical hematopoietic stem cell transplantation (HSTC) in leukemia patients (Ruggeri et al. 2002). In addition, the KIR-HLA constellation is assumed to have an influence on the severity of graft versus host disease (GvHD). Whether these activities of NK cells are clinically important and to what extent these processes are mediated only by KIR-HLA class I interactions remains to be determined. In human populations, KIR gene haplotypes vary in the number and type of KIR genes they contain. Further diversification is observed by expanded allelic polymorphism at the individual genes. In general, KIR haplotypes contain 7–12 genes plus 2 pseudogenes. Extra KIR heterogeneity is provided at the expression level: different subsets of NK cells within an individual express different KIRs. Recently, it was shown that KIR genotyping alone does not seem to be sufficient for donor KIR assessment because of the lack of gene expression in approximately one-fourth of the individuals for one of the inhibitory KIRs that recognize the three major groups of MHC class I ligands (Leung et al. 2005). KIR phenotyping by flow cytometry using monoclonal antibodies is insufficient due to the lack of specific monoclonal antibodies. For trustworthy analysis, one has to combine KIR genotyping with mRNA expression profiling and flow cytometry. Therefore, we developed a new set of sequence-specific primers (SSP). This primer set can be applied to perform either KIR genotyping or mRNA expression profiling despite the high degree of identity of the genes (80–90%, sometimes more than 95%). The primers of each KIR gene (15 genes and 2 pseudogenes) cover all allelic variants annotated by the IPD KIR Sequence Data Base (status quo July 05). Using this primer set, we genotyped 25 individuals, and compared the results with other sets of KIR primers published elsewhere. Additionally, we show the mRNA expression profile employing the same set of new primers. We confirmed these results on the protein level by flow cytometry.

Incompatible Inhibitory KIR Ligands Contribute To Lower Survival Rates in Leukemia Patients Receiving T-Replete Hematopoietic Cell Transplants (HCTs).

Natural killer (NK) cells are becoming increasingly recognized as an important part of the immune system. Killer Ig-like receptors (KIRs) are the major form of receptors used by human NK cells. KIRs transmit either inhibitory or activating signals regulating NK cell activity. Some inhibitory KIRs specifically recognize HLA-A, B or -Cw allotypes on target cells. Thus differences in inhibitory ligand (iKIRL) phenotypes between donors and patients could result in NK cell alloreactivity that further results in different HCT outcomes. To test this hypothesis, the present study evaluated 362 patients with either ALL (99), AML/MDS (141), or CML (122). The patients were transplanted (1996~2003) with T-replete bone marrow, or peripheral blood stem cells from unrelated donors. High resolution HLA typing with DNA-based methods had been prospectively performed for donor selection. The KIR genes of patients and donors were retrospectively typed by a multiplex PCR-SSP method. The cohort was divided into three groups by the HLA and KIR profiles: 247 cases with matched HLA at the antigen level of HLA-A, B, Cw, DRB1, and DQB1 (MH), 64 with mismatched HLA (mMH) and 51 with mMH plus mismatched iKIRL (mMH+miKIRL). A significantly different rate (P=0.009) of estimated one year overall survival was found between the MH (61%, 95%CI 55–67%), the mMH (46%, 34–59%), and the mMH+miKIRL (29%, 18–43%) groups. When analyzing the event free survival, a similar difference (P=0.003) was observed between the MH (57%, 51–63%), the mMH (43%, 32–56%) and the mMH+miKIRL (25%, 15–39%) groups. Further analysis demonstrated a different rate (P=0.02) of relapse between the MH (two-year estimation: 21%, 16–28%), the mMH (16%, 7–31%), and the mMH+miKIRL (36% 18–58%) groups. That the MH group experienced more relapses than the mMH is consistent with the GvL effect. The mMH+miKIRL group experienced the most relapses implied miKIRL undermine the GvL effect. We also tested the influence of lacking iKIRL in patients within the MH group, and found a significant survival difference (P=0.05) among the patients lacking two iKIRLs (n=67, 54% 42%–65%), one iKIRL (n=104, 59% 49%–68%), or none (n=76, 70% 58%–79%). This finding contrasts with the previously reported beneficial role of potential NK cell alloreactivity in T-depleted HCT. Additional studies are required to resolve these issues.

Comparison of Killer Ig-Like Receptor Genotyping and Phenotyping for Selection of Allogeneic Blood Stem Cell Donors

The repertoire of killer Ig-like receptors (KIRs) can be determined at the level of DNA, RNA, or surface protein expression for selection of blood stem cell donors. We compared genotyping and phenotyping of the four inhibitory KIRs that are important in transplantation for leukemia in 73 unrelated persons. In 5 (7%) of the 68 individuals in whom the KIR2DL1 gene was present and in 10 (15%) of the 67 in whom KIR3DL1 was present, the corresponding receptor was not expressed by NK cells, as determined by flow cytometry analysis. In contrast, one or both allelic forms of KIR2DL2/KIR2DL3 were expressed by a high proportion of NK cells in all 73 individuals. However if both KIR2DL2 and KIR2DL3 genes were present, KIR2DL3 was preferentially expressed, as transcripts of KIR2DL2 was not detectable by RT-PCR in 42% of these individuals. In total, repertoire assessment for the four KIRs by genotyping vs phenotyping was not in complete agreement in 18 (25%) of the 73 individuals. Furthermore, among the samples that tested positive for the expression of a certain KIR gene, the levels of transcripts and surface expression varied considerably as measured by both real-time quantitative PCR and flow cytometry analysis. Extension of this comparative analysis to include all 12 KIR family members showed that KIR2DL3 and KIR3DL2 were the only genes whose transcripts were consistently detectable. These results caution the use of genotyping alone for donor selection or leukemia-relapse prognostication because some KIRs may be expressed at a very low level.

The natural killer cell-mediated killing of autologous dendritic cells is confined to a cell subset expressing CD94/NKG2A, but lacking inhibitory killer Ig-like receptors.

The cognate NK-DC interaction in inflamed tissues results in NK cell activation and acquisition of cytotoxicity against immature DC (iDC). This may represent a mechanism of DC selection required for the control of downstream adaptive immune responses. Here we show that killing of monocyte-derived iDC is confined to the NK cell subset that expresses CD94/NKG2A, but not killer Ig-like receptors (KIR). Consistent with these data, the expression of HLA-E (i.e. the cellular ligand of CD94/NKG2A) was down-regulated in iDC. On the other hand, HLA-B and HLA-C down-regulation in iDC was not sufficient to induce cytotoxicity in NK cells expressing KIR3DL1 or KIR2DL. Remarkably, CD94/NKG2A(+)KIR(-) NK cells were heterogeneous in their ability to kill iDC and an inverse correlation existed between their CD94/NKG2A surface density and the magnitude of their cytolytic activity. It is conceivable that the reduced CD94/NKG2A surface density enables these cells to efficiently sense the decrease of HLA-E surface expression in iDC. Finally, most NK cells that lysed iDC did not kill mature DC that express higher amounts of HLA class I molecules (including HLA-E)as compared with iDC. However, a small NK cell subset was capable of killing not only iDC but also mature DC.

Soluble HLA class I induces NK cell apoptosis upon the engagement of killer-activating HLA class I receptors through FasL-Fas interaction

The engagement of the activating isoforms of C-type lectin inhibitory receptor (CLIR) or killer Ig-like receptor (KIR) by their natural ligands, represented by soluble HLA-I (sHLA-I) molecules, induced programmed cell death of natural killer (NK) cells. Indeed, NK cell apoptosis elicited by either putative HLA-E and HLA-F (sHLA-I non-A, -B, -C, and -G) or sHLA-I-Cw4 or -Cw3 from untransfected or -Cw4 or -Cw3 alleles transfected HLA-A(-), B(-), C(-), G(-), E(+), F(+) 721.221 lymphoblastoid cell line, respectively, was blocked by covering the corresponding activating receptor with either anti-CLIR- or anti-KIR-specific monoclonal antibodies (mAbs). After sHLA-I-activating receptor interaction, NK cells produced and released Fas ligand (FasL), which in turn led to NK cell apoptosis by interacting with Fas at the NK cell surface. Blocking anti-Fas mAb, or anti-FasL mAb, inhibited sHLA-I-mediated apoptosis via activating receptor in NK cell clones. This apoptosis was inhibited by NK cell treatment with cyclosporin A, whereas this drug had no effect on activating receptor-mediated activation of cytolysis. Conversely, concanamycin A, an inhibitor of vacuolar type H(+)-adenosine triphosphatase (H(+)-ATPase) of granules, inhibited activating receptor-induced NK cell cytolysis, suggesting that activating receptor-mediated apoptosis and cytolysis can use different intracellular pathways. Furthermore, a large amount of interferon-gamma (IFN-gamma) was detectable in culture supernatant of activating receptor(+) NK cells incubated with the appropriate sHLA-I ligand. Again, cyclosporin A, but not concanamycin A, strongly reduced activating receptor-mediated IFN-gamma production. This suggests that activating receptor-induced apoptosis of NK cells could play a role in eliminating potentially harmful NK cell clones and, at the same time, it leads to production of IFN-gamma, an antiviral cytokine able to amplify immune responses.

Cutting edge: susceptibility to psoriatic arthritis: influence of activating killer Ig-like receptor genes in the absence of specific HLA-C alleles.

NK cell activity is partially controlled through interactions between killer Ig-like receptors (KIR) on NK cells and their respective HLA class I ligands. Independent segregation of HLA and KIR genes, along with KIR specificity for particular HLA allotypes, raises the possibility that any given individual may express KIR molecules for which no ligand is present. Inhibitory receptor genes KIR2DL2/3 and KIR2DL1 were present in nearly all subjects sampled in this study, whereas their respective activating homologs, KIR2DS2 and KIR2DS1, are each present in about half of the subjects. In this work we report that subjects with activating KIR2DS1 and/or KIR2DS2 genes are susceptible to developing psoriatic arthritis, but only when HLA ligands for their homologous inhibitory receptors, KIR2DL1 and KIR2DL2/3, are missing. Absence of ligands for inhibitory KIRs could potentially lower the threshold for NK (and/or T) cell activation mediated through activating receptors, thereby contributing to pathogenesis of psoriatic arthritis.

Biphasic response of NK cells expressing both activating and inhibitory killer Ig-like receptors.

NK cells can co-express inhibitory and activating killer Ig-like receptors (KIR) recognizing the same HLA class I ligand. We present evidence from experiments with NK cells expressing both activating (KIR2DS2) and inhibitory (KIR2DL2 and KIR2DL3) receptors that the activating KIR can function without apparent interference from the inhibitory KIR. These studies used CD158b mAb that is equally reactive with KIR2DS2, KIR2DL2 and KIR2DL3. First, we show using plastic-immobilized CD158b mAb that the activating KIR2DS2 is stimulated, resulting in NK cell division and degranulation. Second, we show using soluble CD158b mAb and FcRII (+) P815 cells that high concentrations of CD158b mAb trigger the inhibitory KIR, whereas low concentrations stimulate the activating KIR2DS2 resulting in NK cell division and cytolysis. These results demonstrate that the activating KIR2DS2 can function on cells co-expressing the inhibitory KIR2DL2 and/or KIR2DL3, indicating the potential for independent function of activating KIR with natural ligand.

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.