Arsenite suppresses IL-2-dependent tumoricidal activities of natural killer cells.

Chronic exposure to arsenic causes cancers in various organs including the skin, liver, lung, and bladder in humans, but the mechanisms of the multi-organ carcinogenicity of arsenic remain unknown. Natural killer (NK) cells play important roles in the immune surveillance and elimination of tumor cells. Although accumulating evidence has indicated that arsenic has immunosuppressive properties, little is known about the effects of arsenic on the tumoricidal functions of NK cells. We examined the effects of arsenite on the cytotoxic activities of human and mouse NK cells toward target tumor cells. Exposure of human NK-92 cells and primary mouse NK cells to sublethal doses of arsenite reduced the IL-2-activated cytotoxic activities toward human K562 cells and murine YAC-1 cells, respectively. NK cells recognize target cells via integrated signals from both activating and inhibitory receptors and induce apoptosis of target cells via a granzyme/perforin system. We found that exposure of NK-92 cells to arsenite diminished the IL-2-activated down-regulation of the inhibitory receptors, KIR2DL2 and KIR2DL3, and the up-regulation of granzyme B and lymphotoxin-α. The IL-2-activated increases in secretion of interferon-γ and IL-10 were also slightly reduced by arsenite. Thus, arsenite suppressed the IL-2-activated cytotoxic activity of NK cells by disrupting multiple pathways required for the recognition and killing of target tumor cells. Our findings provide new insights into the roles of NK cell-mediated tumor immunity in cancer development by arsenic.

KIR downregulation by IL-12/15/18 unleashes human NK cells from KIR/HLA-I inhibition and enhances killing of tumor cells.

To exploit autologous NK cells for cancer immunotherapy, it is highly relevant to circumvent killer cell immunoglobulin-like receptor (KIR)-mediated self-inhibition of human NK cells by HLA-I-expressing tumor cells. Here, we show that stimulation of NK cells with IL-12/15/18 for two days led to downregulation of surface expression of the inhibitory KIR2DL2/L3, KIR2DL1 and KIR3DL1 receptors on peripheral blood NK cells. Downregulation of KIR expression was attributed to decreased KIR mRNA levels which could be re-induced already 3 days after re-culture in IL-2. Reduced KIR2DL2/L3 expression on IL-12/15/18-activated NK cells resulted in less inhibition upon antibody-mediated KIR engagement and increased CD16-dependent cytotoxicity in redirected lysis assays. Most importantly, downregulated KIR2DL2/L3 expression enabled enhanced cytotoxicity of IL-12/15/18-stimulated NK cells against tumor cells expressing cognate HLA-I molecules. NK cells pre-activated with IL-12/15/18 were previously shown to exert potent anti-tumor activity and memory-like long-lived functionality, mediating remission in a subset of acute myeloid leukemia (AML) patients in a clinical trial. Our study reveals a novel mechanism of IL-12/15/18 in improving the cytotoxicity of NK cells by reducing their sensitivity to inhibition by self-HLA-I due to decreased KIR expression, highlighting the potency of IL-12/15/18-activated NK cells for anti-tumor immunotherapy protocols.

The Activating Human NK Cell Receptor KIR2DS2 Recognizes a ß2-Microglobulin-Independent Ligand on Cancer Cells.

The functions of activating members of the killer cell Ig-like receptor (KIR) family are not fully understood, as the ligands for these receptors are largely unidentified. In this study, we report that KIR2DS2 reporter cells recognize a ligand expressed by cancer cell lines. All cancer targets recognized by KIR2DS2 were also recognized by KIR2DL2 and KIR2DL3 reporters. Trogocytosis of membrane proteins from the cancer targets was observed with responding reporter cells, indicating the formation of KIR2DS2 ligand-specific immunological synapses. HLA-C typing of target cells showed that KIR2DS2 recognition was independent of the HLA C1 or C2 group, whereas targets cells that were only recognized by KIR2DL3 expressed C1 group alleles. Anti-HLA class I Abs blocked KIR2DL3 responses toward C1-expressing targets, but they did not block KIR2DS2 recognition of cancer cells. Small interfering RNA knockdown of ß2-microglobulin reduced the expression of class I H chain on the cancer targets by >97%, but it did not reduce the KIR2DS2 reporter responses, indicating a ß2-microglobulin-independent ligand for KIR2DS2. Importantly, KIR2DL3 responses toward some KIR2DS2 ligand-expressing cells were also undiminished after ß2-microglobulin knockdown, and they were not blocked by anti-HLA class I Abs, suggesting that KIR2DL3, in addition to the traditional HLA-C ligands, can bind to the same ß2-microglobulin-independent ligand as KIR2DS2. These observations indicate the existence of a novel, presently uncharacterized ligand for the activating NK cell receptor KIR2DS2. Molecular identification of this ligand may lead to improved KIR-HLA mismatching in hematopoietic stem cell transplantation therapy for leukemia and new, more specific NK cell-based cancer therapies.

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.

KIR3DL2 (CD158k) is a potential therapeutic target in primary cutaneous anaplastic large-cell lymphoma.

BACKGROUND: KIR3DL2, an inhibitory receptor expressed by natural killer cells and a subset of normal CD8(+) T cells, is aberrantly expressed in neoplastic cells in transformed mycosis fungoides and Sézary syndrome. Anti-KIR3DL2 targeted antibody therapy has shown potent activity in preclinical models for these diseases. OBJECTIVES: To examine the expression of KIR3DL2 and its potential use as a therapeutic target in patients with primary cutaneous anaplastic large-cell lymphoma (pcALCL), the most aggressive cutaneous CD30(+) lymphoproliferative disease. METHODS: Samples from 11 patients with pcALCL and three CD30(+) lymphoproliferative disease cell lines - Mac1, Mac2a and Mac2b - were used in KIR3DL2 expression studies using immunohistochemistry, flow cytometry and reverse-transcriptase quantitative polymerase chain reaction. The effect of IPH4102, a monoclonal humanized IgG1 targeting KIR3DL2, was assessed by in vitro cytotoxicity assays against Mac1, Mac2a and Mac2b using allogeneic peripheral blood mononuclear cells as effectors. RESULTS: KIR3DL2 mRNA and protein were found in all human samples of pcALCL, and in the Mac2a and Mac2b cell lines. KIR3DL2 protein expression was present on 85·8 ± 14·0% of CD30(+) skin-infiltrating tumour cells. In vitro functional studies showed that KIR3DL2(+) Mac2a and Mac2b pcALCL lines are sensitive to antibody-derived cytotoxicity mediated by IPH4102, through activation of natural killer cells, in a concentration-dependent manner. CONCLUSIONS: pcALCL tumour cells express KIR3DL2, and we provide preclinical proof of concept for the use of IPH4102, a humanized anti-KIR3DL2 antibody, to treat patients with primary cutaneous CD30(+) ALCL.

Human cytomegalovirus infection promotes rapid maturation of NK cells expressing activating killer Ig-like receptor in patients transplanted with NKG2C-/- umbilical cord blood.

NK cells are the first lymphoid population recovering after allogeneic hematopoietic stem cell transplantation and play a crucial role in early immunity after the graft. Recently, it has been shown that human CMV (HCMV) infection/reactivation can deeply influence NK cell reconstitution after umbilical cord blood transplantation by accelerating the differentiation of mature NKG2A(-) killer Ig-like receptor (KIR)(+) NK cells characterized by the expression of the NKG2C-activating receptor. In view of the hypothesis that NKG2C could be directly involved in NK cell maturation driven by HCMV infection, we analyzed the maturation and function of NK cells developing in three patients with hematological malignancies given umbilical cord blood transplantation from donors carrying a homozygous deletion of the NKG2C gene. We show that HCMV infection can drive rapid NK maturation, characterized by the expansion of CD56(dim)NKG2A(-)KIR(+) cells, even in the absence of NKG2C expression. Interestingly, this expanded mature NK cell subset expressed surface-activating KIR that could trigger NK cell cytotoxicity, degranulation, and IFN-γ release. Given the absence of NKG2C, it is conceivable that activating KIRs may play a role in the HCMV-driven NK cell maturation and that NK cells expressing activating KIRs might contribute, at least in part, to the control of infections after transplantation.

Allelic variation in KIR2DL3 generates a KIR2DL2-like receptor with increased binding to its HLA-C ligand.

Although extensive homology exists between their extracellular domains, NK cell inhibitory receptors killer Ig-like receptor (KIR) 2DL2*001 and KIR2DL3*001 have previously been shown to differ substantially in their HLA-C binding avidity. To explore the largely uncharacterized impact of allelic diversity, the most common KIR2DL2/3 allelic products in European American and African American populations were evaluated for surface expression and binding affinity to their HLA-C group 1 and 2 ligands. Although no significant differences in the degree of cell membrane localization were detected in a transfected human NKL cell line by flow cytometry, surface plasmon resonance and KIR binding to a panel of HLA allotypes demonstrated that KIR2DL3*005 differed significantly from other KIR2DL3 allelic products in its ability to bind HLA-C. The increased affinity and avidity of KIR2DL3*005 for its ligand was also demonstrated to have a larger impact on the inhibition of IFN-γ production by the human KHYG-1 NK cell line compared with KIR2DL3*001, a low-affinity allelic product. Site-directed mutagenesis established that the combination of arginine at residue 11 and glutamic acid at residue 35 in KIR2DL3*005 were critical to the observed phenotype. Although these residues are distal to the KIR/HLA-C interface, molecular modeling suggests that alteration in the interdomain hinge angle of KIR2DL3*005 toward that found in KIR2DL2*001, another strong receptor of the KIR2DL2/3 family, may be the cause of this increased affinity. The regain of inhibitory capacity by KIR2DL3*005 suggests that the rapidly evolving KIR locus may be responding to relatively recent selective pressures placed upon certain human populations.

HLA reduces killer cell Ig-like receptor expression level and frequency in a humanized mouse model.

NK cells use NK cell receptors to be able to recognize and eliminate infected, transformed, and allogeneic cells. Human NK cells are prevented from killing autologous healthy cells by virtue of inhibitory NKRs, primarily killer cell Ig-like receptors (KIR) that bind "self" HLA class I molecules. Individual NK cells stably express a selected set of KIR, but it is currently disputed whether the fraction of NK cells expressing a particular inhibitory KIR is influenced by the presence of the corresponding HLA ligand. The extreme polymorphism of the KIR and HLA loci, with wide-ranging affinities for individual KIR and HLA allele combinations, has made this issue particularly hard to tackle. In this study, we used a transgenic mouse model to investigate the effect of HLA on KIR repertoire and function in the absence of genetic variation inside and outside the KIR locus. These H-2K(b-/-) and H-2D(b-/-) mice lacked ligands for inhibitory Ly49 receptors and were transgenic for HLA-Cw3 and a KIR B haplotype. In this reductionist system, the presence of HLA-Cw3 reduced the frequency of KIR2DL2(+) cells, as well as the surface expression levels of KIR2DL2. In addition, in the presence of HLA-Cw3, the frequency of NKG2A(+) cells and the surface expression levels of NKG2A were reduced. In line with these findings, both transgene-encoded KIR and endogenous NKG2A contributed to the rejection of cells lacking HLA-Cw3. These findings support the idea that HLA influences the human KIR repertoire.

HLA-Cw*0102-restricted HIV-1 p24 epitope variants can modulate the binding of the inhibitory KIR2DL2 receptor and primary NK cell function.

Accumulating evidence suggests an important role for Natural Killer (NK) cells in the control of HIV-1 infection. Recently, it was shown that NK cell-mediated immune pressure can result in the selection of HIV-1 escape mutations. A potential mechanism for this NK cell escape is the selection of HLA class I-presented HIV-1 epitopes that allow for the engagement of inhibitory killer cell immunoglobulin-like receptors (KIRs), notably KIR2DL2. We therefore investigated the consequences of sequence variations within HLA-Cw*0102-restricted epitopes on the interaction of HLA-Cw*0102 with KIR2DL2 using a large panel of overlapping HIV-1 p24 Gag peptides. 217 decameric peptides spanning the HIV-1 p24 Gag consensus sequence were screened for HLA-Cw*0102 stabilization by co-incubation with Cw*0102⁺/TAP-deficient T2 cells using a flow cytometry-based assay. KIR2DL2 binding was assessed using a KIR2DL2-IgG fusion construct. Function of KIR2DL2⁺ NK cells was flow cytometrically analyzed by measuring degranulation of primary NK cells after co-incubation with peptide-pulsed T2 cells. We identified 11 peptides stabilizing HLA-Cw*0102 on the surface of T2 cells. However, only one peptide (p24 Gag209₋218 AAEWDRLHPV) allowed for binding of KIR2DL2. Notably, functional analysis showed a significant inhibition of KIR2DL2⁺ NK cells in the presence of p24 Gag209₋218-pulsed T2 cells, while degranulation of KIR2DL2⁻ NK cells was not affected. Moreover, we demonstrated that sequence variations in position 7 of this epitope observed frequently in naturally occurring HIV-1 sequences can modulate binding to KIR2DL2. Our results show that the majority of HIV-1 p24 Gag peptides stabilizing HLA-Cw*0102 do not allow for binding of KIR2DL2, but identified one HLA-Cw*0102-presented peptide (p24 Gag209₋218) that was recognized by the inhibitory NK cell receptor KIR2DL2 leading to functional inhibition of KIR2DL2-expressing NK cells. Engagement of KIR2DL2 might protect virus-infected cells from NK cell-mediated lysis and selections of sequence polymorphisms that increase avidity to KIR2DL2 might provide a mechanism for HIV-1 to escape NK cell-mediated immune pressure.

Cytomegalovirus reactivation after allogeneic transplantation promotes a lasting increase in educated NKG2C+ natural killer cells with potent function.

During mouse cytomegalovirus (CMV) infection, a population of Ly49H(+) natural killer (NK) cells expands and is responsible for disease clearance through the induction of a "memory NK-cell response." Whether similar events occur in human CMV infection is unknown. In the present study, we characterized the kinetics of the NK-cell response to CMV reactivation in human recipients after hematopoietic cell transplantation. During acute infection, NKG2C(+) NK cells expanded and were potent producers of IFNγ. NKG2C(+) NK cells predominately expressed killer cell immunoglobulin-like receptor, and self-killer cell immunoglobulin-like receptors were required for robust IFNγ production. During the first year after transplantation, CMV reactivation induced a more mature phenotype characterized by an increase in CD56(dim) NK cells. Strikingly, increased frequencies of NKG2C(+) NK cells persisted and continued to increase in recipients who reactivated CMV, whereas these cells remained at low frequency in recipients without CMV reactivation. Persisting NKG2C(+) NK cells lacked NKG2A, expressed CD158b, preferentially acquired CD57, and were potent producers of IFNγ during the first year after transplantation. Recipients who reactivated CMV also expressed higher amounts of IFNγ, T-bet, and IL-15Rα mRNA transcripts. Our findings support the emerging concept that CMV-induced innate memory-cell populations may contribute to malignant disease relapse protection and infectious disease control long after transplantation.

The aberrant expression of stimulatory and inhibitory killer immunoglobulin-like receptors in NK- and NKT-cells contributes to lupus.

BACKGROUND: Killer cell immunoglobulin-like receptors (KIR) contribute to the pathogenesis of multiple auto-immune diseases via the modulation of NK-, NKT- and T-cells. Thus, we want to know whether the expression pattern of KIR is associated with systemic lupus erythematosus (SLE) susceptibility. METHODS: Here, real-time quantitative PCR and fluorescence-activated cell sorting (FACS) were used to measure the stimulatory KIR (sKIR) and inhibitory KIR (iKIR) mRAN and protein levels on NK-, NKT- and T-cells in both SLE patients and healthy controls. RESULTS: In SLE patients, CD158a/h (KIR2DL1/S1) was highly expressed while CD158b/i/j (KIR2DL2/L3/S2, iKIR/iKIR/sKIR) was lowly expressed in NK- and NKT-cells in patients. The expression levels of KIR2DL1 and KIR2DL2 (iKIRs) were decreased while the expression levels of KIR2DS1 (sKIR) were increased in NK- and NKT-cells in the patients. CONCLUSIONS: We found that SLE patients represent aberrant expression of stimulatory and inhibitory KIRs in NK- and NKT-cells. Consequently, these different expression levels of KIRs may contribute to the abnormal function of these cells, which lead to the risk of SLE.

CMV drives clonal expansion of NKG2C+ NK cells expressing self-specific KIRs in chronic hepatitis patients.

Natural killer (NK) cells are affected by infection with human cytomegalovirus (HCMV) manifested by increased expression of the HLA-E binding activating receptor NKG2C. We here show that HCMV seropositivity was associated with a profound expansion of NKG2C(+) CD56(dim) NK cells in patients with chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infection. Multi-color flow cytometry revealed that the expanded NKG2C(+) CD56(dim) NK cells displayed a highly differentiated phenotype, expressed high amounts of granzyme B and exhibited polyfunctional responses (CD107a, IFN-γ, and TNF-α) to stimulation with antibody-coated as well as HLA-E expressing target cells but not when stimulated with IL-12/IL-18. More importantly, NKG2C(+) CD56(dim) NK cells had a clonal expression pattern of inhibitory killer cell immunoglobulin-like receptors (KIRs) specific for self-HLA class I molecules, with predominant usage of KIR2DL2/3. KIR engagement dampened NKG2C-mediated activation suggesting that such biased expression of self-specific KIRs may preserve self-tolerance and limit immune-pathology during viral infection. Together, these findings shed new light on how the human NK-cell compartment adjusts to HCMV infection resulting in clonal expansion and differentiation of educated and polyfunctional NK cells.

KIR2DL2 enhances protective and detrimental HLA class I-mediated immunity in chronic viral infection.

Killer cell immunoglobulin-like receptors (KIRs) influence both innate and adaptive immunity. But while the role of KIRs in NK-mediated innate immunity is well-documented, the impact of KIRs on the T cell response in human disease is not known. Here we test the hypothesis that an individual's KIR genotype affects the efficiency of their HLA class I-mediated antiviral immune response and the outcome of viral infection. We show that, in two unrelated viral infections, hepatitis C virus and human T lymphotropic virus type 1, possession of the KIR2DL2 gene enhanced both protective and detrimental HLA class I-restricted anti-viral immunity. These results reveal a novel role for inhibitory KIRs. We conclude that inhibitory KIRs, in synergy with T cells, are a major determinant of the outcome of persistent viral infection.

Analyses of HLA-C-specific KIR repertoires in donors with group A and B haplotypes suggest a ligand-instructed model of NK cell receptor acquisition.

To determine the influence of KIR and HLA class I polymorphism on human NK cell repertoires, 32 different clonotypes representing all possible combinations of 4 inhibitory KIR and NKG2A were analyzed by multicolor flow cytometry. In donors homozygous for the common group A KIR haplotype, a significant influence of HLA-C ligands was seen: KIR repertoires were dominated by clonotypes expressing a single KIR for the respective cognate ligand, either the C1-specific KIR2DL3 or C2-specific KIR2DL1. In contrast, in donors possessing the polymorphic group B haplotypes, a similar adaptation to cognate HLA-C was lacking. We suggest that this discrepancy is largely the result of a suppressive effect of the group B-specific KIR2DL2 on the frequency of KIR2DL1(+) NK cells. In functional assays, KIR2DL2 not only recognized C1 but also C2 ligands, showing overlapping specificity with KIR2DL1. Moreover, using an NK cell differentiation assay we show sequential acquisition of KIR2DL2 before KIR2DL1 on developing NK cells. Together, these observations are compatible with a ligand-instructed model of NK cell education, in which recognition of HLA class I by an inhibitory receptor (KIR2DL2) suppresses subsequent expression of a second receptor (KIR2DL1) of related specificity. Importantly, the ligand-instructed model fits to the observed KIR repertoires in both broad KIR haplotype groups.

Peptide antagonism as a mechanism for NK cell activation.

Inhibition of natural killer (NK) cells is mediated by MHC class I receptors including the killer cell Ig-like receptor (KIR). We demonstrate that HLA-C binding peptides can function as altered peptide ligands for KIR and antagonize the inhibition mediated by KIR2DL2/KIR2DL3. Antagonistic peptides promote clustering of KIR at the interface of effector and target cells, but do not result in inhibition of NK cells. Our data show that, as for T cells, small changes in the peptide content of MHC class I can regulate NK cell activity.

Quantity of HLA-C surface expression and licensing of KIR2DL+ natural killer cells.

Natural killer (NK) cells require interaction of inhibitory surface receptors with human leukocyte antigen (HLA) ligands during development to acquire functional competence in a process termed "licensing." The quantity of HLA required for this process is unknown. Two polymorphisms affecting HLA-C surface expression (rs9264942 and rs67384697) have recently been identified, and shown to influence progression of HIV infection. We typed a cohort of healthy donors for the two HLA-C-related polymorphisms, KIR2DL1 and KIR2DL3, and their respective HLA-C ligands and analyzed how HLA ligands influenced licensing status of killer cell immunoglobulin-like receptor (KIR)+ NK cells in terms of degranulation and cytokine production in response to HLA-deficient target cells. The presence of respective HLA class I ligands increased the function of KIR2DL1+ and KIR2DL3+ NK cells in a dose-dependent manner. In contrast, neither of the HLA-C-related polymorphisms nor the quantity of cell surface HLA-C had any significant effect on NK cell function. Interestingly, HLA-Cw7-an HLA-C allele with low surface expression-licensed KIR2DL3+ NK cells more strongly than any other KIR2DL3 ligand. The quantity of cell surface HLA-C does not appear to influence licensing of NK cells, and the HLA-C-related polymorphisms presumably influence HIV progression through factors unrelated to NK cell education.

CD158k/KIR3DL2 and NKp46 are frequently expressed in transformed mycosis fungoides.

Malignant Sezary cells express the natural killer (NK) receptors KIR3DL2 (CD158k) and Nkp46 and may co-express activating killer immunoglobulin-like receptors (KIR) that may participate to neoplastic T-cell activation through the JNK pathway. Little is known regarding NK receptor expression in other cutaneous T-cell lymphomas. We studied the expression of KIR and natural cytotoxicity receptor (NCR) transcripts, and KIR3DL1/2 at the protein level, in 16 skin biopsies from 10 patients with transformed mycosis fungoides (tMF). Some KIR and NCR transcripts were found in all cases, with various repertoires. Two to nine different KIR receptors were expressed in a single biopsy. Among them, KIR3DL2 was the most frequent, with the highest level of expression in quantitative analyses and correlated with in situ protein expression, while phosphorylated JNK was never detected. Among NCR, NKp46 was expressed in all investigated cases. The role of KIR3DL2 and NKp46 in tMF oncogenesis remains to be studied.

The Interaction of HLA-C1/KIR2DL2/L3 Promoted KIR2DL2/L3 Single-Positive/NKG2C-Positive Natural Killer Cell Reconstitution, Raising the Incidence of aGVHD after Hematopoietic Stem Cell Transplantation.

NKG2C+ natural killer (NK) cell plays a vital role in CMV infection control after hematopoietic stem cell transplantation (HSCT). However, the modulation on NKG2C+ NK cell reconstitution is still unclear. NK cell education is affected by the interactions of HLA-I/killer immunoglobulin receptor (KIR). Our aim is to figure out which HLA-I/KIR interaction plays a dominant role in NKG2C+ NK education. Based on allogeneic haploidentical HSCT, we investigated the expansion and function of single KIR positive NKG2C+ NK cells via the interaction of KIR with both donor HLA and recipient HLA at days 30, 90, and 180 after HSCT. KIR2DL2/L3 single-positive/NKG2C+ cells were significantly expanded compared with KIR2DL1 or KIR3DL1 single-positive/NKG2C+ cells when donors and recipients were both HLA-C1/C1 or HLA-C1C1BW4 (p < 0.05), with higher NKp30 expression (p < 0.05). Moreover, the proportion of single KIR positive NK cells increased in both NKG2C+/NKG2A- NK cells and conventional NKG2C-/NKG2A- NK cells over time. We also observed that increased proportion of KIR2DL2/L3 single-positive/NKG2C+ NK cells correlated with higher incidence of acute graft-versus-host disease (aGVHD). Our study allows a better understanding of HLA-I/KIR interaction in the NKG2C+ NK cell education after HSCT.

The stage dependent changes in NK cell activity and the expression of activating and inhibitory NK cell receptors in melanoma patients.

BACKGROUND: As NK cell antitumor activity is regulated by the balance between numerous activating and inhibitory receptors the aim of this study was to analyze the changes in these receptors expression as well as the differences in their association with NK cell activity in melanoma patients in different clinical stages. MATERIALS AND METHODS: Forty-three melanoma patients in the three different clinical stages and 17 healthy controls were analyzed for NK cell activity by the standard (51)Cr radioactive assay, as well as for the percentage and absolute number of CD3-CD56+ NK cells and their subsets and the expression of degranulation marker CD107a, activating NKG2D and CD161, and inhibitory KIR, CD158a, and CD158b receptors on CD3-CD56+ NK cells by flow cytometry. IFN-γ and TNF-α PBL production were evaluated by enzyme-linked immunosorbent assay (ELISA). RESULTS: We show that contrary to nonmetastatic, metastatic melanoma patients have significantly impaired NK cell activity, lower CD107a expression, as well as decreased production of IFN-γ and TNF-α. Furthermore, these melanoma patients have a significant decrease in the expression of activating NKG2D receptor, which positively correlates with NK cytotoxicity, and a significant increase in the expression of inhibitory CD158b on CD3-CD56+ NK cells compared with healthy controls. CONCLUSIONS: In this study, we show that metastatic melanoma patients have significant changes in NK cell activity and the expression of activating as well as inhibitory NK cell receptors. These results indicate that only in this patient group, the blocking of inhibitory or enhancing of activating NK cell receptor expression may promote NK cell-mediated cytolysis.

Structural plasticity of KIR2DL2 and KIR2DL3 enables altered docking geometries atop HLA-C.

The closely related inhibitory killer-cell immunoglobulin-like receptors (KIR), KIR2DL2 and KIR2DL3, regulate the activation of natural killer cells (NK) by interacting with the human leukocyte antigen-C1 (HLA-C1) group of molecules. KIR2DL2, KIR2DL3 and HLA-C1 are highly polymorphic, with this variation being associated with differences in the onset and progression of some human diseases. However, the molecular bases underlying these associations remain unresolved. Here, we determined the crystal structures of KIR2DL2 and KIR2DL3 in complex with HLA-C*07:02 presenting a self-epitope. KIR2DL2 differed from KIR2DL3 in docking modality over HLA-C*07:02 that correlates with variabilty of recognition of HLA-C1 allotypes. Mutagenesis assays indicated differences in the mechanism of HLA-C1 allotype recognition by KIR2DL2 and KIR2DL3. Similarly, HLA-C1 allotypes differed markedly in their capacity to inhibit activation of primary NK cells. These functional differences derive, in part, from KIR2DS2 suggesting KIR2DL2 and KIR2DL3 binding geometries combine with other factors to distinguish HLA-C1 functional recognition.