Inhibitory Killer Ig-like Receptors Research Articles

Selection of allogeneic hematopoietic cell transplant donors to optimize natural killer cell alloreactivity

Natural killer (NK) cells are potent mediators of the graft versus leukemia phenomenon critical to the success of allogeneic hematopoietic cell transplantation. Central to calibrating NK effector function via their interaction with class I human leukocyte antigens are the numerous inhibitory killer Ig-like receptors (KIR). The KIR receptors are encoded by a family of polymorphic genes, whose expression is largely stochastic and uninfluenced by human leukocyte antigens genotype. These features provide the opportunity to select hematopoietic cell donors with favorable KIR genotypes that confer enhanced protection from relapse via NK-mediated graft versus leukemia. Over the last 2 decades, a large body of work has emerged examining the use of KIR genotyping to stratify potential donors based on anticipated NK alloreactivity. Overall, these results support KIR-based donor selection for patients undergoing allogeneic hematopoietic cell transplantation for a diagnosis of acute myelogenous leukemia. Despite this, the underlying factors that control NK cell responsiveness are not completely understood, and opportunities remain to refine donor selection using NK cell receptor genotyping. In this review, we will summarize the relevant findings with respect to KIR genotyping as a selection parameter for allogeneic hematopoietic cell donors and address practical considerations with respect to KIR-based selection of donors for patients with myeloid neoplasia.

Impact of the Variable Killer Ig-Like Receptor-Human Leukocyte Antigen Interactions on Natural Killer Cell Cytotoxicity Toward Foreign CD4 T Cells.

Natural killer (NK) cells are known to mount a response against foreign target cells, where the absence of the dominant inhibitory killer Ig-like receptor (KIR)-human leukocyte antigen (HLA) interaction immensely lowers the threshold for NK cell activation. NK cells could thus constitute a vital part in the mucosal defense against cell-associated sexually transmitted diseases. Here, we performed a detailed analysis of hitherto unexplored KIR-HLA-incompatible NK cell interactions. In vitro, healthy NK cells were cocultured with CD4+ T cells derived from human immunodeficiency virus-1 patients, and the KIR-specific NK cell cytotoxicity was measured using flow cytometry. Genotyping of KIR and HLA predicted the KIR-HLA interactions occurring during these 124 allogeneic encounters. KIR2DL1+ NK cells were seen as the strongest intrinsic responders in the absence of their ligand with a 3.2-fold increase in KIR2DL1+ NK cells in the total NK cell response. An association between the size of the alloreactive NK cell population and the amount of CD4+ T cell death (p = 0.0023) and NK cell degranulation (p = 0.0036) was only present in NK cell donors with an activating KIR haplotype. We demonstrate differences in the activating effect of KIR-HLA incompatibility according to the KIR involved, with KIR2DL1 as the strongest responder. An activating KIR haplotype optimized the contribution of KIR-HLA-incompatible NK cells in the total NK cell response.

Human NK Cells Downregulate Zap70 and Syk in Response to Prolonged Activation or DNA Damage.

The extent of NK cell activity during the innate immune response affects downstream immune functions and, ultimately, the outcome of infectious or malignant disease. However, the mechanisms that terminate human NK cell responses have yet to be defined. When activation receptors expressed on NK cell surfaces bind to ligands on diseased cells, they initiate a signal that is propagated by a number of intracellular kinases, including Zap70 and Syk, eventually leading to NK cell activation. We assayed Zap70 and Syk content in NK cells from healthy human donors and identified a subset of NK cells with unusually low levels of these two kinases. We found that this Zap70lowSyklow subset consisted of NK cells expressing a range of surface markers, including CD56hi and CD56low NK cells. Upon in vitro stimulation with target cells, Zap70lowSyklow NK cells failed to produce IFN-γ and lysed target cells at one third the capacity of Zap70hiSykhi NK cells. We determined two independent in vitro conditions that induce the Zap70lowSyklow phenotype in NK cells: continuous stimulation with activation beads and DNA damage. The expression of inhibitory receptors, including NKG2A and inhibitory killer Ig-like receptors (KIRs), was negatively correlated with the Zap70lowSyklow phenotype. Moreover, expression of multiple KIRs reduced the likelihood of Zap70 downregulation during continuous activation, regardless of whether NK cells had been educated through KIR-HLA interactions in vivo. Our findings show that human NK cells are able to terminate their functional activity without the aid of other immune cells through the downregulation of activation kinases.

Donor-Recipient Matching for KIR Genotypes Reduces Chronic GVHD and Missing Inhibitory KIR Ligands Protect against Relapse after Myeloablative, HLA Matched Hematopoietic Cell Transplantation

BackgroundAllogeneic hematopoietic cell transplantation (HCT) can be curative for many hematologic diseases. However, complications such as graft-versus-host disease (GVHD) and relapse of primary malignancy remain significant and are the leading causes of morbidity and mortality. Effects of killer Ig-like receptors (KIR)-influenced NK cells on HCT outcomes have been extensively pursued over the last decade. However, the relevance of the reported algorithms on HLA matched myeloablative HCT with rabbit antithymocyte globulin (ATG) is used for GVHD prophylaxis remains elusive. Here we examined the role of KIR and KIR-ligands of donor-recipient pairs in modifying the outcomes of ATG conditioned HLA matched sibling and unrelated donor HCTMethods and FindingsThe study cohort consisted of 281 HLA matched sibling and unrelated donor-recipient pairs of first allogeneic marrow or blood stem cell transplantation allocated into ‘discovery’ (135 pairs) and ‘validation’ (146 pairs) cohorts. High resolution HLA typing was obtained from the medical charts and KIR gene repertoires were obtained by a Luminex® based SSO method. All surviving patients were followed-up for a minimum of two years. KIR and HLA class I distributions of HCT pairs were stratified as per applicable definitions and were tested for their association with cause specific outcomes [acute GVHD grade II-IV (aGVHD), chronic GVHD needing systemic therapy (cGVHD) and relapse] using a multivariate competing risks regression model as well as with survival outcomes [relapse-free survival (RFS), cGVHD & relapse free survival (cGRFS) and overall survival (OS)] by multivariate Cox proportional hazards regression model. A significant association between KIR genotype mismatching (KIR-B/x donor into KIR-AA recipient or vice versa) and cGVHD was found in both discovery (p = 0.001; SHR = 2.78; 95%CI: 1.50–5.17) and validation cohorts (p = 0.005; SHR = 2.61; 95%CI: 1.33–5.11). High incidence of cGVHD associated with KIR genotype mismatching was applicable to both sibling and unrelated donors and was specific to recipients who had one or two C1 bearing HLA-C epitopes (HLA-C1/x, p = 0.001; SHR = 2.40; 95%CI: 1.42–4.06). When compared with KIR genotype mismatched transplants, HLA-C1/x patients receiving grafts from KIR genotype matched donors had a significantly improved cGRFS (p = 0.013; HR = 1.62; 95%CI: 1.11–2.39). Although there was no effect of KIR genotype matching on survival outcomes, a significantly reduced incidence of relapse (p = 0.001; SHR = 0.22; 95%CI: 0.10–0.54) and improved relapse-free survival (p = 0.038; HR = 0.40; 95%CI: 0.17–0.95) was observed with one or more missing ligands for donor inhibitory KIR among the recipients of unrelated donor transplants.ConclusionsThe present study for the first time presents the beneficial effects of KIR genotype matching in reducing cGVHD in myeloablative transplant setting using HLA matched (sibling and unrelated) donors. The findings offer a clinically applicable donor selection strategy that can help control cGVHD without affecting the risk of relapse and/or identify patients at a high risk of developing cGVHD as potential candidates for preemptive therapy. The findings also affirm the beneficial effect of one or more missing inhibitory KIR ligands in the recipient in reducing relapse and improving a relapse free survival in unrelated donor transplants.

The Size of Activating and Inhibitory Killer Ig-like Receptor Nanoclusters Is Controlled by the Transmembrane Sequence and Affects Signaling

SummarySuper-resolution microscopy has revealed that immune cell receptors are organized in nanoscale clusters at cell surfaces and immune synapses. However, mechanisms and functions for this nanoscale organization remain unclear. Here, we used super-resolution microscopy to compare the surface organization of paired killer Ig-like receptors (KIR), KIR2DL1 and KIR2DS1, on human primary natural killer cells and cell lines. Activating KIR2DS1 assembled in clusters two-fold larger than its inhibitory counterpart KIR2DL1. Site-directed mutagenesis established that the size of nanoclusters is controlled by transmembrane amino acid 233, a lysine in KIR2DS1. Super-resolution microscopy also revealed two ways in which the nanoscale clustering of KIR affects signaling. First, KIR2DS1 and DAP12 nanoclusters are juxtaposed in the resting cell state but coalesce upon receptor ligation. Second, quantitative super-resolution microscopy revealed that phosphorylation of the kinase ZAP-70 or phosphatase SHP-1 is favored in larger KIR nanoclusters. Thus, the size of KIR nanoclusters depends on the transmembrane sequence and affects downstream signaling.

Regulatory and effector functions of gamma-delta (γδ) T cells and their therapeutic potential in adoptive cellular therapy for cancer.

γδ T cells are an important innate immune component of the tumor microenvironment and are known to affect the immune response in a wide variety of tumors. Unlike αβ T cells, γδ T cells are capable of spontaneous secretion of IL-17A and IFN-γ without undergoing clonal expansion. Although γδ T cells do not require self-MHC-restricted priming, they can distinguish "foreign" or transformed cells from healthy self-cells by using activating and inhibitory killer Ig-like receptors. γδ T cells were used in several clinical trials to treat cancer patient due to their MHC-unrestricted cytotoxicity, ability to distinguish transformed cells from normal cells, the capacity to secrete inflammatory cytokines and also their ability to enhance the generation of antigen-specific CD8(+) and CD4(+) T cell response. In this review, we discuss the effector and regulatory function of γδ T cells in the tumor microenvironment with special emphasis on the potential for their use in adoptive cellular immunotherapy.

Abstract 2459: KIR3DL1 and HLA-B subtype combinations predict the efficacy of 3F8 monoclonal antibody therapy for neuroblastoma

Abstract Background: Treatment of high-risk neuroblastoma (NB) with anti-GD2 monoclonal antibody (3F8) recruits Natural Killer (NK) cells for antibody-dependent-cell mediated cytotoxicity (ADCC). NK reactivity is triggered when the signals for activation outweigh inhibition; principal among the inhibitory ligands for NK cells are HLA, detected by inhibitory killer Ig-like receptors (KIR). Among receptor-ligand partnerships, KIR3DL1 and HLA-B exhibit the greatest diversity. Variation in KIR3DL1 surface expression (null, low or high) and dimorphism at HLA-Bw4 amino-acid sequence 80 (isoleucine v. threonine) are associated with varied strengths of interaction. We hypothesized that KIR3DL1/HLA-Bw4 partnerships could influence the outcome of 3F8 treatment by varying the strength of inhibitory signaling. Methods: KIR3DL1 and HLA-B subtype combinations were assessed in a cohort of 245 patients with high-risk NB treated with 3F8 by medium-resolution PCR. Patients were grouped based on strength of interaction expected from KIR3DL1 subtype/HLA-Bw4: Strong-interactors (3DL1-high+Bw4-80I; 3DL1-low+Bw4-80T); weak-interactors (3DL1-high+Bw4-80T; 3DL1-low+Bw4-80I); and the non-interactors, where no KIR3DL1/Bw4 interaction is expected (3DL1-null or KIR3DS1 + any HLA-Bw4; Bw6/Bw6 + any 3DL1) were compared for their influences on overall and progression-free survival. Results: The frequency of KIR3DL1/HLA-B partnerships were: strong-interactors 25.3%, weak-interactors 22.4%, and non-interactors 52.2%. The 5-year progression-free survival (PFS) for strong-interactors, weak-interactors, and non-interactors was 28.6% [95% CI:19.3-42.2%], 34.1% [95% CI:23.5-49.4%] and 52.2% [95% CI:44.1-61.7%], respectively (p = 0.005). The 5-year overall survival (OS) for strong-interactors, weak-interactors, and non-interactors was 49.2% [95% CI: 38.3-63.2%], 44.8% [95% CI:33.3-60.3%] and 63.1% [95% CI:55.2-72.2%] (p = 0.013). When controlling for age, lactate dehydrogenase, and disease status at time of treatment the benefit of non-interacting subtypes was maintained for PFS (HR 0.41, [95% CI: 0.27-0.62], p = <0.001) and OS (HR 0.42, [95% CI:0.26-0.67], p = <0.001). Conclusions: In patients with NB treated with 3F8, non-interacting subtypes are predictive of improved progression-free and overall survival. Comparatively, patients whose KIR3DL1 and HLA-B allele subtypes predict for strong and weak interaction exhibit high and intermediate risk for disease progression. Collectively, our findings support KIR3DL1 and HLA-B subtype analysis for prognostication and indicate disinhibition of NK cells as a therapeutic target. Citation Format: Christopher J. Forlenza, Jeanette E. Boudreau, Junting Zheng, Glenn Heller, Nai-Kong V. Cheung, Katharine C. Hsu. KIR3DL1 and HLA-B subtype combinations predict the efficacy of 3F8 monoclonal antibody therapy for neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2459. doi:10.1158/1538-7445.AM2015-2459

Abstract 1175: Interleukin-15 activates NK and CD8 T cells within the Prostate cancer microenvironment by expanding activatory NK cell receptors and attenuating inhibitory HLA ligands on tumor cells

Abstract Background The prostate cancer microenvironment is profoundly immunosuppressive and NK and T cells infiltrating this environment are rendered anergic or suppressive. Recently, immunomodulatory checkpoints such as PDL1 have been shown to be important in this immunosuppression. Antibodies to such checkpoints have a moderate effect in suppressing tumor growth but many unknown immunosuppressive factors exist on tumor cells. We have shown that prostate cancer cells express many ligands that inhibit NK cell activity by binding to inhibitory Killer Ig like receptors (KIRs). These receptors are critical in modulating NK cell function. We have shown that IL-15, unlike other immunotherapeutic cytokines (e.g IL-12), increases NK and CD8-T-cell activity within the prostate cancer microenvironment. To investigate mechanisms of this activation, we studied effects of IL-15 on inhibitory and activatory receptors on NK cells and their corresponding ligands on prostate cancer cells. Methods The prostate cancer cell-lines PC3 and LNCaP were incubated with non-adherent PBMCs at effector:target ratios of 8:1 and cytokines IL-2 or IL-15. After 1 week, NK-cells in the cocultures were stained with antibodies to inhibitory KIRs (KIR2DL1,KIR2DL2,KIR3DL1) and activatory KIRs (NKp44,NKG2D, NKp46, and DNAM1). Tumor cells in the cocultures were stained with antibodies to inhibitory NK-receptor ligands HLA-class1-BW4 and HLA-G, and activatory receptor ligands Nectin-2 and MICA/MICB. Antibody to HLA-ABC was also used. Staining was followed by flow cytometric analysis. Results IL-15, but not IL-2 inhibited expression of the inhibitory-receptors KIR2DL1 and KIR3DL1 by upto 50%*on NK cells in PBMC-PCa cell cocultures. NKG2D was increased upto 40%* in these cells with IL-15 but not IL-2. On tumor cells, IL-15, but not IL-2 decreased the inhibitory-ligands HLA-Class-1-BW4 by upto 60%* on both LNCaP and PC3 cells. IL-15 also decreased the expression of HLA-G by 75%* on LNCaPs. HLA-A,B,C expression was increased by over 65%* in the two prostate cancer cell lines. Initial data showed that activatory-ligands were not affected on either PC3 or LNCaP tumor cells. * =(p<0.001,1-way-anova, post-hoc Newman-Keuls,n=5). Conclusion One mechanism by which IL-15 increases PCa killing by NK cells in PBMC-PCa cocultures is through upregulating activatory NKG2D and decreasing inhibitory KIR2DL1 and KIR3DL1 receptors. In addition, inhibitory ligands on PCa cells are downregulated, suggesting strong shifts toward NK activation vs inhibition in the cocultures. Increased HLA-A,B,C also favors stronger cytotoxic-T cell recognition. This suggests strong shifts toward NK activation vs inhibition in the cocultures. Increased HLA-A,B,C on the tumor cells also favors stronger cytotoxic-T cell recognition. The molecular basis of these effects are currently being investigated. Citation Format: Christina Alexandra Sakellariou, Oussama Elhage, Osamu Ukimura, Inderbir Gill, Richard Anthony Smith, Prokar Dasgupta, Christine Galustian. Interleukin-15 activates NK and CD8 T cells within the Prostate cancer microenvironment by expanding activatory NK cell receptors and attenuating inhibitory HLA ligands on tumor cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1175. doi:10.1158/1538-7445.AM2014-1175

MP24-17 INTERLEUKIN-15 DOWNREGULATES INHIBITORY NATURAL KILLER(NK) CELL LIGANDS EXPRESSED ON PROSTATE CANCER CELLS RESULTING IN INCREASED NK CELL ACTIVITY IN PROSTATE CANCER-IMMUNE EFFECTOR CELL COCULTURES.

You have accessJournal of UrologyProstate Cancer: Basic Research I1 Apr 2014MP24-17 INTERLEUKIN-15 DOWNREGULATES INHIBITORY NATURAL KILLER(NK) CELL LIGANDS EXPRESSED ON PROSTATE CANCER CELLS RESULTING IN INCREASED NK CELL ACTIVITY IN PROSTATE CANCER-IMMUNE EFFECTOR CELL COCULTURES. Christine Galustian, Christina Sakellariou, Oussama Elhage, Osamu Ukimura, Inderbir Gill, Richard Smith, and Prokar Dasgupta Christine GalustianChristine Galustian More articles by this author , Christina SakellariouChristina Sakellariou More articles by this author , Oussama ElhageOussama Elhage More articles by this author , Osamu UkimuraOsamu Ukimura More articles by this author , Inderbir GillInderbir Gill More articles by this author , Richard SmithRichard Smith More articles by this author , and Prokar DasguptaProkar Dasgupta More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.298AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The prostate cancer microenvironment is profoundly immunosuppressive and NK cells and T cells that infiltrate this environment are rendered anergic or suppressive. Recently, immunomodulatory checkpoints such as PDL1 and CTLA-4 have been shown to be important in creating this immunosuppression. Antibodies to these have a moderate effect in suppressing tumour growth but many unknown immunosuppressive factors exist on tumour cells. We have shown that prostate cancer cells express a wide range of ligands that inhibit NK cell activity by binding to inhibitory Killer Ig like receptors (KIRs). These receptors are critical in modulating the function of NK cells. Also, these cells have a downregulated expression of HLA molecules that render them less susceptible to T cell mediated recognition and killing. We have shown that IL-15, unlike other immunotherapeutic cytokines (e.g IL-12, IL-21), increases NK and CD8-T-cell activity within the prostate cancer microenvironment. We have previously demonstrated that IL-15 upregulates activatory KIRs and downregulates inhibitory KIRs to increase NK cell activity. To further these investigations, we have now examined the effects of IL-15 on ligands on prostate cancer cells that are recognised by activatory and inhibitory KIRs. METHODS PC3 and LNCaP prostate cancer cell-lines were incubated with non-adherent PBMCs at effector:target ratios of 8:1 and cytokines IL-2 or IL-15. After 1 week, Tumour cells were stained with antibodies to inhibitory HLA-class1-BW4 and HLA-G, and activatory Nectin-2 and MICA/MICB KIR-ligands. Antibody to HLA-ABC was also used. Flow cytometric analysis followed staining. RESULTS IL-15, but not IL-2 decreased the inhibitory-ligands HLA-Class-1-BW4 by upto 60%* on both LNCaP and PC3 cells. IL-15 also decreased the expression of HLA-G by 75%* on LNCaP cells. HLA-A,B,C expression was increased by over 65%* in the two prostate cancer cell lines. Initial data showed that activatory-ligands were not affected on either PC3 or LNCaP tumour cells. * =(p<0.001,1-way-anova, post-hoc Newman-Keuls,n=5). CONCLUSIONS IL-15 can downregulate inhibitory NK ligands on prostate cancer cells in addition to its ability to modulate activatory and inhibitory KIRs. This suggests strong shifts toward NK activation vs inhibition in the cocultures. Increased HLA-A,B,C on the tumour cells also favours stronger cytotoxic-T cell recognition. The molecular basis of these effects are currently being investigated. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e265 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Christine Galustian More articles by this author Christina Sakellariou More articles by this author Oussama Elhage More articles by this author Osamu Ukimura More articles by this author Inderbir Gill More articles by this author Richard Smith More articles by this author Prokar Dasgupta More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Mechanism of activation of natural killer and CD8 T cells by interleukin 15 in the prostate cancer microenvironment

BackgroundThe prostate cancer microenvironment profoundly inhibits effector immune-cell activity. We have shown that interleukin (IL) 15, unlike other cytokines proposed for anti-tumour immunotherapy such as IL12 and IL21, can increase natural killer (NK) cell and CD8 T-cell activity within the prostate cancer microenvironment. To investigate mechanisms of this activation, we studied the effects of IL15 on inhibitory and activatory receptors on NK cells and their corresponding ligands on prostate cancer cells. NK receptors known as killer-Ig-like receptors (KIRs) have a crucial role in NK-mediated killing of tumour cells. NK-receptor ligands on tumour cells can attenuate NK activity by binding their corresponding inhibitory receptors. Conversely, increased HLA-related ligand expression can increase the recognition and killing of tumour cells by T cells. MethodsThe prostate cancer cell-lines PC3 and LNCaP were incubated with non-adherent peripheral blood mononuclear cells (PBMCs) at effector-to-target ratios of 8:1 and cytokines IL2 or IL15. After 1 week, NK cells in the co-cultures were stained with antibodies to inhibitory KIRs (KIR2DL1, KIR2DL2, KIR3DL1) and activatory KIRs (NKp44, NKG2D, NKp46, DNAM1). Tumour cells in the co-cultures were stained with antibodies to inhibitory NK-receptor ligands HLA-class1-Bw4 and HLA-G, and activatory receptor ligands Nectin-2, and MICA/B. Antibody to HLA-ABC was also used. Staining was followed by flow cytometric analysis. Groups were compared with one-way ANOVA and post-hoc Newman-Keuls (five repeated experiments). FindingsIL15, but not IL2, inhibited expression of KIR2DL1 and KIR3DL1 by up to 50% (p<0·001) on NK cells in PBMC-prostate cancer cell co-cultures. NKG2D was increased up to 40% (p<0·001) in these cells with IL15 but not IL2. On tumour cells, IL15 decreased HLA-Class-1-Bw4 and HLA-G by 60% (p<0·001) and 75% (p<0·001), respectively. No significant effects were seen on the activatory-ligands whereas HLA-ABC expression was increased by over 65% (p<0·001). InterpretationOne mechanism by which IL15 increases prostate cancer killing by NK cells in PBMC-prosate cancer co-cultures is through upregulating activatory NKG2D and decreasing inhibitory KIR2DL1 and KIR3DL1 receptors. In addition, inhibitory ligands on prostate cancer cells are downregulated, suggesting strong shifts towards NK activation versus inhibition in the co-cultures. Increased HLA-ABC also favours stronger cytotoxic-T-cell recognition. FundingHeathside Trust, UK Medical Research Council, National Institute for Health Research, Prostate cancer UK. The research was funded and supported by the National Institute for Health Research (NIHR) Biomedical Research Centre based at Guy's and St Thomas' NHS Foundation Trust and King's College London.

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.

KIR2DS1-dependent acquisition of CCR7 and migratory properties by human NK cells interacting with allogeneic HLA-C2+ DCs or T-cell blasts

AbstractNatural killer (NK) cells may capture the CCR7 chemokine receptor from allogeneic CCR7+ cells by trogocytosis and acquire migrating properties in response to lymph node chemokines. This event is negatively regulated by inhibitory killer Ig-like receptors (KIRs) and NKG2A. In this study, we analyzed the role of the HLA-C2–specific activating receptor KIR2DS1 in the process of CCR7 uptake by NK cells interacting with different allogeneic CCR7+ cells. Co-incubation of KIR2DS1+ fresh NK cells or NK-cell clones with HLA-C2+ CCR7+ lymphoblastoid cell lines resulted in increased CCR7 uptake. Remarkably, KIR2DS1 expression represented a major advantage for acquiring CCR7 from HLA-C2+ allogeneic dendritic cells (DCs) and T-cell blasts. These findings have important implications in haploidentical hematopoietic stem cell transplantation in which donor-derived (alloreactive) KIR2DS1+ NK cells, upon CCR7 acquisition, become capable of migrating toward lymph nodes, where they may kill patient DCs and T cells, preventing graft-versus-host and host-versus-graft reactions.

Distribution of several activating and inhibitory receptors on CD3−CD56+ NK cells in regional lymph nodes of melanoma patients

BackgroundNatural killer (NK) cells, as the main effector subpopulation of the innate immune system, play an important role in the control of the rise and spread of malignant tumors. Regional lymph nodes (LN) represent the first immunologic barrier to tumor metastasis. Since there are scarce data on NK cells from regional LN of cancer patients, the aim of this study was to investigate the expression of several activating and inhibitory receptors on the entire NK cell population as well as their CD3−CD56dim and CD3−CD56bright functional NK subsets from regional LN of melanoma patients. Materials and methodsMononuclear cells were isolated from 50 regional LN of melanoma patients. The expression of several receptors on NK cells and their functional subsets was analyzed by flow cytometry. ResultsWe show increased percentages of CD3−CD56+ NK cells in involved LN compared with uninvolved LN, mostly in favor of the CD56dim NK cell subset. NK cells in involved LN express similar levels of activating receptor NKG2D, while the level of another activating receptor, CD16, is increased compared with uninvolved LN. Regarding the expression of inhibitory NK cell receptors, we show increased CD158b, but similar low CD158a, inhibitory killer Ig-like cell receptor expression in involved LN compared with uninvolved LN. Furthermore, NK cells in involved compared with uninvolved LN displayed increased CD69 early activation antigen expression. ConclusionsOur results indicate that with tumor infiltration into regional LN of melanoma patients, NK cells, mostly of the CD56dim subset, are recruited into draining LN. The invading NK cells show counterbalance of the increased expression of CD16 activating receptor and increased CD158b inhibitory killer Ig-like cell receptor.

Human Cytomegalovirus (CMV)-Induced Memory-like NKG2C+ NK Cells Are Transplantable and Expand In Vivo in Response to Recipient CMV Antigen

We have previously shown that NKG2C(+) NK cells from CMV naive umbilical cord blood grafts expand preferentially in recipients after CMV reactivation, representing a primary NK cell response after hematopoietic cell transplantation. In this study, recipients of adult donor hematopoietic cell transplantation were assessed to evaluate the role of donor/recipient CMV serostatus on the expression and function of NKG2C(+) NK cells to determine responses to secondary CMV events. Expansion of NKG2C(+) NK cells was seen following clinical CMV reactivation. However, they also expanded in the absence of detectable CMV viremia when both the donor and recipient were CMV seropositive. Upregulation of NKG2C was observed in NK cells from CMV-positive recipients receiving grafts from CMV-seropositive or -seronegative donors. These in vivo-expanded NKG2C(+) NK cells had an increased capacity for target cell-induced cytokine production, expressed an inhibitory killer Ig-like receptor for self-HLA and preferentially acquired CD57. Most importantly, NKG2C(+) NK cells transplanted from seropositive donors exhibit heightened function in response to a secondary CMV event compared with NKG2C(+) NK cells from seronegative donors. We conclude that NKG2C(+) memory-like NK cells are transplantable and require active or latent (subclinical) expression of CMV Ag in the recipient for clonal expansion of NK cells previously exposed to CMV in the donor.

HIV-1 p24 derived epitopes modulate KIR2DL2-binding to HLA-Cw03

Background Recent studies have suggested that HIV-1 can evade Natural Killer (NK)-cell-mediated immunity by mutating viral epitopes to enhance engagement of inhibitory Killer Ig-like receptors (KIRs) expressed on NK cells. However, the precise mechanisms modulating the interaction of inhibitory KIRs and their HLA class I ligands, and the role that HIV-1 epitopes might play in this interaction are not well understood. In this study we investigated whether HLA-Cw3-presented epitopes within HIV-1 p24 Gag can modulate binding of KIR2DL2, an inhibitory KIR, to HLA-Cw03. Methods Using tapasin-deficient 721.220 cell line expressing HLA-Cw*0304 we initially screened for HIV-1 peptides that stabilized HLA-Cw*0304 expression using 222 10mer peptides overlapping by 9 amino acids spanning the entire HIV-1 p24 Gag sequence. Peptides stabalizing HLA-Cw*0304 expression were thereafter investigated for their ability to facilitate binding of a KIR2DL2-IgG fusion construct.

Differential impact of inhibitory and activating Killer Ig-Like Receptors (KIR) on high-risk patients with myeloid and lymphoid malignancies undergoing reduced intensity transplantation from haploidentical related donors

The impact of activating KIR (aKIR) and inhibitory KIR (iKIR) on OS, relapse-related mortality (RRM) and acute GVHD (aGVHD) was prospectively studied in 84 adults with high-risk hematologic malignancies receiving reduced intensity conditioning (RIC) T-cell depleted hematopoietic SCT (HSCT) from haploidentical related donors. In this clinical model, freedom from RRM is dependent on GVL effect. Patients were divided into myeloid (n=49) and lymphoid (n=35) malignancy groups. KIR-ligand and ligand-ligand models were studied in both GVH and rejection directions and statistically correlated with outcome measures. In the myeloid group, OS was higher (P=0.009) and RRM was lower (P=0.036) in patients missing HLA-C group2 ligand to donor iKIR. OS was higher if patients had >1 missing ligand (P=0.018). In lymphoid malignancy, missing ligand to donor KIR had no impact on OS or RRM. However, OS was better with donor aKIR 2DS2 (P=0.028). There was a trend towards shorter OS in recipient with KIR 2DS1, 2DS5 and 3DS1, although sample sizes were too small to provide inferential statistics. Findings in lymphoid malignancy patients should be further studied. These results suggest that the absence of appropriate HLA ligands in the recipient to donor iKIR may induce GVL without aGVHD in myeloid malignancy patients undergoing TCD-RIC transplants.

Bone marrow may be the preferable graft source in recipients homozygous for HLA-C group 2 ligands for inhibitory killer Ig-like receptors

HLA class I molecules participate in natural killer cell regulation by acting as ligands for inhibitory killer cell Ig-like receptors (KIRs). One individual may express one or more inhibitory KIR lacking the corresponding HLA ligand. The role of this 'missing KIR ligand' constellation in hematopoietic SCT (HSCT) remains controversial and depends on incompletely defined transplant variables. We have retrospectively analyzed the effects of missing HLA-C group 1/2 and Bw4 KIR ligands in the recipients on the outcome in 382 HSCT, comparing 118 BMT to 264 PBSC transplants (PBSCT). In the multivariate Cox analysis of PBSCT, poor PFS was observed in homozygous HLA-C group 2 (C2/2) recipients (risk ratio (RR), 1.59; P=0.026). In contrast, C2 homozygosity was not unfavorable after BMT (RR, 0.68; P=0.16). C2 homozygous recipients (n=68) had better PFS after BMT than after PBSCT (RR, 0.17; P=0.001), due to fewer relapses (RR, 0.27; P=0.018). Missing Bw4 favorably influenced PFS after BMT (RR, 0.56; P=0.04), but not after PBSCT. These data suggest opposite effects of missing KIR ligands in BMT vs PBSCT. Larger studies are required to reassess whether BMT should be preferred to PBSCT as an option for C2/C2 recipients.

Extending killer Ig-like receptor function: from HLA class I recognition to sensors of microbial products

Killer Ig-like receptors (KIRs) are human natural killer (NK) receptors that recognize allotypic determinants of human leukocyte antigen (HLA) class I. Inhibitory KIRs discriminate normal cells from tumour or virus-infected cells that have lost or reduced HLA class I expression. Donor NK cell "alloeffector" responses are exploited in haploidentical haematopoietic stem cell transplantation to treat leukaemia. NK cells also express several toll-like receptors (TLRs) that increase NK cell cytotoxicity and cytokine release in response to ligands. Surprisingly, KIR3DL2 binds the TLR ligand CpG-oligodexynucleotides, and together, they are co-internalized and translocated to TLR9-rich early endosomes. This novel KIR-associated function offers clues to understanding the NK cell response to microbial infection, and extends the role played by KIRs in immune defence.

Abstract 5586: NK cell killer Ig-like receptor (KIR) genotype as a novel biomarker for neuroblastoma patients receiving Anti-GD2 monoclonal antibody 3F8

Abstract Background: Natural killer (NK) cells are regulated by inhibitory killer Ig-like receptors (KIRs) that recognize specific HLA class I ligands. KIR-HLA genotypes are tightly linked to NK cell function and clinical outcomes in allogeneic and autologous hematopoietic stem cell transplantation (HSCT). Because NK cells contribute to antibody-dependent cellular cytotoxicity, we hypothesized that specific KIR-HLA genotypes may predict NK cell responsiveness to mAb therapy, and thus, may be useful clinically to identify patients likely to respond to mAb therapy. Methods: 76 children with high risk neuroblastoma received cyclophosphamide-containing induction chemotherapy followed by murine IgG3 monoclonal antibody 3F8 against ganglioside GD2 between 1993 and 2007. HLA and KIR genotyping was performed, and patients were segregated into KIR-HLA groups based on known functional KIR-HLA ligand relationships: KIR2DL2/2DL3 with HLA-CAsn80(HLA-C1 group), KIR2DL1 with HLA-C Lys80(HLA-C2 group), and KIR3DL1 with HLA-Bw4. Overall survival and progression-free survival were estimated by Kaplan-Meier method and hazard ratios by Cox regression. No adjustments were made for multiple comparisons. Comparisons of each end point were based on the log-rank statistics. Results: 63% of the 76 children lacked at least 1 HLA ligand for his/her inhibitory KIR. At 3 years, 80% of patients lacking any KIR ligand (n=46) were alive compared with 69% of patients with all HLA ligands present (n=30) (HR 0.41; 95% CI 0.17-0.98; P=0.04). 23/23 (100%) patients specifically lacking the HLA-Bw4 ligand for KIR3DL1 were alive at 3 years compared with 34/52 (67%) patients exhibiting HLA-Bw4 for KIR 3DL1 (n=52) (HR 0.20; 95% CI 0.05-0.87; P=0.02). Conclusion: KIR-HLA immunogenetics may be a novel biomarker for neuroblastoma patients receiving chemo-immunotherapy. The milieu of HSCT may not be required for NK cells to behave according to “missing ligand,” and KIR-HLA combinations may be predictive of outcome for other malignancies similarly treated with mAb. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5586.

The impact of KIR2DS4 alleles and the expression of KIR in the development of acute GVHD after unrelated allogeneic hematopoietic SCT

The role of killer Ig-like receptors (KIR) in SCT was analyzed. A total of 75 Chinese patients were transplanted with T-depleted hematopoietic stem cells from unrelated donors. Among the 75 donor-recipient pairs, 60 were HLA 10/10 matched and 15 had some mismatches at HLA-C. Transplants from KIR haplotype B/x group donors showed significantly higher overall survival rates compared with those from KIR haplotype A/A donors (relative risk (RR) 3.1 (95% confidence interval (CI) 1.1-8.6), P=0.007). In the haplotype A/A group, a higher risk of acute GVHD (aGVHD) (RR 9.0 (95% CI 1.2-66.9), P=0.01), especially grade III-IV aGVHD (P=0.006), was observed when the donor was homozygous for the full-length expressed KIR2DS4*00101 allele. Real-time PCR showed that a high expression of inhibitory KIR (2DL1 and 3DL1) in the early stages (<90 days) after transplantation correlated with the development of aGVHD (z=2.558, P=0.011). Our findings indicated a significant association of full-length KIR2DS4 or KIR2DL1/3DL1 expression with the occurrence of aGVHD. In aggregate these results suggested that combining KIR and HLA genotyping could help in the selection of transplant donors and improve the outcome of transplantation. Dynamic detection of KIR2DL1/3DL1 expression would be beneficial for prediction of aGVHD after transplantation.