Natural Killer Cell Subpopulations Research Articles

NK Cell Influence on the Outcome of Primary Epstein-Barr Virus Infection.

The herpesvirus Epstein–Barr virus (EBV) was discovered as the first human candidate tumor virus in Burkitt’s lymphoma more than 50 years ago. Despite its strong growth transforming capacity, more than 90% of the human adult population carries this virus asymptomatically under near perfect immune control. The mode of primary EBV infection is in part responsible for EBV-associated diseases, including Hodgkin’s lymphoma. It is, therefore, important to understand which circumstances lead to symptomatic primary EBV infection, called infectious mononucleosis (IM). Innate immune control of lytic viral replication by early-differentiated natural killer (NK) cells was found to attenuate IM symptoms and continuous loss of the respective NK cell subset during the first decade of life might predispose for IM during adolescence. In this review, we discuss the evidence that NK cells are involved in the immune control of EBV, mechanisms by which they might detect and control lytic EBV replication, and compare NK cell subpopulations that expand during different human herpesvirus infections.

OMIP-035: Functional analysis of natural killer cell subsets in macaques.

This panel was developed to measure the functional capability of natural killer (NK) cell subsets in rhesus macaques (Macaca mulatta). It includes markers to determine the frequency of cytokine secreting and cytotoxic NK cell subpopulations in peripheral blood mononuclear cell (PBMC) samples stimulated in vitro with human 721.221 cells. NK cell subsets were defined by the expression of killer cell immunoglobulin-like receptors (KIRs) Mamu-KIR3DL01 and Mamu-KIR3DL05, and differentiation antigens CD16 and CD56. The panel can be used to assess the functional capability of NK cells in a range of normal and pathologic conditions of captive bred rhesus macaques of Indian origin. © 2016 International Society for Advancement of Cytometry.

Melanoma Affects the Composition of Blood Cell-Derived Extracellular Vesicles.

Extracellular vesicles (EVs) are specifically loaded with nucleic acids, lipids, and proteins from their parental cell. Therefore, the constitution of EVs reflects the type and status of the originating cell and EVs in melanoma patient’s plasma could be indicative for the tumor. Likewise, EVs might influence tumor progression by regulating immune responses. We performed a broad protein characterization of EVs from plasma of melanoma patients and healthy donors as well as from T cells, B cells, natural killer (NK) cells, monocytes, monocyte-derived dendritic cells (moDCs), and platelets using a multiplex bead-based platform. Using this method, we succeeded in analyzing 58 proteins that were differentially displayed on EVs. Hierarchical clustering of protein intensity patterns grouped EVs according to their originating cell type. The analysis of EVs from stimulated B cells and moDCs revealed the transfer of surface proteins to vesicles depending on the cell status. The protein profiles of plasma vesicles resembled the protein profiles of EVs from platelets, antigen-presenting cells and NK cells as shown by platelet markers, co-stimulatory proteins, and a NK cell subpopulation marker. In comparison to healthy plasma vesicles, melanoma plasma vesicles showed altered signals for platelet markers, indicating a changed vesicle secretion or protein loading of EVs by platelets and a lower CD8 signal that might be associated with a diminished activity of NK cells or T cells. As we hardly detected melanoma-derived vesicles in patient’s plasma, we concluded that blood cells induced the observed differences. In summary, our results question a direct effect of melanoma cells on the composition of EVs in melanoma plasma, but rather argue for an indirect influence of melanoma cells on the vesicle secretion or vesicle protein loading by blood cells.

The dual role of NK cells in antitumor reactions triggered by ionizing radiation in combination with hyperthermia

ABSTRACTClassical tumor therapy consists of surgery, radio(RT)- and/or chemotherapy. Additive immunotherapy has gained in impact and antitumor in situ immunization strategies are promising to strengthen innate and adaptive immune responses. Immunological effects of RT and especially in combination with immune stimulation are mostly described for melanoma. Since hyperthermia (HT) in multimodal settings is capable of rendering tumor cells immunogenic, we analyzed the in vivo immunogenic potential of RT plus HT-treated B16 melanoma cells with an immunization and therapeutic assay. We focused on the role of natural killer (NK) cells in the triggered antitumor reactions. In vitro experiments showed that RT plus HT-treated B16 melanoma cells died via apoptosis and necrosis and released especially the danger signal HMGB1. The in vivo analyses revealed that melanoma cells are rendered immunogenic by RT plus HT. Especially, the repetitive immunization with treated melanoma cells led to an increase in NK cell number in draining lymph nodes, particularly of the immune regulatory CD27+CD11b− NK cell subpopulation. While permanent NK cell depletion after immunization led to a significant acceleration of tumor outgrowth, a single NK cell depletion two days before immunization resulted in significant tumor growth retardation. The therapeutic model, a local in situ immunization closely resembling the clinical situation when solid tumors are exposed locally to RT plus HT, confirmed these effects. We conclude that a dual and time-dependent impact of NK cells on the efficacy of antitumor immune reactions induced by immunogenic tumor cells generated with RT plus HT exists.

Identification of a subset of human natural killer cells expressing high levels of programmed death 1: A phenotypic and functional characterization

Programmed death 1 (PD-1) is an immunologic checkpoint that limits immune responses by delivering potent inhibitory signals to Tcells on interaction with specific ligands expressed on tumor/virus-infected cells, thus contributing to immune escape mechanisms. Therapeutic PD-1 blockade has been shown to mediate tumor eradication with impressive clinical results. Little is known about the expression/function of PD-1 on human natural killer (NK) cells. We sought to clarify whether human NK cells can express PD-1 and analyze their phenotypic/functional features. We performed multiparametric cytofluorimetric analysis of PD-1+ NK cells and their functional characterization using degranulation, cytokine production, and proliferation assays. We provide unequivocal evidence that PD-1 is highly expressed (PD-1bright) on an NK cell subset detectable in the peripheral blood of approximately one fourth of healthy subjects. These donors are always serologically positive for human cytomegalovirus. PD-1 is expressed by CD56dim but not CD56bright NK cells and is confined to fully mature NK cells characterized by the NKG2A-KIR+CD57+ phenotype. Proportions of PD-1bright NK cells were higher in the ascites of a cohort of patients with ovarian carcinoma, suggesting their possible induction/expansion in tumor environments. Functional analysis revealed a reduced proliferative capability in response to cytokines, low degranulation, and impaired cytokine production on interaction with tumor targets. We have identified and characterized a novel subpopulation of human NK cells expressing high levels of PD-1. These cells have the phenotypic characteristics of fully mature NK cells and are increased in patients with ovarian carcinoma. They display low proliferative responses and impaired antitumor activity that can be partially restored by antibody-mediated disruption of PD-1/programmed death ligand interaction.

PILRα binds an unknown receptor expressed primarily on CD56bright and decidual-NK cells and activates NK cell functions.

Natural Killer (NK) cells are innate immune lymphocytes specializing in recognition and killing of tumors and pathogens, using an array of activating and inhibitory receptors. NK inhibition is mediated by a large repertoire of inhibitory receptors, whereas a limited number of activating NK cell receptors execute NK cell activation. The ligands recognized by the activating receptors are stress-induced, pathogen derived, tumor specific and even self ligands. However, the full spectrum of NK cell receptors and ligands that control NK cell activity remains uncharacterized. Here we demonstrate that Paired Ig-Like type 2 Receptor Alpha (PILRα), binds a distinct human NK cell sub-population present in the peripheral blood and also in the decidua. We further demonstrate that the interaction of NK cells with PILRα expressing targets lead to elevated IFNγ secretion and cytotoxicity. In conclusion, we present here a novel NK activating ligand which binds and activates an unknown NK receptor expressed on a unique NK cell subset.

Cytokines Induce Faster Membrane Diffusion of MHC Class I and the Ly49A Receptor in a Subpopulation of Natural Killer Cells.

Cytokines have the potential to drastically augment immune cell activity. Apart from altering the expression of a multitude of proteins, cytokines also affect immune cell dynamics. However, how cytokines affect the molecular dynamics within the cell membrane of immune cells has not been addressed previously. Molecular movement is a vital component of all biological processes, and the rate of motion is, thus, an inherent determining factor for the pace of such processes. Natural killer (NK) cells are cytotoxic lymphocytes, which belong to the innate immune system. By fluorescence correlation spectroscopy, we investigated the influence of cytokine stimulation on the membrane density and molecular dynamics of the inhibitory receptor Ly49A and its ligand, the major histocompatibility complex class I allele H-2Dd, in freshly isolated murine NK cells. H-2Dd was densely expressed and diffused slowly in resting NK cells. Ly49A was expressed at a lower density and diffused faster. The diffusion rate in resting cells was not altered by disrupting the actin cytoskeleton. A short-term stimulation with interleukin-2 or interferon-α + β did not change the surface density of moving H-2Dd or Ly49A, despite a slight upregulation at the cellular level of H-2Dd by interferon-α + β, and of Ly49A by IL-2. However, the molecular diffusion rates of both H-2Dd and Ly49A increased significantly. A multivariate analysis revealed that the increased diffusion was especially marked in a subpopulation of NK cells, where the diffusion rate was increased around fourfold compared to resting NK cells. After IL-2 stimulation, this subpopulation of NK cells also displayed lower density of Ly49A and higher brightness per entity, indicating that Ly49A may homo-cluster to a larger extent in these cells. A faster diffusion of inhibitory receptors could enable a faster accumulation of these molecules at the immune synapse with a target cell, eventually leading to a more efficient NK cell response. It has previously been assumed that cytokines regulate immune cells primarily via alterations of protein expression levels or posttranslational modifications. These findings suggest that cytokines may also modulate immune cell efficiency by increasing the molecular dynamics early on in the response.

Natural killer cells in highly exposed hepatitis C-seronegative injecting drug users.

Injecting drug use remains the major risk factor for hepatitis C (HCV) transmission. A minority of long-term injecting drug users remain seronegative and aviraemic, despite prolonged exposure to HCV - termed highly exposed seronegative subjects. Natural killer (NK) cells have been implicated in this apparent protection. A longitudinal nested, three group case-control series of subjects was selected from a prospective cohort of seronegative injecting drug users who became incident cases (n = 11), remained seronegative (n = 11) or reported transient high-risk behaviour and remained uninfected (n = 11). The groups were matched by age, sex and initial risk behaviour characteristics. Stored peripheral blood mononuclear cells were assayed in multicolour flow cytometry to enumerate natural killer cell subpopulations and to assess functional activity using Toll-like receptor ligands before measurement of activation, cytokine production and natural cytotoxicity receptor expression. Principal components were derived to describe the detailed phenotypic characteristics of the major NK subpopulations (based on CD56 and CD16 co-expression), before logistic regression analysis to identify associations with exposed, seronegative individuals. The CD56(dim) CD16(+) (P = 0.05, OR 6.92) and CD56(dim) CD16(-) (P = 0.05, OR 6.07) principal components differed between exposed, seronegative individuals and pre-infection samples of the other two groups. These included CD56(dim) CD16(+) and CD56(dim) CD16(-) subsets with CD56(dim) CD16(+) IFN-γ and TNF-α on unstimulated cells, and CD56(dim) CD16(-) CD69(+) , CD107a(+) , IFN-γ and TNF-α following TLR stimulation. The cytotoxic CD56(dim) NK subset thus distinguished highly exposed, seronegative subjects, suggesting NK cytotoxicity may contribute to protection from HCV acquisition. Further investigation of the determinants of this association and prospective assessment of protection against HCV infection are warranted.

Natural Killer Cells and Liver Fibrosis

In the 40 years since the discovery of natural killer (NK) cells, it has been well established that these innate lymphocytes are important for early and effective immune responses against transformed cells and infections with different pathogens. In addition to these classical functions of NK cells, we now know that they are part of a larger family of innate lymphoid cells and that they can even mediate memory-like responses. Additionally, tissue-resident NK cells with distinct phenotypical and functional characteristics have been identified. Here, we focus on the phenotype of different NK cell subpopulations that can be found in the liver and summarize the current knowledge about the functional role of these cells with a special emphasis on liver fibrosis. NK cell cytotoxicity can contribute to liver damage in different forms of liver disease. However, NK cells can limit liver fibrosis by killing hepatic stellate cell-derived myofibroblasts, which play a key role in this pathogenic process. Therefore, liver NK cells need to be tightly regulated in order to balance these beneficial and pathological effects.

CD8+ T cell responses in metastatic melanoma patients receiving an adenovirally antigen engineered dendritic cell vaccine +/- IFN-α

Dendritic cells (DC), the primary antigen presenting cells and stimulators of naive immune cells, are uniquely positioned to promote anti-tumor immunity. We developed a DC vaccine which expresses three full length melanoma antigens tyrosinase, MART-1, and MAGE-A6 engineered with an Ad type 5 adenovirus “AdVTMM2” which can activate CD8+ and CD4+ T cells as well as natural killer (NK) cells. A clinical trial testing this vaccine as well as the potential effects of IFN-α administration post-vaccination has enrolled 36 patients to date (NCT01366144). Peripheral blood banked at baseline, post-DC vaccination, and after either observation or one month of high dose IFN-α was tested for anti-tumor immunity. Here, we present initial immune response testing of the 12 HLA-A2+ patients who were able to be assessed for circulating CD8+ T cell frequencies by HLA-A2-peptide dextramers. Patient PBMCs were analyzed by MHC dextramer binding assay to determine 1) the frequency of CD8+ cells specific to vaccine encoded antigens in the subset of HLA-A2+ patients and 2) potential determinant spreading to antigens not in the vaccine, 3) frequency and co-expression of the checkpoint inhibitor molecules CTLA-4, PD-1, and TIM-3 on CD8+ T cells, and 4) to characterize three NK cell subpopulations. On the CD8+ T cells, PD-1 was the checkpoint molecule most commonly expressed, while CTLA-4 was minimally expressed. TIM-3 was the checkpoint molecule most commonly expressed on all three subpopulations of NK cells. We observed that most patients developed vaccine-encoded antigen-specific responses, and a subset demonstrated determinant spreading to non-vaccine encoded antigens gp100 and/or NY-ESO-1. Expression of checkpoint molecules changed on both T and NK cells through the treatment periods, and the function (by IFNγ ELISPOT) was also assessed. This study will aid in the design of more effective dendritic cell vaccines and adjuvants for metastatic melanoma patients.

Natural Killer Lymphocytes Are Dysfunctional in Kidney Transplant Recipients on Diagnosis of Cancer.

The incidence of cancer is increased after solid organ transplantation. Natural killer (NK) cells are key effectors of the tumor immune response. We conducted a cross sectional multicentre matched case-control study including 42 kidney transplant recipients (KTRs) on diagnosis of cancer and 41 KTRs without cancer. Extensive phenotyping of NK cells populations and functional tests of NK cells were performed. Kidney transplant recipients with cancer had a higher incidence of acute rejection (P = 0.02) and cytomegalovirus (CMV) infection (P = 0.03) than controls. They had more lymphopenia than control KTRs (1020/mm3 +/- 32 vs 1218/mm3 +/- 34; P = 0.001) including a CD4+ lymphopenia (P = 0.01). Total CD3-/CD56+ NK cell counts were similar in both groups. However, KTRs with cancer had a lower frequency of the cytokine-enriched CD56bright NK cell subset (P = 0.001). The percentage of NK cells expressing NKp46 was decreased in KTRs with cancer (45% vs 53 %, P = 0.001). Furthermore, the ability of NK cells to degranulate CD107a+ cytolytic vesicles was reduced (11% vs 22%; P = 0.02), and the percentage of NK cells secreting IFN[gamma] was decreased (7.5% vs 28.8%; P = 0.01) in KTRs with cancer. These results reveal an imbalance between NK cell subpopulations and functional NK cell defects in KTRs at the diagnosis of malignancy, including a decreased expression of NKp46 and decreased numbers of NK cells producing INF[gamma]. This study highlights the role of NKp46, a major activating NK cell receptor, which could be considered as a potential marker during immunological follow-up of KTRs.

Trace Evidence: Identifying Natural Cancer Killers After the Crime

NK cells possess tremendous diversity with respect to receptor repertoire, with the average person having between 6000 and 30,000 different phenotypes (Horowitz et al., 2013). With variegated expression of over 30 activating and inhibitory receptors, identifying which among these many phenotypes is responsible for recognition and elimination of specific viral infections or malignancies has not been feasible in most circumstances. In this issue of EBioMedicine, Krzywinska et al. (Krzywinska et al., 2015) have identified CD45RO as a unique marker of activated natural killer (NK) cells. CD45 is a phosphatase associated with lymphocyte signaling (Tonks et al., 1988, Ledbetter et al., 1988, Charbonneau et al., 1988) which is expressed in several forms that can be discriminated by antibodies to extracellular determinants. The splice variant CD45RO is the short isoform of CD45, which is expressed on memory T cells (Sanders et al., 1988, Thomas and Lefrancois, 1988). An NK cell population expressing both CD45RA and CD45RO was identified in patients with hematological malignancies, and these cells possessed features of having recently been activated. By further demonstrating evidence of recent interaction with leukemic cells in vivo, the authors suggest that CD45RO identifies a subpopulation of NK cells with anti-leukemic activity. If CD45RO proves to be a reliable in vivo marker of activated NK cells, it could be of potential use in identifying or selecting NK cells with context-specific activity (e.g., anti-tumor, anti-viral, auto-immune, decidual). The novel CD45ROpos NK cell subset is identified as mature by being CD56dim and having co-expression of CD16, killer cell immunoglobulin-like receptors (KIR), and the NK maturation marker, CD57. The CD45ROpos NK cells are large in size with increased granularity, indicative of an activated or primed state. Interestingly, these cells have decreased granzyme B content when compared to the CD45RAposROneg expressing NK cells, suggesting recent degranulation. The authors also show that the CD45ROpos NK cells from patients acquire surface antigens specific to the patient's leukemia type, most likely mediated by trogocytosis (Somanchi et al., 2012), suggesting recent interaction with the leukemia cells and possible anti-leukemic activity. It is important to note that although the patients have this activated and mature NK cell subset that appears to interact with their leukemia, these NK cells are not sufficient for clearing the cancer. It is not clear whether this is a result of non-cytotoxic interactions, resistance mechanisms, or inadequate effector numbers. If larger numbers of these NK cell subsets are needed, this raises the possibility of using CD45RO to identify leukemia-reactive subsets that can be expanded ex vivo for therapy (Denman et al., 2012) without a priori knowledge of their receptor repertoire. Determining the NK cell subsets that are tumor-reactive in cancer patients remains a challenge. In non-cancer settings in which the target cells cannot be so easily propagated — or in some cases is unknown — this may represent an approach for identifying the relevant NK cell subsets for mechanistic, diagnostic, or therapeutic purposes. Additional work is needed to determine whether CD45RO is a generalizable marker of recent NK cell activity or is specific to leukemic or malignant cell interactions, whether CD45ROpos NK cells are present at diagnosis, how they are affected by chemotherapy, radiation, and immunotherapy, whether they represent a cytotoxic, regulatory, or exhausted phenotype, and whether quantitation of these NK cells during therapy can be used as a biomarker for survival or response to therapy. Work done in this study has demonstrated an NK cell subset that has both basic and clinical interest, and further research will determine the importance of this subset in human disease. Conflict of Interest The authors declare no conflict of interest.

OMIP-028: activation panel for Rhesus macaque NK cell subsets.

OMIP-028: activation panel for Rhesus macaque NK cell subsets.

Natural Killer Cells: Key Players in Endometriosis.

Endometriosis affects more than 10% of women, causing significant pain and morbidity. It is also a significant cause of infertility. The aetiology of the disease remains an enigma, and the mechanisms responsible for the associated infertility are unclear. A role for immune cells in endometriosis has been postulated, with attention directed towards natural killer (NK) cells and macrophages. NK cells kill tumours and infected cells but also have roles in tissue remodelling in several organs including the uterus and are key to successful pregnancy. Here, we explore evidence (from peer-reviewed published articles) of phenotypic and functional abnormalities in NK cell subpopulations of women with endometriosis. It is clear that peripheral blood NK cells and peritoneal NK cells have reduced cytotoxic function in women with endometriosis. Uterine NK cells have a vital role in infertility, but very little research has been carried out in this area. We propose that abnormal u NK cell activity may contribute to the pathogenesis of endometriosis and its associated infertility and that future research should focus on this complex area.

Natural Killer Cell Immunophenotypic Abnormalities in Secondary Syphilis Patients

Natural Killer Cell Immunophenotypic Abnormalities in Secondary Syphilis Patients

Abstract 1207: Pharmacodynamic evaluation of ADCC mediated effects in humanized IL2 or IL15 NOG mice models

Abstract Humanized mice (i.e. immunodeficient NOG mice engrafted with human hematopoietic stem cells (HSC)) have demonstrated their usefulness for studying the human immune system. These models should help recapitulating some pathology such as viral infection and then are suitable system to evaluate new human specific therapies. Nevertheless, one issue of these models is the incomplete maturation of natural killer (NK) cell subpopulation. This lake of NK cell functionality was solved by the Central Institute for Experimental Animals (CIEA) with the second generation of transgenic NOG mice expressing human IL2 or IL15 (NK-NOG). The serum levels of hIL-2 and hIL-15 in mice were 1-2 and 0.1-0.2 ng/mL, respectively. In these mice, human NK cells differentiated from HSC are well distributed in the mouse body (peripheral blood, bone marrow, spleen…). They expressed various NK receptors such as NKp30, NKp44, NKp46, etc., granzyme A and perforin, indicating their maturity in the mice. They are also fully functional as evidenced using an ex vivo 51Cr release assay demonstrating lysis properties of human NK cells from collected spleen of HSC-humanized mice.In vivo proof of concept studies were successfully performed with humanized NK-NOG mice xenografted with either human Burkitt's disseminated lymphoma cells or with human solid tumors treated with humanized therapeutic antibodies. Prediction of ADCC-mediated antitumor activity of humanized antibodies is now routinely feasible for preclinical evaluation using these new in vivo NK-NOG models. Citation Format: Caroline Mignard, Jean-Francois Mirjolet, Olivier Duchamp, Yasuyuki Ohnishi, Ikumi Katano, Mamoru Ito. Pharmacodynamic evaluation of ADCC mediated effects in humanized IL2 or IL15 NOG mice models. [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 1207. doi:10.1158/1538-7445.AM2014-1207

FEATURES OF IMMUNE REACTIONS IN STRESS CONDITIONS ASSOCIATED ASSOCIATED WITH EXAMS

Abstract. It has been widely accepted that routine psychophysiological stressors may influence immune functioning via their close interactions between nervous, autonomic, endocrine and immune regulatory systems. The aim of this study was to evaluate immune reactions to acute psychophysiological stress in 203 medical students before and after academic exams. The results showed significant decrease of absolute and relative contents CD3+ T cells, natural killer (NK) cell subpopulations and T-NK cells (CD3-CD16+CD56+ and CD3+CD16+CD56+), as well as declined NK cell activity were revealed immediately after exams, as compared to initial values for these indices. Moreover, a significant decrease in CD19+ B cells, CD4+ T-helpers and CD8+ T-cytotoxic lymphocyte counts was found after exams, in comparison with parameters assessed under stress-free conditions, but no differences were observed, when compared with pre-examination values. Serum concentrations of IgA, IgM, IgG were within normal physiological limits. Changes in immunological parameters during acute examination-associated stress depend on characteristics of autonomous and hormonal reactions in humans, and, under particular extreme conditions, such psycho-emotional stress may cause general disturbances of immune reactions. We conclude that acute psycho-physiological stress during the exams causes significant changes in some lymphocyte subpopulations, in particular, natural killers.

Natural Killer Lymphocytes Are Dysfunctional in Kidney Transplant Recipients On Diagnosis of Cancer.

Background: Cancer incidence is increased after kidney transplantation. Natural Killer (NK) cells are key effectors of the tumor immune response through activating receptors. In assessing immunological characteristics of T and NK cells from kidney transplant recipients (KTRs) who develop cancer, we found important deficiencies in NK cells. Patients and Methods: We conducted a prospective multicentre matched case-control study including 42 KTRs on diagnosis of cancer and 41 KTRs without cancer. Extensive phenotyping of T and NK cells populations was performed. Functional tests of NK cells were performed in a subset of patients. Results: KTRs with cancer had a significantly higher incidence of both acute rejection (P=0.02) and CMV infection (P=0.03) than matched controls. First, KTRs with cancer had more lymphopenia than control KTR (1020/mm3+/-32 vs 1218/mm3+/-34; P=0.001) including a significant CD4+ lymphopenia (P=0.01). Total CD3-/CD56+ NK cell counts were similar in KTRs with or without cancer. However, KTRs with cancer had a much lower frequency of the cytokine-enriched CD56bright NK cell subset (P=0.001), and consequently a higher CD56dim/bright ratio (P=0.005). The percentage of NK cells expressing NKp46 was decreased in KTRs with cancer both for all NK cells (44% vs 58%, P=0.01) and for CD56dim and CD56bright subsets. Furthermore, the NK cells showed functional deficiencies. The ability of NK cells to degranulate CD107a+ cytolytic vesicles was reduced in KTRs with cancer (11% vs 22% in control patients; P=0.01). In addition the percentage of NK cells secreting IFNg was decreased (7.5% in KTRs with cancer vs 28.8% in the control group; P=0.01). Conclusion: These results reveal an imbalance between NK cell subpopulations and functional NK defects in KTRs at the onset of malignancy. There was a decreased number of NK cells producing INFg. This study highlights the role of NKp46. Decreased expression of NKp46 may contribute to the initiation of malignancies. NKp46 could be considered as a potentially valuable marker during immunological follow-up of KTRss.

Isolation of Murine Natural Killer Cells

This unit describes the isolation of natural killer (NK) cells from mouse spleen. The basic protocol describes a method for preparing a highly purified NK cell population from mouse spleen by depletion of contaminating cells with selected monoclonal antibodies (MAbs) and magnetic separation. There are several advantages to this negative selection process. One of these is that the NK cells are not coated with antibody and, therefore, are not at risk of functional perturbation by antibody cross-linking. Additionally, negative selection provides a way to isolate diverse subpopulations of NK cells without selectively purifying a specific subpopulation. Following enrichment, NK cell purity can be assessed by cell surface phenotype using flow cytometry.

Nilotinib combined with interleukin-2 mediates antitumor and immunological effects in a B16 melanoma model

The immune system contributes to tumor cell killing which can be enhanced by cancer chemotherapeutics and immune modulatory pharmaceuticals such as tyrosine kinase inhibitors (TKIs). Recently, the beneficial effect of natural killer (NK) cells was demonstrated when combining interleukin-2 (IL-2) with the TKI imatinib. The aim of the present study was to address the antitumor and immunological effects of recently approved TKIs. Therefore, we focused on the comparison of the efficacy between imatinib and nilotinib in combination with IL-2 in a murine B16F10 melanoma model. Both TKIs possessed antitumor activity in vivo. However, the combination of nilotinib and IL-2 showed a superior outcome. Importantly, both the use of immunodeficient Rag2γc-/- mice, which lack T-lymphocytes, B-lymphocytes and NK cells, as well as NK cell-depletion in C57Bl/6 mice reduced the therapeutic effect of nilotinib. Flow cytometry revealed a significant increase in the IFN-γ-producing CD27+ NK cell subpopulation following treatment with nilotinib and IL-2. Furthermore, the therapeutic antitumor effect of nilotinib/IL-2 was completely lost in IFN-γ-/- mice. In summary, we suggest that nilotinib combined with IL-2 confers high antitumor activity involving the subset of IFN-γ-producing CD27+ NK cells. These new insights are of high importance for the understanding and development of immunotherapeutic protocols using TKIs.