Increased Susceptibility of the CD57- NK Cells Expressing KIR2DL2/3 and NKG2C to iCasp9 Gene Retroviral Transduction and the Relationships with Proliferative Potential, Activation Degree, and Death Induction Response.

Nowadays, the use of genetically modified NK cells is a promising strategy for cancer immunotherapy. The additional insertion of genes capable of inducing cell suicide allows for the timely elimination of the modified NK cells. Different subsets of the heterogenic NK cell population may differ in proliferative potential, in susceptibility to genetic viral transduction, and to the subsequent induction of cell death. The CD57-NKG2C+ NK cells are of special interest as potential candidates for therapeutic usage due to their high proliferative potential and certain features of adaptive NK cells. In this study, CD57- NK cell subsets differing in KIR2DL2/3 and NKG2C expression were transduced with the iCasp9 suicide gene. The highest transduction efficacy was observed in the KIR2DL2/3+NKG2C+ NK cell subset, which demonstrated an increased proliferative potential with prolonged cultivation. The increased transduction efficiency of the cell cultures was associated with the higher expression level of the HLA-DR activation marker. Among the iCasp9-transduced subsets, KIR2DL2/3+ cells had the weakest response to the apoptosis induction by the chemical inductor of dimerization (CID). Thus, KIR2DL2/3+NKG2C+ NK cells showed an increased susceptibility to the iCasp9 retroviral transduction, which was associated with higher proliferative potential and activation status. However, the complete elimination of these cells with CID is impeded.

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.

Reduced Expression of Siglec-7, NKG2A, and CD57 on Terminally Differentiated CD56-CD16+ Natural Killer Cell Subset Is Associated with Natural Killer Cell Dysfunction in Chronic HIV-1 Clade C Infection.

HIV-1 viremia has been shown to induce several phenotypic and functional abnormalities in natural killer (NK) cells. To assess immune defects associated with HIV viremia, we examined NK cell function, differentiation status, and phenotypic alterations based on expression of inhibitory and activating receptors on NK cells in HIV-1 subtype C chronically infected participants from Durban, South Africa. NK cell phenotypic profiles were characterized by assessing sialic acid-binding immunoglobulin-like lectin-7 (Siglec-7), NKG2A, and NKG2C markers on frozen peripheral blood mononuclear cells from viremic, antiretroviral therapy (ART)-naive HIV-1 chronically infected participants (n = 23), HIV-1 chronically infected participants who had been on combination antiretroviral therapy (cART) for at least 12 months (n = 23) compared with healthy donors (n = 23). NK cell differentiation was assessed by measurement of killer immunoglobulin receptor (KIR) and NKG2A expression; CD57 and CD107a measurements were carried out in HIV viremic and healthy donors. All phenotypic and functional assessments were analyzed by using multicolor flow cytometry. HIV-1-infected participants displayed greater frequencies of the CD56-CD16+ (CD56negative) NK cell subset compared with healthy donors (p < .0001). Downregulation of Siglec-7 and NKG2A and upregulation of NKG2C were more pronounced in the CD56negative NK cell subset of viremic participants. The CD56negative subset demonstrated a differentiated (KIR+NKG2A-) phenotype with reduced CD57 expression and lower degranulation capacity in HIV-1-infected participants compared with healthy donors. HIV-1 infection induces the expansion of the CD56negative NK cell subset marked by altered receptor expression profiles that are indicative of impaired function and may explain the overall NK cell dysfunction observed in chronic HIV-1 infection.

The Activating NKG2C Receptor Is Significantly Reduced in NK Cells after Allogeneic Stem Cell Transplantation in Patients with Severe Graft-versus-Host Disease.

Natural killer (NK) cells play a central role in the innate immune system. In allogeneic stem cell transplantation (alloSCT), alloreactive NK cells derived by the graft are discussed to mediate the elimination of leukemic cells and dendritic cells in the patient and thereby to reduce the risk for leukemic relapses and graft-versus-host reactions. The alloreactivity of NK cells is determined by various receptors including the activating CD94/NKG2C and the inhibitory CD94/NKG2A receptors, which both recognize the non-classical human leukocyte antigen E (HLA-E). Here we analyze the contribution of these receptors to NK cell alloreactivity in 26 patients over the course of the first year after alloSCT due to acute myeloid leukemia, myelodysplastic syndrome and T cell Non-Hodgkin-Lymphoma. Our results show that NK cells expressing the activating CD94/NKG2C receptor are significantly reduced in patients after alloSCT with severe acute and chronic graft-versus-host disease (GvHD). Moreover, the ratio of CD94/NKG2C to CD94/NKG2A was reduced in patients with severe acute and chronic GvHD after receiving an HLA-mismatched graft. Collectively, these results provide evidence for the first time that CD94/NKG2C is involved in GvHD prevention.

Increases in NKG2C Expression on T Cells and Higher Levels of Circulating CD8+ B Cells Are Associated with Sterilizing Immunity Provided by a Live Attenuated SIV Vaccine.

Vaccines based on live attenuated viruses are highly effective immunogens in the simian immunodeficiency virus (SIV)/rhesus macaque animal model and offer the possibility of studying correlates of protection against infection with virulent virus. We utilized a tether system for studying, in naive macaques and animals vaccinated with a live-attenuated vaccine, the acute events after challenge with pathogenic SIV. This approach allowed for the frequent sampling of small blood volumes without sedation or restraining of the animals, thus reducing the confounding effect of sampling stress. Before challenge, vaccinated animals presented significantly higher levels of proliferating and activated B cells than naive macaques, which were manifested by high expression of CD8 on B cells. After SIV challenge, the only changes observed in protected vaccinated macaques were significant increases in expression of the NK marker NKG2C on CD4 and CD8 T cells. We also identified that infection of naive macaques with SIV resulted in a transient peak of expression of CD20 on CD8 T cells and a constant rise in the number of B cells expressing CD8. Finally, analysis of a larger cohort of vaccinated animals identified that, even when circulating levels of vaccine virus are below the limit of detection, live attenuated vaccines induce systemic increases of IP-10 and perforin. These studies indicate that components of both the innate and adaptive immune systems of animals inoculated with a live-attenuated SIV vaccine respond to and control infection with virulent virus. Persistence of the vaccine virus in tissues may explain the elevated cytokine and B-cell activation levels. In addition, our report underpins the utility of the tether system for the intensive study of acute immune responses to viral infections.

Microchip-Based Single-Cell Imaging Reveals That CD56dimCD57-KIR-NKG2A+ NK Cells Have More Dynamic Migration Associated with Increased Target Cell Conjugation and Probability of Killing Compared to CD56dimCD57-KIR-NKG2A- NK Cells.

NK cells are functionally educated by self-MHC specific receptors, including the inhibitory killer cell Ig-like receptors (KIRs) and the lectin-like CD94/NKG2A heterodimer. Little is known about how NK cell education influences qualitative aspects of cytotoxicity such as migration behavior and efficacy of activation and killing at the single-cell level. In this study, we have compared the behavior of FACS-sorted CD56(dim)CD57(-)KIR(-)NKG2A(+) (NKG2A(+)) and CD56(dim)CD57(-)KIR(-)NKG2A(-) (lacking inhibitory receptors; IR(-)) human NK cells by quantifying migration, cytotoxicity, and contact dynamics using microchip-based live cell imaging. NKG2A(+) NK cells displayed a more dynamic migration behavior and made more contacts with target cells than IR(-) NK cells. NKG2A(+) NK cells also more frequently killed the target cells once a conjugate had been formed. NK cells with serial killing capacity were primarily found among NKG2A(+) NK cells. Conjugates involving IR(-) NK cells were generally more short-lived and IR(-) NK cells did not become activated to the same extent as NKG2A(+) NK cells when in contact with target cells, as evident by their reduced spreading response. In contrast, NKG2A(+) and IR(-) NK cells showed similar dynamics in terms of duration of conjugation periods and NK cell spreading response in conjugates that led to killing. Taken together, these observations suggest that the high killing capacity of NKG2A(+) NK cells is linked to processes regulating events in the recognition phase of NK-target cell contact rather than events after cytotoxicity has been triggered.

NK cell phenotypic modulation in lung cancer environment.

BACKGROUND: Nature killer (NK) cells play an important role in anti-tumor immunotherapy. But it indicated that tumor cells impacted possibly on NK cell normal functions through some molecules mechanisms in tumor microenvironment. MATERIALS AND METHODS: Our study analyzed the change about NK cells surface markers (NK cells receptors) through immunofluorescence, flow cytometry and real-time PCR, the killed function from mouse spleen NK cell and human high/low lung cancer cell line by co-culture. Furthermore we certificated the above result on the lung cancer model of SCID mouse. RESULTS: We showed that the infiltration of NK cells in tumor periphery was related with lung cancer patients' prognosis. And the number of NK cell infiltrating in lung cancer tissue is closely related to the pathological types, size of the primary cancer, smoking history and prognosis of the patients with lung cancer. The expression of NK cells inhibitor receptors increased remarkably in tumor micro-environment, in opposite, the expression of NK cells activated receptors decrease magnificently. CONCLUSIONS: The survival time of lung cancer patient was positively related to NK cell infiltration degree in lung cancer. Thus, the down-regulation of NKG2D, Ly49I and the up-regulation of NKG2A may indicate immune tolerance mechanism and facilitate metastasis in tumor environment. Our research will offer more theory for clinical strategy about tumor immunotherapy.

Increased antitumor effects using IL-2 with anti-TGF-ß reveals competition between mouse NK and CD8 T cells.

Because of increasing interest in the removal of immunosuppressive pathways in cancer, the combination of IL-2 with Abs to neutralize TGF-ß, a potent immunosuppressive cytokine, was assessed. Combination immunotherapy resulted in significantly greater antitumor effects. These were correlated with significant increases in the numbers and functionality of NK cells, NK cell progenitors, and activated CD8 T cells, resulting in the observed antitumor effects. Combination immunotherapy also was accompanied by lesser toxicities than was IL-2 therapy alone. Additionally, we observed a dual competition between NK cells and activated CD8 T cells such that, after immunotherapy, the depletion of either effector population resulted in the increased total expansion of the other population and compensatory antitumor effects. This study demonstrates the efficacy of this combination immunotherapeutic regimen as a promising cancer therapy and illustrates the existence of potent competitive regulatory pathways between NK cells and CD8 T cells in response to systemic activation.

Cytotoxic NKG2C+ CD4 T cells target oligodendrocytes in multiple sclerosis.

The mechanisms whereby immune cells infiltrating the CNS in multiple sclerosis patients contribute to tissue injury remain to be defined. CD4 T cells are key players of this inflammatory response. Myelin-specific CD4 T cells expressing CD56, a surrogate marker of NK cells, were shown to be cytotoxic to human oligodendrocytes. Our aim was to identify NK-associated molecules expressed by human CD4 T cells that confer this oligodendrocyte-directed cytotoxicity. We observed that myelin-reactive CD4 T cell lines, as well as short-term PHA-activated CD4 T cells, can express NKG2C, the activating receptor interacting with HLA-E, a nonclassical MHC class I molecule. These cells coexpress CD56 and NKG2D, have elevated levels of cytotoxic molecules FasL, granzyme B, and perforin compared with their NKG2C-negative counterparts, and mediate significant in vitro cytotoxicity toward human oligodendrocytes, which upregulated HLA-E upon inflammatory cytokine treatment. A significantly elevated proportion of ex vivo peripheral blood CD4 T cells, but not CD8 T cells or NK cells, from multiple sclerosis patients express NKG2C compared with controls. In addition, immunohistochemical analyses showed that multiple sclerosis brain tissues display HLA-E(+) oligodendrocytes and NKG2C(+) CD4 T cells. Our results implicate a novel mechanism through which infiltrating CD4 T cells contribute to tissue injury in multiple sclerosis.

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.

Cytokine production by non-stimulated peripheral blood NK cells and lymphocytes in early-onset severe pre-eclampsia without HELLP.

Preeclampsia involves an exacerbated maternal inflammatory response that suggests a possible role of innate immunity. NK cells can promote this kind of response through cytokine production and the expression of activating or inhibitory receptors. The aims of the present study were to explore cytokine production by peripheral blood mononuclear cells, as well as cytotoxic ability and receptor expression for HLA-E and HLA-G molecules in peripheral natural killer (NK) cells of women with early-onset severe preeclampsia without HELLP (hemolysis, elevated liver enzyme levels and a low platelet count) syndrome. The expression of the ILT2, KIRDL4, NKG2A, and NKG2C receptors and of cytotoxic activity was measured in non-stimulated NK cells, whereas the intracellular expression of IL-4, IL-10, IL-13, IL-12, IFNγ, TNF and VEGF, was assessed in non-stimulated peripheral blood mononuclear cells subsets using flow cytometry. Circulating soluble HLA-G was also determined by ELISA. The intracellular cytokines tested were significantly higher in NK cell subsets from severely preeclamptic women compared with the control group. On the other hand, the percentage of NK cells expressing NKG2A or NKG2C and the cytotoxic activity of NK cells were significantly higher in severely preeclamptic women. Furthermore, there was a significant correlation between urine protein concentration and soluble human leukocyte antigen G (soluble HLA-G) in serum. We conclude that patients with early-onset severe preeclampsia without HELLP syndrome have increased NK cell function related to cytokine production, cytotoxicity and expression of lectin-like receptors such as NKG2.

Blocking the natural killer cell inhibitory receptor NKG2A increases activity of human natural killer cells and clears hepatitis B virus infection in mice.

BACKGROUND & AIMS: We studied the functions of natural killer (NK) cells and the role of the NK cell inhibitory receptor (NKG2A) during hepatitis B virus (HBV) infection in patients and mice. METHODS: We analyzed levels of NKG2A on peripheral blood NK cells from 42 patients with active chronic hepatitis B (CHB), 31 patients with inactive CHB, and 35 healthy volunteers (controls). Five patients with CHB treated with antiviral therapy were also included to evaluate changes in NK cells after HBV titers decreased. We examined the effects of blocking antibodies against NKG2A or its ligand Qa-1 (equivalent to HLA-E in humans) in immunocompetent mice that express HBV from a plasmid and are positive for serum hepatitis B surface antigen (a mouse model of HBV infection). RESULTS: A higher percentage of NK cells from patients with active CHB were positive for NKG2A (38.47%) than from patients with inactive CHB (19.33%; P < .01) or controls (27.96%; P < .05). The percentage of NKG2A(+) cells correlated with serum viral load (r = 0.5457; P < .001). The percentage of NKG2A(+) cells decreased along with HBV load in patients that received antiviral therapy (P < .05). Blocking NKG2A interaction with HLA-E in peripheral NK cells from patients with active CHB increased their cytotoxicity in vitro. NK cells of HBV carrier mice also had higher percentages of NK cells that expressed NKG2A compared with control mice; NKG2A was likely to be up-regulated by production of interleukin-10 by hepatic regulatory CD4(+)CD25(+) T cells. Blocking Qa-1 in these mice promoted viral clearance in an NK cell-dependent manner. CONCLUSIONS: Infection with HBV increases levels of the inhibitory receptor NKG2A on NK cells in mice and humans, and reduces their ability to clear HBV. Reagents designed to block the interaction between NKG2A and HLA-E might be developed to treat CHB infection.

Influence of congenital human cytomegalovirus infection and the NKG2C genotype on NK-cell subset distribution in children.

Human cytomegalovirus (HCMV) has been reported to reshape the NK-cell receptor (NKR) distribution, promoting an expansion of CD94/NKG2C(+) NK and T cells. The role of NK cells in congenital HCMV infection is ill-defined. Here we studied the expression of NKR (i.e., NKG2C, NKG2A, LILRB1, CD161) and the frequency of the NKG2C gene deletion in children with past congenital infection, both symptomatic (n = 15) and asymptomatic (n = 11), including as controls children with postnatal infection (n = 11) and noninfected (n = 20). The expansion of NKG2C(+) NK cells in HCMV-infected individuals appeared particularly marked and was associated with an increased number of LILRB1(+) NK cells in cases with symptomatic congenital infection. Increased numbers of NKG2C(+), NKG2A(+), and CD161(+) T cells were also associated to HCMV infection. The NKG2C deletion frequency was comparable in children with congenital HCMV infection and controls. Remarkably, the homozygous NKG2C(+/+) genotype appeared associated with increased absolute numbers of NKG2C(+) NK cells. Moreover, HCMV-infected NKG2C(+/+) children displayed higher absolute numbers of NKG2A(+) and total NK cells than NKG2C(+/-) individuals. Our study provides novel insights on the impact of HCMV infection on the homeostasis of the NK-cell compartment in children, revealing a modulatory influence of NKG2C copy number.

Memory CD4+ T-cell-mediated protection depends on secondary effectors that are distinct from and superior to primary effectors.

Whether differences between naive cell-derived primary (1°) and memory cell-derived secondary (2°) CD4(+) T-cell effectors contribute to protective recall responses is unclear. Here, we compare these effectors directly after influenza A virus infection. Both develop with similar kinetics, but 2° effectors accumulate in greater number in the infected lung and are the critical component of memory CD4(+) T-cell-mediated protection against influenza A virus, independent of earlier-acting memory-cell helper functions. Phenotypic, functional, and transcriptome analyses indicate that 2° effectors share organ-specific expression patterns with 1° effectors but are more multifunctional, with more multicytokine (IFN-γ(+)/IL-2(+)/TNF(+))-producing cells and contain follicular helper T-cell populations not only in the spleen and draining lymph nodes but also in the lung. In addition, they express more CD127 and NKG2A but less ICOS and Lag-3 than 1° effectors and express higher levels of several genes associated with survival and migration. Targeting two differentially expressed molecules, NKG2A and Lag-3, reveals differential regulation of 1° and 2° effector functions during pathogen challenge.

Fibroblast growth factor-2 enhances NK sensitivity of hepatocellular carcinoma cells.

The roles of fibroblast growth factor-2 (FGF-2) in the hepatocellular carcinoma (HCC) development are still controversial. In this study, we investigated the expression of FGF-2 in chronic hepatitis (CH) type C patients with or without HCC and the immunoregulation of FGF-2 in NK sensitivity of HCC cells. The FGF-2 expressions were detected in the liver tissues of patients, but not in normal liver. The serum FGF-2 levels of the patients with CH, liver cirrhosis (LC) or HCC were significantly higher than those of healthy volunteers. The serum FGF-2 levels of patients decreased with the progression of chronic liver disease. HCC occurrence of LC patients with high levels of serum FGF-2 was significantly lower than that with low levels of serum FGF-2. Proinflammatory cytokines, such as IL-1ß and IL-6, induced FGF-2 expressions in HCC cells and normal hepatocytes. FGF-2 stimulation resulted in increasing the expression of the membrane-bound major histocompatibility complex class I-related chain A (MICA), an NK activating molecule, and decreasing that of human leukocyte antigen (HLA) class I, an NK inhibitory molecule, on HCC cells. This did not occur with normal hepatocytes. Adding anti-FGF receptor-2 neutralizing antibody resulted in inhibiting the change of MICA and HLA class I expressions on FGF-2 stimulated HCC cells. FGF-2 stimulation on HCC cells resulted in increasing NK sensitivity against HCC cells. These findings indicate that FGF-2 produced by HCC cells or normal hepatocytes of chronic liver disease may play critical roles in eliminating HCC cells by innate immunity.

Expansion of a unique CD57⁺NKG2Chi natural killer cell subset during acute human cytomegalovirus infection.

During human CMV infection, there is a preferential expansion of natural killer (NK) cells expressing the activating CD94-NKG2C receptor complex, implicating this receptor in the recognition of CMV-infected cells. We hypothesized that NK cells expanded in response to pathogens will be marked by expression of CD57, a carbohydrate antigen expressed on highly mature cells within the CD56(dim)CD16(+) NK cell compartment. Here we demonstrate the preferential expansion of a unique subset of NK cells coexpressing the activating CD94-NKG2C receptor and CD57 in CMV(+) donors. These CD57(+)NKG2C(hi) NK cells degranulated in response to stimulation through their NKG2C receptor. Furthermore, CD57(+)NKG2C(hi) NK cells preferentially lack expression of the inhibitory NKG2A receptor and the inhibitory KIR3DL1 receptor in individuals expressing its HLA-Bw4 ligand. Moreover, in solid-organ transplant recipients with active CMV infection, the percentage of CD57(+)NKG2C(hi) NK cells in the total NK cell population preferentially increased. During acute CMV infection, the NKG2C(+) NK cells proliferated, became NKG2C(hi), and finally acquired CD57. Thus, we propose that CD57 might provide a marker of "memory" NK cells that have been expanded in response to infection.

KLRG1+NKG2A+ CD8 T cells mediate protection and participate in memory responses during γ-herpesvirus infection.

Functional CD8 T cell effector and memory responses are generated and maintained during murine γ-herpesvirus 68 (γHV68) persistent infection despite continuous presentation of viral lytic Ags. However, the identity of the CD8 T cell subpopulations that mediate effective recall responses and that can participate in the generation of protective memory to a γ-herpesvirus infection remains unknown. During γHV68 persistence, ∼75% of γHV68-specific CD8 T cells coexpress the NK receptors killer cell lectin-like receptor G1 (KLRG1) and NKG2A. In this study, we take advantage of this unique phenotype to analyze the capacity of CD8 T cells expressing or not expressing KLRG1 and NKG2A to mediate effector and memory responses. Our results show that γHV68-specific KLRG1(+)NKG2A(+) CD8 T cells have an effector memory phenotype as well as characteristics of polyfunctional effector cells such us IFN-γ and TNF-α production, killing capacity, and are more efficient at protecting against a γHV68 challenge than their NKG2A(-)KLRG1(-) counterparts. Nevertheless, γHV68-specific NKG2A(+)KLRG1(+) CD8 T cells express IL-7 and IL-15 receptors, can survive long-term without cognate Ag, and subsequently mount a protective response during antigenic recall. These results highlight the plasticity of the immune system to generate protective effector and proliferative memory responses during virus persistence from a pool of KLRG1(+)NKG2A(+) effector memory CD8 T cells.

Up-regulation of NKG2F receptor, a functionally unknown killer receptor, of human natural killer cells by interleukin-2 and interleukin-15.

The NKG2 family receptors are C-type lectin, type II transmembrane molecules, and play important roles in regulation of natural killer (NK) cell functions against tumor and virus. NKG2F is a new member of NKG2 family, and may possibly associate with DAP 12 to activate NK cells. Since lacking available antibody against human NKG2F, the features of NKG2F expression on NK cells remains unclear. In this study, human NKG2F recombinant expression in E. coli was carried out by using pET-28a with a hexahistidine (6x His) tag and a thrombin digestion sequence to the N-terminus of the recombinant protein NKG2F. IPTG (isopropyl-beta-d-thio-galactoside) induction resulted in high expression of recombinant NKG2F protein, which was then purified and identified by anti-His western blotting and LC-MS/MS. Polyclonal antibody was produced by immunization of BALB/c mice with recombinant NKG2F, and then used to detect NKG2F in western blotting and flow cytometry. Our results demonstrated that NKG2F was expressed only by PBMCs but not by human NK cell lines such as NKL and YT at mRNA level. It was observed that NKG2F was expressed on surface of human blood NK cells, and may be up-regulated at mRNA level and protein level after IL-2 or IL-15 stimulation.

IL-7 enhances survival of human CD56bright NK cells.

Lymphoid differentiation and activation critically depend on cytokine stimulation and the interleukin-7 (IL-7) signaling in particular. Although it has been demonstrated that IL-7 may play a role in natural killer (NK) cell maturation, the effect of IL-7 stimulation on mature human NK cells has not been studied. We, therefore, investigated the expression and functional activity of IL-7Ralpha on mature NK populations from adult blood. In this article, we demonstrate that IL-7Ralpha is specifically expressed in the CD56bright noncytotoxic cytokine-producing NK subset. Importantly, this expression is thymus independent, contrary to what is observed in mice. In addition, we show that IL-7Ralpha is expressed at higher levels on NKG2A+CD56bright NK cells. In contrast to IL-15 stimulation, IL-7 does not increase NK cell cytotoxicity, interferon-gamma production, or the expression of activation markers, indicating that these cytokines play different functions in NK homeostasis and activation. However, IL-7 promotes the survival of the CD56bright NK subset and inhibits apoptosis by increasing BCL2 expression. These data should be taken into account when considering the clinical use of IL-7, particularly after stem cell transplantation.

CD8+ NK cells are predominant in chimpanzees, characterized by high NCR expression and cytokine production, and preserved in chronic HIV-1 infection.

HIV-1 infection in humans results in an early and progressive NK cell dysfunction and an accumulation of an "anergic" CD56- CD16+ NK subset, which is characterised by low natural cytotoxicity receptor expression and low cytokine producing capacity. In contrast to humans, chimpanzee NK cells do not display a distinguishable CD56(bright) and CD56(dim) subset but, as shown here, could be subdivided into functionally different CD8+ and CD8- subsets. The CD8+ NK cells expressed significantly higher levels of triggering receptors including NKp46 and, upon in vitro activation, produced more IFN-gamma, TNF-alpha and CD107 than their CD8- counterparts. In addition, chimpanzee CD8- NK cells had relatively high levels of HLA-DR expression, suggestive of an activated state. Killing inhibitory receptors were expressed only at low levels; however, upon in vitro stimulation, they were up-regulated in CD8+ but not in CD8- NK cells and were functionally capable of inhibiting NKp30-triggered killing. In contrast to HIV-1-infected humans, infected chimpanzees maintained their dominant CD8+ NK cell population, with high expression of natural cytotoxicity receptors.