CD56dim Natural Killer Cell Subsets Research Articles

Oncolytic virus treatment differentially affects the CD56dim and CD56bright NK cell subsets in vivo and regulates a spectrum of human NK cell activity.

Natural killer (NK) cells protect against intracellular infection and cancer. These properties are exploited in oncolytic virus (OV) therapy, where antiviral responses enhance anti-tumour immunity. We have analysed the mechanism by which reovirus, an oncolytic dsRNA virus, modulates human NK cell activity. Reovirus activates NK cells in a type I interferon (IFN-I) dependent manner, inducing STAT1 and STAT4signalling in both CD56dim and CD56bright NK cell subsets. Gene expression profiling revealed the dominance of IFN-I responses and identified induction of genes associated with NK cell cytotoxicity and cell cycle progression, with distinct responses in the CD56dim and CD56bright subsets. However, reovirus treatment inhibited IL-15 induced NK cell proliferation in an IFN-I dependent manner and was associated with reduced AKT signalling. In vivo, human CD56dim and CD56bright NK cells responded with similar kinetics to reovirus treatment, but CD56bright NK cells were transiently lost from the peripheral circulation at the peak of the IFN-I response, suggestive of their redistribution to secondary lymphoid tissue. Coupled with the direct, OV-mediated killing of tumour cells, the activation of both CD56dim and CD56bright NK cells by antiviral pathways induces a spectrum of activity that includes the NK cell-mediated killing of tumour cells and modulation of adaptive responses via the trafficking of IFN-γ expressing CD56bright NK cells to lymph nodes.

Functions of NK and iNKT cells in pediatric and adult CVID, ataxia telangiectasia and agammaglobulinemia patients

Primary immune deficiencies (PID) are known to be more than 400 genetic defects caused by the impairment in development and/or functions of the immune system. Common Variable Immunodeficiency (CVID), Ataxia Telangiectasia (AT) and Agammaglobulinemia (AG) are examples of the most common immunodeficiency syndrome. Natural killer (NK) cells are a component of innate immune system and play a major role in the host-rejection of both tumors and virally infected cells. iNKT cells have a role in autoimmune and infectious diseases and controlling of tumor rejection. In this study, NK and iNKT cells and their functions, and intracellular cytokine amount are aimed to determine in patients that suffer CVID, AT and AG. NKp30, NKp46, NKG2D, perforin and granzyme mRNA expression levels were analyzed using RT-PCR. Receptors, cytokine amount of NK cell subset and iNKT were analyzed by flow cytometry. Decreased CD3+ T and elevated NK cell subset in pediatric AT were found. Expression of NKp44 was decreased in adult AG, but not in pediatric patients. Low NKp44 expression in CD3−CD16+CD56dim NK cell subset was found in pediatric AT patients. High HLA-DR, perforin and granzyme expression were found in CD3−CD16+CD56dim NK cell subset of pediatric CVID and AT patients. Alteration of the number of NK subsets, NK receptor expression and cytokine production were observed in pediatric patients compared to healthy subjects.

Inflammatory status might direct ILC and NK cells to IL-17 expressing ILC3 and NK subsets in Behcet's disease

Innate lymphoid cells (ILCs) are lymphoid cells that have important effector and regulatory functions in innate immunity and tissue remodeling. Uncontrolled activation and proliferation of ILCs can contribute to inflammatory autoimmune diseases. Behcet's disease (BD) is a complex systemic inflammatory disorder of unknown etiology. It has been shown that natural killer (NK) cells may play an immunoregulatory role in BD, however the role of ILCs is unknown. In this study, the levels and functions of ILCs and NK cell subsets in BD patients were investigated. Cell surface and cytotoxic granules (perforin and granzyme) expression of NK cells and ILCs were evaluated and labeled according to whole blood lysing protocol in peripheral blood samples obtained from the patients and healthy subjects. Cytokine levels of NK cells were investigated in stimulated peripheral blood mononuclear cells. All data were analyzed by flow cytometry. Total ILC and ILC3+ cells were increased in active BD patients compared to inactive BD patients and healthy subjects. There was no significant difference between the patients and healthy subjects regarding NK cell surface and intracellular molecule expression. Although, an increase in IFN-γ and IL-17, and a decrease in IL-4 levels were observed in CD56dim NK cell subset of BD patients. Recent studies showed increased neutrophilic infiltration and IL-17 secreting Th17 cells in BD patients. It is known that ILC3+cells are similar to Th17 subset regarding their cytokine profile and transcription factor expression patterns. Results of current study may suggest that inflammatory microenvironment in BD patients might direct ILC cells to differentiate into ILC3+ subset, and IL-17 released by NK cells might have a role in neutrophilic infiltration.

Increased Peripheral NKG2A-NKG2D+CD3-CD16+CD56dim NK Cell Subset Was Positively Correlated with Antiphospholipid Antibodies in Patients of Obstetric Antiphospholipid Syndrome

ABSTRACT Obstetric antiphospholipid syndrome (OAPS) is an autoimmune disorder with severe life-threatening complications shown during pregnancy. It has been reported that the increase in CD16+CD56dim natural killer (NK) cells in peripheral blood are risk factors for recurrent miscarriages, but this expression of CD16+CD56dim NK cells in OAPS patients has not been reported, and the mechanism is not clearly illustrated. In this study, we compared the distributional profiles of different NK cell subsets and the expressions of NK cell-activating receptors in peripheral blood of patients with OAPS and healthy women. Our results showed significantly increased NKG2A−NKG2D+ subset and decreased NKG2A+NKG2D− subset in CD3− CD16+CD56dim NK cells, CD3−CD16−CD56bright NK cells and CD56+T cells in OAPS patients compared with those in healthy control women. The CD27−CD11b+ subset significantly increased in CD3−CD16+CD56dim NK cells in OAPS patients compared with those in healthy control women. In addition, the NKG2A−NKG2D+ subset in CD3−CD16 + CD56dim NK subset in triple positivity was higher than single positivity OAPS patients. At the optimal diagnostic threshold established by ROC analysis, using the cut-off of NKG2A−NKG2D+ and CD27−CD11b+ subset in CD3−CD16+CD56dim NK cells is 10.10% and 92.75%, the sensitivity of NKG2A−NKG2D+ and CD27−CD11b+ to detect patients with OAPS compared with healthy control results was 94.1% and 60.8%, and specificity was 84.2% and 89.5%, respectively, with an area under the curve (AUC) of 0.903 and 0.829, respectively. The NKG2A−NKG2D+ subset in CD3−CD16+CD56dim NK cells was positively correlated with the antiphospholipid antibodies lg anti-aCL IgG, lg anti-aCL IgM, lg anti-aCL IgA, lg anti-β2GP1 IgM and Complement 4(C4), while the CD27+CD11b+ subset in CD3−CD16 + CD56dim NK cells was correlated with lg anti-β2GP1 IgG and lg anti-β2GP1 IgA. These results suggested that the NK cytotoxic function enhanced in OAPS patients and unbalanced of NK activating receptors and inhibiting receptors may contribute to the immune pathogenesis of OAPS.

Protocol for Isolation, Stimulation and Functional Profiling of Primary and iPSC-derived Human NK Cells.

Natural killer (NK) cells are innate immune cells, characterized by their cytotoxic capacity, and chemokine and cytokine secretion upon activation. Human NK cells are identified by CD56 expression. Circulating NK cells can be further subdivided into the CD56bright (~10%) and CD56dim NK cell subsets (~90%). NK cell-like cells can also be derived from human induced pluripotent stem cells (iPSC). To study the chemokine and cytokine secretion profile of the distinct heterogenous NK cell subsets, intracellular flow cytometry staining can be performed. However, this assay is challenging when the starting material is limited. Alternatively, NK cell subsets can be enriched, sorted, stimulated, and functionally profiled by measuring secreted effector molecules in the supernatant by Luminex. Here, we provide a rapid and straightforward protocol for the isolation and stimulation of primary NK cells or iPSC-derived NK cell-like cells, and subsequent detection of secreted cytokines and chemokines, which is also applicable for a low number of cells.

Human signal transducer and activator of transcription 5b (STAT5b) mutation causes dysregulated human natural killer cell maturation and impaired lytic function

Patients with signal transducer and activator of transcription 5b (STAT5b) deficiency have impairment in T-cell homeostasis and natural killer (NK) cells which leads to autoimmunity, recurrent infections, and combined immune deficiency. In this study we characterized the NK cell defect in STAT5b-deficient human NK cells, as well as Stat5b-/- mice. We used multiparametric flow cytometry, functional NK cell assays, microscopy, and a Stat5b-/- mouse model to elucidate the effect of impaired and/or absent STAT5b on NK cell development and function. This alteration generated a nonfunctional CD56bright NK cell subset characterized by low cytokine production. The CD56dim NK cell subset had decreased expression of perforin and CD16 and a greater frequency of cells expressing markers of immature NK cells. We observed low NK cell numbers and impaired NK cell maturation, suggesting that STAT5b is involved in terminal NK cell maturation in Stat5b-/- mice. Furthermore, human STAT5b-deficient NK cells had low cytolytic capacity, and fixed-cell microscopy showed poor convergence of lytic granules. This was accompanied by decreased expression of costimulatory and activating receptors. Interestingly, granule convergence and cytolytic function were restored after IL-2 stimulation. Our results show that in addition to the impaired terminal maturation of NK cells, human STAT5b mutation leads to impairments in early activation events in NK cell lytic synapse formation. Our data provide further insight into NK cell defects caused by STAT5b deficiency.

Low-Dose Interleukin-2 Therapy Enhances Cytotoxicity of CD56bright NK Cells in Patients with Chronic Gvhd

Introduction: Natural killer (NK) cells play an important role in defense against infections and cancer. Two major subsets of mature NK cells have been described. CD56bright CD16- NK cells, which normally represent only 5-10% of circulating NK cells, are thought to exhibit less cytolytic activity and greater immune regulatory functions than CD56dim CD16+ NK cells. However, recent studies have reported that cytolytic activity of CD56bright NK cells increases when these cells are stimulated with IL-15or the combination of IL-12, IL-15 and IL-18. Previous studies from our center have shown that daily administration of low-dose IL-2 in patients with chronic graft-versus-host disease (cGVHD) induces selective expansion of CD4+FoxP3+Helios+ regulatory T cells and CD56bright NK cells and improves clinical manifestations of cGVHD. The function of CD56bright NK cells expanded by low-dose IL-2 has not previously been studied.Methods: Single cell mass cytometry (CyTOF) with a panel of 35 metal tagged antibodies was performed on cryopreserved peripheral blood mononuclear cells (PBMC) from 10 adult patients with active cGVHD receiving daily low-dose IL-2 therapy. Patients in this clinical trial received extracorporeal photopheresis (ECP) for 8 weeks prior to starting daily low dose IL-2. ECP therapy (twice weekly) was continued when patients began low-dose IL-2 (1x106 IU/M2/day x 8 weeks). The analytic panel included 26 cell surface markers to identify distinct lymphocyte subsets and 9 intracellular markers to measure functional status and activation of specific signaling pathways. viSNE was used to visualize of high-dimensional data on a two-dimensional map and quantify single cell mass cytometry data. NK cytolytic activity was measured in flow cytometry-based cytotoxicity assays. CD56bright and CD56dim NK cells from 8 adult patients were purified from cryopreserved PBMC by cell sorting and incubated with labeled K562 cells for 4 hours followed by staining with 7-AAD and Annexin-V. E:T ratio of 1:1 was used for incubation with K562 targets.Results: No changes in extracellular or intracellular NK cell markers or quantitative changes in NK cells were observed during the initial 8 week ECP treatment period. Selective expansion of CD56bright NK cells was noted after 1 week of IL-2 therapy (9W) and continued during 8 weeks of daily IL-2 therapy. Increased expression of NKp30, Nkp46, NKG2D, HLA-DR and Ki67 occurred in expanded CD56bright NK cells with peak expression at 1 week after starting IL-2 (9W). At later time points during IL-2 therapy, expression of NKG2D, HLA-DR and Ki67 returned to baseline (Figure 1A). Expression of CD56, CD122 and NKG2A continued to increase during IL-2 treatment. In contrast, expression of CD25 by expanded CD56bright NK cells decreased during IL-2 treatment. Expression of phosphorylated signaling proteins did not change in any NK cell subset during IL-2 treatment. Cytolytic activity was measured in CD56bright and CD56dim NK cell subsets at different times during ECP and IL-2 therapy. After thawing, flow-sorted NK cell subsets were cultured for 16-20 hours with IL-2 (100 IU/ml). Cells were then washed and incubated with tumor targets (K562) for 4 hours and % killing was assessed by flow cytometry. Compared to pre IL-2 treatment, cytolytic activity of CD56bright NK cells increased during IL-2 treatment while cytotoxicity of CD56dim NK cells did not change. Notably, cytotoxicity of CD56bright NK cells became significantly higher than CD56dim NK cells during IL-2 therapy (Figure 1B).Conclusion: Single cell mass cytometry revealed that daily low dose IL-2 therapy induces selective expansion, activation and increased expression of activating NK receptors in CD56bright NK cells. CD56dim NK cells were not affected by IL-2 therapy. In vitro assays revealed that cytolytic activity of CD56bright NK cells increased during IL-2 treatment and exceeded the cytotoxicity of CD56dim NK cells. CD56bright NK cells, traditionally considered to be minimally tumor-responsive, are effectively stimulated by daily low dose IL-2 exposure to enable potent cytotoxicity in response to tumor targets. In patients receiving low-dose IL-2 after allogeneic HSCT, expanded CD56bright NK cells may contribute to graft versus leukemia (GVL) and help prevent relapse after transplant. DisclosuresNikiforow:Kite Pharma: Consultancy. Ho:Jazz Pharmaceuticals: Consultancy. Antin:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Soiffer:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Effect of Age on NK Cell Compartment in Chronic Myeloid Leukemia Patients Treated With Tyrosine Kinase Inhibitors.

Natural killer (NK) cells are a very important component of the innate immune response involved in the lysis of virus infected and tumor cells. Aging has a profound impact in the frequency, phenotype and function of NK cells. Chronic Myeloid Leukemia (CML) is caused by the BCR-ABL gene formation encoding aberrant oncoprotein tyrosine kinase. Treatment with tyrosine kinase inhibitors (TKIs) induces durable deep molecular response. The response to treatment and life expectancy is lower in older patients with chronic phase of CML than in younger patients. In this work we analyse NK cells from TKI-treated CML patients and healthy controls stratified according to age. We have analyzed the expression of NK receptors, activation markers, NK cell differentiation in CD56bright and CD56dim NK cell subsets and the expression of CD107a and IFN-γ in NK cells stimulated with K562. Whereas significant differences on the phenotype and function of NK cells were found between middle-aged (35–65 years old) and elderly (older than 65) healthy individuals, NK cells from TKI-treated CML patients do not show significant differences related with age in most parameters studied, indicating that age is not a limitation of the NK cell recovery after treatment with TKI. Our results also revealed differences in the expression of NK receptors, activation markers and functional assays in NK cells from TKI-treated CML patients compared with age-matched healthy controls. These results highlight the relevance of NK cells in TKI-treated patients and the need of an extensive analysis of the effect of aging on NK cell phenotype and function in these patients in order to define new NK-cell based strategies directed to control CML progression and achieve long-term disease remission after TKI cessation.

Late Development of FcεRγneg Adaptive Natural Killer Cells Upon Human Cytomegalovirus Reactivation in Umbilical Cord Blood Transplantation Recipients.

In human natural killer (NK) cells, human cytomegalovirus (HCMV) has been shown to be a driving force capable of inducing the expansion of a highly differentiated NKG2C+CD57+ subset, persisting over time in both HCMV+ healthy subjects and umbilical cord blood transplantation (UCBT) recipients experiencing HCMV viral reactivation. In HCMV+ healthy subjects, such expanded NK-cells are characterized by epigenetic modifications that modulate their phenotypic and functional characteristics. In particular, an enhanced ADCC activity is detectable in NK cells lacking the signaling protein FcεRγ. Timing and mechanisms involved in the acquisition of HCMV-induced, adaptive-like features by NK cells are currently unknown. In this study, we investigated the de novo acquisition of several adaptive features in NK cells developing after UCBT by monitoring NK-cell differentiation for at least 2 years after transplant. In UCBT recipients experiencing HCMV reactivation, a rapid phenotypic reconfiguration occurred resulting in the expected expansion of CD56dim NKG2C+CD57+ NK cells. However, while certain HCMV-driven adaptive hallmarks, including high KIR, LILRB1, CD2 and low/negative NKG2A, Siglec-7, and CD161 expression, were acquired early after UCBT (namely by month 6), downregulation of the signaling protein FcεRγ was detected at a later time interval (i.e., by month 12). This feature characterized only a minor fraction of the HCMV-imprinted NKG2C+CD57+ CD56dim NK cell subset, while it was detectable in higher proportions of CD57+ NK cells lacking NKG2C. Interestingly, in patients developing a hyporesponsive CD56−CD16bright NK-cell subset, FcεRγ downregulation occurred in these cells earlier than in CD56dim NK cells. Our data suggest that the acquisition of a fully “adaptive” profile requires signals that may lack in UCBT recipients and/or longer time is needed to obtain a stable epigenetic reprogramming. On the other hand, we found that both HCMV-induced FcεRγneg and FcεRγ+ NK cells from these patients, display similar CD107a degranulation and IFN-γ production capabilities in response to different stimuli, thus indicating that the acquisition of specialized effector functions can be achieved before the “adaptation” to HCMV is completed. Our study provides new insights in the process leading to the generation of different adaptive NK-cell subsets and may contribute to develop new approaches for their employment as novel immunotherapeutic tools.

Varicella zoster virus productively infects human natural killer cells and manipulates phenotype.

Varicella zoster virus (VZV) is a ubiquitous human alphaherpesvirus, responsible for varicella upon primary infection and herpes zoster following reactivation from latency. To establish lifelong infection, VZV employs strategies to evade and manipulate the immune system to its advantage in disseminating virus. As innate lymphocytes, natural killer (NK) cells are part of the early immune response to infection, and have been implicated in controlling VZV infection in patients. Understanding of how VZV directly interacts with NK cells, however, has not been investigated in detail. In this study, we provide the first evidence that VZV is capable of infecting human NK cells from peripheral blood in vitro. VZV infection of NK cells is productive, supporting the full kinetic cascade of viral gene expression and producing new infectious virus which was transmitted to epithelial cells in culture. We determined by flow cytometry that NK cell infection with VZV was not only preferential for the mature CD56dim NK cell subset, but also drove acquisition of the terminally-differentiated maturity marker CD57. Interpretation of high dimensional flow cytometry data with tSNE analysis revealed that culture of NK cells with VZV also induced a potent loss of expression of the low-affinity IgG Fc receptor CD16 on the cell surface. Notably, VZV infection of NK cells upregulated surface expression of chemokine receptors associated with trafficking to the skin –a crucial site in VZV disease where highly infectious lesions develop. We demonstrate that VZV actively manipulates the NK cell phenotype through productive infection, and propose a potential role for NK cells in VZV pathogenesis.

Impaired natural killer cell subset phenotypes in human obesity

Obesity is associated with alterations in functionality of immune cells, like macrophages and natural killer (NK) cells, leading to an increased risk for severe infections and several cancer types. This study aimed to examine immune cell populations and functional NK cell parameters focusing on NK cell subset phenotypes in normal-weight and obese humans. Therefore, peripheral blood mononuclear cells (PBMCs) were isolated from normal-weight and obese individuals and analyzed by flow cytometry. Results show no significant changes in the frequency of monocytes, B lymphocytes, or NKT cells but a significantly increased frequency of T lymphocytes in obesity. The frequency of total NK cells was unaltered, whereas the number of low cytotoxic CD56bright NK cell subset was increased, and the number of high cytotoxic CD56dim NK cell subset was decreased in obese subjects. In addition, the frequency of CD56bright NK cells expressing the activating NK cell receptor NKG2D as well as intracellular interferon (IFN)-γ was elevated in the obese study group. In contrast, the frequency of NKG2D- and IFN-γ-positive CD56dim NK cells was lower in obesity compared to normal-weight individuals. Moreover, the expression of the activation marker CD69 was decreased in NK cells, which can be attributed to a reduction of CD69-positive CD56dim NK cells in obese subjects. In conclusion, data reveal an impaired NK cell phenotype and NK cell subset alterations in obese individuals. This NK cell dysfunction might be one link to the higher cancer risk and the elevated susceptibility for viral infections in obesity.

Broad Impairment of Natural Killer Cells from Operationally Tolerant Kidney Transplanted Patients.

The role of natural killer (NK) cells in organ transplantation is controversial. This study aims to decipher their role in kidney transplant tolerance in humans. Previous studies highlighted several modulated genes involved in NK cell biology in blood from spontaneously operationally tolerant patients (TOLs; drug-free kidney-transplanted recipients with stable graft function). We performed a phenotypic, functional, and genetic characterization of NK cells from these patients compared to kidney-transplanted patients with stable graft function under immunosuppression and healthy volunteers (HVs). Both operationally TOLs and stable patients harbored defective expression of the NKp46 activator receptor and lytic molecules perforin and granzyme compared to HVs. Surprisingly, NK cells from operationally TOLs also displayed decreased expression of the CD16 activating marker (in the CD56Dim NK cell subset). This decrease was associated with impairment of their functional capacities upon stimulation, as shown by lower interferon gamma (IFNγ) production and CD107a membranous expression in a reverse antibody-dependent cellular cytotoxicity (ADCC) assay, spontaneous lysis assays, and lower target cell lysis in the 51Cr release assay compared to HVs. Conversely, despite impaired K562 cell lysis in the 51Cr release assay, patients with stable graft function harbored a normal reverse ADCC and even increased amounts of IFNγ+ NK cells in the spontaneous lysis assay. Altogether, the strong impairment of the phenotype and functional cytotoxic capacities of NK cells in operationally TOLs may accord with the establishment of a pro-tolerogenic environment, despite remaining highly activated after transplantation in patients with stable graft function.

Interferon-γ production by tubulointerstitial human CD56bright natural killer cells contributes to renal fibrosis and chronic kidney disease progression

Natural killer (NK) cells are a population of lymphoid cells that play a significant role in mediating innate immune responses. Studies in mice suggest a pathological role for NK cells in models of kidney disease. In this study, we characterized the NK cell subsets present in native kidneys of patients with tubulointerstitial fibrosis, the pathological hallmark of chronic kidney disease. Significantly higher numbers of total NK cells (CD3-CD56+) were detected in renal biopsies with tubulointerstitial fibrosis compared with diseased biopsies without fibrosis and healthy kidney tissue using multi-color flow cytometry. At a subset level, both the CD56dim NK cell subset and particularly the CD56bright NK cell subset were elevated in fibrotic kidney tissue. However, only CD56bright NK cells significantly correlated with the loss of kidney function. Expression of the tissue-retention and -activation molecule CD69 on CD56bright NK cells was significantly increased in fibrotic biopsy specimens compared with non-fibrotic kidney tissue, indicative of a pathogenic phenotype. Further flow cytometric phenotyping revealed selective co-expression of activating receptor CD335 (NKp46) and differentiation marker CD117 (c-kit) on CD56bright NK cells. Multi-color immunofluorescent staining of fibrotic kidney tissue localized the accumulation of NK cells within the tubulointerstitium, with CD56bright NK cells (NKp46+ CD117+) identified as the source of pro-inflammatory cytokine interferon-γ within the NK cell compartment. Thus, activated interferon-γ-producing CD56bright NK cells are positioned to play a key role in the fibrotic process and progression to chronic kidney disease.

Decreased TIM-3 Expression Level of Peripheral Blood Natural Killer Cells in Severe Aplastic Anemia

Severe aplastic anemia (SAA) is a life threatening disease characterized by severe pancytopenia and bone marrow failure. Natural killer (NK) cells are large granular lymphocytes which are one important component of the innate immune system and play a core role in regulation of adaptive immunity. T cell immunoglobulin mucin-3 (TIM-3), a member of the TIM family, appears to play an important negative regulatory role in T cells initial. Now, TIM-3 is widely detected on NK cells, and may contribute as a marker for activation and maturation of NK cells. Our previous studies have confirmed that the decrease of total NK cells, and CD56bright, CD56dim NK cell subsets and the higher expressions of NKp46 and perforin on NK cells may cause the over-function of T lymphocytes and thus lead to hematopoiesis failure in SAA. But, the expression of TIM-3 on NK cells in patients with SAA was still unknown.In this study, we investigated the expression of TIM-3 and its mRNA on NK cells in peripheral blood of untreated and recovered SAA patients by flow cytometry and Real-time PCR. Results showed that the expression of TIM-3 on peripheral blood NK cells in SAA patients before IST was (63.57±12.14) %, which was significantly decreased than that in normal controls (85.62±9.03) % ( p<0.01). The expression of TIM-3 on CD56dim NK cells was (66.41±11.74) % and (83.83±1.59) % separately in SAA patients before IST and normal controls. TIM-3 expressed in SAA patients before IST was lower than that in normal controls ( p<0.01). We also measured TIM-3 expressed on the surface of CD56bright NK cells, but the result showed that there was no statistical difference between SAA patients before IST (61.11±24.99%) and normal controls (62.64±12.06%) (p>0.05). More interesting, the expression of TIM-3 on NK cells was (75.88±12.83) % in SAA patients after IST, which was significantly increased than that in SAA patients before IST, and was no difference with normal controls. As well, we found TIM-3 expression on both CD56dim NK cells and CD56bright NK cells in SAA patients after IST also has a rising trend compared with SAA patients before IST. However, these differences had no statistical significance. Further, we detected TIM-3 mRNA expression in NK cells isolated from peripheral blood. TIM-3 mRNA expression in peripheral blood NK cells from newly diagnosis SAA patients, recovering SAA patients and normal controls was evaluated, respectively. The relative TIM-3 mRNA expression was significantly increased in NK cells in SAA patients after IST compared with SAA patients before IST and normal controls, and this difference had statistical significance. Meanwhile, the relative TIM-3 mRNA expression was lower in NK cells in SAA patients before IST compared with normal controls. However, the difference had no statistical significance. In SAA patients, the expression of TIM-3 on NK cells was positively correlated with the level of WBC (r=0.685, p=0.000), proportion of neutrophil (r=0.825, p=0.000), and proportion of reticulocyte in peripheral blood (r=0.465, p=0.029). And it was negatively correlated with the proportion of lymphocyte in peripheral blood (r=-0.802, p=0.000).According to our series studies, we hypothesize that the lower numbers and dysfunction of NK cells induce the failure of suppressing the over function of DC cells and T cells, that lead damage to healthy hematopoietic stem cells and the onset of SAA. DisclosuresNo relevant conflicts of interest to declare.

Practical NK cell phenotyping and variability in healthy adults.

Human natural killer (NK) cells display a wide array of surface and intracellular markers that indicate various states of differentiation and/or levels of effector function. These NK cell subsets exist simultaneously in peripheral blood and may vary among individuals. We examined variety among selected NK cell receptors expressed by NK cells from normal donors, as well as the distribution of select NK cell subsets and NK cell receptor expression over time in several individual donors. Peripheral blood mononuclear cells were evaluated using flow cytometry via fluorochrome-conjugated antibodies against a number of NK cell receptors. Results were analyzed for both mean fluorescence intensity (MFI) and the percent positive cells for each receptor. CD56(bright) and CD56(dim) NK cell subsets were also considered separately, as was variation in receptor expression in NK cell subsets over time in selected individuals. Through this effort, we provide ranges of NK cell surface receptor expression for a local adult population as well as provide insight into intra-individual variation.

Impaired natural killer cell responses are associated with loss of the highly activated NKG2A(+)CD57(+)CD56(dim) subset in HIV-1 subtype D infection in Uganda.

Objective:Of the predominant HIV-1 subtypes in Uganda, subtype D infection confers a worse prognosis. HIV-1 infection causes perturbations to natural killer (NK) cells, and yet these cells can exert immune pressure on the virus and influence clinical outcome. Here, we studied NK cell activation and function in Ugandans with chronic untreated HIV-1 subtype D infection in comparison to uninfected community matched controls.Methods:Cryopreserved peripheral blood mononuclear cells (PBMCs) from 42 HIV-infected individuals and 28 HIV-negative controls were analysed using eight-colour flow cytometry. NK cell surface expression of CD16, CD56, CD57, HLA-DR and NKG2A were used to investigate activation, maturation and differentiation status. NK cell function was evaluated by measuring interferon-gamma (IFNγ) production in response to K562 cells, or interleukin (IL)-12 and IL-18.Results:CD56dim NK cells from HIV-infected individuals produced less IFNγ in response to IL-12 and IL-18 than did CD56dim NK cells from uninfected controls. Infected individuals had lower levels of CD56dim NK cells coexpressing the differentiation markers NKG2A and CD57 than controls. In addition, their NKG2A+CD57+ CD56dim NK cells displayed elevated activation levels as assessed by HLA-DR expression. Cytokine-induced IFNγ production correlated directly with coexpression of CD57 and NKG2A on CD56dim NK cells.Conclusion:HIV-1 subtype D infection is associated with impaired NK cell responsiveness to cytokines, decline of the NKG2A+CD57+ CD56dim NK cell subset, as well as elevated activation in this subset. These alterations within the NK cell compartment may contribute to immunopathogenesis of HIV-1 subtype D infection in Ugandans.

Abnormalities of quantities and functions of natural killer cells in severe aplastic anemia

Severe aplastic anemia (SAA) is a rare disease characterized by severe pancytopenia and bone marrow failure. Natural killer (NK) cells are large granular lymphocytes derived from hematopoietic stem cells (HSCs) or common lymphoid progenitors (CLP). They play a key role in n the innate immunity and adaptive immune. In this study, the quantitative and functional changes of natural killer (NK) cell subsets in peripheral blood of severe aplastic anemia (SAA) patients before and after immunosuppressive therapy (IST) were investigated. Results showed that the percentage of NK cells and its subsets in peripheral blood lymphocytes was decreased in SAA patients. After IST, the percentage of NK cells and their subsets increased dramatically. The median expressions of CD158a, NKG2D and NKp46 on NK cells were higher in SAA patients compared to that in normal controls, and the expressions of perforin in newly diagnosed and recovery SAA patients were higher than that in controls. Therefore, we concluded that the decrease of total NK cells, and CD56bright, CD56dim NK cell subsets and the higher expressions of NKp46 and perforin on NK cells may cause the over-function of T lymphocytes and thus lead to hematopoiesis failure in SAA.

Dynamics of natural killer cell responses to hepatitis delta virus infection during the course of interferon alpha treatment (P6070)

Abstract Infection with the hepatitis delta virus (HDV) causes the most severe form of viral hepatitis. 15-20 million people are chronically infected with HDV. Hepatitis delta is believed to be an immune-mediated disease however adaptive immune responses against HDV are hardly detectable. This suggests a possible role of natural killer (NK) cells in the pathogenesis of HDV infection. To study this, we collected blood samples from 34 patients with hepatitis delta, 16 out of which were sampled during antiviral treatment with pegylated interferon alpha (peg-IFNalpha). Expression levels of activation and inhibitory NK cell receptors and multi-functional NK cell responses were assessed using 15-color flow cytometry. HDV infected patients exhibited higher baseline frequencies of NK cells. Peg-IFNalpha treatment was associated with a redistribution of NK cell subsets with increased numbers of CD56bright NK cells whereas total and CD56dim NK cells decreased. During treatment the most mature CD56dim NK cell subsets disappeared from the periphery and the frequency of immature CD56dim NK cells increased. Furthermore, the treatment resulted in decreased multi-functional NK cell responses against K562 cells, with and without additional IFNα stimulation. Taken together, we show that HDV infection increases the amount of NK cells. Furthermore our data suggest a reshaping of the NK pool by IFNα during treatment resulting in a loss of mature cells and decreased functional responses.

Toll-like receptors expression and interferon-γ production by NK cells in human sepsis

IntroductionDuring the course of infection, natural killer (NK) cells contribute to innate immunity by producing cytokines, particularly interferon-gamma (IFN-γ). In addition to their beneficial effects against infection, NK cells may play a detrimental role during systemic inflammation, causing lethality during sepsis. Little is known on the immune status of NK cells in patients with systemic inflammatory response syndrome (SIRS) or sepsis in terms of cell surface markers expression and IFN-γ production.MethodsWe investigated 27 sepsis patients and 11 patients with non-infectious SIRS. CD56bright and CD56dim NK cell subsets were identified by flow cytometry and Toll-like receptor (TLR)2, TLR4, TLR9, CX3CR1, CD16 and CD69 expression were analyzed, as well as ex vivo IFN-γ production by NK cells in whole blood samples.ResultsWe first showed that in NK cells from healthy controls, TLR2 and TLR4 expression is mainly intracellular, similarly to TLR9. Intracellular levels of TLR2 and TLR4, in both CD56bright and CD56dim NK cell subsets from sepsis patients, were increased compared to healthy subjects. In addition, the percentage of CD69+ cells was higher among NK cells of sepsis patients. No difference was observed for TLR9, CX3CR1, and CD16 expression. The ex vivo stimulation by TLR4 or TLR9 agonists, or whole bacteria in synergy with accessory cytokines (IL-15+IL-18), resulted in significant production of IFN-γ by NK cells of healthy controls. In contrast, for SIRS and sepsis patients this response was dramatically reduced.ConclusionsThis study reports for the first time an intracellular expression of TLR2 and TLR4 in human NK cells. Surface TLR4 expression allows discriminating sepsis and SIRS. Furthermore, during these pathologies, NK cells undergo an alteration of their immune status characterized by a profound reduction of their capacity to release IFN-γ.

Study on the Quantities and Functions of Natural Killer Cell Subsets in Peripheral Blood of the Patients with Severe Aplastic Anemia,

Abstract 3423 ObjectiveTo investigate the quantitative and functional changes of natural killer(NK) cell subsets in peripheral blood of severe aplastic anemia(SAA) patients before and after immunosuppressive therapy(IST). Methods By means of monoclonal antibody labeling technology, the percentages of NK cells (CD3−CD56+ MCD16+) and their subsets[CD3−CD56bright(CD56bright),CD3−CD56dim(CD56dim),CD3−CD56− CD16+)] in peripheral blood lymphocytes were detected in 42 SAA patients, including 12 patients with newly diagnosed SAA and 30 recovery patients, and 13 normal controls respectively. NK cells inhibitory receptors(CD158a and CD158b),activating receptors(NKG2D and NKp46), perforin and granzyme-β were also detected. Results The percentage of NK cells in peripheral blood lymphocytes was(10.30±6.08)% newly diagnosed patients,(16.47±8.29)% in recovery patients, and(19.45±6.88)% in normal controls. The percentage of NK cells in newly diagnosed SAA patients was lower than that of recovery patients and normal controls(p<0.05),and there was no statistical difference between recovery patients and normal controls(p>0.05).The median percentage of CD56bright cells was 0.11% newly diagnosed SAA patients, 0.68% in recovery patients and 0.53% in normal controls. The median percentage of CD56bright cells in newly diagnosed SAA patients was lower than that of the other two groups (p<0.05),and there was no statistical difference between recovery patients and normal controls(p>0.05).The percentage of CD56dim cells was(9.62±6.04)% in newly diagnosed patients,(13.81±7.52)% in recovery patients and(18.21±7.16)% in normal controls. The percentage of CD56dim cells in newly diagnosed SAA patients was lower than that of normal controls (p<0.05),and there was no statistical difference between the other groups(p>0.05).The median percentage of CD3−CD56−CD16+ cells was 0.37% in newly diagnosed patients, 0.79% in recovery patients and 0.41% in normal controls. The percentage of CD3−CD56−CD16+ cells in recovery SAA patients was higher than that of the other two groups(p<0.05),and there was no statistical difference between newly diagnosed SAA patients and normal controls (p>0.05).The median expressions of CD158a,NKG2D and NKp46 on NK cells were 16.45%,95.68%,88.23% in newly diagnosed SAA patients, 19.34%,96.85%,82.97% in recovery patients and 21.59%,96.13%,40.99% in normal controls. The median expression of NKp46 on NK cells of newly diagnosed and recovery SAA patients were higher than that of healthy individuals(p<0.05).The expression of CD158b on NK cells was(34.66±16.02)% in newly diagnosed patients,(39.13±17.75)% in recovery patients,(41.43±17.43)% in controls, and there was no statistical difference between each groups (p>0.05).The expression of perforin in NK cells cytoplasm was(66.14±20.73)% in newly diagnosed patients,(64.97±21.61)% in recovery patients and (42.11±_27.25)% in controls. The expression of perforin in newly diagnosed and recovery SAA patients were higher than that of controls(p<0.05),and there was no statistical difference between that of the other groups (p>0.05).The median expression of granzyme-β in NK cells cytoplasm is 96.43% in newly diagnosed SAA patients,96.97% in recovery SAA patients,92.88% in controls, and there was no statistical difference between each groups (p>0.05). Conclusions The decrease of the percentage NK cells, CD56bright,CD56dim NK cell subsets and the higher expressions of NKp46 and perforin may cause the over-function of T lymphocytes and thus lead to hematopoiesis failure in SAA≤®. Disclosures:No relevant conflicts of interest to declare.