Profile Of Natural Killer Cells Research Articles

Distinct Human NK Cell Phenotypes and Functional Responses to Mycobacterium tuberculosis in Adults From TB Endemic and Non-endemic Regions.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB), which leads to an estimated 1. 5 million deaths worldwide each year. Although the immune correlates of protection against Mtb infection and TB disease have not been well-defined, natural killer (NK) cells are increasingly recognized as a key component of the innate immune response to Mtb and as a link between innate and adaptive immunity. In this study, we evaluated NK cell phenotypic and functional profiles in QuantiFERON-TB (QFT)+ and QFT− adults in a TB endemic setting in Kisumu, Kenya, and compared their NK cell responses to those of Mtb-naïve healthy adult controls in the U.S. We used flow cytometry to define the phenotypic profile of NK cells and identified distinct CD56dim NK cell phenotypes that differentiated the Kenyan and U.S. groups. Additionally, among Kenyan participants, NK cells from QFT+ individuals with latent Mtb infection (LTBI) were characterized by significant downregulation of the natural cytotoxicity receptor NKp46 and the inhibitory receptor TIGIT, compared with QFT− individuals. Moreover, the distinct CD56dim phenotypic profiles in Kenyan individuals correlated with dampened NK cell responses to tumor cells and diminished activation, degranulation, and cytokine production following stimulation with Mtb antigens, compared with Mtb-naïve U.S. healthy adult controls. Taken together, these data provide evidence that the phenotypic and functional profiles of NK cells are modified in TB endemic settings and will inform future studies aimed at defining NK cell-mediated immune correlates that may be protective against acquisition of Mtb infection and progression to TB disease.

Mesenchymal stem cells alter the frequency and cytokine profile of natural killer cells in abortion-prone mice.

Natural killer cells, which play a pivotal role in the establishment and maintenance of normal pregnancy, are the most abundant leukocytes at the fetomaternal interface that their subsets frequencies and cytokine profile are influential factors in the preservation of the decidual tolerogenic microenvironment. Any imbalance in NK cells' frequency and functions could be associated with pregnancy failure. Mesenchymal stem cells (MSCs) are shown to have immunomodulatory effects on NK cells and their cytokine profile. The purpose of this study is to evaluate the impact of MSCs therapy on the cytokine profiles and subpopulations of NK cells in a murine model of recurrent pregnancy loss. Adipose-derived MSCs were injected intraperitoneally to the abortion-prone mice on Day 4.5 of gestation. The abortion rate was determined after MSCs administration and the frequency and cytokine profiles of the different subsets of NK cells were determined using the flow cytometry. Our results showed that, in abortion-prone mice, the frequency of CD49b+ NK cells was significantly higher than normal pregnant mice that decreased after therapy. We also demonstrated that MSCs downregulated the production of IFN-γ and upregulated IL-4 and IL-10 production by uNK cells. These findings indicate that MSCs can decrease the infiltration of CD49b+ NK cells to the fetomaternal interface and modulate the cytokine profile of NK cells from inflammatory to tolerogenic profile and thereby improve the tolerogenic microenvironment at the fetomaternal interface in benefit of pregnancy maintenance.

Activatory Receptor NKp30 Predicts NK Cell Activation During Controlled Human Malaria Infection.

Natural killer (NK) cells are known to be activated during malaria infection, exhibiting both cytokine production and cytotoxic functions. However, NK cells are heterogeneous in their expression of surface activatory and inhibitory receptors which may influence their response to malaria parasites. Here, we studied the surface marker profile and activation dynamics of NK cells during a Controlled Human Malaria Infection in 12 healthy volunteers. Although there was significant inter-patient variability in timing and magnitude of NK cell activation, we found a consistent and strong increase in expression of the activatory receptor NKp30. Moreover, high baseline NKp30 expression was associated with NK cell activation at lower parasite densities. Our data suggest that NKp30 expression may influence the NK cell response to P. falciparum, explaining inter-patient heterogeneity and suggesting a functional role for this receptor in malaria.

Reinforcing involvement of NK cells in psoriasiform dermatitis animal model.

Psoriasis (Ps) is a chronic inflammatory immune-mediated disease with skin and joint manifestations, characterized by abnormal and rapid proliferation of keratinocytes and infiltration of psoriatic lesions with immune cells. Extensive literature suggests that Ps is a T-cell mediated disease its pathogenesis being highly related to innate and adaptative immune cells. Although natural killer (NK) cells are involved in the inflammatory process of Ps through pro-inflammatory cytokine secretion (tumor necrosis factor α, interferon γ), their role in this pathology is not yet fully elucidated. In order to study the involvement of NK subpopulations in the pathogenesis of Ps we used the imiquimod-based mouse model of psoriasiform dermatitis and NK cells complex phenotype patterns from peripheral blood (PB) and spleen were investigated. Skin inflammation and the disease severity were assessed using in vivo measurements (erythema, desquamation and induration parameters, PASI modified score), splenomegaly assessment and histopathological evaluation. Phenotypic characterization of NK cells in imiquimod (IMQ)-treated mice was performed by flow cytometry, for both PB and spleen cell suspension. A large panel of surface markers was used: maturation and activation markers [cluster of differentiation (CD)49b, CD11b, CD43, CD27, KLRG1, CD335, CD69, CD28, gp49R, CD45R, CD11c] and markers for cytokine receptors (CD25, CD122, CD132). Our experimental data showed important differences in IMQ-treated mouse NK cell phenotype as compared to control group. The maturation markers (CD11b, CD43, CD27, KLRG1) were found increased on NK cells, in periphery and spleen, while CD49b+NK1.1+ was significantly lower, and the alterations correlated with the severity of the disease. Our findings reflect the immune engagement toward activatory profile of NK cells and draw attention to evaluating Ps intensity correlated with the mature profile of circulating NK cells.

Changes in the NK Cell Repertoire Related to Initiation of TB Treatment and Onset of Immune Reconstitution Inflammatory Syndrome in TB/HIV Co-infected Patients in Rio de Janeiro, Brazil-ANRS 12274.

Tuberculosis (TB) is the most common comorbidity and the leading cause of death among HIV-infected individuals. Although the combined antiretroviral therapy (cART) during TB treatment improves the survival of TB/HIV patients, the occurrence of immune reconstitution inflammatory syndrome (IRIS) in some patients poses clinical and scientific challenges. This work aimed to evaluate blood innate lymphocytes during therapeutic intervention for both diseases and their implications for the onset of IRIS. Natural killer (NK) cells, invariant NKT cells (iNKT), γδ T cell subsets, and in vitro NK functional activity were characterized by multiparametric flow cytometry in the following groups: 33 TB/HIV patients (four with paradoxical IRIS), 27 TB and 25 HIV mono-infected subjects (prior to initiation of TB treatment and/or cART and during clinical follow-up to 24 weeks), and 25 healthy controls (HC). Concerning the NK cell repertoire, several activation and inhibitory receptors were skewed in the TB/HIV patients compared to those in the other groups, especially the HCs. Significantly higher expression of CD158a (p = 0.025), NKp80 (p = 0.033), and NKG2C (p = 0.0076) receptors was detected in the TB/HIV IRIS patients than in the non-IRIS patients. Although more NK degranulation was observed in the TB/HIV patients than in the other groups, the therapeutic intervention did not alter the frequency during follow-up (weeks 2–24). A higher frequency of the γδ T cell population was observed in the TB/HIV patients with inversion of the Vδ2+/Vδ2− ratio, especially for those presenting pulmonary TB, suggesting an expansion of particular γδ T subsets during TB/HIV co-infection. In conclusion, HIV infection impacts the frequency of circulating NK cells and γδ T cell subsets in TB/HIV patients. Important modifications of the NK cell repertoire were observed after anti-TB treatment (week 2) but not during the cART/TB follow-up (weeks 6–24). An increase of CD161+ NK cells was related to an unfavorable outcome. Despite the low number of cases, a more preserved NK cell profile was detected in IRIS patients previous to treatment, suggesting a role for these cells in IRIS onset. Longitudinal evaluation of the NK repertoire showed the impact of TB treatment and implicated these cells in TB pathogenesis in TB/HIV co-infected patients.

Flip-flops of natural killer cells in autoimmune diseases versus cancers: Immunologic axis.

Natural killer (NK) cells play an essential role in the immune response to infections, inflammations, and malignancies. Recent studies suggest that NK cell surface receptors and cytokines are the key points of the disease development and protection. We hypothesized that the interactions between NK cell receptors and targeted cells construct an eventual niche, and this niche has an eventual profile in various autoimmune diseases and cancers. The NK cells preactivated with cytokines, such as interleukin-2 (IL-2), IL-12, IL-15, and IL-18 can have higher cytotoxicity; however, the toxic side effect of IL-2 should be considered. The vicissitudes of NK cell profile and its receptors obey the environmental communications and cell interactions. Our vision around the NK cells as an immune axis remained dual, and we still cannot judge the immune responses based on the NK cell flip-flop. A design of eventual niche to monitor the NK cell and targeted cell interaction is needed to strengthen our ability in diagnosis and treatment approaches based on the NK cells. Here, we have reviewed the shifts in the NK cells and their surface receptors in autoimmune diseases, solid tumors, and leukemia, and also discussed the effective chemokines that affect NK cell activation and proliferation. The main aim of this review is to present a broader vision of the NK cell changes in autoimmune disease and cancers.

CD56dim CD16- Natural Killer Cell Profiling in Melanoma Patients Receiving a Cancer Vaccine and Interferon-α.

Natural killer (NK) cells are innate cytotoxic and immunoregulatory lymphocytes that have a central role in anti-tumor immunity and play a critical role in mediating cellular immunity in advanced cancer immunotherapies, such as dendritic cell (DC) vaccines. Our group recently tested a novel recombinant adenovirus-transduced autologous DC-based vaccine that simultaneously induces T cell responses against three melanoma-associated antigens for advanced melanoma patients. Here, we examine the impact of this vaccine as well as the subsequent systemic delivery of high-dose interferon-α2b (HDI) on the circulatory NK cell profile in melanoma patients. At baseline, patient NK cells, particularly those isolated from high-risk patients with no measurable disease, showed altered distribution of CD56dim CD16+ and CD56dim CD16− NK cell subsets, as well as elevated serum levels of immune suppressive MICA, TN5E/CD73 and tactile/CD96, and perforin. Surprisingly, patient NK cells displayed a higher level of activation than those from healthy donors as measured by elevated CD69, NKp44 and CCR7 levels, and enhanced K562 killing. Elevated cytolytic ability strongly correlated with increased representation of CD56dim CD16+ NK cells and amplified CD69 expression on CD56dim CD16+ NK cells. While intradermal DC immunizations did not significantly impact circulatory NK cell activation and distribution profiles, subsequent HDI injections enhanced CD56bright CD16− NK cell numbers when compared to patients that did not receive HDI. Phenotypic analysis of tumor-infiltrating NK cells showed that CD56dim CD16− NK cells are the dominant subset in melanoma tumors. NanoString transcriptomic analysis of melanomas resected at baseline indicated that there was a trend of increased CD56dim NK cell gene signature expression in patients with better clinical response. These data indicate that melanoma patient blood NK cells display elevated activation levels, that intra-dermal DC immunizations did not effectively promote systemic NK cell responses, that systemic HDI administration can modulate NK cell subset distributions and suggest that CD56dim CD16− NK cells are a unique non-cytolytic subset in melanoma patients that may associate with better patient outcome.

Microvesicles from malaria-infected red blood cells activate natural killer cells via MDA5 pathway.

Natural killer (NK) cells provide the first line of defense against malaria parasite infection. However, the molecular mechanisms through which NK cells are activated by parasites are largely unknown, so is the molecular basis underlying the variation in NK cell responses to malaria infection in the human population. Here, we compared transcriptional profiles of responding and non-responding NK cells following exposure to Plasmodium-infected red blood cells (iRBCs) and identified MDA5, a RIG-I-like receptor involved in sensing cytosolic RNAs, to be differentially expressed. Knockout of MDA5 in responding human NK cells by CRISPR/cas9 abolished NK cell activation, IFN-γ secretion, lysis of iRBCs. Similarly, inhibition of TBK1/IKKε, an effector molecule downstream of MDA5, also inhibited activation of responding NK cells. Conversely, activation of MDA5 by liposome-packaged poly I:C restored non-responding NK cells to lyse iRBCs. We further show that microvesicles containing large parasite RNAs from iRBCs activated NK cells by fusing with NK cells. These findings suggest that NK cells are activated through the MDA5 pathway by parasite RNAs that are delivered to the cytoplasm of NK cells by microvesicles from iRBCs. The difference in MDA5 expression between responding and non-responding NK cells following exposure to iRBCs likely contributes to the variation in NK cell responses to malaria infection in the human population.

Expression of NK Cell Surface Receptors in Breast Cancer Tissue as Predictors of Resistance to Antineoplastic Treatment

Background:Currently, one of the most used strategies for the treatment of newly diagnosed patients with breast cancer is neoadjuvant chemotherapy based on the application of taxanes and anthracyclines. However, despite the high number of patients who develop a complete pathological clinical response, resistance and relapse following this therapy continue to be a clinical challenge. As a component of the innate immune system, the cytotoxic function of Natural Killer (NK) cells plays an important role in the elimination of tumor cells. However, the role of NK cells in resistance to systemic therapy in breast cancer remains unclear. The present project aims to evaluate the gene expression profile of human NK cells in breast cancer tissue resistant to treatment with taxanes–anthracyclines.Methods:Biopsies from tumor tissues were obtained from patients with breast cancer without prior treatment. Histopathological analysis and ex vivo exposure to antineoplastic chemotherapeutics were carried out. Alamar blue and lactate dehydrogenase release assays were performed for quantitative analysis of tumor viability. Gene expression profiles from tumor tissues without prior exposure to therapeutic drugs were analyzed by gene expression microarrays and verified by polymerase chain reaction.Results:A significant decrease in gene expression of cell-surface receptors related to NK cells was observed in tumor samples resistant to antineoplastic treatment compared with those that were sensitive to treatment.Conclusion:A decrease in NK cell infiltration into tumor tissue might be a predictive marker for failure of chemotherapeutic treatment in breast cancer.

Cytotoxic activity and calcium signalling in natural killer cells in multiple sclerosis

Objectives Natural killer (NK) cell populations are affected in MS, with reduced circulating numbers and cytotoxic activity, altered cytokine production, and profile between CD56Dim and CD56Bright NK cell populations. Calcium permeable cation channels, transient receptor potential melastatin3 (TRPM3), are expressed on NK cells. However, the role of receptor/calcium interaction in NK cells has not been investigated in MS patients. Furthermore, the role of calcium mobilisation mediating NK lysis through receptors such as TRPM3, has not been studied in MS. Methods We examined NK cell cytotoxic activity and intracellular calcium mobilisation in untreated and alemtuzumab treated MS patients. We examined TRPM3 surface expression as a mechanism for altered NK function in MS. 22 MS (10 untreated, 12 treated alemtuzumab) and 22 healthy controls (HC) were examined. NK cytotoxic activity and calcium signalling were examined under stimulation with ionomycin, pregenelone sulphate (PregS), 2-Aminoethoxydiphenyl borate (2-ABP) and thapsigargin (TG). Receptor expression (TRPM3, CD69, CD107a) was determined on CD56Dim and CD56Bright NK cells. Results There was no difference in NK cytotoxic activity. CD69 expression was significantly lower on CD56Dimcells of untreated MS group when compared to HC (p-value 0.031) and treated MS group (p=0.036) in unstimulated conditions. This effect was abrogated in treated MS group in unstimulated and stimulated conditions. Calcium signalling was increased in CD56Bright cells of treated MS group compared to untreated MS group under stimulation with PregS, 2-ABP and TG. No difference in calcium signalling was observed in CD56Dimcells. TRPM3 expression was increased in untreated MS group compared to HC with PregS alone. Conclusions Altered NK cell profiling may be implicated in MS. Furthermore, alemtuzumab may mediate therapeutic effect via altered expression of CD56Dim and CD56Bright cells. Alemtuzumab alters calcium signalling in CD56Brightcells, however intracellular calcium signalling alone appears insufficient to explain all changes in NK cell profiling in alemtuzumab treated MS patients.

Ibrutinib Potentiated NK Cell-Mediated Cytotoxicity in Mouse Models of B-Cell Lymphomas

Background:Ibrutinib, a first-in-class, once-daily oral inhibitor of Bruton’s tyrosine kinase (BTK), is indicated by the US FDA for the treatment of patients with chronic lymphocytic leukemia/small lymphocytic lymphoma, including patients with deletion 17p, patients with mantle cell lymphoma who have received at least 1 prior therapy, and patients with Waldenström’s macroglobulinemia. In addition to blocking B-cell activation via inhibition of BTK signaling, ibrutinib was reported to modulate immune function by driving TH1 response through IL-2-inducible T-cell kinase (Dubovsky, Blood 2013). Ibrutinib was also shown to reprogram macrophages toward a TH1 phenotype that fostered CD8+ T-cell cytotoxicity in pancreas ductal adenocarcinoma-bearing mice (Gunderson, Cancer Discov 2016). Both the direct and indirect effects on TH1 response may play a role in the therapeutic efficacy of ibrutinib in models of lymphoma and solid tumors (Sagiv-Barfi, PNAS 2015). Another essential component of the immune system with a role against cancer is natural killer (NK) cells involved in the recognition and elimination of tumor cells. In this study, we evaluated the effects of ibrutinib on NK cell-mediated cytotoxicity in mouse models of B-cell lymphoma.Methods:Experiments in TMD8 xenograft models were performed in CB17.SCID and NSG mice, and experiments in an A20 syngeneic model were performed in BALB/c mice. Mice were orally administered ibrutinib (once daily) for a total duration of 2 weeks starting from the time tumors reached a volume of 100-150 mm3. Tumor BTK occupancy was determined by a gel-based probe assay 4 hours after the last dose of ibrutinib, and was normalized to the total BTK level. NK cell-mediated cytotoxicity was also evaluated in mice carrying X-linked spontaneous mutation in BTK, Btk(xid) mice (009361; Jackson Laboratory). For the NK cell cytotoxicity assay, mouse splenocytes were added to PKH67-labeled YAC-1 cells at different effector-to-target ratios and incubated at 37°C for 4 hours. Propidium iodide was added and flow cytometry was performed to determine target cell viability. Human or mouse cytokines/chemokines were quantified using MILLIPLEX® MAP Kit (HCYTMAG-60K-PX38 and MCYTMAG-70K-PX32).Results:In TMD8 ABC-DLBCL models, tumor suppression after ibrutinib treatment was only observed in CB17.SCID mice but not in NSG mice despite tumor BTK occupancy of >90% in both models. As these 2 strains of mice differ in their NK cell profile (NSG mice lack mature T, B, and NK cells whereas CB17.SCID mice are severely deficient in T and B cells), we were interested in studying the immune modulation function of ibrutinib on NK cells. Interestingly, elevated NK cell cytotoxicity was seen in Btk(xid) mice compared to Btk wild-type mice, suggesting a potential role of BTK depletion in NK cell function. Treatment with ibrutinib increased NK cell-mediated cytotoxicity in both syngeneic A20 B-cell lymphoma and TMD8 xenograft models (Figure 1), but did not affect NK cell population (% of NKp46+ or CD3-CD49b+). A20 tumor-bearing mice had lower NK cell cytotoxicity compared to non-tumor-bearing mice, suggesting suppression of NK cell function by tumors. In addition, ibrutinib reduced tumor-derived cytokines IL-6 and IL-10, which are negative regulators of NK cells. IFN-gamma secreted by nontumor cells was increased in the sera of CB17.SCID but not in NSG mice after ibrutinib treatment, providing further evidence of the increased NK function with ibrutinib. The detailed mechanisms of modulation of NK cell function by ibrutinib is currently under investigation.Conclusions:We report herein that ibrutinib enhanced NK cell-mediated cytotoxicity in mouse models of B-cell lymphomas. In addition to its direct effect on BTK inhibition in tumor cells, these data provide further evidence of the immune modulation function of ibrutinib. The role of ibrutinib in NK cell activation as observed in the current study may further expand the potential application of ibrutinib therapy. [Display omitted] DisclosuresKuo:Pharmacyclics, LLC, an AbbVie Company: Employment, Other: Travel, Accommodations, and Expenses, Patents & Royalties: Pharmacyclics, LLC, an AbbVie Company; AbbVie: Equity Ownership. Hsieh:Pharmacyclics, LLC, an AbbVie Company: Employment. Whang:Pharmacyclics, LLC, an AbbVie Company: Employment; AbbVie: Equity Ownership. Huang:Juno: Equity Ownership; Pharmacyclics, LLC, an AbbVie Company: Employment; Five Prime: Equity Ownership; Merrimack: Equity Ownership. Sirisawad:AbbVie: Equity Ownership; Pharmacyclics, LLC, an AbbVie Company: Employment. Chang:AbbVie: Equity Ownership; Pharmacyclics, LLC, an AbbVie Company: Employment, Patents & Royalties: Pharmacyclics, LLC, an AbbVie Company.

Cytokine-Mediated Natural Killer Cells Effects Impair Hematopoietic Stem Cell Function

Natural killer (NK) cells participate in innate and adaptive immune responses, and upon activation rapidly produce cytokines, chemokines, and growth factors, including IFNγ, TNFα, TGFβ, GM-CSF, MIP1α, MIP1β, IL-10, and others, which can affect the function of other hematopoietic cells. Considering the recent evidences that hematopoietic stem cells (HSCs) respond to cytokine signaling, we hypothesized that NK cell-mediated cytokine production could mediate HSC function.By the use of co-cultures of purified Ly5.1 murine NK cells and congenic Ly5.2 HSCs, we concluded that NK activity affects HSC frequency in vitro as well as hematopoietic reconstitution in vivo. Sorted NK cells (CD3- NK1.1+) and HSCs (Lin-, Sca1+, ckithi, CD48-, CD150+) were co-cultured in the presence or absence of IL2 over an OP9 stromal cells layer for 14 to 28 days. After 14 days, the addition of NK cells to HSC cultures resulted in an approximate 2-fold reduction of lineage negative cells (Lin-) recovered cells, as compared to control HSC cultures, as determined by flow cytometry analysis. Lin- counts were even lower in HSC+NK long-term cultures when compared to HSC only cultures. Ly5.1 HSCs and/or Ly5.2 NK cells were injected into sublethally irradiated Ly5.1/2 chimeric mice in a ratio of 105 NK to 103 HSCs per mouse. The addition of IL2-stimulated NK to injected HSCs reduced engraftment from 15.7% to 1.82% when the 16 weeks bone marrow (BM) chimerism was analyzed. In agreement, donor CD45.1 cells contribution to the LSK and HSC subpopulations was reduced in the HSC+NK transplanted mice. To test whether NK depletion from BM grafts would affect HSC function, we performed limiting dilution transplantation assays where whole BM from Ly5.2 mice was submitted to immunonagnetic NK1.1 or IgG depletion and injected into lethally irradiated Ly5.1 animals. Donor chimerism after 8 and 16 weeks of transplant showed that depleting NK cells improves the engraftment ability of HSC in a cell dose-dependent manner. When 25 x104 BM cells were injected, chimerism increased from 40 to more than 90% in NK depleted group. Of note, HSC frequency was 1 in 1595 in the control and 1 in 95 in the NK depleted group.In order to understand the mechanisms by which NK cells could regulate HSCs, we took advantage of a CCAAT/enhancer-binding protein gamma (C/ebpg) knockout (KO) conditional mouse model generated in our laboratory, considering that C/ebpg had been previously shown to regulate NK cytotoxicity. Using similar culture conditions, HSCs and NK cells isolated from control (CT) or Cebpg KO mice were injected into congenic sublethally irradiated recipients. Results showed that Cebpg-deficient NK cells do not harm HSC engraftment as CT NK cells do. For instance, after 8 weeks, the addition of CT non-stimulated and IL-2-stimulated NK cells to normal transplanted HSCs reduced the engraftment from 40% to 20% and 10%, respectively. In contrast, chimerism was not different when HSCs only or HSCs + stimulated KO NK cells were transplanted. Gene expression and cytokine profiles of deficient and normal NK cells revealed the potential players of this HSC-NK regulation. Of these, interferon gamma (IFNg), was lower produced by the C/ebpg deficient NK cells. Therefore, besides controlling NK cytotoxicity, we showed here that C/ebpg also plays a role in the regulation of HSCs by NK-mediated cytokine production.Next, we investigated whether depletion of NK cells from human BM samples would improve transplantation efficiency. NK cells were removed using CD56 antibody and transplanted into sublethally irradiated NSG mice. Sixteen weeks after transplantation, animals were sacrificed and the percentage of human CD45 cells in blood, BM, and spleen demonstrated that NK depletion from human BM favors engraftment.Altogether, these findings provide new insights to the knowledge of HSC regulation by NK cells, which are present in BM transplantation (BMT) grafts. Although the alloreactive effect of NK cells against non-identical tumor cells from BMT recipients is well known, its cytokine-mediated effects over identical progenitor cells from the graft were not previously explored. We show that NK-secreted cytokines harm stem cell function, thus suggesting that depletion of NK cells from BM donor cells preparations can improve stem cell engraftment, particularly in the setting of alternative transplants with limiting cell numbers or non-myeloablative conditioning regimens. DisclosuresNo relevant conflicts of interest to declare.

A population of atypical CD56−CD16+ natural killer cells is expanded in PTSD and is associated with symptom severity

IntroductionPost-traumatic stress disorder (PTSD) has been associated with immune disturbances, including a higher incidence of infections and autoimmune diseases as well as a net pro-inflammatory state. Natural killer (NK) cells, a key component of the innate immune system, have been less well-studied in PTSD despite their importance in immunity. MethodsWe studied two independent samples of combat-exposed male war veterans with or without PTSD, the first (“Discovery Sample”) to generate hypotheses, and the second (“Validation Sample”) to replicate the findings. The Discovery Sample was comprised of 42 PTSD subjects and 42 controls. The Validation Sample was comprised of 25 PTSD subjects and 30 controls. Participants had fasting, morning blood samples collected for examination of the frequency of NK cell subsets, determined by flow cytometry. The current and lifetime Clinician Administered PTSD Scale (CAPS) was used to assess symptom severity. Statistical analyses were adjusted for age and BMI. ResultsPTSD subjects compared to controls had (i) a significantly higher relative frequency of atypical CD56−CD16+ NK cells in the Discovery Sample (p=0.027), which was replicated in the Validation Sample (p=0.004) and the combined sample (p<0.001), and (ii) a non-significantly lower relative frequency of CD56brightCD16− NK cells in the two samples (p=0.082; p=0.118), which became statistically significant in the combined sample (p=0.020). Further, within subjects with PTSD of both samples, the relative frequency of atypical CD56−CD16+ NK cells was near significantly positively correlated with lifetime PTSD severity (p=0.074). DiscussionThis study is the first to characterize NK cell subsets in individuals with PTSD. The results suggest that combat-exposed men with PTSD exhibit an aberrant profile of NK cells with significantly higher frequencies of an atypical population of CD56−CD16+ cells and possibly lower frequencies of the functional CD56brightCD16− NK cell subsets. Higher proportions of dysfunctional CD56−CD16+ cells have been reported in certain chronic viral infections and in senescent individuals. It is possible that this could contribute to immune dysfunctions and prematurely senescent phenotypes seen in PTSD.

Natural killer cells expanded on membrane-bound IL15 or IL21 activate different miRNA transcriptional profiles with functional consequences

MicroRNA (miRNA) are non-coding regulatory RNA that are key posttranscriptional regulatory machinery of cells. miRNA play important role in several physiological functions during embryonic development, differentiation, cancer progression as well as hematopoiesis, and regulation of immune responses. miRNA regulate development of various immune cells including T and Natural killer (NK) cells. Owing to significant interest in adoptive immunotherapy, numerous methods for expanding NK cells have been developed in recent years. We have previously developed a robust platform for ex-vivo expansion of human NK cells using K562 expressing membrane-bound IL21 (K562.mbIL21). The study of the role of miRNA in NK cells biology is an emerging area of research in development, homeostasis and immune responses. However, the role and effect of expansion of NK cells on miRNA and consequent impact on post-transcriptional regulation of protein expression in expanded NK cells remain to be studied. In this study we investigated the role of distinctive miRNA expression profiles that could influence post-transcriptional regulation of gene expression, and consequently differential protein expression in NK cells expanded on K562.mbIL21 platform compared to primary unexpanded NK cells. We had previously shown that K562.mbIL21 platform enables significantly higher expansion of NK cells compared to K562 expressing membrane bound IL15 (K562 mbIL15), therefore in this study we also compared miRNA expression profiles of NK cells expanded on K562.mbIL15 and K562.mbIL21. We used primary protein and miRNA obtained from, human NK cells derived from 4 healthy donors, Pre and post-expansion on K562.mbIL15 and K562.mbIL21 platforms. miRNA expression profiles were analyzed using Nanostring's nCounter. Initial screening of our dataset revealed 13 miRNAs that are differentially expressed by at least 16 folds between fresh and expanded NK cells or mbIL15 and mbIL21. Comparison between the mbIL15 and mbIL21 expanded cells revealed differential expression of miRNAs critical for regulation of cell cycle proteins as well as key cytokines (TNF-a and interferon-g) such as miR-124-3p and miR 146a-5p (76x and 32x lower in mbIL21 expanded cells respectively). Also, analysis of protein expression by RPPA revealed upregulation of cell cycle proteins cyclin D1 and AMPK, and downregulation of pro-apoptotic protein Bim in mbIL21 expanded NK cells compared with mbIL15 and/or primary unexpanded cells. We are currently correlating miRNA expression and proteomic profiles of expanded and unexpanded cells to identify possible targets of miRNA regulation and subsequently study specific roles of these miRNA in this expansion system by overexpression o/blockade of target miRNA.

Phenotypical and functional profiles of natural killer cells exhibiting matrix metalloproteinase-mediated CD16 cleavage after anti-HIV antibody-dependent activation.

Natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC) has been linked to protection from HIV infection and slower progression towards AIDS. However, antibody-dependent activation of NK cells results in phenotypical alterations similar to those observed on NK cells from individuals with progressive HIV infection. Activation of NK cells induces matrix metalloproteinase (MMP)-mediated cleavage of cell surface CD16. In the present study we assessed the phenotype and functional profile of NK cells exhibiting post-activation MMP-mediated CD16 cleavage. We found that NK cells achieving the highest levels of activation during stimulation exhibit the most profound decreases in CD16 expression. Further, we observed that educated KIR3DL1(+) NK cells from human leucocyte antigen (HLA)-Bw4-carrying donors exhibit larger decreases in CD16 expression post-activation than the KIR3DL1(-) NK cell subset containing cells educated via other inhibitory receptor/ligand combinations and non-educated NK cells. Lastly, we assessed the ex-vivo expression of CD16 on educated KIR3DL1(+) NK cells and the KIR3DL1(-) NK cell subset from HLA-Bw4-carrying HIV-uninfected and HIV-infected donors. Suggestive of in-vivo activation of KIR3DL1(+) NK cells during HIV infection, CD16 expression was higher on KIR3DL1(+) than KIR3DL1(-) NK cells in uninfected donors but similar on both subsets in HIV-infected donors. These results are discussed in the context of how they may assist with understanding HIV disease progression and the design of immunotherapies that utilize antibody-dependent NK cell responses.

Activated natural killer cells accelerate liver damage in patients with chronic hepatitis B virus infection.

Emerging evidence indicates that natural killer (NK) cells may contribute to liver injury in patients with hepatitis B virus (HBV) infection. Because HBV infection progresses through various disease phases, the cytolytic profiles of peripheral and intrahepatic NK cells in HBV-infected patients remain to be defined. In this study, we comprehensively characterized intrahepatic and peripheral NK cells in a cohort of HBV-infected individuals, and investigated their impact on liver pathogenesis during chronic HBV infection. The study population included 34 immune-clearance (IC) patients, 36 immune-tolerant (IT) carriers and 10 healthy subjects. We found that the activity of peripheral NK cells from IC patients was functionally elevated compared to IT carriers and controls, and NK cell activation was indicated by an increased expression of CD69, CD107a, interferon (IFN)-γ and tumour necrosis factor (TNF)-α. Further analysis showed that the increased activity of both peripheral and hepatic NK cells was correlated positively with liver injury, which was assessed by serum alanine aminotransferase levels (ALT) and the liver histological activity index (HAI). Interestingly, the frequency of peripheral NK cells was reduced in IC patients (especially those with higher HAI scores of 3-4), but there was a concomitant increase in hepatic NK cells. The functionally activated NK cells are enriched preferentially in the livers of IC patients and skew towards cytolytic activity that accelerates liver injury in chronic hepatitis B (CHB) patients.

The cellular immune system in myelomagenesis: NK cells and T cells in the development of MM and their uses in immunotherapies

As vast strides are being made in the management and treatment of multiple myeloma (MM), recent interests are increasingly focusing on understanding the development of the disease. The knowledge that MM develops exclusively from a protracted phase of monoclonal gammopathy of undetermined significance provides an opportunity to study tumor evolution in this process. Although the immune system has been implicated in the development of MM, the scientific literature on the role and status of various immune components in this process is broad and sometimes contradictory. Accordingly, we present a review of cellular immune subsets in myelomagenesis. We summarize the current literature on the quantitative and functional profiles of natural killer cells and T-cells, including conventional T-cells, natural killer T-cells, γδ T-cells and regulatory T-cells, in myelomagenesis. Our goal is to provide an overview of the status and function of these immune cells in both the peripheral blood and the bone marrow during myelomagenesis. This provides a better understanding of the nature of the immune system in tumor evolution, the knowledge of which is especially significant considering that immunotherapies are increasingly being explored in the treatment of both MM and its precursor conditions.

A Suppressive Microenvironment in Acute Myeloid Leukemia Induces Global Alteration of T and NK Cell Profiles - Evidence for Immune-Editing Effect By Leukemia

While chemotherapy is successful at inducing remission of acute myeloid leukemia (AML), the disease has a high probability of relapse. Immune-surveillance of AML and susceptibility of leukemia cells to both T cell and natural killer (NK) cell attack justifies the application of immune strategies to control residual AML persisting after remission induction. It is critical to understand the immunological landscape of AML at presentation and during the course of treatment to develop a rational approach for the utilization of immunotherapy in improving outcomes in AML. However, the abnormalities of immune function and possible immune deficits in AML have not been fully characterized.To explore cellular immunity in AML, we prospectively collected the samples from 32 patients (median age 63 years; range 24-80) with newly diagnosed AML or high-risk MDS under an IRB approved protocol. Mononuclear cells were isolated from blood and bone marrow at the onset of leukemia, 14-21 days (early phase) and 28-35 days (late phase) after the initiation of induction chemotherapy. Healthy donor blood from age and gender matched controls (n=31, median age 61 years) was concurrently analyzed. Extensive multi-color flow cytometry analysis was performed to characterize T cell subsets [regulatory T cells (Treg), natural killer (NK) cells, NKT cells and γδ T cells] with various functional markers representing T cell exhaustion (PD-1, LAG3, TIM-3, PDL-1), activating and inhibitory NK cell receptors (KIR, Lir-1, NKG2A, NKG2D) and the effector molecule (TRAIL).Both CD4 and CD8 T cell subsets showed phenotypic evidence of exhaustion with elevated expression of PD-1 (CD4: 30.1± 9.4% and CD8 32.8±11.6%: P<0.05), LAG3 (CD4: 28.3± 15.6% and CD8 25.3±16.3%: P<0.001), and TIM3 (CD4: 7.2± 5.0% and CD8 5.4±3.8%: P<0.001) at AML diagnosis compared to healthy controls. The markers of exhaustion were found in all memory subsets (stem cell memory, central memory, effector memory and effector cells) suggesting that T cells were affected during early maturation (p<0.05). Furthermore, a significantly higher frequency of exhausted T cells was found in bone marrow compared with peripheral blood in paired samples (p<0.01). These changes were accompanied with higher frequency of FoxP3+ CD4 cells in both peripheral blood and bone marrow mainly within the HeliosloFoxP3+Treg (p<0.01). Changes persisted in AML patients from the diagnosis to later phase after induction chemotherapy. NK cell subset analysis revealed dynamic NK cell reconstitution after induction chemotherapy: a significantly lower frequency of CD56dimNKG2A+KIR-CD57- NK cells at diagnosis suggesting NK cell maturation arrest, followed by a burst of expansion of CD56brightNK cells early post induction followed by an increase of the CD56dimNKG2A+KIR-CD57- NK subset. Reduced expression of the activating receptor, NKG2D was accompanied with higher expression of Lir-1, a NK cell inhibitory receptor, in all NK cell subsets throughout the course of treatment suggesting a dysfunctional NK cell population. TRAIL expression was significantly reduced in both NKT cells and CD8 cells. Finally, γδ T cells showed significantly lower NKG2D expression at diagnosis.AML is associated with a global abnormality of both innate and adaptive immunity with an increased exhaustion and regulatory phenotype changes across CD4, CD8, NK cell, NKT cells, and γδ T cells. Thus myeloid leukemia cells might create microenvironment that protects them against immune surveillance which persists into early remission through an immune-editing effect. Effective immunotherapy for AML should include strategies to overcome this unfavorable milieu. [Display omitted] DisclosuresSavona:Karyopharm Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity’s Board of Directors or advisory committees; Celgene: Membership on an entity’s Board of Directors or advisory committees; Gilead Sciences, Inc.: Membership on an entity’s Board of Directors or advisory committees; Incyte: Membership on an entity’s Board of Directors or advisory committees; Novartis: Membership on an entity’s Board of Directors or advisory committees.

Profile of natural killer cells after a previous natural Vaccinia virus infection in an in vitro viral re-exposure

The present study compares the profile of NK cells in an in vitro re-exposure by Vaccinia virus (VACV), in groups that have had a previous vaccination or natural infection. Our data suggests that stimulation with VACV triggers a cytotoxic response by NK cells marked by an increase of NCRs: NKp30, NKp44, and NKp46 in infected (vaccinated and unvaccinated) subjects and in non-infected vaccinated patients, when compared with non-infected unvaccinated individuals. However, the degranulation and secretion processes are inhibited in infected (vaccinated and unvaccinated) subjects and in the non-infected vaccinated patients, when compared with non-infected unvaccinated individuals. We demonstrated that stimulation with VACV downregulates the percentage of expression of Perforin, Granzyme A, and CD107a, but upregulate CD94 in infected (vaccinated and unvaccinated) subjects and in non-infected vaccinated patients, when compared with non-infected unvaccinated individuals. Furthermore, the percentage of IFN-γ+ NK cells was significantly lower in non-infected unvaccinated subjects, when compared with infected (vaccinated and unvaccinated) and non-infected vaccinated individuals. Our results also show that the percentage of TNF-α+ NK cells was significantly higher in infected (vaccinated and unvaccinated) subjects and in non-infected vaccinated patients, when compared with non-infected unvaccinated individuals, after in vitro stimulation with UV-inactivated VACV. Our data suggest that the expression of NCRs NKp30, NKp44, NKp46 and cytokines by NK cells are important in the innate response against VACV.

Neutrophil microparticles modulate cytokine production by natural killer cells

Neutrophil microparticles (NMs) are scarce in the circulation but are highly enriched at sites of inflammation and exert profound effects on immune cells. In the present study, we addressed whether NMs modulate cytokine-producing capacity of natural killer (NK) cells. NMs inhibited the production of IFN-γ and TNF-α but enhanced the release of TGF-β1 by IL-2/IL-12-activated NK cells. The inhibitory effect of NMs was strongly attenuated by blockade of phosphatidylserine exposed on NMs. Thus, NMs skew the cytokine profile of NK cells from pro-inflammatory toward anti-inflammatory, potentially favoring the resolution of inflammation.