Natural Killer Subpopulations Research Articles

C/Ebpg (CCAAT/Enhancer Binding Protein Gamma) Balances Cytotoxic and Secretory Potential of Natural Killer Cells

C/EBPs (CCAAT/enhance-binding proteins) are a family of transcription factors involved in a variety of hematopoietic processes, regulating both terminal differentiation and cellular proliferation. Among these, it was previously reported that C/EBP gamma (C/EBPg) has a role in the development of Natural Killer (NK) cells. However, the mechanisms of such regulation are unknown. NK cells are lymphocytes with effector functions of cytotoxicity and production of cytokines, both dependent on a dynamic equilibrium between the expression of activating and inhibitory receptors as well as cytokine receptors. The two functions (cytotoxic and secretory) make NK cells important components of hematopoiesis, able to eliminate susceptible targets as well as recruit other cells to amplify inflammatory responses. With the aim of studying the regulation of NK cells by C/EBPg, we isolated NK cells from transgenic Cebpg knockout (KO) mice and controls to analyze their function. To characterize NK cells, we analyzed their frequency (Lineage-/CD3-/NK1.1+ cells) and the expression of the receptors NKG2D, Ly49D and NKG2A by flow cytometry of splenocytes. Both analyses showed no difference between control or Cebpg KO NK cells. Although the numbers of NK cells and their receptors were similar between Cebpg WT and KO animals, a functional assay that measured NK cell degranulation by CD107a expression after co-incubation with YAC-1 target cells showed that the expression of this marker was 5-times lower in Cebpg KO splenocytes than in controls (CT = 12.44 ± 2.50%; KO = 2.255 ± 0.67%, p=0.007), suggesting that Cebpg deficient NK cells are not fully activated after target cell recognition. In addition, a cytotoxicity assay by flow cytometry was performed using a fluorescent probe (Cell Tracker Orange) that was incorporated to YAC-1 cells upon exposure to sorted and IL-2 activated NK cells in culture. In the 10:1 NK:target cells ratio, Cebpg KO cells were significantly less cytotoxic than NK control cells (CT = 23.36 ± 8.67%; KO = 10.60 ± 1.66%, p=0.038). The other NK:target cells ratios of 5:1 and 1:1 showed the same tendency. In addition, the functional subtypes of these cells were characterized according to the expression of CD27 and CD11b, which allowed the identification of NK subpopulations as immature secretory, mature secretory, cytotoxic or tolerant. The KO animals showed higher percentages of secretory cells (CT = 10.77 ± 5.38%; KO = 12.98 ± 13.63%, p=0.0002) and a reduction of cytotoxic cells in comparison to the NK control cells (CT = 12.22 ± 11.08%; KO = 10.65 ± 3.82% p=0.013). Cytokine levels of IL-2, IL-4, IL-6, IL-10, IL-17α, TNFα and IFNγ, obtained from NK culture supernatants, were measured by flow cytometry, after IL-2 activation. Among these cytokines, the production of IFNγ by Cebpg-deficient NK cells was reduced (CT = 37.68 ± 0.51 pg/mL; KO = 22.34 ± 0.14 pg/mL, p=0.023). Together, these experiments indicate that C/EBPg regulates NK cell cytotoxicity. This may be explained, at least in part, by the reduced frequency of the mature cytotoxic NK subpopulation as compared to the secretory subtypes. Moreover, IFNγ may be an important target for the regulation of NK cell function. Finally, C/EBPg seems to be critical to mediate NK cell functions and not only for their development from the ontogenetic point of view. DisclosuresNo relevant conflicts of interest to declare.

Abnormal Distribution and Function of NK Cells Subsets May Lead to Impaired Tumor Surveillance in a JAK2V617F Myeloproliferative Neoplasm Model

Background: The JAK2V617F mutation is the most common molecular abnormality present in Philadelphia chromosome-negative Classical Myeloproliferative Neoplasms (Ph-neg MPNs), present in approximately 95% of patients with Polycythemia Vera and in almost 50% of patients with primary myelofibrosis and essential thrombocythemia. Besides the genetic mutations, different cells in the bone marrow can modulate the microenvironment and contribute to disease initiation and maintenance. Natural killer (NK) cells are part of innate immunity and are divided into subsets, according the expression of CD27 and CD11b. The immature NK cells, CD27-CD11b- (tolerant), CD27+CD11b-, presenting less cytotoxicity, while the mature NK cells, CD27+CD11b+ and CD27-CD11b+ (cytotoxic), have a great cytotoxic function. Considering that the role of NK cells in Ph-neg MPNs is currently unclear, our hypothesis is that a defect of the NK cells may favor HSC malignant transformation and contribute to MPN development. Aim: We aimed to study their distribution and cytotoxic function in murine primary cells from a MPN transgenic model. Methods: NK cells were quantified by flow cytometry from splenocytes of a pre-established conditional vavCre knockin Jak2V617F (Jak2VF) murine model. Polycythemia and splenomegaly were confirmed in Jak2 wt/V617F vavCre+ (Jak2VF) as compared to Jak2 wt/wt vavCre+ (Jak2WT) control animals. Mice (n= 4 per group) were euthanized between 10-16 weeks of age. For immunophenotyping, spleen was isolated, submitted to red cell lysis, and stained with fluorescence-conjugated antibodies against Ter119, CD19, CD4, CD8 CD3, NK1.1, CD27, CD11b, CD69 and CD107a. All the stainings were performed at 4°C for 20 minutes and acquisition of at least ten thousands events per tube was performed in a FACSCanto™II flow cytometer. For the activation and degranulation assays, splenocytes were cultivated and stimulated with PMA (2.5ug/mL) and ionomicyn (0.7ug/mL) for 3 hours and then submitted to the analysis of CD69and CD107a expression, respectively. The cytotoxicity assay was performed by a co-culture of sorted NK cells with YAC-1 cells previously marked with an impermeable cell tracker, for 3 hours. Then, the target cell death percentage was determined by 7-AAD detection. Results: The analysis of the degranulation marker CD107a in activated NK cells (CD69+) showed that, when compared with the Jak2WT group, the Jak2VF mice presented a decreased frequency of CD107a+ NK cells (87.2% vs 74.1% p=.02). Besides, the ability of Jak2VF NK cells to kill the target cells was reduced when compared to the Jak2WT group (80.1%±17.4 vs 65.5%±8.7 at the 5:1 NK:target cell ratio and 84.9%±11.9 vs 75.3%±0.1 at the 10:1 NK:target cell ratio). The maturation profile showed that, when compared to Jak2WT mice, the frequency of immature NK cells was 4.2-fold increase in Jak2VF animals (8% vs 33% p=.009), and the percentage of these cells that expressed the degranulation marker CD107a was reduced when compared with the Jak2WT group (55.5% vs 42.7% p=.02). In agreement, the frequency of mature NK cells was decreased in Jak2-mutated mice (92% vs 67% p=.009) as compared to controls. Among the mature NK subpopulations, CD107+ cells were lower numbered in Jak2VF mice as compared to Jak2WT cells (94.3% vs 88.9% p=.005). We also observed a differential profile of functional NK subsets between Jak2WT and Jak2VF NK cells. There was an increase of CD27-CD11b- and CD27+CD11b- NK cells in Jak2-mutated mice (5.6% vs 18.3% p=.007 and 2.1% vs 14.6% p=.01, respectively). On the other hand, the percentage of CD27-CD11b+ NK cells in Jak2VF mice was lower than in Jak2WT mice (78.1% vs 49.9% p=.01). In summary, when compared to controls, Jak2-mutated mice presented a reduction in the frequency of NK cells able to release their lytic granules, suggesting a lower cytotoxic function. The reduction of cytotoxicity in Jak2VF NK cells may be explained by two factors: (1) the augment of immature tolerant NK cells, and (2) the reduction of cytotoxicity of the mature subset (CD27-CD11b+). To date, although the functional and maturation subsets of NK cells have been studied in solid tumors, they have not been previously associated with MPNs. Conclusion: Considering that mature and functional NK cells play a critical role in tumor surveillance, it is plausible that an impair of their distribution and function can favor stem cell-derived diseases like Ph-neg MPNs DisclosuresNo relevant conflicts of interest to declare.

Endometrium infection by human herpesvirus-6A: implication in female idiopathic infertility

We first reported HHV-6A DNA presence in 43% of endometrial cells from women with idiopathic infertility, whereas no fertile control women harbored the virus1. Growing evidence confirms this implication1-6. We investigated the effect of HHV-6A infection on the immunological status of the endometrium. This is a case-control study. The sample size guarantees to achieve a 0.8 power with 5% alpha error in detecting differences between groups. Endometrial biopsies, uterine flushing and whole blood samples were collected during the implantation (mid secretory phase) windows in 67 idiopathic infertile women. The presence of HHV-6A infection was evaluated by DNA and mRNA analysis (U22, U42, U94 genes) on endometrial cellular subsets1. We analyzed the endometrial immunological status evaluating: i) the levels of immune-regulatory HLA-G and HLA-E molecules, innate inflammatory (IL-6, IL-8, TNF-alpha), Th1 pro-inflammatory (IFN-gamma, IL-1alpha, IL-1beta, IL-12) and Th2 anti-inflammatory (IL-4, IL-10) cytokines by multiplex ELISA in uterine flushing samples; ii) endometrial receptivity to cytotrophoblasts in an endometrial 3D in vitro model; iii) natural killer (NK) cells and regulatory T cells percentage and immune-phenotype in endometrial biopsies and peripheral blood by flow cytometry. We confirmed the presence of HHV-6A infection in a 40% of idiopathic infertile women. We observed increased levels of IFN-gamma, IL-1beta and IL-12 (p<0.001; Student t test), decreased levels of both IL-4 and IL-10 (p<0.001) in uterine flushing samples from HHV-6A positive women. HLA-G and HLA-E molecules were not expressed on the surface of endometrial epithelial cells from HHV-6A positive women. As a proof of principle, endometrial biopsis positive for HHV-6A infection presented a lower permittivity to cytotrophoblasts invasion. We observed a lower amount of endometrial (e)NK cells in the endometrium of women positive for HHV-6A infection compared with endometrium negative for HHV-6A (p<0.0001; Student T test). In particular, when we analyzed (e)NK subpopulations, we observed a lower percentage of CD56brightCD16- (e)NK cells in women positive for HHV-6A infection (p<0.0001). When we looked at peripheral blood cell subsets, we observed a decrease in CD4+CD25+CD127dim/- regulatory T cells in women positive for HHV-6A infection (p=0.016). The identification of an effect of HHV-6A infection on endometrial immune status opens new perspectives in idiopathic infertile women care management. In addition, it would be possible to select antiviral therapies as novel therapeutic approaches to those idiopathic infertile women characterized by the presence of endometrial HHV-6A infection, to increase their pregnancy rate.

Primary Immunodeficiencies Unravel the Role of IL-2/CD25/STAT5b in Human Natural Killer Cell Maturation.

Natural killer (NK) cells play a pivotal role during immunity against viruses and circumstantial evidence also indicates that they can protect the host against developing tumors. Peripheral blood NK cells comprise CD56brightCD16lo/− cells that constitutively express CD25 (IL-2Rα) and CD56dimCD16hi cells that express CD25 upon activation. Using NK cells from two patients, one with a primary immunodeficiency characterized by a homozygous mutation in CD25 (born in year 2007 and studied since she was 3 years old) and one with a homozygous mutation in STAT5b (born in year 1992 and studied since she was 10 years old), we observed that the absence of IL-2 signaling through CD25 promotes the accumulation of CD56brightCD16high NK cells, and that CD56brightCD16lo, CD56brightCD16high, and CD56dimCD16high NK cells of this patient exhibited higher content of perforin and granzyme B, and proliferation capacity, compared to healthy donors. Also, CD56bright and CD56dim NK cells of this patient exhibited a reduced IFN-γ production in response to cytokine stimulation and increased degranulation against K562 cells. Also, the CD25-deficient patient presented a lower frequency of terminally differentiated NK cells in the CD56dimCD16hi NK subpopulation compared to the HD (assessed by CD57 and CD94 expression). Remarkably, CD56dimCD16high NK cells from both patients exhibited notoriously higher expression of CD62L compared to HD, suggesting that in the absence of IL-2 signaling through CD25 and STAT5b, NK cells fail to properly downregulate CD62L during their transition from CD56brightCD16lo/− to CD56dimCD16hi cells. Thus, we provide the first demonstration about the in vivo requirement of the integrity of the IL-2/CD25/STAT5b axis for proper human NK cell maturation.

Effect of the invivo application of granulocyte colony-stimulating factor on NK cells in bone marrow and peripheral blood.

Granulocyte colony‐stimulating factor (G‐CSF) has been widely used in the field of allogeneic haematopoietic stem cell transplantation (allo‐HSCT) for priming donor stem cells from the bone marrow (BM) to peripheral blood (PB) to collect stem cells more conveniently. Donor‐derived natural killer (NK) cells have important antitumour functions and immune regulatory roles post‐allo‐HSCT. The aim of this study was to evaluate the effect of G‐CSF on donors' NK cells in BM and PB. The percentage of NK cells among nuclear cells and lymphocyte was significantly decreased and led to increased ratio of T and NK cells in BM and PB post‐G‐CSF in vivo application. Relative expansion of CD56bri NK cells led to a decreased ratio of CD56dim and CD56bri NK subsets in BM and PB post‐G‐CSF in vivo application. The expression of CD62L, CD54, CD94, NKP30 and CXCR4 on NK cells was significantly increased in PB after G‐CSF treatment. G‐CSF treatment decreased the IFN‐γ‐secreting NK population (NK1) dramatically in BM and PB, but increased the IL‐13‐secreting NK (NK2), TGF‐β‐secreting NK (NK3) and IL‐10‐secreting NK (NKr) populations significantly in BM. Clinical data demonstrated that higher doses of NK1 infused into the allograft correlated with an increased incidence of chronic graft‐vs‐host disease post‐transplantation. Taken together, our results show that the in vivo application of G‐CSF can modulate NK subpopulations, leading to an increased ratio of T and NK cells and decreased ratio of CD56dim and CD56bri NK cells as well as decreased NK1 populations in both PB and BM.

IL-6/Stat3-Dependent Induction of a Distinct, Obesity-Associated NK Cell Subpopulation Deteriorates Energy and Glucose Homeostasis

Natural killer (NK) cells contribute to the development of obesity-associated insulin resistance. We demonstrate that in mice obesity promotes expansion of a distinct, interleukin-6 receptor (IL6R)a-expressing NK subpopulation, which also expresses a number of other myeloid lineage genes such asthe colony-stimulating factor 1 receptor (Csf1r). Selective ablation of this Csf1r-expressing NK cell population prevents obesity and insulin resistance. Moreover, conditional inactivation of IL6Ra or Stat3 in NK cells limits obesity-associated formation of these myeloid signature NK cells, protecting from obesity, insulin resistance, and obesity-associated inflammation. Also in humans IL6Ra+ NK cells increase in obesity and correlate with markers of systemic low-grade inflammation, and their gene expression profile overlaps with characteristic gene sets of NK cells in obese mice. Collectively, we demonstrate that obesity-associated inflammation and metabolic disturbances depend on interleukin-6/Stat3-dependent formation of a distinct NK population, which may provide a target for the treatment of obesity, metaflammation-associated pathologies, and diabetes.

Assessment of Natural Killer Cell Subsets in HCV Infection and Hepatocellular Carcinoma

Aim of work: In this study the NK cell CD markers testing were offer the possibility and potentiality of some types of NK cells to target HCV and HCC immunotherapy. Background: Natural Killer (NK) cells are important for responding to and destroying cells that have become infected with virus and cancer cells. We evaluated the effects of hepatitis C virus (HCV) only infected patients and HCV-related hepatocellular carcinoma (HCC) of Egyptian cases on peripheral blood NK cell subsets CD56bright and CD56Dim NK cells, CD56dim16+, CD56dim16 and with emphasis on the CD3 cells. Methods: There are NK subpopulations levels that can easily be distinguished by flow-cytometry as depicted. Results: A significant increase in CD3 HCV patients and HCC were observed compared with healthy control individuals.CD56 subsets were non-significantly elevated in HCV only infected patients, but highly significantly lowered in HCV infected cases that associated with HCC, in addition to HCC cases were recorded decreasing levels for CD56 subtypes. Finally the hallmark of this NK cells dysfunction is persistent activation promoting ongoing hepatitis and hepatocytes damage which contribute to the progression of HCC, while being unable to clear HCV due to impaired IFN- γ response. Conclusions: Testing of NK cell subtypes will offers the possibility and potential to target HCC by immunotherapy and personalizing immunotherapy theory in different tumors.

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

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

Altered chemotactic response to CXCL12 in patients carrying GATA2 mutations

GATA2 deficiency formerly described as MonoMAC syndrome; dendritic a cells, monocytes, B cells, and natural killer cell deficiency; familial a myelodysplastic syndrome/acute myeloid leukemia; or Emberger syndrome a encompasses a range of hematologic and nonhematologic anomalies, mainly a characterized by monocytopenia, B lymphopenia, natural killer cell a cytopenia, neutropenia, immunodeficiency, and a high risk of developing a acute myeloid leukemia. Herein, we present 7 patients with GATA2 a deficiency recruited into the French Severe Chronic Neutropenia a Registry, which enrolls patients with all kinds of congenital a neutropenia. We performed extended immunophenotyping of their whole a blood lymphocyte populations, together with the analysis of their a chemotactic responses. Lymphopenia was recorded for B and CD4(+) T cells a in 6 patients. Although only 3 patients displayed natural killer cell a cytopenia, the CD56(bright) natural killer subpopulation was nearly a absent in all 7 patients. Natural killer cells from 6 patients showed a decreased CXCL12/CXCR4-dependent chemotaxis, whereas other lymphocytes, a and most significantly B lymphocytes, displayed enhanced CXCL12induced a chemotaxis compared with healthy volunteers. Surface expression of CXCR4 a was significantly diminished in the patients' natural killer cells, a although the total expression of the receptor was found to be equivalent a to that of natural killer cells from healthy individual controls. a Together, these data reveal that GATA2 deficiency is associated with a impaired membrane expression and chemotactic dysfunctions of CXCR4. a These dysfunctions may contribute to the physiopathology of this a deficiency by affecting the normal distribution of lymphocytes and thus a potentially affecting the susceptibility of patients to associated a infections.

Natural Killer Cell Functional Activity After 4-1BB Costimulation.

Reports show enhancement of CD8 T cells' activity through CD137 (4-1BB) signal; however, not all data proved similar effect in natural killer (NK) cells. Here, the impact of 4-1BB signal on NK cells' function was assessed during short term cultures. To that end, cytokine-activated NK cells were cocultured with adenovirally transduced MCF-7 stimulator cells expressing 4-1BB ligand. Cellular cytotoxicity, cytokine production, and expression of cytotoxicity related genes were assessed after overnight cultures. Sharp decrease of CD56+ and CD56bright NK cells was demonstrated. 4-1BB neither enhanced cellular degranulation nor improved IFN-γ production although it promoted granzyme B, perforin, and FasL gene expression. 4-1BB signal stimulated higher proportions of CD56bright population to degranulate and express CD107a; however, it could not recover killing activity against K562 targets. Our data could not show major promotion in activity of all NK subpopulations. Due to great heterogeneity of NK cells, more investigation is needed to draw a comprehensive conclusion.

Differences in Natural Killer(NK) Reconstitution Between Unmanipulated Haploidentical and HLA Identical Stem Cell Transplantation and Relationship with Citomegalovirus and Graft Versus Host Disease (GVHD). Experience in One Centre in 22 Patients.

Introduction: It´s well known that NK cells exert an important role in postransplant immune reconstitution. Their maturation process through the contact with recipient´s bone marrow stromal cells is crucial for the development of their functional capacities. Previous studies showed the relevance of NK alloreactivity in relapse prevention and the influence of NK subpopulations and expression of activating receptors on CMV infection control, antitumor effects, and graft versus host disease (GVHD). Unmanipulated Haploidentical (Haplo) transplantation with postrasplant cyclophosphamide (Cy) is an emerging alternative for patients (pts) with no identical sibling donor with encouraging results. We previously reported preliminary data of NK cell reconstitution in 7 pts in Haplo setting. Our aim is to contrast our previous data in 22 pts comparing with conventional HLA-identical (HLAid) Stem Cell Transplantation (SCT) and to analyse the relationship between NK reconstitution and CMV reactivation and GVHD.Methods: 22 pts received an Haplo SCT and 7 pts an HLAid SCT in Gregorio Marañon Hospital between January 2013 and April 2014. Peripheral blood was used in all cases. Conditioning regimen comprised fludarabine (Flu), Cy and busulfan (Bux) for Haplo and Flu/Bux or Flu/Melphalan for HLAid SCT. Prophylaxis for GVHD consisted of high dose Cy on +3, +4, cyclosporine (CsA) A and mycophenolatemofetil for Haplo and CsA/Methotrexate for HLAid. Table1 show patients characteristics. For analysis of NK reconstitution and receptor expression multi-colour flow cytometry on FC500 and Navios Beckman Coulter® was used. Total NK cells, CD56 intensity and expression of NKG2A, NKp30, Nkp46 and NKG2D was studied at +15, +30, +60, and +90. For comparison between the two groups Mann–Whitney U-test was used.Results: Median NK cells/mm3 on +15 and +30 were significantly lower on Haplo than HLAid group ( 0 (0-5.5) and 28.5 (12.2-96.5) vs 69.5 (55-134) and 276 (151-422); p<0.05), being similar on + 60 and +90 (86 (49.5-158) and 147 (85-204.5) for Haplo vs 105 (39.2-162.5) and 117 (82-197) for HLAid; p ns). In Haplo pts NK cells reached normal levels on +60, while only 6/22 (27%) on +30. As previously reported NK high percentages of immature CD56bright (CD56b) cells and high expression of immature receptor NKG2A appears on +30, decreasing on +60 and +90 (CD56b 80% (72.5-88.5), NKG2A 88% (80-94) on +30, 39% (25-49) and 73% (62.2-86.7) on +60, 34% (23-43) and 69% (54.7-77.5) on +90. Compared with HLAid patients, the percentage of NKb cells was significantly higher (p<0.05) on Haplo group on +30, +60 and +90 and expression of NKG2A was also higher on +15, +30 and +60 (p<0.05). Interestingly, expression of activating receptors NKP46 and NKG2D linked to NK maturation are only significantly decreased in Haplo group comparing with HLAid on +15 (p<0.05), with appropriate expression and no differences on +30, +60 and +90. In fact, expression of NKP46 tended to be higher in Haplo on +30 (81% (62.5-90) vs 61% (42-71)), with no statistical significance (p 0.06). NKP30 had a low expression, similar in the 2 groups. In Haplo pts, no differences were found in total NK cell between pts with and without CMV reactivation at any time. Patients with acute GVHD had lower NK cells/mm3 at +30 (21.5 (10.7-35) vs 122 (21.7-260.7) and +60 (75(46-137) vs 156 (51-258); p 0.1), but statistical significance was not reached. Proportion of CD56b and NKG2A expression did not show any difference in pts regarding presence of CMV reactivation or GVHD.Conclusions: After Haplo SCT with postransplant Cy NK cell reconstitution seem to be delayed comparing with HLAid sibling SCT, reaching normal levels at +60. As previously reported, we confirm in our series that NK maturation also show a different pattern. In Haplo setting NK cells remains in an immature phenotype with high proportion of NK CD56b and expression of NKG2A much longer than in HLAid pts. Interestingly activating receptors expression, normally linked to maturation is only lower in Haplo pts in +15, but it is similar in both SCT types by day +30 and later, what should translate in appropriate cytotoxic capacity. In Haplo group, those patients with and without CMV reactivation had similar NK cell reconstitution. Patients with GVHD seem to have an impaired early NK reconstitution, but this fact needs to be contrasted.Acknowledgments: Fundación Mutua Madrileña. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Comparative Analysis of NK Receptor and T-Cell Receptor Repertoires in Patients with Chronic Myeloid Leukemia Treated with Different Tyrosine Kinase Inhibitors

Introduction: Given the critical role of BCR–ABL kinase activity in chronic myeloid leukemia (CML), tyrosine kinase inhibitors (TKIs) are currently considered the cornerstone of CML treatment. Previous studies have suggested that TKIs may influence anti-tumor immunity through off-target modulation of different immune effectors. Natural killer (NK) cells, as well as T cells in the context of adaptive immunity, are a key component of the innate immune system, providing first-line defense against virally infected cells and tumors. The activity of NK cells is modulated by a finely-tuned balance between signals received from inhibitory and activating cell surface receptors.Aims: We sought to evaluate the impact of first and second generation TKIs on modulating different NK cell receptors patterns; secondly we studied the effect of a TKIs driven NK subpopulations selection on treatment response. Finally, we analyzed the T cells Vβ-TCR repertoire to identify any restrictions.Materials and Methods: Peripheral blood samples from 25, 9 and 8 chronic phase CML patients treated with imatinib frontline, nilotinib and dasatinib as first or second line therapy, respectively, were collected. Patients characteristics are described below (see table 1). After separation of mononuclear cells (PBMC), the expression of several NK cell receptors (Killer Immunoglobulin-like Receptors, KIR: p70, p140, p58/p50; Killer Lectin-like Receptors, KLR: CD94, NKG2A, NKG2C/A, NKG2D; Natural Cytotoxicity Receptors, NCR: NKp30, NKp44, NKp46, NKp80; Co-receptors: 2B4; LIR1/ILT2, GPR56) and Vβ TCR-repertoire were analized by flow cytometry analysis. Treatment response was assessed with standardized real quantitative polymerase chain reaction and cytogenetics according to ELN recommendations.Results: The leukocyte count was not statistically different between groups (WBC = 5.5 x 109 / L vs. 6.8 x 109 / L vs. 5.6 x 109 / L, p = 0.09, respectively); also lymphocytes, considered either in percentage or absolute number, were comparable (32% vs 26% vs 35%, p = 0.08), as well as the percentage and absolute number of NK cells (20%; 0.37 x 109 / L vs. 15%; 0.26 x 109 / L vs. 24%; 0.54 x 109 / L (p = 0.17, p = 0.10).The analysis of NK receptors expression showed that patients treated with Imatinib exhibited a preferential selection of NK cells subpopulations harboring activating receptors (NKp30, NKp46, NKp80 and NKG2D), while in Dasatinib treated patients an increased expression of KIR (KIR2DL1) receptors was observed (figure 1). Interestingly, these effects were documented also in the absence of lymphocytosis. 44.4% (4 of 9 patients) of patients treated with nilotinib showed preferential expression of Vβ chains, compared with 87.5% of patients treated with dasatinib; no TCR-repertoire restriction was documented in the sole TKI primary resistant patient. 8 out of 17 patients showed a preferential expression of more than oneVβ chain (figure 2). No specific NK receptors profiles were found to be associated with different degrees of treatment response.Conclusions: These preliminary data suggest the existence of a different NKRs and T cell receptor repertoire modulation, mediated by Tyrosine-Kinase Inhibitors. Since no significant correlation between response and specific NK receptor profiles has been demonstrated, TKIs immunomodulatory effect seems secondary compared to direct inhibition of BCR-ABL kinase. However, it’s conceivable that NK and T cells subpopulations selection, induced by TKIs, may become relevant in the immunological control of leukemic disease at the time of drug discontinuation. These observations are currently being investigated on a larger series of patients. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

An effective interferon-gamma-mediated inhibition of hepatitis C virus replication by natural killer cells is associated with spontaneous clearance of acute hepatitis C in human immunodeficiency virus-positive patients

Hepatitis C virus (HCV) coinfection is an increasing health problem in human immunodeficiency virus-positive (HIV(+) ) individuals. However, a considerable proportion of HIV(+) patients manage to overcome acute hepatitis C (AHC) spontaneously. In the present study, we analyzed the role of natural killer (NK) cells in modulating the course of AHC in HIV(+) patients. Twenty-seven HIV(+) patients with AHC (self-limited course: n = 10; chronic course: n = 17), 12 HIV(+) patients with chronic hepatitis C (CHC), 8 HIV monoinfected individuals, and 12 healthy controls were studied. NK cells were phenotypically analyzed by flow cytometry. Interferon-gamma (IFN-γ) secretion, degranulation (CD107a), and anti-HCV (= inhibition of HCV replication) activity of NK subpopulations were analyzed using the HuH7A2 HCVreplicon cell system. NK cell frequency did not differ significantly between HIV(+) patients with chronic and self-limited course of AHC. However, we found NK cells from patients with self-limiting infection to be significantly more effective in inhibiting HCV replication in vitro than NK cells from patients developing CHC. Of note, antiviral NK cell activity showed no significant correlation with NK cell degranulation, but was positively correlated with IFN-γ secretion, and blocking experiments confirmed an important role for IFN-γ in NK cell-mediated inhibition of HCV replication. Accordingly, NK cells from patients that spontaneously cleared the virus displayed a stronger IFN-γ secretion than those developing chronic infection. Finally, we observed high expression of NKG2D and NKp46, respectively, to be associated with self-limiting course of aHCV. Accordingly, we found that blocking of these NK cell receptors significantly impaired antiviral NK cell activity. Our data suggest a strong IFN-γ-mediated antiviral NK cell response to be associated with a self-limited course of AHC in HIV(+) patients.

CD3−CD16−CD56brightImmunoregulatory NK Cells are Increased in the Tumor Microenvironment and Inversely Correlate with Advanced Stages in Patients with Papillary Thyroid Cancer

The innate immune system is the first line of defense and plays a key role in thyroid cancer development. The role of the tumor-infiltrating natural killer (NK) cells is becoming increasingly important in research and potential cancer therapies. NK cell subpopulations, CD3(-)CD16(+)CD56(dim) and CD3(-)CD16(-)CD56(bright), demonstrate a significant role in the tumor immuno-surveillance process. We investigated the distribution of CD3(-)CD16(+)CD56(dim) and CD3(-)CD16(-)CD56(bright) NK subpopulations in tissue and blood samples from patients with papillary thyroid cancer (PTC) and nodular goiter (NG). Twenty-eight patients with PTC, 13 patients with NG, and 50 healthy donors were included in the study. Tissue and blood samples from all patients and blood samples from healthy donors were analyzed for CD3(-)CD16(+)CD56(dim) and CD3(-)CD16(-)CD56(bright) NK cells by flow cytometry. A significant predominance of CD3(-)CD16(+)CD56(dim) cells compared to CD3(-)CD16(-)CD56(bright) NK cells was found in blood samples in all groups (p<0.0001 in PTC, NG, and healthy donors). Increased infiltration by CD3(-)CD16(-)CD56(bright) NK cells was observed in thyroid tissue of patients with PTC, as compared to CD3(-)CD16(+)CD56(dim) NK cells (p=0.046), while CD3(-)CD16(+)CD56(dim) NK cells demonstrated a higher infiltration of NG tissues. CD3(-)CD16(+)CD56(dim) NK cell tissue infiltration positively correlated with advanced stages of PTC. In contrast, the CD3(-)CD16(-)CD56(bright) NK cell population was negatively associated with tumor stage in patients with PTC. CD3(-)CD16(-)CD56(bright) NK cell infiltration seems to be associated with PTC progression. These findings contribute to a better understanding of the immune response in PTC and may lead to novel immunotherapeutic approaches in these patients.

IL-15 and macrophage secretory factors facilitate immune activation of neonatal natural killer cells by lipoteichoic acid

Neonates possess a relatively “naive”, yet inducible immune system. Our hypothesis is that upon strategic antigen exposure, cytokine priming and sensitization by accessory cells, natural killer (NK) cells could be activated to become a functional phenotype. We investigated the in vitro stimulation of cord blood (CB) and adult NK cells upon challenge with lipoteichoic acid (LTA), interleukin (IL)-15 and LTA-primed autologous macrophage-conditioned medium, using CD107a and CD69 phenotypes as indicators of activation. We also examined response of CB macrophages to LTA, in terms of P44/42 extracellular signal-regulated kinases (ERK1/2) activation and cytokine secretion. LTA significantly induced secretion of inflammatory cytokines tumor necrotic factor (TNF)-α, IL-6, IL-12 and activated the upstream signal of ERK1/2 phosphorylation in neonatal macrophages. The magnitude of responses to stimulation differed between neonatal and adult NK cells. Co-stimulation with IL-15 was critical for expansion of the CD69 and CD107a NK subpopulations in both neonatal and adult cells, upon a LTA challenge. NK cell activation could be enhanced by LTA-primed autologous macrophages through secretory factors. Our results indicated that neonatal macrophages and NK cells can evoke immunologic responses to a Gram-positive bacterial antigen. The combinatory priming strategy is relevant for development of novel protocols, such as IL-15 treatment, to compensate for the immaturity of the innate immune system in newborns against bacterial infections.

Natural killer cell functional dichotomy: a feature of chronic viral hepatitis?

Natural killer (NK) cells are involved in innate immune responses to viral infections either via direct cytotoxicity which destroys virus-infected cells or production of immunoregulatory cytokines which modulate adaptive immunity and directly inhibit virus replication. These functions are mediated by different NK subpopulations, with cytotoxicity being generally performed by CD56dim NK cells, whereas CD56bright NK cells are mainly involved in cytokine secretion. NK functional defects are usually combined so that impaired degranulation is often associated with deficient cytokine production. Innate immunity is thought to be relevant in the control of hepatitis virus infections such as hepatitis B virus (HBV) and hepatitis C virus (HCV), and recent findings reproducibly indicate that NK cells in chronic viral hepatitis are characterized by a functional dichotomy, featuring a conserved or enhanced cytotoxicity and a reduced production of interferon (IFN)-γ and tumor necrosis factor-α. In chronic HCV infection this appears to be caused by altered IFN-α signaling resulting from increased signal transducer and activator of transcription 1 (STAT1) phosphorylation, which polarizes NK cells toward cytotoxicity, and a concomitantly reduced IFN-α induced STAT4 phosphorylation yielding reduced IFN-γ mRNA levels. These previously unappreciated findings are compatible on the one hand with the inability to clear HCV and HBV from the liver and on the other they may contribute to understand why these patients are often resistant to IFN-α-based therapies.

CD16+ natural killer cells play a limited role against primary dengue virus infection in tamarins

CD16 is a major molecule expressed on NK cells. To directly assess the role of natural killer (NK) cells in dengue virus (DENV) infection in vivo, CD16 antibody-treated tamarins were inoculated with a DENV-2 strain. This resulted in the transient depletion of CD16(+) NK cells, whereas no significant effects on the overall levels or kinetics of plasma viral loads and antiviral antibodies were observed in the treated monkeys when compared to control monkeys. It remains elusive whether the CD16(-) NK subpopulation could play an important role in the control of primary DENV infection.

Natural killer–dendritic cell interaction in lymphocyte responses in hypersensitivity reactions to betalactams

Betalactams are the commonest cause of allergic reactions mediated by either IgE or T lymphocytes in which innate and adaptive immune systems mediate the earlier stages of immunological responses. One of the links between these systems is related to the interaction between natural killer (NK) and dendritic cells (DC). We have evaluated the role of NK cells and NK-DC interaction in the immunopathological mechanisms of nonimmediate reactions to betalactams. Patients allergic to amoxicillin (AX) (N = 17) and tolerant controls (N = 13) were included. Changes in phenotype (CD69, IFNγ, perforin, and granzyme B) in AX-stimulated NK cells, the cytotoxic activity on mature or immature DC (imDC), and the proliferation and phenotype of NK lymphocytes after culture with AX and DC were determined by flow cytometry. Amoxicillin induced activation and increases of perforin and granzyme B (P = 0.007 and P = 0.041 respectively) but not IFNγ production in NK cells from patients. In NK subpopulations, AX induced a significant enhancement of perforin and granzyme B in CD56(dim) (P = 0.005 and P = 0.002 respectively) and of IFNγ in CD56(bright) (P = 0.001). The cytotoxic phenotype was demonstrated by an increase of annexin V only in imDC (P < 0.001). Amoxicillin also induced an increased NK proliferation with different patterns, cytotoxic or proinflammatory, depending on the presence of imDC or mature DC, respectively. No differences were observed in NK cells from tolerant controls. These data could demonstrate the involvement of NK cells in the immunopathological mechanisms of nonimmediate allergic reactions to AX, showing that both the innate and adaptive immune systems are involved and crosstalk, producing amplification of the harmful effects observed in these drug reactions.

Canine CD8 T cells showing NK cytotoxic activity express mRNAs for NK cell-associated surface molecules

Natural killer (NK) cells have been considered to be a group of lymphocytes lacking clonally distributed receptors for antigens typical of T cells and B cells. In some mammalian species, including humans, a subpopulation of CD8 + peripheral blood lymphocytes (PBLs) exhibits NK activity. This NK subpopulation has not been well characterized in mammals and its characterization is particularly poor in the dog. In this study, we demonstrated that a subset of canine CD8 + cells derived from PBLs and lymphokine (IL-2)-activated killers (LAKs) of PBLs that was CD3 +, CD4 −, CD21 −, CD5 lo, α/βTCR +, and γ/δTCR − contained substantially higher levels of mRNAs for NK cell-related receptors (NKp30, NKp44, NKG2D, 2B4, and CD16 for PBL, and NKG2D and CD56 for LAK) than the corresponding CD8 − cells. This subset of CD8 + lymphocytes derived from LAKs also displayed significantly higher NK cytotoxic activity than the corresponding CD8 − cells. In contrast, CD8 + cells derived from nonstimulated PBLs showed very low levels of NK cytotoxic activity. Our results indicate that, in IL-2-stimulated PBLs, canine CD8 + cells are an important subset associated with NK cytotoxic activity.

Natural killer cell number and phenotype in bovine peripheral blood is influenced by age

Natural killer (NK) cells are critical to the innate defence against intracellular infection. High NK cell frequencies have been detected in human neonates, which may compensate for the relative immaturity of the specific immune response. Additionally, phenotypic subsets of NK cells have been identified in humans with different functional properties. In this study, we examined the age distribution and phenotype of NK populations in bovine peripheral blood, including neonatal animals. We found that the NK cell populations defined by the phenotypes CD3 −CD2 + and NKp46 + largely overlapped, so that the majority of NK cells in bovine peripheral blood were CD3 −CD2 +NKp46 +. The remainder of the NK-like cells comprised two minor populations, CD3 −CD2 +NKp46 − and CD3 −CD2 −NKp46 +; the relative proportions of these varied with age. The lowest frequency of NK cells was recorded in 1-day-old calves, with the highest frequency in day 0 calves. The phenotypic characteristics of CD3 −CD2 + and NKp46 + NK populations were similar; both populations expressed CD45RO, CD45RB, CD11b, CC84, CD8αα and CD8αβ and did not express CD21, WC1, CD14 or γδ TCR. Age-related phenotypic differences were apparent. The phenotypic characteristics of three NK subpopulations were described; a significantly greater proportion of the CD3 −CD2 −NKp46 + population expressed CD8α compared to CD3 −CD2 +NKp46 + cells. Furthermore, a significantly greater proportion of the CD3 −CD2 +NKp46 − population expressed CD8 compared to total CD3 −CD2 + cells. Adult cattle had a significantly higher proportion of perforin + cells compared to calves aged ≤6 weeks. In this age group, the majority of perforin + cells expressed NKp46, while in adults the majority of perforin + cells were NKp46 −. However, the proportion of NKp46 + and CD3 −CD2 + cells that expressed perforin was not significantly different in any age group tested.