Peripheral NK Cell Research Articles

Phenotypically and Functionally Altered T Cell Compartment in DLBCL Patients at Diagnosis and Its Long-Term Modification upon Chemoimmunotherapy Regimen

Background: Peripheral blood lymphopenia at diagnosis and after treatment has been widely reported as a negative prognostic factor in DLBCL. This phenomenon, which reflects host systemic immunity, is still poorly characterized. Very recently, we have described a profound phenotypic and functional alteration of the peripheral NK cell compartment in newly-diagnosed DLBCL patients at diagnosis and upon R-CHOP chemoimmunotherapy, and suggested that the complex dynamics of the immune system plays a pivotal role in the response to chemoimmunotherapy. Since the role of T cell-dependent memory and effector capabilities in long-term cure of chemoimmunotherapy-treated cancer patients is increasingly appreciated, we have focused our studies on their modifications in DLBCL.Aims: To analyze the phenotypic and functional profile of peripheral blood T lymphocyte compartment in DLBCL patients at diagnosis, and to assess the long-term impact of R-CHOP chemoimmunotherapy on T cell populations and functional competence.Patients and Methods: We prospectively compared 32 consecutive newly diagnosed DLBCL patients with 27 healthy, age- and sex-matched controls for: 1) absolute number (/mcl) and percentage (over PBMC) of the main T cell subsets: "conventional CD4+ and CD8+, double positive (CD4+CD8+), double negative (CD4-CD8-), CD56+ innate-like, and FOXP3+CD25bright regulatory T cells (Treg), measured by blood cell count and multi-parameter flow cytometric (FACS) analysis; 2) functional capability of T cell subsets, evaluated as the frequency of IFN gamma (IFNg)-producing cells upon a short-term (6-hr) stimulation with PMA/ionomycin, and the percentage of GrzB+ (cytotoxic granule marker) cells; and 3) plasma concentration of selected cytokines. Patients were analyzed at diagnosis, during and at different timepoints after chemoimmunotherapy completion.Results: DLBCL patients at diagnosis showed lower lymphocyte count (p<.001), mostly due to a selective decrement of CD4+ T cells (p=.003) and B lymphocytes (p=.0001). The absolute number of CD8+ or innate-like CD56+ T cells were not affected. As a result, the relative CD4/CD8 ratio was decreased (p=.009). The absolute number, but not the percentage, of Treg was slightly reduced with respect to controls. While CD8+ and innate-like T cell subsets transiently decreased during R-CHOP cycles, CD4+ T cell and Treg absolute numbers remained significantly lower than controls up to 1 year after therapy completion. As a result, CD4/CD8 ratio also remained lower than controls, up to 1 year after therapy.The phenotypically skewed T cell profile of DLBCL patients at diagnosis was associated with severe functional alterations, as the frequencies of IFNg-producing and Granzyme B-expressing cells were increased in both CD4+ (p=.007 for IFNg+ and p=.005 for GrzB+) and CD8+ (p=.002 for IFNg+ and p<.0001 for GrzB+) conventional T cell subsets. Noteworthy, the functional competence of innate-like CD56+ T cells showed no difference with controls. The frequency of GrzB+ and IFNg-producing CD4+ and CD8+ T cells transiently decreased during therapy, thus becoming comparable to controls, raised again by three months after therapy, and was persistingly higher than controls up to 1 year after therapy completion.Finally, IL-6 and IL-10 plasma levels were significantly elevated in DLBCL at diagnosis (p<.0001 and p=.011, respectively), and gradually normalized, shortly after R-CHOP therapy completion.Interestingly, CD4+ and CD8+ T cell capability to promptly produce IFNg was significantly higher in GC, but not in non-GC DLBCL patients at diagnosis, suggesting that patients T cell functional features may depend on DLBCL phenotype. Moreover, a higher percentage of IFNg+ T cells was observed in patients attaining a long-term remission, but not in resistant or relapsing ones, hinting to the association of immune competence with therapy outcome.Conclusions: T cell-dependent memory and effector capabilities might significantly contribute to the success of chemoimmunotherapy strategies, as suggested by the recently discovered vaccinal effect. The novel information provided by this study may reveal useful for innovative therapeutic approaches more focused on exploiting the dynamics of host immune cells in fighting cancer.*MC Cox and S Battella contributed equally to this work DisclosuresNo relevant conflicts of interest to declare.

Peripheral Blood Immunological Features Associated with Aortic Valve Disease and Ascending Throcic Aorta Aneurysm and Dilation

INTRODUCTION: Ascending thoracic aortic aneurysm (TAA) is a multi-factorial process in which histological modifications and immune-mediated inflammation are closely associated. The predominant role of a Th1-mediated response in influencing aortic wall remodeling, dilation, and aneurysm formation has been suggested by previous studies. Recently, the importance of chemokine receptors for Th1 cells recruitment into vascular inflammatory sites, as well as of the balance between pro- and anti-inflammatory T-cell subsets in influencing the severity of coronary artery disease, have been described.MATERIAL AND METHODS: We evaluated activation markers and chemokine receptors expression on peripheral T-cell and NK cell subsets of subject with aortic valve disease associated with ascending TAA (ascending aortic diameter &gt; 4 cm) and undergoing elective surgery for TAA (Group A), in comparison with patients with aortic valve disease without TAA (ascending aortic diameter &lt; 4 cm) (Group B). Peripheral blood samples from the two groups were also compared for intracellular T-lymphocyte cytokine production, frequency of regulatory T cells (Treg) and soluble levels of cytokine and chemokines. The aortic size index (ASI) was considered a parameter able to reflect aortic pathophysiological modifications leading to aortic dilation.RESULTS: The results demonstrated correlations between ASI values and CCR5 expression on CD3+, CD3+/CD8+, CD4+ and CD4+/CD28- T-cell subsets. In Group A the expression of CCR5 was higher on CD3+/CD8+, CD4+ and CD4+/CD28- T-cell subsets, when compared with Group B. CD4+ and CD4+/CD28- T-cells in Group A showed also a higher expression for the co-stimulatory molecule CD28 and the activation marker CD25, respectively. An increased expression of CXCR3 was found on CD4+, CD3+/CD8+ and CD3+/TCR+ T-cell subsets in Group A. A higher circulating fraction of NK cells, together with a higher NK cell positivity for CX3CR1, were observed in aneurysmatic patients. Intracellular cytokine analysis demonstrated a higher fraction of CD3+/CD4+ T-cells producing IL-17A and IL-10 in Group A, together with a higher intracellular content for IL-21. Finally, a higher soluble level of fractalkine (CX3CL1) has been detected in aneurysm group.CONCLUSION: Results indicate a higher activation state, migratory capacity and cytotoxic potential of peripheral blood NK and T-cell subsets in patients with aortic valve disease associated with ascending TAA, when compared with patients affected by aortic valve disease alone. These findings, together with the observed higher polarization towards a Th17 in patients with aortic aneurysm could suggest the involvement of autoimmune mechanisms leading to cellular loss, inflammation and fibrosis during ascending aortic wall dilation and aneurysmatic progression.Journal of Universal College of Medical Sciences Vol. 3, No. 1, 2015: 11-20

NK Cells are Activated in Amnestic Mild Cognitive Impairment but not in Mild Alzheimer's Disease Patients.

Alzheimerś disease (AD) is a progressive irreversible neurological brain disorder characterized by accumulation of amyloid-β, amyloid plaques, and neurofibrillary tangles. Inflammation and immune alterations have been linked to AD, suggesting that the peripheral immune system plays a role during the asymptomatic period of AD. NK cells participate in innate immune surveillance against intracellular pathogens and malignancy but their role in AD remains controversial. We have investigated changes in peripheral NK cell phenotypes and functions in amnestic mild cognitive impairment (aMCI, n = 10), patients with mild AD (mAD, n = 11), and healthy elderly controls (n = 10). Patients selected according to NINCDS-ADRDA criteria were classified using neuropsychological assessment tests. Phenotype analysis revealed differences in expression of CD16 (increased in mAD), NKG2A (decreased in aMCI), and TLR2 and TLR9 (both decreased in mAD). Functional assays revealed that NK cell killing activity and degranulation (CD107 expression) were unchanged in the three groups. In contrast, expression of the CD95 receptor was increased in aMCI and mAD. Granzyme B expression and cytokine production (TNFα, IFNγ) were increased in aMCI but not in mAD. CCL19- but not CCL21-dependent chemotaxis was decreased in aMCI and mAD, despite the fact that CCR7 expression was increased in aMCI. Our data suggest that the number of alterations observed in peripheral NK cells in aMCI represent an activation state compared to mAD patients and that may reflect an active immune response against a still to be defined aggression.

Peripheral natural killer cell maturation depends on the transcription factor Aiolos.

Natural killer (NK) cells are an innate lymphoid cell lineage characterized by their capacity to provide rapid effector functions, including cytokine production and cytotoxicity. Here, we identify the Ikaros family member, Aiolos, as a regulator of NK-cell maturation. Aiolos expression is initiated at the point of lineage commitment and maintained throughout NK-cell ontogeny. Analysis of cell surface markers representative of distinct stages of peripheral NK-cell maturation revealed that Aiolos was required for the maturation in the spleen of CD11b(high)CD27(-) NK cells. The differentiation block was intrinsic to the NK-cell lineage and resembled that found in mice lacking either T-bet or Blimp1; however, genetic analysis revealed that Aiolos acted independently of all other known regulators of NK-cell differentiation. NK cells lacking Aiolos were strongly hyper-reactive to a variety of NK-cell-mediated tumor models, yet impaired in controlling viral infection, suggesting a regulatory function for CD27(-) NK cells in balancing these two arms of the immune response. These data place Aiolos in the emerging gene regulatory network controlling NK-cell maturation and function.

NK Cell Activation as a Functional Marker for the Individual Response to Immunosuppression.

Background: NK cells might be involved in graft rejection due to their cytotoxic capacities but they bear also protective potential. After kidney transplantation, CD16 is down- regulated on CD56dim NK cells, which alters the peripheral NK cell repertoire remarkably (Neudörfl et. al, 2013). NK cell degranulation is accompanied by transient CD16 loss, this alteration can either reflect the functional status or be caused by immunosuppressive medication (IS). The effect of different immunosuppressive regimes (CNI vs. mTORi) on NK cell function has not been assessed completely yet. Hence, we aimed to investigate the underlying mechanisms with special attention to CD16 surface expression, IFN-g production and NFATc1 regulation. Methods: PBMCs of healthy donors were incubated with medium containing either Cyclosporine A, Tacrolimus, Sirolimus, Everolimus, Myphenolic acid (5μM) or control solvent, stimulated by PMA/Ionomycin or left untreated for 6 or 24h and analyzed by flow cytometry for CD16, IFN-g and NFATc1 expression. Results: CD16 surface expression was significantly reduced after stimulation in all donors. IS influenced this down modulation in two different ways: In the first donor type, IS did not change this activation-induced reduction. In the second type, CNI but not the other inhibitors were able to prevent CD16 down-regulation after 6h of stimulation. The inhibitors alone had no effect on CD16 surface expression. CNI diminished significantly IFN-g induction after stimulation while the other inhibitors had only little effect. Regarding the role of the calcineurin-NFAT pathway in NK cells, we observed an increase of total NFATc1 protein after stimulation in T, B and NK cells of all donors, which was blocked by CNI. However, extent and kinetics of this NFATc1 regulation differed between donors. Conclusions: Our studies provide an important insight into NK cell signaling and their behavior under IS. NFATc1 plays an important role not only in T cell but also in NK cell physiology. Furthermore, surface expression of CD16 and IFN-g induction are regulated independently of each other and CNI and mTORi differ in their impact on both parameters. Importantly, CD16 down-regulation seems to be associated with a greater sensitivity of NK and T cell responses to mTORi. Taken together, our studies support the development of markers that characterize individual responsiveness to and functional status under IS. Our aim is to further develop these functional assays for selection of an optimized IS in the future.

Diagnostic Accuracy of a Defined Immunophenotypic and Molecular Genetic Approach for Peripheral T/NK-cell Lymphomas

The diagnosis of peripheral T-cell and NK-cell lymphomas (PTNKCL) is difficult with few standards for required ancillary studies. We evaluated a series of PTNKCLs using a tiered approach to immunohistochemistry and molecular genetic characterization to document diagnostic accuracy and clinical relevance. Seven hematopathologists reviewed 374 cases that included PTNKCL and non-PTNKCL cases to mimic diagnostic practice. Cases received tier 0, 1, and 2 diagnoses by 3 independent pathologists, on the basis of hematoxylin and eosin stains and progressive immunohistochemistry panels. A tier 2b diagnosis was rendered when gene rearrangement data were available, and a final consensus diagnosis was rendered after discussion of each case. Across all 374 cases, consensus agreement was 92.5%. For PTNKCLs, World Health Organization subclassification was possible in 16.5%, 37.1%, 82.8%, and 85.9% of individual reviewer diagnoses at tier 0, 1, 2, and 2b, respectively. Gene rearrangement contributed to a change in diagnosis in 51 of 647 (8%) individual reviews. Following this algorithm may provide prognostic information on the basis of individual marker expression in common PTNKCL types (CD4 in peripheral T-cell lymphoma, not otherwise specified and PD-1 in angioimmunoblastic T-cell lymphoma). This evidence-based approach to the diagnosis of PTNKCL informs practicing pathologists, clinical trial designers, and policy-makers regarding required ancillary studies.

THU0127 Pharmacodynamics of A Novel JAK1 Selective Inhibitor in Rat Arthritis and Anemia Models and in Healthy Human Subjects

BackgroundAnti-cytokine therapies have become the mainstay of treatment for rheumatoid arthritis (RA) disease symptoms and can arrest disease progression. Despite numerous treatment options there are still many RA patients who...

Perturbation of NK cell peripheral homeostasis accelerates prostate carcinoma metastasis

The activating receptor NK cell group 2 member D (NKG2D) mediates antitumor immunity in experimental animal models. However, whether NKG2D ligands contribute to tumor suppression or progression clinically remains controversial. Here, we have described 2 novel lines of "humanized" bi-transgenic (bi-Tg) mice in which native human NKG2D ligand MHC class I polypeptide-related sequence B (MICB) or the engineered membrane-restricted MICB (MICB.A2) was expressed in the prostate of the transgenic adenocarcinoma of the mouse prostate (TRAMP) model of spontaneous carcinogenesis. Bi-Tg TRAMP/MICB mice exhibited a markedly increased incidence of progressed carcinomas and metastasis, whereas TRAMP/MICB.A2 mice enjoyed long-term tumor-free survival conferred by sustained NKG2D-mediated antitumor immunity. Mechanistically, we found that cancer progression in TRAMP/MICB mice was associated with loss of the peripheral NK cell pool owing to high serum levels of tumor-derived soluble MICB (sMICB). Prostate cancer patients also displayed reduction of peripheral NK cells and high sMIC levels. Our study has not only provided direct evidence in "humanized" mouse models that soluble and membrane-restricted NKG2D ligands pose opposite impacts on cancer progression, but also uncovered a mechanism of sMIC-induced impairment of NK cell antitumor immunity. Our findings suggest that the impact of soluble NKG2D ligands should be considered in NK cell-based cancer immunotherapy and that our unique mouse models should be valuable for therapy optimization.

Natural killer cell impairment is associated with liver fibrotic progression in chronic HBV infection (P3361)

Abstract Natural killer (NK) cells have been implicated in inducing fibrosis remission in mice model; however, their roles in fibrotic progression remain to be elucidated in liver fibrosis (LC) patients. We comprehensively characterized peripheral and intrahepatic NK cells in 50 chronic hepatitis B (CHB) patients and 68 HBV-associated LC patients as well as 35 healthy subjects (HC). Both peripheral and intrahepatic NK cell frequencies in LC and CHB patients were significantly decreased, and they expressed lower levels of TRAIL, activation marker, activation receptors and granzyme and perforin and higher levels of inhibitory receptors in LC patients than those from CHB patients, thus displaying the decreased CD107a and interferon (IFN)-γ production and reduced killing capacity against hepatic stallete cells (HSCs). In vitro, HSCs-derived TGF-β could suppress NK cytolytic activity, and blockade of TGF-β significantly enhanced NK cell-derived CD107a and IFN-γ production. The immunohistochemical staining showed that NKp46-positive cells were more enriched in the α-SMA-negative area in livers from LC patients. Finally, NK cell cytolytic activity was also correlated negatively with liver fibrosis scores in HBV infected patients, which is further confirmed by the longitudinal follow-up of LC patients. Our findings may facilitate the rational development of immunotherapeutic strategies to enhance NK activity while limiting or abolishing liver fibrosis in chronic HBV infection.

Natural killer cells and their activation status in normal pregnancy.

Increased peripheral blood-activated NK cell counts are associated with increased risk of miscarriage and failed in vitro fertilization treatment. However, assessment of activated peripheral NK cells in normal and pathological pregnancies beyond implantation and early miscarriage has not been described. Total CD69 expressing NK cells counts were measured by flow cytometry in healthy women with singleton pregnancies, including 45 at 11+6–13+6 weeks' gestation, 46 at 20+0–22+4 weeks, and 42 at 31+6–33+5 weeks. The number of peripheral blood NK cells decreased, whereas the percentage of activated CD69 expressing NK cells increased from the first to the third trimester of pregnancy. This study shows the course of peripheral blood NK cells and activated CD69 expressing NK cells in uncomplicated nulliparous singleton pregnancies. This is a first step in understanding their implication in pathological pregnancies.

Peripheral T-cell and NK-cell lymphomas in the WHO classification: pearls and pitfalls

Peripheral T-cell and NK-cell lymphomas are functionally, pathologically, and clinically complex. Most nodal T-cell lymphomas belong to the adaptive immune system, whereas many extranodal T-cell and NK-cell lymphomas are derived from innate immune cells. The pathological manifestations often reflect the functional attributes of the neoplastic cells. Several forms of peripheral T-cell lymphoma are derived from T-follicular helper cells (TFH), and include angioimmunoblastic T-cell lymphoma, the follicular variant of peripheral T-cell lymphoma, not otherwise specified, and primary cutaneous small/medium CD4-positive T-cell lymphoma. TFH-derived neoplasms are often associated with atypical and clonal B-cell proliferations, which take a number of forms, sometimes mimicking classical Hodgkin's lymphoma, and sometimes showing marked plasmacytic differentiation. Most extranodal T-cell lymphomas are cytotoxic and often arise in mucosal-associated sites. They can be derived from either αβ or γδ cytotoxic T cells, and include subcutaneous panniculitis-like T-cell lymphoma, and enteropathy-associated T-cell lymphomas, both Type I and Type II. Type I enteropathy-associated lymphomas occur in association with celiac disease, whereas Type II lymphomas are more often sporadic. For some T-cell lymphomas, such as hepatosplenic T-cell lymphoma, immunophenotypic heterogeneity is seen within a single disease entity. New data are emerging on the molecular pathogenesis of T-cell and NK-cell lymphoma, but most tumor types remain poorly characterized.

Functional Significance of CD57 Expression on Human NK Cells and Relevance to Disease

Historically, human NK cells have been identified as CD3−CD56+CD16± lymphocytes. More recently it has been established that CD57 expression defines functionally discrete sub-populations of NK cells. On T cells, CD57 expression has been regarded as a marker of terminal differentiation and (perhaps wrongly) of anergy and senescence. Similarly, CD57 expression seems to identify the final stages of peripheral NK cell maturation; its expression increases with age and is associated with chronic infections, particularly human cytomegalovirus infection. However, CD57+ NK cells are highly cytotoxic and their presence seems to be beneficial in a number of non-communicable diseases. The purpose of this article is to review our current understanding of CD57 expression as a marker of NK cell function and disease prognosis, as well as to outline areas for further research.

Bi(o)communications among peripheral blood fractions: A focus on NK and NKT cell biology in rheumatoid arthritis

Rheumatoid Arthritis (RA) is an autoimmune disease with unknown pathophysiology involving many interwoven signalling cascades. ROS, NK and NKT cells might be crucial in the disease severity of RA of which the role of NK and NKT cells are controversial in literature. However, the role of oxidative stress, its impact on NK and NKT cell immunobiology and disease activity (DAS28) is largely unknown. Therefore, we studied the role of oxidative stress and NK cell subsets in the pathogenesis of RA. The state of oxidative stress in various peripheral blood fractions, percentage NK and NKT cell expression, their altered apoptotic signaling pathways involving mitochondrial membrane potential, FAS associated death domain (FADD) mediated pathways and DNA damage were analyzed. Results indicated a state of profound oxidative stress in the peripheral blood of RA patients where percentage of NK and NKT cell subsets diminished while ROS levels increased. The depolarized mitochondrial membrane potential, FAS, FASL and active caspase-3 positive NK and NKT cell subsets were considerably elevated in patients. The DNA damage, assessed as percentage of DNA in comet tail, was significantly elevated. Findings of the present work indicate increased apoptosis of peripheral NK and NKT cells in the diseased condition. PBMC and RBC are the major sites of enhanced oxidative stress. The state of oxidative stress and altered immunobiology of NK and NKT cells strongly correlated with Disease activity score. The present study strongly supports the protective role of NK cell subsets in the pathogenesis of RA.

Immunohistochemical detection of CD3 in T-cell lymphomas: superior sensitivity of rabbit monoclonal 2GV6 antibody compared to mouse monoclonal F7·2·38 antibody

Immunohistochemical detection of cluster of differentiation 3 (CD3) expression is important for the diagnosis of peripheral T-cell and NK-cell lymphomas. We compared CD3 staining intensity and percentage of CD3-positive cells in 28 T-cell lymphomas was compared using mouse anti-human CD3 antibody (clone F7·2·38) and rabbit anti-human CD3 antibody (clone 2GV6). Analysis was performed on tissue microarray sections containing 12 peripheral T-cell lymphomas unspecified, 4 angioimmunoblastic T-cell lymphomas, 6 adult T-cell leukemia/lymphomas, and 6 anaplastic large T-cell lymphomas. Compared to mouse antibody, CD3 staining intensity and the fraction of CD3-positive cells increased significantly with rabbit antibody in the vast majority of the T-cell lymphomas analyzed. The mean staining intensity of positive cells (weak: +1; moderate: +2; strong: +3) of all 28 lymphomas rose from 2·1 with mouse antibody to 2·5 with rabbit antibody. Notably, the mean fraction of strongly positive cells (+3) was 17·7% with mouse antibody and increased considerably to 49·1% with rabbit antibody. In addition, there was an overall 8% increase in CD3-positive cells if rabbit antibody was used. The results show that detection of CD3 expression in T-cell lymphomas with rabbit antibody represents an improved standard by increasing sensitivity and staining intensity.

NK Cell Subsets in Renal Transplantation

Introduction: Following kidney transplantation, the involvement of NK cells in rejection episodes or immunological tolerance is discussed controversially. The peripheral NK cell repertoire of KTx patients at 3 or 6 months post Tx was investigated in parallel to the protocol biopsy for histopathological evaluation of the graft. The alterations were correlated to the biopsy status (“unsuspicious”, T cell-mediated rejection TCMR or borderline changes) and immunosuppression, respectively. Methods: Peripheral blood lymphocytes of KTx patients (N=100) at the time of the protocol biopsy at 3 or 6 month after Tx were isolated after informed consent of the patients. In addition to the “truecount” quantification of T, B, NK cells (cell/μl), the composition of the NK cell repertoire was determined by flow cytometry for the expression of several NK receptors including KIR and CD94/NKG2. In addition to NK cells, receptor expression was also addressed in T cell subsets. In some cases, protocol biopsies and peripheral blood was available at multiple time points after Tx and, thus, changes over time and immunosuppression (CsA vs. Tac vs. mTOR inhibitors) could be defined. Results: The numbers of NK cells/μl blood differed between patients after KTx with “unsuspicious” grafts and TCMR or borderline histopathology although not at a significant level. However, the distribution of CD56bright vs. CD56dim NK cells in these patient cohorts displayed significant differences compared to healthy donors. In addition, the NK subset analyses revealed substantial differences in the numbers of KIR+ NK cells between the patient cohorts. Conclusions: In our first analyses with patients between 3 and 12 months after KTx, we could demonstrate that the numbers of NK cells in peripheral blood differs between patients with kidney grafts unsuspicious of rejection and biopsy-proven TCMR or borderline changes. In addition, a more detailed NK cell subset analysis revealed significant differences in NK subset distribution between the patient cohorts and also, for some subsets, the immunosuppressive treatment groups mTOR inhibitors, in particular. Therefore, we assume that the composition of peripheral NK cells represents an important hallmark of the immune status, rejection vs. tolerance, in kidney transplantation. (This work was funded by the IFB-Tx, DFG TRR77, A3 snd SFB738 B8 projects and the HGF Alliance Immmunotherapy).

MicroRNA-Deficient NK Cells Exhibit Decreased Survival but Enhanced Function

NK cells are innate immune lymphocytes important for early host defense against infectious pathogens and malignant transformation. MicroRNAs (miRNAs) are small RNA molecules that regulate a wide variety of cellular processes, typically by specific complementary targeting of the 3'UTR of mRNAs. The Dicer1 gene encodes a conserved enzyme essential for miRNA processing, and Dicer1 deficiency leads to a global defect in miRNA biogenesis. In this study, we report a mouse model of lymphocyte-restricted Dicer1 disruption to evaluate the role of Dicer1-dependent miRNAs in the development and function of NK cells. As expected, Dicer1-deficient NK cells had decreased total miRNA content. Furthermore, miRNA-deficient NK cells exhibited reduced survival and impaired maturation defined by cell surface phenotypic markers. However, Dicer1-deficient NK cells exhibited enhanced degranulation and IFN-γ production in vitro in response to cytokines, tumor target cells, and activating NK cell receptor ligation. Moreover, a similar phenotype of increased IFN-γ was evident during acute MCMV infection in vivo. miRs-15a/15b/16 were identified as abundant miRNAs in NK cells that directly target the murine IFN-γ 3'UTR, thereby providing a potential mechanism for enhanced IFN-γ production. These data suggest that the function of miRNAs in NK cell biology is complex, with an important role in NK cell development, survival, or homeostasis, while tempering peripheral NK cell activation. Further study of individual miRNAs in an NK cell specific fashion will provide insight into these complex miRNA regulatory effects in NK cell biology.

MicroRNA-Deficient Murine NK Cells Exhibit Impaired Development and Survival but Enhanced IFN-γ Production In Vitro and In Vivo

Abstract 357NK cells are innate immune lymphocytes important for early host defense against infectious pathogens and malignant transformation. MicroRNAs (miRNAs) are small regulatory RNAs that control a wide variety of cellular processes by specific targeting of mRNA 3’UTRs. The Dicer1 gene encodes a conserved enzyme essential for miRNA processing, and Dicer1 deficiency leads to a global defect in miRNA biogenesis. While miRNA expression and regulation of adaptive T and B lymphocytes are well established, their role in the regulation of NK cell biology remains unclear. We postulated that miRNAs serve an essential role in orchestrating NK cell development and activation. To test this hypothesis, we combined lymphocyte-restricted hCD2-Cre transgenic, Rosa26-YFP-Cre-reporter, and Dicer1 ‘floxed’ mice. In this model, 25–50% of Dicer1 wt/wt NK cells are YFP+ marking expression of Cre. As expected, YFP+ NK cells from Dicer1 fl/fl and fl/wt mice were confirmed to excise Dicer1, and exhibit decreased total miRNA content based on Nanostring profiling and real-time qPCR (Dicer1 fl/fl: P<0.001, fl/wt: P<0.01). MiRNA-deficient Dicer1 fl/fl mice exhibited reduced YFP+ NK percentages (spleen Dicer1 fl/fl: 14±4%, fl/wt: 35±7%, wt/wt 36±7%, P<0.001) as well as reduced absolute numbers of YFP+ NK cells [spleen Dicer1 fl/fl: 3.4±0.6×10E5, fl/wt: 6.3±1.7×10E5, wt/wt 6.1±.99×10E5, P<0.01]. In addition, Dicer1 fl/fl mice had reductions NK cell precursors in the BM (stage 2–3 NK precursors mean decrease 70±14% in Dicer1 fl/fl compared to wt/wt, P <0.01). Further, Dicer1 fl/fl NK cells exhibited reduced survival ex vivo when cultured in medium (P<0.01), low dose- (P<0.01), or high dose-IL-15 (P<0.01). These data collectively indicate that Dicer1-dependent miRNAs regulate NK cell development and homeostasis, and the net effect of miRNA loss is impaired NK development and/or survival. However, in our model Dicer1-deficient mature NK cells exhibited enhanced functionality; a finding that contrasts to less NK selective miRNA-deficient NK cell models (Bezman et al. J Immunol 185:3835, 2010). Degranulation (CD107a+, a surrogate for cytotoxicity) was enhanced in vitro in response to YAC-1 tumor target cells (P<0.05) and activating NK cell receptor ligation (P<0.001). This was unlikely due to alteration in activating NK cell receptor expression since the surface density of NKG2D and NKp46 were not affected by miRNA deficiency. Moreover, interferon-gamma (IFN-γ) production was enhanced in vitro in miRNA-deficient NK cells in response to IL-12+IL-15 (P<0.01), YAC-1 tumor target cells (P<0.01), and activating NK cell receptor ligation (P<0.001). Further, evaluation of NK cells 36 hours after infection with MCMV resulted in significantly increased IFN-γ production (% NK YFP+IFN-γ+) in Dicer1 fl/fl (64± 4.9%) vs. fl/wt (52±11%, p <0.01) or vs. wt/wt (41±6%, p <0.001) in vivo. MiRs-15/16 were identified as abundant miRNAs in NK cells that had reduced expression in Dicer1 fl/fl NK cells, and are predicted to target the murine IFN-γ 3’UTR. This targeting was validated in vitro, by transfecting 293T cells with miRNA-15/16 or control over-expression vectors and a sensor plasmid that places luciferase under the control of the murine IFN-γ 3’UTR (34% decrease, P<0.01). Moreover, the targeting was direct, since miR-15/16 targeting of IFN-γ was abrogated after mutation of two predicted binding sites in the IFN-γ 3’UTR. These data indicated that miR-15/16 may regulate IFN-γ translation by resting NK cells. Thus, our study suggests that the function of miRNAs in NK cell biology is complex, with an important role in NK cell development, survival and/or homeostasis, while tempering peripheral NK cell activation. Further study of individual miRNAs in an NK cell specific fashion will provide insight into these complex miRNA regulatory effects in NK cell development/survival and effector function. Disclosures:No relevant conflicts of interest to declare.

Activating NK cell receptor expression/function (NKp30, NKp46, DNAM‐1) during chronic viraemic HCV infection is associated with the outcome of combined treatment

Specific NK cell killer inhibitory receptor (KIR):HLA haplotype combinations have been associated with successful clearance of acute and chronic HCV infection. Whether an imbalance of activating NK cell receptors also contributes to the outcome of treatment of chronic HCV infection, however, is not known. We studied peripheral NK cell phenotype and function in 28 chronically viraemic HCV genotype I treatment-naïve patients who underwent treatment with pegylated IFN-α and ribavirin. At baseline, chronically infected patients with sustained virological response (SVR) had reduced CD56(bright) CD16(+/-) cell populations, increased CD56(dull) CD16(+) NK cell proportions, and lower expression of NKp30, DNAM-1, and CD85j. Similarly, reduced NK cell IFN-γ production but increased degranulation was observed among nonresponding (NR) patients. After treatment, CD56(bright) CD16(+/-) NK cell numbers increased in both SVR and NR patients, with a parallel significant increase in activating NKp30 molecule densities in SVR patients only. In vitro experiments using purified NK cells in the presence of rIL-2 and IFN-α confirmed upregulation of NKp30 and also of NKp46 and DNAM-1 in patients with subsequent SVR. Thus, differences in patient NK cell receptor expression and modulation during chronic HCV-1 infection are associated with subsequent outcome of standard treatment. Individual activating receptor expression/function integrates with KIR:HLA genotype carriage to determine the clearance of HCV infection upon treatment.

Characteristics of peripheral NK cells in hepatocellular carcinoma patients

Functional defects in NK cells have been proposed to be responsible for the impairment of anti-tumor immune responses. However, it remained unclear whether the function of NK cells were impaired in patients with hepatocellular carcinoma. To address this issue, we analyzed the frequency and function of peripheral NK cell subsets in hepatocellular carcinoma (HCC) patients. 35 HCC patients and 24 healthy controls (HC) were enrolled in the study. Peripheral NK frequency was analyzed using flow cytometry. In addition, the capacity of NK cells to produce IFN gamma and to lyse K562 cells was evaluated. In contrast with the healthy controls, the frequency of peripheral NK cells in hepatocellular carcinoma patients was decreased (12.19%+/-10.85% vs 24.01%+/-8.78%, u = 4.01, probability value less than 0.01), while the frequency of CD56(bright)CD16(neg) NK cells was increased (0.62%+/-0.39% vs 0.48%+/-0.28%, u = 1.96, probability value less than 0.05), and the frequency of CD56(dim)CD16(pos) NK cells was significantly decreased (11.59%+/-7.49% vs 22.66%+/-8.84%, u = 3.92, probability value less than 0.01). In addition, peripheral NK cells from HCC patients exhibited decreased capacity to produce IFN gamma (effective cells 13.31%) and to lyse K562 cells (mixed ratio 30:1, 10:1, 1:1, effective cells 16.72%+/-7.33% vs 26.29%+/-12.36%, u = 2.52, P less than 0.05, 8.01%+/-4.40% vs 13.09%+/-5.03%, u = 3.32, probability value less than 0.05, 3.51%+/-2.82% vs 3.42%+/-1.64%, u = 1.56, probability value more than 0.05, respectively) as compared with healthy subjects. Anti-tumor activity of NK cells in HCC patients was impaired.

Road Transportation Stress Promptly Increases Bovine Peripheral Blood Absolute NK Cell Counts and Cortisol Levels

Livestock transportation effects on the number of circulating leukocytes have been reported. However, data related specifically to the relation between acute stress levels during transport and leukocyte differentiation, including lymphocyte subsets, are lacking. This study was undertaken to evaluate the distribution of peripheral blood leukocyte differential counts, CD25+ lymphocytes and NK cells in calves subjected to truck transportation on different road types. Healthy Japanese Black calves were divided into three treatments: 1) those moved around in a mountainous area (Group M); 2) those moved around on flatland (Group F); and 3) those that were not transported (control). The plasma cortisol levels in Group M increased during transport. The increase was significantly higher at the end of transport than in the controls (P<0.05); a slight increase was noted in Group F. Total leukocytes and the neutrophil to lymphocyte ratio in Group M were elevated with neutrophilia at 2 hr post-transport (P<0.05); the former levels remained higher than those in the controls for 4 hr. The numbers of lymphocytes, monocytes, eosinophils and CD25+ lymphocytes remained unchanged throughout the observations. The number of circulating NK cells in Group M increased during transport and peaked shortly after transport (P<0.05). Subsequent to these time points, the counts in Group F showed a trend toward elevation. The circulating NK cell counts were positively correlated with the plasma cortisol level during transport (M, r=0.755, P<0.0005; F, r=0.653; P<0.005). These results suggest that circulating NK cells might be more rapidly mobilized than other leukocytes. Therefore, they might reflect acute stress levels in calves during road transportation.