Syntaxin 11 is required for NK and CD8+ T‐cell cytotoxicity and neutrophil degranulation

Syntaxin 11 (STX11) controls vesicular trafficking and is a key player in exocytosis. Since Stx11 mutations are causally associated with a familial hemophagocytic lymphohistio‐cytosis, we wanted to clarify whether STX11 is functionally important for key immune cell populations. This was studied in primary cells obtained from newly generated Stx11^?/? mice. Our data revealed that STX11 is not only widely expressed in different immune cells, but also induced upon LPS or IFN‐γ treatment. However, Stx11 deficiency does not affect macrophage phagocytic function and cytokine secretion, mast cell activation, or antigen presentation by DCs. Instead, STX11 selectively controls lymphocyte cytotoxicity in NK and activated CD8⁺ T cells and degranulation in neutrophils. Stx11^?/? NK cells and CTLs show impaired degranulation, despite a comparable activation, maturation and expression of the complex‐forming partners MUNC18–2 and VTI1B. In addition, Stx11^?/? CTLs and NK cells produce abnormal levels of IFN‐γ. Since functional reconstitution rescues the defective phenotype of Stx11^?/? CTLs, we suggest a direct, specific and key role of STX11 in controlling lymphocyte cytotoxicity, cytokine production and secretion. Finally, we show that these mice are a very useful tool for dissecting the role of STX11 in vesicular trafficking and secretion.     

Natural Killer Cell Subsets and Their Functional Activity in Behçet’s Disease

Background: Behçet’s disease (BD) is a rare, chronic autoinflammatory disorder of unknown origin. Natural killer (NK) cells are one of the major immunoregulatory cell groups of the innate immune system, but their role in BD pathogenesis is not well documented.

Objectives: We aimed to investigate the role of NK cell subsets and their cytokine secretion and cytotoxic activity in patients with BD.

Patients and methods: The study group consisted of BD patients who had only mucocutaneous involvement, and they were compared with healthy subjects. BD patients were divided into two groups according to their frequencies of oral ulcerations. NK cell cytotoxicity was determined using CD107a expression and a CFSE-based cytotoxicity test. Expression of NK cell receptors and surface markers and the intracellular IL-5, IL-10, IL-17, and IFN-γ levels in CD16⁺ NK cells were assessed by flow cytometry.

Results: Although the cytokine secretion pattern was different, no difference was obtained in cytotoxic activity, expression of activatory receptors, or degranulation of NK cells.

Conclusion: Increases in NK1/NK2 ratio and CD16⁺IFN-γ⁺ NK1 cells might support the idea of a biased IFN-γ dominant immune response in the mucocutaneous involvement of BD pathogenesis. Although the cytokine secretion pattern was different, no difference was obtained in cytotoxic activity, expression of activatory receptors, or degranulation of NK cells.     

Intrahepatic and peripheral blood phenotypes of natural killer and T cells: differential surface expression of killer cell immunoglobulin‐like receptors

Deep characterization of the frequencies, phenotypes and functionalities of liver and peripheral blood natural killer (NK), natural killer T (NKT) and T cells from healthy individuals is an essential step to further interpret changes in liver diseases. These data indicate that CCR7, a chemokine essential for cell migration through lymphoid organs, is almost absent in liver NK and T cells. CD56^bright NK cells, which represent half of liver NK cells, showed lower expression of the inhibitory molecule NKG2A and an increased frequency of the activation marker NKp44. By contrast, a decrease of CD16 expression with a potential decreased capacity to perform antibody‐dependent cellular cytotoxicity was the main difference between liver and peripheral blood CD56^dim NK cells. Liver T cells with an effector memory or terminally differentiated phenotype showed an increased frequency of MAIT cells,T‐cell receptor‐γδ (TCR‐γδ) T cells and TCR‐αβ CD8⁺ cells, with few naive T cells. Most liver NK and T cells expressed the homing markers CD161 and CD244. Liver T cells revealed a unique expression pattern of killer cell immunoglobulin‐like receptors (KIR) receptors, with increased degranulation ability and higher secretion of interferon‐γ. Hence, the liver possesses a large amount of memory and terminally differentiated CD8⁺ cells with a unique expression pattern of KIR activating receptors that have a potent functional capacity as well as a reduced amount of CCR7, which are unable to migrate to regional lymph nodes. These results are consistent with previous studies showing that liver T (and also NK) cells likely remain and die in the liver.     

NK cells from tuberculous pleurisy express high ICAM‐1 levels and exert stimulatory effect on local T cells

Tuberculous pleurisy, one of the most common manifestations of extrapulmonary tuberculosis, is characterized by a T‐cell‐mediated hypersensitivity reaction along with a Th1 immune profile. In this study, we investigated functional cross‐talk among T and NK cells in human tuberculous pleurisy. We found that endogenously activated pleural fluid‐derived NK cells express high ICAM‐1 levels and induce T‐cell activation ex vivo through ICAM‐1. Besides, upon in vitro stimulation with monokines and PAMP, resting peripheral blood NK cells increased ICAM‐1 expression leading to cellular activation and Th1 polarization of autologous T cells. Furthermore, these effects were abolished by anti‐ICAM‐1 Ab. Hence, NK cells may contribute to the adaptive immune response by a direct cell‐contact‐dependent mechanism in the context of Mycobacterium tuberculosis infection.     

Sorafenib paradoxically activates the RAS/RAF/ERK pathway in polyclonal human NK cells during expansion and thereby enhances effector functions in a dose‐ and time‐dependent manner

Natural killer (NK) cells play a major role in host immunity against leukaemia and lymphoma. However, clinical trials applying NK cells have not been as efficient as hoped for. Patients treated with rapidly accelerated fibrosarcoma (RAF) inhibitors exhibit increased tumour infiltration by immune cells, suggesting that a combination of RAF inhibitors with immunotherapy might be beneficial. As mitogen‐activated protein kinases (MAPKs) such as raf‐1 proto‐oncogene, serine/threonine kinase (CRAF) regulate NK cell functions, we performed an in‐vitro investigation on the potential of clinically relevant short‐acting tyrosine kinase inhibitors (TKIs) as potential adjuvants for NK cell therapy: NK cells from healthy human blood donors were thus treated with sorafenib, sunitinib or the pan‐RAF inhibitor ZM336372 during ex‐vivo expansion. Functional outcomes assessed after washout of the drugs included cytokine production, degranulation, cytotoxicity, apoptosis induction and signal transduction with/without target cell contact. Paradoxically, sorafenib enhanced NK cell effector functions in a time‐ and dose‐dependent manner by raising the steady‐state activation level. Of note, this did not lead to NK cell exhaustion, but enhanced activity against target cells such as K562 or Daudis mediated via the RAS/RAF/extracellular‐regulated kinase (ERK) pathway, but not via protein kinase B (AKT). Our data will pave the path to develop a rationale for the considered use of RAF inhibitors such as sorafenib for pre‐activation in NK cell‐based adoptive immune therapy.     

Uptake of poly-dispersed single-walled carbon nanotubes and decline of functions in mouse NK cells undergoing activation

The interaction of poly-dispersed acid-functionalized single-walled carbon nanotubes (AF-SWCNT) with NK cells undergoing activation was examined. Exposure to AF-SWCNT during NK activation in vitro by interleukin (IL)-2, and in vivo by Poly(I:C) significantly lowered cytotoxic activity generated against YAC-1 tumor cells. Recoveries of spleen NK1.1⁺ cells as well as the activated subset of NK cells (NK1.1⁺CD69⁺ cells) were significantly reduced by the AF-SWCNT exposure. The proportion of apoptotic NK cells (NK1.1⁺ phosphatidylserine⁺) in the spleen cell preparations activated in vitro was also significantly elevated. Expression levels of CD107a [for assessing NK cell degranulation] as well as of FasL marker [mediating non-secretory pathway of NK cell killing] were significantly lower in cells exposed to AF-SWCNT during the activation phase. Intracellular levels of interferon (IFN)-γ and tumor necrosis factor (TNF)-α in the cells were also significantly reduced. Fluorescent AF-SWCNT (FAF-SWCNT) were internalized by the NK cells and uptake was significantly greater in activated cells. Confocal microscopy indicated the internalized FAF-SWCNT were localized to the cytoplasm of the NK cells. These results indicated that AF-SWCNT were internalized by NK cells and caused a general down-regulation of a variety of parameters associated with NK cell cytotoxicity and other cellular functions.     

Inhibitory 2B4 contributes to NK cell education and immunological derangements in XLP1 patients

X‐linked lymphoproliferative disease 1 (XLP1) is an inherited immunodeficiency, caused by mutations in SH2D1A encoding Signaling Lymphocyte Activation Molecule (SLAM)‐associated protein (SAP). In XLP1, 2B4, upon engagement with CD48, has inhibitory instead of activating function. This causes a selective inability of cytotoxic effectors to kill EBV‐infected cells, with dramatic clinical sequelae. Here, we investigated the NK cell education in XLP1, upon characterization of killer Ig‐like receptor (KIR)/KIR‐L genotype and phenotypic repertoire of self‐HLA class I specific inhibitory NK receptors (self‐iNKRs). We also analyzed NK‐cell cytotoxicity against CD48⁺ or CD48^? KIR‐ligand matched or autologous hematopoietic cells in XLP1 patients and healthy controls. XLP1 NK cells may show a defective phenotypic repertoire with substantial proportion of cells lacking self‐iNKR. These NK cells are cytotoxic and the inhibitory 2B4/CD48 pathway plays a major role to prevent killing of CD48⁺ EBV‐transformed B cells and M1 macrophages. Importantly, self‐iNKR defective NK cells kill CD48^? targets, such as mature DCs. Self‐iNKR^? NK cells in XLP1 patients are functional even in resting conditions, suggesting a role of the inhibitory 2B4/CD48 pathway in the education process during NK‐cell maturation. Killing of autologous mature DC by self‐iNKR defective XLP1 NK cells may impair adaptive responses, further exacerbating the patients’ immune defect.     

Siglec‐7 Defines a Highly Functional Natural Killer Cell Subset and Inhibits Cell‐Mediated Activities

Sialic acid‐binding immunoglobulin‐like lectin‐7 (Siglec‐7) is an inhibitory receptor expressed on natural killer (NK) cells. In this study, we investigated the relationship between Siglec‐7 expression and NK cell functions. Siglec‐7 was highly expressed on NK cells and was preferentially expressed by mature NK cells from peripheral blood of healthy adults. Siglec‐7⁺ NK cells displayed higher levels of activating receptors CD38, CD16, DNAM1, NKp30 and NKp46, but lower levels of inhibitory receptors such as NKG2A and CD158b, compared with Siglec‐7^– NK cells. Functional tests showed that Siglec‐7⁺ NK cells displayed more CD107a degranulation and IFN‐γ production than Siglec‐7^– NK cells. Siglec‐7 inhibited NK cell functions when interacting with specific antibodies. These data suggest that Siglec‐7 defines a highly functional NK cell subset and suppresses NK cell‐mediated functions when cross‐linked with specific antibodies.     

1,25‐Dihydroxy‐vitamin D3 regulates NK‐cell cytotoxicity,cytokine secretion,and degranulation in women with recurrent pregnancy losses

Vitamin D has a pivotal role in regulating immune responses by promoting Th2 immune responses and suppressing Th1 responses. Propensities to a Th1 immune response and increased NK‐cell levels and cytotoxicity have been reported in women with recurrent pregnancy losses (RPL). In women with RPL, vitamin D deficiency is prevalent; however, the effect of vitamin D on NK cells is largely unknown. In this study, we demonstrated that CD69⁺ activating receptor expression on NK cells was significantly decreased by incubation with 1,25(OH)₂D₃ in a dose‐dependent manner, while CD158a and CD158b inhibitory receptor expression was upregulated. The degranulation marker CD107a was significantly downregulated on NK cells following incubation with 1,25(OH)₂D₃. NK‐cell conjugation with K562 target cells was not affected by 1,25(OH)₂D₃; however, depolarization of perforin granules in conjugated NK cells was significantly increased. TLR4 expression on NK cells was significantly decreased and TNF‐α and IFN‐γ production was significantly reduced by 1,25(OH)₂D₃ through interference with NF‐κB. Our results suggest 1,25(OH)₂D₃ has immune regulatory effects on NK cell cytotoxicity, cytokine secretion and degranulation process as well as TLR4 expression. Potential therapeutic application of 1,25(OH)₂D₃ for dysregulated NK‐cell immunity should be explored in the future.     

Influence of autologous dendritic cells on the in-vitro expansion and functions of peripheral blood NK cells

Abstract

Context: Allogeneic reactive NK cells were previously shown to exert a graft-versus-leukemia (GVL) effect during allogeneic hematopoietic stem cell transplantation, as well as reduce the incidence of graft-versus-host disease (GVHD).

Objective: We used autologous immature DCs as feeder cells for the in-vitro expansion of NK cells and studied the function of the NK cell cultures.

Materials and methods: NK cells were cultured for 15 days in the presence of autologous, immature DCs. Fold expansion, killing activity and expression of IFN-γ, perforin and granzyme B were evaluated.

Results: The highest NK cell expansion efficiency was observed when the ratio of NK cells:DCs was 2:1 and when cells were cultured in a contact-dependent manner. The killing activity of NK cells was highest when the NK:DC ratio was 10:1. NK cell cultures exhibited a significant upregulation in the mRNA expression of IFN-γ, perforin and granzyme B when the ratio of NK cells to DCs was 10:1.

Discussion: We successfully amplified NK cells using autologous immature DCs derived from human peripheral monocytes after induction as feeder cells. The use of autologous immature DCs for ex-vivo expansion of NK cells can be clinically applied to overcome limitations, such as the small number of NK cells in peripheral blood, and the high cost of NK cell sorting. Transfusion of allogeneic reactive NK cells has been suggested as a potential adjunctive therapeutic strategy after transplantation.

Conclusion: Autologous immature DCs can be used as feeder cells for ex-vivo expansion of functional NK cells.     

DIFFERENTIAL STAT5 ACTIVATION AND PHENOTYPIC MARKER EXPRESSION BY IMMUNE CELLS FOLLOWING LOW LEVELS OF ETHANOL CONSUMPTION IN MICE

ABSTRACT

Ethanol has been recognized as an immunosuppressive agent for many years. Effects of high levels of ethanol consumption on immune functions have been extensively studied, but little is known about the effects of low levels (scuh as 5% ethanol) of ethanol consumption. Herein we report that exposure of mice to 5% ethanol for 4–8 weeks decreases IL-2-augmented splenic NK cell activity, decreases the numbers of NK cells in spleen and liver, decreases the number of granulocytes (Gr-1⁺) in bone marrow and spleen, and decreases the percentages of B cells in liver. In contrast, the percentages of CD4⁺CD8⁺ thymocytes, CD4⁺CD8^? splenocytes, CD4⁺CD8^? liver nonparenchymal cells, CD3⁺ splenocytes, and CD3⁺ bone marrow cells were increased. Furthermore, exposure to 5% ethanol increases STAT5 activation in T cells and liver cells while decreases STAT5 activation in NK cells. Taken together, these findings suggest that low levels of ethanol consumption can differentially modulate immune cells in thymus, spleen, bone marrow and liver, which may be due to differential regulation of STAT5 activation by ethanol.     

Increased number and function of natural killer cells in human immunodeficiency virus 1‐positive subjects co‐infected with herpes simplex virus 2

Natural killer (NK) cells bridge the interface between innate and adaptive immunity and are implicated in the control of herpes simplex virus 2 (HSV‐2) infection. In subjects infected with human immunodeficiency virus 1 (HIV‐1), the critical impact of the innate immune response on disease progression has recently come into focus. Higher numbers of NK cells are associated with lower HIV‐1 plasma viraemia. Individuals with the compound genotype of killer cell immunoglobulin‐like receptor (KIR) 3DS1 and human leucocyte antigen (HLA)‐Bw4‐80I, or who have alleles of KIR3DL1 that encode proteins highly expressed on the NK cell surface, have a significant delay in disease progression. We studied the effect of HSV‐2 co‐infection in HIV‐1‐infected subjects, and show that HSV‐2 co‐infection results in a pan‐lymphocytosis, with elevated absolute numbers of CD4⁺ and CD8⁺ T cells, and NK cells. The NK cells in HSV‐2 co‐infected subjects functioned more efficiently, with an increase in degranulation after in vitro stimulation. The number of NK cells expressing the activating receptors NKp30 and NKp46, and expressing KIR3DL1 or KIR3DS1, was inversely correlated with HIV‐1 plasma viral load in subjects mono‐infected with HIV‐1, but not in subjects co‐infected with HSV‐2. This suggests that HSV‐2 infection mediates changes within the NK cell population that may affect immunity in HIV‐1 infection.     

NK cells in autoimmune diseases: Linking innate and adaptive immune responses

The pathogenesis of autoimmunity remains to be fully elucidated, although the contribution of genetic and environmental factors is generally recognized. Despite autoimmune conditions are principally due to T and B lymphocytes, NK cells also appear to play a role in the promotion and/or maintenance of altered adaptive immune responses or in peripheral tolerance mechanisms.Although NK cells are components of the innate immune system, they shows characteristics of the adaptive immune system, such as the expansion of pathogen-specific cells, the generation of long-lasting “memory” cells able to persist upon cognate antigen encounter, and the possibility to induce an increased secondary recall response to re-challenge.Human NK cells are generally identified as CD56⁺ CD3^?, conversely CD56⁺ CD3⁺ cells represent a mixed population of NK-like T (NK T) cells and antigen-experienced T cells showing the up-regulation of several NK cell markers. CD56^dim constitute about 90% of NK cells in the peripheral blood, they are mature and involved in cytotoxicity responses; CD56^bright instead are more immature, mostly involved in cytokine production, having only a limited role in cytolytic responses, keen to leave the blood vessels as the principal population observed in lymph nodes. NK cells have been identified also in non-lymphoid tissues since, in pathologic conditions, they can quickly reach the target organs. A cross-talk between NK with dendritic cells and T cells is established throughout different receptor-ligand bindings.Several studies support the correlation between NK cell number and/or functional alterations, such as a defective cytotoxic activity and several autoimmune conditions. Among the different autoimmune pathologies and even within the same disease, NK cell function is significantly different either promoting or even protecting against the onset of the autoimmune condition.In this Review, we discuss recent literature supporting the role played by NK cells, as a bridge between innate and adaptive immunity, in the onset of autoimmune diseases.     

Rheumatoid arthritis

Summary: Therapeutic efficacy of depleting B cells or blocking T‐cell costimulation in rheumatoid arthritis (RA) has confirmed the critical pathogenic role of adaptive immune responses. Yet, RA preferentially affects elderly individuals, in whom adaptive immunity to exogenous antigens begins to fail. Here, we propose that senescence of the immune system is a risk factor for RA, with chronic inflammation resulting from the accumulation of degenerate T cells that have a low threshold for activation and utilize a spectrum of novel receptors to respond to microenvironmental cues. The process of immunosenescence is accelerated in RA and precedes the onset of disease, the acceleration, in part, being conferred by the HLA‐DR4 haplotype. Naive CD4⁺ T cells in RA are contracted in diversity and restricted in clonal burst. Senescence of effector CD4⁺ T cells is associated with the loss of CD28 and the de novo expression of KIR2DS2, NKG2D, and CX₃CR1, all of which function as costimulatory molecules and reduce the threshold for T‐cell activation. The synovial microenvironment promotes chronic persistent immune responses by facilitating ectopic lymphoid neogenesis, such as the formation of aberrant germinal centers. With the propensity to develop complex lymphoid architectures and to provide optimal activation conditions for senescent CD4⁺ T cells, the synovium becomes a natural target for pathogenic immune responses in prematurely aged individuals.     

PGE2 expression by HPV6/11-induced respiratory papillomas blocks NK cell activation in patients with recurrent respiratory papillomatosis

Recurrent respiratory papillomatosis (RRP), a rare chronic disease caused primarily by human papillomavirus types 6 and 11, consists of repeated growth of premalignant papillomas in the airway. RRP is characterized by multiple abnormalities in innate and adaptive immunity. Natural killer (NK) cells play important roles in immune surveillance and are part of the innate immune responses that help prevent tumor growth. We identified that papillomas lack classical class I MHC and retain nonclassical class I MHC expression. Moreover, in this study, we have identified and characterized the mechanism that blocks NK cell targeting of papilloma cells. Here, we show for the first time that the PGE₂ secreted by papilloma cells directly inhibits NK cells activation/degranulation principally through the PGE₂ receptor EP2, and to a lesser extent through EP4 signaling. Thus, papilloma cells have a potent mechanism to block NK cell function that likely supports papilloma cell growth.     

TLR‐Stimulated Eosinophils Mediate Recruitment and Activation of NK Cells In Vivo

Eosinophils like many myeloid innate immune cells can provide cytokines and chemokines for the activation of other immune cells upon TLR stimulation. When TLR‐stimulated eosinophils were inoculated i.p. into wild‐type mice, and NK cells were rapidly recruited and exhibited antitumour cytotoxicity. However, when mice depleted of CD11c⁺ cells were used, a marked decrease in the number of recruited NK cells was observed. We postulated that CpG or LPS from the injected eosinophils could be transferred to host cells, which in turn could recruit NK cells. However, by inoculating mice deficient in TLR4 or TLR9 with LPS or CpG‐stimulated eosinophils respectively, NK cell recruitment was still observed alongside cytotoxicity and IFNγ production. CpG stimulation of eosinophils produced the pro‐inflammatory cytokine IL‐12 and the chemokine CXCL10, which are important for NK cell activation and recruitment in vivo. To demonstrate the importance of CXCL10 in NK cell recruitment, we found that CpG‐stimulated eosinophils pretreated with the gut microbial metabolite butyrate had reduced expression and production of CXCL10 and IL‐12 and concomitantly were poor at recruitment of NK cells and inducing IFNγ in NK cells. Therefore, eosinophils like other innate immune cells of myeloid origin can conceivably stimulate NK cell activity. In addition, products of the gut microbiota can be potential inhibitors of NK cell.     

Establishing the reference intervals of NK cell functions in healthy adults

Natural killer (NK) cells play a key role in host defense against microbial pathogens. Establishing the reference intervals (RIs) of NK cell functions would be valuable in assessing the immune status of hosts. We evaluated the NK cell activity in healthy adults. We further established and validated the RIs of representative NK cell functions. Flow cytometry was used to evaluate the cytokine production and CD107a degranulation of NK cells. Levels of soluble IFN-γ in the culture supernatants were evaluated by ELISA. Our results demonstrated that the intracellular IFN-γ production of NK cells was positively correlated with CD107a expression and soluble IFN-γ levels. There were no significant differences in NK cell functions between different age and gender groups. The mean values and RIs of representative NK cell functions are as following: IFN-γ⁺ NK cells (%): 28.09 (11.3–51.95); CD107a⁺ NK cells (%): 17.90 (9.852–27.56); soluble IFN-γ (pg/ml): 330.4 (41.38–717.8). In addition, the intracellular IFN-γ production and degranulation activity of NK cells in patients with colorectal cancer were significantly lower than that in healthy adults. Our study has established the RIs of NK cell functions in healthy adults, which might be used for monitoring the immune status of the hosts.