IFN‐λ‐mediated IL‐12 production in macrophages induces IFN‐γ production in human NK cells

With increasing interest in alternative options to interferon‐alpha‐based treatments, IFN‐λ has shown therapeutic promise in a variety of diseases. Although the antiviral activity of IFN‐λ has been extensively studied, there is limited knowledge regarding the immunological functions of IFN‐λ and how these differ from those of other classes of IFNs. In this study, we investigated the effects of IFN‐λ on primary human NK cells, both in a direct and indirect capacity. We demonstrate that in contrast to interferon‐alpha, IFN‐λ is unable to directly stimulate NK cells, due to the absence of IFN‐λ receptor chain 1 (IFN‐λR1) on NK cells. However, IFN‐λ, in combination with TLR4 challenge, is able to induce the production of select members of the IL‐12 family of cytokines in monocyte‐derived macrophages. We further show that through macrophage‐mediated IL‐12 production, IFN‐λ is able to indirectly affect NK cells and ultimately induce IFN‐γ production.     

Soluble HLA‐I‐mediated secretion of TGF‐β1 by human NK cells and consequent down‐regulation of anti‐tumor cytolytic activity

Soluble HLA class I (sHLA‐I) molecules can regulate survival of NK cells and their anti‐tumor killing activity. Herein, we have analysed whether interaction of sHLA‐I with CD8 and/or different isoforms of killer Ig‐like receptors (KIR) induced secretion of transforming growth factor (TGF)‐β1. CD8⁺KIR^? NK cell clones secreted TGF‐β1 upon the interaction of sHLA‐I with CD8 molecule. sHLA‐Cw4 or sHLA‐Cw3 alleles engaging inhibitory isoforms of KIR, namely KIR2DL1 or KIR2DL2, strongly downregulated TGF‐β1 production elicited through CD8. On the other hand, sHLA‐Cw4 or sHLA‐Cw3 alleles induced secretion of TGF‐β1 by ligation of stimulatory KIR2DS1 or KIR2DS2 isoforms. TGF‐β1 strongly reduced NK cell‐mediated tumor cell lysis and production of pro‐inflammatory cytokines such as TNF‐α and IFN‐γ. Also, TGF‐β1 inhibited NK cell cytolysis induced by the engagement of stimulatory receptors including NKG2D, DNAM1, 2B4, CD69, NKp30, NKp44 and NKp46. The IL‐2‐dependent surface upregulation of some of these receptors was prevented by TGF‐β1. Furthermore, TGF‐β1 hampered IL‐2‐induced NK cell proliferation but not IL‐2‐mediated rescue from apoptosis of NK cells. Depletion of TGF‐β1 restored all the NK cell‐mediated functional activities analysed. Taken together these findings suggest that sHLA‐I antigens may downregulate the NK cell‐mediated innate response by inducing TGF‐β1 release.     

B‐cell co‐receptor CD72 is expressed on NK cells and inhibits IFN‐γ production but not cytotoxicity

NK cells have two main functions, namely cell‐mediated cytotoxicity and production of cytokines. Multiple inhibitory receptors that regulate NK‐cell cytotoxicity have been characterized whereas little is known about receptors regulating cytokine production. Here we report that CD72, which is considered to be an important co‐receptor regulating B‐cell activation, is also expressed on mouse NK cells. NK cells expressing high levels of CD72, upon stimulation with IL‐12 and IL‐18 or target cells, produce significantly less IFN‐γ than those expressing low levels of CD72, whereas both subsets are equally cytotoxic. Ectopic expression of CD72 in the murine NK‐cell line KY2 inhibits cytokine‐induced IFN‐γ production, and the inhibitory effect is diminished by mutations in the inhibitory motifs in the intracellular domain or replacement of the extracellular domain of CD72. Thus, CD72 is an inhibitory receptor on NK cells regulating cytokine production.     

Enhancement of cytokine‐driven NK cell IFN‐γ production after vaccination of HCMV infected Africans

Human cytomegalovirus (HCMV) infection drives the phenotypic and functional differentiation of NK cells, thereby influencing the responses of these cells after vaccination. NK cell functional differentiation is particularly advanced in African populations with universal exposure to HCMV. To investigate the impact of advanced differentiation on vaccine‐induced responses, we studied NK‐cell function before and after vaccination with Trivalent Influenza Vaccine (TIV) or diphtheria, tetanus, pertussis, inactivated poliovirus vaccine (DTPiP) in Africans with universal, lifelong HCMV exposure. In contrast to populations with lower prevalence of HCMV infection, no significant enhancement of NK‐cell responses (IFN‐γ, CD107a, CD25) occurred after in vitro re‐stimulation of post‐vaccination NK cells with TIV or DTPiP antigens compared to pre‐vaccination baseline cells. However, both vaccinations resulted in higher frequencies of NK cells producing IFN‐γ in response to exogenous IL‐12 with IL‐18, which persisted for up to 6 months. Enhanced cytokine responsiveness was restricted to less differentiated NK cells, with increased frequencies of IFN‐γ⁺ cells observed within CD56^brightCD57^?, CD56^dimCD57^?NKG2C^? and CD56^dimCD57^?NKG2C⁺ NK‐cell subsets. These data suggest a common mechanism whereby different vaccines enhance NK cell IFN‐γ function in HCMV infected donors and raise the potential for further exploitation of NK cell “pre‐activation” to improve vaccine effectiveness.     

PolyI:C plus IL‐2 or IL‐12 induce IFN‐γ production by human NK cells via autocrine IFN‐β

NK lymphocytes and type I IFN (IFN‐α/β) are major actors of the innate anti‐viral response that also influence adaptive immune responses. We evaluated type I IFN production by human NK cells in response to polyI:C, a potent type I IFN‐inducing TLR3 agonist. PolyI:C plus IL‐2/IL‐12 induced IFN‐β (but not IFN‐α) mRNA expression and protein production by highly pure human NK cells and by the human NK cell line NK92. Neutralizing anti‐IFNAR1 or anti‐IFN‐β Ab prevented the production of IFN‐γ induced by polyI:C plus IL‐2/IL‐12. Similarly, IFN‐γ production induced by polyI:C plus IL‐12 was reduced in NK cells isolated from IFNAR1^?/? compared with WT mice. The ability of polyI:C plus IL‐12 to induce IFN‐γ production was related to an increase of TLR3, Mda5 and IFNAR expression and by an increase of STAT1 and STAT4 phosphorylation. Collectively, these data demonstrate that NK cells, in response to polyI:C plus IL‐2/IL‐12, produce IFN‐β that induce, in an autocrine manner, the production of IFN‐γ and thereby highlight that NK cells may control the outcome of protective or injurious immune responses through type I IFN secretion.     

Significant NK cell activation associated with decreased cytolytic function in peripheral blood of HIV-1-infected patients

One of the hallmarks of HIV-1 infection is represented by the finding of massive T cell activation in peripheral blood lymphocytes of infected patients. An impairment of NK cell function during HIV-1 infection is also detected, and is associated with decreased expression of natural cytotoxicity receptors (NCR). In this study we tried to determine whether also NK cells are affected by relevant activation and whether this could be associated with decreased NK cell function. In 18 viremic HIV-1-infected patients, freshly drawn purified peripheral NK cells displayed significant levels of activation with an incomplete pattern (HLA-DR(+)CD69(+)CD25(-)NKp44(-)). Activated (HLA-DR(+)CD69(+)) peripheral NK cells expressed an NCR(dull) phenotype as determined by cytofluorometric analysis in all the patients, and did not derive from a homogeneous/oligoclonal expansion in vivo as analyzed by expression of HLA-specific inhibitory NK cell receptors. As determined by cytotoxicity assays, activated NK cells showed a decreased cytolytic function in HIV-1-infected patients. Thus, the decrease in NK cell function observed during HIV-1 infection is associated not only with decreased NCR expression, but also with significant and incomplete NK cell activation in vivo. These results suggest a consistent continuous involvement of the innate immune response in the failure to control viral replication.     

Innate IFN‐γ promotes development of experimental autoimmune encephalomyelitis: A role for NK cells and M1 macrophages

The role of IFN‐γ in the pathogenesis of autoimmune diseases is controversial. Although Th1 cells can induce experimental autoimmune encephalomyelitis (EAE), IFN‐γ can suppress Th17 cells that are pathogenic in EAE. Here we show that NK cells provide an early source of IFN‐γ during development of EAE. Depletion of NK cells or neutralization of IFN‐γ delayed the onset of EAE and was associated with reduced infiltration of IL‐17⁺ and GM‐CSF⁺ T cells into the CNS. In the passive transfer model, immune cells from myelin oligodendrocyte glycoprotein (MOG)‐immunized IFN‐γ^?/? mice failed to induce EAE, despite producing IL‐17 and GM‐CSF. The macrophages expressed markers of M2 activation and the T cells had low very late antigen‐4 (VLA‐4) expression and failed to infiltrate the CNS. Addition of recombinant IFN‐γ to immune cells from the IFN‐γ^?/? mice activated M1 macrophages and restored VLA‐4 expression, migratory, and encephalitogenic activity of T cells. Furthermore, treatment of recipient mice with anti‐VLA‐4 neutralizing antibody abrogated EAE induced by transfer of T cells from WT mice. Our findings demonstrate IFN‐γ‐producing T cells are not required for development of EAE, but NK cell‐derived IFN‐γ has a key role in promoting M1 macrophage expansion and VLA‐4‐mediated migration of encephalitogenic T cells into the CNS.     

Statins inhibit NK‐cell cytotoxicity by interfering with LFA‐1‐mediated conjugate formation

Inhibitors of the 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase, commonly referred to as statins, are inhibitors of cholesterol biosynthesis. They are broadly used for treating hypercholesterolemia and for prevention of cardio‐ and cerebrovascular diseases. Recent publications show that statins also act as immunomodulatory drugs. Here, we show that lipophilic statins inhibit NK‐cell degranulation and cytotoxicity. This effect was reversible by addition of substrates of isoprenylation, but not by addition of cholesterol. In NK‐target cell conjugates intracellular Ca²⁺ flux was unaffected by statin treatment. However, statins strongly reduced the amount of conjugate formation between NK and target cells. This inhibition was paralleled by a statin‐dependent inhibition of LFA‐1‐mediated adhesion and a reduction of NK‐cell polarization. This demonstrates that statins impair the formation of effector–target cell conjugates resulting in the disruption of early signaling and the loss of NK‐cell cytotoxicity.     

Comprehensive analysis of HIV Gag‐specific IFN‐γ response in HIV‐1‐ and HIV‐2‐infected asymptomatic patients from a clinical cohort in The Gambia

Majority of HIV‐2‐infected individuals meet the criteria of long‐term non‐progressors. This has been linked to superior qualitative HIV‐2‐specific cellular immune responses that correlate with viral control. However, it is unknown whether this is due to frequent targeting of immunodominant Gag epitopes in HIV‐2 than HIV‐1 infection. We describe a comprehensive comparison of the magnitude, breadth and frequency of Gag responses and the degree of cross‐recognition of frequently targeted, immunodominant Gag peptides in a cross‐sectional study of asymptomatic HIV‐1‐ and HIV‐2‐infected individuals. Fresh PBMC from 20 HIV‐1‐ and 20 HIV‐2‐infected patients with similar CD4⁺ T‐cell counts (p=0.36) were stimulated with pools of HIV‐1 and/or HIV‐2 Gag peptides in an IFN‐γ ELISPOT assay. We found no difference in the cumulative magnitude of IFN‐γ responses (p=0.75) despite significantly lower plasma viral loads in HIV‐2‐infected people (p<0.0001). However, Gag211–290 was targeted with significantly higher magnitude in HIV‐2‐infected subjects (p=0.03) although this did not correlate with viral control. There was no difference in frequently targeted Gag peptides, the breadth, immunodominance or cross‐recognition of Gag peptide pools between the two infections. This suggests that other factors may control viral replication in HIV‐2 infection.     

IL‐18‐induced expression of high‐affinity IL‐2R on murine NK cells is essential for NK‐cell IFN‐γ production during murine Plasmodium yoelii infection

Early production of pro‐inflammatory cytokines, including IFN‐γ, is essential for control of blood‐stage malaria infections. We have shown that IFN‐γ production can be induced among human natural killer (NK) cells by coculture with Plasmodium falciparum infected erythrocytes, but the importance of this response is unclear. To further explore the role of NK cells during malaria infection, we have characterized the NK‐cell response of C57BL/6 mice during lethal (PyYM) or nonlethal (Py17XNL) P. yoelii infection. Ex vivo flow cytometry revealed that NK cells are activated within 24 h of Py17XNL blood‐stage infection, expressing CD25 and producing IFN‐γ; this response was blunted and delayed during PyYM infection. CD25 expression and IFN‐γ production were highly correlated, suggesting a causal relationship between the two responses. Subsequent in vitro experiments revealed that IL‐18 signaling is essential for induction of CD25 and synergizes with IL‐12 to enhance CD25 expression on splenic NK cells. In accordance with this, Py17XNL‐infected erythrocytes induced NK‐cell CD25 expression and IFN‐γ production in a manner that is completely IL‐18‐ and partially IL‐12‐dependent, and IFN‐γ production is enhanced by IL‐2. These data suggest that IL‐2 signaling via CD25 amplifies IL‐18‐ and IL‐12‐mediated NK‐cell activation during malaria infection.     

CCR6 and NK1.1 distinguish between IL‐17A and IFN‐γ‐producing γδ effector T cells

γδ T cells are a potent source of innate IL‐17A and IFN‐γ, and they acquire the capacity to produce these cytokines within the thymus. However, the precise stages and required signals that guide this differentiation are unclear. Here we show that the CD24^low CD44^high effector γδ T cells of the adult thymus are segregated into two lineages by the mutually exclusive expression of CCR6 and NK1.1. Only CCR6⁺ γδ T cells produced IL‐17A, while NK1.1⁺ γδ T cells were efficient producers of IFN‐γ but not of IL‐17A. Their effector phenotype correlated with loss of CCR9 expression, particularly among the NK1.1⁺ γδ T cells. Accordingly, both γδ T‐cell subsets were rare in gut‐associated lymphoid tissues, but abundant in peripheral lymphoid tissues. There, they provided IL‐17A and IFN‐γ in response to TCR‐specific and TCR‐independent stimuli. IL‐12 and IL‐18 induced IFN‐γ and IL‐23 induced IL‐17A production by NK1.1⁺ or CCR6⁺ γδ T cells, respectively. Importantly, we show that CCR6⁺ γδ T cells are more responsive to TCR stimulation than their NK1.1⁺ counterparts. In conclusion, our findings support the hypothesis that CCR6⁺ IL‐17A‐producing γδ T cells derive from less TCR‐dependent selection events than IFN‐γ‐producing NK1.1⁺ γδ T cells.     

Activated monocytes augment TRAIL‐mediated cytotoxicity by human NK cells through release of IFN‐γ

Natural killer (NK) cells are innate lymphocytes that are able to directly kill tumor cells through different mechanisms including ligation of TNF‐related apoptosis‐inducing ligand (TRAIL) receptors. Zoledronic acid (ZA) is a bisphosphonate known to upregulate the expression of TRAIL on human γδ T cells. Here, we investigated whether exposure to ZA would upregulate TRAIL expression on human NK cells and augment their cytotoxicity against tumor cells. When cocultured with monocytes, treatment with ZA and IL‐2 resulted in a significant upregulation of TRAIL expression on human NK cells (p = 0.002). Consequently, ZA‐primed NK cells were significantly more cytotoxic against TRAIL sensitive tumor cells (p < 0.0001). In the presence of ZA and IL‐2, monocytes produced high levels of IFN‐γ; when cultured in the presence of neutralizing antibodies to IFN‐γ, TRAIL expression and TRAIL‐mediated cytotoxicity of NK cells were significantly reduced. Furthermore, in tumor‐bearing SCID/Beige mice, a significant delayed tumor progression and prolonged survival was observed after infusion of ZA‐primed NK cells compared with that observed in mice infused with unprimed NK cells. These findings represent a novel approach to potentiate TRAIL‐mediated apoptosis by adoptively infused NK cells that could improve the outcome in patients with cancer.     

Increased detection of proliferating,polyfunctional, HIV‐1‐specific T cells in DNA‐modified vaccinia virus Ankara‐vaccinated human volunteers by cultured IFN‐γ ELISPOT assay

Induction of a long‐term immunological memory, which can expand and defend the host upon pathogen encounter, is the “holy grail” of vaccinology. Here, using a sensitive cultured IFN‐γ ELISPOT assay, we show that 50% (15 out of 30) of healthy, HIV‐1/2‐uninfected volunteers who received pTHr.HIVA DNA prime‐modified vaccinia virus Ankara. HIVA boost vaccine regimen 1 to 3 1/2 years ago had detectable HIV‐1‐specific T‐cell responses. These T cells, predominantly of the CD4⁺ subtype, could proliferate and produce multiple cytokines in response to in vitro peptide stimulation. Peptide mapping studies showed that the vaccine‐induced CD4⁺ T cells were mostly directed toward epitopes targeted in HIV‐1‐infected individuals. In addition, we used the same assay to re‐evaluate 51 volunteers from past vaccine trial IAVI‐006 and corrected the previously reported 10% of vaccine responders to 50%. Thus, we confirmed that cultured assays are a valuable tool for studying T‐cell memory. These results are discussed in the context of the current state‐of‐affairs of the HIV‐1 vaccine field.     

IFN‐γ production by CD27+ NK cells exacerbates Listeria monocytogenes infection in mice by inhibiting granulocyte mobilization

Natural killer (NK) cells are key components of the immune system involved in several immune reactions, including the clearance of intracellular pathogens. When activated, NK cells rapidly secrete particular cytokines that activate innate immunity and facilitate development of adaptive responses. Conflicting reports on the role of NK cells during infection by Listeria monocytogenes can be found in the literature. Here, we demonstrate that during lethal infection by L. monocytogenes, activation of NK cells via the costimulatory molecule CD27 leads to excessive IFN‐γ production. This impairs innate anti‐bacterial host defenses by inducing downregulation of CXCR2 on granulocytes and consequently inhibiting their recruitment to the sites of infection. The use of antibodies to block CD27 signaling or to deplete IFN‐γ was sufficient to rescue mice from lethal challenge by L. monocytogenes. Our findings contribute to a better understanding of the importance of CD27 signaling in activation of NK cells and should provide new ways of interfering with infections.     

CEACAM1 on activated NK cells inhibits NKG2D‐mediated cytolytic function and signaling

Carcinoembryonic antigen‐related cell adhesion molecule 1 (CEACAM1) is expressed on activated natural killer (NK) cells wherein it inhibits lysis of CEACAM1‐bearing tumor cell lines. The mechanism for this is unknown. Here, we show that interleukin‐2‐induced expression of CEACAM1 on both mouse and primary human NK cells impairs the ability of NK gene complex group 2 member D (NKG2D) to stimulate cytolysis of CEACAM1‐bearing cells. This process requires the expression of CEACAM1 on the NK cells and on the tumor cells, which is consistent with the involvement of trans‐homophilic interactions between CEACAM1. Mechanistically, co‐engagement of NKG2D and CEACAM1 results in a biochemical association between these two surface receptors and the recruitment of Src homology phosphatase 1 by CEACAM1 that leads to dephosphorylation of the guanine nucleotide exchange factor Vav1 and blockade of downstream signaling that is associated with the initiation of cytolysis. Thus, CEACAM1 on activated NK cells functions as an inhibitory receptor for NKG2D‐mediated cytolysis, which has important implications for understanding the means by which CEACAM1 expression adversely affects tumor immunity.