mTORC2 regulates multiple aspects of NKT‐cell development and function

Invariant NKT (iNKT) cells bridge innate and adaptive immunity by rapidly secreting cytokines and lysing targets following TCR recognition of lipid antigens. Based on their ability to secrete IFN‐γ, IL‐4 and IL‐17A, iNKT‐cells are classified as NKT‐1, NKT‐2, and NKT‐17 subsets, respectively. The molecular pathways regulating iNKT‐cell fate are not fully defined. Recent studies implicate Rictor, a required component of mTORC2, in the development of select iNKT‐cell subsets, however these reports are conflicting. To resolve these questions, we used Rictor^fl/fl CD4cre⁺ mice and found that Rictor is required for NKT‐17 cell development and normal iNKT‐cell cytolytic function. Conversely, Rictor is not absolutely required for IL‐4 and IFN‐γ production as peripheral iNKT‐cells make copious amounts of these cytokines. Overall iNKT‐cell numbers are dramatically reduced in the absence of Rictor. We provide data indicating Rictor regulates cell survival as well as proliferation of developing and mature iNKT‐cells. Thus, mTORC2 regulates multiple aspects of iNKT‐cell development and function.     

The pro‐Th2 cytokine IL‐33 directly interacts with invariant NKT and NK cells to induce IFN‐γ production

IL‐33 has recently been identified as a cytokine endowed with pro‐Th2 functions, raising the question of its effect on invariant natural killer T cell (iNKT), which are potent IL‐4 producers. Here, we report a two‐fold increase of iNKT‐cell counts in spleen and liver after a 7‐day treatment of mice with IL‐33, which results from a direct effect, given that purified iNKT cells express the T1/ST2 receptor constitutively and respond to IL‐33 by in vitro expansion and functional activation. Conversely to the expected pro‐Th2 effect, IL‐33 induced a preferential increase in IFN‐γ rather than IL‐4 production upon TCR engagement that depended on endogenous IL‐12. Moreover, in combination with the pro‐inflammatory cytokine IL‐12, IL‐33 enhanced IFN‐γ production by both iNKT and NK cells. Taken together these data support the conclusion that IL‐33 can contribute as a co‐stimulatory factor to innate cellular immune responses.     

Stress‐activated dendritic cells interact with CD4+ T cells to elicit homeostatic memory

Evidence is presented that thermal or oxidizing stress‐activated DC interact with CD4⁺ T cells to induce and maintain a TCR‐independent homeostatic memory circuit. Stress‐activated DC expressed endogenous intra‐cellular and cell surface HSP70. The NF‐κB signalling pathway was activated and led to the expression of membrane‐associated IL‐15 molecules. These interacted with the IL‐15 receptor complex on CD4⁺ T cells, thus activating the Jak3 and STAT5 phosphorylation signalling pathway to induce CD40 ligand expression, T‐cell proliferation and IFN‐γ production. CD40 ligand on CD4⁺ T cells in turn re‐activated CD40 molecules on DC, inducing DC maturation and IL‐15 expression thereby maintaining the feedback circuit. The proliferating CD4⁺ T cells were characterized as CD45RA^? CD62L⁺ central memory cells, which underwent homeostatic proliferation. The circuit is independent of antigen and MHC‐class‐II‐TCR interaction as demonstrated by resistance to TCR inhibition by ZAP70 inhibitor or MHC‐class II antibodies. These findings suggest that stress can activate a DC‐CD4⁺ T‐cell interacting circuit, which may be responsible for maintaining a homeostatic antigen‐independent memory.     

Direct recognition of LPS by human but not murine CD8+ T cells via TLR4 complex

LPS comprises a major PAMP and is a key target of the immune system during bacterial infection. While LPS can be recognised by innate immune cells via the TLR4 complex, it is unknown whether T lymphocytes, especially CD8⁺ T cells are also capable of doing so. We report here that naïve human CD8⁺ T cells, after activation by TCR stimulation, express surface TLR4 and CD14. These activated CD8⁺ T cells can then secrete high concentrations of IFN‐γ, granzyme and perforin in response to LPS. These effects can be specifically inhibited using siRNA for TLR4. Furthermore, LPS can synergise with IL‐12 to polarise the CD8⁺ T cells into cytotoxic T‐cell 1 (Tc1) that produce IFN‐γ but not IL‐4, with or without TCR activation. Moreover, CD8⁺CD45RO⁺ memory T cells constitutively expressed TLR4 and markedly enhanced IFN‐γ production when stimulated with LPS. In contrast, activated murine CD8⁺ T cells lack TLR4 and CD14 expression and fail to respond to LPS for proliferation and cytokine production. Thus, human but not murine CD8⁺ T cells are able to directly recognise bacterial LPS via LPS receptor complex and TLR4 provides a novel signal for the activation of effector and memory human CD8⁺ T cells.     

The A2aR adenosine receptor controls cytokine production in iNKT cells

The purine nucleoside adenosine is an important anti‐inflammatory molecule, inhibiting a variety of immune cells by adenosine receptor‐mediated mechanisms. Invariant NKT (iNKT) cells recognize glycolipids presented on CD1d molecules and produce vigorous amounts of cytokines upon activation, hence regulating immune reactions. The mechanisms polarizing their cytokine pattern are elusive. Previous studies demonstrated that adenosine can suppress IFN‐γ production by iNKT cells. We describe the expression of all four known adenosine receptors A1R, A2aR, A2bR and A3R on mouse iNKT cells. We show that IL‐4 production in primary mouse iNKT cells and a human iNKT line is efficiently inhibited by A2aR blockade with an inverse relation to IL‐4. These data are supported by A2aR‐deficient mice, which exhibit largely decreased levels of IL‐4, IL‐10 and TGF‐β concomitantly with an increase of IFN‐γ upon α‐galactosylceramide administration in vivo. While A2aR inhibits other lymphocyte populations, A2aR is required for the secretion of IL‐4 and IL‐10 by iNKT cells. These data suggest adenosine:A2aR‐mediated mechanisms can control the cytokine secretion pattern of iNKT cells.     

T‐cell receptor activation of human CD4+ T cells shifts the innate TLR response from CXCL8hiIFN‐γnull to CXCL8loIFN‐γhi

Toll‐like receptors (TLRs) play a major part in providing innate immunity against pathogenic microorganisms. Recent studies show that these receptors are also expressed on T cells, which are the sentinels of adaptive immunity. Here, we have investigated the regulatory role of the T‐cell receptor in the functioning of these innate receptors in T cells. We show that freshly isolated human CD4⁺ T cells readily secrete the neutrophil chemoattractant CXCL8 upon activation with the TLR ligands Pam3CSK and flagellin. In contrast, TCR‐activated cells secrete considerably less CXCL8 but start producing IFN‐γ upon stimulation with TLR agonists in the absence of concomitant TCR engagement. These T cells show increased activation of p38 and JNK MAP‐kinases in response to TLR stimulation, and inhibition of p38 abrogates TLR‐induced IFN‐γ secretion. The shifting of the T‐cell innate immune response from CXCL8^hiIFN‐γ^null in freshly isolated to CXCL8^loIFN‐γ^hi in activated T cells is also observed in response to endogenous innate stimulus, IL‐1. These results suggest that the innate immune response of human CD4⁺ T cells switches from a proinflammatory to an effector type following activation of these cells through the antigen receptor.     

West Nile virus‐infected human dendritic cells fail to fully activate invariant natural killer T cells

West Nile virus (WNV) infection is a mosquito‐borne zoonosis with increasing prevalence in the United States. WNV infection begins in the skin, and the virus replicates initially in keratinocytes and dendritic cells (DCs). In the skin and cutaneous lymph nodes, infected DCs are likely to interact with invariant natural killer T cells (iNKTs). Bidirectional interactions between DCs and iNKTs amplify the innate immune response to viral infections, thus controlling viral load and regulating adaptive immunity. iNKTs are stimulated by CD1d‐bound lipid antigens or activated indirectly by inflammatory cytokines. We exposed human monocyte‐derived DCs to WNV Kunjin and determined their ability to activate isolated blood iNKTs. DCs became infected as judged by synthesis of viral mRNA and Envelope and NS‐1 proteins, but did not undergo significant apoptosis. Infected DCs up‐regulated the co‐stimulatory molecules CD86 and CD40, but showed decreased expression of CD1d. WNV infection induced DC secretion of type I interferon (IFN), but no or minimal interleukin (IL)?12, IL‐23, IL‐18 or IL‐10. Unexpectedly, we found that the WNV‐infected DCs stimulated human iNKTs to up‐regulate CD69 and produce low amounts of IL‐10, but not proinflammatory cytokines such as IFN‐γ or tumour necrosis factor (TNF)‐α. Both CD1d and IFNAR blockade partially abrogated this iNKT response, suggesting involvement of a T cell receptor (TCR)–CD1d interaction and type I interferon receptor (IFNAR) signalling. Thus, WNV infection interferes with DC–iNKT interactions by preventing the production of proinflammatory cytokines. iNKTs may be a source of IL‐10 observed in human flavivirus infections and initiate an anti‐inflammatory innate response that limits adaptive immunity and immune pathology upon WNV infection.     

IFN‐γ stimulates CpG‐induced IL‐10 production in B cells via p38 and JNK signalling pathways

The production of IL‐10, a potent immunosuppressive cytokine, must be strictly regulated to ensure a balanced immune response. IFN‐γ, a key cytokine in multiple immune processes and pathologies, is known as an inhibitor of IL‐10 production by monocytes and macrophages, but also has some regulatory functions. In the present study, we explored the role of IFN‐γ on Toll‐like receptor (TLR)‐induced IL‐10 production in murine peritoneal and spleen cells and in human peripheral blood mononuclear cells. IFN‐γ inhibited IL‐10 production induced by TLR2, TLR3, TLR4 and TLR7/8 agonists, but stimulated IL‐10 production when cells were triggered with CpG oligodeoxynucleotides, a specific TLR9 agonist. The stimulatory effect of IFN‐γ on TLR9‐induced IL‐10 was restricted to B cells. In line with the increased IL‐10, B cells stimulated with CpG and IFN‐γ profoundly inhibited CD4 T cell proliferation. Further research into the mechanisms involved, revealed that the mitogen‐activated protein kinases p38 and JNK are essential players in this stimulatory effect, and that the phosphatase MKP1 – an inhibitor of p38 and JNK activity – is downregulated after combined stimulation with IFN‐γ and CpG. Our data may represent a novel immunoregulatory role of IFN‐γ in B cells after triggering of TLR9, by stimulating IL‐10 production.     

CD161++CD8+ T cells,including the MAIT cell subset,are specifically activated by IL‐12+IL‐18 in a TCR‐independent manner

CD161⁺⁺CD8⁺ T cells represent a novel subset that is dominated in adult peripheral blood by mucosal‐associated invariant T (MAIT) cells, as defined by the expression of a variable‐α chain 7.2 (Vα7.2)‐Jα33 TCR, and IL‐18Rα. Stimulation with IL‐18+IL‐12 is known to induce IFN‐γ by both NK cells and, to a more limited extent, T cells. Here, we show the CD161⁺⁺ CD8⁺ T‐cell population is the primary T‐cell population triggered by this mechanism. Both CD161⁺⁺Vα7.2⁺ and CD161⁺⁺Vα7.2^? T‐cell subsets responded to IL‐12+IL‐18 stimulation, demonstrating this response was not restricted to the MAIT cells, but to the CD161⁺⁺ phenotype. Bacteria and TLR agonists also indirectly triggered IFN‐γ expression via IL‐12 and IL‐18. These data show that CD161⁺⁺ T cells are the predominant T‐cell population that responds directly to IL‐12+IL‐18 stimulation. Furthermore, our findings broaden the potential role of MAIT cells beyond bacterial responsiveness to potentially include viral infections and other inflammatory stimuli.     

Invariant NKT cells and CD1d+ cells amass in human omentum and are depleted in patients with cancer and obesity

Invariant NKT (iNKT) cells recognize lipid antigens presented by CD1d and respond rapidly by killing tumor cells and release cytokines that activate and regulate adaptive immune responses. They are essential for tumor rejection in various mouse models, but clinical trials in humans involving iNKT cells have been less successful, partly due to their rarity in humans compared with mice. Here we describe an accumulation of functional iNKT cells in human omentum, a migratory organ with healing properties. Analysis of 39 omental samples revealed that T cells are the predominant lymphoid cell type and of these, 10% expressed the invariant Vα24Jα18 TCR chain, found on iNKT cells, higher than in any other human organ tested to date. About 15% of omental hematopoietic cells expressed CD1d, compared with 1% in blood (p<0.001). Enriched omental iNKT cells killed CD1d⁺ targets and released IFN‐γ and IL‐4 upon activation. Omental iNKT‐cell frequencies were lower in patients with severe obesity (p=0.005), and with colorectal carcinoma (p=0.004) compared with lean healthy subjects. These data suggest a novel role for the omentum in immune regulation and tumor immunity and identify it as a potential source of iNKT cells for therapeutic use.     

TLR8‐mediated activation of human monocytes inhibits TL1A expression

TLR play important roles in inflammation and innate immune response to pathogens. TLR8 recognizes ssRNA and induces NF‐κB via MyD88 signaling. TL1A is a member of the TNF superfamily that markedly enhances IFN‐γ production by IL‐12/IL‐18‐stimulated peripheral and mucosal CD4⁺ T cells. TL1A expression is increased in the mucosa of patients with inflammatory bowel disease and is considered a key mediator of Crohn's disease (CD). We have previously shown that TL1A is strongly induced by immune complexes (IC) but not TLR ligands in antigen‐presenting cells. However, a potential interaction between these pro‐inflammatory signaling pathways has not been investigated. IC‐induced TL1A expression of monocytes was potently inhibited by a TLR8 or TLR7/8 ligand (R848) in a dose‐dependent manner. Furthermore, when co‐cultured with CD4⁺ T cells, TLR8 ligands inhibited TL1A production, resulting in almost complete inhibition of IFN‐γ production by the CD4⁺ T cells. Furthermore, we demonstrate that IFN‐α is not required for this suppressive effect by TLR8 signaling. Our data demonstrate for the first time a direct interaction between TLR and TL1A signaling pathways. TLR8 activation may be an important, novel pathway for targeted treatment of Th1‐mediated diseases, such as CD.     

Innate,antigen‐independent role for T cells in the activation of the immune system by Propionibacterium acnes

Propionibacterium acnes is a human commensal but also an opportunistic pathogen. In mice, P. acnes exerts strong immunomodulatory activities, including formation of intrahepatic granulomas and induction of LPS hypersensitivity. These activities are dependent on P. acnes recognition via TLR9 and subsequent IL‐12‐mediated IFN‐γ production. We show that P. acnes elicits IL‐12p40 and p35 mRNA expression in macrophages, and IFN‐γ mRNA in liver CD4⁺ T cells and NK cells. After priming with P. acnes, CD4⁺ T cells serve as the major IFN‐γ mRNA source. In the absence of CD4⁺ T cells, CD8⁺ T cells (regardless of antigenic specificity) or NK cells can produce sufficient IFN‐γ to induce the P. acnes‐driven immune effects. Moreover, in the absence of αβT cells, γδT cells also enable the development of strongly enhanced TNF‐α and IFN‐γ responses to LPS and intrahepatic granuloma formation. Thus, under microbial pressure, different T‐cell types, independent of their antigen specificity, exert NK‐cell‐like functions, which contribute decisively to the activation of the innate immune system.     

IL‐18 skews the invariant NKT‐cell population via autoreactive activation in atopic eczema

Atopic eczema (AE) is a chronic relapsing inflammatory skin disease where the commensal yeast Malassezia can act as a microbial trigger factor. Malassezia activates human DC to produce IL‐18, an innate cytokine that is elevated in serum of AE patients; however, the precise role of IL‐18 in human AE etiology is unknown. Herein, we investigated the effect of IL‐18 on the human invariant NKT (iNKT) cell compartment in AE. We found that IL‐18 was a potent activator of human iNKT‐cells and promoted a pro‐inflammatory CD1d‐dependent response, even in the absence of exogenous ligands. Chronic activation via IL‐18 on the other hand was inhibitory and skewed the iNKT‐cell pool by selectively suppressing CD4⁺ iNKT‐cells. This was mimicked in AE patients where the proportion of CD4⁺ iNKT‐cells was reduced in peripheral blood and coincided with elevated plasma levels of IL‐18. Furthermore, a reduced CD4⁺ iNKT‐cell pool was associated with elevated IgE levels in plasma, and the plasma levels of IL‐18 correlated with both total IgE and disease severity in the AE patients. Based on these findings, we propose that IL‐18‐mediated activation and subsequent dysregulation of the CD1d‐restricted iNKT‐cells plays a role in the pathogenesis of human AE.     

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.     

Sympathetic neural signaling via the β2‐adrenergic receptor suppresses T‐cell receptor‐mediated human and mouse CD8+ T‐cell effector function

Postganglionic sympathetic neurons innervate secondary lymphoid organs and secrete norepinephrine (NE) as the primary neurotransmitter. NE binds and signals through five distinct members of the adrenergic receptor family. In this study, we show elevated expression of the β2‐adrenergic receptor (ADRB2) on primary human CD8⁺ effector memory T cells. Treatment of both human and murine CD8⁺ T cells with NE decreased IFN‐γ and TNF‐α secretion and suppressed their cytolytic capacity in response to T‐cell receptor (TCR) activation. The effects of NE were specifically reversed by β 2‐specific antagonists. Adrb2^?/? CD8⁺ T cells were completely resistant to the effects of NE. Further, the ADRB2‐specific pharmacological ligand, albuterol, significantly suppressed effector functions in both human and mouse CD8⁺ T cells. While both TCR activation and stimulation with IL‐12 + IL‐18 were able to induce inflammatory cytokine secretion, NE failed to suppress IFN‐γ secretion in response to IL‐12 + IL18. Finally, the long‐acting ADRB2‐specific agonist, salmeterol, markedly reduced the cytokine secretion capacity of CD8⁺ T cells in response to infection with vesicular stomatitis virus. This study reveals a novel intrinsic role for ADRB2 signaling in CD8⁺ T‐cell function and underscores the novel role this pathway plays in adaptive T‐cell responses to 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.     

Leishmania donovani‐induced expression of signal regulatory protein α on Kupffer cells enhances hepatic invariant NKT‐cell activation

Signal regulatory protein α (SIRPα) and its cognate ligand CD47 have been documented to have a broad range of cellular functions in development and immunity. Here, we investigated the role of SIRPα–CD47 signalling in invariant NKT (iNKT) cell responses. We found that CD47 was required for the optimal production of IFN‐γ from splenic iNKT cells following exposure to the αGalCer analogue PBS‐57 and in vivo infection of mice with Leishmania donovani. Surprisingly, although SIRPα was undetectable in the liver of uninfected mice, the hepatic iNKT‐cell response to infection was also impaired in CD47^?/? mice. However, we found that SIRPα was rapidly induced on Kupffer cells following L. donovani infection, via a mechanism involving G‐protein‐coupled receptors. Thus, we describe a novel amplification pathway affecting cytokine production by hepatic iNKT cells, which may facilitate the breakdown of hepatic tolerance after infection.     

TLR2 engagement on CD4+ T cells enhances effector functions and protective responses to Mycobacterium tuberculosis

We have previously demonstrated that mycobacterial lipoproteins engage TLR2 on human CD4⁺ T cells and upregulate TCR‐triggered IFN‐γ secretion and cell proliferation in vitro. Here we examined the role of CD4⁺ T‐cell‐expressed TLR2 in Mycobacterium tuberculosis (MTB) Ag‐specific T‐cell priming and in protection against MTB infection in vivo. Like their human counterparts, mouse CD4⁺ T cells express TLR2 and respond to TLR2 costimulation in vitro. This Th1‐like response was observed in the context of both polyclonal and Ag‐specific TCR stimulation. To evaluate the role of T‐cell TLR2 in priming of CD4⁺ T cells in vivo, naive MTB Ag85B‐specific TCR transgenic CD4⁺ T cells (P25 TCR‐Tg) were adoptively transferred into Tlr2^?/? recipient C57BL/6 mice that were then immunized with Ag85B and with or without TLR2 ligand Pam₃Cys‐SKKKK. TLR2 engagement during priming resulted in increased numbers of IFN‐γ‐secreting P25 TCR‐Tg T cells 1 week after immunization. P25 TCR‐Tg T cells stimulated in vitro via TCR and TLR2 conferred more protection than T cells stimulated via TCR alone when adoptively transferred before MTB infection. Our findings indicate that TLR2 engagement on CD4⁺ T cells increases MTB Ag‐specific responses and may contribute to protection against MTB infection.