Fine specificity of natural killer T cells against GD3 ganglioside and identification of GM3 as an inhibitory natural killer T-cell ligand

GD3, a ganglioside expressed on melanoma, is the only tumour-associated glycolipid described to date that can induce a CD1d-restricted natural killer T (NKT)-cell response. We analysed the fine specificity of GD3-reactive NKT cells and discovered that immunization with GD3 induced two populations of GD3-reactive NKT cells. One population was CD4+ CD8- and was specific for GD3; the other population was CD4- CD8- and cross-reacted with GM3 in a CD1d-restricted manner, but did not cross-react with GM2, GD2, or lactosylceramide. This indicated that the T-cell receptors reacting with GD3 recognize glucose-galactose linked to at least one N-acetyl-neuraminic acid but will not accommodate a terminal N-acetylgalactosamine. Immunization with GM2, GM3, GD2, or lactosylceramide did not induce an NKT-cell response. Coimmunization of GM3-loaded antigen-presenting cells (APCs) with GD3-loaded APCs suppressed the NKT-cell response to GD3 in a CD1d-restricted manner. This suppressive effect was specific for GM3 and was a local effect lasting 2-4 days. In vitro, GM3-loaded APCs also suppressed the interleukin-4 response, but not the interferon-gamma response, of NKT cells to alpha-galactosylceramide. However, there was no effect on the T helper type 2 responses of conventional T cells. We found that this suppression was not mediated by soluble factors. We hypothesize that GM3 induces changes to the APC that lead to suppression of T helper type 2-like NKT-cell responses.     

Activation of invariant natural killer T cells in regional lymph nodes as new antigen‐specific immunotherapy via induction of interleukin‐21 and interferon‐γ

Invariant natural killer T (iNKT) cells play important immunoregulatory functions in allergen‐induced airway hyperresponsiveness and inflammation. To clarify the role of iNKT cells in allergic rhinitis (AR), we generated bone marrow‐derived dendritic cells (BMDCs), which were pulsed by ovalbumin (OVA) and α‐galactosylceramide (OVA/α‐GalCer‐BMDCs) and administered into the oral submucosa of OVA‐sensitized mice before nasal challenge. Nasal symptoms, level of OVA‐specific immunoglobulin (IgE), and T helper type 2 (Th2) cytokine production in cervical lymph nodes (CLNs) were significantly ameliorated in wild‐type (WT) mice treated with OVA/α‐GalCer‐BMDCs, but not in WT mice treated with OVA‐BMDCs. These anti‐allergic effects were not observed in Jα18^–/– recipients that lack iNKT cells, even after similar treatment with OVA/α‐GalCer‐BMDCs in an adoptive transfer study with CD4⁺ T cells and B cells from OVA‐sensitized WT mice. In WT recipients of OVA/α‐GalCer‐BMDCs, the number of interleukin (IL)‐21‐producing iNKT cells increased significantly and the Th1/Th2 balance shifted towards the Th1 dominant state. Treatment with anti‐IL‐21 and anti‐interferon (IFN)‐γ antibodies abrogated these anti‐allergic effects in mice treated with α‐GalCer/OVA‐BMDCs. These results suggest that activation of iNKT cells in regional lymph nodes induces anti‐allergic effects through production of IL‐21 or IFN‐γ, and that these effects are enhanced by simultaneous stimulation with antigen. Thus, iNKT cells might be a useful target in development of new treatment strategies for AR.     

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.     

Adipose invariant natural killer T cells

Adipose tissue is a dynamic organ that makes up a substantial proportion of the body; in severe obesity it can account for 50% of body mass. Details of the unique immune system resident in human and murine adipose tissue are only recently emerging, and so it has remained a largely unexplored and unappreciated immune site until now. Adipose tissue harbours a unique collection of immune cells, which often display unusual functions compared with their counterparts elsewhere in the body. These resident immune cells are key to maintaining tissue and immune homeostasis, yet in obesity their chronic aberrant stimulation can contribute to the inflammation and pathogenesis associated with obesity. Anti‐inflammatory adipose‐resident lymphocytes are often depleted in obesity, whereas pro‐inflammatory immune cells accumulate, leading to an overall inflammatory state, which is a key step in the development of obesity‐induced metabolic disease. A good example is invariant natural killer T (iNKT) cells, which make up a large proportion of lymphocytes in human and murine adipose tissue. Here, they are unusually poised to produce anti‐inflammatory or regulatory cytokines, however in obesity, iNKT cells are greatly reduced. As iNKT cells are potent transactivaors of other immune cells, and can act as a bridge between innate and adaptive immunity, their loss in obesity represents the loss of a major regulatory population. Restoring iNKT cells, or activating them in obese mice leads to improved glucose handling, insulin sensitivity, and even weight loss, and hence represents an exciting therapeutic avenue to be explored for restoring homeostasis in obese adipose tissue.     

Lower numbers of natural killer T cells in HIV-1 and Mycobacterium leprae co-infected patients

Natural killer T (NKT) cells are a heterogeneous population of lymphocytes that recognize antigens presented by CD1d and have attracted attention because of their potential role linking innate and adaptive immune responses. Peripheral NKT cells display a memory-activated phenotype and can rapidly secrete large amounts of pro-inflammatory cytokines upon antigenic activation. In this study, we evaluated NKT cells in the context of patients co-infected with HIV-1 and Mycobacterium leprae. The volunteers were enrolled into four groups: 22 healthy controls, 23 HIV-1-infected patients, 20 patients with leprosy and 17 patients with leprosy and HIV-1-infection. Flow cytometry and ELISPOT assays were performed on peripheral blood mononuclear cells. We demonstrated that patients co-infected with HIV-1 and M. leprae have significantly lower NKT cell frequencies [median 0.022%, interquartile range (IQR): 0.007-0.051] in the peripheral blood when compared with healthy subjects (median 0.077%, IQR: 0.032-0.405, P < 0.01) or HIV-1 mono-infected patients (median 0.072%, IQR: 0.030-0.160, P < 0.05). Also, more NKT cells from co-infected patients secreted interferon-γ after stimulation with DimerX, when compared with leprosy mono-infected patients (P = 0.05). These results suggest that NKT cells are decreased in frequency in HIV-1 and M. leprae co-infected patients compared with HIV-1 mono-infected patients alone, but are at a more activated state. Innate immunity in human subjects is strongly influenced by their spectrum of chronic infections, and in HIV-1-infected subjects, a concurrent mycobacterial infection probably hyper-activates and lowers circulating NKT cell numbers.     

Autoreactive natural killer T cells: promoting immune protection and immune tolerance through varied interactions with myeloid antigen-presenting cells

Natural killer T (NKT) cells are innate T lymphocytes that are restricted by CD1d antigen‐presenting molecules and recognize lipids and glycolipids as antigens. NKT cells have attracted attention for their potent immunoregulatory effects. Like other types of regulatory lymphocytes, a high proportion of NKT cells appear to be autoreactive to self antigens. Thus, as myeloid antigen‐presenting cells (APCs) such as monocytes, dendritic cells (DCs) and myeloid‐derived suppressor cells (MDSCs) constitutively express CD1d, NKT cells are able to interact with these APCs not only during times of immune activation but also in immunologically quiescent periods. The interactions of NKT cells with myeloid APCs can have either pro‐inflammatory or tolerizing outcomes, and a central question is how the ensuing response is determined. Here we bring together published results from a variety of model systems to highlight three critical factors that influence the outcome of the NKT–APC interaction: (i) the strength of the antigenic signal delivered to the NKT cell, as determined by antigen abundance and/or T‐cell receptor (TCR) affinity; (ii) the presence or absence of cytokines that costimulate NKT cells [e.g. interleukin (IL)‐12, IL‐18 and interferon (IFN)‐α]; (iii) APC intrinsic factors such as differentiation state (e.g. monocyte versus DC) and Toll‐like receptor (TLR) stimulation. Together with recent findings that demonstrate new links between NKT cell activation and endogenous lipid metabolism, these results outline a picture in which the functions of NKT cells are closely attuned to the existing biological context. Thus, NKT cells may actively promote tolerance until a critical level of danger signals arises, at which point they switch to activating pro‐inflammatory immune responses.     

Endogenous lipid antigens for invariant natural killer T cells hold the reins in adipose tissue homeostasis

The global obesity epidemic and its associated co‐morbidities, including type 2 diabetes, cardiovascular disease and certain types of cancers, have drawn attention to the pivotal role of adipocytes in health and disease. Besides their ‘classical’ function in energy storage and release, adipocytes interact with adipose‐tissue‐resident immune cells, among which are lipid‐responsive invariant natural killer T (iNKT) cells. The iNKT cells are activated by lipid antigens presented by antigen‐presenting cells as CD1d/lipid complexes. Upon activation, iNKT cells can rapidly secrete soluble mediators that either promote or oppose inflammation. In lean adipose tissue, iNKT cells elicit a predominantly anti‐inflammatory immune response, whereas obesity is associated with declining iNKT cell numbers. Recent work showed that adipocytes act as non‐professional antigen‐presenting cells for lipid antigens. Here, we discuss endogenous lipid antigen processing and presentation by adipocytes, and speculate on how these lipid antigens, together with ‘environmental factors’ such as tissue/organ environment and co‐stimulatory signals, are able to influence the fate of adipose‐tissue‐resident iNKT cells, and thereby the role of these cells in obesity and its associated pathologies.     

A case of natural killer/T cell lymphoma of the subcutis resembling subcutaneous panniculitis-like T cell lymphoma

A case of nasal type natural killer (NK)/T cell lymphoma of the subcutis showing clinical and morphological features that resemble subcutaneous panniculitis-like T cell lymphoma (SPTCL) is presented. A 73-year-old man presented with swelling of the left arm and was diagnosed with panniculitis by a dermatologist. It was concluded from a skin biopsy specimen that the patient had non-Hodgkin's lymphoma of the large cell, NK/T cell type because the neoplastic cells showed polyclonal CD3 immunoreactivity. Treatment with interferon-gamma was initiated, but the patient died of disseminated intravascular coagulation and multiple organ failure 2 months after the initial symptoms appeared. However, involvement of additional organs by the lymphoma was not apparent clinically. An autopsy was not performed. A routinely stained section of the biopsy skin specimen revealed massive necrosis of the subcutaneous fat, karyorrhexis admixed with reactive histiocytes, and large atypical lymphoid cells. Immunoreactivity for polyclonal CD3 was present in the perinuclear region, but absent in the neoplastic cell membranes. CD56, CD45RO (UCHL-1), CD43 (MT1), CD45 (leukocyte common antigen), and the cytotoxic molecules perforin, granzyme B and TIA-1 were positive, but CD20 (L26), CD4, CD8, and betaF1 were negative. Epstein-Barr virus (EBV) mRNA was detected in the nuclei of neoplastic cells by in situ hybridization. Subcutaneous panniculitis-like T cell lymphoma is reported to be an EBV-negative, clonal T cell neoplasm. Although this case showed clinical and morphological features that resembled SPTCL, perinuclear polyclonal CD3 staining and membranous CD56 reactivity seen in neoplastic cells were suggestive of NK cells. Furthermore, the neoplastic cells were positive for EBV. This case is considered to be a NK/T cell lymphoma of the subcutis resembling SPTCL. It is believed that it is important to recognize such a tumor because patients may undergo a fulminant clinical course, despite the tumor being localized in the subcutaneous adipose tissue.     

Strategy of lipid recognition by invariant natural killer T cells: 'one for all and all for one'

Invariant natural killer T (iNKT) cells are evolutionarily conserved lipid-reactive T cells that bridge innate and adaptive immune responses. Despite a relatively restricted T-cell receptor (TCR) diversity, these cells respond to a variety of structurally distinct foreign (i.e. microbial or synthetic) as well as host-derived (self-) lipid antigens presented by the CD1d molecule. These multi-tasking lymphocytes are among the first responders in immunity, and produce an impressive array of cytokines and chemokines that can tailor the ensuing immune response. Accordingly, iNKT cells play important functions in autoimmune diseases, cancer, infection and inflammation. These properties make iNKT cells appealing targets in immune-based therapies. Yet, much has to be learned on the mechanisms that allow iNKT cells to produce polarized responses. Responses of iNKT cells are influenced by the direct signals perceived by the cells through their TCRs, as well as by indirect co-stimulatory (and potentially co-inhibitory) cues that they receive from antigen-presenting cells or the local milieu. A decade ago, biochemists and immunologists have started to describe synthetic lipid agonists with cytokine skewing potential, paving a new research avenue in the iNKT cell field. Yet how iNKT cells translate various antigenic signals into distinct functional responses has remained obscure. Recent findings have revealed a unique and innate mode of lipid recognition by iNKT cells, and suggest that both the lipid antigen presented and the diversity of the TCR modulate the strength of CD1d-iNKT TCR interactions. In this review, we focus on novel discoveries on lipid recognition by iNKT cells, and how these findings may help us to design effective strategies to steer iNKT cell responses for immune intervention.     

Cytotoxic T lymphocyte and natural killer cell responses to non-typeable Haemophilus influenzae

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells have a key role in host defence against infectious pathogens, but their response to bacteria is not well characterized. Non-typeable Haemophilus influenzae is a major cause of respiratory tract infection including otitis media, sinusitis, tonsillitis and chronic bronchitis (especially in chronic obstructive pulmonary disease and bronchiectasis). This bacterium is also present in the pharynx of most healthy adults. The primary factor that may determine whether clinical disease occurs or not is the nature of the lymphocyte response. Here we examined the CTL cell and NK cell responses to nontypeable H. influenzae in healthy control subjects and in subjects who had bronchiectasis and recurrent bronchial infection with this bacterium. Cells were stimulated with live H. influenzae and intracellular cytokine production and release of cytotoxic granules measured. Control subjects had significantly higher levels of interferon gamma production by both CTL and NK cells, while levels of cytotoxic granule release were similar in both groups. The main lymphocyte subsets that proliferated in response to H. influenzae stimulation were the CTL and NK cells. The results suggest that CTL and NK cell responses may be important in preventing disease from nontypeable H. influenzae infection.     

Dissociated expression of natural killer 1.1 and T‐cell receptor by invariant natural killer T cells after interleukin‐12 receptor and T‐cell receptor signalling

Invariant (i) natural killer T (NKT) cells become undetectable after stimulation with α-galactosylceramide (α-GalCer) or interleukin (IL)-12. Although down-modulation of surface T-cell receptor (TCR)/NKR-P1C (NK1.1) expression has been shown convincingly after stimulation with α-GalCer, it is unclear whether this also holds true for IL-12 stimulation. To determine whether failure to detect iNKT cells after IL-12 stimulation is caused by dissociation/internalization of TCR and/or NKR-P1C, or by block of de novo synthesis of these molecules, and to examine the role of IL-12 in the disappearance of iNKT cells after stimulation with α-GalCer, surface (s)/cytoplasmic (c) protein expression, as well as messenger RNA (mRNA) expression of TCR/NKR-P1C by iNKT cells after stimulation with α-GalCer or IL-12, and the influence of IL-12 neutralization on the down-modulation of sTCR/sNKR-P1C expression by iNKT cells after stimulation with α-GalCer were examined. The s/cTCR⁺s/cNKR-P1C⁺ iNKT cells became undetectable after in vivo administration of α-GalCer, which was partially prevented by IL-12 neutralization. Whereas s/cNKR-P1C⁺ iNKT cells became undetectable after in vivo administration of IL-12, s/cTCR⁺ iNKT cells were only marginally affected. mRNA expression of TCR/NKR-P1C remained unaffected by α-GalCer or IL-12 treatment, despite the down-modulation of cTCR and/or cNKR-P1C protein expression. By contrast, cTCR⁺cNKR-P1C⁺ sTCR⁻ sNKR-P1C⁻ iNKT cells and cNKR-P1C⁺ sNKR-P1C⁻ iNKT cells were detectable after in vitro stimulation with α-GalCer and IL-12, respectively. Our results indicate that TCR and NKR-P1C expression by iNKT cells is differentially regulated by signalling through TCR and IL-12R. They also suggest that IL-12 participates, in part, in the disappearance of iNKT cells after stimulation with α-GalCer by down-modulating not only sNKR-P1C, but also sTCR.     

Depletion of CD4+CD25+ regulatory T cells enhances natural killer T cell‐mediated anti‐tumour immunity in a murine mammary breast cancer model

Both invariant natural killer T (NK T) cells and CD4⁺CD25⁺ T regulatory cells (T_regs) regulate the immune system to maintain homeostasis. In a tumour setting, NK T cells activated by α‐galactosylceramide (α‐GalCer) execute anti‐tumour activity by secreting cytokines. By contrast, T_regs intrinsically suppress antigen‐specific immune responses and are often found to be elevated in tumour patients. In this study, we have shown that T_regs regulate NK T cell function negatively in vitro, suggesting a direct interaction between these cell types. In a murine mammary tumour model, we demonstrated that administration of either α‐GalCer or anti‐CD25 antibody alone markedly suppressed tumour formation and pulmonary metastasis, and resulted in an increase in the survival rate up to 44% (from a baseline of 0%). When treatments were combined, depletion of T_regs boosted the anti‐tumour effect of α‐GalCer, and the survival rate jumped to 85%. Our results imply a potential application of combining T_reg cell depletion with α‐GalCer to stimulate NK T cells for cancer therapy.     

Lack of anti-tumour reactivity despite enhanced numbers of circulating natural killer T cells in two patients with metastatic renal cell carcinoma

Natural killer T (NK T) cells play a central role as intermediates between innate and adaptive immune responses important to induce anti-tumour reactivity in cancer patients. In two of 14 renal cell carcinoma (RCC) patients, treated with interferon (IFN)-α, we detected significantly enhanced numbers of circulating NK T cells which were typed phenotypically and analysed for anti-tumour reactivity. These NK T cells were T cell receptor (TCR) Vα24/Vβ11(+), 6B11(+) and bound CD1d tetramers. No correlation was observed between NK T frequencies and regulatory T cells (T(regs)), which were also enhanced. NK T cells expressed CD56, CD161, CD45RO and CD69 and were predominantly CD8(+), in contrast to the circulating T cell pool that contained both CD4(+) and CD8(+) T cells, as is found in healthy individuals. It is unlikely that IFN-α triggered the high NK T frequency, as all other patients expressed low to normal NK T numbers. A parallel was observed in IFN-α-related increase in activation of NK T cells with that in conventional T and non-T cells. Normal interleukin (IL)-7, IL-12 and IL-15 plasma levels were found. In one of the patients sporadic NK T cells were detected at the tumour site. α-Galactosylceramide (αGalCer) stimulation of peripheral blood mononuclear cells or isolated NK T cell lines from both patients induced IFN-γ, but no IL-4 and no response towards autologous tumour cells or lysates. The clinical course of disease in both patients was not exceptional with regard to histological subtype and extent of metastatic disease. Therefore, despite a constitutive high peripheral frequency and in vitroαGalCer responsiveness, the NK T cells in the two RCC patients did not show anti-tumour responsiveness.     

Miscarriage induced by adoptive transfer of dendritic cells and invariant natural killer T cells into mice

Unexpected fetal loss is one of the common complications of pregnancy; however, the pathogenesis of many miscarriages, particularly those not associated with infections, is unknown. We previously found that activated DEC‐205⁺ dendritic cells (DCs) and NK1.1⁺ invariant natural killer T (iNKT) cells are recruited into the myometrium of mice when miscarriage is induced by the intraperitoneal administration of α‐galactosylceramide (α‐GalCer). Here we demonstrate that the adoptive transfer of DEC‐205⁺ bone marrow‐derived DCs cocultured with α‐GalCer (DEC‐205⁺ BMDCs‐c/w‐α‐GalCer) directly induced marked fetal loss by syngeneic pregnant C57BL/6 (B6) mice and allogeneic mice (B6 (♀) × BALB/c (♂)), which was accompanied by the accumulation of activated iNKT cells in the myometrium. Further, the adoptive transfer of NK1.1⁺ iNKT cells obtained from B6 mice injected with α‐GalCer facilitated miscarriages in syngeneic Jα18^(?/?) (iNKT cell‐deficient) mice. These results suggest that DEC‐205⁺ DCs and NK1.1⁺ iNKT cells play crucial roles required for the initiation of fetal loss associated with stimulation by glycolipid antigens and sterile inflammation.     

TCR Valpha14 natural killer T cells function as effector T cells in mice with collagen-induced arthritis

Natural killer (NK) T cells are a unique, recently identified cell population and are suggested to act as regulatory cells in autoimmune disorders. In the present study, designed to investigate the role of NKT cells in arthritis development, we attempted to induce arthritis by immunization of type II collagen (CIA) in Jalpha281 knock out (NKT-KO) and CD1d knock out (CD1d-KO) mice, which are depleted of NKT cells. From the results, the incidence of arthritis (40%) and the arthritis score (1.5 +/- 2.2 and 2.0 +/- 2.7) were reduced in NKT-KO and CD1d-KO mice compared to those in respective wild type mice (90%, 5.4 +/- 3.2 and 2.0 +/- 2.7, P < 0.01). Anti-CII antibody levels in the sera of NKT-KO and CD1d-KO mice were significantly decreased compared to the controls (OD values; 0.32 +/- 0.16 and 0.29 +/- 0.06 versus 0.58 +/- 0.08 and 0.38 +/- 0.08, P < 0.01). These results suggest that NKT cells play a role as effector T cells in CIA. Although the cell proliferative response and cytokine production in NKT-KO mice after the primary immunization were comparable to those in wild type mice, the ratios of both activated T or B cells were lower in NKT-KO mice than wild type mice after secondary immunization (T cells: 9.9 +/- 1.8% versus 16.0 +/- 3.4%, P < 0.01, B cells: 4.1 +/- 0.5% versus 5.1 +/- 0.7%, P < 0.05), suggesting that inv-NKT cells contribute to the pathogenicity in the development phase of arthritis. In addition, IL-4 and IL-1beta mRNA expression levels in the spleen during the arthritis development phase were lower in NKT-KO mice, while the IFN-gamma mRNA expression level was temporarily higher. These results suggest that inv-NKT cells influence cytokine production in arthritis development. In conclusion, inv-NKT cells may promote the generation of arthritis, especially during the development rather than the initiation phase.     

In vivo activation of invariant V alpha 14 natural killer T cells by alpha-galactosylceramide sequentially induces Fas-dependent and -independent cytotoxicity

The present study was designed to clarify the cytotoxic capacities of invariant V alpha 14 natural killer T (iNKT) cells activated in vivo. We found that as early as 2 h after a single injection of alpha-galactosylceramide (alpha-GalCer), sorted iNKT splenocytes from treated mice kill Fas-transfected target cells. The implication of the Fas pathway in this lysis was strengthened by both the blockage of cytotoxicity in the presence of anti-Fas ligand (FasL) monoclonal antibody (mAb) and the up-regulation of FasL expression on iNKT cells. Sorted NK cells did not participate in the lytic activity at this time point. Yet, they became cytotoxic later on, 24 h post-treatment, when target cell lysis was mainly independent of the Fas pathway. This type of cell killing was predominant at this later time point, even though iNKT cells conserved a slight Fas-dependent cytotoxicity. NK cells failed to acquire the ability to kill target cells when IFN-gamma production in alpha-GalCer-injected mice was blocked by anti-IFN-gamma mAb, underscoring the major role of this cytokine. In conclusion, our findings provide the first direct evidence that iNKT cells can exert Fas-dependent cytotoxicity very shortly after in vivo alpha-GalCer activation and later, through IFN-gamma secretion, enable NK cells to kill target cells in a Fas-independent pathway.     

Turned on by danger: activation of CD1d-restricted invariant natural killer T cells

CD1d-restricted invariant natural killer T (iNKT) cells bear characteristics of innate and adaptive lymphocytes, which allow them to bridge the two halves of the immune response and play roles in many disease settings. Recent work has characterized precisely how their activation is initiated and regulated. Novel antigens from important pathogens have been identified, as has an abundant self-antigen, β-glucopyranosylcaramide, capable of mediating an iNKT-cell response. Studies of the iNKT T-cell receptor (TCR)-antigen-CD1d complex show how docking between CD1d-antigen and iNKT TCR is highly conserved, and how small sequence differences in the TCR establish intrinsic variation in iNKT TCR affinity. The sequence of the TCR CDR3β loop determines iNKT TCR affinity for ligand-CD1d, independent of ligand identity. CD1d ligands can promote T helper type 1 (Th1) or Th2 biased cytokine responses, depending on the composition of their lipid tails. Ligands loaded into CD1d on the cell surface promote Th2 responses, whereas ligands with long hydrophobic tails are loaded endosomally and promote Th1 responses. This information is informing the design of synthetic iNKT-cell antigens. The iNKT cells may be activated by exogenous antigen, or by a combination of dendritic cell-derived interleukin-12 and iNKT TCR-self-antigen-CD1d engagement. The iNKT-cell activation is further modulated by recent foreign or self-antigen encounter. Activation of dendritic cells through pattern recognition receptors alters their antigen presentation and cytokine production, strongly influencing iNKT-cell activation. In a range of bacterial infections, dendritic cell-dependent innate activation of iNKT cells through interleukin-12 is the dominant influence on their activity.     

T helper type 2-polarized invariant natural killer T cells reduce disease severity in acute intra-abdominal sepsis

Sepsis is characterized by a severe systemic inflammatory response to infection that is associated with high morbidity and mortality despite optimal care. Invariant natural killer T (iNK T) cells are potent regulatory lymphocytes that can produce pro- and/or anti-inflammatory cytokines, thus shaping the course and nature of immune responses; however, little is known about their role in sepsis. We demonstrate here that patients with sepsis/severe sepsis have significantly elevated proportions of iNK T cells in their peripheral blood (as a percentage of their circulating T cells) compared to non-septic patients. We therefore investigated the role of iNK T cells in a mouse model of intra-abdominal sepsis (IAS). Our data show that iNK T cells are pathogenic in IAS, and that T helper type 2 (Th2) polarization of iNK T cells using the synthetic glycolipid OCH significantly reduces mortality from IAS. This reduction in mortality is associated with the systemic elevation of the anti-inflammatory cytokine interleukin (IL)-13 and reduction of several proinflammatory cytokines within the spleen, notably interleukin (IL)-17. Finally, we show that treatment of sepsis with OCH in mice is accompanied by significantly reduced apoptosis of splenic T and B lymphocytes and macrophages, but not natural killer cells. We propose that modulation of iNK T cell responses towards a Th2 phenotype may be an effective therapeutic strategy in early sepsis.     

Increased killer immunoglobulin-like receptor expression and functional defects in natural killer cells in lung cancer

Frequencies of natural killer (NK) cells from patients with non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC) did not differ from healthy controls. A higher proportion of NK cells from NSCLC patients expressed the killer immunoglobulin-like receptor (KIR) CD158b than in controls (P = 0.0004), in the presence or absence of its ligand, HLA-C1. A similar result was obtained for CD158e in the presence of its ligand HLA-Bw4 in NSCLC patients (P = 0.003); this was entirely attributable to the Bw4I group of alleles in the presence of which a fivefold higher percentage of CD158e(+) NK cells was found in NSCLC patients than controls. Proportions of CD158b(+) NK cells declined with advancing disease in NSCLC patients. Expression of NKp46, CD25 and perforin A, and production of interferon-γ following stimulation with interleukin-12 and interleukin-18, were all significantly lower in NK cells from NSCLC patients than in controls. Both NK cell cytotoxicity and granzyme B expression were also reduced in lung cancer patients. Increased inhibitory KIR expression would decrease NK cell cytotoxic function against tumour cells retaining class I HLA expression. Furthermore, the reduced ability to produce interferon-γ would restrict the ability of NK cells to stimulate T-cell responses in patients with lung cancer.     

Presentation of alpha-galactosylceramide by murine CD1d to natural killer T cells is facilitated by plasma membrane glycolipid rafts

CD1 molecules are non-polymorphic major histocompatibility complex class I-related proteins that bind and present glycolipid antigens to T-cell antigen receptors (TCR) expressed by alphabeta T cells or natural killer-like T cells (NKT). Anti-metastatic properties of NKT cells reactive to the CD1d-binding antigen alpha-galactosylceramide (alpha-GalCer) are now being explored as a contributor to tumour cell killing. In this study, we tested the hypothesis that presentation of alpha-GalCer by murine CD1d (mCD1d) to mCD1d-restricted NKT cells was facilitated by plasma membrane glycolipid rafts. Confocal microscopy of mCD1d-transfected A20 B cells (A20mCD1d) demonstrated that mCD1d was raft-localized. This observation was confirmed by immunoblotting of raft fractions isolated on sucrose density gradients. Raft disruption by the cholesterol-binding agent nystatin, or short-chain ceramides, inhibited presentation of low concentrations of alpha-GalCer to NKT cells. Inhibition of antigen presentation was reversed by treatment of A20mCD1d cells with higher alpha-GalCer concentrations, or removal of raft-disrupting agents. These data indicate that partitioning of mCD1d into membrane rafts increases the capacity of antigen-presenting cells to present limiting quantities of glycolipid antigens, perhaps by stabilizing mCD1d/antigen structures on the plasma membrane and optimizing TCR engagement on NKT cells.