Different subsets of natural killer T cells may vary in their roles in health and disease

Natural killer T cells (NKT) can regulate innate and adaptive immune responses. Type I and type II NKT cell subsets recognize different lipid antigens presented by CD1d, an MHC class‐I‐like molecule. Most type I NKT cells express a semi‐invariant T‐cell receptor (TCR), but a major subset of type II NKT cells reactive to a self antigen sulphatide use an oligoclonal TCR. Whereas TCR‐α dominates CD1d‐lipid recognition by type I NKT cells, TCR‐α and TCR‐β contribute equally to CD1d‐lipid recognition by type II NKT cells. These variable modes of NKT cell recognition of lipid–CD1d complexes activate a host of cytokine‐dependent responses that can either exacerbate or protect from disease. Recent studies of chronic inflammatory and autoimmune diseases have led to a hypothesis that: (i) although type I NKT cells can promote pathogenic and regulatory responses, they are more frequently pathogenic, and (ii) type II NKT cells are predominantly inhibitory and protective from such responses and diseases. This review focuses on a further test of this hypothesis by the use of recently developed techniques, intravital imaging and mass cytometry, to analyse the molecular and cellular dynamics of type I and type II NKT cell antigen‐presenting cell motility, interaction, activation and immunoregulation that promote immune responses leading to health versus disease outcomes.     

Inhibition of CD1d‐mediated antigen presentation by the transforming growth factor‐β/Smad signalling pathway

CD1d‐mediated lipid antigen presentation activates a subset of innate immune lymphocytes called invariant natural killer T (NKT) cells that, by virtue of their potent cytokine production, bridge the innate and adaptive immune systems. Transforming growth factor (TGF‐β) is a known immune modulator that can activate the mitogen‐activated protein kinase p38; we have previously shown that p38 is a negative regulator of CD1d‐mediated antigen presentation. Several studies implicate a role for TGF‐β in the activation of p38. Therefore, we hypothesized that TGF‐β would impair antigen presentation by CD1d. Indeed, a dose‐dependent decrease in CD1d‐mediated antigen presentation and impairment of lipid antigen processing was observed in response to TGF‐β treatment. However, it was found that this inhibition was not through p38 activation. Instead, Smads 2, 3 and 4, downstream elements of the TGF‐β canonical signalling pathway, contributed to the observed effects. In marked contrast to that observed with CD1d, TGF‐β was found to enhance MHC class II‐mediated antigen presentation. Overall, these results suggest that the canonical TGF‐β/Smad pathway negatively regulates an important arm of the host's innate immune responses – CD1d‐mediated lipid antigen presentation to NKT cells.     

Severe functional impairment and elevated PD‐1 expression in CD1d‐restricted NKT cells retained during chronic HIV‐1 infection

Invariant CD1d‐restricted NKT cells play important roles in regulating both innate and adaptive immunity. They are targeted by HIV‐1 infection and severely reduced in number or even lost in many infected subjects. Here, we have investigated the characteristics of NKT cells retained by some patients despite chronic HIV‐1 infection. NKT cells preserved under these circumstances displayed an impaired ability to proliferate and produce IFN‐γ in response to CD1d‐restricted lipid antigen as compared with cells from uninfected control subjects. HIV‐1 infection was associated with an elevated expression of the inhibitory programmed death‐1 (PD‐1) receptor (CD279) on the CD4^? subset of NKT cells. However, blocking experiments indicated that the functional defects in NKT cells were largely PD‐1‐independent. Furthermore, the elevated PD‐1 expression and the functional defects were not restored by anti‐retroviral treatment, and the NKT cell numbers in blood did not recover significantly in response to treatment. The functional phenotype of NKT cells in these patients suggests an irreversible immune exhaustion due to chronic activation in vivo. The data demonstrate a severe functional impairment in the remaining NKT‐cell compartment in HIV‐1‐infected patients, which limits the prospects to mobilize these cells in immunotherapy approaches in patients.     

Targeting SIRP‐α protects from type 2‐driven allergic airway inflammation

The interplay between innate and adaptive immune responses is essential for the establishment of allergic diseases. CD47 and its receptor, signal regulatory protein α (SIRP‐α), govern innate cell trafficking. We previously reported that administration of CD47^+/+ but not CD47^−/− SIRP‐α⁺ BM‐derived DC (BMDC) induced airway inflammation and Th2 responses in otherwise resistant CD47‐deficient mice. We show here that early administration of a CD47‐Fc fusion molecule suppressed the accumulation of SIRP‐α⁺ DC in mediastinal LN, the development of systemic and local Th2 responses as well as airway inflammation in sensitized and challenged BALB/c mice. Mechanistic studies highlighted that SIRP‐α ligation by CD47‐Fc on BMDC did not impair Ag uptake, Ag presentation and Ag‐specific DO11.10 Tg Th2 priming and effector function in vitro, whereas in vivo administration of CD47‐Fc or CD47‐Fc‐pretreated BMDC inhibited Tg T‐cell proliferation, pinpointing that altered DC trafficking accounts for defective Th priming. We conclude that the CD47/SIRP‐α axis may be harnessed in vivo to suppress airway SIRP‐α⁺ DC homing to mediastinal LN, Th2 responses and allergic airway inflammation.     

Understanding the Regulatory Roles of Natural Killer T Cells in Rheumatoid Arthritis: T Helper Cell Differentiation Dependent or Independent?

Rheumatoid arthritis (RA) is the most common chronic systemic autoimmune disease. This disease is thought to be caused by pathogenic T cells. Th1, Th2, Th17 and Treg cells have been implicated in the pathogenesis of RA. These Th cells differentiate from CD4+ T cells primarily due to the effects of cytokines. Natural killer T (NKT) cells are a distinct subset of lymphocytes that can rapidly secrete massive amount of cytokines, including IL‐2, IL‐4, IL‐12 and IFN‐γ. Numerous studies showed that NKT cells can influence the differentiation of CD4+ T cells via cytokines in vitro. These findings suggest that NKT cells play an important role in RA by polarizing Th1, Th2, Th17 and Treg cells. In view of the complexity of RA, we discussed whether NKT cells really influence the development of RA through regulating the differentiation of Th cells.     

Non‐glycosidic compounds can stimulate both human and mouse iNKT cells

Invariant natural killer T (iNKT) cells recognize CD1d/glycolipid complexes and upon activation with synthetic agonists display immunostimulatory properties. We have previously described that the non‐glycosidic CD1d‐binding lipid, threitolceramide (ThrCer) activates murine and human iNKT cells. Here, we show that incorporating the headgroup of ThrCer into a conformationally more restricted 6‐ or 7‐membered ring results in significantly more potent non‐glycosidic analogs. In particular, ThrCer 6 was found to promote strong anti‐tumor responses and to induce a more prolonged stimulation of iNKT cells than does the canonical α‐galactosylceramide (α‐GalCer), achieving an enhanced T‐cell response at lower concentrations compared with α‐GalCer both in vitro, using human iNKT‐cell lines and in vivo, using C57BL/6 mice. Collectively, these studies describe novel non‐glycosidic ThrCer‐based analogs that have improved potency in iNKT‐cell activation compared with that of α‐GalCer, and are clinically relevant iNKT‐cell agonists.     

Multiple tissue‐specific isoforms of sulfatide activate CD1d‐restricted type II NKT cells

The glycosphingolipid sulfatide (SO₃‐3Galβ1Cer) is a demonstrated ligand for a subset of CD1d‐restricted NKT cells, which could regulate experimental autoimmune encephalomyelitis, a murine model for multiple sclerosis, as well as tumor immunity and experimental hepatitis. Native sulfatide is a mixture of sulfatide isoforms, i.e. sulfatide molecules with different long‐chain bases and fatty acid chain lengths and saturation. Here, we demonstrate that sulfatide‐specific CD1d‐restricted murine NKT hybridomas recognized several different sulfatide isoforms. These included the physiologically relevant isoforms C24:1 and C24:0, major constituents of the myelin sheet of the nervous system, and C16:0, prominent in the pancreatic islet β‐cells. The most potent sulfatide isoform was lysosulfatide (lacking a fatty acid). Shortened fatty acid chain length (C24:1 versus C18:1), or saturation of the long fatty acid (C24:0), resulted in reduced stimulatory capacity, and fatty acid hydroxylation abolished the response. Moreover, sulfatide was not responsible for the natural autoreactivity toward splenocytes by XV19 T hybridoma cells. Our results reveal a promiscuity in the recognition of sulfatide isoforms by a CD1d‐restricted NKT‐cell clone, and suggest that sulfatide, a major component of the myelin sheet and pancreatic β‐cells, is one of several natural ligands for type II CD1d‐restricted NKT cells.     

CD1d-mediated antigen presentation to natural killer T (NKT) cells

CD1d molecules are lipid antigen-presenting molecules. They are involved in presenting these antigens to a unique subpopulation of T cells called natural killer T (NKT) cells, which have the capacity to produce both T helper (Th) 1 and Th2 cytokines. Thus, it is possible that the antigens presented by CD1d and/or the level at which they are presented could have profound effects on the immunoregulation of autoimmune and infectious diseases, as well as cancer. Because of the ability of CD1d-binding ligands to modulate NKT cell responses, targeting CD1d-mediated antigen presentation as a novel approach for new therapies in these and other diseases holds great promise.     

Allele‐sensitive mutant,Itkas, reveals that Itk kinase activity is required for Th1, Th2, Th17, and iNKT‐cell cytokine production

Itk^?/? mice exhibit defects in the activation, development, and function of CD4⁺ and CD8⁺ T cells and iNKT cells. These and other defects in these mice make it difficult to uncouple the developmental versus functional requirement of Itk signaling. Here, we report an allele‐sensitive mutant of Itk (Itkas) whose catalytic activity can be selectively inhibited by analogs of the PP1 kinase inhibitor. We show that Itkas behaves like WT Itk in the absence of the inhibitor and can rescue the development of Itk^?/? T cells in mice. Using mice carrying Itkas, we show using its inhibitor that Itk activity is required not only for Th2, Th17, and iNKT‐cell cytokine production, but also surprisingly, for Th1 cytokine production. This work has important implications for understanding the role of Itk signaling in the development versus function of iNKT cells, Th1, Th2, and Th17 cells.     

Acidification-dependent activation of CD1d-restricted natural killer T cells is intact in cystic fibrosis

CD1d‐restricted natural killer T (NKT) cells are emerging as critical regulators of the immune response to infectious agents, including Pseudomonas aeruginosa; and therapies to augment NKT‐cell activation may represent a novel approach to treat chronic, antibiotic‐resistant bacterial infections. We examined the capacity of dendritic cells (DCs) from people with cystic fibrosis (CF) to activate NKT cells. Our study was motivated by three lines of evidence: (i) NKT cells play a critical role in clearing P. aeruginosa infection; (ii) activation of NKT cells requires acidification‐dependent processing of glycolipid antigens within the endolysosomal compartment; and (iii) endolysosomal acidification may be reduced in CF. We demonstrated that NKT‐cell activation was dependent upon intact organelle acidification as inhibitors of the vacuolar (H⁺)‐ATPases prevented DCs from activating NKT cells with two glycolipid antigens, α‐galactosylceramide and galactose‐galactosylceramide. In contrast, cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel dysfunction had no significant biological impact on the capacity of DCs to activate NKT cells. Dendritic cells from subjects with CF and DCs treated with the thiazolidinone CFTR_inh‐172 inhibitor showed no reduction in their ability to activate NKT cells. Based on these data, we find no evidence for an inherent defect in glycolipid antigen presentation to NKT cells in CF subjects.     

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.     

Regulation of immune responses by CD1d-restricted natural killer T cells

Natural killer T (NKT) cells are a unique subset of T lymphocytes that share receptor structures and properties with conventional T lymphocytes and natural killer (NK) cells. NKT cells are specific for glycolipid antigens such as the marine sponge-derived agent α-galactosylceramide (α-GalCer) presented by the major histocompatibility complex (MHC) class I-like molcule CD1d. My laboratory has evaluated the function of NKT cells by generating and analyzing CD1d-deficient mice. These studies showed that CD1d expression is required for NKT cell development, but not absolutely necessary for the generation of polarized T helper (Th) cell responses. Further, we have studied the in vivo response of NKT cells toα-GalCer stimulation and the capacity of α-GalCer to modulate innate and adaptive immune responses. Our results revealed that, quickly following administration of α-GalCer, NKT cells expand and produce cytokines, trans-activate a variety of innate and adaptive immune cells, and promote Th2 responses that are capable of suppressing Th1-dominant autoimmunity. Our findings indicate that NKT cells play a regulatory role in the immune response and that specific activation of these cells may be exploited for therapeutic purposes.     

The adaptor protein SAP regulates type II NKT‐cell development,cytokine production,and cytotoxicity against lymphoma

CD1d‐restricted NKT cells represent a unique lineage of immunoregulatory T cells that are divided into two groups, type I and type II, based on their TCR usage. Because there are no specific tools to identify type II NKT cells, little is known about their developmental requirements and functional regulation. In our previous study, we showed that signaling lymphocytic activation molecule associated protein (SAP) is essential for the development of type II NKT cells. Here, using a type II NKT‐cell TCR transgenic mouse model, we demonstrated that CD1d‐expressing hematopoietic cells, but not thymic epithelial cells, meditate efficient selection of type II NKT cells. Furthermore, we showed that SAP regulates type II NKT‐cell development by controlling early growth response 2 protein and promyelocytic leukemia zinc finger expression. SAP‐deficient 24αβ transgenic T cells (24αβ T cells) exhibited an immature phenotype with reduced Th2 cytokine‐producing capacity and diminished cytotoxicity to CD1d‐expressing lymphoma cells. The impaired IL‐4 production by SAP‐deficient 24αβ T cells was associated with reduced IFN regulatory factor 4 and GATA‐3 induction following TCR stimulation. Collectively, these data suggest that SAP is critical for regulating type II NKT cell responses. Aberrant responses of these T cells may contribute to the immune dysregulation observed in X‐linked lymphoproliferative disease caused by mutations in SAP.     

Constitutive macropinocytosis allows TAP-dependent major histocompatibility compex class I presentation of exogenous soluble antigen by bone marrow-derived dendritic cells

Dendritic cells expanded from mouse bone marrow (BMDC) with granulocyte/macrophage-colony-stimulating factor have potent T cell-stimulatory properties both in vitro and in vivo. This has been well documented for major histocompatibility complex (MHC) class II-restricted responses, and more recently using peptide-loaded and protein-pulsed DC for CD8 responses following adoptive transfer in mice. An unresolved question concerns the capacity of BMDC to present exogenous antigen on MHC class I molecules, an unconventional mode of MHC class I loading for which there is now considerable evidence, particularly in macrophages. Here, we show that BMDC exhibit high levels of macropinocytosis driven by constitutive membrane ruffling activity. Up to one-third of actively ruffling and macropinocytosing BMDC transferred pinocytosed horseradish peroxidase into the cytosol following a 15-min pulse, suggesting that they might be capable of presenting exogenous soluble antigen on MHC class I molecules. We show that BMDC presented exogenous ovalbumin to a T cell hybridoma more effectively, more rapidly, and at lower exogenous antigen concentrations than BM macrophages on a cell-for-cell basis. Presentation was TAP dependent, brefeldin A sensitive, and blocked by inhibitors of proteasomal processing, demonstrating use of the classical MHC class I pathway. Although effective presentation of exogenous antigen by BMDC occurred in the absence of agents which stimulate macropinocytosis, treatment with phorbol myristate acetate (PMA) enhanced both pinocytosis and MHC class I presentation by BMDC. Finally, PMA-stimulated BMDC exposed to exogenous ovalbumin in vitro were able to prime an antigen-specific cytotoxic T lymphocyte response following adoptive transfer in vivo.     

Inhibition of R5‐tropic HIV type‐1 replication in CD4+ natural killer T cells by γδ T lymphocytes

After the development of highly active anti‐retroviral therapy, it became clear that the majority of emergent HIV‐1 is macrophage‐tropic and infects CD4⁺, CCR5‐expressing cells (R5‐tropic). There are three distinct cell populations, R5‐tropic, HIV‐1‐susceptible CD4⁺ cells: (i) natural killer T (NKT) cells, (ii) dendritic cells and macrophages, and (iii) tissue‐associated T cells residing primarily at mucosal surfaces. We have confirmed that CD4⁺ NKT cells derived from peripheral blood mononuclear cells (PBMCs) predominantly express CCR5 rather than CXCR4, whereas the reverse is true for CD4⁺ T cells derived from circulating PBMCs, and that R5‐tropic HIV‐1 expands efficiently in the CD4⁺ NKT cells. Moreover, when PBMCs depleted of CD8α⁺ cells were stimulated in the presence of α‐galactosylceramide (α‐GalCer) and R5‐tropic HIV‐1 [NL(AD8)], the production of HIV‐1 virions was not suppressed, whereas, similar to the untreated PBMCs, depletion of CD8β⁺ cells from PBMCs significantly inhibited virion production. These findings suggest that CD8αα⁺ but not CD8αβ⁺ cells may have the ability to inhibit R5‐tropic HIV‐1 replication in CD4⁺ NKT cells. Here, we show that co‐culturing R5‐tropic HIV‐1‐infected CD4⁺ NKT cells with CD8αα⁺ γδ T cells, in particular Vγ1Vδ1 cells, but not with CD8αα⁺ NKT cells or CD8αα⁺ dendritic cells, inhibits HIV‐1 replication mainly by secreting chemokines, such as macrophage inflammatory proteins 1α and 1β and RANTES. Collectively, these results indicate the importance of CD8αα⁺ γδ T cells in the control of R5‐tropic HIV‐1 replication and persistence in CD4⁺ NKT cells.     

The interaction between invariant Natural Killer T cells and the mucosal microbiota

The surface of mammalian bodies is colonized by a multitude of microbial organisms, which under normal conditions support the host and are considered beneficial commensals. This requires, however, that the composition of the commensal microbiota is tightly controlled and regulated. The host immune system plays an important role in the maintenance of this microbiota composition. Here we focus on the contribution of one particular immune cell type, invariant Natural Killer T (iNKT) cells, in this process. The iNKT cells are a unique subset of T cells characterized by two main features. First, they express an invariant T‐cell receptor that recognizes glycolipid antigens presented by CD1d, a non‐polymorphic major histocompatibility complex class I‐like molecule. Second, iNKT cells develop as effector/memory cells and swiftly exert effector functions, like cytokine production and cytotoxicity, after activation. We outline the influence that the mucosal microbiota can have on iNKT cells, and how iNKT cells contribute to the maintenance of the microbiota composition.