CD1d-independent activation of mouse and human iNKT cells by bacterial superantigens

Invariant NKT (iNKT) cells are infrequent but important immunomodulatory lymphocytes that exhibit CD1d-restricted reactivity with glycolipid Ags. iNKT cells express a unique T-cell receptor (TCR) composed of an invariant α-chain, paired with a limited range of β-chains. Superantigens (SAgs) are microbial toxins defined by their ability to activate conventional T cells in a TCR β-chain variable domain (Vβ)-specific manner. However, whether iNKT cells are directly activated by bacterial SAgs remains an open question. Herein, we explored the responsiveness of mouse and human iNKT cells to a panel of staphylococcal and streptococcal SAgs and examined the contribution of major histocompatibility complex (MHC) class II and CD1d to these responses. Bacterial SAgs that target mouse Vβ8, such as staphylococcal enterotoxin B (SEB), were able to activate mouse hybridoma and primary hepatic iNKT cells in the presence of mouse APCs expressing human leukocyte antigen (HLA)-DR4. iNKT cell-mediated cytokine secretion in SEB-challenged HLA-DR4-transgenic mice was CD1d-independent and accompanied by a high interferon-γ:interleukin-4 ratio consistent with an in vivo Th1 bias. Furthermore, iNKT cells from SEB-injected HLA-DR4-transgenic mice, and iNKT cells from SEB-treated human PBMCs, showed early activation by intracellular cytokine staining and CD69 expression. Unlike iNKT cell stimulation by α-galactosylceramide, stimulation by SEB did not induce TCR downregulation of either mouse or human iNKT cells. We conclude that Vβ8-targeting bacterial SAgs can activate iNKT cells by utilizing a novel pathway that requires MHC class II interactions, but not CD1d. Therefore, iNKT cells fulfill important effector functions in response to bacterial SAgs and may provide attractive targets in the management of SAg-induced illnesses.     

Another view of T cell antigen recognition: cooperative engagement of glycolipid antigens by Va14Ja18 natural T(iNKT) cell receptor [corrected

Va14Ja18 natural T (iNKT) cells rapidly elicit a robust effector response to different glycolipid Ags, with distinct functional outcomes. Biochemical parameters controlling iNKT cell function are partly defined. However, the impact of iNKT cell receptor beta-chain repertoire and how alpha-galactosylceramide (alpha-GalCer) analogues induce distinct functional responses have remained elusive. Using altered glycolipid ligands, we discovered that the Vb repertoire of iNKT cells impacts recognition and Ag avidity, and that stimulation with suboptimal avidity Ag results in preferential expansion of high-affinity iNKT cells. iNKT cell proliferation and cytokine secretion, which correlate with iNKT cell receptor down-regulation, are induced within narrow biochemical thresholds. Multimers of CD1d1-alphaGalCer- and alphaGalCer analogue-loaded complexes demonstrate cooperative engagement of the Va14Ja18 iNKT cell receptor whose structure and/or organization appear distinct from conventional alphabeta TCR. Our findings demonstrate that iNKT cell functions are controlled by affinity thresholds for glycolipid Ags and reveal a novel property of their Ag receptor apparatus that may have an important role in iNKT cell activation.     

The autophagy machinery restrains iNKT cell activation through CD1D1 internalization

Invariant natural killer T (iNKT) cells are innate T cells with powerful immune regulatory functions that recognize glycolipid antigens presented by the CD1D protein. While iNKT cell-activating glycolipids are currently being explored for their efficacy to improve immunotherapy against infectious diseases and cancer, little is known about the mechanisms that control CD1D antigen presentation and iNKT cell activation in vivo. CD1D molecules survey endocytic pathways to bind lipid antigens in MHC class II-containing compartments (MIICs) before recycling to the plasma membrane. Autophagosomes intersect with MIICs and autophagy-related proteins are known to support antigen loading for increased CD4⁺ T cell immunity. Here, we report that mice with dendritic cell (DC)-specific deletion of the essential autophagy gene Atg5 showed better CD1D1-restricted glycolipid presentation in vivo. These effects led to enhanced iNKT cell cytokine production upon antigen recognition and lower bacterial loads during Sphingomonas paucimobilis infection. Enhanced iNKT cell activation was independent of receptor-mediated glycolipid uptake or costimulatory signals. Instead, loss of Atg5 in DCs impaired clathrin-dependent internalization of CD1D1 molecules via the adaptor protein complex 2 (AP2) and, thus, increased surface expression of stimulatory CD1D1-glycolipid complexes. These findings indicate that the autophagic machinery assists in the recruitment of AP2 to CD1D1 molecules resulting in attenuated iNKT cell activation, in contrast to the supporting role of macroautophagy in CD4⁺ T cell stimulation.     

Innate-Like Control of Human iNKT Cell Autoreactivity via the Hypervariable CDR3β Loop

Invariant Natural Killer T cells (iNKT) are a versatile lymphocyte subset with important roles in both host defense and immunological tolerance. They express a highly conserved TCR which mediates recognition of the non-polymorphic, lipid-binding molecule CD1d. The structure of human iNKT TCRs is unique in that only one of the six complementarity determining region (CDR) loops, CDR3β, is hypervariable. The role of this loop for iNKT biology has been controversial, and it is unresolved whether it contributes to iNKT TCR:CD1d binding or antigen selectivity. On the one hand, the CDR3β loop is dispensable for iNKT TCR binding to CD1d molecules presenting the xenobiotic alpha-galactosylceramide ligand KRN7000, which elicits a strong functional response from mouse and human iNKT cells. However, a role for CDR3β in the recognition of CD1d molecules presenting less potent ligands, such as self-lipids, is suggested by the clonal distribution of iNKT autoreactivity. We demonstrate that the human iNKT repertoire comprises subsets of greatly differing TCR affinity to CD1d, and that these differences relate to their autoreactive functions. These functionally different iNKT subsets segregate in their ability to bind CD1d-tetramers loaded with the partial agonist α-linked glycolipid antigen OCH and structurally different endogenous β-glycosylceramides. Using surface plasmon resonance with recombinant iNKT TCRs and different ligand-CD1d complexes, we demonstrate that the CDR3β sequence strongly impacts on the iNKT TCR affinity to CD1d, independent of the loaded CD1d ligand. Collectively our data reveal a crucial role for CDR3β for the function of human iNKT cells by tuning the overall affinity of the iNKT TCR to CD1d. This mechanism is relatively independent of the bound CD1d ligand and thus forms the basis of an inherent, CDR3β dependent functional hierarchy of human iNKT cells.     

Adipocytes harbor a glucosylceramide biosynthesis pathway involved in iNKT cell activation

BackgroundNatural killer T (NKT) cells in adipose tissue (AT) contribute to whole body energy homeostasis.ResultsInhibition of the glucosylceramide synthesis in adipocytes impairs iNKT cell activity.ConclusionGlucosylceramide biosynthesis pathway is important for endogenous lipid antigen activation of iNKT cells in adipocytes.SignificanceUnraveling adipocyte-iNKT cell communication may help to fight obesity-induced AT dysfunction.Overproduction and/or accumulation of ceramide and ceramide metabolites, including glucosylceramides, can lead to insulin resistance. However, glucosylceramides also fulfill important physiological functions. They are presented by antigen presenting cells (APC) as endogenous lipid antigens via CD1d to activate a unique lymphocyte subspecies, the CD1d-restricted invariant (i) natural killer T (NKT) cells. Recently, adipocytes have emerged as lipid APC that can activate adipose tissue-resident iNKT cells and thereby contribute to whole body energy homeostasis. Here we investigate the role of the glucosylceramide biosynthesis pathway in the activation of iNKT cells by adipocytes.UDP-glucose ceramide glucosyltransferase (Ugcg), the first rate limiting step in the glucosylceramide biosynthesis pathway, was inhibited via chemical compounds and shRNA knockdown in vivo and in vitro. β-1,4-Galactosyltransferase (B4Galt) 5 and 6, enzymes that convert glucosylceramides into potentially inactive lactosylceramides, were subjected to shRNA knock down. Subsequently, (pre)adipocyte cell lines were tested in co-culture experiments with iNKT cells (IFNγ and IL4 secretion).Inhibition of Ugcg activity shows that it regulates presentation of a considerable fraction of lipid self-antigens in adipocytes. Furthermore, reduced expression levels of either B4Galt5 or -6, indicate that B4Galt5 is dominant in the production of cellular lactosylceramides, but that inhibition of either enzyme results in increased iNKT cell activation. Additionally, in vivo inhibition of Ugcg by the aminosugar AMP-DNM results in decreased iNKT cell effector function in adipose tissue.Inhibition of endogenous glucosylceramide production results in decreased iNKT cells activity and cytokine production, underscoring the role of this biosynthetic pathway in lipid self-antigen presentation by adipocytes.     

The ins and outs of CD1 molecules: bringing lipids under immunological surveillance

An emerging area of investigation is the role of lipids as immunological antigens. CD1 glycoproteins comprise a family of molecules that are specialized for presenting lipids, glycolipids and lipopeptides to T lymphocytes. Variations in the cytoplasmic tail sequences of CD1 isoforms lead to differential association with adaptor proteins and consequently divergent routes of intracellular trafficking, resulting in surveillance of distinct cellular sites for binding lipid antigens. CD1 molecules efficiently gain access to lipids from intracellular microbial pathogens in endosomal compartments, and the trafficking and lipid-binding specialization of CD1 isoforms may correlate with the endosomal segregation of structurally distinct lipids. Endosomal trafficking is also critical for CD1d molecules to load antigenic self-lipids that are presented to autoreactive CD1d-restricted natural killer (NK)T cells and is required for the positive selection of these unique T cells. Recent studies reveal a key role for accessory proteins that facilitate the uptake of lipid antigens by CD1 molecules. These include lysosomal lipid-transfer proteins, such as the saposins, and apolipoprotein E, the major serum factor that binds and delivers extracellular lipids to antigen-presenting cells. These advances in understanding the CD1 lipid antigen presentation system raise new considerations about the role of the immune response in lipid-related diseases.     

iNKT cells in microbial immunity: recognition of microbial glycolipids

Natural killer T cells expressing an invariant T cell antigen receptor (iNKT cells) are cells of the innate immune system. After recognizing glycolipid antigens presented by CD1d molecules on antigen presenting cells (APCs), iNKT cells rapidly produce large quantities of cytokines, thereby stimulating many types of cells. Recent studies have described several mechanisms of iNKT cell activation and the contribution of these cells to antimicrobial responses. iNKT cells can be activated by endogenous antigens and/or inflammatory cytokines from APCs. However, iNKT cells also recognize certain microbial glycolipids by their invariant T cell antigen receptor (TCR), and they contribute to pathogen clearance in certain microbial infections. These findings indicate that the iNKT TCR is useful for detecting certain microbial pathogens. Moreover, recent studies suggest that iNKT cell glycolipid antigens may be useful in antimicrobial therapy and vaccines.     

Synthesis and Th1-immunostimulatory activity of α-galactosylceramide analogues bearing a halogen-containing or selenium-containing acyl chain

A novel series of CD1d ligand α-galactosylceramides (α-GalCers) were synthesized by incorporation of the heavy atoms Br and Se in the acyl chain backbone of α-galactosyl-N-cerotoylphytosphingosine. The synthetic analogues are potent CD1d ligands and stimulate mouse invariant natural killer T (iNKT) cells to selectively enhance Th1 cytokine production. These synthetic analogues would be efficient X-ray crystallographic probes to disclose precise atomic positions of alkyl carbons and lipid–protein interactions in KRN7000/CD1d complexes.     

CD1d presentation of glycolipids

The CD1 family of antigen-presenting molecules consists of five members, CD1a to e. Of these molecules CD1d has been the subject of much interest over the past 10 years following the discovery that this molecule presents antigens to a group of T cells known as invariant natural killer T cells (iNKT). iNKT cells carry an invariant T cell receptor which contains homologous gene segments in mouse and man. iNKT cells are positively selected in the thymus in the same manner as major histocompatibility complex restricted T cells, except iNKT cells require CD1d to be presented by thymocytes rather than epithelial cells. Once in peripheral organs, iNKT cells appear to play multiple roles in host defence against pathogens and cancer. If the numbers of iNKT cells are not correctly regulated it can result in autoimmune disorders, such as diabetes. The ligands for iNKT cells have been the subject of much research but identifying physiologically relevant candidate ligands for positive selection or activation has proved technically very challenging. This is largely due to the fact that the ligands for iNKT cells are lipids. The lipid ligands for thymic selection and some of those involved in peripheral activation are self-derived. Glycosphingolipids are suggested to be the class of lipid for iNKT cell thymic development. For peripheral activation it appears multiple classes of self-derived lipids may play a role, in addition to pathogen-derived lipids. This review will cover essential background to iNKT cell and CD1d biology with emphasis on the candidate iNKT cell ligands proposed to date.     

Amide analogues of CD1d agonists modulate iNKT-cell-mediated cytokine production

Invariant natural killer T (iNKT) cells are restricted by the non-polymorphic MHC class I-like protein, CD1d, and activated following presentation of lipid antigens bound to CD1d molecules. The prototypical iNKT cell agonist is α-galactosyl ceramide (α-GalCer). CD1d-mediated activation of iNKT cells by this molecule results in the rapid secretion of a range of pro-inflammatory (Th1) and regulatory (Th2) cytokines. Polarization of the cytokine response can be achieved by modifying the structure of the glycolipid, which opens up the possibility of using CD1d agonists as therapeutic agents for a range of diseases. Analysis of crystal structures of the T-cell receptor-α-GalCer-CD1d complex led us to postulate that amide isosteres of known CD1d agonists should modulate the cytokine response profile upon iNKT-cell activation. To this end, we describe the synthesis and biological activity of amide analogues of α-GalCer and its non-glycosidic analogue threitol ceramide (ThrCer). All of the analogues were found to stimulate murine and human iNKT cells by CD1d-mediated presentation to varying degrees; however, the thioamide and carbamate analogues of ThrCer were of particular interest in that they elicited a strongly polarized cytokine response (more interferon-gamma (IFN-γ), no interleukin-4 (IL-4)) in mice. While the ThrCer-carbamate analogue was shown to transactivate natural killer (NK) cells, a mechanism that has been used to account for the preferential production of IFN-γ by other CD1d agonists, this pathway does not account for the polarized cytokine response observed for the thioamide analogue.     

Efficient activation of Valpha14 invariant NKT cells by foreign lipid antigen is associated with concurrent dendritic cell-specific self recognition

A burst release of cytokines by Valpha14 invariant NKT (iNKT) cells upon their TCR engagement critically regulates innate and adaptive immune responses. However, it remains unclear in vivo why iNKT cells respond efficiently to microbial or intracellular lipid Ags that are at low levels or that possess suboptimal antigenicity. We found that dendritic cells (DCs) potentiated iNKT cells to respond to a minimal amount of ligand alpha-galactosylceramide (alphaGalCer) through CD1d-dependent autoreactive responses that require endosomal processing and CD1d trafficking. The ability of potentiation of NKT cells was DC specific and did not depend on costimulatory signals and IL-12 production by DCs. However, DCs that failed to synthesize a major endogenous lipid Ag isoglobotrihexosylceramide were unable to potentiate NKT cells for efficient activation. Further analysis showed that differences in the level and pattern of endogenous lipid Ag presentation differentiate DCs and B cells for effective potentiation and subsequent activation of iNKT cells in the presence of an exogenous Ag. Thus, CD1d-dependent potentiation by DCs may be crucial for iNKT cell-mediated immunity against infectious agents.     

Expression of CD1d molecules by human schwann cells and potential interactions with immunoregulatory invariant NK T cells

CD1d-restricted NKT cells expressing invariant TCR alpha-chains (iNKT cells) produce both proinflammatory and anti-inflammatory cytokines rapidly upon activation, and are believed to play an important role in both host defense and immunoregulation. To address the potential implications of iNKT cell responses for infectious or inflammatory diseases of the nervous system, we investigated the expression of CD1d in human peripheral nerve. We found that CD1d was expressed on the surface of Schwann cells in situ and on primary or immortalized Schwann cell lines in culture. Schwann cells activated iNKT cells in a CD1d-dependent manner in the presence of alpha-galactosylceramide. Surprisingly, the cytokine production of iNKT cells stimulated by alpha-galactosylceramide presented by CD1d+ Schwann cells showed a predominance of Th2-associated cytokines such as IL-5 and IL-13 with a marked deficiency of proinflammatory Th1 cytokines such as IFN-gamma or TNF-alpha. Our findings suggest a mechanism by which iNKT cells may restrain inflammatory responses in peripheral nerves, and raise the possibility that the expression of CD1d by Schwann cells could be relevant in the pathogenesis of infectious and inflammatory diseases of the peripheral nervous system.     

IFN-gamma-producing human invariant NKT cells promote tumor-associated antigen-specific cytotoxic T cell responses

CD1d-restricted invariant NKT (iNKT) cells can enhance immunity to cancer or prevent autoimmunity, depending on the cytokine profile secreted. Antitumor effects of the iNKT cell ligand alpha-galactosylceramide (alphaGC) and iNKT cell adoptive transfer have been demonstrated in various tumor models. Together with reduced numbers of iNKT cells in cancer patients, which have been linked to poor clinical outcome, these data suggest that cancer patients may benefit from therapy aiming at iNKT cell proliferation and activation. Herein we present results of investigations on the effects of human iNKT cells on Ag-specific CTL responses. iNKT cells were expanded using alphaGC-pulsed allogeneic DC derived from the acute myeloid leukemia cell line MUTZ-3, transduced with CD1d to enhance iNKT cell stimulation, and with IL-12 to stimulate type 1 cytokine production. Enhanced activation and increased IFN-gamma production was observed in iNKT cells, irrespective of CD4 expression, upon stimulation with IL-12-overexpressing dendritic cells. IL-12-stimulated iNKT cells strongly enhanced the MART-1 (melanoma Ag recognized by T cell 1)-specific CD8(+) CTL response, which was dependent on iNKT cell-derived IFN-gamma. Furthermore, autologous IL-12-overexpressing dendritic cells, loaded with Ag as well as alphaGC, was superior in stimulating both iNKT cells and Ag-specific CTL. This study shows that IL-12-overexpressing allogeneic dendritic cells expand IFN-gamma-producing iNKT cells, which may be more effective against tumors in vivo. Furthermore, the efficacy of autologous Ag-loaded DC vaccines may well be enhanced by IL-12 overexpression and loading with alphaGC.     

Binding strength and dynamics of invariant natural killer cell T cell receptor/CD1d-glycosphingolipid interaction on living cells by single molecule force spectroscopy

Invariant natural killer T (iNKT) cells are a population of T lymphocytes that play an important role in regulating immunity to infection and tumors by recognizing endogenous and exogenous CD1d-bound lipid molecules. Using soluble iNKT T cell receptor (TCR) molecules, we applied single molecule force spectroscopy for the investigation of the iNKT TCR affinity for human CD1d molecules loaded with glycolipids differing in the length of the phytosphingosine chain using either recombinant CD1d molecules or lipid-pulsed THP1 cells. In both settings, the dissociation of the iNKT TCR from human CD1d molecules loaded with the lipid containing the longer phytosphingosine chain required higher unbinding forces compared with the shorter phytosphingosine lipid. Our findings are discussed in the context of previous results obtained by surface plasmon resonance measurements. We present new insights into the energy landscape and the kinetic rate constants of the iNKT TCR/human CD1d-glycosphingolipid interaction and emphasize the unique potential of single molecule force spectroscopy on living cells.     

Innate recognition of cell wall β-glucans drives invariant natural killer T cell responses against fungi

iNKT cells are innate T lymphocytes recognizing endogenous and foreign lipid antigens presented in the MHC-like molecule CD1d. The semi-invariant iNKT cell TCR can detect certain bacterial and parasitic lipids and drive iNKT cell responses. How iNKT cells respond to fungi, however, is unknown. We found that CD1d-deficient mice, which lack iNKT cells, poorly control infection with the fungal pathogen Aspergillus fumigatus. Furthermore, A. fumigatus rapidly activates iNKT cells in vivo and in vitro in the presence of APCs. Surprisingly, despite a requirement for CD1d recognition, the antifungal iNKT cell response does not require fungal lipids. Instead, Dectin-1- and MyD88-mediated responses to β-1,3 glucans, major fungal cell-wall polysaccharides, trigger IL-12 production by APCs that drives self-reactive iNKT cells to secrete IFN-γ. Innate recognition of β-1,3 glucans also drives iNKT cell responses against Candida, Histoplasma, and Alternaria, suggesting that this mechanism may broadly define the basis for antifungal iNKT cell responses.     

Distinct and overlapping effector functions of expanded human CD4+, CD8α+ and CD4-CD8α- invariant natural killer T cells

CD1d-restricted invariant natural killer T (iNKT) cells have diverse immune stimulatory/regulatory activities through their ability to release cytokines and to kill or transactivate other cells. Activation of iNKT cells can protect against multiple diseases in mice but clinical trials in humans have had limited impact. Clinical studies to date have targeted polyclonal mixtures of iNKT cells and we proposed that their subset compositions will influence therapeutic outcomes. We sorted and expanded iNKT cells from healthy donors and compared the phenotypes, cytotoxic activities and cytokine profiles of the CD4(+), CD8α(+) and CD4(-)CD8α(-) double-negative (DN) subsets. CD4(+) iNKT cells expanded more readily than CD8α(+) and DN iNKT cells upon mitogen stimulation. CD8α(+) and DN iNKT cells most frequently expressed CD56, CD161 and NKG2D and most potently killed CD1d(+) cell lines and primary leukemia cells. All iNKT subsets released Th1 (IFN-γ and TNF-α) and Th2 (IL-4, IL-5 and IL-13) cytokines. Relative amounts followed a CD8α>DN>CD4 pattern for Th1 and CD4>DN>CD8α for Th2. All iNKT subsets could simultaneously produce IFN-γ and IL-4, but single-positivity for IFN-γ or IL-4 was strikingly rare in CD4(+) and CD8α(+) fractions, respectively. Only CD4(+) iNKT cells produced IL-9 and IL-10; DN cells released IL-17; and none produced IL-22. All iNKT subsets upregulated CD40L upon glycolipid stimulation and induced IL-10 and IL-12 secretion by dendritic cells. Thus, subset composition of iNKT cells is a major determinant of function. Use of enriched CD8α(+), DN or CD4(+) iNKT cells may optimally harness the immunoregulatory properties of iNKT cells for treatment of disease.     

The complementarity determining region 2 of BV8S2 (V beta 8.2) contributes to antigen recognition by rat invariant NKT cell TCR

Invariant NKT cells (iNKT cells) are characterized by a semi-invariant TCR comprising an invariant alpha-chain paired with beta-chains with limited BV gene usage which are specific for complexes of CD1d and glycolipid Ags like alpha-galactosylceramide (alpha-GalCer). iNKT cells can be visualized with alpha-GalCer-loaded CD1d tetramers, and the binding of mouse CD1d tetramers to mouse as well as to human iNKT cells suggests a high degree of conservation in recognition of glycolipid Ags between species. Surprisingly, mouse CD1d tetramers failed to stain a discrete cell population among F344/Crl rat liver lymphocytes, although comprised iNKT cells are indicated by IL-4 and IFN-gamma secretion after alpha-GalCer stimulation. The arising hypothesis that rat iNKT TCR recognizes alpha-GalCer only if presented by syngeneic CD1d was then tested with the help of newly generated rat and mouse iNKT TCR-transduced cell lines. Cells expressing mouse iNKT TCR reacted to alpha-GalCer presented by rat or mouse CD1d and efficiently bound alpha-GalCer-loaded mouse CD1d tetramers. In contrast, cells expressing rat iNKT TCR responded only to alpha-GalCer presented by syngeneic CD1d and bound mouse CD1d tetramers only poorly or not at all. Finally, CD1d-dependent alpha-GalCer reactivity and binding of mouse CD1d tetramers was tested for cells expressing iNKT TCR comprising either rat or mouse AV14 (Valpha14) alpha-chains and wild-type or mutated BV8S2 (Vbeta8.2) beta-chains. The results confirmed the need of syngeneic CD1d as restriction element for rat iNKT TCR and identified the CDR2 of BV8S2 as an essential site for ligand recognition by iNKT TCR.     

Specific Deletion of LDL Receptor-Related Protein on Macrophages Has Skewed In Vivo Effects on Cytokine Production by Invariant Natural Killer T Cells

Expression of molecules involved in lipid homeostasis such as the low density lipoprotein receptor (LDLr) on antigen presenting cells (APCs) has been shown to enhance invariant natural killer T (iNKT) cell function. However, the contribution to iNKT cell activation by other lipoprotein receptors with shared structural and ligand binding properties to the LDLr has not been described. In this study, we investigated whether a structurally related receptor to the LDLr, known as LDL receptor-related protein (LRP), plays a role in iNKT cell activation. We found that, unlike the LDLr which is highly expressed on all immune cells, the LRP was preferentially expressed at high levels on F4/80⁺ macrophages (MΦ). We also show that CD169⁺ MΦs, known to present antigen to iNKT cells, exhibited increased expression of LRP compared to CD169^- MΦs. To test the contribution of MΦ LRP to iNKT cell activation we used a mouse model of MΦ LRP conditional knockout (LRP-cKO). LRP-cKO MΦs pulsed with glycolipid alpha-galactosylceramide (αGC) elicited normal IL-2 secretion by iNKT hybridoma and in vivo challenge of LRP-cKO mice led to normal IFN-γ, but blunted IL-4 response in both serum and intracellular expression by iNKT cells. Flow cytometric analyses show similar levels of MHC class-I like molecule CD1d on LRP-cKO MΦs and normal glycolipid uptake. Survey of the iNKT cell compartment in LRP-cKO mice revealed intact numbers and percentages and no homeostatic disruption as evidenced by the absence of programmed death-1 and Ly-49 surface receptors. Mixed bone marrow chimeras showed that the inability iNKT cells to make IL-4 is cell extrinsic and can be rescued in the presence of wild type APCs. Collectively, these data demonstrate that, although MΦ LRP may not be necessary for IFN-γ responses, it can contribute to iNKT cell activation by enhancing early IL-4 secretion.     

Structural features of the acyl chain determine self-phospholipid antigen recognition by a CD1d-restricted invariant NKT (iNKT) cell

Little is known about the antigen specificity of CD1d-restricted T cells, except that they frequently recognize CD1d-expressing antigen-presenting cells in the absence of exogenous antigen. We previously demonstrated that the 24.8.A iNKT cell hybridoma was broadly reactive with CD1d-transfected cell lines and recognized the polar lipid fraction of a tumor cell extract. In the present study, the antigen recognized by the 24.8.A iNKT cell hybridoma was purified to homogeneity and identified as palmitoyl-oleoyl-sn-glycero-3-phosphoethanolamine (16:0-18:1 PE). The 24.8.A iNKT cell hybridoma recognized synthetic 16:0-18:1[cis] PE, confirming that this phospholipid is antigenic. Recognition correlated with the degree of unsaturation of the acyl chains. Using a panel of synthetic PEs, the 24.8.A iNKT cell hybridoma was shown to be activated by PEs that contained at least one unsaturated acyl chain. The configuration of the double bonds was important, as the 24.8.A iNKT cell hybridoma recognized unsaturated acyl chains in the cis, but not the trans, configuration. PEs with multiple double bonds were recognized better than those with a single double bond, and increasing acyl chain unsaturation correlated with increased binding of PE to CD1d. These data illustrate the potential importance of the acyl chain structure for phospholipid antigen binding to CD1d.