Harnessing Invariant NKT Cells in Vaccination Strategies

REVIEWS

Harnessing invariant NKT cells in vaccination strategies

Vincenzo Cerundolo, Jonathan D. Silk, S. Hajar Masri and Mariolina Salio Abstract | To optimize vaccination strategies, it is important to use protocols that can ‘jump-start’ immune responses by harnessing cells of the innate immune system to assist the expansion of antigen-specific B and T cells. In this Review, we discuss the evidence indicating that invariant natural killer T (iNKT) cells can positively modulate dendritic cells and B cells, and that their pharmacological activation in the presence of antigenic proteins can enhance antigen-specific B- and T-cell responses. In addition, we describe structural and kinetic analyses that assist in the design of optimal iNKT-cell agonists that could be used in the clinical setting as vaccine adjuvants.

Prime–boost vaccination Current vaccination strategies that are based on the use of (PAMPs), such as Toll‑like receptors (TLRs), and from Repeated immunizations that recombinant viruses are failing to elicit T‑cell responses other antigen‑responsive cells in the environment that are administered when a single that are similar to those generated by natural infections. provide additional activating signals, such as the ligation application of a vaccine is The limited success of recombinant viruses as delivery of CD40. Over the past few years, it has been shown insufficient, using the same vectors for vaccines is mainly due to their high immuno‑ that invariant natural killer T (iNKT) cells, a subset of vaccine preparation + (homologous prime–boost) genicity, which results in an overwhelming immune αβ T‑cell receptor (αβTCR) T cells that are restricted by or using different vaccine response to the viral‑vector proteins rather than the CD1d molecules, can modulate DC and B‑cell activity, preparations (heterologous recombinant protein antigens. Although heterologous and can increase DC‑induced B‑ and T‑cell responses prime–boost) to sequentially prime–boost vaccination strategies are designed to overcome (FIG. 1a). It has also been shown that iNKT cells can stimulate a stronger immune response. Prior exposure to the immunodominance of vector‑specific T‑cell responses, amplify TLR‑derived signals. So, it is thought that com‑ one vaccine strain can elicit current priming strategies, such as DNA priming, are binations of compounds that activate iNKT cells and antibody and T‑cell responses proving ineffective for the generation of large numbers of compounds that stimulate immune cells through TLRs, to shared epitopes following antigen‑specific T‑cell responses in humans1–3. could potentially provide effective vaccine adjuvants. exposure to a second vaccine Advances in molecular technology have allowed the In this Review, we summarize the evidence indicat‑ strain, increasing the efficacy of heterologous prime–boost design of synthetic protein vaccines, which provide a level ing that the activation of iNKT cells bridges innate and regimens. of specificity that has not been possible with traditional adaptive immune responses, resulting in the expansion vaccines that are based on live attenuated pathogens or of antigen‑specific B‑ and T‑cell responses, properties whole inactivated organisms. Such specificity is offering that could be used to optimize vaccination strategies. a basis for the design of T‑cell therapies for infectious diseases and cancer. Optimization of such vaccination Activation of iNKT cells strategies to ensure the induction of immune responses iNKT cells are a unique population of T cells with that are specific for recombinant protein antigens is of immuno modulatory properties that link innate and paramount importance and requires a better understand‑ adaptive immune responses (BOX 1; FIG. 1a). iNKT cells ing of the signals that coordinate immune responses develop in the thymus from haematopoietic precursors Tumour Immunology Group, to infection. In an effort to overcome the limitation and are selected by CD1d molecules on the surface of Weatherall Institute of + + Molecular Medicine, of immunodominant virus‑specific T‑cell responses, CD4 CD8 double positive (DP) cortical thymocytes. University of Oxford, compounds that mimic these signals and activate innate iNKT‑cell development follows a pathway that branches Oxford, OX3 9DU, UK. immune responses should be used as adjuvants for from mainstream T‑cell development and leads to the Correspondence to V.C. vaccinations and in combination with subunit vaccines. acquisition of a memory‑activated phenotype and the e‑mail: vincenzo.cerundolo@ Initiation of dendritic cell (DC) activity during capacity to rapidly secrete cytokines following engage‑ imm.ox.ac.uk doi:10.1038/nri2451 infection occurs through the integration of a series of ment of their TCR. The molecular mechanisms of the Published online instructive signals: from the pathogen itself, through unique iNKT‑cell developmental pathway are being 12 December 2008 receptors for pathogen‑associated molecular patterns revealed and have been extensively reviewed4,5.

b a Antibody secretion

iNKT cell

B cell TCR CD40 IFNγ and IL-4 Microbial-derived CD40L lipid CD1d CD1d CD40L CD40 Lipid TCR Lysosome TCR CD1d DC DC IL-4? iNKT cell Microorganism CD40L CD27 CD70 CD40 TCR OX40 OX40L CD1d c MDSC IFNγ IL-12 iNKT cell

Endogenous + + TCR NK cell CD8 CD4 IL-12, IL-18 lipid NOS2 production T cell T cell and type I IFN Arginase 1 production CD1d

TLR DC

Proliferation Lysosome Lytic function Lytic function IFNγ production Cytokine production

Figure 1 | Natural killer T cells interact with and modulate the function of many different cell types. a | Invariant natural killer T (iNKT) cells directly and indirectly modulate the function of many other cell types, such as NK cells and T cells. These interactions are bidirectional, as iNKT cells receive signals from antigen-presentingNa turcellse Re (APCs)views and| Immunolog vice y versa. Signals can be received through cell-surface receptors, such as T-cell receptors (TCRs) recognizing glycolipid–CD1d complexes, co-stimulatory receptors, such as CD40, CD27 and OX40 recognizing their ligands CD40L, CD70 and OX40L, respectively, as well as through soluble mediators, such as cytokines. Activated iNKT cells inhibit the function of myeloid-derived suppressor cells (MDSCs), which suppress immune responses with nitric oxide synthase 2 (NOS2) and arginase 1. b | APCs infected with microorganisms can process microbial glycolipids and present them by CD1d molecules, which results in iNKT-cell activation. c | Signalling through Toll-like receptors (TLRs) leads to the presentation of endogenous glycolipids by CD1d molecules that, together with the secretion of interleukin-12 (IL-12), activate iNKT cells. TLR-induced release of soluble mediators, such as IL-12, IL-18 and type I interferons (IFNs), can also activate iNKT cells in a CD1d-independent manner. DC, dendritic cell.

unlike conventional CD4+ and CD8+ T cells, and non‑immunogenic glycosphingolipids, such as iNKT cells are activated by endogenous or exog‑ tetrasaccharide‑containing glycosphingolipids11, in enous lipid ligands (FIG. 1b,c), for example, those of the cell wall of any given Sphingomonas strain may Sphingomonas spp. and Borrelia burgdorferi 6–10, which serve as an immune‑evasion mechanism11. During are recognized directly by the iNKT‑cell TCR when B. burgdorferi infection the lipid antigens that are recog‑ bound to CD1d. (FIG. 1b). This recognition is independ‑ nized by iNKT cells are galactosyl diacylglycerols (FIG. 2). ent of TLR‑mediated activation of antigen‑presenting Similar to results obtained with different Sphingomonas cells (APCs) or interleukin‑12 (IL‑12) secretion. strains, B. burgdorferi‑dependent iNKT‑cell activation Sphingomonas spp. are Gram‑negative bacteria and varies depending on the length and saturation of the have a cell wall that lacks lipopolysaccharide (LPS) galactosyl diacylglycerol acyl chains6. Other microbial but is rich in α‑linked glycosphingolipids, which can lipids that have been reported to activate subsets of vary in the complexity and sequence of the α‑linked iNKT cells are the Leishmania donovani surface glyco‑ sugar, the acyl chain and the sphingoid base of the conjugate lypophosphoglycan12 and the Mycobacterium ceramide lipid11. It has been suggested that the balance leprae phosphatidylinositol tetramannoside (PIM4)13 between immunogenic glycosphingolipids, such as (FIG. 2), although iNKT‑cell recognition of PIM4 has α‑glucuronosylceramides, α‑galacturonosylceramides been shown only with purified PIM4 (ReF. 13) and not (including the Sphingomonas cell‑wall antigen PBS‑30)7,8, with synthetic PIM4 (ReF. 6).

Box 1 | Phenotype of invariant natural killer T cells Natural killer T (NKT) cells are a heterogeneous population of cells that express a T‑cell receptor (TCR) and are restricted by the CD1d molecule98. Most NKT cells (known as invariant NKT cells; iNKT cells) express an invariant TCR α‑chain (Vα14–Jα18 in mice and the homologous Vα24–Jα18 in humans) that is paired with a semi‑invariant TCR β‑chain (Vβ2, Vβ7 or Vβ8.2 in mice and Vβ11 in humans). iNKT cells can be identified by staining with multimeric complexes of CD1d molecules loaded with the lipid α‑galactosylceramide (α‑GalCer)99–101. In mice iNKT cells are either CD4+ or double negative (DN) for co‑receptor expression, whereas in humans a CD8+ subset of iNKT cells also exists. It has been shown that these subsets have slightly different functional properties: human DN and CD8+ iNKT cells are highly cytolytic and + 102,103 produce T helper 1 (TH1)‑type cytokines, whereas CD4 iNKT cells produce both TH1‑ and TH2‑type cytokines and differentially regulate dendritic‑cell activity104. In addition, the antitumour activity of iNKT cells has been shown to apply mainly to the liver DN subset in two mouse models105. Although iNKT‑cell maturation in the periphery has previously been associated with the acquisition of NK1.1 expression, a population of mature NK1.1– iNKT cells has recently been reported106. Furthermore, a CD4–NK1.1– subset that produces interleukin‑17 (IL‑17) and constitutively expresses IL‑23 and retinoic‑acid‑receptor‑related orphan receptor‑γt (RORγt) has been identified58,107. IL‑17 secretion may contribute to their pathogenic role in several diseases108,109. iNKT cells are found in abundance in the thymus, spleen, liver and bone marrow, which may be explained by their

expression of a chemokine receptor profile that is similar to that of TH1 cells which home to inflamed tissues. Few iNKT cells express lymph‑node homing receptors110–113.

In addition to direct recognition of bacterial lipids, as shown by the increased susceptibility of iNKT‑ human and mouse iNKT cells can be activated indirectly cell‑deficient mice to methylcholanthrene‑induced (for example, during Salmonella enterica infection) by tumours; these mice have both an earlier onset and stimulatory cytokines, such as IL‑12 and type I interfer‑ a higher incidence of tumours26. Although there is ons (IFNs). These are released by DCs following TLR compelling evidence that iNKT cells have an impor‑ signalling8,14–16 and amplify the weak activating signals tant role in immunosurveillance, the mechanisms that are generated from the ligation of self glycolipids by which iNKT cells control tumour growth remain (BOX 2; FIG. 1c). APC‑derived cytokines can also acti‑ unclear. Indeed, direct recognition of tumour cells by vate iNKT cells irrespectively of TCR engagement by iNKT cells is not required for rejection, as tumours lipid–CD1d complexes17. that lack CD1d expression can be rejected in wild‑type In addition to bacterial infections and TLR‑dependent mice27, which indicates that iNKT cells might promote signalling events, activation and expansion of iNKT cells tumour rejection indirectly. It is probable that the

can be achieved with pharmacological compounds that iNKT‑cell‑dependent production of T helper 1 (TH1)‑ bind to CD1d molecules and are recognized by the type cytokines18, which favour NK‑cell activation28 and iNKT‑cell TCR. The first compound to be identified inhibit tumour angiogenesis29, support tumour elimi‑ as an iNKT‑cell activator was α‑galactosylceramide nation. Recent results showing that iNKT cells can (α‑GalCer) (FIG. 2), which is an active component of counterbalance the suppressive activity of myeloid‑ glycosphingolipid extracts from the marine sponge Agelas derived suppressor cells (MDSCs)30 provide further mauritianus and has been shown to have antitumour insights into the mechanisms by which iNKT cells activity in the mouse B16 melanoma model18,19. might contribute to tumour immunosurveillance In the past few years several analogues of α‑GalCer (BOX 3). Indeed, higher numbers of MDSCs are found have been synthesized that can activate iNKT cells in the spleen and ascites fluid of tumour‑bearing both in vivo and in vitro (reviewed in ReF. 20) (FIG. 2). iNKT‑cell‑deficient mice than wild‑type mice31. The results of experiments carried out using these Stimulation of iNKT cells through recognition compounds revealed that pharmacological activa‑ of the α‑GalCer–CD1d complex results in the rapid

tion of iNKT cells is a more rapid and efficient way production of TH1‑ and TH2‑type cytokines, such as to activate iNKT cells than the use of TLR agonists or IFNγ and IL‑4 (ReFs 18,32), and the increased expres‑ microbial injection6–8,21. However, synthetic compounds sion of CD40 ligand (CD40L)33 (FIG. 1a), which induces that activate iNKT cells should be used cautiously, as DC maturation34,35. As DC maturation is required for overstimulation of iNKT cells can result in iNKT‑cell the initiation of adaptive immunity, it was proposed anergy22,23 and lysis of α‑GalCer‑loaded APCs following that co‑injection of iNKT‑cell agonists and antigens TCR engagement24,25. could be used to expand antigen‑specific responses. Consistent with this hypothesis, we and others showed iNKT cells bridge innate and adaptive immunity that stimulation of iNKT cells with α‑GalCer in vivo B16 melanoma A widely used experimental Recently, it has become clear that iNKT cells have significantly increases immune responses to co‑injected 35–39 mouse melanoma. B16 an important role in bridging innate and adaptive antigens . Cytokines, such as tumour‑necrosis factor melanoma is poorly immune responses, and that activation of iNKT cells (TNF), type I IFNs and IFNγ, further enhance DC immunogenic and therefore is results in rapid DC and B‑cell maturation and NK‑cell maturation in trans34,38 (FIG. 1a). It has recently been difficult to eliminate. Largely activation (FIG. 1a). Initial studies showed that injection shown that upregulation of the expression of CD70 because of this, B16 α (ReF. 40) 41 melanoma is a good model of ‑GalCer could promote the survival of mice with and OX40 ligand by mature DCs is impor‑ 9 for testing cancer melanoma . It was subsequently found that iNKT cells tant for co‑stimulating iNKT cells and for promoting immunotherapies. are important mediators of cancer immunosurveillance, antigen‑specific CD8+ T‑cell responses.

a Natural ligands O O

HN HO O OH O HO O O OH HO O O HO OH OH O OH O HO OH HO O HO Isoglobotrihexosylceramide (endogenous) HO O O Glycolipid II (B. burgdorferi) OH HO O O Phosphatidylinositol tetramannoside HO OH O OH (M. leprae) HO O OH O O HO OO– O HN HO OH O P OH OH HO O O O HO O O HO O O OH HO O OH OH HO HO O HO O α-Glucuronosylceramide (Sphingomonas spp.) OH HO b Synthetic ligands O O

HN HN OH OH OH O O O OH OH HO OH α-Galactosylceramide (synthetic or HO OH Threitolceramide marine sponge) HO O C20:2 HO

HN OH OH O O

HO OH OH α-C-galactosylceramide OCH9 O HO O

HN HN OH H2 OH OH OH C O O O

OH HO OH HO OH OH HO HO Figure 2 | Examples of invariant-natural-killer-T-cell agonists. a | Numerous invariant natural killer T (iNKT)-cell natural agonists have been characterized. Isoglobotrihexosylceramide is an endogenous glycolipid115. Examples of microbial ligands include Borrelia burgdorferi α-galactosyldiacylglycerol (glycolipid II), Sphingomonas spp. α-glucuronosylceramide (PBS-30) and Mycobacterium leprae phosphatidylinositol tetramannoside (PIM4)6–8,13. b | Synthetic Nature Reviews | Immunology ligands include α-galactosylceramide, which can also be obtained from the marine sponge Agelas mauritianus18, α-C- galactosylceramide61, OCH9 (ReF. 59), C20:2 (ReF. 63) and threitolceramide25.

The adjuvant effect of iNKT‑cell stimulation is further inducing B‑cell maturation, higher antibody titres and increased by the addition of TLR agonists38,42, suggesting expansion of the B‑cell memory pool25,42,44,45. More that signals transmitted by iNKT cells to DCs can amplify recently, B‑cell receptor (BCR)‑mediated uptake of the effect of pathogen‑induced signalling. Indeed, TLR CD1d‑restricted antigens was shown to be an effec‑ and CD40 signalling seem to be highly coordinated, as tive means of enhancing iNKT‑cell‑dependent B‑cell triggering of CD40 in vivo with CD40‑specific antibod‑ responses in vivo46,47. Targeting of iNKT‑cell ligands to ies in the absence of microbial stimulation results in low antigen‑specific B cells can be achieved by using anti‑ levels of IL‑12p70 production. Similarly, inflammatory gen and iNKT‑cell agonists bound to particles, such signals in the absence of TLR stimulation fail to prime as silica beads46, or by synthesizing B‑cell antigens functional CD4+ T‑cell responses43. Clearly, the timely linked to α‑GalCer47. This mediates the production provision of iNKT‑cell‑derived signals is important for of high titres of specific IgM and early class‑switched optimizing DC and, consequently, T‑cell responses. antibodies. These results indicate that iNKT cells In addition to promoting the generation of potent can facilitate B‑cell activation in response to particu‑ antigen‑specific CD4+ and CD8+ T‑cell responses35,37–39, late iNKT‑cell agonists, thereby enhancing specific iNKT‑cell activation can also provide help to B cells, antibody responses.

Box 2 | Invariant-natural-killer-T-cell endogenous ligands The nature of the antigen (or antigens) mediating invariant natural killer T (iNKT)‑cell selection in the thymus is still uncertain. Evidence from a β‑glucosylceramide‑synthase‑deficient cell line that cannot stimulate Vα14+ iNKT cells suggested that the natural ligand is a glycosphingolipid114. The endogenous glycosphingolipid isoglobotrihexosyl‑ ceramide (iGb3) (FIG. 2), an agonist for human and mouse iNKT cells115, was initially considered to be the only natural selecting ligand for these cells, as mice deficient in the lysosomal enzyme β‑hexosaminidase, which generates iGb3 from iGb4, had impaired iNKT‑cell development115. However, subsequent work revealed that mice lacking other lysosomal enzymes that are involved in glycosphingolipid catabolism and in cholesterol transport have severe defects in iNKT‑cell development (reviewed in ReF.116). It is probable that altered lipid trafficking and lysosomal processing, which are common to all these mutant mice, account for the defect in iNKT‑cell development. Indeed, analysis of iGb3‑synthase‑deficient mice did not reveal any anomaly in the number or function of iNKT cells117. iGb3 was not detected by a sensitive high‑performance liquid chromatography in the thymi of these mice or in their dendritic cells (DCs)118, despite an earlier report that it may mediate the recognition of lipopolysaccharide‑matured DCs8. Finally, iGb3 was not detected in any human tissue118, which is consistent with the observation that humans lack a functional iGb3 synthase owing to several mutations in the gene that encodes it119. More recently, however, the presence of iGb4 has been detected in the human thymus using mass spectrometry120. Therefore, the biochemical identification of iGb3 remains an open question. The identity of the lipid (or lipids) that mediates iNKT‑cell activation in the periphery is also under investigation. It has recently been shown that Toll‑like receptor (TLR)‑mediated activation of DCs enhances the expression of transcripts of several glycosyltransferases that are involved in the biosynthesis of glycosphingolipids15,16, and mouse TLR‑stimulated DCs produce a charged β‑linked glycosphingolipid that can activate iNKT cells together with type I interferon15. Other endogenous antigens that have been reported to activate subsets of mouse iNKT cells are phospholipids121 and the gangliosides GD3 and GM3, which are often overexpressed in melanomas and other tumours of neuroectodermal origin122.

The activation of iNKT cells with α‑GalCer and the iNKT‑cell‑deficient mice abolished the suppressive activity subsequent expansion of antigen‑specific B and T cells of MDSCs by reducing the activity of nitric oxide synthase following injection of recombinant proteins is achiev‑ and arginase 1, thereby rescuing the ability of these mice able only within a short time period; maximal efficacy to clear influenza A virus infection. we also showed that is observed only when the antigen and α‑GalCer are myeloid cells with a suppressive phenotype were present administered simultaneously35. Consistent with a role in the peripheral blood of individuals that were infected for DCs in enhancing iNKT‑cell activation in vivo, it was with influenza A virus and that iNKT cells could abol‑ also shown that the duration of cytokine production fol‑ ish their suppressive phenotype. These results highlight lowing α‑GalCer injection was significantly increased by a new role for iNKT cells in modulating immuno logical injecting α‑GalCer‑loaded bone‑marrow‑derived DCs, suppressive mechanisms that are activated during acute compared with the injection of free α‑GalCer48,49. inflammatory processes30. iNKT‑cell‑dependent induction of antigen‑specific B‑ and T‑cell responses can be observed following the Cytokine secretion and iNKT-cell anergy injection of α‑GalCer through several routes, including Activation of iNKT cells results in TCR downregula‑ intranasal50,51 and oral administration38,52. By contrast, tion, proliferation and prolonged cytokine secretion55–57. because of the dichotomy between rapid iNKT‑cell‑ In addition to the secretion of IL‑4 and IFNγ, activated dependent DC maturation and the length of time that iNKT cells have also been shown to produce IL‑2, IL‑3, is required for the expression of antigens encoded by IL‑5, IL‑6, IL‑9, IL‑10, IL‑13, IL‑17, IL‑21, TNF, trans‑ plasmid DNA, it remains unclear whether intramuscular forming growth factor‑β (TGFβ) and granulocyte/ injection of plasmid DNA with α‑GalCer can result in macrophage colony‑stimulating factor (GM‑CSF), as well the enhancement of antigen‑specific immune responses. as a wide range of chemokines5,58. It is becoming clear

However, recent results indicate that intramuscular co‑ that the repertoire of TH1‑ and TH2‑type cytokines that administration of α‑GalCer and plasmid DNA encoding is produced by iNKT cells is modulated by the strength Cytokine storm the HIv‑1 protein Gag can facilitate the expansion of of iNKT‑cell TCR signalling events24,59–61 and by the type of A strong systemic immune Gag‑specific immune responses53. APC presenting the iNKT‑cell agonists49,62,63. Activation response that results in the α release of high levels of In addition to the ability of iNKT cells to facilitate of iNKT cells by strong agonists such as ‑GalCer, which inflammatory mediators (such DC maturation and B‑cell activation, we have recently has a high affinity for the iNKT‑cell TCR and a long half‑ as cytokines, oxygen free shown that iNKT cells can increase antigen‑specific life24,64,65, results in high levels of cytokine production. radicals and coagulation immune responses by regulating the suppressive activity This is further amplified by iNKT‑cell‑dependent DC factors). Both pro‑inflammatory + + 54 cytokines (such as of CD11b GR1 MDSCs , which increase in number maturation and NK‑cell activation. As such an exces‑ 30 tumour‑necrosis factor, during inflammation . we observed greater numbers sive production of cytokines could potentially cause a interleukin‑1 (IL‑1) and IL‑6) of MDSCs during infection with influenza A virus in cytokine storm, the activation of iNKT cells needs to be and anti‑inflammatory iNKT‑cell‑deficient and CD1d‑deficient mice than tightly regulated. cytokines (such as IL‑10 and wild‑type mice, suggesting that iNKT cells have a role in Importantly, overstimulation of iNKT cells by strong IL‑1 receptor antagonist) are 24,25 increased in the serum of regulating the frequency and activity of MDSCs during agonists results in DC lysis and activation‑induced 30 patients experiencing a infection with this virus . Consistent with these find‑ iNKT‑cell anergy (defined as unresponsiveness to sub‑ cytokine storm. ings, we showed that adoptive transfer of iNKT cells to sequent stimuli). iNKT‑cell anergy has been observed

Box 3 | Non-invariant natural killer T cells thorough characterization of different binding surfaces, all of which can be recognized by the semi‑invariant A second population of natural killer T (NKT) cells exists (known as type II NKT cells), TCRs of iNKT cells. which is CD1d restricted but does not express an invariant T‑cell receptor (TCR) and Borg and colleagues72 crystallized a human iNKT‑ lacks reactivity to α‑galactosylceramide. Type II NKT cells were first described in 1995 cell TCR with an α‑GalCer–CD1d complex, providing (ReF. 123), but their characterization has been limited because of the lack of specific markers and agonistic reagents. More recently, a fraction of type II NKT cells has been insight into the interactions between the molecules and shown to be reactive to the self‑glycolipid sulphatide and has been detected by the the chain residues that are involved in the process. They ability of these cells to bind sulphatide‑loaded CD1d tetramers124. Type II NKT cells described a unique ‘lock‑and‑key’ mode of interac‑ have an important immune‑regulatory role in infection, autoimmune disease and tion, whereby the iNKT‑cell TCR docked in parallel tumour immunosurveillance (recently reviewed in ReF.125). In contrast to invariant to the F′‑channel end of the CD1d antigen‑binding iNKT cells, type II NKT cells suppress antitumour immunity, thereby promoting the pocket72 (FIG. 3a). This is different to the interaction activation of myeloid‑derived suppressor cells through the production of interleukin‑13. that has been described for TCR–peptide–MHC complexes, in which both the hypervariable segments complementarity‑determining region 3α (CDR3α) after injection of α‑GalCer22,23, of bacteria that can and CDR3β of the TCR adopt a more central position stimulate iNKT cells and of certain TLR ligands21,66. relative to the binding groove (FIG. 3b). The CDR2β loop The molecular mechanisms that control iNKT‑cell of the iNKT‑cell TCR mainly interacts with the CD1d unresponsiveness are still ill defined. However, iNKT‑ backbone, and germline‑encoded residues of both the cell unresponsiveness is similar to conventional T‑cell CDR1α and CDR3α loops contribute to the recognition anergy67 in that it is long lasting (up to 4–6 weeks), it of α‑GalCer by interacting with CD1d and the bound can be reversed by the administration of IL‑2 and it is α‑GalCer molecule. This form of recognition resembles cell intrinsic21–23,66. IL‑12 is required for bacterium‑ but that of a pattern‑recognition receptor and explains not α‑GalCer‑induced iNKT‑cell unresponsiveness21, the use of the invariant vα24–jα18 segments by all and alteration of the expression of NK‑cell receptors iNKT‑cell TCRs (vα14–jα18 in mice)72,76,77. may contribute to the hyporesponsive phenotype Several research groups have carried out a series of that is observed in some experimental systems21,68. kinetic and functional experiments to assess the role Following restimulation of anergic iNKT cells, there is of the polar head and the length and saturation of the a bigger reduction in the secretion of IFNγ than of IL‑4 α‑GalCer alkyl chains in controlling the rate of dis‑ (ReFs 21,22), which may explain the efficacy of repeated sociation of lipids that are bound to CD1d molecules injection of α‑GalCer in ameliorating autoimmunity in and the affinity of binding of lipid‑specific TCRs. The

disease models that are driven by a TH1‑cell response, knowledge derived from the crystallographic studies such as type 1 diabetes. has provided a framework to understand the properties It is worth noting that, in contrast to the injection of of the different compounds. In the next sections, we sum‑ free α‑GalCer, repeated injections of α‑GalCer‑loaded marize some of the conclusions that have been drawn DCs do not seem to induce iNKT‑cell unresponsiveness from these experiments. in mice48 and humans69. Modifications of α‑GalCer lipid chains. The results from Structure and function of iNKT-cell agonists experiments carried out using either α‑GalCer analogues Combined structural, kinetic and functional studies on or bacterium‑derived iNKT‑cell agonists that had modi‑ the molecular mechanisms of iNKT‑cell activation are fied lipid tails suggested three conclusions. First, the paving the way for the identification of optimal iNKT‑ length of the acyl and phytosphingosine chains affects cell agonists that could be used as vaccine adjuvants. The the stability of the lipid–CD1d complex6,11,24,78–82. Second, crystal structures of human and mouse CD1d in complex the level of saturation of the alkyl chains influences the with α‑GalCer70 (FIG. 3) or the variant agonist PBS‑25 rate of lipid dissociation from CD1d molecules24,63. (ReF. 71), which has an 8‑carbon acyl chain instead of the Third, the length of the lipid chain occupying the long 26‑carbon fatty‑acid chain of α‑GalCer, revealed F′ channel modulates the affinity of the iNKT‑cell TCR a narrow and highly hydrophobic antigen‑binding for the glycolipid–CD1d complex24,59,80–82. groove that is characterized by two channels, A′ and F′. The effect of lipid length and saturation on the stability α‑GalCer saturates the binding capacity of the CD1d of the lipid–CD1d complex is consistent with structural binding groove, the 26 carbon atoms of the acyl chain features of the A′ channel, which follows a curved path filling the A′ channel and the 18 carbon atoms of the circumventing a ‘pole’ formed by residues cysteine 12 phytosphingosine chain that fits into the F′channel70,72 and phenylalanine 70 (ReFs 70–72)(FIG. 3c). These results (FIG. 3c). The structure of the CD1d binding groove is suggest that a preformed kink in the acyl chain caused T-cell anergy A state of T‑cell markedly different from that of the shallower, wider by the presence of unsaturated bonds might stabilize unresponsiveness to and multi‑pocketed grooves of the classical MHC mol‑ binding of the lipid by favouring the tightly curved con‑ stimulation with antigen. ecules (FIG. 3d), which only accommodate the anchoring formation that is required for binding to the A′ channel. T‑cell anergy can be induced side‑chain residues of presented peptide antigens73. Indeed, compounds with unsaturated bonds in the acyl by stimulation with a large In addition to α‑GalCer70,72 and PBS‑25 (ReF. 71), the chain occupying the A′ channel can be directly loaded amount of specific antigen in the absence of the structures of CD1d in complex with iNKT‑cell ago‑ onto cell‑surface‑expressed CD1d molecules, resulting in 74 engagement of co‑stimulatory nists α‑galacturonosylceramide and the endogenous their presentation by non‑professional APCs and in a bias molecules. 75 63,83,84 ligand isoglobotrihexosylceramide (iGb3) provide a towards the secretion of TH2‑type cytokines .

a b The effect of variations in the length of the lipid tail Cα that occupies the F′ channel on iNKT‑cell TCR‑binding Cβ TCR affinity is consistent with results from analyses using iNKT-cell TCR site‑directed mutagenesis of human77 and mouse76 iNKT‑cell TCRs. These analyses revealed that the region directly above the F′ channel provides a binding hot Vα Vα Vβ spot for the iNKT‑cell TCR. These results suggests that Vβ α-GalCer iNKT cells recognize both the group head and the length Peptide of lipid antigens, thereby ensuring greater specificity of antigen recognition. In agreement with the results that highlight the impor‑ tance of the length of the lipid chains occupying the CD1d HLA-A2 β2m F′ channel, stimulation of mouse iNKT cells with the com‑ β2m pound OCH9 (FIG. 2), which has a shorter phytosphingo‑ sine chain, induced higher levels of IL‑4 secretion than stimulation with α‑GalCer and was effective at treating

c d mouse models of TH1‑cell‑mediated autoimmune Lipid chains disorders, such as experimental autoimmune encephalo‑ myelitis59,85. Although OCH9 does not alter the cytokine CDR F70 profile of human iNKT cells24 and does not skew antigen‑ F84 loops specific T‑cell responses towards IL‑4 secretion when Peptide used to promote iNKT‑cell‑dependent T‑cell activation in mice38, in other experimental systems it has been shown 131 N HLA-A2 that early secretion of IL‑4 by iNKT cells is required to V98 C12 V116 + 86 F114 prime naive CD4 T cells to differentiate into TH2 cells . A′ channel F′ channel In summary, these results indicate that the length efα-GalCer Threitolceramide of lipid chains occupying the F′channel should be 18 carbons and the acyl chain occupying the A′ channel F29 CDR2α F29 should be 26 carbons and saturated to obtain optimal stability of the loaded CD1d molecule and fully exploit CDR3α CDR1α S30 S30 F51 F51 its binding capacity. This ensures prolonged duration G96 G96 3'-OH 3'-OH of iNKT‑cell stimulation and optimal presentation by 4'-OH 4'-OH professional APCs. R95 2'-OH R95 2'-OH Modifications of the α‑GalCer polar head. Three hydro‑ α1 W153 W153 gen bonds between human CD1d and α‑GalCer at the α2 junction of the two alkyl chains and the polar head group serve to anchor α‑GalCer in a distinct orientation and Figure 3 | Structural features of the interactions between the T-cell receptors position it within the lipid‑binding groove70. The 2′‑OH of invariant natural killer T cells and lipid–CD1d complexes.Nature a,b Re vie| Comparisonws | Immunolog of y of the galactose ring, which is crucial for the antigenic‑ the structure of the complex comprising α‑galactosylceramide (α-GalCer)–CD1d and the ity of α‑GalCer, forms a hydrogen bond with aspartic invariant natural killer T (iNKT)-cell T-cell receptor (TCR) with the structure of the complex of acid 151. The 3′‑OH on the sphingosine chain forms a the JM22-TCR with peptide 58–66 from influenza virus matrix protein in the binding groove hydrogen bond with aspartic acid 80 and the glycosidic of an HLA-A2 molecule. Significant differences were revealed in TCR recognition of the linkage 1′‑O of α‑GalCer forms the third hydrogen bond antigen. c | The side view of α-GalCer bound to the CD1d groove; channels A′ and F′ are to threonine 154. Surprisingly, a C‑glycosidic variant of indicated in grey, α-GalCer is indicated in orange and key residues of CD1d are indicated in α α blue. d | A side view of peptide 157–165 from the NY-ESO-1 tumour antigen (yellow) in the ‑GalCer ( ‑C‑GalCer), in which the glycosidic link‑ ′ α (FIG. 2) binding groove of the HLA-A2 molecule (grey) is shown with complementarity-determining age 1 ‑O of ‑GalCer is replaced by CH2 , was a region (CDR) loops from the cognate TCR above the peptide. Structures c and d highlight more potent adjuvant than α‑GalCer in mouse models the depth of the channels in CD1d molecules that anchor the fatty-acid tails (c) compared of malaria and metastatic melanoma61 and could gener‑ with the shallow peptide-binding groove in MHC class I molecules (d). e | Hydrogen bonds ate more prolonged immune responses and greater levels that stabilize the TCR–α-GalCer–CD1d complex are shown. f | Molecular modelling of the of IFNγ secretion. This was possibly because its binding TCR–threitolceramide–CD1d complex is shown25, based on the structure shown in part e. to CD1d in DCs was more stable and it also induced The numbers of stabilizing hydrogen bonds with F29, S30 and G96 residues in the TCR are increased NK‑cell activation61,87. Although no kinetic shown in parts e and f. The human CD1d molecule is indicated in green, the ligands are measurements of α‑C‑GalCer have been carried out, it indicated in magenta and CDR1α and CDR3α of the iNKT-cell TCR are indicated in yellow is possible that the C‑glycosidic linkage may provide a and cyan, respectively. β2m, β2-microglobulin; C, constant region; V, variable region. Part a is reproduced, with permission, from ReF.72  (2007) Macmillan Publishers Ltd. more rigid glycosyl head group and/or increase resist‑ All rights reserved. Part b is reproduced, with permission, from ReF. 126  (2003) Macmillan ance to enzymatic degradation, which would make its 61 Publishers Ltd. All rights reserved. Part c is reproduced, with permission, from ReF.70 association with CD1d more stable . Further studies are  (2005) Macmillan Publishers Ltd. All rights reserved. Part d is reproduced, with permission, therefore warranted to clarify the mechanisms that are from ReF. 127  (2005) The Rockefeller University Press. Parts e and f are reproduced, with responsible for this increased potency of the C‑glycosidic permission, from ReF. 25  (2008) The American Association of Immunologists. variant of α‑GalCer.

Initial experiments carried out to establish the con‑ Injection of α‑GalCer‑loaded DCs. So far, four Phase I tribution of the glycosidic portion of α‑GalCer to iNKT‑ clinical trials have been carried out using DCs pulsed cell activation showed that substitution of galactose with with α‑GalCer. In the first Phase I trial autologous glucose reduced activation and that mannosylceramide immature monocyte‑derived DCs that had been pulsed or β‑GalCer were not recognized by iNKT cells18. with α‑GalCer were administered intravenously to Although more recent results have shown that β‑linked patients with metastatic tumours92. Similarly to the glycosphingolipids, such as some forms of β‑anomeric results obtained from intravenous immunization with GalCer, can stimulate iNKT cells88,89, it remains unclear α‑GalCer91, the frequency of iNKT cells transiently whether these results were due to the potential contami‑ decreased 1–2 days after treatment. A transient decrease nation by α‑linked glycosphingolipid that can occur in the frequency of NK, B and T cells was also observed. during the synthesis of these compounds. Additional Increased serum levels of IFNγ and IL‑12 were detected studies correlating structure with function indicated after vaccination, along with the activation of NK and that the glycolipid–CD1d–TCR interaction tolerates a T cells. Interestingly, although adoptively transferred small molecule at the carbon 6 position on the sugar of DCs were initially found in the lungs, they migrated α‑GalCer, but that iNKT‑cell activation is inhibited by to the liver and spleen within 24 hours92. A decrease other substitution patterns (reviewed in ReF. 20). in tumour markers in the serum of two patients was until recently, all the known iNKT‑cell agonists also reported. contained a glycosidic group in the polar head. we have In the second clinical trial, patients with non‑small‑ now shown that iNKT cells can also recognize com‑ cell lung cancer were immunized intravenously up to pounds that lack a sugar head and have non‑glycosidic four times with more than 5 x 107 immature DCs per m2 linkages between the hydrophilic head and the ceramide that were loaded with α‑GalCer93. Injections were gen‑ moiety25 (FIGs 2,3f). These compounds comprise a new erally well tolerated and a significant increase in the class of iNKT‑cell agonists, and this broadens the range frequency of iNKT cells after two injections of DCs was of compounds that can be recognized by these cells. Of reported in one patient. The levels of mRNA encoding particular interest is threitolceramide, which is similar to IFNγ in circulating iNKT cells were also increased. α‑GalCer in that it stably binds CD1d molecules owing to A marked increase in iNKT‑cell number was the optimal length of its lipid chains and it is likely to form observed in another Phase I clinical trial, in which five the three hydrogen bonds with CD1d molecules because patients with myeloma were immunized three times of the presence of 4′‑OH, 3′‑OH and 2′‑OH residues in the intravenously with mature DCs69. The first injection head group25 (FIG. 3f). we have shown that threitolceramide used unpulsed DCs to establish a baseline for the fre‑ can induce optimal DC maturation and antigen‑specific quency of iNKT cells, and subsequent injections were B‑ and T‑cell priming, but does not cause extensive iNKT‑ carried out with α‑GalCer‑loaded DCs. Injection of cell‑dependent lysis of DCs, as is observed with α‑GalCer. α‑GalCer‑loaded DCs led to an increase of more than This is probably because of its weaker binding affinity to 100‑fold in the number of iNKT cells in some patients, the iNKT‑cell TCR25. Furthermore, iNKT cells that have which was detectable for up to 6 months after vaccina‑ been activated by threitolceramide recover more quickly tion. In addition, the investigators detected increased from anergy than those stimulated by α‑GalCer25. serum levels of the cytokines that are associated with DC maturation, such as IL‑12, and an increase in the Harnessing iNKT cells in the clinic frequency of cytomegalovirus (CMv)‑specific CD8+ The evidence indicating that the harnessing of mouse T cells. These data indicated that injection of DCs loaded iNKT cells increases antigen‑specific immune responses with α‑GalCer can lead to the activation of APCs in vivo, by bridging innate and adaptive immunity provides the resulting in enhanced antigen‑specific T‑cell responses, basis for designing immunotherapeutic protocols to and suggested that iNKT‑cell agonists may also facilitate potentiate immune responses against pathogens and the expansion of antigen‑specific responses in humans, tumours, and possibly to modulate autoimmune responses as was previously described in mice. This study also (reviewed in ReF. 90). In this section, we summarize the showed that following immunization with α‑GalCer‑ clinical trials that have been carried out so far. loaded DCs, three patients had clinically relevant decreases in the level of monoclonal immunoglobulins Intravenous injection of α‑GalCer. A dose‑escalation (M protein) in the serum or urine and one patient study using α‑GalCer was carried out in patients showed stabilization of the disease69. with solid tumours, who were injected intravenously with Finally, patients with head and neck cancer were 50–4,800 mg per m2 α‑GalCer91. Consistent with results vaccinated with α‑GalCer‑loaded autologous imma‑ obtained in mouse models, the frequency of iNKT cells ture DCs, which were administered into the nasal sub‑ in the blood decreased following injection, presumably mucosa94. The immunizations induced the expansion as a result of activation‑induced TCR downregulation of iNKT cells and enhanced NK‑cell activity in some or of their migration to surrounding tissues following patients. In this study, the cells used were a mixture of activation by α‑GalCer. α‑GalCer‑dependent activa‑ APCs that were derived from peripheral‑blood mono‑ tion of iNKT cells led to increased levels of IFNγ, IL‑12 nuclear cells, and the percentage of DCs administered and GM‑CSF in the serum of a few of the patients, and in each case varied from 30% to 55%. A partial reduction in this depended on the frequency of iNKT cells in the tumour size was reported in one patient and stabilization pre‑vaccination samples91. of disease was observed in five out of nine patients.

Immunization with in vitro expanded iNKT cells. should be carried out to assess whether results that have Similarly to the clinical trials that have been carried been obtained in animal models, which show that iNKT‑ out using in vitro expanded peptide‑specific T cells for cell activation can enhance antigen‑specific B‑ and T‑cell immuno therapy (reviewed in ReF. 95), a clinical trial responses, can be extended to humans. was performed using adoptively transferred iNKT cells. Although numerous synthetic CD1d‑binding ligands Patients with non‑small‑cell lung cancer received two have been described, it is important to ensure that the intravenous injections of up to 5 x 107 cells per m2 in vitro‑ iNKT‑cell agonists used in the clinic can be repeatedly activated (with α‑GalCer and IL‑2) iNKT cells96. There injected into patients without inducing iNKT‑cell over‑ were no severe adverse events in any of the patients, activation, resulting in iNKT‑cell anergy and cytokine and in some cases increased frequencies of iNKT cells storm. This could be achieved by identifying iNKT‑cell were detected in blood samples post‑immunization. agonists that have a reduced affinity for the iNKT‑cell TCR Administration of activated iNKT cells also induced IFNγ and at the same time ensure optimal DC maturation. production in vivo, mainly from NK cells. Nevertheless, The next generation of iNKT‑cell agonists should be none of the patients had any notable positive clinical capable of targeting specific APC populations to ensure response to this immunotherapy. preferential binding to DCs and/or antigen‑specific B cells. These properties could be achieved by either Conclusions and future perspectives modifying the structure of the synthetic iNKT‑cell Since the discovery of iNKT cells over 20 years ago, it agonists to facilitate loading by either DCs or B cells has become clear that iNKT cells have extremely ver‑ (as it was initially shown in ReF. 63) or by coupling the satile responses to activation. The challenge remains to iNKT‑cell agonists to cell‑type‑specific antibodies. understand how to activate and manipulate the differ‑ Consistent with the latter possibility, recent data showed ent subpopulations of iNKT cells and how to translate that targeting of iNKT‑cell ligands to antigen‑specific these results into the clinical setting to control the B cells can be enhanced by linking them to known B‑cell presentation of iNKT‑cell agonists by different APCs antigens. This method uses BCR‑mediated uptake and in different microenvironments both quantitatively subsequent presentation of the glycolipid antigen in the and qualitatively. context of CD1d and enhancement of B‑cell activity46,47. To date, no clinical trials have been carried out in In addition, it has been shown that sustained activation which iNKT‑cell agonists are co‑injected with subunit and tumour‑cell targeting of iNKT cells using a fusion vaccines and only one clinical trial has looked indirectly protein consisting of α‑GalCer–CD1d and scFv antibody at the ability of α‑GalCer to enhance the response of fragment specific for human epidermal growth‑factor the adaptive immune cells in the clinic69. Clinical trials receptor 2 induces an antitumour effect97.

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