Longitudinal analysis of invariant natural killer T cell activation reveals a cMAF-associated transcriptional state of NKT10 cells

Innate T cells, including CD1d-restricted invariant natural killer T (iNKT) cells, are characterized by their rapid activation in response to nonpeptide antigens, such as lipids. While the transcriptional profiles of naive, effector and memory adaptive T cells have been well studied, less is known about transcriptional regulation of different iNKT cell activation states. Here, using single cell RNA-sequencing, we performed longitudinal profiling of activated iNKT cells, generating a transcriptomic atlas of iNKT cell activation states. We found that transcriptional signatures of activation are highly conserved among heterogeneous iNKT cell populations, including NKT1, NKT2 and NKT17 subsets, and human iNKT cells. Strikingly, we found that regulatory iNKT cells, such as adipose iNKT cells, undergo blunted activation, and display constitutive enrichment of memory-like cMAF+ and KLRG1+ populations. Moreover, we identify a conserved cMAF-associated transcriptional network among NKT10 cells, providing novel insights into the biology of regulatory and antigen experienced iNKT cells.

Expansion of invariant natural killer T cells from systemic lupus erythematosus patients by alpha-Galactosylceramide and IL-15

CD1d-restricted invariant natural killer T cells (iNKT cells) may play an important role in the pathogenesis of systemic lupus erythematosus (SLE). Interleukin (IL)-15 is a pro-inflammatory cytokine which is over-expressed in SLE patients. In the present study, we investigated the iNKT cell expansion of mononuclear cells (MNCs) from SLE patients following 10 days’ culture with α-galactosylceramide (α-Galcer) and /or IL-15. We sought to determine the phenotypic and functional characteristics of the expanded iNKT cells compared to healthy controls and correlated with disease activity. We observed that 1. The percentages of Vα24+/Vβ11+ iNKT cells following 10-day incubation was lower in SLE groups compared to controls; 2. The percentages and absolute numbers of Vα24+/Vβ11+ iNKT cells were expanded by α-galactosylceramide (α-Galcer), and further enhanced with IL-15 in SLE patient, but the effect of IL-15 was much lower than controls; 3.IL-15 +α-Galcer expanded CD3+/CD56+ NKT-like cells from SLE patients, especially with active disease 4. The CD161+ Vα24+/Vβ11+ iNKT cells in SLE were more responsive to α-Galcer stimulation than the CD161- counterpart; 5. IL-15 decreased apoptosis of α-Galcer activated SLE iNKT cells; 6. IL-15 enhanced CD69, CD1d and CD11a expression on α-Galcer treated iNKT cells; 7. The IL-4 production of iNKT cells was decreased in SLE patients compared to controls; 8. IL-15 increased IFN-γ and IL-4 production of SLE iNKT cells; 8. IL-15 failed to augment the ability of iNKT cells to aid NK-mediated K562 cytolysis in SLE patients; 9. CD161 positivity, granzyme B and perforin expression of α-Galcer+IL-15 expanded iNKT cells correlated with C3 levels in SLE patients. Taken together, our results demonstrated numeric and functional deficiency of iNKT cells and their response to IL-15 in SLE patients. Our finding may provide insight for using adoptive iNKT cell therapy in autoimmune diseases.

Assessment of combined vaccination and immune modulation as an anti-tumour therapy

<p>Glioblastoma multiforme (GBM) is a common and lethal type of brain cancer, with a very poor prognosis. Current therapy consisting of surgical resection, radiation and chemotherapy produces a median survival of only 12-15 months. Therefore, there is a need to develop new therapeutic approaches for the treatment of GBM. This thesis investigates a new series of synthetic cancer vaccines, conjugating tumour-associated antigens (TAAs) to an isomer of ɑ-Galactosylceramide (ɑ-GalCer), a potent invariant natural killer T (iNKT) cell agonist with documented adjuvant activity. Upon antigen encounter, activated iNKT cells are capable of licensing dendritic cells (DCs) through CD40:CD40L interactions and cytokine production. The licensed DCs subsequently stimulate potent CD8⁺ T cell responses, capable of killing cancerous tissue. Conjugation of ɑ-GalCer to the TAA-derived peptide was achieved via an enzymatically cleavable linker sensitive to cathepsin B activity. This strategy allows co-delivery of the active components, with the rationale that the same DC will be able to co-present both ɑ-GalCer for iNKT cell activation, and peptide to induce an enhanced CD8⁺ T cell responses. The conjugate vaccines assessed in this thesis were able to induce iNKT cell activation and produce CD8⁺ T cell cytoxicity. However, this did not correlate with in vivo antitumour activity, as the vaccine that incorporated the TAA survivin, produced minimal cytotoxicity but potent anti-tumour responses against an implantable model of glioma. Enhancing T cell-mediated immune responses has been validated by immune checkpoint inhibition for the treatment of cancer. However, many patients do not respond to the therapy. It is thought that this subset of patients may lack pre-existing T cell responses, which are required for the efficacy of checkpoint inhibition. Therefore, there is considerable interest in whether the use of vaccines that stimulate T cell activation can improve responses to checkpoint blockade and other immune modulating drugs. The survivin vaccine was combined with the immune checkpoint blockade inhibitors ɑ-PD-1, ɑ-CTLA-4 and ɑ-LAG-3, the co-stimulatory agent a-4-1BB, or administered with T regulatory cell (TREG) depletion, to reveal the immunogenicity of the vaccine. This research revealed that combining the survivin vaccine with the immune checkpoint inhibitor ɑ-CTLA-4 improved overall survival of mice, compared to the vaccine alone. This finding suggests that this combined therapy may be a useful immunotherapeutic strategy for the treatment of GBM.</p>

Assessment of combined vaccination and immune modulation as an anti-tumour therapy

<p>Glioblastoma multiforme (GBM) is a common and lethal type of brain cancer, with a very poor prognosis. Current therapy consisting of surgical resection, radiation and chemotherapy produces a median survival of only 12-15 months. Therefore, there is a need to develop new therapeutic approaches for the treatment of GBM. This thesis investigates a new series of synthetic cancer vaccines, conjugating tumour-associated antigens (TAAs) to an isomer of ɑ-Galactosylceramide (ɑ-GalCer), a potent invariant natural killer T (iNKT) cell agonist with documented adjuvant activity. Upon antigen encounter, activated iNKT cells are capable of licensing dendritic cells (DCs) through CD40:CD40L interactions and cytokine production. The licensed DCs subsequently stimulate potent CD8⁺ T cell responses, capable of killing cancerous tissue. Conjugation of ɑ-GalCer to the TAA-derived peptide was achieved via an enzymatically cleavable linker sensitive to cathepsin B activity. This strategy allows co-delivery of the active components, with the rationale that the same DC will be able to co-present both ɑ-GalCer for iNKT cell activation, and peptide to induce an enhanced CD8⁺ T cell responses. The conjugate vaccines assessed in this thesis were able to induce iNKT cell activation and produce CD8⁺ T cell cytoxicity. However, this did not correlate with in vivo antitumour activity, as the vaccine that incorporated the TAA survivin, produced minimal cytotoxicity but potent anti-tumour responses against an implantable model of glioma. Enhancing T cell-mediated immune responses has been validated by immune checkpoint inhibition for the treatment of cancer. However, many patients do not respond to the therapy. It is thought that this subset of patients may lack pre-existing T cell responses, which are required for the efficacy of checkpoint inhibition. Therefore, there is considerable interest in whether the use of vaccines that stimulate T cell activation can improve responses to checkpoint blockade and other immune modulating drugs. The survivin vaccine was combined with the immune checkpoint blockade inhibitors ɑ-PD-1, ɑ-CTLA-4 and ɑ-LAG-3, the co-stimulatory agent a-4-1BB, or administered with T regulatory cell (TREG) depletion, to reveal the immunogenicity of the vaccine. This research revealed that combining the survivin vaccine with the immune checkpoint inhibitor ɑ-CTLA-4 improved overall survival of mice, compared to the vaccine alone. This finding suggests that this combined therapy may be a useful immunotherapeutic strategy for the treatment of GBM.</p>

Liver X receptors regulate natural killer T cell population and antitumor activity in the liver of mice

The nuclear receptors liver X receptor α (LXRα) and LXRβ are lipid sensors that regulate lipid metabolism and immunity. Natural killer T (NKT) cells, a T cell subset expressing surface markers of both natural killer cells and T lymphocytes and involved in antitumor immunity, are another abundant immune cell type in the liver. The potential function of the metabolic regulators LXRα/β in hepatic NKT cells remains unknown. In this study, we examined the role of LXRα and LXRβ in NKT cells using mice deficient for LXRα and/or LXRβ, and found that hepatic invariant NKT (iNKT) cells are drastically decreased in LXRα/β-KO mice. Cytokine production stimulated by the iNKT cell activator α-galactosylceramide was impaired in LXRα/β-KO hepatic mononuclear cells and in LXRα/β-KO mice. iNKT cell-mediated antitumor effect was also disturbed in LXRα/β-KO mice. LXRα/β-KO mice transplanted with wild-type bone marrow showed decreased iNKT cells in the liver and spleen. The thymus of LXRα/β-KO mice showed a decreased population of iNKT cells. In conclusion, LXRα and LXRβ are essential for NKT cell-mediated immunity, such as cytokine production and hepatic antitumor activity, and are involved in NKT cell development in immune tissues, such as the thymus.

The synthesis and biological evaluation of glycosphingolipids for improved cancer immunotherapy

<p>The immune system plays a crucial role in providing the first line of defence against invading pathogens such as bacteria, viruses and parasites. It is activated when immune cells known as dendritic cells (DCs) detect specific molecules that are foreign to the host, and present them to T cells. This in turn causes the activation of T cells, which marks the start of an immune response leading to the clearance of the invader. The pathogen-derived molecules recognised by the immune cells are typically peptides and their role as activators of the immune system is well established. While T cells were originally thought to only recognise peptide antigens, it is now evident that T cells are also able to recognise nonpeptide antigens. Recognition of non-peptide antigens confers protection against pathogens that have cell surfaces that are highly functionalised with carbohydrate moieties, such as glycolipids, glycopeptides and polysaccharides. Specifically, glycosphingolipids (GSLs) can activate invariant Natural Killer T (iNKT) cells via their T cell receptor (TCR) when presented by the CD1d molecule found on the surface of DCs. α-Galactosylceramide (α-GalCer 1, Figure 1), a synthetic analogue of a GSL extracted from the marine sponge Agelas mauritianus, was discovered to be a potent stimulator of iNKT cells when presented by CD1d. α-GalCer is currently being used in clinical trials as an adjuvant to boost the activation of immune cells during cancer immunotherapy. Although the molecular interaction of α-GalCer with CD1d and iNKT cells is well established, it is not fully understood how the glycolipid interacts with different subsets of DCs. Greater understanding of the fate of the glycolipid during cancer immunotherapy will provide crucial information on how the current therapy can be improved. In this thesis, the design and synthesis of two fluorescent α-GalCer probes, dansyl-α-GalCer (2, Figure 1) and BODIPY-α-GalCer (3, Figure 1) is reported. Dansyl-α-GalCer was able to activate DCs and iNKT cells in a similar fashion to the parent glycolipid α-GalCer. Its activity was CD1d-dependent and DCs that have taken up α-GalCer in vitro can be detected by flow cytometry. Unfortunately, the fluorescence of dansyl-α-GalCer was too weak to be detected by fluorescent microscopy due to photobleaching of the dye. Accordingly, another α-GalCer probe bearing a brighter fluorescent group, BODIPY, was synthesised. The α-GalCer probes were made via two synthetic strategies and the benefits and shortcomings of each synthetic route are discussed. Isoglobotrihexosylceramide (4, iGb3, Figure 2) is another GSL known to activate iNKT cells. Like α-GalCer, it is presented by DCs in the context of a CD1d molecule. iGb3 contains a sphingosine lipid backbone β-linked to a trisaccharide head group, which is in contrast to the α-linked phytosphingosine lipid found on α-GalCer. Despite the structural difference, iGb3 can stimulate iNKT cells, though to a lesser extent than α-GalCer. The intriguing activity of iGb3 provides a platform to further investigate the molecular interactions between CD1d, glycolipid and TCR of iNKT cell. The crystal structure of iGb3 in complex with mouse CD1d and TCR of mouse iNKT cell show compelling evidence that the terminal galactose moiety is crucial for the observed activity and this is attributed to the hydrogen bond between the 6´´´-OH and Thr159 on the CD1d. To unambiguously determine the importance of the hydrogen bond conferred by 6´´´-OH, 6´´´-deoxy-iGb3-sphingosine (5, Figure 2) was synthesised. 6´´´-deoxy-iGb3-sphinganine 6 was also synthesised to study the role of the double bond on the sphingosine backbone. A novel synthetic route for the synthesis of iGb3 analogues was established. This allowed for the expedient synthesis of 6´´´-deoxy-iGb3 derivatives that will subsequently be crystallised with CD1d and TCR of iNKT cell, to provide further insight into the structural requirements of β-linked GSLs. Studies have also revealed that the length and saturation of the N-acyl chain of GSLs greatly influences their activity. It is speculated that varying the length of the acyl chain affects the processing and loading of the glycolipid onto CD1d and also TCR binding affinity. To this end, the syntheses of a series of acyl chain analogues of iGb3, including the shorter chain homologue C12:0 7 (Figure 3) and the unsaturated C20:2 derivative 8 are reported. A divergent synthetic route was employed, whereby a common intermediate from the synthesis of 6´´´-deoxyiGb3 was used. This allowed for efficient syntheses of the acyl chain analogues that will facilitate a greater understanding of the structure-activity relationships. Taken together, the GSLs synthesised provide crucial insight into how they modulate the immune system and will guide future optimisation of cancer immunotherapy regimes.</p>

The Synthesis of Carbohydrates for the Treatment of Disease

<p>In this thesis I investigated two aspects of glycobiology. In the first, I investigated the potential of α-GalCer analogues to be used in cancer immunotherapy. Two 4-deoxy α-GalCer analogues, with either a sphinganine or a sphingosine base, were synthesised using a convergent strategy. The α-GalCer sphinganine derivative was synthesised in 14 steps from D-arabinose, and in an overall 13% yield. The α-GalCer sphingosine analogue was synthesised in 13 steps also in 13% yield. Biological analysis revealed that both 4-deoxy analogues possessed comparable activity to α-GalCer in mice, however demonstrated significantly reduced hNKT cell activity. The reduced activity was attributed to species-specific differences in iNKT cell glycolipid recognition rather than reduced CD1d presentation. From these results we suggest that glycolipids developed for potent CD1d-iNKT cell activity in humans should contain a ceramide base with the 4-hydroxyl present. The second part of this thesis focused on protecting group free methodology for the synthesis of sugar mimetics that have proven potential as glycosidase inhibitors. In this work I developed an efficient, high yielding and diastereoselective strategy for the synthesis of a number of five and six membered azasugars. This strategy utilises two novel reaction methodologies. The first enabled the stereoselective formation of cyclic carbamates from olefinic amines, the transition states controlling the stereoselectivity during this reaction are discussed. The second reaction facilitated the synthesis of primary amines without the need for protecting groups, the scope of this reductive amination methodology is also investigated. The five membered azasugars 1,4-dideoxy-1,4-imino-Dxylitol, 1,4-dideoxy-1,4-imino-L-lyxitol, 1,4-dideoxy-1,4-imino-L-xylitol and 1,2,4-trideoxy-1,4-imino-L-xylitol were prepared in 5 steps, in good overall yields (57%, 55%, 54% and 48% respectively), and without the need for protecting groups. The six membered azasugar DGJ was prepared over six steps in 33% yield using similar methodology. The synthesised compounds were also tested for anti-tubercular activity using a BCG alamar blue assay.</p>