DECLARATION OF ALL CONTRIBUTIONS TO THE LITERATURE REVIEW
The original concept of this research project was devised by Professor Jamie Rossjohn, my supervisor and head of the Rossjohn Lab : Centre for Advanced Molecular Imaging at Monash University, which focuses on molecular interactions in immunity. Draft of the literature review was written by myself and edited by Dr. Jérôme Le Nours, my co-supervisor. Other published materials taken and cited for the content of this literature review was credited to the original authors through the in-text citations and reference list.
Introduction
1. Human Immune Surveillance
Our immediate environment poses evidently various potential threats to our body and those include a variety of harmful substances such as pathogens (bacteria, viruses, and parasites).1 Thus, we require a rigorous system of defence to protect our body and prevent unnecessary harm.1 Our immune system has consistently and remarkably adapted to protect itself from those pathogenic threats in order to maintain our wellbeing.1 To achieve this our immune system comprises a complex network of cells, organs, tissues, proteins, and molecules (Figure 1) that collectively constitutes the primary line of defence against various pathogens.1 Typically our immune system has evolved into two major interconnected types of defence mechanism to counter these microbial threats that consist of an innate and an adaptive response.1, 2
The innate immunity is often described as the “first line of defence” because it provides a rapid and non-specific response upon encountering a foreign entity.2 However, it does not have the ability to generate immunologic memory, unlike its complementary associate.2 The weaponry of the innate arm comprises physical epithelial barriers, mast cells, Natural Killer cells (NK), leukocytes (e.g. neutrophils), and macrophages (Figure 1).2, 3 The adaptive immune response is specific and develops after being exposed to pathogens-derived molecules (antigens or Ag), encompassing proteins, lipids, metabolites, and carbohydrates.2 Two key immune cell populations (T- and B-cells) within the adaptive arm of the immune system recognise such antigens (Figure 1).2 The B-cells generate antibodies that interact with antigens in their intact form, whereas the T-cells possess receptors on their cell surface named T-cell receptors (TCRs) that recognise fragments of antigens. In T-cell immunity, fragments of antigens are presented on the cell surface of antigen presenting cells (APCs) by specific antigen-presenting molecules that include the Major Histocompatibility Complex molecules (MHC).2 Among the conventional T-cell subsets, there exists typically two main groups that comprise the CD8+ killer T-cells and CD4+ helper T-cells. Both CD8+ and CD4+ T-cell populations recognize peptide-derived antigens that are presented by the highly polymorphic MHC class I and II molecules, respectively.2 However, in recent years, two additional unconventional T-cell populations termed as Natural Killer T-cells (NKT) and Mucosal-associated invariant T-cells (MAIT) have been identified and characterized.4 NKT cells play key roles in T- cell mediated immunity by bridging the gap between the innate and adaptive immune systems (Figure 1) functioning as ‘innate-adaptive hybrids’ and they are also implicated in a wide range of diseases or conditions including allergy, autoimmunity, and more importantly tumour immunity (cancer).5, 6 They endow profound immunomodulatory potential and therefore can either enhance or suppress an immune response.7, 8 Thus, harnessing NKT cells would be of great benefit in designing immunotherapeutic targets in the treatment of various human diseases such as cancer.
Figure 1. Diagram of the two arms of immune response: Innate and Adaptive. The diagram describes the population of immune cells within each immune defensive arm, with the Natural Killer T cells (NKT cells) bridging the two immune responses. From Dranoff G., Nat. Rev, 2004.9
2. Natural Killer T-Cells (NKT)
NKT cells represent a potent distinct subtype of T-cells that can induce a broad array of immune responses.7 They originated and developed from the thymus before maturing in the periphery and start to fulfil their immune functions by recognizing specific antigens and releasing cytokines (Figure 2).7 They share a characteristic feature of the NK cells by expressing the NK cell surface markers CD161 (in humans) and NK1.1 (in mice).10, 11 However, they differ from regular NK cells by also expressing T-cell surface receptors (TCRs), a common attribute of the classical αβ T-cell lineage.11
Figure 2. Diagram of the NKT cell interactions with various immune cells. NKT cells will recognize antigens and release cytokines to stimulate various immune responses. From Brennan PJ. et al., Nature Reviews Immunology, 2013.7
2.1 NKT T-cell Receptors (NKT TCR)
During the process of T-cell development and positive selection in the thymus, the generation of functional TCRs is achieved by random rearrangement of α- and β-chains (Figure 3A). The αβ TCR is an heterodimer composed of an α- and β-chain, each chain is composed of a variable (V) and a constant (C) domain that adopt an immunoglobulin-like architecture (Ig like) (Figure 3B). The V domain in the TCR α-chain comprises of two regions encoded by the variable (V; TRAV) and joining (J; TRAJ) gene segments, whereas the TCR β-chain V domain consists of three regions encoded by variable (V; TRBV), diversity (D; TRBD) and joining (J; TRBJ) gene segments.12 In each chain, within the variable domain comprise three complementary-determining regions (CDRs): CDR1α, CDRα2, CDR3α (α- chain), CDR1β, CDR2β and CDR3β (β-chain) that define the antigen binding locus of the TCR (Figures 3B and 3C).13 Specifically, the CDR1 and CDR2 loops of the α- and β- chains are encoded within the TRAV and TRBV regions, respectively, whilst the CDR3 loop is encoded at the junction of TRAV and TRAJ regions in the TCR α-chain or TRBV, TRBD and TRBJ regions in the TCR β-chain.14 The diversity of the TCR repertoire is due to the generation of different germline-encoded CDR1, CDR2 gene sequences in combination with varied non-germline encoded CDR3 gene segments as a result of somatic recombination event.15 As such, a combinatorial diversity as a consequence of various permutations and combinations of V, D and J gene sequences and a junctional diversity due to the lack of precision and inclusion of non-templated-encoded nucleotides at V(D)J junctions results in TCR diversification.16, 17
Figure 3. Illustration of the genetic mapping and structural properties of NKT TCR. A) Schematic representation of somatic recombination of gene segments of the α- and β-chains of a TCR. Adapted from Turner et al., 2006.18 ; B) 3D structure of an NKT TCR, CDR loops in the variable segments are labelled; C) A schematic representation of the structure of TCR.
Here, the NKT TCRs are responsible for the molecular recognition of lipid-based antigens that are presented by the non-polymorphic antigen presenting MHC class I-like molecule, CD1d.11, 19 Upon recognition of the lipid-based antigens by the NKT TCRs, the NKT cells are rapidly activated leading to the secretion of an array of pro- inflammatory (Th1) cytokines such as interferon-γ (IFNγ) and tumour necrosis factor (TNF); and anti-inflammatory (Th2) cytokines including interleukin-4 (IL-4), IL-10 and IL-13.20, 21 Based on the TCR repertoire usage and antigen specificity, NKT TCRs can be classified into two main classes that are named type I and type II NKT TCRs.11, 14 And more recently, additional subsets of NKT TCRs named “atypical” TCRs have also been identified and characterized (Table 1).22, 23
Table 1. Classification and characteristics of NKT cells. Adapted from Macho-Fernandez and Brigl, Frontiers in immunology, 2015.24
2.1.1 Type I iNKT TCR
The type I NKT TCR uses an invariant α-chain that pairs with a limited subset of β-chains and hence is known as invariant NKT TCR (iNKT). In humans, type I NKT TCR expresses TRAV*10+ TRAJ*18+ (Vα24-Jα18) TCR α-chain and pair with TRBV*25-1+ (Vβ11) β-chain, whereas in mice the orthologous chain expresses TRAV*11+TRAJ*18+ (Vα14Jα18) α-chain pairing with the limited subset of TCR β-chains (TRBV*13 (Vβ8.2), TRBV*29 (Vβ7), or TRBV*1 (Vβ2).14, 25 Type I NKT TCRs can also be defined by their ability to recognize α-Galactosylceramide (α-GalCer) (FIGURE 4), a glycosphingolipid from the marine sponge Agelas mauritianus, thus makes α-GalCer a prototypical ligand for the activation of type I NKT cells.26, 27
Figure 4. The chemical structure of the prototypical lipid ligand α-Galactosylceramide (α-GalCer). From Schmieg J. et al., The Journal of Experimental Medicine, 2003.28
2.1.2 Type II NKT TCR
Unlike, type I TCR, type II NKT TCRs expresses a more diverse TCR gene repertoire and are also defined by their inability to recognize α-GalCer.14, 29 However, type II NKT TCRs are responsive towards a sulphated glycolipid antigen named “sulphatide” (found in neuronal tissues).14 As previously stated, the TCR were observed to have a wide range of variety but they tend to be more biased towards both α- and β- chains V segments: Vα3, Vα8, Vβ3 and Vβ8; it also exhibits a highly conserved CDR3B region.14, 29 One of the first analogue of the type II NKT cell in mice is a hybridoma called XV19.29 The XV19 TCR’s gene arrangements of the α-chain is TRAV7D-4*02–TRAJ26*01 (Vα1-Jα26) with its CDR3α expressing a CAASEQNNYAQGLTF sequence.29 Meanwhile, the β-chain expresses TRBV3-TRBD2-TRBJ2.1 (Vβ16-Dβ2-Jβ2.1) with its CDR3β demonstrated a CASSFWGAYAEQFF sequence.29
2.1.3 Atypical type I NKT TCRs
Another type of NKT cell is considered the ones that does not fit the population of NKT cell type I and II. These atypical NKT cells have been found to recognize glycolipid antigens such as α-GalCer, presented by the antigen presenting molecule: CD1d.22, 23 This fact corresponds the type I NKT for its specificity towards glycolipid antigens presented by CD1d. Nevertheless, the TCR chain gene expressions are varied from one atypical NKT TCR to another; moreover, there have only been a handful of discovered types up until now. Examples of these are atypical NKT TCRs that expresses TRAV21-TRAJ8-TRBV7–8, TRAV12-3-TRAJ27-TRBV6-, and a canonical Vα10-Jα50 TCR α-chain that has a stronger affinity with a microbial glycolipid antigen namely, α-glucosylceramide (α-GlcCer).22, 23 These novel discoveries of diverse TCR chains confirms the hypothesis of specific antigen-docking mode and specificity for respective structure of TCR chains.23
3. The Major Histocompatibility Complex (MHC)
3.1 MHC class I and II
The Major Histocompatibility Complex (MHC) molecules are highly polymorphic cell surface receptors that are expressed on most of the immune cells.26, 30 They have been classified into two main groups: the MHC class I and Class II (MHC I and II), and form the most polymorphic gene clusters in the genome of mice and humans. In humans, MHC genes are located on the chromosome 6 and encode for over 100 HLA-I (Human Leukocyte Antigen) and HLA-II genes (FIGURE 5).31 The MHC I and II antigen presenting receptors present peptide antigens to CD8+ and CD4+ T-cells, respectively, and play a critical role in the adaptive arm of our immune system.2 The remarkable allelic polymorphism exhibited by MHC ensures a “defence grid” such that no microbes can evade the immune surveillance.31 Besides pathogen protection, the allelic polymorphism in MHC molecules can have a vast influence on the various physiological conditions such as TCR selection, development, tumour immunity and autoreactivity.31, 32
Figure 5. Genomic arrangements of the human MHC. Class I and Class II MHC is encoded within the purple and green area respectively. From Adams EJ. et al., Annual Review Immunology, 2013.26
For a long time, it was assumed that T-cell mediated immunity was solely driven by the peptide antigens presented by MHC class I and II molecules. However, more than 20 years ago, a new and distinct family of antigen presenting molecules known as Cluster of Differentiation 1 (CD1) gene family studies was identified.33
3.2 MHC I-like molecules: The Cluster of Differentiation 1 (CD1) family
The CD1 family of genes consists of five CD1 isoforms (CD1A, B, C, D and E) in humans and two orthologues of CD1D in mice.34 . In humans and mice, the CD1 genes are encoded on chromosome 1 and 3 of their respective genomes and are therefore located outside the gene locus of the classical MHC I and II molecules (Figure 5).33 Based on the genetic arrangement and expression pattern, the human CD1 genes have been classified into three main groups: the group 1 (CD1A, B, and C), group 2 (CD1D) and group 3 (CD1E).35-39 Furthermore, as opposed to MHC I molecules, CD1 molecules exhibit lower allelic polymorphism and present lipids as cognate antigens for T-cell recognition and activation.35-39 Structurally, the CD1 glycoproteins share the overall architecture of the MHC I molecules whereby an heavy chain that is composed of three extracellular domains (α1-, α2- and α3- domains) are associated non-covalently to the β2-microglobulin (β2m) molecule (Figure 6B).35-39 However, the α1- and α2-domains that are formed by two helices form an hydrophobic binding groove that accommodates the lipid-based antigens as opposed to the more hydrophilic peptide antigens binding groove of the MHC class I. In general, the majority of lipid-based Ags are amphipathic molecules with polar head groups (carbohydrate, peptide, inorganic ester) that are linked to lipid anchors formed from fatty acids or other aliphatic hydrocarbon chains.35-39 Typically, the CD1 molecules present these lipid-based Ags by sequestering the lipid anchors within the hydrophobic cleft, with each type of human CD1 protein possessing a highly distinct Ag-binding cleft (Figure 6A).40 For instance, the CD1a and CD1b glycoproteins have a different structural architecture.40 The clefts are markedly different in molecular volume (1280 vs 2230 Å3), the number of named pockets (2 or 4) and the connectivity of individual pockets.35, 38, 41 For example, CD1a has one linear “tube” comprised of two pockets (A’ and F’) while CD1b has four named pockets (A’, F’, C’ and T’) that are broadly interconnected (Figure 6A).35, 38, 41
Figure 6. Illustrations of the binding grooves and the molecular structure of CD1 family. A) Schematic representations of the binding grooves of the family of CD1 molecules. From Pereira et al., Journal of Immunology Research, 2016.40 ; B) The three-dimensional crystal structure of the mouse CD1d glycoprotein. From Barral DC. et al., Nature Review Immunology, 2007.42
Whilst the group 1 CD1 molecules present a wide variety of self- and microbial lipids that are recognized by some identified T cell subsets e.g. “germline-encoded mycolyl lipid-reactive” (GEM) T cells for CD1b, the CD1d glycoprotein presents lipid-based antigens that are recognized by iNKT cells.43, 44 More recently, another distinct human T cell subset termed as γδ T cells was also shown to recognize CD1d in a lipid antigen dependent manner.45, 46 Surprisingly, these cells in contrast to iNKT and type II NKT cells can recognize the lipid antigens α-GalCer and sulfatide.47-49 Structurally, the CD1d glycoprotein was the first three-dimensional crystal structure to be determined in the CD1 group of antigens presenting molecules (Figure 6B) and revealed that the α1- and α2-helices have evolved to form an hydrophobic groove (~1750 Å3) that is composed of two distinct pockets named A’- and F’-pockets (Figure 6A).36, 39
4. Molecular recognition of CD1d-lipid-based antigens by NKT TCRs
4.1 Type I NKT TCR molecular recognition
The ternary crystal structure of a human type I NKT TCR in complex with CD1d presented the prototypical type I lipid antigen α-GalCer provided the first insights into the molecular mechanism that underpins the recognition of CD1d-lipid Ags by a type NKT I TCR.48 The ternary structure revealed a complete distinct docking mode relative to the ones observed in the TCR-MHC-peptide ternary complexes.48 Here, the type I NKT TCR adopted a parallel mode of docking whereby the TCR positioned over the F’-pocket of the CD1d binding groove. At the NKT TCR/CD1d-α-GalCer interface, the CDR loops of the TCR α-chain dominated the molecular interactions.48 The galactose head group of the α-GalCer antigen protruded out the CD1d binding cleft and was exclusively contacted by residues belonging to the CDR1α and CDR3α loops.14, 48 The NKT TCR β-chain contribution to the molecular TCR/CD1d-α-GalCer interface was more limited and the CDR2β loop dominated the interactions with the human CD1d.48 Importantly, all the subsequent mouse and human type I NKT TCR CD1d-lipid Ags ternary complexes crystal structures revealed a similar recognition strategy (Parallel docking mode of the TCR over CD1d).14, 50, 51 Collectively, those structural insights revealed that the type I NKT TCRs function like an innate-like pattern recognition receptors that engage the lipid-based antigens in a conserved mode of molecular recognition that is dominated by the germline encoded CDR loops region.
4.2 Type II NKT TCR molecular recognition
The ternary crystal structures of the mouse XV19 type II NKT TCR in complex with CD1d presenting the sulfatide/lysosulfatide lipid antigens provided the first molecular insights into the recognition of CD1d presenting lipid-based antigens by type II NKT TCRs.29, 49 The three-dimensional structures revealed a novel and distinct recognition strategy relative to the well-characterized type I NKT TCRs mode of recognition. Here, the type II NKT TCR docked orthogonally over the extreme end of the A’-pocket of the CD1d binding cleft whereby the α- and β-chains of the XV19 TCR contributed equally to the interactions at the CD1d-lipid/TCR binding interface.29, 49 The non-germline encoded CDR3α and CDR3β dominated the molecular interactions. 29, 49 This clearly contrasted to what has been observed in the type I NKT TCR-CD1d-Ags ternary complexes structures whereby the TCR α-chain and the germline encoded CDR loops were the key contributors to the molecular interactions at the CD1-lipid/TCR interface.51 Thus, the XV19 NKT TCR-CD1d-lipid Ags ternary crystal structures provided immediate molecular insights into the mechanism that underpins the recognition of lipid-based Ags by type II NKT TCR and highlighted the contrasting mode of recognition of the type I and type II NKT TCRs of lipid-based antigens presented by CD1d.
Figure 7. Three-dimensional illustration describing interaction of type I NKT-TCR-α-GalCer-CD1d complex (top). A) Structure of the general complex demonstrating how each component is geometrically placed. B) A more detailed depiction of how CDR loops are suspended around the heavy chain α helices of CD1d and the antigen presented (dashed lines portrays α-GalCer that is placed parallelled with the α chains). C) A 3D footprint picturing attachment of antigen with the CDRs of TCR and enclosing CD1d structure. From Rossjohn J. et.al., Nature Review Immunology, 2012.14 Three-dimensional illustration portraying the interaction of type II XV19 NKT-TCR-sulfatide-CD1d complex (bottom). D) A picture of the common ternary structure of the complex. E) An amplified image of the structure around the pockets of CD1d where the CDR loops will interact and coil around structures of sulfatide and CD1d (the dashed lines describes where Vα and Vβ domains of the TCR mainly sits). F) A 3D footprint illustrating the attachment of sulfatide with The CDR loops inside the CD1d structure. From Patel O. et al., Nature Immunology, 2012.29
5. Tumour T-cell mediated immunity
According to the Global Cancer Statistics of 2012, cancer was responsible for the death of 8.2 millions people all over the world, and 14.1 millions of cancer cases were diagnosed that year.52 The implication of NKT cells in tumour immunity was first discovered whereby studies demonstrated that NKT cells were activated in the presence of the prototypical lipid-based Ag, α-GalCer (Figure 8A), that resulted in the inhibition of tumour growth in the B16-bearing mice model.53, 54 Type I iNKT cells promote tumour immunity and is dependent on its Th1-type cytokine IFN-γ production, whereas type II NKT cells suppressed tumour immunity through IL-13 secretion.21 It has also been shown that iNKT cells are implicated in natural tumour immunosurveillance without requiring the introduction of exogenous antigens such as α-GalCer.21 For instance, using CD1d knockout (KO) mice that then lacks all NKT cells, and Jα18 KO mice (no iNKT cells and significant less diversity in the TCR α-chain repertoire), tumour susceptibility was observed in methylcholanthrene induced tumour model. Protection was then restored by adoptive transfer of purified iNKT liver cells.21, 55, 56 Interestingly, in individuals that suffer from prostate cancer and solid tumours, it was shown that circulating iNKT cell frequency declined significantly that the production of IFN-γ was subsequently lowered providing further evidence of their role in tumour immunity.57-59
6. Tumour lipid-based antigens: The gangliosides and fucosylceramides families
There are several lipid-based antigens that are structurally analogous to the α-GalCer that is originally tumour-derived and are important targets for NKT cells. Two of these tumour Ags are called Ganglioside and Fucosylceramide. Gangliosides and Fucosylceramides are group of antigens that are widely and aberrantly expressed by tumours, such as in the case of several types of cancer.60, 61 They are agonist ligands for NKT TCR that activates the anti-tumour or tumour suppression immune response from the inflammatory cascade generated by NKT cells.60, 61
Gangliosides are a distinct type of glycosphingolipids (GSLs) because structurally they contain sialic-acid (SA) components, and variations of these will determine the class of ganglioside based on the amount of its SA chains embedded in the hydrophobic head group.60 Their glycan head group are often more flexible and can undergo conformational changes.60 As an analogue of α-GalCer, gangliosides are also a fundamental molecule for cell recognition and signalling that can elicit a wide range of response.60 This is due to their ability to interact with both sugars and protein molecules because of their chemical properties (Figure 8B).60 Recent findings indicate that the gangliosides are overly expressed in the presence of malignant cells, and from this we know that it may be used as tumour markers.60 Although, the potent class of gangliosides that will induce immunostimulatory events are still under research.60
The fucosylceramides are also molecules derived from numerous types of cancerous cells; the first to be discovered is from adenocarcinoma.61 They are proven to be antigenic and are fundamental in the biological activity of tumour cells.61 Moreover, when tested in mice, there are evidence of fucosylceramide that elicits IFNγ and lymphocyte proliferation; this is further proof of its immunostimulatory capabilities.61 When tested for its capacity to activate NKT cells, it shows a considerable power to stimulate response, although less adequate when compared to α-GalCer.61 Looking at the chemical structure, their head group is much more simpler than that of gangliosides (Figure 8C). But generally, they both possess a lipid polar head group and a lipid aliphatic tail group.
Figure 8. Chemical structures of lipid-based antigens. Mainly, the structures consist of a lipid polar head group (shaded in blue) and a lipid aliphatic tail group (shaded in green) A) A prototypical lipid-based Ag derived from marine sponges called α-GalCer, generally presented by CD1d and recognized by type I NKT TCR. Adapted from Kaer and Luc Van, Nat Rev Immunol, 2005.62 B) One of the tumour lipid-based Ag known as Ganglioside, class GM1. Adapted from Krengel U., et al. Frontiers in Immunology, 2014.60 C) Another tumour lipid-based Ag known as Fucosylceramide; the image above illustrates a type of fucosylceramide: α-FucosylCeramide. Adapted from Veerapen N, et al., Immunol Cell Biol,2010.61
PROJECT OUTLINE
Background
In T-cell mediated immunity, an important goal is to understand how our immune T-cells can recognize external threats and therefore act as the body’s main line of defence. This knowledge is crucial because if the process is unravelled, we may be able to utilize their characteristics and create a potent and effective immunotherapy. The human immune system itself is already equipped with two defensive arms, (innate and adaptive) in order to recognize and eliminate harmful entities in the body that include cancerous or tumour cells. NKT cells are part of both arms and have been implicated in tumour immunity by recognizing tumour lipid-based antigens that are aberrantly expressed by a variety of cancer cells. Although we already know that the NKT cells can be stimulated by lipid-based antigens that are specifically presented by the MHC class I-like CD1d, there is very little known fact on the molecular presentation of tumour lipid-based antigens by CD1d and their subsequent recognition by NKT TCRs. Therefore, getting the molecular insights into the recognition and presentation mechanisms may be highly beneficial in order to design the immunotherapeutics for cancer.
Research Question
What are the molecular mechanisms that underpin the recognition of tumour lipid-based antigens?
Aims and Hypothesis
Aims 1: To gain molecular insights into the presentation of tumour lipid-based Antigens (Gangliosides and Fucosylceramides) by the CD1d molecule.
Hypothesis 1: The polar head group of the tumour Antigen will be exposed for TCR recognition whilst the aliphatic tails will be embedded in the hydrophobic CD1d binding groove.
Aim 2: To assess the responsiveness (affinity measurements by Surface Plasmon Resonance) of CD1d presenting tumour lipid-based Antigens (Gangliosides and Fucosylceramides) against a panel of NKT TCRs (Type I and “atypical” TCRs) available in the laboratory.
Hypothesis 2: The NKT TCRs will display fine specificity towards the tumour lipid-based Antigens.
Aim 3: To gain molecular insights into the recognition of tumour lipid-based Antigens by type I NKT TCR.
Hypothesis 3: Recognition strategy may be similar with NKT TCR type I.
Methodology
The research includes methods associated with biochemical and biophysical technique including protein production, expression, and purification, Surface Plasmon Resonance (SPR), and X-ray crystallography. The first step is to produce the proteins that are needed in the project (CD1d and NKT TCR); CD1d using baculovirus insect cells protein expression system, while NKT TCR uses E.coli. After both protein samples are obtaines, contaminants needed to be discarded in order to get a pure protein. Thus, these samples will undergo protein purification process using various chromatography methods (Ni/NTA Chromatography, Size Exclusion Chromatography, Ion Exchange Chromatography). Then, the lipid antigen of interest (tumour lipid) will be loaded into the CD1d protein using “in vitro” lipid loading techniques. Meanwhile, several different NKT TCR samples will be tested for their affinity using the Surface Plasmon Resonance (SPR). Finally, both proteins will be crystallized and sent to the Australian Synchroton where the protein crystals will be exposed to intense x-ray beam, resulting in a diffraction pattern. This diffraction pattern will be reconstructed back into the final 3D crystal structures using computerized technique.
Expected Outcomes
• The study will provide the molecular basis for the presentation of tumour lipid antigens by CD1d and their recognition by NKT TCR.
• The study will provide a greater understanding of the fine specificity of the human NKT TCR towards tumour antigens.
• The study may ultimately yield to novel reagents and approaches for tumour immunotherapy.