Periodic Reporting for period 4 - Crosstag (Unravelling cross-presentation pathways using a chemical biology approach)
Periodo di rendicontazione: 2019-11-01 al 2020-04-30
Immune therapies are currently being pursued to reinvigorate the immune reaction against tumours. This is not trivial, as the right cytotoxic T-cells must be activated against a tumour-specific antigen to get the right response; all in the face of a tumour attempting to switch the resulting immune response back off.
One method for enhancing the cytotoxic anti-tumour response is by targeting receptors on certain immune cells that can result in the enhanced activation of these cytotoxic T-cells. One of these is the mannose receptor. However, this glycoprotein-binding receptor appears to have two functions: it can either potently enhance cytotoxic T-cell activation, or, in certain cases, reduce the activation of these T-cells when a vaccine is targeted towards it. Current tools, such as anti-MR antibodies and randomly glycosylated ligands fail to selectively enhance cytotoxic T-cell activation, as it appears that the receptor can distinguish between subtle differences in sugar coatings on the proteins it enhances.
The main aim of this proposal is to determine what structural parameters of the glycoprotein antigen result in this enhanced cytotoxic T-cell activation. Furthermore, using new imaging techniques based on super resolution microscopy, it is the aim of the proposal to look at how the cells activating the cytotoxic T-cells treat vaccines differently in case of these different vaccines.
The outcome of this proposal will hopefully be an improved understanding of how we can enhance this cytotoxic T-cell response. This, in the face of the recent breakthroughs in cancer immunotherapy, will lead to better, cheaper immunotherapies for cancer.
One method for enhancing the cytotoxic anti-tumour response is by targeting receptors on certain immune cells that can result in the enhanced activation of these cytotoxic T-cells. One of these is the mannose receptor. However, this glycoprotein-binding receptor appears to have two functions: it can either potently enhance cytotoxic T-cell activation, or, in certain cases, reduce the activation of these T-cells when a vaccine is targeted towards it. Current tools, such as anti-MR antibodies and randomly glycosylated ligands fail to selectively enhance cytotoxic T-cell activation, as it appears that the receptor can distinguish between subtle differences in sugar coatings on the proteins it enhances.
The main aim of this proposal is to determine what structural parameters of the glycoprotein antigen result in this enhanced cytotoxic T-cell activation. Furthermore, using new imaging techniques based on super resolution microscopy, it is the aim of the proposal to look at how the cells activating the cytotoxic T-cells treat vaccines differently in case of these different vaccines.
The outcome of this proposal will hopefully be an improved understanding of how we can enhance this cytotoxic T-cell response. This, in the face of the recent breakthroughs in cancer immunotherapy, will lead to better, cheaper immunotherapies for cancer.
The following results were achieved during the project.
On the one hand a library of biologically traceable single glycoform ligands are being synthesised that target different aspects of the biology of the mannose receptor. - with controlled variation in glycan nature, stoichiometry and positioning - for the MR and study differences in uptake, routing and antigen presentation. We have made a series of different antigens that contain bioorthogonal groups for imaging. We have made some cancer long peptide vaccines, as well as model protein antigens, and proteins related to auto-immune emergence. We have also made a series of carbohydrates targeting different binding domains of the carbohydrate receptors and constructs of these antigens modified with some of these sugars. Immunological evaluation of these proteins yielded some surprising and exciting results. The protein antigens modified with multiple bioorthogonal groups could be used to track the antigen without affecting the handling of the protein. The immunological properties of the peptide antigens were rather eratic. This lead to the hypothesis that factors, such as peptide solubility and aggregation-proneness, played key roles in the antigenicity of the antigens. Site specific glycosylation of these peptides altered antigen presentation and cross-presentation in a lectin-independent manner further supporting this hypothesis.
These results were further compounded when we focused the technique on the study of the multiple sclerosis-autoantigen myelin oligodendrocyte glycoprotein (MOG). When we made peptides of this antigen that carried either glycans at the native glycosylation site and/or citrullines in the dominant T-cell epitope, we found that some of these peptides aggregated in an amyloid like fashion. This could be in part rescued by glycosylation, but only for specific glycans. The aggregates that formed from these constructs were also toxic to microglia and neurons, which could explain some of the pathology observed in primary progressive multiple sclerosis.
The 2nd aim of the proposal was to develop a new imaging technique that will allow the subcellular tracking of the antigens as it being handled differently by the receptors. For this we have now developed an approach that allows the super-positioning of a super-resolution microscopy image of the bioorthogonal antigens on an 75 nanometer thin section of a cell and place it on top of an electron microscopy image of this same thin section. This technique called bioorthogonal correlative light electron microscopy allows us to get the most information on the location of antigen as it is being processed in the cell. We first optimised it (for technical reasons) to show the degradation and processing of bacteria in immune cells. This part of the project has allowed to show e. coli degradation, but also the persistence of salmonella in intracellular vacuoles. We have now even used the technique to show the effect of various drug cocktails on the intracellular survival of the tuberculosis bacillus in immune cells. In this latter manuscript - which is currently under review - we show that multiple different click reactions can be combined in this technique, and that the approach is compatible with classical labelling strategies, such as antibody and fluorescent protein labelling. We have also produced the first example of the combination of this technique with super-resolution microscopy.
On the one hand a library of biologically traceable single glycoform ligands are being synthesised that target different aspects of the biology of the mannose receptor. - with controlled variation in glycan nature, stoichiometry and positioning - for the MR and study differences in uptake, routing and antigen presentation. We have made a series of different antigens that contain bioorthogonal groups for imaging. We have made some cancer long peptide vaccines, as well as model protein antigens, and proteins related to auto-immune emergence. We have also made a series of carbohydrates targeting different binding domains of the carbohydrate receptors and constructs of these antigens modified with some of these sugars. Immunological evaluation of these proteins yielded some surprising and exciting results. The protein antigens modified with multiple bioorthogonal groups could be used to track the antigen without affecting the handling of the protein. The immunological properties of the peptide antigens were rather eratic. This lead to the hypothesis that factors, such as peptide solubility and aggregation-proneness, played key roles in the antigenicity of the antigens. Site specific glycosylation of these peptides altered antigen presentation and cross-presentation in a lectin-independent manner further supporting this hypothesis.
These results were further compounded when we focused the technique on the study of the multiple sclerosis-autoantigen myelin oligodendrocyte glycoprotein (MOG). When we made peptides of this antigen that carried either glycans at the native glycosylation site and/or citrullines in the dominant T-cell epitope, we found that some of these peptides aggregated in an amyloid like fashion. This could be in part rescued by glycosylation, but only for specific glycans. The aggregates that formed from these constructs were also toxic to microglia and neurons, which could explain some of the pathology observed in primary progressive multiple sclerosis.
The 2nd aim of the proposal was to develop a new imaging technique that will allow the subcellular tracking of the antigens as it being handled differently by the receptors. For this we have now developed an approach that allows the super-positioning of a super-resolution microscopy image of the bioorthogonal antigens on an 75 nanometer thin section of a cell and place it on top of an electron microscopy image of this same thin section. This technique called bioorthogonal correlative light electron microscopy allows us to get the most information on the location of antigen as it is being processed in the cell. We first optimised it (for technical reasons) to show the degradation and processing of bacteria in immune cells. This part of the project has allowed to show e. coli degradation, but also the persistence of salmonella in intracellular vacuoles. We have now even used the technique to show the effect of various drug cocktails on the intracellular survival of the tuberculosis bacillus in immune cells. In this latter manuscript - which is currently under review - we show that multiple different click reactions can be combined in this technique, and that the approach is compatible with classical labelling strategies, such as antibody and fluorescent protein labelling. We have also produced the first example of the combination of this technique with super-resolution microscopy.
We have progressed beyond the state of the art and the proposal w.r.t. the imaging method. The initial plan was to only look at super-resolution images of the antigen. However, by now being able to combine it with electron microscopy, we not only obtain information on the antigen, even as it is being degraded, we can place it in the context of the organelles in the cells where these antigens are found.
By the end of the project, I hope to have met all goals, and imaged the different routes that antigens take depending on the glycan-mannose receptor interaction; both for cancer vaccine antigens, as well as for auto-immune antigens.
By the end of the project, I hope to have met all goals, and imaged the different routes that antigens take depending on the glycan-mannose receptor interaction; both for cancer vaccine antigens, as well as for auto-immune antigens.