Periodic Reporting for period 4 - DC_Nutrient (Investigating nutrients as key determinants of DC-induced CD8 T cell responses)
Reporting period: 2022-11-01 to 2024-04-30
A new immunoregulatory axis has emerged in recent years demonstrating that cellular metabolism is crucial in controlling immune responses. This regulatory axis is acutely sensitive to nutrients that fuel metabolic pathways and support nutrient sensitive signalling pathways. My recent research demonstrates that nutrients are dynamically controlled and are not equally available to all immune cells. However, studying nutrient availability and uptake with single cell resolution is hampered by limitations in technology. This research programme will generate innovative new technologies to measure the local distribution of nutrients, including amino and fatty acids, to be visualised and quantified within single cells. These technologies will pioneer a new era of in vivo nutrient analysis. Nutrient deprivation of antigen presenting DC will then be investigated (using our new technologies) in response to various stimuli within the inflammatory lymph node and correlated to CD8 T cell responses. We will generate state-of-the-art transgenic mice to specifically knock-down nutrient transporters in DC to prove that the availability of these nutrients to antigen presenting DC is a key mechanism for controlling CD8 T cells responses. This would be a paradigm shifting discovery that would open new horizons for the study of nutrient-regulated immune responses.
This work is important for society because understanding when and where nutrients are limiting or in abundance is important in the development of new immunotherapeutic strategies.
The overall objectives of this current study are:
(1) Develop and validate innovate single-cell approaches for measuring nutrient uptake in vivo.
(2) Investigate nutrient availability to DCs in inflammatory LNs and associated CD8 T cell responses.
(3) Prove nutrient deprivation of antigen presenting DC regulates CD8 T cell responses in vivo.
This work is important for society because understanding when and where nutrients are limiting or in abundance is important in the development of new immunotherapeutic strategies.
The overall objectives of this current study are:
(1) Develop and validate innovate single-cell approaches for measuring nutrient uptake in vivo.
(2) Investigate nutrient availability to DCs in inflammatory LNs and associated CD8 T cell responses.
(3) Prove nutrient deprivation of antigen presenting DC regulates CD8 T cell responses in vivo.
Objective 1:
--We have developed and validated a novel single cell assay that accurately reports upon the uptake through the primary glutamine transporter Slc1a5 using flow cytometry and confocal imaging, using bioorthogonal (CLICK) amino acid analogues.
The process of developing this new technology has brought us a new appreciation of the specificity of nutrient transporters and the extent to which amino acids can be modified without affecting the affinity for the relevant transporter. Based on this we have moved away from the idea of developing uptake assays for an individual amino acid to developing assays for specific amino acid transporters. This increased knowledge will not be applied to developing additional assays for other nutrient transporters. Injection of these CLICK-amino acid in vivo can be performed to measure the uptake through Slc1a5 within the in vivo niche and allowing for the first time the single cell quantification of nutrient competition in complex immune microenvironments.
We have also applied this technology to measure the uptake of fatty acids (FAs) into immune cells using fatty acid analogues with a cyclopropene group that is a minimal bioorhtogonal handle that can be used to CLICK a flourphore to transported FAs. Using this technology and FAs of different chain lenght and saturation we are not gauging the FA preferences of immune cells in different situations.
Objective 2:
--We have optimised the conditions for studying dendritic cells subsets directly ex vivo after isolation from murine spleen. We show that cDC1 starved of glucose while activated with CpG have a significantly increased capacity for the activation of T cells responses. This has lead to the hypothesis that this approach may be a applied to improve the efficacy of cDC tumour vaccination protocols, which is an angle we are currently pursuing. cDC1 are much more sensitive to being starved of glutamine and their function is impaired under these conditions.
Objective 3:
--We have completed proteomic analysis of the 3 major DC subsets (cDC1, cDC2, pDC) +/- stimulation with CpG in vivo. This analysis has provided quantitative data on the proteins copies for over 6000 proteins in each of the DC subsets. This data has revealed novel details about DC metabolism in vivo that contradicts much of the previous metabolism research in bone marrow derived DCs (a DC model now accepted to be rather flawed). These analyses show that cDC1 have a metabolic signature that is polarised towards amino acid and lipid metabolism rather than that of glucose. Disrupting the uptake of amino acids through Slc7a5 pharmacologically was found to affect the cross presentation of antigens by cDC1. This was linked to the requirement for Slc7a5 transported amino acids to support mTORC1 activity, which in turn regulated the acidity of the lysosome (an important factor for the processing of antigens for cross presentation).
--We then generated mice that allow for the deletion of Slc7a5 in cDC1. The data show that cDC1 lacking the expression of Slc7a5 also show alterations in antigen cross presentation leading to reduced CD8 T cell responses.
--We have developed and validated a novel single cell assay that accurately reports upon the uptake through the primary glutamine transporter Slc1a5 using flow cytometry and confocal imaging, using bioorthogonal (CLICK) amino acid analogues.
The process of developing this new technology has brought us a new appreciation of the specificity of nutrient transporters and the extent to which amino acids can be modified without affecting the affinity for the relevant transporter. Based on this we have moved away from the idea of developing uptake assays for an individual amino acid to developing assays for specific amino acid transporters. This increased knowledge will not be applied to developing additional assays for other nutrient transporters. Injection of these CLICK-amino acid in vivo can be performed to measure the uptake through Slc1a5 within the in vivo niche and allowing for the first time the single cell quantification of nutrient competition in complex immune microenvironments.
We have also applied this technology to measure the uptake of fatty acids (FAs) into immune cells using fatty acid analogues with a cyclopropene group that is a minimal bioorhtogonal handle that can be used to CLICK a flourphore to transported FAs. Using this technology and FAs of different chain lenght and saturation we are not gauging the FA preferences of immune cells in different situations.
Objective 2:
--We have optimised the conditions for studying dendritic cells subsets directly ex vivo after isolation from murine spleen. We show that cDC1 starved of glucose while activated with CpG have a significantly increased capacity for the activation of T cells responses. This has lead to the hypothesis that this approach may be a applied to improve the efficacy of cDC tumour vaccination protocols, which is an angle we are currently pursuing. cDC1 are much more sensitive to being starved of glutamine and their function is impaired under these conditions.
Objective 3:
--We have completed proteomic analysis of the 3 major DC subsets (cDC1, cDC2, pDC) +/- stimulation with CpG in vivo. This analysis has provided quantitative data on the proteins copies for over 6000 proteins in each of the DC subsets. This data has revealed novel details about DC metabolism in vivo that contradicts much of the previous metabolism research in bone marrow derived DCs (a DC model now accepted to be rather flawed). These analyses show that cDC1 have a metabolic signature that is polarised towards amino acid and lipid metabolism rather than that of glucose. Disrupting the uptake of amino acids through Slc7a5 pharmacologically was found to affect the cross presentation of antigens by cDC1. This was linked to the requirement for Slc7a5 transported amino acids to support mTORC1 activity, which in turn regulated the acidity of the lysosome (an important factor for the processing of antigens for cross presentation).
--We then generated mice that allow for the deletion of Slc7a5 in cDC1. The data show that cDC1 lacking the expression of Slc7a5 also show alterations in antigen cross presentation leading to reduced CD8 T cell responses.
This project has made some important progress beyond the state or the art:
(1) development of a novel single cell assay for uptake through the amino acid transporter Slc1a5, which is one of the most important amino acid transporters in immune cells. This technology has now been expanded to measure FA uptake into single immune cells. In addition, new bioorthogonal amino acid analogues have been generated by our chemist collaborators that are taken up into activated immune cells. We are currently determining the specificity of uptake in the hope that this will provide a new single cell assay for a different amino acid transporter.
(2) the generation of a new transgenic mouse model that allows for the inducible down-regulation of a single amino acid transporter in a single cell type (in our case cDC1 cells)
Final results:
(1) The discovery that short term starvation of cDC1 of glucose can lead to enhanced induction of T cell responses. This provides a new route towards enhancing tcDC1 tumour vaccinations strategies.
(2) Proteomic analysis of in vivo cDC subsets has lead to a re-evaluation of the metabolic configurations of these cells under correct physiological conditions. Using the single cell metabolic analysis toolbox that we have developed we generated an accurate picture of these metabolic configurations of these DC subsets. Focusing on the cDC1 cells we showed that amino acid uptake is essential to support core cDC1 functions including cross presentation and the activation of CD8 T cells. Mechanistically, we show that the uptake of large neutral amino acids through Slc7a5 supports signalling through the kinase mTORC1 that regulates the acid conditions within the lysosome. This changes the rate of peptide degradation and ultimately the pool of peptides of a suitable size available fore presentation on MHCI molecules.
(3) We have developed our single cell uptake assay for Slc1a5 to be applied in complex in vivo microenvironments. This involved injecting the bioorthogonal amino acid in vivo followed by the harvesting of tissues, isolation of immune cells and the CLICK-addition of a fluorphore to quantify the amount of the CLICK probe transprted into each individual cell in vivo within the niche that each given cell occupied. We now have functional assays to measure the uptake of additional nutrients including various types of fatty acids, which are also being tested in vivo.
(1) development of a novel single cell assay for uptake through the amino acid transporter Slc1a5, which is one of the most important amino acid transporters in immune cells. This technology has now been expanded to measure FA uptake into single immune cells. In addition, new bioorthogonal amino acid analogues have been generated by our chemist collaborators that are taken up into activated immune cells. We are currently determining the specificity of uptake in the hope that this will provide a new single cell assay for a different amino acid transporter.
(2) the generation of a new transgenic mouse model that allows for the inducible down-regulation of a single amino acid transporter in a single cell type (in our case cDC1 cells)
Final results:
(1) The discovery that short term starvation of cDC1 of glucose can lead to enhanced induction of T cell responses. This provides a new route towards enhancing tcDC1 tumour vaccinations strategies.
(2) Proteomic analysis of in vivo cDC subsets has lead to a re-evaluation of the metabolic configurations of these cells under correct physiological conditions. Using the single cell metabolic analysis toolbox that we have developed we generated an accurate picture of these metabolic configurations of these DC subsets. Focusing on the cDC1 cells we showed that amino acid uptake is essential to support core cDC1 functions including cross presentation and the activation of CD8 T cells. Mechanistically, we show that the uptake of large neutral amino acids through Slc7a5 supports signalling through the kinase mTORC1 that regulates the acid conditions within the lysosome. This changes the rate of peptide degradation and ultimately the pool of peptides of a suitable size available fore presentation on MHCI molecules.
(3) We have developed our single cell uptake assay for Slc1a5 to be applied in complex in vivo microenvironments. This involved injecting the bioorthogonal amino acid in vivo followed by the harvesting of tissues, isolation of immune cells and the CLICK-addition of a fluorphore to quantify the amount of the CLICK probe transprted into each individual cell in vivo within the niche that each given cell occupied. We now have functional assays to measure the uptake of additional nutrients including various types of fatty acids, which are also being tested in vivo.