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.
