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Investigating nutrients as key determinants of DC-induced CD8 T cell responses

Periodic Reporting for period 2 - DC_Nutrient (Investigating nutrients as key determinants of DC-induced CD8 T cell responses)

Reporting period: 2019-11-01 to 2021-04-30

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. The local regulation of the DC nutrient microenvironment by neighbouring cells has profound effects on DC function and T cell responses. Nutrient deprived DC have altered signalling (decreased mTORC1 activity), increased pro-inflammatory functions (IL12 and costimulatory molecule expression) and induce enhanced T cell responses (proliferation, IFN production). However, proving this, particularly in vivo, is a major challenge as the tools to investigate nutrient dynamics within complex microenvironments have not yet been developed. This research programme will generate innovative new technologies to measure the local distribution of glucose, glutamine and leucine (all of which control mTORC1 signalling) to be visualised and quantified. 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 for glucose, glutamine, or leucine in DC to definitively 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. For instance, a recent study by Prof. Powell et al, showed the benefit of inhibiting glutamine metabolism within tumours as this led to increased glutamine availability for T cells and ultimately improved T cells antitumour responses.

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 Sllc1a5 using flow cytometry and confocal imaging.
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.

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 glutamine and lipid metabolism rather than that of glucose.
--We have generated all the transgenic cassettes required for generating mice that allow for the inducible down-regulation of amino acid transport through Slc7a5 specifically in cDC1. These transgenic mice are now being crossed with each other to generate experimental mice containing all 3 cassettes. These experimental mice will be then be validated and used to test the hypothesis that nutrient starvation of cDC1 in vivo reshapes cDC1-induced T cell responses.

While we have made important progress across all three objectives this project has also suffered from significant delays due to:
(1) the Covid-19 pandemic, which has delayed the production of the transgenic models that we are generating and caused the cancellation of a large number of experiments. This is because the lockdowns imposed in Ireland had a particularly large impact upon the activities within our animal research facilities.
(2) Significant periods of ill health experienced by the PI and one of the postdoctoral researchers.
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.
(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)

Expected results:
(1) The discovery that short term starvation of cDC1 of glucose can lead to enhanced induction of T cell responses is now being utilized to improve cDC1 tumour vaccinations strategies. We expect to make significant progress towards this in the next phase of this project. The benefit of this approach is that it could easily applied to improve the current strategies in use for cancer patients as it simply involves a change to the culturing conditions.
(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 expect to generate an accurate picture of these metabolic configurations that will allow for a new understanding of how these DC subsets can be regulated by metabolic microenvironments in health and disease.
(3) Development of further single cell uptake assays for additional nutrient transporters to further enable the metabolic analysis of immune cells in complex in vivo microenvironments.