Tissue resident memory CD8 T cells (TRM) protect against infections. In the lungs, TRM offer critical protection against influenza. Skin TRM display a unique metabolic profile compared to other memory cell subsets. They undergo enhanced fatty acid oxidation and oxidative phosphorylation as compared with other cells. It is unclear if this programming holds true in the lung. We hypothesize that lung CD8 T cells with different integrin profiles will utilize unique metabolic programs. Differential expression and pathway analyses were conducted on RNAseq datasets, demonstrating an increased reliance on fatty acid oxidation in TRM compared to circulating memory CD8 T cells. This was most clear when a novel pathway analysis software ‘fluximplied’ that we created was used. Metabolic models were constructed to verify these findings, functional assays were conducted, and the levels of oxidative phosphorylation, mitochondrial mass, and neutral lipids were measured in addition to fatty acid uptake, glucose uptake, and fatty acid oxidation Seahorse assays, confirming a lipid-centric metabolism of TRM. It is unclear if this lipid-centric nature of TRM is a correlate of TRM phenotype or essential to TRM development. We developed an ex vivo model where naive T cells were differentiated with cytokines and metabolites into integrin expressing subsets. Higher amounts and more time with lipid-rich media increased expression of TRM markers. Lastly, in a T cell transfer model, we demonstrate that naive T cells that uptake more fatty acids have more TRM progeny than their low-uptake counterparts after transfer and influenza infection. In summary we demonstrate lung TRM have unique metabolic profiles and that lipids impact TRM differentiation patterns. This work was funded by a Program Project grant through the National Institutes of Health, National Institute of Allergy and Infectious Diseases P01-AI102851 and through the National Institutes of Health training grant T32GM007356-46 and the National Institutes of Health training grant T32HL066988-20.

Tissue resident memory CD8 T cells (TRM) principally reside in peripheral non-lymphoid tissues and confer protection against a variety of illnesses ranging from infections to cancers. The functions of different CD8 T cell subsets in the skin have been linked with distinct metabolic pathways and differ from other CD8 T cell subsets. For example, they undergo fatty acid oxidation and oxidative phosphorylation to a greater degree than circulating memory and naïve cells. However, it is unclear if this programming is universal. Our work demonstrates TRM are not homogenous and, based on integrin expression, exist as subsets with distinct gene expression and function. We hypothesize that lung TRM with different integrin profiles will utilize unique metabolic programs. First, differential expression and pathway analysis was conducted on RNAseq datasets yielding important differences between subsets. Next, metabolic models were constructed and interrogated. Functional metabolite uptake assays were conducted, and the levels of oxidative phosphorylation, mitochondrial mass, and neutral lipids were measured. Furthermore, T cell differentiation studies were conducted varying concentrations of metabolites to investigate the causal relationship in TRM development, demonstrating that, in vitro, lipid conditions impact memory cell maintenance generally, and that glucose concentration impacts TRM-like differentiation specifically, with no effect on central memory-like T cell differentiation. In summary, a diverse set of analyses were utilized to demonstrate lung TRM have unique metabolic profiles and that metabolic microenvironments alter differentiation in vitro.