ABSTRACT: Metabolic and nutrient-sensing pathways play an important role in controlling the efficacy of effector T cells. Oxygen is a critical regulator of cellular metabolism. However, during immune responses T cells must function in oxygen-deficient, or hypoxic, environments. Here, we used high resolution mass spectrometry to investigate how hypoxia configures the proteome of CD8+ cytotoxic T lymphocytes (CTLs). We identified and quantified over 7,600 proteins and discovered that hypoxia increased the abundance of a selected number of proteins in CTLs. This included glucose transporters, metabolic enzymes, transcription factors, cytolytic effector molecules, checkpoint receptors and adhesion molecules. While some of these proteins may augment the effector functions of CTLs, others may limit their cytotoxicity. These data provide a comprehensive resource for understanding the magnitude of the CTL response to hypoxia and emphasise the importance of oxygen-sensing pathways for controlling CD8+ T cells. Additionally, this study provides new understanding about how hypoxia may promote the effector function of CTLs, while contributing to their dysfunction in some contexts. For this project, P14 cytotoxic T cells (CTLs) were generated from T cells from the spleens of mice. CD8+ T cells were activated for 2 days with the P14-T cell receptor (TCR) cognate ligand (peptide gp33-41 from Lymphocytic Choriomeningitis Virus, LCMV) in the presence of 20 ng/ml IL 2 (Proleukin) and 2 ng/ml IL-12 (Peprotech). Cells were grown in a humidified incubator with a gas atmosphere of 18% oxygen and 5% CO2 and a temperature of 37�C. After 48 hours, the activated CD8+ T cells were removed from TCR stimulation and were cultured in RPMI 1640, with 10% foetal bovine serum (FBS), supplemented with 100 units/ml penicillin-G, 100 �g/ml streptomycin, 50 �M-mercaptoethanol and 20 ng/ml IL-2 (Proleukin). Each day, the T cells were counted and split to 500,000 cells per ml with fresh IL-2 being added to a final concentration of 20 ng/ml. CTLs were differentiated in IL-2 for 4 days, before being counted and re-suspended at a concentration of 300,000 cells per ml in RPMI with 10% foetal bovine serum, 50 units/ml penicillin-G, 50 �g/ml streptomycin, 50 �M-mercaptoethanol and 20 ng/ml IL-2. A volume of 8 mls of the cell suspension was plated per well of a 6 well plate, and the CTLs were either maintained in a gas atmosphere of 18% oxygen and 5% CO2 or transferred to an atmosphere of 1% O2 and 5% CO2 in a Galaxy 48 R incubator (Eppendorf) for 24 hours. Three biological replicates (CTLs generated from three separate spleens) were analysed in this experiment."
Sarah Ross 
