Extended Data Fig. 10: LAG3 deletion doesn’t affect single cytokine production or metabolic capacity.
From: Autoreactive CD8+ T cells are restrained by an exhaustion-like program that is maintained by LAG3
The consequences of LAG3 deletion were evaluated by flow cytometry to phenotype intra-islet CD8+ T cells for cytokine production, metabolic capacity, and antigen specificity. (a-d) Flow cytometry was performed on 8-week-old female Lag3∆TM and Cre Controls taking cells from ndLN, pLN and islets. (a-b) lymphocytes were stimulated ex vivo for 5 hours with PMA, ionomycin, and brefeldin A and then assessed for cytokine production and degranulation. CD107a, GzmB, Tnfα and IFNγ were quantified (2 independent experiments n = 6-7 per genotype). Cytokine production is unchanged between genotypes, though dual cytokine production, an indicator of polyfunctionality, IFNγ+Gzmb+, is increased in Lag3∆TM (ndLN, pLN, Islet p = .0083, 0.035, 0.44) (b). (c) Lymphocytes were isolated from islets, ndLN, and pLN, cultured in serum free media for 37 degrees C in the presence of GlucoseCy5, CellROX, or MitoSOX, for 30 mins, surface stained including TMRE and MitoTracker, and analyzed by flow cytometry (2 independent experiments, n = 2-6 per genotype). Lag3L/L-YFP.NOD controls were included in this experiment to control for fluorescent protein expression that may overlap with metabolic markers. (a-c) Each data point corresponds to a single mouse. A two-sided nonparametric Mann-Whitney statistical test was preformed where P = * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. Unlabeled indicates not statistically significant. Graphs portray the median. (d) representative flow plots of tetramer staining in 8-week-old female Lag3∆TM and Cre Controls intra-islet CD8+ T cells.
