Journal: Nature Communications
Article Title: The tumor suppressor menin prevents effector CD8 T-cell dysfunction by targeting mTORC1-dependent metabolic activation
Figure Lengend Snippet: The glutamine-α-KG axis is involved in the dysfunction in menin KO CD8 T cells. a A representative staining profile of CD62L/CD27 on the cell surface of the WT and menin KO effector CD8 T cells on day 7. The percentages of cells are indicated in each quadrant. Naive CD8 T cells were activated and cultured for 3 days under normal (Ctrl), glutamine-deprived (dGln), or glutamine-deprived supplemented with DM-α-KG (dGln/α-KG) conditions for 3 days, and then the cells were further expanded with IL-2 under normal conditions for an additional 4 days. An analysis was performed on day 7 after the initial anti-TCR-β/CD28 stimulation. b Representative staining profiles of CD226 and PD-1 on the cell surface of the cells in a . c The ELISA results for IL-6, IL-10, and OPN in the supernatants of the cells in a restimulated with immobilized anti-TCR-β for 16 h are shown with standard deviation ( n = 3: biological replicates). d The results of the quantitative RT-PCR analysis of the pro-inflammatory enzymes in the cells in a . The results are presented relative to the expression of Cd3ε mRNA with the standard deviations ( n = 3: technical replicates). e The percentages of SA β-galactosidase (SA β-Gal)-positive cells on day 12 after the initial anti-TCR-β/CD28 mAb stimulation are shown with the standard deviation ( n = 3: biological replicates). f A 1:1 mixture of WT OT-1 Tg effector CD8 T (Thy1.1 + )/ menin KO OT-1 Tg effector CD8 T cells under normal conditions (Thy1.2 + ) or WT (Thy1.1 + )/ menin KO under glutamine-deprived conditions (Thy1.2 + ) was adoptively transferred into WT congenic (Thy1.1 + Thy1.2 + ) mice. Twenty days after the transfer, the mice were infected with Lm -OVA to activate the donor cells. The donor cells were collected from the spleen on day 5 after Lm -OVA infection and analyzed by FACS. The absolute number of donor cells in the spleen is shown (mean ± SD, n = 4 per group: biological replicates). * p
Article Snippet: Antibodies used for intracellular and cell-surface staining were as follows: anti-Osteopontin-phycoerythrin (PE) mAb (cat#IC808P; R & D Systems, Minneapolis, MN, USA), anti-IFN-γ-allophycocyanin (APC) mAb (cat#554413; BD Bioscience, San Jose, CA, USA), anti-IFN-γ-fluorescein isothiocyanate (FITC) mAb (cat#554411; BD Bioscience), IL-2-APC mAb (cat#JES6-5H4; TONBO Biosciences, San Diego, CA, USA), anti-Phospho-S6 (Ser235/236)-Alexa Fluor 647 mAb (cat#4851; Cell Signaling Technology, Danvers, MA, USA), anti-Phospho-S6 (Ser240/244) Alexa Fluor 647 mAb (cat#5044; Cell Signaling Technology), anti-S6 ribosomal protein Alexa Fluor 647 mAb (cat#5548; Cell Signaling Technology), anti-CD27-PE mAb (cat#558754; BD Bioscience), anti-CD62L-APC mAb (cat#20-0621; TONBO Biosciences), anti-CD226-APC mAb (cat#128809; BioLegend, San Diego, CA, USA), and anti-PD-1-PE mAb (cat#551892; BD Bioscience).
Techniques: Staining, Cell Culture, Enzyme-linked Immunosorbent Assay, Standard Deviation, Quantitative RT-PCR, Expressing, Mouse Assay, Infection, FACS