Metformin effects on metabolism and proliferation are independent of AMPK in T cell effectors.
(A) Cell suspensions of lymph nodes from AMPKα1fl/fl or AMPKα1fl/fl CD4-Cre+ mice bearing the OT-I TCR were activated with 1 nM SIINFEKL and treated as described in Figure 1A. CD8pos cells were analysed by flow cytometry for TCR-induced changes in cell size using forward and side scatter (FSC and SSC, respectively). (B) Lymph node cells from AMPKα1fl/fl or AMPKα1fl/fl CD4-Crepos mice bearing OT-I TCR were labelled with CFSE or Cell Tracer Violet, respectively, and mixed in a one-to-one-ratio. Co-cultures were then activated with 1 nM SIINFEKL for up to 72 h in the absence or presence of 10 mM metformin. CD8pos cells were analysed for proliferation-induced dilution of loaded fluorescent dyes using flow cytometry. Data are representative of three independent experiments. (C) In-vitro-generated AMPKα1fl/fl or AMPKα1fl/fl CD4-Crepos CTL were treated without or with metformin [10 mM] for 48 h. The starting cell number was 2×105. Proliferation was assessed over the period of treatment by cell counts using flow cytometry. Bar graph displays pooled data of four independent culture experiments. Two-way ANOVA with Bonferroni’s multiple comparisons test was used to determine statistical differences with **p<0.01 and ***p<0.001 between treatments. (D) One million IL-2-differentiated AMPKα1fl/fl and AMPKα1fl/fl CD4Crepos CTL were cultured in absence or presence of metformin [10 mM] for 48 h and subjected to [3H] 2-DG uptake or lactic acid production assays. Bar graphs display pooled data of three independent mice analysed in triplicate and are mean ± SD.