Extended Data Fig. 10 |. LKB1 couples TCA cycle and serine metabolism to Th17 effector function.

a) Principal component analysis (PCA) of the transcriptome of control and Opa1^CD4Cre CD4⁺ T_N cells transfected with Cas9 and indicated gRNAs, cultured 4 days in Th17 conditions (n = 3 biological and transfection replicates). b) Volcano plots of the differentially expressed genes (DEG, FC > 1, p-value < 0.001) for the comparisons: control gStk11 vs. control gCtl (left) and Opa1^CD4CregStk11 vs. Opa1^CD4CregCtl (right). Blue dots annotate the highest 40 DEG. c, d, KEGG pathway and Gene Ontology (GO) annotation analysis from DEG (FC>1, p-value<0.001) between (c) control gStk11 vs. control gCtl or (d) Opa1^CD4CregStk11 vs. Opa1^CD4CregCtl. e) Intracellular serine in control and Opa1^CD4Cre CD4⁺ T_N cells transfected with Cas9 and indicated gRNAs (n = 6 transfection replicates, 2 independent experiments) by LC-MS. f) Left, schematic shows 2-HG production from primary and promiscuous enzymatic reactions and the inhibitors used for targeting its production. Right, CD4⁺ T_N cells from control and Opa1^CD4Cre mice were cultured under Th17 conditions for 1 day followed by 48 h inhibitor treatment. IL-17A expression was analyzed by flow cytometry in restimulated cells and expressed as log2 FC inhibitor-treated vs. vehicle-treated cells (CBR, AGI, NHI-2, GSK864: n = 7 per group; BAY, LW6: n = 3 per group). Graph summarizes data from 2 experiments. g) Kinetics of fractional labelling of glutamate (m + 5), α-ketoglutarate (m + 4), malate (m + 4) and fumarate (m+4) in control and Opa1^CD4Cre CD4⁺ T cells transfected with Cas9 and indicated gRNAs cultured for 4 days in Th17 conditions and incubated with [U-¹³C]-Glutamine for the indicated times (n = 3 biological and transfection replicates). Data are means ± s.e.m. Two-sided šidák’s test (f) or Tukey’s test (e, g). Exact P values are indicated.