Extended Data Figure 2. HIF-1α-dependent metabolic alterations underlie S-2HG production in CD8+ T-lymphocytes.
a/ Example calculation of intracellular 2HG concentration.
b/ 1H-NMR analysis for 2HG from CD8+ T-lymphocytes cultured as in Fig. 2b.
c/ Deletion efficiency of Hif1α or Hif2α in CD8+ T-lymphocytes, isolated from Hif1αfl/fldlckcre or Hif2αfl/fldlckcre mice. n=4 mice.
d/ Total 2HG levels, normalized to viable cell count or protein content, in Hif1αfl/fl and Hif1αfl/fldlckcre CD8+ T-lymphocytes cultured as in Fig. 2b. n=4 mice per genotype.
e/ Total 2HG levels, normalized to viable cells or protein content, in Hif2αfl/fl and Hif2αfl/fldlckcre CD8+ T-lymphocytes cultured as in Fig. 2b. n=4 mice per genotype.
f/ Illustration outlining the workflow for metabolite extraction, deletion efficiency and viability experiments in Hif1αfl/fl, Hif1αfl/fldlckcre, Hif2αfl/fl and Hif2αfl/fldlckcre CD8+ T-lymphocytes. Also shown are viability measurements at day 4. n=4 mice per genotype.
g/ Total amount of 2HG in Hif1αfl/fl (n=6) and Hif1αfl/fldlckcre (n=7) CD8+ T-lymphocytes, at indicated times following activation. n≥4 mice per time point.
h/ Heat map indicating qPCR measurement of expression of enzymes involved in central carbon metabolism in CD8+ T-lymphocytes cultured as in Fig. 2b; n=4 mice per condition.
i/ LC-MS/MS quantification of total intracellular succinate, fumarate and malate levels in CD8+ T-lymphocytes isolated from C57BL/6J mice and cultured as in Fig. 2b; n=7 mice.
j/ Heat map indicating qPCR measurement, in Hif1αfl/fl (n=4) and Hif1αfl/fldlckcre (n=3) CD8+ T-lymphocytes growing in 1% oxygen, of expression of enzymes implicated in the hypoxic production of S-2HG.
k/ qPCR validation of shRNA-knockdowns in CD8+ T-lymphocytes isolated from C57BL/6J mice.
l/ LC-MS/MS quantification of S- and R-2HG in CD8+ T-lymphocytes isolated from C57BL/6J mice, with shRNA-mediated knockdown of Mdh1; n=4 pools of 4 mice per pool.
m/ LC-MS/MS quantification of S- and R-2HG in CD8+ T-lymphocytes isolated from C57BL/6J mice, with shRNA-mediated knockdown of Mdh2; n=4 pools of 4 mice per pool.
n/ LC-MS/MS quantification of R-2HG in CD8+ T-lymphocytes isolated from C57BL/6J mice, with shRNA-mediated knockdown of Ldha; n=4 pools of 4 mice per pool.
o/ Validation of Pdk1-FLAG and Ldha-FLAG expression in Hif1αfl/fldlckcre CD8+ T-lymphocytes by immunoblot analysis for FLAG.
p/ LC-MS/MS quantification of total intracellular glutamate levels in CD8+ T-lymphocytes cultured as in Fig. 2b; n=7 mice.
q-r/ LC-MS/MS quantification of total intracellular glutamate levels in Hif1αfl/fl, Hif1αfl/fldlckcre, Hif2αfl/fl and Hif2αfl/fldlckcre CD8+ T-lymphocytes cultured as in Fig. 2b; n=4 mice per genotype.
s/ Immunoblot of cytosolic fractions for phospho-PDH E1α (S232) and total PDH-E1α in CD8+ T-lymphocytes cultured in 1% oxygen in the presence of the indicated concentration of DCA for 48h.
t/ Total intracellular concentration of 2HG in CD8+ T-lymphocytes from C57BL/6J mice cultured as in Fig. 2b and treated with 5mM DCA for the latter 48h of culture; n=4 mice.
u/ Total intracellular amount of 2HG, nomralized to viable cell count or protein content in CD8+ T-lymphocytes from C57BL/6J mice cultured as in Fig. 2b and treated with 5mM DCA for the latter 48h of culture; n=4 mice.
v/ Total intracellular amount of glutamate in CD8+ T-lymphocytes from C57BL/6J mice cultured as in Fig. 2b and treated with 5mM DCA for the latter 48h of culture; n=4 mice.
Two-way ANOVA for grouped data (d, e, f, q, r, t, u, v). Paired t-test for matched comparisons (i, p), one-way ANOVA for multiple matched comparisons (g, l, m, n). Error bars denote s.d. and each dot represents an individual mouse in g, i and p. ns= non-significant, *p<0.05, **p<0.01, *** p<0.0001. Experiments were performed with indicated numbers of mice from multiple occasions. For immunoblot source images, see Supplementary Fig. 1.