Extended Data Fig. 5: LDHA-dependent lactate production is essential for CD40 signal.
(a–b) Extracellular lactate production in BMDMs stimulated with or without FGK45 in the absence or presence of etomoxir (Eto) (a), or AMPK inhibitor (iAMPK) (b) under glucose-deplete media. (c–d) Extracellular lactate levels (c) and the production of proinflammatory cytokines (d) in Cas9-expressing BMDMs harboring control sgRNA (Ctrl) and LGHA sgRNAs (LDHA sgRNA-1 and -2) stimulated with or without FGK45 mAb. (e) Illustration of the design for indicated mice follwed with tumor engraftment and then FGK45 treatment. (f) Representative flow plot (left) and quantitative results (right) of LDHA expression in TAMs isolated from tumor-bearing mice with indicated genetypes. (g) qPCR analysis of mRNA expression of anti-inflammatory marker genes ins TAM isolated from tumor-bearing WT (Ctrl) and LdhAcKO mice. (h) Representative immunoblots of indicated proteins in wild type BMDMs (Ctrl) and LDHA-KO BMDM (LDHAcKO) stimulated with or without FGK45 mAb for ndicated periods. (i–j) Chromatin-immunoprecipitation (ChIP) analysis of acetylated histone H3 (AcH3) (i) or acetylated histone H4 (AcH4) (j) in Cas9-expressing BMDMs harboring control sgRNA (Ctrl) and LdhA gRNAs (LDHA sgRNA-1 and -2) with or without FGK45 mAb stimulation after real-time qPCR of enhancer/ promoter reguons on the proinflammatory marker gene loci. Data are normalized to input. (k) qPCR analysis of mRNA expression of proinflammatory marker genes in wld type BMDMs (Ctrl) and LDHA-KO BMDMs (LDHAcKO) stimulated with or without FGK45 mAb in the absence or presence of lactate (L; 10 mM) or sodium acetate (A; 5 mM) under glucose-deplete media. (a–d, i–k) Representative data from three individual experiments (n = 3 per group). (e–g) Representative pooled data from two individual experiments with n = 5 mice per group in (f) and n = 6 for WT-PBS, n = 8 for WT-FGK45, n = 5 for LDHAcKO-PBS and n = 8 for LDHAcKO-PBS in (g). All data are presented as mean ± SD and analyzed by a two-tailed Student’s t-test.
