Fig. 4. LA is increased in the AML supernatant inhibiting T cell proliferation.
(A) Relative abundance of LA in fresh RPMI compared to T cell supernatant after 48-hour cell culture in fresh medium or medium originating from 48-hour AML cell culture or in 48-hour AML cell culture supernatant determined by liquid chromatography–mass spectrometry (LC-MS). Statistical analysis of six to nine biologically independent experiments (means ± SEM) is shown. P values were determined using one-way ANOVA, ****P < 0.0001. (B) Concentration of lactate in fresh RPMI compared to T cell culture supernatants after 48-hour culture alone, with AML cells or with BMDCs, or in medium originating from 48-hour AML/BMDC cell culture, determined by NMR analysis. Statistical analysis of seven to nine biologically independent experiments (means ± SEM) using Kruskal-Wallis test is shown. (C) pH measurements of T cell culture supernatants after 48-hour culture in fresh medium or medium originating from 48-hour AML/BMDC cell culture. Statistical analysis of three to six biologically independent experiments (means ± SEM) is shown. P values were determined using one-way ANOVA. (D) Relative abundance of LA in the serum of patients with AML during remission and at the time of relapse after allo-HCT obtained by LC-MS (n = 7). (E) Relative abundance of LA in the serum of patients with AML at two time points in remission after allo-HCT obtained by LC-MS (n = 16). Each data point in (D) and (E) represents the measurement of an individual patient at the indicated time point; n values represent individual patients. P values were determined using the two-sided Student’s paired t test. (F) ECAR measured at baseline and in response to glucose, oligomycin, and 2-DG for CD8+ T cells isolated from 48-hour cell culture treated with indicated concentrations of LA. Graph is representative of five biologically independent experiments. (G) ECAR of CD8+ T cells (n = 5). The n values represent biologically independent experiments (means ± SEM). P values were determined using one-way ANOVA. (H) Cell cycle analysis of CD8+ T cells. Statistical analysis of five biologically independent experiments (means ± SEM) is shown. P values were determined using two-way ANOVA showing P values of G0 phases. (I) Proliferation assay of T cells treated with LA as described in (F) or at a similar pH achieved using HCl (pH 7.0) for 72 hours. Statistical analysis (left) of percentage of proliferating cells of four biologically independent experiments (means ± SEM) and representative histograms (right) is shown. P value was determined using one-way ANOVA. ****P < 0.0001. (J) Total lysates of CD8+ T cells after CD3/28 stimulation and treatment with 15 mM LA for the indicated time points were analyzed by Western blotting. Graph depicts quantification of pS6K. Statistical analysis of three biologically independent experiments is shown. For quantification, all proteins were normalized to the corresponding loading control 14-3-3. P values were calculated using two-way ANOVA.