Figure 6.
Metabolic analysis of H-2kb+CD8+ T cells derived from the vehicle or MDM2 inhibitor-treated leukemia-bearing mice. (A) Extracellular acidification rate (ECAR) of CD8+ T cells isolated from the spleen on day 12 following allo-HCT (C57BL/6 BM plus C57BL/6 T cells) of WEHI-3B leukemia-bearing BALB/c mice. Recipient mice were treated either with vehicle or MDM2 inhibitor RG-7112, as indicated. For each replicate, normalization to the ECAR baseline value was performed. Mean value ± standard error of the mean (SEM) from n = 4 biologically independent replicates; each replicate was generated by pooling the spleens from 2 mice. P values were calculated using a 2-sided unpaired Student t test. (B) Glycolysis (calculated as the difference between ECAR after glucose injection and basal ECAR) and glycolytic capacity (calculated as the difference between ECAR after oligomycin injection and basal ECAR) of CD8+ T cells isolated from BMT recipients as described in (A). Mean value ± SEM from n = 4 biologically independent replicates; each replicate was generated by pooling the spleens from 2 mice. P values were calculated using a 2-sided unpaired Student t test. (C) CD8+ T cells were enriched from the spleens of allo-HCT recipient mice and treated with an MDM2 inhibitor. Polar metabolites were extracted and measured by Liquid chromatography–mass spectrometry (LC-MS) as described in the supplemental Methods from n = 8 mice treated with vehicle and n = 7 mice treated with MDM2 inhibitor. Absolute abundance of metabolites from the pyrimidine biosynthesis pathway. Pathway scheme created with Biorender.com. *P < .05 and **P < .01. (D) CD8+ T cells were enriched from the spleens of allo-HCT recipient mice and treated with an MDM2 inhibitor. Polar metabolites were extracted and measured by LC-MS as described in the supplemental Methods from n = 8 mice treated with vehicle and n = 7 mice treated with an MDM2-inhibitor. Heatmap of the 27 significantly regulated metabolites between the MDM2 inhibitor and vehicle (P < .05). The color scale indicates the normalized concentration in each sample. (E) Volcano plot of 100 metabolites analyzed with a targeted approach isolated from CD8+ T cells that were enriched from the spleens of allo-HCT–recipient mice, treated with MDM2 inhibitor or vehicle. Polar metabolites were extracted and measured by LC-MS as described in the supplemental Methods from n = 8 mice treated with vehicle and n = 7 mice treated with an MDM2-inhibitor. P values were calculated using the unpaired 2-tailed Student t-test.

Metabolic analysis of H-2kb+CD8+ T cells derived from the vehicle or MDM2 inhibitor-treated leukemia-bearing mice. (A) Extracellular acidification rate (ECAR) of CD8+ T cells isolated from the spleen on day 12 following allo-HCT (C57BL/6 BM plus C57BL/6 T cells) of WEHI-3B leukemia-bearing BALB/c mice. Recipient mice were treated either with vehicle or MDM2 inhibitor RG-7112, as indicated. For each replicate, normalization to the ECAR baseline value was performed. Mean value ± standard error of the mean (SEM) from n = 4 biologically independent replicates; each replicate was generated by pooling the spleens from 2 mice. P values were calculated using a 2-sided unpaired Student t test. (B) Glycolysis (calculated as the difference between ECAR after glucose injection and basal ECAR) and glycolytic capacity (calculated as the difference between ECAR after oligomycin injection and basal ECAR) of CD8+ T cells isolated from BMT recipients as described in (A). Mean value ± SEM from n = 4 biologically independent replicates; each replicate was generated by pooling the spleens from 2 mice. P values were calculated using a 2-sided unpaired Student t test. (C) CD8+ T cells were enriched from the spleens of allo-HCT recipient mice and treated with an MDM2 inhibitor. Polar metabolites were extracted and measured by Liquid chromatography–mass spectrometry (LC-MS) as described in the supplemental Methods from n = 8 mice treated with vehicle and n = 7 mice treated with MDM2 inhibitor. Absolute abundance of metabolites from the pyrimidine biosynthesis pathway. Pathway scheme created with Biorender.com. *P < .05 and **P < .01. (D) CD8+ T cells were enriched from the spleens of allo-HCT recipient mice and treated with an MDM2 inhibitor. Polar metabolites were extracted and measured by LC-MS as described in the supplemental Methods from n = 8 mice treated with vehicle and n = 7 mice treated with an MDM2-inhibitor. Heatmap of the 27 significantly regulated metabolites between the MDM2 inhibitor and vehicle (P < .05). The color scale indicates the normalized concentration in each sample. (E) Volcano plot of 100 metabolites analyzed with a targeted approach isolated from CD8+ T cells that were enriched from the spleens of allo-HCT–recipient mice, treated with MDM2 inhibitor or vehicle. Polar metabolites were extracted and measured by LC-MS as described in the supplemental Methods from n = 8 mice treated with vehicle and n = 7 mice treated with an MDM2-inhibitor. P values were calculated using the unpaired 2-tailed Student t-test.

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