Figure 2.
Transcriptomic differences between reconstituting 8-OHdGhiand 8-OHdGloT cells. (A) CD3+ T cells of patients who underwent 8-OHdGhi (n = 4) and 8-OHdGlo (n = 5) allo-SCT were isolated using FACS-based sorting, followed by RNA isolation and RNA sequencing. (B) Volcano plot of differentially expressed genes (each dot represents 1 gene) between 8-OHdGhi and 8-OHdGlo T cells. Transcripts significantly overrepresented (ie, adjusted P ≤ .1 and log2 fold change ≥1.5) in 1 of both groups are annotated (orange box for 8-OHdGlo and red box for 8-OHdGhi T cells). (C) The PCA of differentially expressed genes shows distinct clustering of samples based on the 8-OHdG level. (D) GSEA of differential gene expression between 8-OHdGhi and 8-OHdGlo T- cells. The graph depicts significantly enriched pathways found in human MSigDB hallmark sets and c2 KEGG gene sets (version 7.1). Size of gene set is annotated to the right. Bar length corresponds to NES. (E) HD-derived CD3+ T cells were activated for 72 hours with anti-CD2, anti-CD3, and anti-CD28 beads and treated twice per day with 10 μM H2O2. Phosphorylated isoforms of AKT (pAKT), of AMPK (pAMPK), and of mTOR (pmTOR), MYC, and HIF1-α were measured as the MFI using FACS (n = 5). Data are expressed as the fold change of expression in T cells pretreated with hydrogen peroxide, relative to untreated T cells (+H2O2 vs −H2O2). (F) Heat map shows differentially regulated cell-cycle–related genes as determined from the RNA seq analysis of 8-OHdGhi and 8-OHdGlo T cells. Only regulated genes with an adjusted P ≤ .1 together with a log2 fold change ≥0.585 or lesser than or equal to −0.585 were selected. The color scale represents the row-wise z score of gene expression. (G) Proportion of proliferating Ki67+ shown for 8-OHdGhi and 8-OHdGlo CD3+ T cells measured at different time points after allo-SCT, using FACS. P value: ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. “n” indicates the sample number; bars represent the standard error of the mean. GSEA, gene set enrichment analysis; NES, normalized enrichment score. PCA, principal component analysis.

Transcriptomic differences between reconstituting 8-OHdGhiand 8-OHdGloT cells. (A) CD3+ T cells of patients who underwent 8-OHdGhi (n = 4) and 8-OHdGlo (n = 5) allo-SCT were isolated using FACS-based sorting, followed by RNA isolation and RNA sequencing. (B) Volcano plot of differentially expressed genes (each dot represents 1 gene) between 8-OHdGhi and 8-OHdGlo T cells. Transcripts significantly overrepresented (ie, adjusted P ≤ .1 and log2 fold change ≥1.5) in 1 of both groups are annotated (orange box for 8-OHdGlo and red box for 8-OHdGhi T cells). (C) The PCA of differentially expressed genes shows distinct clustering of samples based on the 8-OHdG level. (D) GSEA of differential gene expression between 8-OHdGhi and 8-OHdGlo T- cells. The graph depicts significantly enriched pathways found in human MSigDB hallmark sets and c2 KEGG gene sets (version 7.1). Size of gene set is annotated to the right. Bar length corresponds to NES. (E) HD-derived CD3+ T cells were activated for 72 hours with anti-CD2, anti-CD3, and anti-CD28 beads and treated twice per day with 10 μM H2O2. Phosphorylated isoforms of AKT (pAKT), of AMPK (pAMPK), and of mTOR (pmTOR), MYC, and HIF1-α were measured as the MFI using FACS (n = 5). Data are expressed as the fold change of expression in T cells pretreated with hydrogen peroxide, relative to untreated T cells (+H2O2 vs −H2O2). (F) Heat map shows differentially regulated cell-cycle–related genes as determined from the RNA seq analysis of 8-OHdGhi and 8-OHdGlo T cells. Only regulated genes with an adjusted P ≤ .1 together with a log2 fold change ≥0.585 or lesser than or equal to −0.585 were selected. The color scale represents the row-wise z score of gene expression. (G) Proportion of proliferating Ki67+ shown for 8-OHdGhi and 8-OHdGlo CD3+ T cells measured at different time points after allo-SCT, using FACS. P value: ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. “n” indicates the sample number; bars represent the standard error of the mean. GSEA, gene set enrichment analysis; NES, normalized enrichment score. PCA, principal component analysis.

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