RNAseq of allogeneic CD4⁺ T_n and T_em cells indicates an overall upregulation of glycolytic enzymes in alloreactive T_em cells. (A) FACS-purified allogeneic T_em and T_n cells from the spleen (>0.5 million cells per sample) were pooled from multiple study animals on day 14. RNA was extracted from pooled samples of each cell type (n = 1 for T_n, n = 3 for T_em), and the library was generated using the HyperPrep RNA-Seq Kit. All samples were sequenced on a HiSeq4000 (Illumina), and the reads were trimmed, mapped to the reference genome, and normalized to the library size as counts per million. The shaded squares indicate an increased (red) or decreased (blue) expression in T_em cells over T_n cells, and log2 fold-change values are indicated in panel B. (C) CD4⁺ T cells were isolated from the spleen of syngeneic and allogeneic animals on day 14 posttransplant and analyzed via flow cytometry to analyze the protein expression of GLUT1, HK2, and GAPDH in naive CD4⁺ and T_em cells. The ratio of the median fluorescence intensity of T_em to T_n cells is displayed as mean ± SEM; n = 7 (syn)/10 (allo) animals; Welch’s ANOVA test with Dunnett’s T3 multiple comparison test. (D) Gene expression module score of glycolysis genes and (E) TCA cycle genes in a single-cell sequencing data set of CD4⁺ cells isolated from the liver of syngeneic and allogeneic mice on day 14 posttransplant quantifying the up- or downregulation of a predefined set of genes (see also supplemental Table 3). Data were generated from 3 separately processed biological replicates for each condition that were pooled for the bioinformatic analysis. Two-way ANOVA with Sidak’s multiple comparison test; bar graphs represent mean ± SEM. (F) Quantification of the protein expression of GLUT1, HK2, and GAPDH in T-cell subsets isolated from the liver on day 14 as described in panel C. Aco2, aconitase 2; Aldoa, fructose-bisphosphate aldolase A; Cs, citrate synthase; Eno1, enolase 1; Fh, fumarate hydratase; Gpi1, glucose phosphate isomerase 1; Gpt, glutamate pyruvate transaminase; Hk1, hexokinase 1; Idh, isocitrate dehydrogenase 1; Ldha, lactose dehydrogenase A; Mdh, malate dehydrogenase; MFI, mean fluorescence intensity; Ogdh, oxoglutarate dehydrogenase; Pck2, phosphoenolpyruvate carboxykinase 2; Pdk1, pyruvate dehydrogenase kinase 1; Pdp1, pyruvate dehydrogenase phosphatase 1; Pfkp, phosphofructokinase; Pgk1, phosphoglycerate kinase 1; Pgm1, phosphoglucomutase1; Slc2a1, solute carrier family 2 member 1 (GLUT1); Slc2a3, solute carrier family 2 member 3 (GLUT3); Slc16a1, solute carrier family member 16 (MCT1); Slc16a3, solute carrier family 2 member 1 (MCT4); Sucla, succinyl-CoA ligase; Sudh, succinate dehydrogenase. *P < .05, **P < .01, ***P < .001.