1. Volume 45, Issue 2, Pages 358-373 (August 2016)
Bioenergetic Insufficiencies Due to Metabolic Alterations Regulated by the Inhibitory Receptor PD-1 Are an Early Driver of CD8+ T Cell Exhaustion 
Bertram Bengsch, Andy L. Johnson, Makoto Kurachi, Pamela M. Odorizzi, Kristen E. Pauken, John Attanasio, Erietta Stelekati, Laura M. McLane, Michael A. Paley, Greg M. Delgoffe, E. John Wherry 
Immunity 
Volume 45, Issue 2, Pages (August 2016)
DOI: /j.immuni
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2. Immunity 2016 45, 358-373DOI: (10.1016/j.immuni.2016.07.008)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 1
Altered Regulation of Metabolic Genes Early and Late during Clone 13 Infection
Microarray profiling of LCMV GP33-specific CD8+ T cells in LCMV Arm or clone 13 infection (GSE41867) analyzed for (A) number of differentially expressed genes (2-fold or 4-fold cutoff).
(B) GSEA analysis for KEGG glycolysis and gluconeogenesis pathways and OXPHOS pathways at day 8 or day 30 p.i. Negative enrichment score (ES) indicates enrichment in Arm infection.
(C) GSEA of the indicated KEGG metabolic pathways at day 8 and day 30 p.i.
(D) Heatmap of leading edge genes driving gene set enrichment of KEGG metabolic pathways at day 8 p.i. See also Figure S1.
Immunity  , DOI: ( /j.immuni )
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4. Figure 2
Suppression of CD8+ T Cell Mitochondrial Respiration and Glycolysis Early in Chronic Infection
Metabolic flux profiling was performed on purified CD8+ T cells from mice day 8 p.i. with LCMV Arm (blue) or clone 13 (red). (A) OCR and ECAR at baseline.
(B) OCR obtained during mitochondrial stress test, performed by injection of Oligomycin (Oligo), mitochondrial decoupler FCCP, Cpt1a inhibitor Etomoxir (Eto) to interrogate FAO or ETC inhibitors Antimycin A/Rotenone (Ant/Rot).
(C and D) MRC and SRC of CD8+ T cells from day 8 p.i. was determined after FCCP injection.
(E) Compared to (2B), 2-DG injection was used to assess glucose metabolism.
(F) The effect of CPT1a inhibition on MRC and (G) the contribution of FA metabolism (i.e., Eto-sensitive MRC) is plotted for CD8+ T cells from Arm versus clone 13.
(H) The effect of 2-DG injection on mitochondrial respiration and (I) the contribution of glucose (i.e., the 2-DG-sensitive MRC).
(J) Glucose uptake measured by incorporation of glucose analog 2-NBDG and Glut-1 expression (K) of naive CD8+ T cells and LCMV-specific P14 T cells isolated on day 8 p.i. of Arm versus clone 13 infection. Data are representative of three independent experiments (3–5 mice per experimental group). ∗ indicates p < 0.05, ∗∗p < 0.01, ∗∗∗p < by unpaired Student’s t test. Error bars are mean ± SEM. See also Figures S2 and S3.
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5. Figure 3
Depolarized Mitochondria in Virus-Specific CD8+ T Cells in Early Clone 13 Infection
Mitochondrial profiling of virus-specific CD8+ T cells at day 8 after Arm (blue) or clone 13 (red) infection. Live, singlet CD8+ CD45.1+ P14 cells were analyzed (A) Mitotracker Green (MTG) staining of P14 cells at day 8 p.i.
(B) Virus-specific CD8+ T cells with depolarized mitochondria identified as positive for MTG and negative for Mitotracker DeepRed (MTDR) (triangle gate).
(C) Electron microscopy of sorted P14 cells from uninfected, Arm, or clone 13 infected mice (day 8 p.i.). Cells with representative mitochondrial morphology are shown.
(D) Representative plots of virus-specific CD8+ T cells at day 8 p.i. of clone 13 with depolarized (red) versus regularly polarized mitochondria (gray).
(E) ROS in P14 CD8+ T cells measured using MtSox. Cells were costained for Annexin V and amine-reactive L/D reagent. Gated on live singlet CD8+ CD45.1+ P14 cells, gates for MtSox and ROS are indicated on the left.
(F) Amnis Imagestream analysis (60X magnification). Cells were gated on congenic CD45.1+ P14 and MTG versus MTDR staining was used to identify cells with depolarized mitochondria. Arrows indicate a representative cell staining with regular mitochondrial polarization (upper) versus depolarized mitochondria (lower). Nuclear integrity assessed by Hoechst Data are representative of three experiments (Imagestream: one experiment) with 3–5 mice in each experimental group. ∗∗∗ indicates p < by unpaired Student’s t test. Error bars are mean ± SEM. See also Figure S4.
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6. Figure 4
MTOR Inhibition Improves the Mitochondrial Phenotype during Early Clone 13 Infection
Role of mTOR in developing Tex cells.
(A) Time course of ex vivo pS6 in P14 CD8+ T cells during LCMV Arm or clone 13 infection.
(B) Time course of pS6 following 20 min GP33 stimulation in vitro. Gated on live singlet CD8+ P14.
(C) Clone 13 infected mice were treated daily from day 5–8 p.i with 300 μg/kg rapamycin (Rapa) or PBS (control) and analyzed on day 8 p.i. Frequencies of CD45.1+ P14 cells are shown. Gated on live singlet CD8+ T cells (D) P14 CD8+ T cells analyzed for expression of PD-1 and CD44 (E) Representative histograms for PD-1 or MTG of P14 CD8+ T cells from control or Rapa treated mice.
(F) Mitochondrial mass (MTG), depolarization (MTG+MTDR−), and ROS production (MtSox+) following Rapa treatment.
(G) Glucose uptake by 2-NBDG fluorescence.
(H) Functional profile of virus-specific T cells after GP33 peptide stimulation. Degranulating (CD107a+) P14 cells producing IFN-γ, TNF, IL-2, MIP1α, and frequency of polyfunctional cells is shown. Data are gated on live singlet CD8+CD45.1+ P14 cells. Data indicate n = 3–5 mice per experimental group and are representative of two independent experiments. ∗ indicates p < 0.05, ∗∗p < 0.01 by unpaired Student’s t test. Error bars are mean ± SEM.
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7. Figure 5
Mitochondrial Mass, mTOR Signaling and Low Mitochondrial Bioenergetics during LCMV Clone 13 Infection
Mitochondrial phenotype, respiratory function and mTOR signaling of P14 cells analyzed after day 8 p.i. of LCMV Arm (blue) or clone 13 (Cl13) (red) infection.
(A) Normalized mitochondrial mass indicated by relative units (RU) of MTG fluorescence, and (B) representative histograms of MTG on day 8 and day 35 p.i. are shown gated on live singlet P14 cells.
(C) The fraction of depolarized mitochondria for P14 CD8+ T cells on day 35 p.i. on similar scale as in Figure 2.
(D and E) LCMV-specific CD8+ T cells were sorted after day 35 of Arm and clone 13 infection and mitochondrial stress test performed by injection of Oligo, FCCP, Eto, or Ant/Rot as in Figure 2.
(F) C57Bl/6 mice were depleted of CD4+ T cells, P14 cells adoptively transferred and infected with clone 13. Mice were treated with PD-L1 blocking or isotype control antibody from day 22–35 p.i. Mitochondrial mass was determined in P14 CD8+ T cells from control or treated mice by MTG.
(G) Representative data for glucose uptake by P14 CD8+ T cells (left column) from isotype (upper) or anti-PDL1 (lower) treated mice by 2-NBDG fluorescence. Glucose uptake in PD-1Int and PD-1Hi subsets (gated by PD-1 versus CD27) of virus-specific CD8+ T cells (right panel).
(H) Glucose uptake for the PD-1Int and PD-1Hi subsets in (G) is quantified. Kinetic data in (A) summarizes five experiments (3–5 mice per experimental group). Data in (B) are representative of three experiments (n = 3 per group). Data in (D and E) represent pooled data from one experiment. Data in (G and H) represent an experiment with n = 10 mice.∗ indicates p < 0.05, ∗∗∗p < by unpaired Student’s t test. Error bars are mean ± SEM. See also Figure S5.
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8. Figure 6
PD-1 Controls Metabolic Dysregulation of Tex Cells in Early and Established Chronic LCMV Infection
(A) Congenically distinct Pdcd1+/+ (CD45.1+CD45.2−) and Pdcd1−/− (CD45.1+CD45.2+) P14 CD8+ T cells were cotransferred into naive (CD45.1−CD45.2+) mice, followed by infection with clone 13 (cl13). The frequency of Pdcd1+/+ versus Pdcd1−/− P14 cells at day 8 p.i. is shown.
(B–F) Cotransferred Pdcd1+/+ and Pdcd1−/− P14 CD8+ T cells analyzed for glucose uptake (B), mitochondrial mass (C), mitochondrial depolarization (D), and functional parameters (F) at day 8 p.i. with cl13. (E) Representative electron microscopy on sorted cotransferred Pdcd1+/+ and Pdcd1−/− P14 cells.
(G–J) Metabolic flux was performed on negatively selected, magnetic bead purified CD8+ T cells from naive uninfected, Arm or cl13 infected WT or Pdcd1−/− mice at d8 p.i. (G) Basal respiration. (H) Glycolytic stress test measuring ECAR performed by injecting glucose to glucose-starved cells, followed by Oligo. (I) Basal glycolysis was calculated by the increase in ECAR post glucose injection for Pdcd1−/− clone 13 infection. (J) Glycolytic capacity was determined after Oligo injection ∗ indicates p < 0.05, ∗∗p < 0.01, ∗∗∗p < by unpaired (G–J) or paired (A–F) Student’s t test. Error bars are mean ± SEM. See also Figure S5.
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9. Figure 7
PGC1α Overexpression Improves Metabolism of Tex Cells and Restores Function
Expression of PGC1α was analyzed in developing Tex cells.
(A) Cotransferred Pdcd1+/+ and Pdcd1−/− P14 CD8+ T cells were analyzed for PGC1α expression at day 8 p.i. with clone 13.
(B) GSEA of genes in the KEGG PPAR pathway at day 8 p.i. with Arm or clone 13.
(C) WT P14 cells were transduced with Ppargc1a RV and potential-dependent mitochondrial MTDR staining and glucose uptake analyzed at 48 hr and 96 hr post T cell activation.
(D) RV-transduced P14 cells were transferred into mice at day 1 of clone 13 infection and transduced P14 cells analyzed at day 8 for PGC1α expression, glucose uptake, PD-1 expression, and polyfunctionality.
(F) Mitochondrial polarization analysis was performed at day 8 p.i. with clone 13. Data indicate three independent experiments (n = 3–5 mice per group per experiment); ∗ indicates p < 0.05, ∗∗p < 0.01, by paired (A) or unpaired (D and E) Student’s t test. Error bars are mean ± SEM.
Immunity  , DOI: ( /j.immuni )
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