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1 Supplementary figure 1 Nature Medicine: doi:1.138/nm.275

CLUSTER BY SELF-ORGANIZING MAPS SELECTED PATHWAY ANALISYS TERMS Cluster : up-regulated genes in acute patients Cell cycle/dna repair Fatty acid metabolism/heme biosynthesis Signaling

Endocytosis Oxidative phosphorylation/electron transport chain Proteasome/lysosome Protein proces.

Integrin-linked kinase signaling Clusters 3 and : up-regulated genes in chronic patients 3 Krueppel-associated box/zinc-finger Hedgehog Glycine-serine-threonine metabolism Hypoxic and

2 CLUSTER BY SELF-ORGANIZING MAPS SELECTED PATHWAY ANALISYS TERMS Cluster : up-regulated genes in acute patients Cell cycle/dna repair Fatty acid metabolism/heme biosynthesis Signaling (IL-1, PI3K, MAPK) 1 Cluster 1: genes up-regulated in acute and down-regulated in chronic patients Class I PI3K signaling events mediated by AKT Regulation of actin cytoskeleton Endocytosis Oxidative phosphorylation/electron transport chain Proteasome/lysosome Protein proces. in endoplasmic reticulum/metabolism of proteins 2 Cluster 2: down-regulated genes in chronic patients Metabolism/Metabolism of vitamins and cofactors RNA transport Translation factors Integrin-linked kinase signaling Clusters 3 and : up-regulated genes in chronic patients 3 Krueppel-associated box/zinc-finger Hedgehog Glycine-serine-threonine metabolism Hypoxic and oxygen homeostasis regul. of HIF-1a Ribosome Mucin type O-glycan biosynthesis Cluster 5: down-regulated genes in acute patients 5 Ribosomal proteins LincRNA/Non-protein coding transcripts Signaling by GPCR ACUTE RESOLVED CHRONIC ACUTE/RESOLVED CHRONIC/RESOLVED Nature Medicine: doi:1.138/nm.275

3 Supplementary figure 2 Nature Medicine: doi:1.138/nm.275

RESPONSE P53 INDEPENDENT G1 S DNA DAMAGE CHECKPOINT

ACTIVATION OF NF-KB IN B CELLS SIGNALING BY B CELL

ER PHAGOSOME PATHWAY RNA POL I RNA POL III

ERYTHEMATOSUS TRANSCRIPTION RNA POL I PROMOTER

CONTAINING NUCLEOSOMES AT THE CENTROMERE RNA POL I

PHOSPHORYLATION PACKAGING OF TELOMERE ENDS

4 a MITOCHONDRION ES = -.33 p =.9 ES = -.9 p <1 - PROTEASOME b PROTEASOME DNA DAMAGE RESPONSE P53 INDEPENDENT G1 S DNA DAMAGE CHECKPOINT PROTEASOME REGULATION ORNITHINE DECARBOX. ACTIVATION OF NF-KB IN B CELLS SIGNALING BY B CELL RECEPTOR BCR P53 DEPENDENT G1 DNA DAMAGE RESPONSE ER PHAGOSOME PATHWAY RNA POL I RNA POL III MITOCHONDRIAL SYSTEMIC LUPUS TRANSCRIPTION ERYTHEMATOSUS TRANSCRIPTION RNA POL I PROMOTER OPENING TRANSCRIPTION DEPOSITION OF NEW CENPA CONTAINING NUCLEOSOMES AT THE CENTROMERE RNA POL I TRANSCRIPTION MITOCHONDRION OXIDATIVE PHOSPHORYLATION PACKAGING OF TELOMERE ENDS RESPIRATORY ELECTRON TRANSPORT ENOS ACTIVATION AND REGULATION RESPIRATORY ELECTRON TRANSP. ATP SYNTHESIS BY CHEMIOSMOTIC COUPL. HEAT PRODUCTION BY UNCOUPLING PROT. DNA DAMAGE RESPONSE UP UP Node color DOWN Node border color DOWN Chr vs Res Chr vs FLUh ES = -.32 p =.3 TRANSCRIPTION ES = -.5 p = FLUh Chr Nature Medicine: doi:1.138/nm.275

5 Supplementary figure 3 Nature Medicine: doi:1.138/nm.275

6 Counts CD8 JC1 FL-2 Anti-CyC mab MFI Dextr+CD8+ cells Anti-ATP5O mab MFI Dextr+CD8+ cells Normalized to mode a Cytochrome c ATP5O 2,5 1, p=.3 p=.15 HC Res Chr 1,5 6 2 p=.5 p=.5 HC Res Chr Chr HC Chr Res Anti-CyC mab MFI Anti-ATP5O mab MFI b Valinomycin Unstimulated Stimulated Chr 1.5% 3% HC 2%.6% Dextramer JC1 FL-1 CCCP Unstimulated Stimulated Chr 99.9% 7.3% 23.% Res 99.9% 1.1%.8% DiOC6 Nature Medicine: doi:1.138/nm.275

7 Supplementary figure Nature Medicine: doi:1.138/nm.275

8 Ratio MFI stim./unstim. Dextr+CD8+ cells by Proteostat staining Counts Proteostat ratio no acute p= p= unstimulated anti-cd3 stimulated.5. HC n=15 Ratio Prot healthy Res n=7 Ratio Prot res Chr n=7 Ratio Prot cron Proteostat HC Res Chr Nature Medicine: doi:1.138/nm.275

9 Supplementary figure 5 Nature Medicine: doi:1.138/nm.275

ANNV MFI/ DEXTR+ CELLS % DEXTRAMER+ % ANNV+7AAD+ % DEXTRAMER+ % ANNV+7AAD+ % DEXTRAMER+ % ANNV+7AAD+ %

MitoQ MitoTempo Pt. n..9% 3.65% 2.39% 7.85% 1.9% 62.59% HBV-specific FLU-specific 1.65% 38.3% 2.

7% 62.2% 7.% 8.53% 8.51% 71.7% Pt. n.36 HBV-specific.19% 2.8%.21% 7.3% 1.8%.37% 62.8% 68.95% 7.2% 6.

10 ANNV MFI/ DEXTR+ CELLS % DEXTRAMER+ % ANNV+7AAD+ % DEXTRAMER+ % ANNV+7AAD+ % DEXTRAMER+ % ANNV+7AAD+ % DEXTRAMER+ % ANNV+7AAD+ % DEXTRAMER+ % ANNV+7AAD+ CD8 Dextramer CD8 Dextramer CD8 Dextramer a No antioxidant MitoQ MitoTempo Pt. n..9% 3.65% 2.39% 7.85% 1.9% 62.59% HBV-specific FLU-specific 1.65% 38.3% 2.% 3% 1 % 33% Pt. n.15 HBV-specific No expansion No IFN- detection.5% No IFN- detection.15% 67% FLU-specific 6.7% 62.2% 7.% 8.53% 8.51% 71.7% Pt. n.36 HBV-specific.19% 2.8%.21% 7.3% 1.8%.37% 62.8% 68.95% 7.2% 6.5% 6% 75% FLU-specific Dextramer IFN Dextramer IFN Dextramer IFN b Pt. n.25 c Pt. n.2 Pt. n.37 Pt. n.25 Pt. n.9 Pt. n No MTQ MTT antioxidant No MTQ MTT antioxidant No MTQ MTT antioxidant No MTQ MTT antioxidant No MTT antioxidant % DEXTRAMER+ % DEXTR+ANNV+7AAD+ Nature Medicine: doi:1.138/nm.275

11 Supplementary figure 6 Nature Medicine: doi:1.138/nm.275

12 a Fold change Chronic / Resolved b Fold change Chronic / Healthy PDCD CD CTLA PDCD1 CD2 KLRG BATF 3,5 3 2,5 2 1,5 1,5 EOMES BATF EOMES 1,8,6,,2 TBX21,5,,3,2,1 CD28 1,8,6,,2 -,2 TBX21 1,8,6,,2 -,2 CD28 qpcr Microarray Nature Medicine: doi:1.138/nm.275

13 Supplementary figure 7 Nature Medicine: doi:1.138/nm.275

Acute vs. Resolved NES = 1.936 p <1 - FDR <1 - NES = 1.66 p <1 - FDR =.

14 Acute vs. Resolved NES = p <1 - FDR <1 - NES = 1.66 p <1 - FDR =.33 Kaech day8 effector vs memory CD8 T cell UP (Kaech SM et al, 22) Effector vs memory CD8 T cell UP (Wherry EJ et al, 27) Chronic vs. Resolved Chronic vs. Healthy NES = p <1 - FDR =.9 NES = p <1 - FDR =.19 Exhausted vs memory CD8 T cell DN (Wherry EJ et al, 27) Nature Medicine: doi:1.138/nm.275

15 Supplementary figure 8 Nature Medicine: doi:1.138/nm.275

16 Microarray Fold change Microarray Fold change Microarray Fold change Microarray Fold change Microarray Fold change Microarray Fold change Microarray Fold change Microarray Fold change Microarray Fold change a R 2 =.65 p= qpcr ATP5D expression Chr/Res Ac/Res PSMD expression 15 3, ,5 2 5 R 2 =.72 1,5 p=.2 1, qpcr Chr/Res Ac/Res b R 2 =.75 p= qpcr TIMM1 expression,15,1,5 Chr/Res 2 15 HSPA1A expression 1 5 R 2 =.91 p= qpcr Chr/Res 15 SUMO1 expression,93 15 HSPA1L expression R 2 =.57 p= qpcr,92,91,9,899 Chr/Res 1 5 R 2 =.72 p= qpcr Chr/Res 15 1 PSMB8 expression 5 R 2 =.75 p= qpcr,,3,2,1 Chr/Res 15 1 TIMM23 expression 5 R 2 =.56 p= qpcr,2,1 Chr/Res 15 NDUFA6 expression,2 1 5 R 2 =.59 p= qpcr,1 Chr/Res qpcr Microarray Nature Medicine: doi:1.138/nm.275

17 SUPPLEMENTARY FIGURE LEGENDS Supplementary Figure 1. Pathway analysis of gene clusters differentially regulated in acute, resolved and chronic hepatitis B patients. Heat-map representation of differentially expressed genes identified by ANOVA in the three patient groups, subdivided into six expression patterns by self-organizing maps; up-regulated and down-regulated genes are shown in red and green, respectively. The barplots on the right compare mean gene expression levels in acute and chronic patients with those of resolved patients that served as a common reference. Selected pathway and gene ontology terms significantly associated (FDR.5) with misregulated transcripts in individual clusters are listed on the right-side of each gene cluster. Supplementary Figure 2. Comparison of exhausted HBV-specific CD8 cells from chronic patients with healthy subject FLUspecific CD8 cells. (a) Gene sets associated to the indicated processes, retrieved from GSEA comparison of chronic vs. resolved patients (see Fig. 3), are similarly enriched in the chronic patient vs. healthy subject comparison; enrichment values for each gene set are indicated on the left. FDR = false discovery rate. (b) Network (Cytoscape enrichment map) visualization of the concordance between the dysregulated (mainly down-regulated) gene sets and pathways revealed by GSEA comparison of exhausted CD8 cells with HBV- or FLUspecific CD8 cells from resolved and healthy subjects. The results of the chronic vs. resolved and the chronic vs. healthy comparisons are represented as red (up-regulated) to blue (down-regulated) colors in the center and the rim of each node, respectively, as indicated; a generalized and largely concordant downregulation is apparent. Supplementary Figure 3. Validation of mitochondrion-related gene dysregulation. (a) Expression levels of the electron transport chain proteins CyC (left) and ATP5O (right) represented as MFI values of target-specific mabs measured in FLU-specific cells from healthy controls (HC) and in HBV-specific cells from resolved (Res) and chronic patients (Chr). Representative examples of CyC and ATP5O electron transport chain protein expression in chronic patients (Chr) as compared to healthy controls (HC) and to resolved patients (Res) are also shown. (b) Dot-plots (top) and histograms (bottom) showing the fraction of stimulated cells undergoing MMP depolarization, determined on two representative CD8 cell samples by JC1 and DiOC6 staining, respectively. Cells treated with valinomycin or CCCP are shown as positive controls for MMP depolarization. Supplementary Figure. Functional validation of proteasome-related gene dysregulation. Aggresome quantification, expressed as the ratio of ProteoStat MFI measured in overnight anti-cd3-stimulated versus unstimulated dextramer+ cells from healthy controls (HC), resolved (Res) and chronic (Chr) patients. A representative example is illustrated on the right. Nature Medicine: doi:1.138/nm.275

18 Supplementary Figure 5. Effect of MT-antioxidants on cytokine production, proliferation and viability of virus-specific CD8 cells. (a) PBMCs were stimulated with HLA-A2-restricted HBV or FLU peptides in the presence or absence of MitoQ or MitoTempo; T cells were stained with the corresponding HLA class-i dextramers prior to flow-cytometric analysis. The dot-plots represent IFNγ production by HBV-, or FLU-specific CD8 cells from three chronic hepatitis B patients. Preferential targeting of HBV-specific CD8 cells by MTantioxidants is apparent; of note, expansion of HBV-specific CD8 cells from patient (Pt) n. 15 was only observed in MT-antioxidanttreated cultures. (b) Ex-vivo annexin V staining, expressed as MFI, of unstimulated (left bar), anti-cd3-stimulated (middle bar) and MitoTempo-treated/anti-CD3 stimulated (right bar) HBV-specific CD8 cells from a chronic patient. (c) Frequencies of dextramer+ and annexin V+7AAD+/dextramer+ CD8 cells from chronic patients in untreated and antioxidant-treated T cell lines expanded in vitro by a 1-days stimulation with the HBV core peptide. An increase in the frequency of dextramer+ cells, associated with a reduction of annexin V+ expression upon MT-antioxidant treatment is apparent. Supplementary Figure 6. qpcr validation of selected genes previously associated to T cell exhaustion. (a) Upregulation of exhaustion-related inhibitory co-receptors and transcription factor genes 5 (Paley, M.A. et al. Science 338, (212); Quigley, M. et al. Nat Med 16, (21)) in HBV-specific CD8 cells from chronic HBV patients compared to patients with resolved infection. Relative mean expression levels (n=3) measured by qpcr or microarray are represented by white and grey bars, respectively. On the bottom, the same is shown for two genes (CD28 co-receptor and TBX21/T-bet transcription factor) which are down-regulated in CD8 cells from chronic HBV patients 5, (Paley, M.A. et al. Science 338, (212); Kurktschiev, P.D. et al. J Exp Med 211, (21)). (b) Same as (a) for the comparison between HBV-specific CD8 cells from chronic HBV patients and FLU-specific CD8 cells from healthy controls. Supplementary Figure 7. GSEA comparison of the chronic HBV transcript dataset with gene sets from the Immunologic signatures (MSigDB, C7) database. Dysregulated genes revealed by the acute HBV vs resolved patients comparison are significantly associated to the effector vs memory CD8 T cells gene sets identified in the LCMV-mouse model of chronic infection. Dysregulated genes that emerged from the comparison of chronic HBV vs. resolved patients (left) and chronic HBV patients vs. healthy subjects (right) are significantly associated to the previously published Exhausted vs memory CD8 T cell DN gene set. Enrichment values for each gene-set are indicated; FDR = false discovery rate. Supplementary Figure 8. Validation of selected genes dysregulated in CD8 cells from hepatitis B patients by Real-Time qpcr. (a) qpcr analysis of two genes (ATP5D and PSMD) up-regulated in acute vs. resolved and down-regulated in chronic vs. resolved comparisons. Spearman s correlation plots of expression levels derived from microarray (log 2 ) and qpcr (log Ct ) analyses are shown on the left. The bar-plots on the right show the ratios between mean expression levels derived from the chronic vs. resolved (Chr/Res) and the acute vs. resolved (Ac/Res) comparisons, measured by microarrays (grey bars) or Real-Time qpcr (white bars). (b) Same as (a) for a subset of genes down-regulated (TIMM1, SUMO1, PSMB8, NDUFA6 and TIMM23) or up-regulated (HSPA1A, HSPA1L) in the chronic vs. resolved (Chr/Res) comparison. Nature Medicine: doi:1.138/nm.275

19 Supplementary Table Chronic patient clinical details Chronic HBV patients Gender (M/F) 27/15 Age* 2.5 (2-66) HBV-DNA * (IU/ml) 2.35x1 5 (.3 9.6x1 8 ) ALT * (U/L) 5.5 (11-351) HBV genotype 36 gen D 3 gen A 2 gen C 1 gen E eag neg HLA-A POS * Median and range shown Nature Medicine: doi:1.138/nm.275

20 Figure 3 statistical details. Panel C (left). Mann Whitney test: Chr vs HC, n1=7, n2=8, p=.3, U=.; Chr vs Res, n1=7, n2=7, p=.6. U=.; Chr vs Ac, n1=7, N2=3, p=.167, U=.. Panel C (right). Mann Whitney test: Chr vs HC, n1=9, n2=7, p=.2, U=.; Chr vs Res, n1=9, n2=6, p=., U=.; Chr PD1+ vs HC, n1=5, N2=7, p=.1,u=.; Chr PD1+ vs Res, n1=5,n2=6, p=., U=.. Panel D. Mann Whitney test: Chr vs HC, n1=16, n2=17, p<.1, U=2.; Chr vs Res, n1=16, n2=9, p=.55, U=22.5; Chr PD1+ vs HC, n1=9, n2=17, p=.15, U=2.; Chr PD1+ vs Res, n1=9, n2=9, p=., U=17.. Panel E (top). Wilcoxon signed rank test: Chr vs HC, number of pairs=1, p=.39, W=53; Chr vs Res, number of pairs=1, p=.371,w=1. Panel E. Wilcoxon signed rank test: (left) HC Unstimulated vs Stimulated, number of pairs=1, p=.2, W=55; (middle) Res Unstimulated vs Stimulated, number of pairs=1, p=.39, W=53; (right) Chr Unstimulated vs Stimulated, number of pairs=9, p=.6523, W=-9. Nature Medicine doi:1.138/nm.275

21 Figure statistical details. Panel A (top left). Wilcoxon signed rank test: %IFNg+ CD8+ MitoQ treated vs untreated, number of pairs=27, p<.1, W=-315; %TNFa+ CD8+ MitoQ treated vs untreated, number of pairs=27, p=.19, W=-235; %IFNg+ TNFa+ CD8+ MitoQ treated vs untreated, number of pairs=27, p<.1, W=-269. Panel A (top right). Wilcoxon signed rank test: fold increase %IFNg+ CD8+ MitoQ treated vs untreated, n=27, p<.1, W=299; fold increase %TNFa+ CD8+ MitoQ treated vs untreated, n=27, p=.2, W=275; fold increase %IFNg+ TNFa+ CD8+ MitoQ treated vs untreated, n=27, p<.1, W=233. Panel A (bottom left). Wilcoxon signed rank test: %IFNg+ CD8+ MitoTempo treated vs untreated, number of pairs=27, p<.1, W=-366; %TNFa+ CD8+ MitoTempo treated vs untreated, number of pairs=27, p<.1, W=-319; %IFNg+ TNFa+ CD8+ MitoTempo treated vs untreated, number of pairs=27, p<.1, W=-291. Panel A (bottom right). Wilcoxon signed rank test: fold increase %IFNg+ CD8+ MitoTempo treated vs untreated, n=27, p<.1, W=37; fold increase %TNFa+ CD8+ MitoTempo treated vs untreated, n=27, p<.1, W=36; fold increase %IFNg+ TNFa+ CD8+ MitoTempo treated vs untreated, n=27, p<.1, W=39. Panel B (top). Wilcoxon signed rank test: fold increase %IFNg+ CD3+ MitoQ treated vs untreated, n=27, p<.1, W=38; fold increase %TNFa+ CD3+ MitoQ treated vs untreated, n=27, p=.22, W=26; fold increase %IFNg+ TNFa+ CD3+ MitoQ treated vs untreated, n=27, p=.2, W=29. Panel B (bottom). Wilcoxon signed rank test: fold increase %IFNg+ CD3+ MitoTempo treated vs untreated, n=27, p<.1, W=317; fold increase %TNFa+ CD3+ MitoTempo treated vs untreated, n=27, p=.28, W=182; fold increase %IFNg+ TNFa+ CD3+ MitoTempo treated vs untreated, n=27, p=.2, W=259. Nature Medicine doi:1.138/nm.275

22 Figure 6 statistical details. Panel A (left). Wilcoxon signed rank test: fold increase %IFNg+ CD8+ HBV vs FLU MitoQ treated, n=9, p<.39, W=35.; fold increase %TNFa+ CD8+ HBV vs FLU MitoQ treated, n=9, p=.27, W=37.; fold increase %IFNg+ TNFa+ CD8+ HBV vs FLU MitoQ treated, n=9, p=.195, W=39.. Panel A (right). Wilcoxon signed rank test: fold increase %IFNg+ CD8+ HBV vs FLU MitoTempo treated, n=9, p<.195, W=39.; fold increase %TNFa+ CD8+ HBV vs FLU MitoTempo treated, n=9, p=.273, W=37.; fold increase %IFNg+ TNFa+ CD8+ HBV vs FLU MitoTempo treated, n=9, p=.5, W=33.. Nature Medicine doi:1.138/nm.275

HD1 (FLU) HD2 (EBV) HD2 (FLU)

ramer staining + anti-pe beads ramer staining a HD1 (FLU) HD2 (EBV) HD2 (FLU).73.11.56.46.24 1.12 b CD127 + c CD127 + d CD127 - e CD127 - PD1 - PD1 + PD1 + PD1-1 1 1 1 %CD127 + PD1-8 6 4 2 + anti-pe %CD127