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Supplementary Figure S1
ATL cells CD4 CD7 CD25 CD3 CD5 CD4 Include normal Tregs CD7 CD25 CD5 CD4
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Supplementary Figure S2 A B
Methylation status of CTLA4 exon 2 Methylation status of HELIOS intron 5. Healthy donor CD4+CD25- conventional T cells Healthy donor CD4+CD25- conventional T cells Healthy donor Regulatory T cells Healthy donor Regulatory T cells Primary ATL cells Primary ATL cells 1 16 1 16 2 17 2 17 3 18 3 18 4 19 4 19 5 20 5 20 6 21 6 21 7 22 7 22 8 23 8 23 9 24 9 24 Not available 10 25 10 25 11 26 11 26 12 12 13 13 14 14 15 15 HTLV-1-infected cell lines HTLV-1-infected cell lines MT-1 MT-1 MJ MJ MT-2 MT-2 ATL-43T ATL-43T MT-4 MT-4 Hut102 Hut102 CpG demethylation 0% 100%
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Supplementary Figure S3 A
Hypomethylation Methylation ATL cells MJ Hut102 Isotype 100 99.7% 24.2% Cell Numbers (% Max) MFI =17401 MFI =328 FOXP3 100 20000 80 15000 60 % of FOXP3+ cells MFI of FOXP3 10000 40 5000 20 Hypomethylation Methylation Hypomethylation Methylation B C Hypomethylation Methylation FOXP3/β-ACTIN 2 4 8 6 Methylation 5 10 15 20 FOXP3/β-ACTIN Hypomethylation P=.01 3
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Supplementary Figure S4
Isotype ATL cells A NS 15000 Hypomethylation Methylation 100 10000 MFI of CD25 Cell Numbers (% Max) 5000 MFI =5134 MFI =2163 CD25 Hypomethylation Methylation Regulatory T cells B NS 4000 Hypomethylation Methylation 100 3000 Cell Numbers (% Max) MFI of CTLA4 2000 MFI =914 MFI =1667 1000 CTLA4 Hypomethylation Methylation Regulatory T cells C NS Hypomethylation Methylation 800 100 600 MFI of GITR Cell Numbers (% Max) 400 MFI =391 MFI =345 200 GITR Hypomethylation Methylation Regulatory T cells D NS 15000 Hypomethylation Methylation 100 10000 MFI of CCR4 Cell Numbers (% Max) 5000 MFI =6482 MFI =4831 CCR4 Hypomethylation Methylation Regulatory T cells
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Supplementary Figure S4
Isotype ATL cells NS E 4000 Hypomethylation Methylation 3000 100 MFI of HELIOS 2000 Cell Numbers (% Max) 1000 MFI =1286 MFI =1539 HELIOS Hypomethylation Methylation Regulatory T cells F NS 100 Hypomethylation Methylation 80 100 60 1.64% 63.4% % of Ki67+ cells Cell Numbers (% Max) 40 20 Ki67 Hypomethylation Methylation Regulatory T cells
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Treg cells from healthy donor
Supplementary Figure S5 Isotype ATL cells A NS 30 Hypomethylation Methylation CD4+ from healthy donor 100 20 0.70% 5.86% 16.0% % of IFN-γ+ cells Cell Numbers (% Max) 10 IFN-γ Hypomethylation Methylation B NS Methylation 30 Hypomethylation CD4+ from healthy donor 100 0.80% 11.3 % 2.32 % 20 % of IL-4+ cells Cell Numbers (% Max) 10 IL-4 Hypomethylation Methylation NS C 30 Hypomethylation Methylation CD4+ from healthy donor 100 20 0.26% 2.66 % 0.34 % % of IL-17+ cells Cell Numbers (% Max) 10 Hypomethylation Methylation IL-17 D NS Hypomethylation Methylation Treg cells from healthy donor 30 100 0.65% 10.8% 8.85% 20 Cell Numbers (% Max) % of IL-10+ cells 10 IL-10 Hypomethylation Methylation 6
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Supplementary Figure S5 E
NS Hypomethylation Methylation CD4+ from healthy donor 30 100 0.64% 3.89% 18.0% 20 Cell Numbers (% Max) % of IL-2+ cells 10 IL-2 Hypomethylation Methylation F NS Hypomethylation Methylation CD4+ from healthy donor 30 100 14.7% 0.28% 3.52% 20 Cell Numbers (% Max) % of TNF-α+ cells 10 TNF-α Hypomethylation Methylation 7
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Supplementary Figure S6
NS P=0.02 120 80 CTLA4/β-ACTIN 40 Hypomethylation Methylation Regulatory T cells CD4+ conventional T cells B NS P=0.02 60 40 HELIOS/β-ACTIN 20 Hypomethylation Methylation Regulatory T cells CD4+ conventional T cells C NS P=0.009 120 80 EOS/β-ACTIN 40 Hypomethylation Methylation Regulatory T cells CD4+ conventional T cells
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Supplementary Figure S7
Responder cells : ATL cells Responder alone 1:1 1:1 1:1 1:1 88.2% 40.0% 39.2% 71.3% 58.3% MJ ATL-43T MT-1 Hut102 * * * % of proliferative cells Responder alone MJ ATL-43T MT-1 Hut102 * * * Total numbers of responder cells Responder alone MJ ATL-43T MT-1 Hut102
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A B Supplementary Figure S8 NS 8000 6000 1800 1600 500 tax / 18S rRNA
400 300 200 100 Hypomethylation Methylation Regulatory T cells CD4+ conventional T cells B NS 100 90 5 1 0.6 HBZ / 18S rRNA 0.4 0.2 Hypomethylation Methylation Regulatory T cells CD4+ conventional T cells
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Supplementary Figure S9
1.0 Methylation Hypomethylation 0.8 P=.1 0.6 Overall survival 0.4 0.2 1000 2000 3000 4000 Days after diagnosis (days) B 1.0 CD45RA-FOXP3high > 20% 0.8 CD45RA-FOXP3high < 20% P=.14 0.6 Overall survival 0.4 0.2 1000 2000 3000 4000 Days after diagnosis (days) C 1.0 CD45RA-FOXP3high > 20% CD45RA-FOXP3high < 20% 0.8 P=.16 0.6 Overall survival 0.4 0.2 1000 2000 3000 4000 Days after diagnosis (days)
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Supplementary Figure S10
Healthy donor Fr I: 2.3% Fr II: 6.2% Fr III: 5.1% FOXP3 CD45RA Case F1 Case F5 0.1% 56% 35% 0% 7% 92% Case F9 0% 2% 21% Case F13 0% 17% 7% 0.1% 1% Case F2 0% 0% 13% 4% Case F6 0% 44% 20% Case F10 Case F14 0.2% 31% 28% 0% 18% 79% Case F3 0% 92% 7% Case F7 0% 0.2% Case F11 Case F4 0% 35% 7% Case F8 0% 41% 29% Case F12 0% 32% 38%
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