BCR-ABL - LSK BCR-ABL + LKS - (%)

Marker Clone BCR-ABL + LSK (%) BCR-ABL + LKS - (%) BCR-ABL - LSK (%) P value vs. BCR-ABL + LKS - P value vs. BCR-ABL - LSK CD2 RM2-5 12.9 ± 3.6 36.7 ± 6.5 19.3 ± 2.4 0.01 0.10 CD5 53-7.3 13.9 ± 3.2 20.8 ± 3.8 35.8 ± 10.3 0.12 0.04 CD9 KMC8 98.7 ± 0.29 98.0 ± 0.78 99.0 ± 0.63 0.30 0.66 CD11b M1/70 27.6 ± 2.7 5.95 ± 0.93 35.1 ± 4.7 0.0004 0.12 CD13 R3-242 36.5 ± 1.1 45.0 ± 6.2 22.1 ± 6.4 0.13 0.04 CD19 ID3 11.1 ± 3.8 45.9 ± 6.0 12.4 ± 2.8 0.002 0.72 CD24 M1/69 82.1 ± 4.9 98.8 ± 0.2 51.7 ± 5.4 0.04 0.004 CD25 3C7 23.6 ± 10.5 2.6 ± 1.9 2.0 ± 1.2 0.05 0.04 CD31 MEC13.3 98.7 ± 1.1 91.2 ± 0.95 97.7 ± 2.8 0.002 0.65 CD34 RAM34 59.2 ± 5.9 20.3 ± 7.6 81.8 ± 9.5 0.005 0.05 CD40 3/23 7.5 ± 5.6 1.8 ± 0.3 20.2 ± 6.8 0.29 0.11 CD41 MWReg30 43.9 ± 7.2 58.6 ± 4.6 42.5 ± 6.0 0.07 0.84 CD43 S7 58.4 ± 12.9 80.7 ± 2.1 29.6 ± 13.2 0.13 0.09 CD44 IM7 96.7 ± 1.9 100 ± 0.0 98.6 ± 1.2 0 0.30 CD47 Miap301 99.9 ± 0.09 99.8 ± 0.05 100 ± 0.0 0.42 0.37 CD48 HM48-1 94.5 ± 5.0 99.5 ± 0.33 95.8 ± 2.4 0.30 0.76 CD49e 5H10-27 72.1 ± 9.5 93.2 ± 6.1 84.1 ± 8.5 0.002 0.06 CD51 RMV-7 97.4 ± 0.79 86.1 ± 2.6 96.4 ± 0.94 0.004 0.31 CD52 BTG-2G 11.1 ± 4.0 43.7 ± 12.6 12.8 ± 4.9 0.001 0.55 CD62L MEL-14 100 ± 0.0 7.2 ± 1.1 100 ± 0.0 0 1.0 CD69 H1.2F3 15.4 ± 4.5 23.4 ± 3.9 32.8 ± 3.7 0.13 0.01 CD71 C2 90.5 ± 3.9 82.4 ± 7.7 96.5 ± 3.2 0.006 0.02 CD90 30-H12 81.1 ± 7.6 32.0 ± 12.7 86.7 ± 5.0 0.009 0.43 Fas Jo2 22.5 ± 8.8 51.5 ± 3.3 30.0 ± 5.0 0.01 0.35 CD105 MJ7/18 100 ± 0.05 14.3 ± 1.9 99.9 ± 0.05 0.0002 0.52 Mpl AMM2 75.9 ± 2.3 77.8 ± 2.0 68.8 ± 5.7 0.10 0.42 M-CSFR AFS98 91.8 ± 1.6 87.7 ± 0.37 88.8 ± 2.6 0.03 0.25 CD127 SB/199 6.8 ± 5.0 17.6 ± 3.8 10.7 ± 7.0 0.07 0.55 CD133 13A4 92.2 ± 0.3 88.7 ± 0.4 81.4 ± 4.6 0.008 0.03 Flt3 A2F10.1 7.1 ± 3.3 36.7 ± 7.3 40.8 ± 3.2 0.006 0.0005 CD144 BV13 95.4 ± 0.39 88.5 ± 1.77 89.8 ± 6.30 0.006 0.28

CD150 TC15-12F12.2 24.8 ± 7.6 21.4 ± 5.5 11.3 ± 6.0 0.63 0.12 Alcam ALC-48 3.5 ± 2.5 49.7 ± 6.8 3.3 ± 1.2 0.0008 0.93 CXCR4 2B11/CXCR4 97.8 ± 1.0 99.9 ± 0.1 88.4 ± 6.3 0.10 0.11 EPCR RCR-16 90.5 ± 0.90 89.6 ± 0.45 84.7 ± 2.1 0.27 0.02 Tie2 TEK4 83.2 ± 10.5 80.3 ± 12.1 81.6 ± 9.6 0.69 0.80 PD-L1 10F.9G2 42.5 ± 14.0 66.9 ± 8.7 52.9 ± 7.5 0.10 0.20 Flk1 Avas 12alpha1 11.0 ± 3.5 41.6 ± 11.9 10.1 ± 6.8 0.002 0.80 Jam1 H202-106 99.4 ± 0.28 92.1 ± 0.2 96.8 ± 1.2 <0.0001 0.04 N-cad D13077 1 94.2 ± 1.7 86.9 ± 4.0 90.1 ± 0.5 0.08 0.03 EpCAM G8.8 100 ± 0.0 3.38 ± 1.6 100 ± 0.0 0 1.00 PCLP-1 10B9 77.6 ± 2.7 85.0 ± 1.3 30.7 ± 3.5 0.03 0.0001 PLVAP MECA-32 6.9 ± 2.9 7.4 ± 3.2 7.9 ± 2.3 0.87 0.73 Supplemental Table 1. Screen for markers specifically expressed on CML LSK cells. Bone-marrow cells from CML mice were collected, stained for various surface proteins using fluorphore- or biotin-conjugated antibodies, and then analyzed by FACS (n = 3 6). Shown are percentages of cells positive for the indicated markers in the BCR-ABL + LSK, BCR-ABL + LKS -, and BCR-ABL - LSK fractions. P values were calculated based on the twotailed Student s t-test comparing BCR-ABL + LSK vs BCR-ABL - LSK cells or BCR-ABL + LSK vs BCR-ABL + LKS - cells. Among the markers tested, only CD25 was specifically expressed on BCR-ABL + LSK cells. Supplemental Table 2. Gene sets used for GSEA. GSE3982_EOSINOPHIL_VS_BASOPHIL_UP GSE3982_EOSINOPHIL_VS_BASOPHIL_DN GSE3982_MAST_CELL_VS_BASOPHIL_UP GSE3982_MAST_CELL_VS_BASOPHIL_DN GSE3982_DC_VS_BASOPHIL_UP GSE3982_DC_VS_BASOPHIL_DN GSE3982_MAC_VS_BASOPHIL_UP GSE3982_MAC_VS_BASOPHIL_DN GSE3982_NEUTROPHIL_VS_BASOPHIL_UP

GSE3982_NEUTROPHIL_VS_BASOPHIL_DN GSE3982_BCELL_VS_BASOPHIL_UP GSE3982_BCELL_VS_BASOPHIL_DN GSE3982_BASOPHIL_VS_EFF_MEMORY_CD4_TCELL_UP GSE3982_BASOPHIL_VS_EFF_MEMORY_CD4_TCELL_DN GSE3982_BASOPHIL_VS_CENT_MEMORY_CD4_TCELL_UP GSE3982_BASOPHIL_VS_CENT_MEMORY_CD4_TCELL_DN GSE3982_BASOPHIL_VS_NKCELL_UP GSE3982_BASOPHIL_VS_NKCELL_DN GSE3982_BASOPHIL_VS_TH1_UP GSE3982_BASOPHIL_VS_TH1_DN GSE3982_BASOPHIL_VS_TH2_UP GSE3982_BASOPHIL_VS_TH2_DN GSE3982_CTRL_VS_IGE_STIM_MAST_CELL_UP GSE3982_CTRL_VS_IGE_STIM_MAST_CELL_DN GSE3982_EOSINOPHIL_VS_MAST_CELL_UP GSE3982_EOSINOPHIL_VS_MAST_CELL_DN GSE3982_MAST_CELL_VS_DC_UP GSE3982_MAST_CELL_VS_DC_DN GSE3982_MAST_CELL_VS_MAC_UP GSE3982_MAST_CELL_VS_MAC_DN GSE3982_MAST_CELL_VS_NEUTROPHIL_UP GSE3982_MAST_CELL_VS_NEUTROPHIL_DN GSE3982_MAST_CELL_VS_BCELL_UP GSE3982_MAST_CELL_VS_BCELL_DN GSE3982_MAST_CELL_VS_EFF_MEMORY_CD4_TCELL_UP GSE3982_MAST_CELL_VS_EFF_MEMORY_CD4_TCELL_DN GSE3982_MAST_CELL_VS_CENT_MEMORY_CD4_TCELL_UP GSE3982_MAST_CELL_VS_CENT_MEMORY_CD4_TCELL_DN GSE3982_MAST_CELL_VS_NKCELL_UP GSE3982_MAST_CELL_VS_NKCELL_DN GSE3982_MAST_CELL_VS_TH1_UP GSE3982_MAST_CELL_VS_TH1_DN GSE3982_MAST_CELL_VS_TH2_UP GSE3982_MAST_CELL_VS_TH2_DN

Supplemental References 1 Toyama, H., Arai, F., Hosokawa, K., Ikushima, Y. M. & Suda, T. N-cadherin + HSCs in fetal liver exhibit higher long-term bone marrow reconstitution activity than N-cadherin - HSCs. Biochem. Biophys. Res. Commun. 428, 354-359 (2012).

Supplemental Figure 1. CD25 is highly expressed in the CD34 - Flt3 - LSK fraction of CML model mice. (A B) Flow-cytometric analysis of the frequency (A; means ± s.d.) and number (B; means ± s.d.) of CD25 + cells in the CD34 - Flt3 - LSK fraction of CML model mice (red histogram) and control mice (gray histogram) (CML, n = 4; GFP, n = 4). (C) Flow-cytometric analysis of the frequency of CD25 + cells in LSK, LSK -, LKS -, LS - K -, and Gr-1/Mac-1 + cells of CML model mice (means ± s.d., n = 7). *P < 0.05 and **P < 0.01. Supplemental Figure 2. Mast-cell-related genes are highly expressed in CD25 + LSK cells. A set of genes with an expression pattern similar to that of CD25. Genes expressed in CD25 + LSK, CD25 - LSK, LKS -, and Gra-1 + Mac-1 + cells from BCR-ABL + spleens were analyzed by microarray. Supplemental Figure 3. Variations in Sca-1 staining among antibody clones. Frequencies of CML LSK, CD25 - F - LSK, CD25 + F - LSK, or CD25 + F + LSK fractions detected by the Sca-1 antibody (clone E13-161.7) used throughout this study and another Sca-1 antibody,

clone D7 (means ± s.d., n = 3). Supplemental Figure 4. CD25 + F - LSK cells exhibit higher colony-forming capacity and a predisposition to mast-cell differentiation. (A) Colony-forming capacity of CD25 + F + LSK, CD25 + F - LSK, or CD25 - F - LSK cells from CML model mice was examined in methyl cellulose supplemented with cytokines (means ± s.d., n = 3). (B) Single cell-derived colony counts and cellular morphology of CD25 + F + LSK, CD25 + F - LSK, or CD25 - F - LSK cells in liquid culture. Upper, Number of colonies derived from single CD25 + F + LSK, CD25 + F - LSK, or CD25 - F - LSK cells in the presence or absence of 100 ng/ml IL-2 or 50 ng/ml SCF plus 10 ng/ml of IL-3. Lower, cell morphology in liquid culture. Images were obtained and analyzed using a microscope (IX70; Olympus), UplanApo 20x/0.70 objective lens (Olympus), UPIanApo 40 /0.85 objective lens (Olympus) and DP Controller (Olympus). **P < 0.01. Supplemental Figure 5. Apoptosis, homing capacity, and cell-cycle status of distinct subsets of cells in the CML LIC population. (A) Annexin V + cells in normal LSK, CML CD25 - F - LSK, CD25 + F - LSK, and CD25 + F + LSK

fractions (means ± s.d., n = 3). **P < 0.01. (B C) Homing capacity of BCR-ABL transduced CD25 + LSK or CD25 - LSK cells (2 10 4 per sample) to bone marrow (B) or spleen (C) 16 hr after BMT (n = 3; means ± SD). (D) Flow-cytometric analysis of the cell cycle in CML CD25 - F - LSK, CD25 + F - LSK, or CD25 + F + LSK fractions (means ± s.d., n = 4). *P < 0.05. Supplemental Figure 6. TGF-β2 is highly expressed in CD25 + LSK cells and increases the number of CML LSK cells. (A) Quantitative PCR (qpcr) of TGF-β2 and TGF-βR1 expression in the indicated fractions from CML model mice. Each value was normalized to β-actin expression and is expressed as fold induction compared to the corresponding level in the control group (means ± s.d, n = 4). (B) Number of LSK cells after ex vivo culture of CD25 + F - LSK or CD25 - F - LSK cells with 10 ng/ml TGF-β1 or TGF-β2 for 8 days (means ± s.d., n = 4 5). *P < 0.05 and **P < 0.01. Supplemental Figure 7. Expression of IL-2 receptor components in CML and normal primitive subpopulations. (A) Flow-cytometric analysis of CD122 (IL-2Rβ) and CD132 (common γ-chain) in the

indicated fractions (red histograms; means ± s.d., n = 4). Gray histograms are isotype controls. (B) Flow-cytometric analysis of CD122 (IL-2R ) and CD132 (common -chain) expression in normal bone-marrow fractions, including LT-HSC (CD34 - Flt3 - LSK), LSK, and GMP (Lin - c- Kit + Sca-1 - CD34 + FcγRII/III + ) (orange histograms). Gray histograms indicate isotype control staining. Numbers indicate the CD122- or CD132-positive fraction (means ± s.d., n = 3). Supplemental Figure 8. Surface marker profiles of IL-2 + cells in CML mice. Intracellular flow-cytometric analysis of IL-2 + cells in CML spleen (red histogram: IL-2 PE staining, gray histogram: isotype control) in combination with anti-cd8a, anti-b220, and anti- Gr-1 antibodies (mean ± s.d., n = 3). Supplemental Figure 9. Acceleration of leukemia by IL-2. (A) Survival of CML mice injected with vehicle or IL-2 (n = 7). (B) CD25 + F - LSK or CD25 - F - LSK cells from CML model mice (3 10 3 cells per sample) were cultured for 16 days. Calculated cell number in LSK fractions (means ± s.d.) are shown (n = 5). *P < 0.05 and **P < 0.01. (C) Colony numbers from CFU-C assay of three independent human CML samples with or

without human IL-2 (100U/ml) or anti-human CD25 neutralizing antibody (10 g/ml) (means ± s.d., n = 3). *P < 0.05. Supplemental Figure 10. Peripheral blood parameters of Il2ra -/- LSK derived CML mice after transplantation. Blood parameters were examined 11 days after transplantation (n = 6). Supplemental Figure 11. Characterization of Il2Ra -/- CML mice and Il2Ra -/- mice. (A C) BCR-ABL + myeloid cell number in bone marrow (BM) or spleen (Sp) (A), BCR-ABL + cell frequency (B) and number (C) in Il2Ra +/+ or Il2Ra -/- CML mice (11 days after transplantation) (means ± s.d., n = 7). **P < 0.01. (D) LSK frequency in Il2Ra +/+ or Il2Ra -/- BM (means ± s.d., n = 4 5). Supplemental Figure 12. Characterization of CML model mice treated with anti IL-2 monoclonal antibody. Peripheral blood status (A), BCR-ABL + myeloid cell number in bone marrow (BM) or spleen (Sp) (B), BCR-ABL + myeloid cell frequency in peripheral blood (PB) (C), spleen weight (D), BCR-ABL + cell frequency (E) and number (F) in bone marrow or spleen, and number of bone-

marrow LSK cells (G) in CML model mice treated with isotype antibody (n = 4) or anti-il-2 monoclonal antibody (n = 7) (11 days after transplantation) (means ± s.d.). *P < 0.05, **P < 0.01. Supplemental Figure 13. Effect of anti-cd25 monoclonal antibody on CML model mice treated with nilotinib. Spleen weight (A), BCR-ABL + cell frequency in bone marrow (B) and spleen (C), and number of bone-marrow LSK cells in bone marrow (D) and spleen (E) in CML model mice treated with either vehicle or nilotinib in conjunction with either isotype antibody or anti-cd25 monoclonal antibody (11 days after transplantation) (n = 5; means ± s.d.). *P < 0.05, **P < 0.01. Supplemental Figure 14. CD25 expression in untreated CML or normal human hematopoietic stem/progenitor cells. (A) Another pair of representative profiles for CD25 expression in freshly isolated bonemarrow CD34 + CD38 - (red histogram) and CD34 + CD38 + (blue histogram) fractions from an untreated CML CP patient or a lymphoma patient without bone-marrow involvement (related to Figure 6D).

(B) Flow-cytometric profiles for CD25 expression in the frozen bone-marrow CD34 + CD38 - (red histogram) and CD34 + CD38 + (blue histogram) fractions from untreated CML CP patients (n = 3). Numbers indicate CD25 + cells in CD34 + CD38 - fractions. Supplemental Figure 15. Gene set enrichment analysis on human CML and B-ALL samples. (A B) Gene set enrichment analysis was applied to human leukemia samples. CML BC (n = 33) was compared to CML CP (n = 57) (A), and CD25 + B-ALL (n = 43) was compared to CD25-low or -negative B-ALL (n = 151) (B). Supplemental Figure 16. Revised model of CML LIC formation. Proposed model indicating distinct roles for IL-2 and CD25 + LSK cell-derived TGF-β in maintaining the leukemia-initiating capacity of CML LSK cells.

Supplemental Figure 1 A BCR-ABL + CD34 - Flt3 - LSK gated B Count CD25 47.1±16.2%* Number of cells (x10 3 ) 7 6 5 4 3 2 1 0 GFP * CML C ** CD25 positive cells (%) LSK LSK - LKS - LS - K - Gr-1 /Mac-1 +

Supplemental Figure 2

Supplemental Figure 3 LSK CD25 - F - LSK CD25 + F - LSK CD25 + F + LSK P=0.51 P=0.49 P=0.30 P=0.85 Frequency (%) Frequency (%) Frequency (%) Frequency (%)

Supplemental Figure 4 A B

Supplemental Figure 5 A ** B P=0.45 C P=0.22 Annexin V + (%) Number of cells Number of cells Normal LSK CD25 - F - LSK CD25 + F - LSK CD25 + F + LSK CD25 - LSK CD25 + LSK CD25 - LSK CD25 + LSK D Normal LSK CD25 - F - LSK CD25 + F - LSK CD25 + F + LSK Ki67 23.4 40.1 ±9.1% ±11.3% G1 S/G2/M 26.9 ±5.0% 55.5 ±4.1% 36.4 ±8.6% 53.8 ±8.2% 67.1 ±9.3% 15.1 ±5.8% G0 36.5 ± 3.1% 17.6 ± 4.6% 9.7 ± 2.7%* 17.8 ± 6.7% Hoechst 33342

Supplemental Figure 6 A B

Supplemental Figure 7 A B

Supplemental Figure 8 Splenic MNCs Cell number 1.4±1.1% CD8a 58.5±10.2% Cell number 87.6±5.1% IL-2-PE B220 Gr-1

Supplemental Figure 9 A Survival rate (%) Vehicle IL-2 P=0.029 B Cell number ** ** * ** CD25 + F + LSK CD25 + F - LSK CD25 - F - LSK Time post BMT (days) Vehicle IL-2 CD25 - F - LSK Vehicle IL-2 CD25 + F - LSK C * Colony number * *

Supplemental Figure 10 WBC (x10 3 / l) RBC (x10 4 / l) Hb (g/dl) HCT (%) WBC (x10 3 / l) 40 35 30 25 20 15 10 5 P = 0.0076 RBC (x10 4 / l) 1000 800 600 400 200 P = 0.0082 Hb (g/dl) 16 14 12 10 8 6 4 2 P = 0.039 HCT (%) 60 50 40 30 20 10 P = 0.019 0 0 0 Il2ra +/+ Il2ra -/- Il2ra +/+ Il2ra -/- Il2ra +/+ Il2ra -/- Il2ra +/+ Il2ra -/- 0 70 MCV (fl) P = 0.86 20 MCH (pg) P = 0.91 35 MCHC (g/dl) P = 0.99 30 Plt (x10 4 / l) P = 0.15 60 30 25 MCV (fl) 50 40 30 20 MCH (pg) 15 10 5 MCHC (g/dl) 25 20 15 10 Plt (x10 4 / l) 20 15 10 10 5 5 0 0 0 Il2ra +/+ Il2ra -/- Il2ra +/+ Il2ra -/- Il2ra +/+ Il2ra -/- Il2ra +/+ Il2ra -/- 0

Supplemental Figure 11 A Number of cells (x10 7 ) P=0.0008 P=0.00008 P=0.0001 P=0.00005 Il2Ra +/+ Il2Ra -/- BCR-ABL + BM granulocyte Il2Ra +/+ Il2Ra -/- BCR-ABL + BM monocyte Il2Ra +/+ Il2Ra -/- Il2Ra +/+ Il2Ra -/- BCR-ABL + Sp granulocyte BCR-ABL + Sp monocyte B Frequency (%) ** ** C Number of cells (x10 7 ) ** ** D Frequency (%) LSK P=0.069 Il2Ra +/+ Il2Ra -/- Bone marrow Il2Ra +/+ Il2Ra -/- Spleen Il2Ra +/+ Il2Ra -/- Bone marrow Il2Ra +/+ Il2Ra -/- Spleen Il2Ra +/+ Il2Ra -/-

A Number of cells (x10 7 ) Supplemental Figure 12 anti-il-2 P value WBC (/ul) 28750±9946 27167±1657 0.854 RBC (x10 4 /ul) 838±102 1032±26 0.031 Hb (g/dl) 12.5±1.7 16.2±0.8 0.002 HCT (%) 40.0±7.0 52.3±1.9 0.037 MCV (fl) 47.6±2.7 50.7±1.0 0.017 MCH (pg) 14.9±0.4 15.7±0.5 0.043 Plt (x10 4 /ul) 33.0±9.6 46.0±7.7 0.056 B P=0.96 P=0.038 P=0.38 P=0.012 C Frequency in PB (%) * anti IL-2 BCR-ABL + granulocyte * anti IL-2 BCR-ABL + monocyte D Spleen weight (mg) P=0.19 anti IL-2 anti IL-2 anti IL-2 BCR-ABL + BM granulocyte BCR-ABL + BM monocyte E Frequency (%) ** ** BCR-ABL + Sp granulocyte anti IL-2 F anti IL-2 BCR-ABL + Sp monocyte Number of cells (x10 7 ) * * G Number of cells (x10 3 ) * * CD25 + F + LSK CD25 + F - LSK CD25 - F - LSK anti IL-2 anti IL-2 Bone marrow Spleen anti IL-2 Bone marrow anti IL-2 Spleen anti IL-2

Supplemental Figure 13 A ** B * C ** * * Spleen weight (mg) GFP + frequency in BM (%) GFP + frequency in Sp (%) +vehicle +Nilo Anti CD25 +Nilo +vehicle Anti CD25 +Nilo +Nilo Anti CD25 +vehicle +Nilo +Nilo D CD25 + F + LSK CD25 + F - LSK CD25 - F - LSK E CD25 + F + LSK CD25 + F - LSK CD25 - F - LSK Number of cells (BM) Number of cells (Sp) +vehicle +Nilo Anti CD25 +Nilo +vehicle +Nilo Anti CD25 +Nilo

Supplemental Figure 14 A CD38 Count Count B CD34 CD25 CML Case 3 CML Case 4 CML Case 5 35.8% 0.88% 0 % Count CD25

Supplemental Figure 15 A CML BC vs CML CP B B-ALL CD25 positive vs B-ALL CD25 low or negative

Supplemental Figure 16