precursor all. · nature immunology: doi:10.1038/ni.3160 supplementary figure 1 high levels of...
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Nature Immunology: doi:10.1038/ni.3160
Supplementary Figure 1
High levels of AICDA and RAG1 mRNA at the time of diagnosis predict poor clinical outcomes for patients with B cell precursor ALL. (a) Left: Comparison of overall survival probabilities of ALL patients segregated into two categories based on their median AICDA expression levels (ECOG, n=215). P value was calculated by logrank test. Center: Comparison of overall survival probabilities of ALL patients segregated into two categories based on their median RAG1 expression levels (ECOG, n=215). P value calculated by logrank test. Right: Comparison of relapse-free survival probabilities of ALL patients segregated into two categories based on their median RAG1 expression levels (ECOG, n=215). P value calculated by logrank test. (b) Comparison of AICDA expression levels at diagnosis and relapse in matched sample pairs (P9906 COG, n=49) of childhood ALL patients. P-value was calculated using paired Wilcoxon two sided t test.
Nature Immunology: doi:10.1038/ni.3160
Supplementary Figure 2
Aicda and Rag are upregulated during early B cell development upon loss of IL-7R signaling (small pre-BII cell stage).
(a) Quantitative RT-PCR showing Aicda mRNA levels upon reconstitution of Blnk into Blnk-/- pre-B cells (n=3, mean ± s.d.). (b) Aicda mRNA levels measured by qRT-PCR before and after 24 hours of IL-7 withdrawal (n=3, mean ± s.d.). (c)
Nature Immunology: doi:10.1038/ni.3160
Rag1 mRNA levels measured by qRT-PCR (n=3, mean ± s.d.) before and after IL-7 withdrawal in mouse pre-B cells. (d) Aicda mRNA levels measured by qRT-PCR after retroviral expression of a constitutively active form of Foxo3a (Foxo3aCA) or empty vector (EV) in pre-B cells, in the presence or absence of IL-7 (n=3, mean ± s.d.).
Nature Immunology: doi:10.1038/ni.3160
Supplementary Figure 3
Small pre-BII cells from Aicda-GFP and Aicda-Cre × Rosa 26-LSL-eYFP reporter mice respond to inflammatory signals from LPS by upregulating Aicda.
Nature Immunology: doi:10.1038/ni.3160
(a) Change in percentage of Aicda-GFP+ cells with time, in the presence and absence of LPS, before and after differentiation to small pre-BII stage. One representative experiment out of three is shown. (b) Aicda-GFP pre-B cells upregulate expression of Aicda, Rag1 and Rag2 at the small pre-BII stage in the context of inflammatory signals like LPS (GFP+ κLC+ cells). One representative experiment out of three is shown. (c, d) Change in percentage of Aicda-Cre eYFP+ cells with time, in the presence and absence of LPS, before and after differentiation to the small pre-BII stage. Experiments from two independent bone marrows are shown.
Nature Immunology: doi:10.1038/ni.3160
Supplementary Figure 4
Evidence for concurrent activity of RAG1-RAG2 and AID in single pre-B cell clones.
Diagrammatic representation of cooperation between AID (somatic hypermutation), and RAG1-RAG2 activities (VH replacement) in the clonal evolution of a pediatric pre-B ALL patient.
Nature Immunology: doi:10.1038/ni.3160
Supplementary Figure 5
Cooperation among RAG1 and RAG2 and AID promotes clonal evolution towards pre-B ALL.
Schematic: Loss of IL-7R signaling at small pre-BII makes a pre-B cell vulnerable to acquisition of genetic changes by activation of AID, RAG1 and RAG2.
Nature Immunology: doi:10.1038/ni.3160
Supplementary Figure 6
Flow cytometry to sorting human cord blood B cell clones transduced to express AID (iRFP670), RAG1 (eGFP) and RAG2(dsRedE2).
Lentiviral vectors encoding Aicda (pCL6-Aicda-IRES-iRFP670-wo), Rag1 (pCL6-Rag1-IRES-eGFP-wo), Rag2 (pCL6-Rag2-IRES-dsRedExpress2-wo) and the corresponding empty vector (EV) controls were introduced into EBV-transformed human CD19+ cord blood B cells by the transduction protocol described in Materials and Methods. Cells were eithertransduced with EVs, Aicda alone, Rag1 and Rag2 combination or Aicda, Rag1 and Rag2. After 4 days, living EBV cord blood B cells, stained with DAPI, that were triple positive for eGFP, iRFP670 and dsRedExpress2 were single cell sortedinto 96well plates using a 488nm(525/50), 640nm(670/30), 561nm(582/15) and 355nm(450/50) configuration on an BDAriaII Sorter. 3D graphics were generated with WinList (Verity Software House).
Nature Immunology: doi:10.1038/ni.3160
Supplementary Figure 7
Verification of the overexpression of AID, RAG1 and RAG1 alone or in combination in human cord blood B cell clones by fluorescence microscopy
Lentiviral vectors encoding Aicda (pCL6-Aicda-IRES-iRFP670-wo), Rag1 (pCL6-Rag1-IRES-eGFP-wo), Rag2 (pCL6-Rag2-IRES-dsRedExpress2-wo) and the corresponding empty vector (EV) controls were introduced into EBV-transformed human CD19+ cord blood B cells by the transduction protocol described in Materials and Methods. Cells were either transduced with EVs, Aicda alone, Rag1 and Rag2 combination or Aicda, Rag1 and Rag2. The transduction of the cells for Rag1 (eGFP) and Rag2 (dsRedExpress2) was verified by immunofluorescence microscopy. Transduction of Aicda (iRFP670) was verified by flow cytometry (Figure S6).
Nature Immunology: doi:10.1038/ni.3160
Supplementary Figure 8
AID and RAG are required for the leukemic transformation of ETV6-RUNX1 pre-B cell clones in the context of repeated inflammatory stimulation.
Mice that had become terminally ill (Aicda+/+ Rag1+/+ No IL-7+LPS group) were sacrificed and bone marrow and spleenswere analyzed by flow cytometry. Verification of leukemia as the cause of terminal illness was carried out by flow cytometry measurement of the percentage of CD19+/ ETV6-RUNX1 GFP+ cells in the bone marrow and spleen of all the sacrificed mice in the group of LPS and IL-7 withdrawal.
mice that were injected with Aicda+/+ Rag1+/+ pre-B cells after repetitive stimulation with
Nature Immunology: doi:10.1038/ni.3160
Supplementary Figure 9
Immunohistochemical analysis of ETV6-RUNX1 pre-B ALL infiltration in congenic recipient mice.
Mice that had become terminally ill (Aicda+/+
Rag1+/+
No IL7+LPS group) were sacrificed and bone marrow and spleens were analyzed by flow cytometry. Pre-B ALL as the cause of terminal illness was verified byimmunohistochemistry and leukemic infiltrates in spleen (top) and liver were visualized. H&E staining andimmunohistochemistry for CD19
+ pre-B cell infiltration (and isotype control staining) were performed on
spleen and liver sections of all the sacrificed mice in the group of mice that were injected with Aicda+/+
Rag1+/+
pre-B cells after repetitive stimulation with LPS and IL7 withdrawal.
Table S1: Mutation frequency of VH region in pre-B cells from human bone marrow.
Average hypermutation frequency of VH region = 26.2 x 10-3 bp
Clone VH DH JH Mut Freq (x10-3
bp)
1 VH1-3 D1-7 JH4 34.3
2 VH1-24 D1-26 JH4 2.9
3 VH1-18 D2-15 JH4 37.1
4 VH1-2 D2-2 JH6 25.7
5 VH1-24 D2-2 JH6 57.1
6 VH1-69 D2-2 JH6 8.6
7 VH1-18 D2-2 JH6 68.6
8 VH1-8 D2-2 JH6 17.1
9 VH1-46 D3-10 JH6 37.1
10 VH1-8 D3-22 JH4 34.3
11 VH1-46 D3-22 JH5 0.0
12 VH1-58 D3-22 JH5 34.3
13 VH1-46 D3-3 JH5 11.4
14 VH1-2 D3-9 JH4 14.3
VH1-2 D3-9 JH4 8.6
VH1-2 D3-9 JH4 11.4
15 VH1-46 D4-17 JH4 22.9
16 VH1-2 D4-17 JH4 31.4
17 VH1-3 D5-12 JH6 14.3
18 VH1-24 D5-24 JH4 22.9
Nature Immunology: doi:10.1038/ni.3160
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Table S2: SHM and CSR of VH region in pre-B cells from fetal liver.
Average hypermutation frequency of VH region = 14.1 x 10-3 bp
VH DH JH Constant Mut freq (x10-3bp) JH Constant Region
VH1-46 D4-17 JH4 Cµ→Cγ1 20.0 TGACTACTGG CAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCC
VH3-23 D1-14 JH4 Cµ→Cα 25.7 GACTACTGG CATCCCCGACCAGCCCCAAGGTCTTCCCGCTGAGCCT
VH3-23 D1-20 JH4 Cµ 0.0 ACTACTTTGACTACTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA
VH3-23 D1-7 JH6 Cµ 14.3 CTACTACTACGGTATGGACGTCTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA
VH3-23 D3-10 JH4 Cµ 14.3 GACTACTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA
VH3-23 D3-3 JH4 Cµ→Cγ1 17.1 TTGACTACTGG AGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCT
VH3-23 D4-11 JH6 Cµ→Cγ3 8.6 TTACTACTACTACTGCGGTATGGACGTCTGG GGCGCCCTGCTCCAGGAGCACCTCTGGGGGCACAGCG
VH3-23 D6-19 JH4 Cµ 17.1 TGACTACTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA
VH3-23 D6-6 JH4 Cµ 0.0 ACTACTTTGACTACTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA
VH3-30 D4-17 JH4 Cµ 8.6 ACTACTTTGACTACTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA
VH3-30 D6-13 JH6 Cµ→Cγ1 45.7 CTACTACAACGGTATGGACGTCTGG CCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACA
VH3-30-3 D1-26 JH6 Cµ→Cγ1 14.3 TACTACTCCTACTACGGTATGGACGTCTGG TCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCT
VH3-33 D6-19 JH5 Cµ 8.6 AACTGGTTCGACCCCTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA
VH3-7 D1-26 JH4 Cµ 20.0 GACTACTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA
VH4-34 D6-13 JH5 Cµ 14.3 ACCCCTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA
VH4-39 D3-10 JH4 Cµ 11.4 TACTTTGACTACTGG GGGGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA
VH6-1 D2-8 JH2 n.d. 14.3 ACTGGTACTTCGATCTCTGG
VH3-11 D3-9 JH3 Cµ→Cγ1 8.6 GCTTTTGATATCTGG CCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACC
VH3-30 D6-6 JH4 Cµ 11.4 TTTGACTACTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA
VH3-30-3 D6-13 JH4 Cµ 11.4 TTTGACTACTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA
VH3-33 D7-27 JH4 Cµ 11.4 ACTTTGACTACTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA
VH3-49 D3-22 JH4 Cµ 20.0 TGACTACTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA
VH3-7 D2-15 JH3 Cµ 2.9 GATGCTTTTGATATCTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA
VH3-7 D2-21 JH4 Cµ 25.7 TGACTACTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA
VH3-73 D4-23 JH4 Cµ→Cγ1 17.1 TTGACTACTGG CCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACC
VH3-9 D4-11 JH4 Cµ 2.9 ACTACTTTGACTACTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA
VH4-34 D4-11 JH3 Cµ→Cγ1 25.7 GATGCTTTAGATATCTGG CTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCC
VH4-59 D2-15 JH3 Cµ→Cγ1 8.6 GATGCTTTTGATGTCTGG CCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACC
VH4-59 D6-19 JH4 Cµ 20.0 TTTGACTACTGG GAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA
VH6-1 D3-10 JH3 Cµ 5.7 GCTTTTGATATCTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA
VH6-1 D6-13 JH5 Cµ 14.3 ACTGGTTCGACCCCTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA
VH6-1 D7-27 JH4 Cµ 11.4 TACGTTGACTACTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA
VH1-2 D6-13 JH4 Cµ 14.3 GACTACTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA
VH1-3 D2-15 JH4 Cµ 2.9 ACTACTTTGACTACTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA
VH1-69 D3-10 JH6 Cµ 2.9 ACTACTACTACTACGGTATGGACGTCTGG GGGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA
VH2-5 D6-13 JH4 Cµ→Cγ1 77.1 CTTTGACTACTGG CCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACC
VH3-11 D1-20 JH4 Cµ 8.6 TTTGACTACTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA
VH3-11 D3-10 JH1 Cµ 5.7 TGAATACTTCCAGCACTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA
VH3-11 D3-10 JH4 Cµ 17.1 TTGACTACTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA
VH3-11 D6-19 JH4 Cµ 8.6 TACTTTGACTACTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA
VH3-23 D1-1 JH4 Cµ 8.6 CTTTGACTACTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA
VH3-23 D1-1 JH4 Cµ 14.3 TTTGACTACTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA
VH3-23 D1-26 JH6 Cµ 34.3 ACGGTATGGACGTCTGG GGGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA
VH3-30 D3-9 JH4 Cµ 0.0 ACTACTTTGACTACTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA
VH3-30 D6-13 JH4 Cµ 14.3 TTTGACTACTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA
VH3-33 D3-10 JH2 Cµ 8.6 ACTGGTACTTCGATCTCTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA
VH3-33 D3-9 JH6 Cµ 8.6 TACTACTACTACGGTATGGACGTCTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA
VH3-66*03 D6-19 JH1 Cµ 2.9 GCTGAATACTTCCAGCACTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA
VH3-7 D1-26 JH4 Cµ→Cγ1 20.0 ACTACTGG CCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACC
VH3-7 D2-2 JH5 Cµ 8.6 AACTGGTTCGACCCCTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA
VH3-7 D6-19 JH6 Cµ 14.3 ATTACTACTACTACTACGGTATGGACGTCTGG GGGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA
VH3-74 D2-8 JH4 Cµ→Cγ1 8.6 CTTTGACTACTGG CCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACC
VH6-1 D1-26 JH3 Cµ 8.6 GCTTTTGATATCTGG GAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA
Nature Immunology: doi:10.1038/ni.3160
Table S3: SHM and VH replacement in ETV6-RUNX1 ALL patients (n = 13). 7/13 cases positive for VH
replacement. Cryptic RSSs are underlined.
Average mutation frequency of VH = 6.4 x 10-3 bp
Average mutation frequency of JH = 34.5 x 10-3 bp
Patient Allele VH DH JH
VH Mut
Freq
(x10-3
bp)
JH Mut
Freq
(x10-3
bp) VH-N DH-N-JH
REH 1 VH3-15 D3-10 JH6 8.0 66.9 AGCCGTGTATTACTGTACCACAGGG ATGGTTCGGGGAGTTATTATACTACTACTACGGTATGGACGTCTGG
LAX13 1 VH3-33 D3-22 JH6 8.2 96.8 GCTGTGTATTACTGTGCGAGAGA CTCTCAGAGTACTACTACGGTATGGACGTCTGG
PD4009a 1 VH1-69 D3-3 JH6 5.5 80.6 GCCGTGTATTACTGTGCGAGAGAGGCGAGTCCTCCCGAGAGATTGAGGGCGGGG ACGATTTTTGGAGTGGTTATTCTACTACTACGGTATGGACGTCTGG
VH2-70 D3-3 JH6 0.0 0.0 CAGCCACGTATTACTGTGCACGGCCCCGCGAGTCCTCCCGAGAGATTGAGGGCGGGG ACGATTTTTGGAGTGGTTATTCTACTACTACGGTATGGACGTCTGG
VH3-66 D3-3 JH6 0.0 0.0 ACGGCTGTGTATTACTGTGCGAGTCCTCCCGAGAGATTGAGGGCGGGG ACGATTTTTGGAGTGGTTATTCTACTACTACGGTATGGACGTCTGG
PD4020a 1 VH5-10 D3-10 JH4 12.9 122.4 CTCGGACACCGCCATGTATTACTCCTAGCGAGACACCTT TTACTATGGTTCGGGGAGTTATTATAACTACTGG
2 VH1-3 D6-6 JH4 15.3 272.5 ACACGGCTGTGTATTACTGTGCGTGGGCC TATAGCAGCTACCCGG
VH2-5 D6-6 JH4 0.0 0.0 CACAGCCACATATTACTGTGCACTCACGTGGGCC TATAGCAGCTACCCGG
VH3-21/VH3-48 D6-6 JH4 0.0 0.0 GCTGTGTATTACTGTGCGAGAGAGAGTGGGCC TATAGCAGCTACCCGG
VH3-23 D6-6 JH4 0.0 0.0 GCCTTGTATTACTGTGCAAGAGAGGGCGTGGGCC TATAGCAGCTACCCGG
VH3-30 D6-6 JH4 0.0 0.0 GACACGGCTGTGTATTACTGTGCCGGAAGCGACC TATAGCAGCTACCCGG
VH3-33 D6-6 JH4 0.0 0.0 GCTGTGTATTACTGTGCGAGAGATTCTCTTCGTGGGCC TATAGCAGCTACCCGG
VH3-7 D6-6 JH4 0.0 0.0 GGCCGTGTATTACTGTGCGAGAGCAGGGTGGGCC TATAGCAGCTACCCGG
PD4021a 1 VH2-70 D2-2 JH5 4.3 0.0 CCACGTATTACTGTGCACGGATAGT ATAATGTAGTAGTACCAGCTGCTATACAACTGGTTCGACCCCTGG
PD4038a 1 VH2-70 D3-16 JH4 3.8 145.8 GCCACGTATTACTGTGCACGGAT GGGGAGAGACGCGAGACCCATCGAGAGATCGGGGCGAAAGAGGCTACTGG
VH3-66 D3-16 JH4 0.0 0.0 ACACGGCTGTGTATTACTGTGCGTTAG GGGGAGAGACGCGAGACCCATCGAGAGATCGGGGCGAAAGAGGCTACTGG
VH4-61 D3-16 JH4 0.0 0.0 GGCCGTGTATTACTGTGCGAGAG GGGGAGAGACGCGAGACCCATCGAGAGATCGGGGCGAAAGAGGCTACTGG
2 VH2-70 D5-12 JH4 6.9 155.1 GACACAGCCACGTATTACTGTGCCTATTCGACCCCGCGAGACTAGAGACGCGAGACCCATCGA GAGATCGGGGCGAAAGAGGCTACTGG
VH3-64 D5-12 JH4 0.0 0.0 GGACATGGCTGTGTATTACTGTGGGGGGCGGCGCGAGACCCATCGA GAGATCGGGGCGAAAGAGGCTACTGG
VH3-66 D5-12 JH4 0.0 0.0 ACGGCTGTGTATTACTGTGCGAGGACCCTCCGAGAGACGCGAGACCCATCGA GAGATCGGGGCGAAAGAGGCTACTGG
VH3-72 D5-12 JH4 0.0 0.0 GCCGTGTATTACTGTGCTAGAGAAGAAAGACGCGAGACCCATCGA GAGATCGGGGCGAAAGAGGCTACTGG
VH4-59 D5-12 JH4 0.0 0.0 GCCGTGTATTACTGTGCGAGAGACGCGAGACCCATCGA GAGATCGGGGCGAAAGAGGCTACTGG
VH4-61 D5-12 JH4 0.0 0.0 GGCCGTGTATTACTGTGCGAGAGTCCCTCCTAGCCCATCGA GAGATCGGGGCGAAAGAGGCTACTGG
PD3952a 1 VH2-70D D3-22 JH6 18.0 32.3 CACGTATTACTGTGCACGGATACTCAGC GTATTACTATGATAGTAGTGGTTATTCCCAAGGCATTACTACTACTACTACGGTATGGACGTCTGG
VH3-30/VH3-33 D3-22 JH6 0.0 0.0 TGAGGACACGGCTGTGAGGACTA ---------TGATAGTAGTGGTTATTCCCAAGGCATTACTACTACTACTACGGTATGGACGTCTGG
VH3-66 D3-22 JH6 0.0 0.0 CGGCTGTGTATTACTGTGCGAGAGAGCGGAC GTATTACTATGATAGTAGTGGTTATTCCCAAGGCATTACTACTACTACTACGGTATGGACGTCTGG
VH3-73 D3-22 JH6 0.0 0.0 GCCGTGTATTACTGTACTAGACAGAGC GTATTACTATGATAGTAGTGGTTATTCCCAAGGCATTACTACTACTACTACGGTATGGACGTCTGG
VH4-55 D3-22 JH6 0.0 0.0 ACGGCCGTGTATTACTGTGCGAGC GTATTACTATGATAGTAGTGGTTATTCCCAAGGCATTACTACTACTACTACGGTATGGACGTCTGG
2 VH3-21 D3-22 JH6 0.0 0.0 CACGGCTGTGTATTACTGTGCGAAAAGCG ATTACTATGATAGTAGTGGTGAACCATACTACTACGGTATGGACGTCTGG
PD3961a 1 VH3-30 D2-2 JH6 19.8 83.0 GGCTGTGTATTACTGTGCGAAAGGGGGG GGATATTGTAGTAGTACCAGCTGCTATGGGGGGCTACTACTACTACGGTATGGACGTCTGG
VH3-7 D2-2 JH6 0.0 0.0 GGCTGTGTATTACTGTGCGAAAGGGGGG GGATATTGTAGTAGTACCAGCTGCTATGGGGGGCTACTACTACTACGGTATGGACGTCTGG
VH4-34 D2-2 JH6 0.0 0.0 GCTGTGTATTACTGTGCGAAAGGGGGG GGATATTGTAGTAGTACCAGCTGCTATGGGGGGCTACTACTACTACGGTATGGACGTCTGG
2 VH3-7 D7-27 JH4 22.9 71.4 GACACGGCTGTGTATTACTGTGC CTCAGAGCGGCTACTGG
PD4022a 1 VH1-2 JH6 16.1 100.7
2 VH3-7 D2-2 JH6 9.3 57.2 GGCTGTGTATTACTGTGCGAGAGTGGG CCACCTCTACTACTACTACGGTATGGACGTCTGG
PD4024a 1 VH2-70 D3-10 JH5 4.8 82.9 GCCACGTATTACTGTGCACGGATCCCTGACTC ATTACTATGGTTCAGGGAGTCTGGTTCGACCCCTGG
VH3-71 D3-10 JH5 0.0 0.0 CGGCTGTGTATTACTGTGCGAGAAGTC ATTACTATGGTTCAGGGAGTCTGGTTCGACCCCTGG
PD4036a 1 VH6-1 D3-10 JH6 4.2 48.4 GCTGTGTATTACTGTGCAAGAGAGTT GTATTACTATGGTTCGGGGAGTTAGGGGGGTAAGTACTACTACTACTACATGGACGTCTGG
2 VH3-30/VH3-33/ VH3-7 D2-21 JH6 116.0 50.4 AAGGACACGGCCATGTATTACTGTACCAGAGAGGTCCCCCG GTGGTGGTGACTGCTATTCCTCTACTACTACGGTATGGACGTCTGG
PD4037a 1 VH1-69 D2-2 JH1 3.6 19.2 GCCGTGTATTACTGTGCGAGAGACCC GATATTGTAGTAGTACCAGCTGCTATACCCTGAATACTTCCAGCACTGG
VH2-70 D2-2 JH1 0.0 0.0 CAGCCACGTATTACTGTGCACGGCCTTGACGAGAGATCCCC GATATTGTAGTAGTACCAGCTGCTATACCCTGAATACTTCCAGCACTGG
VH2-70D D2-2 JH1 0.0 0.0 ACAGCCACGTATTACTGTGCCCGCCGGACGAGAGATCCCC GATATTGTAGTAGTACCAGCTGCTATACCCTGAATACTTCCAGCACTGG
VH3-69-1 D2-2 JH1 0.0 0.0 GCTGTGTATTACTGTGCGAGAGAAGATAGATGAGACATCCCC GATATTGTAGTAGTACCAGCTGCTATACCCTGAATACTTCCAGCACTGG
920527-4278 1 VH6-1 JH6 0.0 34.5
Nature Immunology: doi:10.1038/ni.3160
Table S4: SHM and VH replacement in Ph-negative ALL patients (n = 58). 2/58 cases positive for VH
replacement in non-ETV6-RUNX1 leukemic subgroups.
Average mutation frequency of VH = 14 x 10-3 bp
Average mutation frequency of JH = 101.4 x 10-3 bp
Average mutation frequency of VH = 18.4 x 10-3 bp
Average mutation frequency of JH = 159.3 x 10-3 bp
Average mutation frequency of VH = 25.3 x 10-3 bp
Average mutation frequency of JH = 110.5 x 10-3 bp
Table S4a: SHM and VH replacement leukemia patients with common MLL and TCF3-PBX1
rearrangements (n = 5). Cryptic RSSs are underlined.
Table S4b: SHM and VH replacement in hyperdiploid leukemia patients (n = 17). Cryptic RSSs are
underlined.
Table S4c: SHM and VH replacement in leukemia patients with uncommon karyotypes (n = 10).
Cryptic RSSs are underlined.
Patient Cytogenetics VH DH JH
Mut Freq
VH (x10-3
bp)
Mut Freq
JH (x10-3
bp) VH-N DH-N-JH
P902486/96 MLL-AF4 VH5-a D2-15 JH6 7.8 150.9 CGCCATGTATTACTGTGCGAGAC TATTGTAGTGGTGGTAGCTGCCGCCGAGTACTACTACTACGGTATGGACGTCTGG
P901941/96 MLL-ENL VH2-5 D3-10 JH6 0.0 241.9 CACATATTACTGTGCACACAGATGGGATTT TATTACTATGGTTCGGGGAGTTATTATACCCTTACTACTACTACGGTATGGACGTCTGG
P902032/95 TCF3-PBX1 VH1-8 JH4 12.3 0.0
VH3-66 JH4 8.3 0.0
P900469/95 TCF3-PBX1 VH6-1 D1-26 JH6 0.0 132.1 CTGTGTATTACTGTGCAAGAGAT GTAGAGTGGGAGCTACTTACTACTACTACGGTATGGACGTCTGG
Patient 20 MYC-IGH VH3-74 D6-13 JH4 55.6 83.3 CTCTATATTACTGTGTAAGAGACGGAG ATACCACCAGTTGGTCTTTTGACTACTGG
Patient Cytogenetics VH DH JH
Mut Freq
VH (x10-3
bp)
Mut Freq
JH (x10-3
bp) VH-N DH-N-JH
B273/98 Hyperdiploid VH2-5 D2-15 JH4 7.4 210.5 ACATATTACTGTGCACACAGACCAAATTCCTTCTCGA GTAGTGGTGGTAGCTGCTTGAGACTACTGG
B2033/97 Hyperdiploid VH1-58 D3-3 JH5 11.7 73.2 CCGTGTATTACTGTGCGGCAGAAG TACGATTTTTGGAGTGGTTATTATATCAACTGGTTCGACCCCTGG
P90100/96 Hyperdiploid VH3-9 D3-3 JH6 12.2 132.1 CTTGTATTACTGTGCAAAAGAT GTATTACGATTTTTGGAGTGGTTATTATACTACTACTACGGTATGGACGTCTGG
P901756/96 Hyperdiploid VH2-5 D5-12 JH5 15.0 285.7 ACATATTACTGTGCACACAGACAAGGGG ATAGTGGCTACCCGTATAAAGACCTGG
B960/97jfr1224 Hyperdiploid VH4-34 D3-16 JH4 0.0 184.2 GCTGTGTATTACTGTGCGAGAGTCTAGGTC TGATTACGTTTGGGGGAGTTATCGATTGAGATCTGACTACTGG
c901275/94 Hyperdiploid VH6-1 JH6 4.7 200.0
c27012-91 Hyperdiploid VH1-3 D6-6 JH4 0.0 261.9 CTGTGTATTACTGTGCGAGAGAGG ATAGCAGCTCGCCCTTAAACTGG
VH6-1 D2-8 JH6 0.0 277.8 CTGTGTATTACTGTGCAAGAGAGC TGTACTAATGGTGTATGCTATAAGAGGAAAACGGTATGGACGTCTGG
P901348/95 Hyperdiploid VH4-34 D7-27 JH4 4.2 184.2 CTGTGTATTACTGTGCGAGAGGAG CTAACTGGGGTGACCGTGGGCCGACTACTGG
P902781/96 Hyperdiploid VH3-23 D2-2 JH6 4.1 113.2 CCGTATATTACTGTGCGAAAGACCTCCGGG AGTAGTACCAGCTGCTATTACTACTACTACGGTATGGACGTCTGG
B675/97 Hyperdiploid VH4-34 D6-19 JH4 0.0 52.6 GGCTGTGTATTACTGTGCGAGATGCCGATCCTTCT GTATAGCAGTGGCTGGTACTTTGACTACTGG
P90131/96 Hyperdiploid VH3-53 D4-23 JH4 4.1 105.3 CCGTGTATTACTGTGCGAGAGAA ACTGTGGGGATTCCCCTTTGACTACTGG
P902300/95 Hyperdiploid VH6-1 JH6 33.0 172.4
P900978/95 Hyperdiploid VH3-9 D1-26 JH4 8.2 256.4 TTGTATTACTGTGCAAAAGATAATAGATAGA TGGGAGCTACTACTTTGACTACTGG
c900162/94 Hyperdiploid VH3-30 D3-10 JH6 4.1 264.2 GCTGTGTATTACTGTGCGAGAGGAGGG GGTTTCGGGATACTACGGTATGGACGTCTGG
Patient 5 Hyperdiploid VH3-13 JH5 24.6 196.1
Patient 10 Hyperdiploid VH3-7 D3-10 JH5 85.1 16.0 CGGCTGTGTATTACTGTGCGAG GGGGACAACTGGTTCGACCCCTGG
VH3-66 D2-15 JH4 73.7 166.7 GCTGTGTATTACTGTGCGAGAGTT TGGTATCAGCTAATGCCCCGGGACTCCTGG
VH4-34 49.3 0.0
Patient 18 Hyperdiploid VH4-39 D3-10 JH6 13.7 80.6 GCGGACACGGCTGTGTATTACCCCCCCGTAAGTGG TTATTAACGTTCGGGTCTTTGGCTACTACTACTACGGTATGGACGTCTGG
VH1-2 D2-2 JH6 31.0 112.9 ACGGCCGTGTATTACTGTGCGACACGGTGTTGATAGC TATTGTAGTAGTACCAGCTGCTATAGGTTTCGGCTACTACTACGGTATGGACGTGTGG
Patient Cytogenetics VH DH JH
Mut Freq
VH (x10-3
bp)
Mut Freq
JH (x10-3
bp) VH-N DH-N-JH
hösten-95 Other VH1-3 D7-27 JH4 16.5 210.5 CTGTGTATTACTGTGCGAGAGATTGGGT TAACTGGGGATGACTACTGG
B1057/97 Other VH3-74 JH4 24.5 285.7
P900667/96 Other VH3-33 D2-2 JH6 8.2 150.9 GGCTGTGTATTACTGTGCGAGACCCC AGGAGAAACAGAATACTACTACTACGGTATGGACGTCTGG
VH4-4 D4-11 JH4/JH6 33.5 138.9 GCGGACACGGCCGTGTACAGTA CTACATGGACGTCTGG
c900891/94 Other VH6-1 JH6 4.2 0.0
c900024/94 Other VH2-70 3.7 0.0
P901693/95 Other VH3-21 8.2 0.0
c26170/87 Other VH3-23 D3-10 JH4 0.0 250.0 ACGGCCGTATATTACTGTGCGA GAGGTATTACGATTTTTGATGGG
Patient 3 Other VH4-61 D3-3 JH4 27.5 62.5 GCCCTGTATTACTGTGCGGGAG TTTGGGGCTGGGCTACTTCCTTTGACTACTGG
Patient 17 Other VH4-34 D3-22 JH4 42.1 104.2 GGCTGTTTATTACTGTGCGAGATGCGG CTATCATAGTAGTGGTTATTACTACGGACCGCCGTTCACTACTGG
Patient 23 Other VH3-64D D1-14 JH5 69.4 102.6 CACGCCTGTGTATTACTGTGTG CCACGGG
VH7-4-1 D6-19 JH2 45.5 18.9 TGTCCTCTATTACTGTGCGAGAGTCAGCC ATAGTAGTGGCTCCCCACTACTGGTACTTCGATCTCTGG
VH1-18 D3-3 JH6 45.5 112.9 CCGTGTATTACTGTGCGAGAGACAGACAGAC TACGATTTTTGGAGTGGTTATTAAAGTTGACTACTACTACGGTATGGACGTCTGG
Nature Immunology: doi:10.1038/ni.3160
Average mutation frequency of VH = 15.2 x 10-3 bp
Average mutation frequency of JH = 107.6 x 10-3 bp
Table S4: SHM and VH replacement in Ph-negative leukemia patients (n = 58). 2/58 cases positive for
VH replacement in non-ETV6-RUNX1 leukemic subgroups.
Table S4d: SHM and VH replacement leukemia patients with normal or undetermined karyotypes (n =26).
Cryptic RSSs are underlined.
Patient Cytogenetics VH DH JH
Mut Freq
VH (x10-3
bp)
Mut Freq
JH (x10-3
bp) VH-N DH-N-JH
B192/98 Normal VH4-34 D2-2 JH4 12.6 333.3 CTGTGTATTACTGTGCGAGAGGCCCACCTGGATATTCGC TGTAGTAGTACCAGCTGCTATGGTTCGGGGAGTTATTATTTGACTACTGG
P902775/95 Normal VH3-33 JH6 0.0 277.8
Patient 1 n.d. VH7-81 D3-22 JH6 115.8 129.0 GAGGACACTGCCGGCAGGTCCAACCCA AGTGGTTATTACCCAGCCCTACTACTACTACATGGACGTCTGG
Patient 29 n.d. VH3-71 D1-26 JH5 27.4 19.6 AGGACACGGCCGTGTTCCGGTGGG GTAGGAACTACGTCGGGCCCCCATCCGGAGTGAAAACTGGTTCGACCCCTGG
Ber 10 n.d. VH1-8 JH4/JH5 38.2 228.6
VH1-8 34.7 0.0
VH1-8 JH4/JH5 34.7 58.8
VH1-8 JH4/ JH5 38.2 0.0
Ber 71 n.d. VH1-8 D2-15 JH6 0.0 32.3 CCGTGTATTACTGTGCGAGAGGGGGG ----ATTGTAGTGGTGGTAGCTGCTACTCGGGGGCCCCTCACTACTACTACTACTACATGGACGTCTGG
VH1-2 D2-15 JH6 0.0 64.5 CCGTGTATTACTGTGCGAGAGAGTTTCTAT GGATATTGTAGTGGTGGTAGCTGCTACTCGGGGGCCCCTCACTACTACTACTACTACATGGACGTCTGG
VH1-2 D2-15 JH6 0.0 32.3 CGGCCGTGTATTACTGTGCGAGCCTG GGGTATTGTAGTGGTGGTAGCTGCTACTCGGGGGCCCCTCACTACTACTACTACTACATGGACGTCTGG
VH1-2 D2-15 JH6 0.0 129.0 CCGTGTATTACTGTGCGAGAGA AGGATATTGTAGTGGTGGTAGCTGCTACTCGGGGGCCCCTCACTACTACTACTACATGGACGTCTGG
Ber 77 n.d. VH1-69 D6-19 JH4 0.0 41.7 CGGCCGTGTATTACTGTGCGAGCCGAC GTATAGCAGTGGCTAACCTCAACTTTGACTACTGG
VH3-30 3.5 0.0
VH1-18 D3-10 JH3 24.3 0.0 CCGTGTATTACTGTGCGAGAGACACGACCCC ATTACTATGGTTCGGGGAGAATTTTGATGCTTTTGATATCTGG
VH1-8 D3-3 JH4 0.0 125.0 CCGTGTATTACTGTGCGAGAGG TTTTCGGACAGCAGAGGACCTTGGCTACTGG
Ber 82 n.d. VH3-11 D3-22 JH6 0.0 32.3 CTGTGTATTACTGTGCGAGAGAGGGGGAGGGTTTTT GTATTACTATGATAGTAGTGGTTATTACTACCCCTTATTACTACTACTACTACGGTATGGACGTCTGG
VH3-13 D3-22 JH6 0.0 0.0 CTGTGTATTACTGTGCAAGAGAGGGTGATCGGGGGAGGGTTTTT GTATTACTATGATAGTAGTGGTTATTACTACCCCTTATTACTACTACTACTACGGTATGGACGTCTGG
VH3-13 D3-22 JH6 0.0 32.3 CTGTGTATTACTGTGCAAGAGAGGGTGATCGGGGGAGGGTTTTT GTATTACTATGATAGTAGTGGTTATTACTACCCCTTATTACTACTACTACTACGGTATGGACGTCTGG
Ber 100 n.d. VH1-18 D2-21 JH4 0.0 187.5 GCCGTGTATTACTGTGCGAGAGCCTCT GTGGTGACTGCTATGGGGTACTGG
Ber 101 n.d. VH1-45 D3-16 JH5 3.5 156.9 ACACAGCCATGTATTACTGTGCC TGGGGGGCGGTTCGACCCCTGG
VH3-30 D5-18 JH4 10.5 20.8 ACACGGCTGTGTATTACTGTGCCCCCTCCCCCCC CAGCTATGGTTACCTACTTTGACTACTGG
VH1-18 D3-22 JH3 48.6 120.0 CCATGTATAACTGTGCGAGAGAGCCGC GATATTAGTGCTTCTTACCGTTTGATATCTGG
Ber 107 n.d. VH3-23 D4-11 JH4 0.0 104.2 GCCGTATATTACTGTGCGAAAGGCTTGCCTTTGAAGAC TGACTTCAAAGATTGACTACTGG
VH1-2 D3-22 JH3 0.0 20.0 CCGTGTATTACTGTGCGAGAGAGCGATCCTTAGAAAG TTACTATGATAGTAGTGGTAATGCTTTTGATATCTGG
Ber 111 n.d. VH1-2 D5-18 JH4 0.0 145.8 CCGTGTATTACTGTGCGAGAGAA GGATACAGCTATGGTGCCTGG
VH3-9 D3-22 JH4 0.0 62.5 GGCCTTGTATTACTGTGCAAAACCTT TATTACTATGATAGTAGTGGTTATTAAAGGGTGCTTTGACTACTGG
Ber 117 n.d. VH1-69 D3-22 JH4 0.0 62.5 CACGGCCGTGTATTACTGTGCGGGGT ATAGTAGTGGTTATTACTCGGCCCCGGGGGACTTTGACTACTGG
VH1-8 D2-2 JH5 13.9 0.0 GCCGTGTATTACTGTGCGAGAGAACGACACTTT TACTGTAGTAGTCCCGGCTGCTATACGGGCGGCCCTCGTGCACAACTGGTTCGACCCCTGG
VH7-4-1 D3-22 JH4 24.3 187.5 GCCGTCTACTACTGTGCGAGAGTAAC TAGTAGTTACCTAGCCCTCTGG
VH1-69 D2-15 JH5 3.5 98.0 CCGTGTATTACTGTGCGAGAGAGCGCTGA GGTGGTAGCTGCTACTCACTGGTTCGACCCCTGG
Ber 127 n.d. VH1-69 D3-10 JH6 6.9 16.1 CCGTGTATTACTGTGCGAGAGAG ATGGTTCGGGGAGTTATTATCTACTGGGAACTACTACTACTACTACGGTATGGACGTCTGG
VH3-13 D4-17 JH6 3.5 32.3 GCTGTGTATTACTGTGCAAGAGCTTA TACGGTGACTACGGGGATTACTACTACTACTACGGTATGGACGTCTGG
VH1-18 D2-8 JH6 0.0 96.8 CGGCCGTGTATTACTGTGCGAGGG CTGGGGGTGGGCCATCACCCTACTACTACGGTATGGACGTCTGG
Ber 137 n.d. VH1-8 D6-19 JH6 0.0 161.3 CCGTGTATTACTGTGCGAGAGG GGTATAGCAGTGGCTGGTACCACATGGACGTCTGG
VH1-2 D3-22 JH6 0.0 209.7 CGGCCGTGTATTACTGTGCGAGTATCGGGG ATTACTATGATAGTAGTGGTTACGTATTAGACATGGACGTCTGG
VH1-2 D2-15 JH4 59.0 104.2 CCGTTTATTACTGTGCGAGAGATCCAC TAGTCTTGGCTTTGGCTACTGG
VH1-2 D3-10 JH4 3.5 145.8 CCGTGTATTACTGTGCGACAGAAAC TATTACTATGGTTCGCTACATGGACGTCTGG
VH1-2 D2-15 JH4 86.8 95.2 GCCATATTTTACTGTGCGACAGTGGAGGCGGTCGCAG TGTAGTGGTGGTAGCTGCTCAATCTCTACTACTACGGTATGGACGTCTGG
VH3-30 D3-10 JH5 6.9 39.2 CTGTGTATTACTGTGCGAAAGAAGGA GGTTCAGGGAGTTTTCTAACTGGTTCGACCCCTGG
VH1-69 D3-3 JH6 0.0 158.7 CCGTGTATTACTGTGCGAGAGAAGGTGTCGGAG ACGATTTTTGGAGTGGTTATTATGGGGCGGCGGGCTACTACTACGGTATGGACGTCTGG
VH1-2 D5-18 JH4 3.5 104.2 ACACGGCCGTGTATTACTGTGCCCTAGGGG GGATACAGCTATGGTACCCTTTTGACTACTGG
Ber 72 n.d. VH1-18 D3-3 JH4 0.0 83.3 GCCGTGTATTACTGTGCGAGAGTGGGACCGAGG GGAGGGGTTTACTTTGACTACTGG
VH6-1 D6-19 JH6 0.0 193.5 CGGCTGTGTATTACTGTGCAAGTGGGGAGGC GTATAGCAGTGGCTGGATATACGGTATGGACGTCTGG
VH3-15 D2-2 JH4 0.0 208.3 CCGTGTATTACTGTACCACAGA TTGTAGTAGTACCAGCTGCTATGCCCACTTGTAGACTGG
VH3-15 D2-2 JH4 0.0 166.7 CCGTGTATTACTGTACCACAGA TTGTAGTAGTACCAGCTGCTATGCCCACTTGTAGACTGG
Ber 75 n.d. VH1-NL1 D3-22 JH4 66.2 104.2 TCGTGTATTCCTGTGTGAGAGAG TACTATGATAGTAGTGGTTGTCTAGCCGGACTACTGG
VH3-30 D1-1 JH4 0.0 270.8 GCTGTGTATTACTGTGCGAAAGTA GGTAGAATAAGCGACCCCGGGGTCTGAGGCGGTACTGG
VH1-2 D3-22 JH2 49.8 113.2 CGGCCGTGCATTACTGTGCGAGGTGCAAAGCATG TGATAGAAGTGATTATCTTACGCGACCCCTATTACAACGACACGTACTTCGATCTCTGG
Ber 83 n.d. VH1-46 D5-24 JH4 0.0 83.3 CGGCCGTGTATTACTGTGCGAGGG GTAGAGATGGCTACAATTATAACTACTGG
VH3-53 D3-9 JH6 3.5 129.0 GCCGTGTATTACTGTGCGAGAGGGGGGG ATTACGATATTTTGACTGGTTATTATGCCCGTACCACTACTACGGTATGGACGTCTGG
VH1-2 D6-19 JH4 0.0 125.0 CCGTGTATTACTGTGCGAGAGATCCC ATAGCAGTGGCTGGTACTCCACCGACTACTGG
VH1-2 D2-2 JH4 0.0 0.0 GGCCGTGTATTACTGTGCGAGATCCATTTCT CTGCTAGACAAGTCGACCCATAAACTAACTACTTTGACTACTGG
Ber 94 n.d. VH1-2 D3-22 JH5 0.0 98.0 CCGTGTATTACTGTGCGAGAGAGGGGC TATTACTATGATAGTAGTGGTTATATCTAAGGGGGGTGGTTCGACCCCTGG
Ber 102 n.d. VH6-1 JH6 37.0 0.0 GGACAGGGGGGAACCTGGGGGT ATGGACGTCTGG
VH1-8 D2-15 JH5 3.5 98.0 CGGCCGTGTATTACTGTGCGAGTTGCCCCC GTGGTAGCTGCTACTTACAGGGGTTCGACCCCTGG
VH6-1 D3-16 JH4 0.0 130.4 GCTGTGTATTACTGTGCAAGAGCCGGAACCATTAAACGATATTTTGACTGGTTATTATAAA GTATTATGATTACATTTGGGGGAGTTATCGTTTCTCTGACTACTGG
Ber 103 n.d. VH1-45 D2-21 JH5 3.5 78.4 GCCATGTATTACTGTGCAAGATTTTT GTGACTGCCCCCAGGGGGTCTGGTTCGACCCCTGG
VH1-45 D2-21 JH5 6.9 78.4 GCCATGTATTACTGTGCAAGATTTTT GTGACTGCCCCCAGGGGGTCTGGTTCGACCCCTGG
Ber 113 n.d. VH1-2 D6-6 JH6 114.6 161.3 GGCCGTATATTACTGCGCGAGA AGCAGTTCGTCGACCATTTTCTACCACGGCATGGACGTCTGG
VH1-8 D1-26 JH6 0.0 145.2 GGCCGTGTATTACTGTGCGAGACCCGTCCTC AGTGGGAGCTACTTCTACGGTATGGACGTCTGG
VH3-15 D6-13 JH1 61.2 230.8 CAGCCGTCTATTACTGTACCACGTACAGGTTTGGGAAC ATAGCAGCAGCTCTCAGTGTAAAGAAGTGG
Ber 115 n.d. VH1-8 D3-9 JH5 3.5 98.0 GGACACGGCCGTGTATTACTGTTCCTTTCGGCGGAGAATTGG ATTACGATATTTTGACTGGTTATGTACTGGTTCGACCCCTGG
VH1-69 D1-1 JH6 52.1 193.5 GGCCGTGTATTACTGTGCGAGAT CACCTGGAGCGCAAGGGAACTACTACAATATGGACGTCTGG
VH1-18 D1-26 JH4 41.7 83.3 GGCCGTGTATTACTGTGCGAGATTAA GTTTAGTGGGAGTTTCCATTGACTACTGG
VH3-30 D1-26 JH5 0.0 235.3 ACGGCTGTGTATTACTGTGCGAACGACGC TACAGTGGGAGCTACTACGG
Ber 129 n.d. VH1-3 D6-13 JH1 0.0 134.6 CTGTGTATTACTGTGCGAGAGAGTG ATAGCAGCGGCTGGTACTACTTCCAGCACTGG
VH1-8 D2-2 JH5 41.7 39.2 CCGTGTATTATTGTGCGAGAGGGAGG TTGTACTAGTACCAGTTGCTAAGTCTACAGTCCGGGTATAGCAGTGGCTGGCCTGAACTGGTTCGACCCCTGG
Ber 131 n.d. VH6-1 D2-2 JH6 0.0 161.3 GCTGTGTATTACTGTGCAAGAGGGGCG TATTGTAGTAGTACCAGCTGCTATGATGGGTGGGGTACTACGGTATGGACGTCTGG
VH3-53 D6-19 JH6 0.0 177.4 ACACGGCCGTGTATTACTGTGCCCGCT TAGAAGGGGCCTACTACGGTATGGACGTCTGG
VH3-53 D6-19 JH6 0.0 145.2 ACACGGCCGTGTATTACTGTGCCCGCT TAGAAGGGGCCTACTACGGTATGGACGTCTGG
Ber 125 n.d. VH3-13 D3-9 JH5 0.0 39.2 CGGCTGTGTATTACTGTGCAAGTCGGGGGG GATATTTTGACTGGTTATTAAGGGGGAAACTGGTTCGACCCCTGG
VH1-69 D6-6 JH4 34.7 166.7 CCGTGTATTACTGTGCGAGAGAC GTAGAGCCCCTAGTAGGCGGGGGTCTGGACGTCTGG
VH1-69 D3-16 JH4 0.0 125.0 CCGTGTATTACTGTGCGAGAGAGGAGAGT TATTATGATTACGTTTGGGGGAGCCTATGGGCTTTTGACTACTGG
Nature Immunology: doi:10.1038/ni.3160
Table S5: Clonal Evolution in MLL-AF4 patientCell Allele 1 Allele 2
VH DH JH VH DH JH
Mut
Freq VH
(x10-3
bp)
Mut
Freq JH
(x10-3
bp) VH-N DH-N-JH
1 D3-15 JH6 VH4-31 D2-2 JH6-3 3.3 40.0 TGTGCGAGAGATGGGGTTTGAGC GGGTATTGTAGTAGTACCAGCTGCTACTACTACTACTACTTACATGGACGTCTGG
2 D3-15 JH6 VH4-31 D2-2 JH6-3 6.7 40.0 TGTGCGAGAGATGGGGTTTGAGC GGGTATTGTAGTAGTACCAGCTGCTACTACTACTACTACTTACATGGACGTCTGG
3 D3-15 JH6 VH4-31 D2-2 JH6-3 0.0 40.0 TGTGCGAGAGATGGGGTTTGAGC GGGTATTGTAGTAGTACCAGCTGCTACTACTACTACTACTTACATGGACGTCTGG
4 D3-15 JH6 VH4-31 D2-2 JH6-3 6.7 0.0 TGTGCGAGAGATGGGGTTTGAGC GGGTATTGTAGTAGTACCAGCTGCTACTACTACTACTACTTACATGGACGTCTGG
5 D3-15 JH6 VH1-2 D2-2 JH6-3 40.0 100.0 TGTGTTCTTCCCTAGGA ---TATTGTAGTAGTACCAGCTGCTACTACTACTACTACTTACATGGACGTCTGG
6 D3-15 JH6 VH1-2 D2-2 JH6-3 46.7 100.0 TGTGTTCTTCCCTAGGA ---TATTGTAGTAGTACCAGCTGCTACTACTACTACTACTTACATGGACGTCTGG
7 D3-15 JH6 VH1-2 D2-2 JH6-3 36.7 100.0 TGTGTTCTTCCCTAGGA ---TATTGTAGTAGTACCAGCTGCTACTACTACTACTACTTACATGGACGTCTGG
16 D3-15 JH6 VH7-4 D2-2 JH6-3 10.0 120.0 TGTGTTCTTCCCTAGGA ---TATTGTAGTAGTACCAGCTGCTACTACTACTACTACTTACATGGACGTCTGG
17 D3-15 JH6 VH7-4 D2-2 JH6-3 10.0 100.0 TGTGTTCTTCCCTAGGA ---TATTGTAGTAGTACCAGCTGCTACTACTACTACTACTTACATGGACGTCTGG
8 D3-15 JH6 VH3-13 D2-2 JH6-3 3.3 60.0 TGTGCAAGAGAGGGTTTGAGC GGGTATTGTAGTAGTACCAGCTGCTACTACTACTACTACTTACATGGACGTCTGG
9 D3-15 JH6 VH3-13 D2-2 JH6-3 0.0 80.0 TGTGCAAGAGAGGGTTTGAGC GGGTATTGTAGTAGTACCAGCTGCTACTACTACTACTACTTACATGGACGTCTGG
10 D3-15 JH6 VH3-13 D2-2 JH6-3 0.0 40.0 TGTGCAAGAGAGGGTTTGAGC GGGTATTGTAGTAGTACCAGCTGCTACTACTACTACTACTTACATGGACGTCTGG
11 D3-15 JH6 VH7-4 D2-2 JH6-3 20.0 60.0 TGTGCGAGAGGGTTTGAGC GGGTATTGTAGTAGTACCAGCTGCTACTACTACTACTACTTACATGGACGTCTGG
12 D3-15 JH6 VH7-4 D2-2 JH6-3 13.3 60.0 TGTGCGAGAGGGTTTGAGC GGGTATTGTAGTAGTACCAGCTGCTACTACTACTACTACTTACATGGACGTCTGG
13 D3-15 JH6 VH7-4 D2-2 JH6-3 13.3 80.0 TGTGCGAGAGGGTTTGAGC GGGTATTGTAGTAGTACCAGCTGCTACTACTACTACTACTTACATGGACGTCTGG
14 D3-15 JH6 VH7-4 D2-2 JH6-3 13.3 40.0 TGTGCGAGAGGGTTTGAGC GGGTATTGTAGTAGTACCAGCTGCTACTACTACTACTACTTACATGGACGTCTGG
15 D3-15 JH6 VH7-4 D2-2 JH6-3 10.0 40.0 TGTGCGAGAGGGTTTGAGC GGGTATTGTAGTAGTACCAGCTGCTACTACTACTACTACTTACATGGACGTCTGG
18 D3-15 JH6 VH2-5 D2-2 JH6-3 3.3 20.0 TGTGCACACCCCCCCGGGGGGGGTTTGAGC GGGTATTGTAGTAGTACCAGCTGCTACTACTACTACTACTTACATGGACGTCTGG
19 D3-15 JH6 VH2-5 D2-2 JH6-3 3.3 60.0 TGTGCACACCCCCCCGGGGGGGGTTTGAGC GGGTATTGTAGTAGTACCAGCTGCTACTACTACTACTACTTACATGGACGTCTGG
20 D3-15 JH6 VH2-5 D2-2 JH6-3 3.3 20.0 TGTGCACACCCCCCCGGGGGGGGTTTGAGC GGGTATTGTAGTAGTACCAGCTGCTACTACTACTACTACTTACATGGACGTCTGG
21 D3-15 JH6 VH2-5 D2-2 JH6-3 6.7 20.0 TGTGCACACCCCCCCGGGGGGGGTTTGAGC GGGTATTGTAGTAGTACCAGCTGCTACTACTACTACTACTTACATGGACGTCTGG
22 D3-15 JH6 VH2-5 D2-2 JH6-3 6.7 20.0 TGTGCACACCCCCCCGGGGGGGGTTTGAGC GGGTATTGTAGTAGTACCAGCTGCTACTACTACTACTACTTACATGGACGTCTGG
23 D3-15 JH6 VH2-5 D2-2 JH6-3 6.7 40.0 TGTGCACACCCCCCCGGGGGGGGTTTGAGC GGGTATTGTAGTAGTACCAGCTGCTACTACTACTACTACTTACATGGACGTCTGG
24 D3-15 JH6 VH2-5 D2-2 JH6-3 6.7 40.0 TGTGCACACCCCCCCGGGGGGGGTTTGAGC GGGTATTGTAGTAGTACCAGCTGCTACTACTACTACTACTTACATGGACGTCTGG
25 D3-15 JH6 VH2-5 D2-2 JH6-3 10.0 40.0 TGTGCACACCCCCCCGGGGGGGGTTTGAGC GGGTATTGTAGTAGTACCAGCTGCTACTACTACTACTACTTACATGGACGTCTGG
26 D3-15 JH6 VH2-5 D2-2 JH6-3 6.7 20.0 TGTGCACACCCCCCCGGGGGGGGTTTGAGC GGGTATTGTAGTAGTACCAGCTGCTACTACTACTACTACTTACATGGACGTCTGG
Cell Allele 1 Allele 2
VH DH JH VH DH JH
Mut
Freq VH
(x10-3
bp)
Mut
Freq JH
(x10-3
bp) VH-N DH-N-JH
27 Germline VH3-7 D3-22 JH6-2 3.3 20.0 TGTGCGAGAGACG TATTACTATGATAGTAGTGGTTATTACCAGGGTATTACTACTACTACTACTTACATGGACGTCTGG
28 Germline VH3-30 D3-22 JH6-2 3.3 0.0 TGTGCAAGAGATA TATTACTATGATAGTAGTGGTTATTACCAGGGTATTACTACTACTACTACTTACATGGACGTCTGG
29 Germline VH3-30 D3-22 JH6-2 10.0 0.0 TGTGCGAG TATTACTATGATAGTAGTGGTTATTACCAGGGTATTACTACTACTACTACTTACATGGACGTCTGG
30 Germline VH3-30 D3-22 JH6-2 10.0 0.0 TGTGCGAG TATTACTATGATAGTAGTGGTTATTACCAGGGTATTACTACTACTACTACTTACATGGACGTCTGG
31 Germline VH3-30 D3-22 JH6-2 10.0 0.0 TGTGCGAG TATTACTATGATAGTAGTGGTTATTACCAGGGTATTACTACTACTACTACTTACATGGACGTCTGG
32 D3-15 JH6 VH6-1 D3-22 JH6-2 0.0 0.0 TGTGCAAGAGATCCGTATAGCAGTGGCTGG TATTACTATGATAGTAGTGGTTATTACCAGGGTATTACTACTACTACTACTTACATGGACGTCTGG
33 D3-15 JH6 VH6-1 D3-22 JH6-2 3.3 20.0 TGTGCAAGAGATCCGTATAGCAGTGGCTGG TATTACTATGATAGTAGTGGTTATTACCAGGGTATTACTACTACTACTACTTACATGGACGTCTGG
34 D3-15 JH6 VH6-1 D3-22 JH6-2 3.3 20.0 TGTGCAAGAGATCCGTATAGCAGTGGCTGG TATTACTATGATAGTAGTGGTTATTACCAGGGTATTACTACTACTACTACTTACATGGACGTCTGG
35 D3-15 JH6 VH6-1 D3-22 JH6-2 3.3 20.0 TGTGCAAGAGATCCGTATAGCAGTGGCTGG TATTACTATGATAGTAGTGGTTATTACCAGGGTATTACTACTACTACTACTTACATGGACGTCTGG
36 D3-15 JH6 VH6-1 D3-22 JH6-2 0.0 20.0 TGTGCAAGAGATCCGTATAGCAGTGGCTGG TATTACTATGATAGTAGTGGTTATTACCAGGGTATTACTACTACTACTACTTACATGGACGTCTGG
37 D3-15 JH6 VH6-1 D3-22 JH6-2 3.3 20.0 TGTGCAAGAGATCCGTATAGCAGTGGCTGG TATTACTATGATAGTAGTGGTTATTACCAGGGTATTACTACTACTACTACTTACATGGACGTCTGG
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Supplementary Table 6: Characteristics of ETV6-RUNX1 patient samples used for IGH sequencing studies in
Supplementary Table 3.
Ref No.
ETV6-
RUNX1
sample
Diagnosis
age
(months)
BM
blasts
(%)
ETV6-RUNX1 breakpoint
Sample used
for IGH
sequencing
51 REH 180 100
46(44-47)<2n>X, -X, +16, del(3)(p22),
t(4;12;21;16)(q32;p13;q22;q24.3)-inv(12)(p13q22),
t(5;12)(q31-q32;p12), der(16)t(16;21)(q24.3;q22)
sideline with inv(5)der(5)(p15q31),+18
carries t(12;21) and del(12) producing respective
ETV6-RUNX1 fusion and deletion of residual ETV6
Cell line
LAX13 96 80 47,XX,t(12;15)(p13;q15),+21[6]/46,XX[14] Xenograft
(Passage 3)
23 PD4009a 44 96 chr12:g.o12024743_chr21:36311593bkpt
chr12:g.12024661_chr21:o36311621bkpt Diagnostic
23 PD4020a 49 85 Diagnostic
23 PD4021a 58 80 chr12:g.o12031874_chr21:36356100bkpt Diagnostic
23 PD4038a 62 96 chr12:g.o12028634_chr21:36404913bkpt
chr12:g.12028601_chr21:o36404683bkpt Diagnostic
23 PD3952a 30 63 chr12:g.12035173_chr21:o36347548bkpt Diagnostic
23 PD3961a 23 40 Diagnostic
23 PD4022a 43 92 Diagnostic
23 PD4024a 34 94 chr12:g.o12032290_chr21:36338458bkpt
chr12:g.12032237_chr21:o36338430bkpt Diagnostic
23 PD4036a 30 96 chr12:g.o12034799_chr21:36420621bkpt Diagnostic
23 PD4037a 46 86 chr12:g.o12035488_chr21:36418785bkpt
chr12:g.12035482_chr21:o36420169bkpt Diagnostic
43 920527-4278 n.d. n.d. 46,XY,t(2;14)(q23;q32); t(12;21) Diagnostic
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Supplementary Table 7: Characteristics of patient samples used for IGH sequencing studies in
Supplementary Table 4.
Ref No. Leukemia
sample
Diagnosis
age
(months)
Karyotype Cytogenetics Sample used for
IGH sequencing
43 aP902486/96 n.d. 46,XY,t(4;11)(q21;q23) MLL-AF4 Diagnostic
43 aP901941/96 n.d. 46,XX,t(11;19)(q23;p13) MLL-ENL Diagnostic
43 aP902032/95 n.d. 46,XX,t(1;19)(q23;p13) TCF3-PBX1 Diagnostic
43 aP900469/95 n.d. 46,XY,t(1;15)(q11;p11),t(1;19)(q2
3;p11),del(6)(q21) TCF3-PBX1 Diagnostic
43 bPatient 20 144 46,XY,del(2)(p2),t(8;14)(q24;q32) MYC-IGH Diagnostic
43 aB273/98 n.d. 55,XY,+X,+4,+5,+6,-
10,+14,+18,+21,inc[4] Hyperdiploid Diagnostic
43 aB2033/97
n.d. 54-
55,XX,+X,+4,+6,+?9,+14,+15,+17
,+21
Hyperdiploid Diagnostic
43 aP90100/96 n.d. 51-55,XX,+6,+14,+21,+22,inc Hyperdiploid Diagnostic
43 aP901756/96 n.d. 55,XX,+6,+14,+18,+19,+21,+21,in
c Hyperdiploid Diagnostic
43 aB960/97jfr12
24
n.d. 52-
55,XY,+X,+4,+6,+?14,+17,+21,+2
1
Hyperdiploid Diagnostic
43 ac901275/94 n.d. 55,XX,+X,+3,+6,+10,+14,+18,inc Hyperdiploid Diagnostic
43 ac27012-91 n.d. 56,XX,+X,+X,+3,+4,+9,+10,+13+
18,+19,+21 Hyperdiploid Diagnostic
43 aP901348/95
n.d. 53-
55,XX,+X,+4,+5,+6,+14,+17,+21,
+21,+mar
Hyperdiploid Diagnostic
43 aP902781/96 n.d. 51-
54,XX,+X,+6,+8,+14,+17,+18,inc Hyperdiploid Diagnostic
43 aB675/97
n.d. 50-
56,+X,+4,+6,+10,+17,+18,+18,+2
1,inc
Hyperdiploid Diagnostic
43 aP90131/96 n.d. 57-61,XX,+2,+3,+6,+8,+10,+14,-
16,-17+19,+20,+21,+22,inc Hyperdiploid Diagnostic
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43 aP902300/95
n.d. 54,XY,+X,+4,del(10)(p11),+21,+2
1,+4mar/52-
55,XY,+X,+4,+13,+19,+21,+4mar
Hyperdiploid Diagnostic
43 aP900978/95 n.d. 54,XX,+X,+7,+18,+21,+4mar Hyperdiploid Diagnostic
43 ac900162/94
n.d. 59,XXY,+Y,-1,-2,-3,-7,-8,-9,-10,-
11,-12,-13,-16,-19,-20,+21,-
22,+2mar
Hyperdiploid Diagnostic
52 bPatient 5 36 High hyperdiploid clone Hyperdiploid Diagnostic
52 bPatient 10 48 High hyperdiploid clone with
del(6)(q2) Hyperdiploid Diagnostic
52 bPatient 18 84 High hyperdiploid clone with
trisomy 14 Hyperdiploid Diagnostic
43 ahösten-95 n.d. 46,XX,del(1)(q?) Other Diagnostic
43 aB1057/97
n.d. 47,XY,t(1;12)(p32;p12),add(6)(q2
1),t(7;7)(q22;q36),add(11)(q21),m
ar,inc
Other Diagnostic
43 aP900667/96 n.d. 46,XY,t(3;12)(p11;p13)[15]/46,XY
[23] Other Diagnostic
43 ac900891/94 n.d. 47-48,XX,+X,+?21 Other Diagnostic
43 ac900024/94 n.d. 46,XY,der(12)t(12;14)(p12;q11) Other Diagnostic
43 aP901693/95 n.d. 46,XY,del(9)(p11),del(17)(p11)[20
] Other Diagnostic
43 ac26170/87 n.d. 46,XX,t(1;12)(p31;?q)),del(11)(q1
4) Other Diagnostic
52 bPatient 3 24 46,XY,t(9;15)(p2;q1) Other Diagnostic
52 bPatient 17 72 46,XX,del(6)(q2?q5) Other Diagnostic
52 bPatient 23 168 46,XY,t(17;22)(q25;q11) Other Diagnostic
43 aP902775/95 n.d. 46,XX Normal Diagnostic
43 aB192/98 n.d. 46,XX Normal Diagnostic
52 bPatient 1 24 n.d. N/A Diagnostic
52 bPatient 29 252 n.d. N/A Diagnostic
Ber 10 n.d. n.d. N/A Diagnostic
Ber 71 n.d. n.d. N/A Diagnostic
Ber 77 n.d. n.d. N/A Diagnostic
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Ber 82 n.d. n.d. N/A Diagnostic
Ber 100 n.d. n.d. N/A Diagnostic
Ber 101 n.d. n.d. N/A Diagnostic
Ber 107 n.d. n.d. N/A Diagnostic
Ber 111 n.d. n.d. N/A Diagnostic
Ber 117 n.d. n.d. N/A Diagnostic
Ber 127 n.d. n.d. N/A Diagnostic
Ber 137 n.d. n.d. N/A Diagnostic
Ber 72 n.d. n.d. N/A Diagnostic
Ber 75 n.d. n.d. N/A Diagnostic
Ber 83 n.d. n.d. N/A Diagnostic
Ber 94 n.d. n.d. N/A Diagnostic
Ber 102 n.d. n.d. N/A Diagnostic
Ber 103 n.d. n.d. N/A Diagnostic
Ber 113 n.d. n.d. N/A Diagnostic
Ber 115 n.d. n.d. N/A Diagnostic
Ber 129 n.d. n.d. N/A Diagnostic
Ber 131 n.d. n.d. N/A Diagnostic
Ber 125 n.d. n.d. N/A Diagnostic
Nature Immunology: doi:10.1038/ni.3160
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Supplementary Table 8: Antibodies used in this study.
Immunoblot
Antigen Clone ID Specificity Dilution Source
AID L7E7 Mouse/Human 1:1000 Cell Signaling Technology
β-Actin C4 Mouse/Human 1:10,000 Santa Cruz Biotechnology
FOXO4 9472 Mouse/Human 1:1000 Cell Signaling Technology
RAG-1 Mouse/Human 1:1000 A kind gift from David
Schatz Laboratory
STAT5 3H7 Mouse/Human 1:1000 Cell Signaling Technology
Phospho-Y694 STAT5 14H2 Mouse/Human 1:1000 Cell Signaling Technology
RAG-1 EPRAGR1 Mouse 1:1000 Abcam
RAG-2 EPRAGR239 Mouse 1:1000 Abcam
Flow cytometry
Surface Antigen Clone ID Specificity Source
CD19 1D3 Mouse BD
IL-7Rα A7R34 Mouse eBioscience
Igκ Light Chain 187.1 Mouse BD
IL-2Rα (CD25) 7D4 Mouse BD
B220 RA3-6B2 Mouse/Human BD
c-Kit 2B8 Mouse BD
IgM R6-60.2 Mouse BD
CD19 HIB19 Human Biolegend
Igκ Light Chain G20-193 Human BD
Igλ Light Chain JDC-12 Human BD
Nature Immunology: doi:10.1038/ni.3160
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Supplementary Table 9: Sequences of oligonucleotide primers used in this study.
Quantitative RT-PCR
Hprt_F 5’-GGGGGCTATAAGTTCTTTGC-3’
Hprt_R 5’-TCCAACACTTCGAGAGGTCC-3’
Rag2_F 5’-GCAGATGGTAACAGTGGGTC-3’
Rag2_R 5’-ATTGCAGGCTTCAGTTTGAG-3’
Rag1_F 5’-TAACAACCAAGCTGCAGACA-3’
Rag1_R 5’-CCTCTGAGGAATCCTTCTCC-3’
Aicda_F 5’- AAATGTCCGCTGGGCCAA-3’
Aicda_R 5’- CATCGACTTCGTACAAGGG-3’
AICDA F 5’–TCCTTTTCACTGGACTTTGG–3’
AICDA R 5’–GACTGAGGTTGGGGTTCC–3’
COX6B F 5’-AACTACAAGACCGCCCCTTT-3’
COX6B R 5’-GCAGCCAGTTCAGATCTTCC-3’
Somatic hypermutation of human VH region
Forward primer mix (VH mix)
VH1_F 5’-CAGTCTGGGGCTGAGGTGAAGA-3’
VH2_F 5’-GTCCTRCGCTGGTGAAACCCACACA-3’
VH3_F 5’-GGGGTCCCTGAGACTCTCCTGTGCAG-3’
VH4_F 5’-GACCCTGTCCCTCACCTGCRCTGTC-3’
VH5_F 5’-AAAAAGCCCGGGGAGTCTCTGARGA-3’
VH6_F 5’-ACCTGTGCCATCTCCGGGGACAGTG-3’
Reverse primer mix (JH mix)
3’JH1.2.4.5_R 5’-ACCTGAGGAGACGGTGACCAGGGT-3’
3’JH3_R 5’-ACCTGAAGAGACGGTGACCATTGT-3’
3’JH6_R 5’-ACCTGAGGAGACGGTGACCGTGGT-3’
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Supplementary Table 10a: Retroviral vectors used in the study.
Constitutive expression Inducible activation
MSCV Cre-IRES-GFP
MSCV Blnk-IRES-GFP35 MSCV-ERT2-Puro
MSCV BlnkY96F-IRES-GFP35 MSCV-Cre-ERT2-Puro53 (Addgene)
MSCV ETV6-RUNX1-IRES-GFP
MSCV ETV6-RUNX1 ∆RHD-IRES-GFP
MSCV Foxo1CA-IRES-GFP35
MSCV IRES-GFP35
MSCV Foxo3aCA IRES-CD9054
MSCV IRES-CD9054
Supplementary Table 10b: Lentiviral vectors used in the study.
Constitutive expression
pCL6-Aicda-IRES-iRFP670-wo
pCL6-Rag1-IRES-eGFP-wo
pCL6-Rag2-IRES-dsRedExpress2-wo
Transfections of the above retroviral and lentiviral constructs were performed as discussed in the online methods
section.
Nature Immunology: doi:10.1038/ni.3160