Supplementary Materials and Methods
Animals. NOD/Ltsz-scid/scid (NOD-SCID) and NOD.Cg-Prkdc(scid)Il2rg(tm1Wjll)/SzJ (NSG)
mice were housed in pathogen-free animal facilities of CEA, Fontenay-aux-Roses, France. NODSCID and NSG mice were irradiated at 3 Gy and anesthetized with isoflurane before intravenous
injection (IV). All experimental procedures were done in compliance with French Ministry of
Agriculture
regulations
(animal
facility
registration
number:
A920322)
for
animal
experimentation.
Human T-ALL, umbilical cord blood and thymus samples. All primary samples were obtained
after informed consent of the patients in accordance with national ethic rules. White blood cells
were isolated by ficoll centrifugation, immuno-phenotyped and utilized directly or frozen in Fetal
Calf Serum (FCS) containing 10% DMSO and 25ng/ml recombinant human (rh) IL-7. CD34+ cells
were isolated from fresh UCB or infant thymuses using CD34 immunomagnetic purification (CD34
microbead kit, Myltenyi Biotech, Paris, France). T-ALL samples were either from patients or
xenografts of NOD-SCID or NSG mice (see Suppl Table). In the last case, T-ALL cells (human
CD45+/CD7+) constituted more than 90% of cells contained in mouse hematopoietic organs.
Culture conditions. Peripheral blood T-ALL were cultured as previously described (1) . Briefly,
T-ALL cells were cultured in complete T-ALL medium containing reconstituted alpha-MEM
supplemented with 10% FCS (06450, Stem Cell Technologies, Vancouver, Canada) and 10%
human AB serum (J Boy, Reims, France), in presence of stem cell factor (rhSCF, 50ng/ml, Amgen,
Neuilly-sur-Seine, France), rhFlt3-L (20ng/ml, Diaclone, Besançon, France), Insulin (20nM,
Sigma) and
rhIL-7 (10ng/ml, R&D System). In specific experiments, GSI (N-[N-(3,5-
difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester; DAPT, Calbiochem, Strasbourg,
France ; 10µM) was added in the culture medium either during the overall culture period. GSI was
diluted into DMSO and control cultures included DMSO in medium.
Cell surface staining. Cells were stained with Phycoerythrin (PE-), PE-Cyanin 5 (PC5-) and
Allophycocyanin (APC-) conjugated mouse anti-human specific CD45, CD7, CD4 and CD8
monoclonal antibodies (1:25 dilution; Beckman Coulter, Villepinte, France). Cell-surface markers
and EGFP expression was monitored on a FACScalibur (BD Biosciences, Le Pont de Claix,
France).
Packaging and vectors. Vector plasmids (pTRIP/∆U3-EF1α-GFP, pTRIP/∆U3-MND-GFP and
pTRIP/∆U3-SFFV-GFP), encapsidation plasmid (p8.91), VSV-G-expressing (phCMV-G) plasmid
(2) and IL-7 cDNA fragment fused with the surface subunit of the amphotropic MLV env gene
(G/IL-7SUx) were used (2,3).
Production of lentiviral vector particles. Lentiviral particles were produced by transient calcium
phosphate co-transfection of 293T cells (3 flasks, 1.8x106 cells/175cm2 flask, 3 days prior
transfection) with 100 µg of the pTRIP/U3-EF1α (or -MND or -SFFV)-GFP vector construct, 100
µg of either p8.91 packaging construct and 70 µg of the phCMV-G env construct. In the case of
HIV-1-derived vectors co-pseudotyped with VSV-G and G/IL-7SUx chimeric glycoproteins, an
equimolar quantity of envelope-expressing plasmids was used. Transfection medium was replaced
16 hours after transfection by T-ALL medium. Viral supernatant was harvested 48 hours later, lowspeed centrifuged, filtered, buffered with 1% of HEPES and treated with 10 µg/ml of DNAse and 1
mM of MgCl2. Infectious titers were determined by doing limiting dilution of LVs on Jurkat cells.
Titers were comprised between 107 and 108 transduction unit/ml. Multiplicities of infection (MOIs)
are indicated.
Transduction of human T-ALL samples: Final experimental protocol (Fig 1E). Fresh or
thawed T-ALL samples were co-cultured during 48 hours on MS5-DL1 in complete T-ALL
medium. Then, blasts and MS5-DL1 cells were vigorously mixed, a cell suspension was made and
filtered two times on 70 m nylon filters (SEFAR NITEX 03-80/41) in order to eliminate clumps
of MS5-DL1 cells. This cell suspension was then incubated in plastic wells of tissue culture treated
plates during 30 to 60 minutes and supernatant containing blast cells was gently recovered. This
step allows remaining MS5-DL1 cells to be mostly depleted. Blasts were then incubated for
transduction in immobilized human DL1 (Ig-DL1, 2.5 µg/mL) pre-coated wells (4). Blasts were
centrifugated (1500g) during 2 hours in presence of 5 µg/mL protamine sulfate and G/IL-7SUx
pseudotyped LVs at indicated MOI. The final selected vector construct is the pTRIP/∆U3-MNDGFP vector. Transduction further proceeded during 48 hours at 37°C. Blasts were washed twice in
PBS and EGFP expression was evaluated using FACS analysis. EGFP+ blasts were sorted on a
FACSaria (BD Biosciences) and used in functional assays.
Transduction conditions of UCB CD34+ cells. Freshly purified UCB CD34+ cells (106 cells/mL)
were incubated with LVs in 100% BIT serum-free medium (Stem Cell Technologies, Vancouver,
Canada) plus stem cell factor (SCF; 100 ng/mL), Flt3-ligand (Flt3-L; 100 ng/mL), thrombopoietin
(TPO; 10ng/mL) and interleukin 3 (IL-3; 60ng/mL) during 3 days. At the end of the transduction
period, cells were washed twice in PBS and EGFP expression was monitored by FACS analysis.
Transduction conditions of human thymus CD34+ cells. Freshly purified human thymus CD34+
cells (106 cells/mL) were incubated with the LVs in complete T-ALL medium during 3 days. At
the end of the transduction period, cells were washed twice in PBS and EGFP expression was
monitored by FACS analysis.
Assessment of in vitro proliferation. was determined using the APC-BrdU/7-AAD flow kit (BD
Biosciences). Briefly, cells (106 cells/mL) were incubated over night with 10 mM BrdU. The day
after, cells were washed and stained with a dilution of 1/50 APC-labeled antibodies directed against
BrdU. 7-AAD (0.25µg/106 cells) was mixed to the cells 10 minutes before FACS analysis.
Supplementary Reference List
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