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Baboon envelope pseudotyped lentiviral vectors: a highly efficient new tool to genetically manipulate T-cell acute lymphoblastic leukaemia-initiating cells.
Caroline Costa1+, Guillaume Hypolite2+, Ornellie Bernadin1+, Camille Lévy1, Francois-Loïc Cosset1, Vahid Asnafi2, Elizabeth Macintyre2, Els Verhoeyen1,3 and Melania Tesio2
SUPPLEMENTAL DATA
Supplementary figure 1
Supplementary figure 2
Supplementary figure 3
Supplementary figure 4
Supplementary figure 5
Supplementary figure 6
Supplementary table 1
Supplementary table 2
Supplementary table 3
Supplementary table 4
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A. LDL-R, CD150, CD46, ASCT1 and ASCT2 mRNA levels in primary T-ALL cells. Results are expressed as fold change compared to CD7 levels, set as 1 (dotted line).
B-C. Representative FACS plots showing LDLR, CD150 and CD46 expression on CD45+CD7+ blasts (A) and on LIC-enriched CD34+ blasts (B) from a patient-derived sample (UPN763).
D. ASCT1 and ASCT2 expression in LIC-enriched CD34+ blasts in basal conditions (not stimulated) or following 3 days of culture on OP9-DL1 cells in the presence of cytokines and insulin. Data are expressed as fold change to ASCT1 and ASCT2 mRNA levels detected in unstimulated healthy T-cells, set here to 1. * p0.05 (one-tailed Student t test)
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ASCT1 and ASCT2 expression in patients-derived T-ALL cells according to the following different oncogenic abnormalities: NOTCH1/FBXW7 activating mutations (N/F mut) (A-D), PTEN deletions/mutations (PTEN altered) (B-E), CALM-AF10 fusion transcript (CA), SIL-TAL1 fusion transcript (S-T) and TLX1/3 overexpression (TLX1/3) (C-F). Data are expressed as fold change to ASCT1 and ASCT2 mRNA levels detected in unstimulated healthy T-cells, set here to 1.
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GFP expression analysed 4 days post-transduction on leukemic CD45+CD7+ blasts (UPN149, UPN105, UPN489, UPN735, UPN525) transduced with VSVG, HF or BaEV-pseudotyped lentiviral vectors using a range of MOIs (1, 5, 10 and 20). * p0.05, *** p0.001 (one-tailed Student t test).
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A-B. Percentage of GFP expression (A) and GFP mean fluorescent intensity (B) evaluated two and six days post-transduction (UPN633, UPN613).
C-D. Percentage GFP expression (C) and vector copy numbers (D) in blasts (UPN633, UPN613) transduced with increasing MOIs.
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Competitive xenografts. Experimental procedure (A) and vector copy numbers observed in bone marrow cells issued from secondary transplants (B).
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Experimental procedure followed during non-competitive xenografts (A) and during limiting dilutions assays (B).
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Supplementary Table 1
Oncogenic abnormalities and maturation arrest stage of primary T-ALL blasts analysed during in vitro experiments
Patient Origina Maturationarrest stage
Oncogenicabnormalities
UPN525 Xenograft IMG RAS mutationUPN534 Xenograft Pre- PTEN deletionUPN584 Xenograft TCR FBXW7 mutation
SIL-TAL1 fusion transcript
UPN727 Xenograft TCR NOTCH1 mutationFBXW7 mutation
UPN149 Xenograft TCR TLX3 overexpressionNOTCH1/FBXW7
mutation
UPN656 Xenograft TCR NOTCH1 mutation
TLX3 overexpressionPTEN mutation
UPN633 Xenograft TCR PTEN mutationUPN613 Xenograft TCR NOTCH1 mutation
UPN727 Patient-derived
Pre-FBXW7 mutation
PTEN deletionTLX1 overexpression
UPN763 Patient-derived
IMD RAS mutation
a Xenograft means patient-derived T-ALL cells amplified in NSG mice (1 to 2 passages) and patient-derived means T-ALL cells transduced upon direct isolation from patient’s samples.
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Supplementary table 2. Competitive xenografts into NSG mice.
Engraftment of transduced and non-transduced clones into two serial cohorts of NGS mice.
PRIMARY
T-ALL
MATURATIONARREST STAGE &
ONCOGENIC ABNORMALITIES
GFP EXPRESSION IN CD45+CD7+ BLASTS
INJECTED BLASTS
XENOGRAFTED MICE
UPN525IMG
RAS mutation82.5 %
Primary Secondary
Blood: 73.6 ± 6.3 (n=2)
87.2 ± 0.3 (n=5)
Bone marrow: 76.9 ± 2.65 (n=2)
79.4 ± 1.9 (n=5)
Spleen: 73.5 ± 2.75 (n=2)
83.6 ± 2.4 (n=5)
UPN534
Pre-
PTEN deletion49.3 %
Blood: 41.8 (n=1) 44.7 ± 4.2 (n=3)
Bone marrow: 42.9 (n=1) 45.6 ± 4.3 (n=3)
Spleen: 43.2 (n=1) 46.3 ± 1.9 (n=3)
UPN584
TCR
FBXW7 mutationSIL-TAL1 fusion
transcript
59.4%
Blood: 63.0 ± 2.4 (n=2)
61.6 ± 5.7 (n=4)
Bone marrow: 60.7 ± 6.1 (n=2)
55.5 ± 2.7 (n=4)
Spleen: 55.4 ± 1.26 (n=2)
39 ± 9.6 (n=4)10
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Supplementary table 3. Non competitive xenografts
Summary of non competitive xenograft experiments showing the engraftment of sorted GFP negative and GFP positive blasts as well as the percentage of GFP expression in two serial cohorts of NSG mice.
T-ALLMATURATION
ARREST STAGE &
ONCOGENIC ABNORMALITI
ES
PRIMARYXENOGRAFTS
SECONDARYXENOGRAFTS
UPN525
IMG
RAS mutationBlood:
Bone marrow:
Spleen:
Engraftment GFP-
blasts
51 ± 1.3 (n=3)
80.9 ± 2.8 (n=3)
41.4 ± 2.7 (n=3)
Engraftment
GFP+ blasts
45.9 ± 2.3 (n=3)
69.2 ± 7.4 (n=3)
58.9 ± 0.2 (n=3)
% GFPexpressio
n
99.5 ± 0.15
(n=3)
98.8 ± 0.26
(n=3)
99.5 ± 0.05
(n=3)
Engraftment GFP-
blasts
54.3 ± 3.4 (n=3)
82.9 ± 4.8 (n=3)
88.4± 0.6 (n=3)
Engraftment
GFP+ blasts
60.4 ± 3.9 (n=3)
91.9 ± 6.6 (n=3)
90.6 ± 0.7 (n=3)
% GFPexpressio
n
99 ± 0.5 (n=3)
97 ± 0.9 (n=3)
96.5 ± 0.35
(n=3)
Blood: 31.2 ± 5.7 51 ± 17.5 99 ± 1.8 41.5 ± 3.4 50.5 ± 3.0 98.4 ± 0.2
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UPN534
Pre-
PTEN deletionBone
marrow:
Spleen:
(n=2)
77.5 ± 0.15
(n=2)
81.7 ± 5.7 (n=2)
(n=2)
82.3 ± 2.75
(n=2)
85.8 ± 1.7 (n=2)
(n=2)
89.9 ± 0.32
(n=2)
98.7 ± 0.08
(n=2)
(n=3)
89.1 ± 3.3 (n=3)
87.1 ± 1.9 (n=3)
(n=4)
94.2 ± 2.1 (n=4)
86.3± 1.2 (n=4)
(n=4)
96.9 ± 0.2 (n=4)
97.9 ± 0.1 (n=4)
UPN584
TCR
FBXW7 mutation,
SIL-TAL1 fusion transcript
Blood:
Bone marrow:
Spleen:
41.9 ± 1.4 (n=2)
87.8 ± 0.9 (n=2)
63.7 ± 3.3 (n=2)
45.4 ± 2.3 (n=2)
85.5 ± 1.9 (n=2)
59.8 ± 1.4 (n=2)
95 ± 1.3(n=2)
96.9 ± 0.95
(n=2)
97.8 ± 0.9 (n=2)
83.7 ± 1.6 (n=2)
96.5 ± 2.1 (n=2)
86.5 ± 1.3 (n=2)
81 ± 1.5 (n=2)
93 ± 1.7(n=2)
88.8 ± 1.1 (n=2)
98 ± 0.8 (n=2)
99 ± 2.1 (n=2)
98 ± 0.5 (n=2)
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Supplementary table 4. Limiting dilution assay
Cell dose Blasts Number of
injected mice
Number of
engrafted mice
Time to death
(days)
5 x10^5 GFP- 3 3/3 33, 34, 37
GFP+ 3 3/3 35, 37, 38
5 x10^4 GFP- 6 6/6 37,39,44,46,50,62
GFP+ 6 6/6 38, 39, 39, 43, 46,68
1 x10^4 GFP- 8 7/8 40, 43, 47, 52, 53, 61, 68
GFP+ 6 5/6 40, 42, 58, 61, 78
1 x10^3 GFP- 5 2/5 70, 82
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GFP+ 5 3/5 58, 60,110
1 x10^2 GFP- 5 0/5 n/a
GFP+ 5 0/5 n/a
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