supplementary materials for · 2015. 2. 2. · table s1. characteristics of patients included in...
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www.sciencetranslationalmedicine.org/cgi/content/full/7/273/273ra13/DC1
Supplementary Materials for
In vivo tracking of T cells in humans unveils decade-long survival and activity of genetically modified T memory stem cells
Luca Biasco,* Serena Scala, Luca Basso Ricci, Francesca Dionisio, Cristina Baricordi, Andrea Calabria, Stefania Giannelli, Nicoletta Cieri, Federica Barzaghi, Roberta Pajno,
Hamoud Al-Mousa, Alessia Scarselli, Caterina Cancrini, Claudio Bordignon, Maria Grazia Roncarolo, Eugenio Montini, Chiara Bonini, Alessandro Aiuti*
*Corresponding author. E-mail: [email protected] (L.B.); [email protected] (A.A.)
Published 4 February 2015, Sci. Transl. Med. 7, 273ra13 (2015) DOI: 10.1126/scitranslmed.3010314
The PDF file includes:
Patient characteristics Fig. S1. CD4+/CD8+ frequency ratio in all groups of individuals. Fig. S2. FMO controls and gating strategy for TSCM identification. Fig. S3. Analysis of CD8+ cell composition and TSCM frequencies in ADA, BMT, and HSC-GT patients versus BMT-treated individuals with other primary immunodeficiencies. Fig. S4. In vivo and in vitro analyses of TSCM contribution in CD4+ cells. Fig. S5. TSCM absolute numbers in HDs and patients treated with BMT, HSC-GT, or PBL-GT+ERT. Fig. S6. In vitro IFN-γ production of sorted T cell subsets from HDs and GT patients. Fig. S7. In vitro analyses of sorted TN and TSCM from HD. Fig. S8. Phenotypical and functional characterization of in vitro generated TSCM. Fig. S9. Vector marking of sorted T cell subsets from PBL-GT+ERT patients. Fig. S10. Sorting scheme and IS retrieval of sorted T cell subpopulations from GT-treated patients. Fig. S11. IS analyses of T cell subpopulations from PBL-GT+ERT patients. Fig. S12. IS analyses of T cell subpopulations from HSC-GT patients. Fig. S13. Overlaps among single closest genes to insertion sites collected in vivo from PBL-GT+ERT and HSC-GT patients. Fig. S14. Top 10 enriched (binomial ranking) biological processes and MSig pathways associated to hit genes in PBL-GT+ERT (PBL-GT) and HSC-GT. Fig. S15. ISs detected over time in T central and T effector memory cells.
Table S1. Characteristics of patients included in this study. Table S2. Percentages of T cell subpopulations on CD8+ cells in BMT-, HSC-GT–, and PBL-GT+ERT–treated patients. Table S3. Frequencies of TSCM on CD8+ CD62L+CD45RA+ cells in BMT, HSC-GT, and PBL-GT individuals. Table S4. TREC content in BMT, HSC-GT, and PBL-GT patients. Table S5. CD8+ cell composition after 6 days of CD3/CD28+ rhIL2 stimulation of sorted T cell subsets. Table S6. Number of unique integrations retrieved for each T cell subset per patient.
1
Supplementary Materials
Patients characteristics
ADA-SCID patients were enrolled in different GT clinical trials using autologous transduced
PBL or HSC. Clinical trials were approved by San Raffaele Scientific Institute Ethical
Committee and Italian National regulatory authorities and patients were enrolled following
parents’ informed consent. Cells were transduced with a retroviral vector encoding human
ADA cDNA under the MLV long terminal repeat (LTR) promoter (GIADAl)(29). Vector
production and transduction protocol for PBL and HSC have been previously described(29,
31). PBL-GT patients were treated between 1992 and 1998 with repeated infusions of
autologous transduced PBL (clinical trial #NCT00599781). Three of the four patients studied
in this manuscript (PBL-GT Pt1, 2 and 3) have been previously described (28, 29); PBL-GT
Pt4 received similar treatment (manuscript in preparation). All patients were maintained on
ERT, with the exception of Pt3 who discontinued it for a period for 6 months.
The HSC-GT patients studied in this work (n=10) were treated between 2002 and 2011 with a
single infusion of autologous transduced CD34+ cells in the context of two clinical trials
(#NCT00599781 and #NCT00598481) or under compassionate use (Pt18). Five patients (Pt3,
Pt5, Pt6, Pt9 and Pt10) were previously described, while the other 5 are all ADA-SCID
patients with confirmed mutation and lack of HLA-identical sibling donor (Table S1 and
manuscript in preparation). Since April 2012 Glaxo Smith Kline (GSK) has become the
sponsor of a long-term follow up clinical trial (study 115611) under which ADA-SCID patients
with HSC-GT are followed long-term.
The group of pediatric allogeneic BMT is composed by 10 patients affected by different
variants of primary immunodeficiencies, including 4 ADA-SCID patients (Table S1). Patients
received BMT from different donor type with or without preparatory conditioning (Table S1).
ERT Pt1 is a late onset male ADA-SCID patient diagnosed at 22 months after birth and
carries a mild phenotype. ERT Pt2 is an early onset female ADA-SCID affected individual
diagnosed at 1 month after birth with a more severe phenotype. The lengths of ERT
supplementation at the time of analyses were 1 year for Pt1 and 5 years for Pt2. Biological
samples were obtained from patients, with approval of the San Raffaele Scientific Institute’s
2
or Bambino Gesù Hospital Ethics Committee and consent from parents or subjects. PB from
pediatric or adult healthy subjects was obtained on the occasion of other blood testing after
informed consent in the context of a research protocol established at San Raffaele Scientific
Institute.
Supplementary Figure and Table legends
Fig. S1. CD4+/CD8+ frequency ratio in all groups of individuals.
Ratio of CD4+ and CD8+ cells frequencies on CD3+ cells in HD Ped, HD Ad, BMT, HSC-
GT and PBL-GT+ERT individuals
HD Ped HD Ad BMT HSC-GT PBL-GT
+ERT
CD
4/C
D8
ratio
Fig. S1
HDped HDad BMTHSC-GTPBL-GT0
1
2
3
4
3
Fig. S2. FMO controls and gating strategy for T SCM identification.
Dot plots graphs showing both the FMO controls for each marker (displayed in red) and the
full stained sample (displayed in black). Gating on CD3+ cells, we investigated CD8+ and
CD4+ T cells subpopulations based on the expression of CD62L, CD45RA and CD95
markers. On the histograms are represented the expression of CD95, IL2Rβ, IL7Rα markers
in the full stained CD62L+CD45RA+ (black) and TEM (gray) cells, in comparison with the
respective TEM FMO controls. The FMO controls showed no spread of the other
fluorescences in the channel of interest. The gates for markers positivity were stringently set
to clearly dissect among discrete subpopulations.
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FSC-A
FS
C-H
SS
C-A
FSC-A
SS
C-A
CD3
CD8
CD
4
SS
C-H
SSC-A
CD8
CD
4
CD95
CD45RA
CD
62L
CD45RA
CD
62L
CD45RA
CD
62L
CD45RA
CD
62L
IL-2Rβ CD95 IL-7Rα
CD8 CD4
FMO CD3
FMO CD8 FMO CD4
FMO CD45RA FMO CD45RA FMO CD62L FMO CD62L
FMO CD95 FMO CD95 FMO IL-2Rβ FMO IL-7Rα
% o
f eve
nts
% o
f eve
nts
% o
f eve
nts
% o
f eve
nts
FMO sample T EM Full stained T EM Full stained CD45RA+/CD62L+
Fig. S2
4
Fig. S3. Analysis of CD8 + cell composition and T SCM frequencies in ADA, BMT, and
HSC-GT patients versus BMT-treated individuals with other primary
immunodeficiencies.
(A) Stacked bars graph showing composition of CD8+ T-cell compartment in BMT patients
with primary immunodeficiencies, ADA-SCID BMT patients and ADA-SCID HSC-GT treated
patients. (B) Column graph showing TSCM frequency on CD3+CD8+CD45RA+CD62L+ cells in
the same groups of individuals. (PID, primary immunodeficiencies). (Statistical analysis:
Mann-Whitney test; *P<0.05; PID BMT vs ADA HSC-GT Pvalue= 0.0160)
BMT BMT ada hsc-gt0
20
40
60
80
100
% o
f TS
CM
on
CD
8+C
D62
L+C
D45
RA
+ ce
lls
PID BMT ADA BMT ADA HSC-GT
*
ns
Fig. S3
CD62L&
CD45RA&
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0
103
104
105
TCM&
TEM& T
EMRA&
A B
BMT PID BMT ADA HSC-GT0
25
50
75
100
PID BMT ADA BMT ADA HSC-GT
% o
f CD
8+ c
ells
5
Fig. S4. In vivo and in vitro analyses of T SCM contribution in CD4 + cells.
(A) Representative plots of CD4+ T cells of a BMT, an ADA-SCID HSC-GT and a PBL-
GT+ERT treated patients and three HD Ad subjects. (B) Frequencies of CD4+ T-cell subsets
in pediatric (n=22) and adult (n=52) HD, BMT (n=10), ADA-SCID HSC-GT (n=10) and PBL-
GT+ERT (n=4) treated patients. (C) Relative TSCM frequency on
CD3+CD4+CD45RA+CD62L+ cells in each group of individuals. Red dots identify BMT treated
0102 103 104 105
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0
25
50
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100
0
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100
0
25
50
75
100
Pre-culture 96h 11d 18d 72h
% o
f CD
4+ c
ells
pre culture 96h 7d 15d
0
25
50
75
100
% o
f CD
4+ c
ells
0
25
50
75
100
% o
f CD
4+ c
ells
Pre-culture 96h 11d 18d Pre-culture 96h 11d 18d
TSCM NAIVE CM EM TEMRATCM TSCM TN TEM TEMRA TSCM NAIVE CM EM TEMRATCM TSCM TN TEM TEMRA
TSCM NAIVE CM EM TEMRATCM TSCM TN TEM TEMRA
% o
f CD
4+ c
ells
D
E F
HD Ped HD Ad BMT HSC-GT PBL-GT +ERT
% o
f CD
4+ c
ells
CD62L&
CD45RA&
% o
f TS
CM
on
CD
62L+
CD
45R
A+
cel
ls
HD Ped HD Ad BMT HSC-GT PBL-GT
+ERT
**
*
****
****
*
A B
C
HD Ad
HD Ad
HD Ad PBL-GT ERT
HSC-GT
BMT
TCM
TEM
TEMRA
CM
TE
TSCM
TN
Fig. S4
36.4 50.1
13.2 0.24
17.3 79.2
2.27 0.69
36.3 50.3
13.1 0.23
34.5 42.1
21.5 1.86
25.7
5.12
69
0.11
43.1 7.96
45.5 3.41
6
ADA-SCID patients. (D) CD4+ cells composition during in vitro culture as described in fig. 3A.
(E-F) Column graphs showing the relative frequencies of CD4+ T-cell subsets from sorted
TSCM and TN during in vitro culture (n=6). (Statistical analysis: Mann-Whitney test; *P<0.05;
**P<0.01; ****P<0.0001. HD Ped vs HSC-GT Pvalue= <0.0001; HD Ad vs HSC-GT Pvalue=
<0.0001; BMT vs HSC-GT Pvalue= 0.0115; HD Ped vs PBL-GT+ERT Pvalue= 0.0212; HD Ad
vs PBL-GT+ERT Pvalue= 0.0052; )
7
Fig. S5. T SCM absolute numbers in HDs and patients treated with BMT, HSC-GT, or PBL-
GT+ERT.
Dot plot showing the absolute number of TSCM (cells/uL) measured in pediatric and adult HD
in comparison with BMT, HSC-GT and PBL-GT+ERT treated subjects. The number of TSCM
was calculated for each individual on the basis of peripheral blood lymphocytes cell count and
relative TSCM frequency.
BMT HSC-GT PBL-GT
+ERT
Fig. S5
TS
CM a
bsol
ute
num
ber
(cel
ls/u
L)
0
50
100
150
200
250
HD Ad HD Ped
8
Fig. S6. In vitro IFN- γ production of sorted T cell subsets from HDs and G T patients.
(A) Graph showing the percentage of IFNγ+ cells in sorted TN, TSCM, TCM and TEM from
HD (n=3), HSC-GT (n=2) and PBL-GT+ERT (n=3) individuals after 6 hours of stimulation with
PMA/Ionomycin. (B) Dot plot displaying the percentage of IFNγ+ cells in sorted T-cell
subpopulations upon PMA/Ionomycin stimulation in HD and GT treated patients. (Statistical
analysis: Mann-Whitney test; *P<0.05; **P<0.01 ***P<0.001. TN vs TSCM Pvalue= 0.0013;
TN vs TCM Pvalue= 0.0007; TN vs TEM Pvalue= 0.0007; TSCM vs TEM Pvalue= 0.0047;
TCM vs TEM Pvalue= 0.0207.)
HD HSC-GT PBL-GT0
2
4
6
20
40
60
80
100
HD Ad HSC-GT PBL-GT+ERT
% o
f IFNγ+
cel
ls
NAIVE TSCM CM EM
0.1
1
10
100
TN TSCM TCM TEM
% o
f IF
Nγ+
cel
ls
(Log
sca
le)
TCM
TEM
CM
TSCM
TN
A B
Fig. S6
** ***
***
** *
9
Fig. S7. In vitro analyses of sorted T N and TSCM from HD.
(A) Graph showing relative fold increase of sorted TSCM and TN T-cell subsets in vitro
calculated on absolute number of plated cells at time 0. The cells were cultured as described
in 3A (US, unstimulated cells). (B) Relative frequencies of TSCM in CD8+ cells from originally
sorted TSCM and TN after 11 and 18 days of culture as described for 3A. (Statistical analysis:
Mann-Whitney test; *P<0.05; TN vs TSCM at 11 days Pvalue= 0.0317)
0
2
4
6
8
10
Fig. S7
0 96h 7 days 11 days 14 days
TSCM US + IL2
TSCM PHA/IL2 + IL2
TN US + IL2
TN PHA/IL2 + IL2
Fol
d in
crea
se
0
25
50
75
100
*
TN TSCM TN TSCM
11days 18days
Originally sorted populations
A
B
% o
f TS
CM
on
CD
8+ c
ells
10
Fig. S8. Phenotypical and functional characterizati on of in vitro generated T SCM.
(A) Experimental scheme for functional and phenotypical characterization of in vitro generated
TSCM. T cell subtypes were sorted (1st sorting) from 3 HD PBMC, stimulated for 6 hours with
PMA/Ionomycin and analyzed for the IFNγ production. Sorted TN were also stimulated 96
hours with PHA+IL-2 and cultured in presence of IL-2 for additional 7 days as described in
Fig. 3A. At 96h and 11 days of culture, cells were analyzed for the expression of CD69 and
HLA-DR activation markers. At the end of culture (11 days), T cell subsets generated from
11
originally purified TN cells were sorted (2nd sorting) and were both stimulated with
PMA/Ionomycin for IFNγ production analysis and cultured for 6 days with OKT3+IL-2 to
evaluate their ability of proliferate in absence of co-stimulatory signals.
(B) Plots on the left in the upper part of the panel show the sorting strategy for T cell subsets
isolation from PBMC (1st sorting). Plot in the lower part displays the newly generated Tscm
from originally purified CD95- TN cells after 11 days of culture as described by the panel A
(2nd sorting). The histograms show the IFNγ expression upon PMA/Ionomycin stimulation of
sorted T cell subsets from freshly isolated PBMC and from 11 days cultured TN cells. The
graph on the right summarizes the data of IFNγ positive cells from 3 healthy donors.
(B) Histograms showing the dilutions of Cell TraceTM-Violet proliferation marker upon 6 days
OKT3+IL-2 stimulation in in vitro generated TSCM (red), TCM (light blue) and TEM (dark
blue), in comparison with the unstimulated control (grey).
(C) Overlaid histograms show the expression of CD69 (first row) and HLA-DR (second row)
activation markers in T-cell subsets from HD (first column) and sorted CD95- naïve T cells
after 96h (second column) and at the end of culture (third column).
12
Fig. S9. Vector marking of sorted T cell subsets fr om PBL-GT+ERT patients.
Graphs showing the percentage of transduction estimated by qPCR of sorted TN, TSCM,
TCM, TEM from PBL-GT+ERT Pt1 (A) and Pt2 (B). Vector-marked TN cells were
undetectable.
TN TSCM TCM TEM TN TSCM TCM TEM
% o
f tra
nsdu
ctio
n
% o
f tra
nsdu
ctio
n
PBL-GT+ERT Pt1 PBL-GT+ERT Pt2
Fig. S9
A B
0.1
1
10
100
0.1
1
10
100
13
Fig. S10. Sorting scheme and IS retrieval of sorted T cell subpopulations from GT-
treated patients.
Representative FACS plots of T-cell subpopulations showing (A) sorting strategy and (B)
post-sorting analysis. (C) Spreadex gel runs of LAM-PCR amplicons generated
(representative results with AciI restriction enzyme) from PBL-GT+ERT (PBL-GT) and HSC-
GT samples (IC= internal vector control fragment).
Fig. S10
Sorting scheme : PBL-GT+ERT Sorting scheme : HSC-GT
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CD
62L
CD45RA
SS
C-A
CD95
TCM
TEM
TSCM
CD
62L
CD45RA
SS
C-A
CD95
TN
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<Pacific Blue-A>
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0.9480.0143
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<PE-A>
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C-A
4.74
95.3
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<A
PC
-A>
96 0.148
2.24e-33.83
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<Pacific Blue-A>
0
103
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105
<A
PC
-A>
7.74 7.81e-3
0.050892.2
TN TSCM TCM TEM
CD
62L
CD45RA CD95 CD45RA CD95 CD45RA CD45RA
CD
62L
CD
62L
CD
62L
TS
CM
TC
M
TE
M
TS
CM
TC
M
TE
M
TS
CM
TC
M
TE
M
Pt1 Pt2 Pt3
Ladd
er
400bp 300bp
200bp
100bp
50bp
IC
TS
CM
TC
M
TE
M
Pt3
TN
TS
CM
TC
M
TE
M
Pt5
TN
TS
CM
TC
M
TE
M
Pt6
TN
TS
CM
TC
M
TE
M
Pt14
TN
TS
CM
TC
M
TE
M
Pt15
TN
IC
Ladd
er
400bp 300bp
200bp
100bp
50bp
PBL-GT+ERT HSC-GT Pre-infusion
A
B
C
14
Fig. S11. IS analyses of T cell subpopulations from PBL-GT+ERT patients.
Analyses of insertion sites sharing, clonal contribution within each subtype and on total
analyzed cells performed on individual PBL-GT patients. The graphical representation is the
same used in Fig. 5A-D
Pt1
Pt3
Pt2
Pt4
19
181 33
17
99 37
223
246 277
3
11 18
0.02.55.07.5
10.012.515.017.520.0
2550
75
100
% o
f an
alyz
ed T
cel
ls
MIR4785EPHA6
0.02.55.07.5
10.012.515.017.520.0
25
50
75
100
% o
f an
alyz
ed T
cel
ls
U2AF2
0.02.55.07.5
10.012.515.017.520.0
2550
75
100
% o
f an
alyz
ed T
cel
ls
0.02.55.07.5
10.012.515.017.520.0
25
50
75
100
% o
f an
alyz
ed T
cel
ls NFKBIA
RBM26
NAIVE TSCM CM EM ECTN
Fig. S11
A B C TN TSCM TCM TEM
TSCM TCM TEM
TSCM TCM TEM
TSCM TCM TEM
TSCM TCM TEM
15
Fig. S12. IS analyses of T cell subpopulations from HSC-GT patients.
Analyses of insertion sites sharing, clonal contribution within each subtype and on total
analyzed cells performed on individual HSC-GT patients. The graphical representation is the
same used in Fig. 5A-D
0.02.55.07.5
10.012.515.017.520.0
25
50
75
100
% o
f an
alyz
ed T
cel
ls
NBR1
TRY6TRY6TRY6
EN
0.02.55.07.5
10.012.515.017.520.0
255075
100
% o
f an
alyz
ed T
cel
ls
ZDHHC8P1RPS6KA3
ARHGAP25
CACNG2
0.02.55.07.5
10.012.515.017.520.0
25
50
75
100
% o
f an
alyz
ed T
cel
ls
CCR3NDUFV2
0.02.55.07.5
10.012.515.017.520.0
25
50
75
100
% o
f an
alyz
ed T
cel
ls
FGD4
FGD4
FGD4
FGD4
Pt3
Pt6
Pt5
Pt14
Pt15
98 134
205 126
129 71
164 150
55 41
25 30
21 15
12 18
48 42
88 70 0.02.55.07.5
10.012.515.017.520.0
25
5075
100
% o
f an
alyz
ed T
cel
ls
C14orf166B
DUSP10
NAIVE TSCM CM EM Fig. S12 A B C
TSCM TCM TEM TN
TSCM TCM TEM TN
TSCM TCM TEM TN
TSCM TCM TEM TN
TSCM TCM TEM TN
TSCM TCM TEM TN
16
Fig. S13. Overlaps among single closest genes to in sertion sites collected in vivo from
PBL-GT+ERT and HSC-GT patients.
Word clouds besides each circle show the intensity of IS clustering for each gene locus
hosting an IS inside or in its proximity (the bigger the gene name the higher the number of IS
PBL-GT+ERT
HSC-GT
557
726
82
SSBP3 RGS3 UBXN11 FGD4
PKN2 RUNX3 DNMT3A TMTC4
DUSP10 RCSD1 LOC84931 NFKBIA
IL2RA DDX18 ARHGAP15 SNX29
ADRBK1 FHIT RYBP LYRM1
BCL9L FKBP5 CPEB2 LPIN2
SELPLG MAD1L1 KCNIP4 BCL2
FOXN3 C9orf123 MRPL1 PPP5C
EVL PHF2 TRIO CARD8
LINC00221 C10orf54 SV2C PTPRA
SNUPN MICAL2 SSBP2 RNF24
TBCD EFCAB4B CYFIP2 PLCG1
C18orf1 CRTC3 TAGAP PTPN1
ARHGEF18 ENO3 EEPD1 TIAM1
TAF1B KDM6B CNTNAP2 IFNAR1
RUNX1 NTN1 ST3GAL1 CYTH4
DYRK1A MACROD2 SHB FAM9C
PDE4D SFI1 KLF6 LOC286442
TNFAIP3 SH3KBP1 ARID5B FAM133A
ZC3H12D RERE SIPA1
TRY6 VPS13D CCND2
Fig. S13
17
in its locus). The name of the 82 genes found hit in T-cell subtypes of both groups of patients
are reported at the intersection between the circles.
18
Fig. S14. Top 10 enriched (binomial ranking) biolog ical processes and MSig pathways
associated to hit genes in PBL-GT+ERT (PBL-GT) and HSC-GT.
Color intensity on heatmaps (from white to red or darkblue) represents the relative enrichment
for each functional category as 1/Binomial-PValue(35).
19
Fig. S15. ISs detected over time in T central and T effector memory cells. Integrations
detected in TCM and TEM subpopulations in all PBL-GT individuals on two independent
timepoints (Time 1 and Time2) after last infusion. Graphical representation is the same as the
one shown in Fig. 6B.
95 14 164
Time 1 Time 2
58 15 81
Time 1 Time 2
CM insertions overtime
EM insertions overtime
Fig. S15
20
Table S1 . Characteristics of patients included in this study.
Abbreviations: HSC-GT, hematopoietic stem cells gene therapy; PBL-GT, peripheral blood
lymphocytes gene therapy; BMT, bone marrow transplant; ERT, enzyme replacement
therapy; FU, follow up.
Table S1
PBL-GT+ERT Sex Age at onset (mo) ERT treatment Age at first
infusion (yr)
Years of FU at analysis (after last infusion)
% of VP CD4+ cells
% of VP CD8+ cells
Pt1 F 12 PEG-ADA 3,5 11 7,8 0,6
Pt2 M 24 PEG-ADA 2 11 10,2 1,8
Pt3 F 2PEG-ADA
(discontinued for 6 months)
0,3 12 100 100
Pt4 M 5 PEG-ADA 0,3 11 20,4 14,7
HSC-GT Sex Age at onset (mo)
previous treatment (lenght)
Age at GT (yr)
Years of FU at analysis
% of VP CD4+ cells
% of VP CD8+ cells
Pt3 M 2 Haplo-BMT 1 10 59,2 58,3
Pt5 F 2 PEG-ADA (1.25Y)
1,6 9 86,8 79,4
Pt6 M 1 PEG-ADA (5.3Y) 5,6 8 100 81,9
Pt9 M 5 PEG-ADA (0.8Y) 1,4 6 100 100
Pt10 F 3 Haplo-BMT PEG-ADA (1Y)
1,8 4 100 100
Pt11 M 4 PEG-ADA (0.7Y) 1,6 5 99,4 93,8
Pt13 M 1 PEG-ADA (2mo) 0,5 5 86,2 97,9
Pt14 M 2 PEG-ADA (6Y) 6,2 4 100 100
Pt15 F 14 PEG-ADA (1Y) 2,4 5 72 87,2
Pt18 M 1 PEG-ADA 2 2 100 5,6
BMT Sex Diagnosis Age at BMT (mo)
years of FU at analysis
chimerism in T cells
chimerism in Myeloid
cellsconditioning
Pt1 M SCID (γ-chain) 7 10y 100% donor 100% host No
Pt2 M SCID (JAK-3) 13 8y 100% donor >95% donor Busulfan-Cyclophosphamide
Pt3 FAgammaglobulinemia
and B, NK, DC e monocytes deficit
97 3y 100% donor >95% donorThiotepa,Treosulfan,
Fludarabine
Pt4 F SCID (JAK-3) 7 9y >95% donor >85% host No
Pt5 M CGD 49 7y 100% donor >95% donor Busulfan-Cyclophosphamide
Pt6 F Omenn Syndrome 7 2y >95% donor >95% donorThiotepa,Treosulfan,
Fludarabine
Pt7 M ADA-SCID 1 7y 100% donor 100% host No
Pt8 M ADA-SCID 5 6y >95% donor 100% host No
Pt9 M ADA-SCID 4 5y 100% donor >90% donor Busulfan, Fludarabine
Pt10 F ADA-SCID 60 10y 85% donor 10% donor No
21
Table S2. Percentages of T cell subpopulations on C D8+ cells in BMT-, HSC-GT– and
PBL-GT+ERT–treated patients.
CD62L+ CD45RA+ TCM TEM TEMRA
BMT Pt1 6,7 23,5 65,0 4,9
BMT Pt2 76,1 9,1 10,5 4,2
BMT Pt3 54,6 9,5 31,4 4,3
BMT Pt4 89,0 1,7 2,7 6,2
BMT Pt5 65,1 13,1 15,2 4,3
BMT Pt6 81,5 5,7 5,6 6,1
BMT Pt7 50,6 15,3 18,7 15,4
BMT Pt8 14,2 26,8 48,9 10,2
BMT Pt9 63,6 5,8 16,2 13,6
BMT Pt10 33,5 11,9 38,2 13,6
HSC-GT Pt3 28,2 22,0 34,8 12,4
HSC-GT Pt5 15,6 13,9 45,8 23,0
HSC-GT Pt6 8,4 26,5 54,6 7,6
HSC-GT Pt9 45,2 6,5 21,7 26,6
HSC-GT Pt10 44,2 9,8 11,4 34,6
HSC-GT Pt11 12,3 0,1 1,0 85,5
HSC-GT Pt13 28,3 8,3 25,3 35,7
HSC-GT Pt14 40,9 9,9 20,6 25,9
HSC-GT Pt15 27,9 9,1 39,4 22,2
HSC-GT Pt18 45,7 7,4 15,9 30,4
PBL-GT+ERT Pt1 40,5 12,1 16,9 27,9
PBL-GT+ERT Pt2 5,2 2,2 79,1 11,2
PBL-GT+ERT Pt3 9,3 73,8 14,9 1,6
PBL-GT+ERT Pt4 11,7 11,8 41,5 32,5
% on CD8+ cells
Table S2
22
Table S3. Frequencies of T SCM on CD8 + CD62L+CD45RA+ cells in BMT, HSC-GT, and
PBL-GT individuals.
% of TSCM on CD62L+CD45RA+ cells
BMT Pt1 65,0
BMT Pt2 17,1
BMT Pt3 16,9
BMT Pt4 12,0
BMT Pt5 16,4
BMT Pt6 6,7
BMT Pt7 47,8
BMT Pt8 66,1
BMT Pt9 7,0
BMT Pt10 16,9
HSC-GT Pt3 31,3
HSC-GT Pt5 58,2
HSC-GT Pt6 73,0
HSC-GT Pt9 42,4
HSC-GT Pt10 57,6
HSC-GT Pt11 58,6
HSC-GT Pt13 72,1
HSC-GT Pt14 44,4
HSC-GT Pt15 55,9
HSC-GT Pt18 53,9
PBL-GT+ERT Pt1 64,8
PBL-GT+ERT Pt2 66,9
PBL-GT+ERT Pt3 98,5
PBL-GT+ERT Pt4 95,0
Table S3
23
Table S4 TREC content in BMT, HSC-GT, and PBL-GT p atients.
The amount of TREC was estimated per 100ng of DNA from purified CD3+CD45RA+ cells
24
Table S5. CD8+ cell composition after 6 days CD3/CD28 + rhIL2 stimulation of sorted T
cell subsets.
The first column of the table shows the sorted subpopulations. Each row displays the
percentage of the resulted T cell subsets after 6 days of CD3/CD28+rhIL2 stimulation.
HD Ad CD62L+CD45RA+ TCM TEM TEMRATN 9,8 45,6 35,0 9,6
TSCM 21,9 32,9 29,8 15,4TCM 0,2 25,2 72,7 2,0TEM 0,5 17,3 80,6 1,6
%"on"CD8+"cells
HSC-GT Pt15 CD62L+CD45RA+ TCM TEM TEMRATN 8,0 26,4 58,3 7,3
TSCM 4,9 54,8 35,9 4,4TCM 1,4 15,3 80,6 4,2TEM 0,0 0,0 96,8 3,2
%"on"CD8+"cells
PBL-GT Pt1 CD62L+CD45RA+ TCM TEM TEMRATN 9,6 56,1 28,3 5,8
TSCM 22,6 25,4 21,3 30,7TCM 0,2 50,1 49,6 0,2TEM 0,3 22,3 76,9 0,6
%"on"CD8+"cells
Table S5
25
Table S6. Number of unique integrations retrieved for each T cell subset per patient.
Table S6
PBL-GT
Pt2 pre-infusion
TSCM CM EM 6 18 26
Pt4 pre-infusion
TSCM CM EM 14 27 7
Pt1 6-8 years after last infusion
TSCM CM EM 19 181 33
Pt2 7-12 years after last infusion
TSCM CM EM 17 99 37
Pt3 7-12 years after last infusion
TSCM CM EM 223 246 277
Pt4 9 years after last infusion
TSCM CM EM 3 11 18
HSC-GT
Pt3 10 years after GT
NAIVE TSCM CM EM 98 134 205 126
Pt5 6 years after GT
NAIVE TSCM CM EM 129 71 164 150
Pt6 8 years after GT
NAIVE TSCM CM EM 55 41 25 30
Pt14 5 years after GT
NAIVE TSCM CM EM 21 15 12 18
Pt15 4 Years after GT
NAIVE TSCM CM EM 48 42 88 70