Supplementary Materials for · 2015. 2. 2. · Table S1. Characteristics of patients included in this study. Table S2. Percentages of T cell subpopulations on CD8 + cells in BMT-,

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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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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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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Pre-culture 96h 11d 18d 72h

% o

f CD

4+ c

ells

pre culture 96h 7d 15d

0

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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

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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

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CD45RA CD95 CD45RA CD95 CD45RA CD45RA

CD

62L

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62L

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62L

TS

CM

TC

M

TE

M

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M

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M

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CM

TC

M

TE

M

Pt1 Pt2 Pt3

Ladd

er

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IC

TS

CM

TC

M

TE

M

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TN

TS

CM

TC

M

TE

M

Pt5

TN

TS

CM

TC

M

TE

M

Pt6

TN

TS

CM

TC

M

TE

M

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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

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cel

ls

MIR4785EPHA6

0.02.55.07.5

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cel

ls

U2AF2

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alyz

ed T

cel

ls

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25

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% 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

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ls

NBR1

TRY6TRY6TRY6

EN

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ls

ZDHHC8P1RPS6KA3

ARHGAP25

CACNG2

0.02.55.07.5

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ls

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0.02.55.07.5

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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

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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


TSCM CM EM 17 99 37


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







Pt15 4 Years after GT


Supplementary Materials for · 2015. 2. 2. · Table S1. Characteristics of patients included in this study. Table S2. Percentages of T cell subpopulations on CD8 + cells in BMT-,

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