www.sciencetranslationalmedicine.org/cgi/content/full/3/111/111ra120/DC1

Supplementary Materials for

The PDL1-PD1 Axis Converts Human TH1 Cells into Regulatory T Cells

Shoba Amarnath,* Courtney W. Mangus, James C. M. Wang, Fang Wei, Alice He, Veena Kapoor, Jason E. Foley, Paul R. Massey, Tania C. Felizardo, James L. Riley,

Bruce L. Levine, Carl H. June, Jeffrey A. Medin, Daniel H. Fowler

*To whom correspondence should be addressed. E-mail: samarnath@mail.nih.gov

Published 30 November 2011, Sci. Transl. Med. 3, 111ra120 (2011) DOI: 10.1126/scitranslmed.3003130

The PDF file includes:

Fig. S1. Primary human T cells engraft after transduction with lentiviral vector expressing PDL1 and TMPK/CD19 fusion protein. Fig. S2. TMPK cell fate safety switch is functional in vitro and in vivo. Fig. S3. PDL1-expressing effector T cells alter the balance of TH1 and Treg cells in vivo. Fig. S4. TH1 cells expanded from naïve human CD4

+ T cells and human pathogenic TH1 cells are responsive to PDL1-mediated conversion. Fig. S5. PDL1-expressing T cells: Evaluation of reverse signaling in vitro and in vivo during exposure to TH1 cells. Fig. S6. TH1 cells on coculture with PDL1, IL-10, TGF-β, or Treg exhibit decreased effector activity independent of PD1 up-regulation or FOXP3 expression. Fig. S7. PD1 siRNA knocks down PD1 expression and reduces SHP1/2 in human TH1 cells.

a

5’ LTR Ψ RRE cPPT EF1-ALPHA CD19 TMPK IRES PDL1op WPRE 3’ LTR

5’ LTR Ψ RRE cPPT EF1-ALPHA CD19 TMPK WPRE 3’ LTR

b

10 1 10 2 10 3 10 4

10 1 10 2

10 3

10 4 0.06%

99%

10 1 10 2 10 3 10 4

10 1

10 2

10 3

10 4 24%

76%

10 1 10 2 10 3 10 4

10 1

10 2

10 3

10 4 24%

76%

CD4

CD19

10 1 10 2 10 3 10 4

10 1

10 2

10 3

10 4 26% 27%

6%

10 1 10 2 10 3 10 4

10 1

10 2

10 3

10 4 1.5% 0.5%

27%

10 1 10 2 10 3 10 4

10 1

10 2

10 3

10 4 0.7% 0%

0.4%

CD19

PDL1

e

d c

0

10

20

30

% C

D19

+PD

L1+

NT TMPK TMPK-PDL1

0

10

20

30

% C

D4+

CD19

+

NT TMPK TMPK-PDL1

10 0 10 1 10 2 10 3 10 4 0

10

20

30 1.1%

10 0 10 1 10 2 10 3 10 4 0

10

20

30 76.7%

PDL1

NT T

NT T 0

10

20 30

40 50

#hCD

19+P

DL1

+(x1

03) *

NT T 0

25

50

75

100

125

#hCD

19+P

DL1

+ (x

104 ) *

NT T NT T 0

10

20

30

#hCD

19+P

DL1

+ (x

103 )

**

NT T0

1

2

3

#hCD

19+P

DL1

+ (x

103 )

*

NT T0

20

40

60

80

#hCD

19+P

DL1

+ (x

103 ) *

#hCD

19+P

DL1

+ (x

103 )

NT T0

510 15 20 25

*

#hCD

19+P

DL1

+ (x

103 )

NT T0

25

50

75

*

Supplemental Figure 1 :Primary human T cells engraft after transduction with lentiviral vector expressing PDL1 and TMPK/CD19 fusion protein . (a) Control-LV construct encodes expression of TMPK.CD19 fusion protein using EF-1a promoter (top panel); therapeutic-LV construct also encodes PDL1 after an IRES sequence (bottom panel). (b) Post-transduction co-expression of PDL1 and fusion protein. Non-transduced cells expressed CD4 in the absence of CD19 and PDL1 (top and bottom left panels). Control-LV transduced cells expressed CD19 (top and bottom middle panels). PDL1-LV transduced cells co-expressed CD19 and PDL1 (top and bottom right panels). Pooled results from n=7 normal donors are shown. (c) Representative flow plots showing in vivo transgene expression. Transduced cells were enriched by flow sorting and adoptively transferred to rag2-/-cγ-/- hosts; (d) absolute number of transgene-expressing cells was quantified at day 5 post-transfer in spleen (left panel) and bone marrow (right panel) (NT, non-transduced; T, therapeutic-LV transduced; pooled results, n=5 per cohort). Harvested T cells were co-stimulated ex vivo and expanded for 5 days to assess transgene expression stability of cells harvested from spleen (bottom panel) and bone marrow (bottom panel). (e) Persistent in vivo transgene expression. Transduced and enriched human T cells were adoptively transferred; transgene-expressing cells were quantified at day 20 post-transfer in spleen (top left panel) and bone marrow (top right panel). Harvested T cells were co-stimulated ex vivo and expanded for 7 days and transgene expression was determined on cells harvested from spleen (bottom left panel) and bone marrow (bottom right panel). For each panel, n=5 per cohort unless otherwise indicated; * indicates p≤0.05.

Supplemental Figure 2 :TMPK cell fate safety switch is functional in vitro and in vivo. Human primary T cells were transduced with PDL1-LV or control eGFP-LV; PDL1-LV cells were then enriched by flow sorting. Transduced T cells were treated ex vivo with AZT (100 µM; three days) and cell death was measured (Annexin V, 7AAD). (a) Representative plots show eGFP expression (top panel), and cell death markers in eGFP-negative (bottom left panel) and eGFP-positive (bottom right panel) cells. (b) Representative plots show CD19 expression (top panel), and cell death markers in CD19-negative (bottom left panel) and CD19-positive (bottom right panel) cells. (c) PDL1-LV-transduced T cells exposed to AZT ex vivo lack engraftment potential. Transduced T cells were exposed to AZT (100 µM; three days); live cells were then sorted and adoptively transferred to NOD/SCID hosts to determine engraftment potential (at day 5 post-transfer). Engraftment of PDL1-LV-transduced T cells not exposed to AZT was compared to engraftment of non-transduced or eGFP-transduced T cells exposed to AZT (left panel); engraftment of PDL1-LV transduced T cells either exposed or not exposed to AZT ex vivo (right panel). (d) T cells transduced with PDL1-LV were adoptively transferred to NOD/SCID hosts that received AZT therapy (top left panel) (50 mg/kg/d; twice per, absolute number of splenic T cells engrafted was determined for recipients (n=5 per cohort) of PDL1.TMPK transduced T cells with or without in vivo AZT treatment (bottom left panel) and PDL1.TMPK -transduced T cells with or without AZT treatment (bottom right panel). For each panel, * indicates p ≤ 0.05.

a

d

b

10 1

10 2

10 3

10 4

83% 12%

10 1 10 2 10 3 10 4

10 1

10 2

10 3

10 4 1.4% 0.6%

7.3%

10 1 10 2 10 3 10 4

10 1

10 2

10 3

10 4 9.5% 5.2%

8.8%

AV

GFP

CD4

7AA

D

10 1

10 2

10 3

10 4

21% 74%

10 1 10 2 10 3 10 4

10 1

10 2

10 3

10 4 1.4% 1.2%

14%

10 1 10 2 10 3 10 4

10 1

10 2

10 3

10 4 0.2% 15.4%

65%

AV

7AA

D

CD19

CD4

c

AZT - + TMPK TMPK

0

10

20 *

#h-C

D45

+ CD

19+

(x10

4 )

NT TMPK GFP 0

10

20

30

AZT + + -

#h-C

D45

+ (x

104 )

TMPK TMPK 0

10 20 30 40 50

AZT - +

#h-C

D45

+hCD

19-

(x10

6 )

10 1 10 2 10 3 10 4

10 1

10 2

10 3

10 4 2.2% 64%

30% 10 1 10 2 10 3 10 4

10 1

10 2

10 3

10 4 0.1% 1.7%

92%

CD4

CD19

TMPK TMPK 0

1

2

AZT - +

#h-C

D45

+ h-

CD19

+ (x

106 )

*

a

b

0

1

2 *

CFSE+ Th1eff

TeffTMPK TeffPDL1

#hCF

SE+I

FNγ (

x103

)

#hCF

SE+I

FNγ-

Foxp

3+ (x

103 )

0

0.5

1.0

1.5 **

CFSE+ Th1eff

TeffTMPK TeffPDL1

0

25

50

75

#h-C

D45

+ CF

SE+

(x10

3 )

CFSE+ Th1eff

TTMPK TPDL1 10 10 10 10

10 2

10 3

10 4

10 5 88.2%

10.5%

hCD45

CFSE

c

Supplemental Figure 3

PDL1-expressing effector T cells alter the balance of TH 1 and T cells in vivo. Human Th1 cells

were CFSE-labeled and adoptively transferred either alone or with control-LV or PDL1-LV

transduced T cells. (a) Th1 cell splenic engraftment at 24 hr post-infusion is shown (left panel,

representative data; right panel, pooled data of n=3 per cohort). In vivo plasticity of Th1 cells was

monitored by assessment of cytokine secretion and FOXP3 expression by i.c. flow cytometry.

Absolute numbers of splenic Th1 cells expressing IFN-g were quantified (b); Th1 cells expressing

FOXP3 in the absence of IFN-g were also enumerated (c). (* indicates p ≤ 0.05).

2 3 4 5

egr

#h-B

7H4

(x10

3 )

0

0.5

1

CD4 CD8 Teff.PDL1

Th1eff

CD4 CD8 Teff.PDL1

Th1eff

0

#h-H

VEM

(x10

3 )

1

2

3

a b

c

#hTN

F-Fo

xp3

+(x1

03)

0

1

2

3

4

5

CD4 CD8 Teff.PDL1

Th1eff

d

e

#hB7

H4(

x103

)

0

0.5

1

K K.PDL1

Th1eff

#hH

VEM

(x10

3 )

0

1

2

3

4

K K.PDL1 Th1eff

f

K K.PDL1 Th1eff

IL2+

Foxp

3-(x

103 )

0

1

2

#hTN

F+Fo

xp3-

(x10

3 )

0

10

20

30

40

K K.PDL1 Th1eff

#hTN

F-Fo

xp3+

(x10

3 )

K K.PDL1 Th1eff

0 12 3 4 5 6 7

#h-B

7H4

(x10

3 )

0

5

10

15

20

25

K K.PDL1 Th1eff

**

0 10 20 30 40 50 60 70

#h-H

VEM

( x10

3 )

K K.PDL1 Th1eff

**

#hIF

Nγ-

Foxp

3+

(x10

3 )

0 1 2 3 4 5 6 7

K K.PDL1 Th1eff

**

#hIL

2+Fo

xp3-

(x

103 )

05

10 15 20

25

K K.PDL1 Th1eff

*

K.PDL1

#hIL

2-Fo

xp3+

(x10

3 )

0 1 2 3 4 5 6 7

K Th1eff

*

#hTN

F+Fo

xp3-

(x10

3 )

0

25

50

75

100

125

K K.PDL1 Th1eff

*

#hTN

F-Fo

xp3+

(x10

3 )

0 1 2 3 4 5 6

K K.PDL1 Th1eff

*

#hIF

Nγ-

Foxp

3+ (x

103 )

0

1

2

3

CD4 CD8 Teff.PDL1

Th1eff

** *

#hIL

2+Fo

xp3-

(x10

3 )

0 0.1

0.2

0.3 0.4

0.5

CD4 CD8 Teff.PDL1

Th1eff

**

#hTN

F+Fo

xp3-

(x10

3 )

0

5

10

CD4 CD8 Teff.PDL1

Th1eff

** **

#hIL

2-Fo

xp3+

(x10

3 )

0

10

20

CD4 CD8 Teff.PDL1

Th1eff

**

K.PDL1 IL

2-Fo

xp3+

(x10

3 )

0

10

20

KTh1eff

*

K.PDL1

#hIF

Nγ-

Foxp

3+ (x

103 )

0

0.5

1

KTh1eff

*

Supplemental Figure 4

TH1 cells expanded from naïve human CD4+ T cells and human pathogenic TH1 cells are responsive to

PDL1-mediated conversion. Polarized Th1 cells were adoptively transferred into NSG mice along with

irradiated K562 cells (K) or K562 cells expressing PDL1 (K.PDL1). (a) At day 5 post transfer,

splenocytes were harvested and Th1 cells were characterized for co-inhibitory molecules, such as

B7H4 (left panel) and HVEM (right panel). (b) splenocytes were stimulated ex vivo with PMA and

ionomycin for 4 hrs and cytokine production was measured using flow cyometry. Absolute numbers of

IFN-γ-FOXP3+, IL2- FOXP3+, TNF-FOXP3+ , IL2+FOXP3- and TNF+FOXP3-are shown. Human

pathogenic Th1 cells were utilized in an additional in vivo experiment along with CD4+ T cells

expressing PDL1 or CD8+ T cells expressing PDL1. (c) At day 5 post transfer, splenocytes were

harvested and absolute numbers of hB7H4 (left panel) and hHVEM (right panel) were quantified. (d)

Absolute numbers of IFN-γ-FOXP3+, IL2+FOXP3-, IL2- FOXP3+ , TNF-FOXP3+ and TNF+FOXP3- is

also shown. (e) Using the same pathogenic Th1 cells another transplant was performed where

irradiated K562 cells (K) or K562 cells expressing PDL1 (K.PDL1) were adoptively transferred. At day

5 after transfer, splenocytes were harvested and absolute numbers of hB7H4 (left panel) and hHVEM

(right panel) is shown. (f) Absolute numbers of IFN-γ-FOXP3+, IL2+FOXP3-, IL2-Foxp3+, TNF+FOXP3- and TNF-FOXP3+ were quantified. * indicates p ≤ 0.05, ** indicates p ≤ 0.005.

a

b

%Fo

xp3+

0 5

10 15 20 25

GFP PDL1 GFP PDL1 CD4 CD8

c

d

#hH

VEM

(x10

2 )

0

10

20

GFP CD8

PDL1 CD4

#hIF

Nγ(

x102

)

0

1

2

3

GFP CD8

PDL1 CD4

e

f

%H

VEM

+

0

10

20

30

40

GFP CD8

PDL1 CD4

0

0.1

0.2

0.3

GFP CD8

PDL1 CD4

%B7

H4+

%G

alec

tin-9

+

0 10 20 30 40 50

GFP CD8

PDL1 CD4

%CD

80+

0

5

10

GFP CD8

PDL1 CD4

%Tb

et+

0 1 2 3 4 5 6 7

GFP CD8

PDL1 CD4

%Fo

xp3

0

10

20

30

GFP CD8

PDL1 CD4

%IF

Nγ

0

10

20

GFP CD8

PDL1 CD4

%hv

em+

0

5

10

GFP PDL1 GFP PDL1 CD8 CD4

** **

%B7

H4+

GFP PDL1 GFP PDL1 CD8 CD4

0

5

10 ** **

GFP PDL1 GFP PDL1 CD4 CD8

%G

alec

tin-9

+

0

20

40

60

** **

%CD

80+

0

10

20

GFP PDL1 GFP PDL1 CD4 CD8

** %

Tbet

+

0 5

10 15 20 25

GFP PDL1 GFP PDL1 CD4 CD8

**

**

%IF

Nγ+

0

20 40

60 80

GFP PDL1 GFP PDL1 CD4 CD8

** **

GFP CD8

PDL1 CD4

0 #hB

7H4(

x102

)

5

10

**

#hG

alec

tin-9

(x10

2 )

GFP CD8

PDL1 CD4

0 1 2 3 4 5 6 7 **

GFP CD8

PDL1 CD4

#hCD

80(x

102 )

0

1

2

**

GFP CD8

PDL1 CD4

#hTb

et(x

102 )

0

10

20

30 **

#hFo

xp3(

x102

)

0

5

10

GFP CD8

PDL1 CD4

**

Supplemental Figure 5 PDL1 expressing T cells: valuation of reverse signaling in vitro and in vivo during exposure to T 1 cells. Human CD4+ and CD8+ T cells were transduced with either GFP LV or PDL1 LV. (a) Stably transduced T cells were then characterized for the expression of coinhibitory molecules such as HVEM, B7H4, galectin-9 and CD80 . (b) Transfected T cells were then stimulated with PMA and ionomycin for 4hrs following which they were characterized for transcription factors TBET, FOXP3 and IFN-γ expression. (c) Human transduced T cells were co-injected along with Th1 cells into immuno-compromised murine hosts. Splenocytes were harvested at day 5 and LV transduced cells were characterized by gating on CD45+GFP+ or CD45+CD8+PDL1+ or CD45+CD4+PDL1+ populations. Absolute numbers of HVEM, B7H4, galectin 9 and CD80 are shown. (d) Splenocytes were stimulated ex vivo and absolute numbers transduced human T cells expressing TBET, FOXP3 and IFN- γ were quantified. (e) In vitro experiments were set up where transduced CD8+ T cells and CD4+ T cells were co-cultured with CFSE+ Th1 cells for 3 days. On day 3 post transfer, transduced cells were gated as the CFSE- population and characterized for the expression of HVEM, B7H4, galectin 9 and CD80. (f) Intracellular flow cytometry was performed to further characterize TBET, FOXP3 and IFN- γ expression of the transduced cells post co-culture (* indicates p ≤ 0.05, ** indicates p ≤ 0.005).

- E H

%CD

80

0

12 3 4 5 6

PDL1 IL10 TGF- β TREG Th1+3/28

Smad2

P-Smad2

Donor#1 Donor#2

3/ 2 8 + + + + + + PDL1 + + + + TGFβ + +

GFP PDL1 GFP PDL1 CD8 CD4

%p-

STAT

1

0 10 20 30 40 50 60

GFP CD8

PDL1 CD4

%p-

STAT

1

0

5

10

GFP CD8

PDL1 CD4

%p-

STAT

4

0

5

10

a

b

c

d

e

%p-

STAT

1

0

5 10

15 20

25

PDL1 IL10 TGF-β TREG Th1+3/28

** **

PDL1 IL10 TGF- β TREG Th1+3/28

%p-

STAT

4

0 5

10 15 20 25 30 35

**

%PD

1

0

50

100

PDL1 IL10 TGF- β TREG Th1+3/28

**

%Tb

et

0

10

20

30

PDL1 IL10 TGF- β TREG

Th1+3/28

* **

**

0

**

%IF

Nγ

50

100

PDL1 IL10 TGF- β TREG Th1+3/28

**

%p-

STAT

4 0

25

50

75

100

GFP PDL1 GFP PDL1 CD8 CD4

*

*

Supplementary Figure 6 TH1 cells on coculture with PDL1, IL-10, TGF- or Tre g exhibit decreased effector activity independent of PD1 up-regulation or FOXP3 expression. Human Th1 cells were labeled with CFSE and then co-cultured with either PDL1 beads, rhIL10 (100ng/ml), rhTGF- b (2ng/ml) or TREG cells (1:1; TREG:Th1 ratio) for 3 days. In some cultures, transduced CD4 or CD8 T cells expressing PDL1 was added to the co-culture. (a) STAT1 signaling of the Th1 cells after co-culture was measured (left panel). STAT1 signaling on LV transduced T cells was also measured before co-culture (middle panel) and after co-culture (right panel). (b) Similarly STAT4 signaling was also measured in Th1 cells following co-culture (left panel) and on LV transduced T cells were also measured before co-culture (middle panel) and after co-culture (right panel). (c) Th1 cells were also characterized for PDL1 binding ligands PD1 (left panel) and CD80 (right panel) following co-culture. (d) At day 3, after co-culture Th1 cells were restimulated and flow cytometry was performed to measure expression of TBET (left panel) and IFN-g (right panel). (e) In order to detect TGF-b signaling, Th1 cells were either stimulated with beads coated with antibodies to CD3 and CD28 alone or in the presence of PDL1 coated beads or with rhTGF-b for 6 hrs. Protein lysates were prepared after 6 hrs and western blotting analysis was performed to detect p-smad2 signaling. Representative blots from 2 donors are shown along with total Smad2 protein expression. * indicates p ≤ 0.05, ** indicates p ≤ 0.005.

b

PD1

No SiRNA

10 0 10 1 10 2 10 3 10 40

10

20

30

40

# Ce

lls

29%

PD1 SiRNA

10 0 10 1 10 2 10 3 10 40

10

20

30

40

# Ce

lls

99%

Flow sorted PD1kd Th1 effectors

PD1

a b

10 0 10 1 10 2 10 3 10 4 0

10

20

30

# Ce

lls

92%

SHP-1

SHP-2

c

B-actin

SHP2

%p-

STAT

1

0

10

20

30

40

50

60

Th1eff+rhIFN+SiRNA PDL1Fc beads

PD1 SHP1

*** **

**

Neg. Neg.

d

Supplementary Figure 7 PD1 siRNA knocks down PD1 expression and reduces SHP1/2 in human TH1 cells. Human Th1 cells were expanded ex vivo and then were treated with siRNA for PD1. (a) Representative flow cytometry data for control Th1 cells (left panel) and Th1 cells treated with siRNA for PD1 (right panel). (b) Representative data of PD1 expression of Th1 cells after flow sorting prior to adoptive transfer. (c) Th1 cell SHP1/2 protein expression by western blot analysis with SHP1 or SHP2 siRNA treatment. (d) Untreated Th1 cells, Th1 cells with PD1 knockdown, and Th1 cells treated with SHP1/2 SiRNA (n=5 donors) were evaluated for STAT1 activation. For each panel: * indicates p ≤ 0.05, ** indicates p ≤ 0.005, *** indicates p ≤ 0.0005).