www.sciencemag.org/cgi/content/full/science.aaf0683/DC1

Supplementary Material for

Rescue of exhausted CD8 T cells by PD-1–targeted therapies is CD28-dependent

Alice O. Kamphorst, Andreas Wieland, Tahseen Nasti, Shu Yang, Ruan Zhang, Daniel L. Barber, Bogumila T. Konieczny, Candace Z. Daugherty, Lydia Koenig, Ke Yu, Gabriel

L. Sica, Arlene H. Sharpe, Gordon J. Freeman, Bruce R. Blazar, Laurence A. Turka, Taofeek K. Owonikoko, Rathi Pillai, Suresh S. Ramalingam, Koichi Araki, Rafi Ahmed*

*Corresponding author. Email: rahmed@emory.edu

Published 9 March 2017 as Science First Release

DOI: 10.1126/science.aaf0683

This PDF file includes:

Materials and Methods Supplementary Text Figs. S1 to S9 References

2

Materials and Methods

NSCLC patient samples:

Institutional review board approval was obtained prior to study enrollment and written

informed consent was obtained from all donors. For blood samples collections, we

approached NSCLC patients treated at Winship Cancer Institute who were initiating

therapy with anti-PD-1 or anti-PD-L1 blocking antibodies on clinical trials or as standard

of care therapy to participate in our study. Patients received anti-PD-1 therapy

pembrolizumab/MK-3475 (10 mg/kg) or nivolumab (3mg/kg), or anti-PD-L1 therapy

MPDL3280A (atezolizumab) (1200mg) as intravenous infusion every 2-3 weeks until

disease progression or unacceptable side effect. Peripheral blood samples were collected

before infusion (baseline) and at second treatment into cell preparation tubes with sodium

citrate (BD). Peripheral blood mononuclear cells were isolated according to standard

protocol and immediately stained or frozen for posterior analysis. Lung tumor specimens

were obtained from 17 patients with NSCLC that underwent surgical resection as

standard care. Samples collected were comprised of 4 squamous cell carcinomas (stage

II-III) and 13 adenocarcinomas (stage IA-IV). Tumors were harvested in cold L-15

Leibovitz media (HyClone), minced into small pieces and digested for 1h at 37°C with

shaking with an enzymatic cocktail of Collagenase I (125mg/1L), DNase I (50mg/1L),

Collagenase IV (125mg/1L), Collagenase II (125mg/1L), Elastase (50mg/1L)

(Worthington). Single cell suspensions were obtained, and after RBC ACK lysis, cells

were stained for immediate analysis by flow cytometry.

3

Mice and infections:

C57BL/6, Balb/c, B6 CD45.1, CD28KO and Rosa26Cre-ERT2 mice were purchased from the

Jackson Laboratory. CD28KO were bred to CD45.1 P14 TCR transgenic mice in house.

Rosa26Cre-ERT2 mice were bred to CD28f/f mice (27), and CD45.1 P14 TCR transgenic mice

in house. Mice were infected at 6-8 weeks of age and mixed bone marrow chimera mice

were infected 8-12 weeks after irradiation and reconstitution. For acute LCMV infection

mice were given 2x105 plaque forming units (PFU) LCMV Arm by intraperitoneal route

(i.p.). For chronic LCMV infection mice were given 500 µg of the CD4-depleting

antibody GK1.5 i.p. (BioXcell) 1 day prior to infection and again on the day of infection

with 2x106 PFU LCMV clone 13 by intravenous route (i.v.) (9). Mice were analyzed 45

to 95 days post-infection. Viral load was assayed by plaque assays on Vero E6 cells as

previously described (28). All mice were used in accordance with the Emory University

Institutional Animal Care and Use Committee Guidelines.

Adoptive cell transfers:

For experiments using P14 T cells, 1-2 days before infection, 2-5 × 103 P14 T cells were

transferred i.v. Cell transfer consisted of CD8 T cell-enriched fraction (CD8 T cell

isolation kit, Miltenyi Biotec) or total splenocytes isolated from naïve P14 mice. In co-

transfer experiments of CD28KO and WT P14, 3-4 fold more CD28KO cells were

transferred to achieve a 1:1 KO to WT ratio in chronically infected mice.

For mixed bone marrow chimera experiments, recipient mice (at least 8-week old)

received one dose of 550 rad. Bone marrow cells were isolated from donor mice and

4

prepared into single cell suspensions. 5-10 x106 bone marrow cells depleted of CD4 and

CD8 T cells (Milteny Biotec) were transferred i.v. and mice were rested for 2-3 months

to allow for reconstitution before infection.

In vivo tumor model:

Balb/c mice were inoculated subcutaneously with 0.2 x 106 CT26 colon carcinoma cells

in 25% matrigel and depleted of CD4 T cells by i.p. injection of 250 µg GK1.5 antibody.

(BioXcell). CD4 depletion was maintained for the duration of the experiment by repeated

injections of 250 µg GK1.5 antibody every 7-10 days. CD4 depletion was performed to

restrict our study on CD8 T cells and avoid potential effects on CD4 Treg cells by B7-

blocking antibodies that would interfere with interpretation of the results (29). Mice were

randomized into different treatment groups when CT26 tumor reached 150 mm3

(±70mm3), between 11-14 days after inoculation. Mice whose tumors exceeded

acceptable limits (19 mm3 diameter or ulcerated tumors) were euthanized and removed

from the study. Tumors were measured every 2-4 days using a caliper and tumor growth

was calculated as volume of ellipsoid (length x width x height x 0.52).

In vivo treatments:

For blockade of the PD-1 pathway, 200 µg anti-mouse PD-L1 antibody (10F.9G2,

prepared in house) or 200 µg anti-mouse PD-1 antibody (29F.1A12, prepared in house)

was administered i.p. every 3 days.. Where indicated we also treated mice with 200 µg

anti-mouse-PD-1 clone muDX400 (Merck), administered i.p. every 3-5 days. For

5

blockade of B7 molecules, 500 µg CTLA-4-Ig or 200 µg anti-mouse B7-1 (16-10A1,

BioXcell) and 200 µg anti-mouse B7-2 (GL-1, BioXcell) were administered i.p. a few

hours before the first dose of anti-PD-L1 or anti-PD-1, and then every 3 days. Final

analyses were performed 14 days after treatment initiation, unless indicated otherwise.

In vivo conditional CD28 gene deletion was achieved in Rosa26Cre-ERT2 CD28f/f cells by

tamoxifen administration to LCMV chronically infected mice. Tamoxifen (Sigma-

Aldrich) was dissolved in ethanol, diluted 1:20 in sunflower oil, and sonicated for 5 min

at 37°C. Mice were injected daily with 1 mg tamoxifen i.p. for 5 consecutive days, then

rested for a minimum of 7 days before further treatment.

Antibodies and flow cytometry:

Human samples: Peripheral blood mononuclear cells were stained with anti-CD8 (RPA-

T8), -CD4 (RPA-T4), -CD3 (UCHT1), -CD45RA (2H4LDH11LDB9), -CCR7 (150503),

-PD-1 (EH12.2H7), -CD28 (CD28.2), -HLA-DR (l243), -CD38 (HIT2) and -Ki67 (B56).

After anti-PD-1 treatment, PD-1 expression was detected by combining anti-human IgG4

biotin (HP-6025, Sigma) with anti-PD-1 antibody. Live/Dead cell discrimination was

performed using fixable Yellow Dead Cell Stain Kit (Life Technology). Intracellular

staining for Ki-67 was performed using Foxp3 Fixation Kit (eBioscience). Antibodies

were purchased from BD Bioscience, Biolegend, eBioscience or Beckman Coulter.

Single cell suspensions obtained from lung tumor were stained with anti-CD8 (RPA-T8),

-CD4 (RPA-T4), -CD3 (UCHT1), -PD-1 (EH12.2H7), -CD28 (CD28.2) from BD or

6

Biolegend; anti-TIM-3 (344823) from R&D. Live/Dead cell discrimination was

performed using fixable Yellow Dead Cell Stain Kit (Life Technology).

Murine tissues: Single cell suspensions were obtained from blood, spleen, lung and liver

as previously described (29). Single cell suspensions were stained with anti-CD8α (53-

6.7), -CD4 (RM4-5), -CD45.1 (A20), -CD45.2 (104), -CD44 (IM7), anti-CD28 (E18) and

-PD-1 (RMP1-30 or 29F.1A12) from BD, eBioscience or Biolegend; TIM-3 (215008)

from R&D systems. Dead cells were excluded by gating out cells positive for Live/Dead

fixable dead cell stain (Invitrogen). LCMV MHC class I tetramers were prepared and

used as previously described (30). LCMV-specific responses were assessed by re-

stimulating splenocytes with 0.1 μg/ml of GP33, GP276, NP396, NP118 or NP235

LCMV peptides in the presence of GolgiPlug (brefeldin A) and GolgiStop (monesin) for

5h at 37°C. Intracellular staining for IFN-γ (XMG1.2), TNF-α (MP6-XT22), and

granzyme B (GB12 or GB11) was performed with Cytofix/Cytoperm kit (BD

Biosciences). Intracellular staining for Ki-67 (B56, BD Pharmingen) was performed with

Foxp3/ Transcription Factor Staining Buffer Set (eBioscience) according to

manufacturer’s instructions.

Samples were acquired with a Becton Dickinson FACS Canto or LSRII and analyzed

using FlowJo (Treestar).

Statistical analysis:

All data were analyzed using Prism 6 (GraphPad).

7

CD28CD44

Db G

P27

6

DbGP276 memoryDbGP276 exhausted

acute LCMVchronic LCMV naive CD44lo CD8 T cells

4.16 3.33

NS******

A B

Exhausted Memory Naive0

500

1000

1500

CD

28 M

FI

C

Fig. S1. Exhausted CD8 T cells express similar levels of CD28 than naïve cells, but lower levels

than memory CD8 T cells. C57BL/6 mice were infected with acute LCMV Arm or

chronic LCMV clone13. Splenocytes were analyzed at least 45 days post-infection when

CD8 T cells have differentiated into memory cells after acute infection or exhausted cells

after chronic infection. (A) Gating on LCMV-DbGP276-specific cells after gating on

splenic CD8 T cells. Data are from representative mice from 4 independent experiments

with 3-6 mice per group. (B) CD28 expression on memory (red) or exhausted (blue)

LCMV-DbGP276-specific CD8 T cells. Dashed black line shows CD28 expression on

naïve CD44lo CD8 T cells. Data are from representative mice from 4 independent

experiments with 3-6 mice per group. (C) Mean fluorescence intensity as in B.

Representative data from one experiment (N=3) out of 4 independent experiments. Error

bars indicate SEM. ANOVA with Sidak’s correction for multiple comparisons.

***P<0.001.. NS = not significant.

8

A

_PD-L1 analysisafter 14d

ChronicLCMV

UNT _PD-L10

1000

2000

3000

CD

28 M

FI

BP = 0.82

DbGP276

Fig. S2 Rescue by PD-1 therapy does not increase CD28 expression on LCMV-specific CD8 T

cells. (A) Experimental layout. (B) CD28 mean fluorescence intensity (MFI) on LCMV-

DbGP276-specific CD8 T cells in the spleen of chronically infected mice. Data show

compiled data from two experiments and is representative from 4 independent

experiments with 3-5 mice per group. Error bars indicate SEM. Unpaired t-test.

9

BA

103

104

105

106

UNT _PD-L1 _PD-L1 +CTLA-4-Ig

Db G

P27

6/lu

ngNS

**

104

105

106

IFN

-a+ c

ells

/spl

een

UNT _PD-L1 _PD-L1 +CTLA-4-Ig

NS******

0.97

UNT _PD-L1

IFN

-a

CD44

_PD-L1+CTLA-4-Ig

8.46 1.09

C

S3

Fig. S3 CTLA-4-Ig abrogates anti-PD-L1-mediated rescue of exhausted CD8 T cells.

At least 45 days after establishment of chronic infection with LCMV clone 13, indicated

groups of mice were treated with anti-PD-L1 as a single agent or in combination with

CTLA-4-Ig. Analysis was performed after 14 days of treatment. (A) Number of LCMV-

DbGP276-specific CD8 T cells in the lung. Data show one representative of 2

independent experiments, with at least 4 mice per group. Error bars indicate SEM. (B)

Number of IFN-γ–producing CD8 T cells in the spleen after ex vivo restimulation with

LCMV GP276 peptide. Data show one representative of 2 independent experiments, with

at least 4 mice per group. Error bars indicate SEM. (C) Frequencies of CD8 T cells

producing IFN-γ in the spleen after ex vivo restimulation with a pool of LCMV peptides.

Data show representative mice of 2 independent experiments, with at least 4 mice per

group. ANOVA with Sidak’s correction for multiple comparisons. *P<0.05, ***P<0.001.

NS = not significant.

10

UNT _PD-L1 _PD-L1+_B7

_B7102

103

104

105

P14

/ 106 l

ung

cells

S4

******** ****

NS

Fig. S4 Transient B7 blockade prevents rescue of LCMV-specific exhausted CD8 T cells by PD-

1 therapy, but has no major impact on the maintenance of LCMV-specific exhausted CD8

T cells during chronic infection. Experiment layout as in Fig. 1K. Graph shows frequency

of P14 cells in lung. Data show compilation of 2 independent experiments, with 3- 4 mice

per group. ANOVA with Sidak’s correction for multiple comparisons. Error bars indicate

SEM. ****P<0.0001. NS = not significant.

11

UNT _B7104

105

106

107

108

PFU

/g s

plee

n

104

105

106

107

108

PFU

/g li

ver

104

105

106

107

108

PFU

/g lu

ng

UNT _B7 UNT _B7

NS(P=0.7224)

NS(P=0.1026)

NS(P=0.7587)

S5

Fig. S5

Transient blockade of the B7-pathway in mice with established LCMV chronic infection

does not affect viral load. Graphs show viral load in different organs after a two-week B7

blockade regimen in mice with established LCMV chronic infection (as in Fig. 1A). Data

show compiled data from two independent experiments with 4-5 mice per group. Error

bars indicate SEM. Unpaired t-test.

12

spl

een

DbGP33 DbGP276 D

lung

C

UNT _PD-1 _PD-1 +_B7

UNT _PD-1 _PD-1 + _B7

AUNT

Db G

P27

6

CD44

B

UNT

_PD-1

_PD-1+ _B7

IFN-a + cells/spleen

S6

5.1 27.2 4.2

104

105

106

107

102

103

104

105

0

5

10

15

20

25

Ki-6

7+ (%

Tet

ram

er+)

DbGP33 DbGP276

UNT _PD-1 _PD-1 + _B7

UNT _PD-1 _PD-1 + _B7

104 105 106

GP33GP276

*******

IFN

-a

CD44

UNT _PD-1 _PD-1 + _B70.914.01.3

E

NS******* NS

*******

NS******

NS******

NS* *

NS* *

NSNS

_PD-1 _PD-1 + _B7

Fig. S6

CD8 T cell rescue by PD-1 therapy also relies on B7 costimulation. At least 45 days after

establishment of LCMV clone 13 chronic infection, indicated groups of mice were

treated for 2-weeks with anti-PD-1 (clone 29F.1A12, rat IgG2a) as a single agent or in

combination with anti-B7-1 and anti-B7-2. (A) Frequencies of LCMV-DbGP276-specific

CD8 T cells in the spleen. Data show representative mice from 3 independent

experiments with 4-5 mice per group. (B) Numbers of LCMV-DbGP33 and LCMV-

DbGP276-specific CD8 T cells in different organs. Data show one representative

experiment of 3 independent experiments with 4-5 mice per group. Error bars indicate

SEM. (C) Ki-67 expression on LCMV-DbGP33 and LCMV-DbGP276-specific CD8 T

13

cells in the spleen. Data show one representative experiment of 2 independent

experiments with 4-5 mice per group. Error bars indicate SEM. (D) Numbers of IFN-γ–

producing CD8 T cells in the spleen after ex vivo restimulation with the indicated LCMV

peptides. . Data show one representative experiment of 3 independent experiments with

4-5 mice per group. Comparisons are between treated groups and untreated mice. Error

bars indicate SEM. (E) Frequencies of CD8 T cells producing IFN-γ in the spleen after ex

vivo restimulation with a pool of LCMV peptides. Data show representative mice from 3

independent experiments with 4-5 mice per group. ANOVA with Sidak’s correction for

multiple comparisons. *P<0.05, ***P<0.001, ****P<0.0001. NS = not significant.

14

spl

een

DbGP276

lung

AUNT _PD-1

Db G

P27

6

CD44

_PD-1+_B7

B

NS**

1.22 11.42 1.00 1.35_B7

103

104

105

106

107 **

**

2

103

104

105 NS****

***

102

103

104

105

106

liver

NS****

**

103

104

105

106

GP276 peptide stimulation

Iso _PD-1 _PD-1+_B7

Iso+_B7

NS

***

***

*** IF

N-a+

cel

ls/sp

leen

0

10

20

30

40

Iso _PD-1 _PD-1+_B7

Iso+_B7

Iso _PD-1 _PD-1+_B7

Iso+_B7

IFN

-a+ (%

Db G

P276

)

C

D

10

DbGP276 functionality

NS*

****

S7

Fig. S7 B7-costimulation is required for CD8 T cell rescue by PD-1 therapy. At least 45 days

after LCMV clone 13 chronic infection, indicated groups of mice were treated for 2-

weeks with anti-PD-1 (clone muDX400, mouse IgG1), isotype control mouse IgG1 or

combination with anti-B7-1 and anti-B7-2 blocking antibodies. (A) Frequencies of

LCMV-DbGP276-specific CD8 T cells in the spleen. Data show representative mice from

2 independent experiments with 4-5 mice per group. (B) Numbers of LCMV-DbGP276-

15

specific CD8 T cells in different organs. Data show compiled data from 2 independent

experiments with 4-5 mice per group each. Error bars indicate SEM. (C) Numbers of

IFN-γ–producing CD8 T cells in the spleen after ex vivo restimulation with LCMV

GP276 peptide. Data show compiled data from 2 independent experiments with 4-5 mice

per group each. Error bars indicate SEM. (D) Frequencies of LCMV-DbGP276-specific

CD8 T cells producing IFN-γ after ex vivo restimulation with LCMV GP276 peptide.

Data show compiled data from 2 independent experiments with 4-5 mice per group each.

Error bars indicate SEM. ANOVA with Sidak’s correction for multiple comparisons.

*P<0.05, **P<0.01, ***P<0.001. NS = not significant.

16

B CWT P14 CD28KO P14

_PD-L1

POST

PRE

UNT

CD45.2

CD

45.1

UNT _PDL1 UNT _PDL1

D

WT P14 CD28KO P14

F

WT P14 CD28KO P14UNT _PDL1

WT

/KO

ra

tio in lungs

G

59.4 40.6

63.6 36.4

51.3 48.7

92.7 7.33

102

103

104

105 *

UNT _PDL1

1

2

4

8

16

32

64

*E

102

103

104

105

UNT _PDL1 UNT _PDL1

*

1

2

4

8

16

32

64*

0 5 10 1510

1

102

103

104

Days after treatment

_PDL1

UNT

0 5 10 1510

1

102

103

104

Days after treatment

_PDL1

UNT

P1

4/

10

6 P

BM

C

P1

4/

10

6 P

BM

C

P1

4/

10

6 s

ple

en

ce

lls

P1

4/

10

6 lu

ng

ce

lls

WT

/KO

ra

tio in s

ple

en

chronic LCMV

CD28KO P14 (CD45.1/.2)

+ WT P14 (CD45.1/.1)

1d

B6 CD45.2/2

A

45d anti-PD-L1

(14d)

NS NS

WT KO WT KO

WT KO WT KO

Fig. S8 CD28 deficient LCMV-specific CD8 T cells fail to expand following blockade of the PD-

1 pathway during chronic infection. (A) Experiment layout. (B) Frequencies of P14 cells

in blood pre and post anti-PD-L1 treatment. Data are from one representative experiment

of 3 independent experiments, with at least 3 mice per group. Error bars indicate SEM.

(C) Gating strategy to quantify WT and CD28KO P14 cells in blood. Data show

representative mice, pre and post-treatment of data summarized in B. (D) Frequencies of

P14 cells in spleen. Data are from one representative experiment of 3 independent

experiments, with at least 3 mice per group. Error bars indicate SEM. (E) WT to

CD28KO P14 ratio in the spleen. Data are from one representative experiment of 3

17

independent experiments, with at least 3 mice per group. Error bars indicate SEM. (F)

Frequencies of P14 cells in lung. Data are from one representative experiment of 3

independent experiments, with at least 3 mice per group. Error bars indicate SEM. (G)

WT to CD28KO P14 ratio in the lung. Data are from one representative experiment of 3

independent experiments, with at least 3 mice per group. Error bars indicate SEM. D and

F, Unpaired t-test, E and G, Mann Whitney test. *P<0.05. NS = not significant.

18

B

E

CD8 TILS

64.0

18.3

PD-1

TIM

-3

75.1

24.9

23.4

76.6

Tim-3+ PD-1+ Tim-3neg PD-1+

CD8 TILS

PD-1

CD

28

CD

28+

(CD

8 %

)

CD3+CD8+ NSCLC TILS

CD

28

51.84

26.1287.43

41.17

CD8

D

Early stageNSCLCpatient

Lungresection surgery

ATIL

isolation for analysis

C

Tim-3neg

PD-1+Tim-3+

PD-1hi

**

0

20

40

60

80

100

CD

28+

(% )

Pt475 Pt658

Pt263 Pt649

0

20

40

60

80

100

Fig. S9 CD28 expression on CD8 T cells infiltrating NSCLC tumors. (A) Study design (N=17).

(B) CD28 expression on CD8 TILs from 4 representative patients (Pt). (C) As, in B, but

graph shows data from all 17 patients. Error bars indicate SEM. (D) Gating on TIM-3neg

PD-1+ and TIM-3+ PD-1+CD8 TILs, and expression of CD28 on those T cell subsets in

a representative tumor. (E) Summary of the data as in D (N=14, three tumor samples

containing less than 2% TIM-3+ cells among CD8 T cells were omitted from this

analysis). ** P<0.01 (Paired t-test).

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