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: [email protected]
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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