Post on 24-Jan-2021
Supplemental Materials
Robust anti-tumor effects of combined anti-CD4 depleting antibody and anti-PD-1/PD-L1
immune checkpoint antibody treatment in mice
Satoshi Ueha, Shoji Yokochi, Yoshiro Ishiwata, Haru Ogiwara, Krishant Chand, Takumi
Nakajima, Kosuke Hachiga, Shigeyuki Shichino, Yuya Terashima, Etsuko Toda, Francis HW
Shand, Kazuhiro Kakimi, Satoru Ito, and Kouji Matsushima
Supplementary Tables
Table S1. Antibodies for flow cytometry and immunohistostainingAntibody Clone Antibody CloneBrdU Bu20a ��������� �����
CD3 145-2C11 ����� �������
CD4 RM4-5 PD-1/CD279 RMP1-30
CD8a 53-6.7 PD-L1/CD274 10F.9G2
CD11b M1/70 IFNγ XMG1.2
CD19 1D3 TNFα MP6-XT22
CD31 390 Siglec-H 551
CD44 IM7 Ly-6C HK1.4
CD45 30-F11 Ly-6G 1A8
CD45.2 104 TCRVβ13 MR12-3
B220 RA3-6B2 CD271 (human) C40-1457 orME20.4
NK1.1 PK136 �0-16.%��-,1/-*0
CD90.1 OX-7 �-20%��'�� ��������
CD137 17B5 �!1��'�� ����
���� ���� �!1��'��! ��
�)+� ����� �!1��'��" �����
���������� � ��/+%,)!,��!+01%/��'� ���� �
+�"0�4%/%�.2/#(!0%$�&/-+����")-0#)%,#%���)-�%'%,$��%�)-0#)%,#%�-/��-,"-")-0#)%,#%���*%5!��*2-/�*!"%*%$�0%#-,$!/6��"0�!,$�01/%.1!3)$),0�4%/%�.2/#(!0%$�&/-+�)&%�1%#(,-*-')%0�
Table S2. Primers and probes used in real-time PCRGene Sequence (5'-3') Dye
Forward tctcattcctgcttgtggcReverse gctggcaccactagttggttProbe aattcgagtgacaagcctgtagcc
Forward ctcaagtggcatagatgtggaagaReverse gagataatctggctctgcaggattProbe tcttggatatctggaggaactggcaa
Forward cgtcattttctgcctcatcctReverse tggtcttagattccggattcagProbe aagcttgaaatcatccctgcgagcc
Forward aatctgtggctaccggtggtaReverse ttctgcaggtggaagagctgProbe tggttctggtggctctggttggaa
Forward cgcctggtacaaaaacctccaReverse ccgtgataaagtgcgtgccaProbe ctcagagccctcccccgcaact
Forward gctgctcactgtgaaggaagtReverse tggggaatgcattttaccatForward aaatcgtggtccccaagcReverse tcctcatgttttgggaactatctForward cggtgcagatttccaagaagReverse ggacttcaactccagagtagcc
Gzmb
SYBR greenCd274
Rps3
Tnf
FAM probe
Ifng
Cxcl10
Fasl
Prf1
Supplementary Figures
Supplementary Figure S1. Generation of B16F10-∆hLNGFR transfected cells.
B16F10 cells expressing the truncated form of human low-affinity nerve growth factor receptor
(∆hLNGFR) were generated by retroviral transduction and 2 subsequent rounds of in vivo
passaging. (A) C57BL/6 mice were inoculated subcutaneously with various numbers of
B16F10-∆hLNGFR cells and tumor growth was monitored. Data represent mean ± SE of 5 mice
per group. (B) Single-cell suspensions prepared from day 14 B16F10-∆hLNGFR or B16F10
subcutaneous tumors were analyzed for the expression of ∆hLNGFR on CD45– CD31– tumor
cells. (C) Immunofluorescent staining of a representative section from a day 14
B16F10-∆hLNGFR subcutaneous tumor. Green, ∆hLNGFR; red, propidium iodide (PI). Scale
bar represents 50 µm.
0 10 3 10 4 10 50
20
40
60
80
100
¨K/1*)5%��)���¨K/1*)5%��)��
CD45��CD31�A B C
¨K/1*)5�PI
����0
$;
� 5 1� 15 2� 25�
���
����
����
����
����
Days�afteU�tumoU�LnoculatLon
TumoU�volume�(mm3 )
�����4������4���2���4
Supplementary Figure S2. Optimization of the timing and dose for the administration of
anti-CD4 mAb monotherapy.
B16F10 (A, D–E), LLC (B) or Colon 26 (C) tumor cells were inoculated subcutaneously into
the flanks of C57BL/6 or BALB/c mice. (A–C) Tumor-bearing mice were administered with
200 µg of anti-CD4 depleting mAb on the day(s) indicated and tumor growth was monitored. (D,
E) Mice bearing B16F10 tumors were administered with successive doses of anti-CD4 depleting
mAb on days 5 and 9 after tumor inoculation. (D) Tumor volumes on day 12. (E) Frequency of
CD4+ cells among spleen cells on day 7. Data represent mean ± SE of 8 (A–C), 5 (D) or 3 (E)
mice per group. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared to control).
0 5 10 15 20 250
400
800
1200
Days after tumor inoculation
Tum
or v
olum
e (m
m3 )
ControlαCD4 d -2αCD4 d 0αCD4 d 3αCD4 d 5αCD4 d 9
0 10 20 300
500
1000
1500
Days after tumor inoculation
Tum
or v
olum
e (m
m3 )
ControlαCD4 d -2αCD4 d 0αCD4 d 3αCD4 d 5αCD4 d 9
0 3.1 12.5 50 2000
10
20
30
(%) C
D4+
cells
am
ong
sple
nocy
tes
0 3.1 12.5 50 2000
500
1000
1500
Tum
or v
olum
e (m
m3 )
A� B� C�B16F10 (C57BL/6)� LLC (C57BL/6)� Colon26 (BALB/c)�
Anti-CD4 mAb (µg)�
*�
Anti-CD4 mAb (µg)�
**�
***�***� ***�
**�**�**�
**�**�
D� E�
0 5 10 15 20 250
500
1000
1500
2000
2500
Days after tumor inoculation
Tum
or v
olum
e (m
m3 )
ControlαCD4 d -2αCD4 d 0αCD4 d 3αCD4 d 5αCD4 d 9
**�**�**�**�
Supplementary Figure S3. Anti-CD4 mAb treatment decreases numbers of CD4+ T cells
and pDCs in the blood, lymph nodes, spleen and tumor.
B16F10 tumor-bearing mice were treated with anti-CD4 mAb on day 5 after tumor inoculation,
and leukocyte populations in blood, draining lymph node (dLN), non-dLN, spleen and tumor
were analyzed by flow cytometry. (A) Flow cytometry plots showing the frequency of CD4+ T
cells and siglec-H+ pDCs among whole splenocytes on day 9. (B) Numbers of CD4+ T cells and
siglec-H+ pDCs in tissue compartments on day 7. (C) Numbers of CD4+ T cells and pDCs in the
spleen after anti-CD4 mAb treatment. (D) LLC tumor-bearing mice were administrated with
anti-CD4 mAb on day 5 and 9, and the frequency of CD4+ T cells among blood CD45+ cells
was analyzed on days 0, 2, 7, 15, 21 and 28 after 1st Ab administration. (B–D) Data are
representative of at least four independent experiments. Data represent mean ± SE of 3–4 mice
per group. Numbers in flow cytometry plots indicate mean frequencies within the total
Siglec-H
CD
4
CD
25
Foxp3
spleen (day 9 after tumor inoculation)
Control
CD11b- CD19-CD11b- CD19-
Siglec-H- CD4+
_CD4
CD4+ T
(15.7)
pDC
(0.21)
CD4+ T
(0.01)
pDC
(0.07)
A B
Days after tumor inoculation
CD4+ T cells spleen
pDCs
Cel
l num
ber
8 10 12 14
104
105
106 Control_CD4
* *
8 10 12 14
104
105
106
107
108 Control_CD4
*** *** ***
C D
Control
_CD4
101
102
103
104
105
106
107
108
CD
4+ T
Cell n
um
ber
*** ***
******
**
day 7 after tumor inoculation
Foxp3+
CD25+
Foxp3+
CD25-
Control
_CD4
**
***
blo
od
dLN
ndLN
spleen
tum
or
101
102
103
104
105
106
pD
C n
um
ber
Control_CD4
0 10 20 300
10
20
30
Days after Ab administration
(%) C
D4+
cells
am
ong
bloo
d C
D45
+ ce
lls
4
_CD4(2nd)
splenocyte population. *, P < 0.05; **, P < 0.01; ***. P < 0.001 (compared to control).
Supplementary Figure S4. Intravascular staining distinguishes the intravascular and
parenchymal leukocyte fractions in tumor tissue.
Mice bearing B16F10 tumors were injected intravenously with fluorochrome-conjugated
anti-CD45.2 mAb 3 min prior to the collection of tissues. (A) Flow cytometry plots showing the
CD45.2 intravascular staining (IVS)-positive fraction among the CD45+ cell and CD45+
CD11b– CD19– NK1.1– CD8+ T cell populations. IVS-CD45.2+ or IVS-CD45.2– CD8+ T cells
were further analyzed for the expression of PD-1 and CD137 to identify the tumor-reactive
population. (B) Frequency of PD-1+ CD137– or PD-1+ CD137+ cells among IVS-CD45.2+ or
IVS-CD45.2– CD8+ T cells. Data represent mean ± SE of 4 mice and are representative of at
least 4 independent experiments. Numbers in flow cytometry plots indicate mean frequencies
within parental populations.
0 103 104 1050
30K
60K
90K
120K
0 103 104 1050
30K
60K
90K
120K
0 103 104 105
0
103
104
105
0 103 104 1050
30K
60K
90K
120K
0 103 104 105
0
103
104
105
0 103 104 105
0
103
104
105
CD11b/
CD19/NK1.1
CD
8
ivs-CD45.2
SS
C
PD-1
CD
137
CD45
CD45+ IVS-
(80.0)IVS+
(18.6)
IVS-
(83.6)IVS+
(15.5)(2.0)
(4.6)
(19.6)
(29.3)
CD45+CD8+IVS-
CD45+CD8+IVS+
CD45+
CD45+
Live cells (Control tumor)
CD8+
SS
C
CD45+CD8+
PD-1+
CD137-
PD-1+
CD137+
0
10
20
30
40
50
(%) C
D8+ T
cel
ls
IVS(+)IVS(-)
A B
Supplementary Figure S5. Anti-CD4 mAb treatment enhances the killing activity of
tumor-infiltrating CD8+ T cells.
(A) Experimental procedure. For the preparation of target cells, B16F10 (B16) or LLC tumor
cells were treated with 10 U/mL recombinant mouse IFNγ (R&D) for 24 h. B16F10 tumor cells
were pulsed with hgp100 peptide (hgp10025–33; KVPRNQDWL, 1 µg/mL) for 3 h. The cells
were then stained with 0.5 µM CFSE and used as targets in a killing assay. Effector cells were
prepared from the tumor or dLN of mice bearing B16F10 tumors with or without anti-CD4
mAb treatment and adoptively transferred with one million Pmel-1 CD8+ T cells on day –1.
Tumor-infiltrating cells or dLN cells were prepared on day 14 and depleted of cells positive for
CD4, CD11b, B220, NK1.1, TER-119, MHC class II and gp38/podoplanin using biotinylated
mAbs and a MACS iMAG separator system (Miltenyi Biotec). The negative fraction of the
Target: LLC
Target:1 x 104 cells / well
Intracellular cleaved caspase 3 staining
Flow cytometry analysis
Coculture (4 hr)
Effector:1 x, 3.3 x, 10 x 104 cells / well
Pmel-1 CD8+ effector T cells (Te)
(hgp100-pulsed bone marrow DC primed)
B16F10
A
B
C
B16F10 (d 0)
_CD4 mAb (d 5 & 9)
Tumor cells (d 14)
Dump- enrichment
(CD4, CD11b, B220, NK1.1,
TER119, MHC II, gp38)
Dump- enrichment
(CD4, CD11b, B220, NK1.1,
TER119, MHC II)
CD8+ enrichment
(Pmel-1 CD8+ transfer (d -1))
LLC
IFNa 10 U/mL 24hr
hgp100 1 +g/mL 3hr
IFNa 10 U/mL 24hr
CFSE labelingCFSE labeling
dLN cells (d 14)
0 102
103
104
105
0
50K
100K
150K
200K
250K
0 103
104
105
0
50K
100K
150K
200K
250K
0 103
104
105
0
50K
100K
150K
200K
250K
0 103
104
105
0
50K
100K
150K
200K
250K
0 103
104
105
0
50K
100K
150K
200K
250K
CFSE
CFSE+ B16F10 cells
B16F10 / tumor CD8+
coculture Pmel-1 CD8+ Te no-T cell controlB16 control
tumor CD8+
B16 _CD4
tumor CD8+
FS
C
FS
C
Intracellular cleaved caspase 3
Effector : tumor CD8+
B16 Control
B16 _CD4
Pmel-1
Target: B16F10
Effector: dLN CD8+
B16 Control
B16 _CD4
Pmel-1
Effector: tumor CD8+
B16 _CD4
Pmel-1
1 3.3 10
0
10
20
30
55
60
65
Effector / target
(%) cC
as3
+ / C
FS
E+
1 3.3 10
0
10
20
30
55
60
65
Effector / target
(%) cC
as3
+ / C
FS
E+
1 3.3 10
0
10
20
30
55
60
65
Effector / target
(%) cC
as3
+ / C
FS
E+
tumor-infiltrating cells was further enriched for CD8+ cells using an autoMACS machine. For
the positive control in the killing assay, Pmel-1 splenocytes were stimulated with
hgp100-pulsed bone marrow DCs, which were prepared by standard protocol, for 3.5 days to
obtain Pmel-1 effector CD8+ T cells. The enriched CD8+ cells were cocultured with target tumor
cells at the indicated effector / target ratios for 4 h. After incubation, the cells were subjected to
intracellular staining of cleaved caspase 3 (cCas3, Cell Signaling Technology) and then
analyzed by flow cytometry. (B) Representative flow cytometry plots showing the coculture of
B16F10 and enriched CD8+ T cells, and showing intracellular cCas3 in CFSE+ tumor cells. (C)
The proportion of cCas3+ cells among CFSE+ tumor cells in the coculture of B16F10 cells and
B16F10 tumor-infiltrating CD8+ T cells (left), B16F10 cells and B16F10 dLN CD8+ T cells
(center), and LLC cells and B16F10 tumor-infiltrating CD8+ T cells. The no-T cell control
background was subtracted from the results. Tumor-infiltrating CD8+ T cells in CD4+
cell-depleted B16F10-bearing mice showed killing activity against B16F10 tumor cells but not
against LLC tumor cells.
Supplementary Figure S6. Anti-CD4 mAb treatment systemically increases the number of
CD8+ CD44hi PD-1+ T cells.
Mice bearing LLC or Colon 26 tumors were injected intraperitoneally with anti-CD4 mAb on
days 5 and 9 after tumor inoculation. (A) Tumor weight and (B and C) the number of CD8+
CD44hi PD-1+ T cells in the tumor, blood, dLN, ndLN and spleen were analyzed on day 14. (D)
Immunophenotype of tumor-infiltrating IVS-CD45– CD8+ T cells. Gray filled histograms
indicate isotype control staining of control CD8+ T cells; gray lines and black lines indicate
CD8+ T cells from control and anti-CD4 mAb treated mice, respectively. Cells were fixed and
permeabilized before CTLA4 (IC-CTLA4) staining. Tumor-infiltrating cells were pooled from
5 mice per group. Anti-CD4 mAb administration increased the proportion of cells
positive for LAG3, Tim3 and CTLA4 among CD8+ T cells from LLC and Colon 26
tumors. (A–C) Data represent mean ± SE of 5 mice per group. *, P < 0.05; **, P < 0.01; ***, P
< 0.001 (compared to control).
A B C
D
Control
LAG3 Tim3PD-1 CD137 BTLA IC-CTLA4GITR
_CD4
Isotype (Control) Control _CD4
LLC
Colon 26
blood dL
Nnd
LNsp
leen
103
104
105
106
0
1
2
3
CD
8+ C
D44
hi P
D-1
+
T ce
lls (
x103 )
/ mg
tumor
CD
8+ C
D44
hi P
D-1
+
T ce
lls
0
50
100
150
200Tu
mor
wei
ght (
mg)
LLC
***
*
***
*
blood dL
Nnd
LNsp
leen
104
105
106
CD
8+ C
D44
hi P
D-1
+
T ce
lls
0
2
4
6C
D8+
CD
44hi
PD
-1+
T ce
lls (
x103 )
/ mg
tumor 0
50
100
150
200
Tum
or w
eigh
t (m
g)
Colon 26**
**
*
Supplementary Figure S7. Anti-CD4 mAb treatment causes proliferation of tumor-specific
CD8+ T cells.
(A-H) Melanoma antigen specific TCR (TCRVα1Vβ13; Pmel-1) expressing CD8+ T cells with
the Thy1.1 congenic marker were adoptively transferred one day prior to B16F10 tumor
inoculation. Tumor-bearing mice were injected intraperitoneally with anti-CD4 mAb on days 5
and 9, and the number of Pmel-1 CD8+ T cells in the blood, draining LN (dLN), non-draining
LN (ndLN), spleen and tumor were analyzed by flow cytometry on days 9 and 14 after tumor
inoculation. Mice were injected intraperitoneally with BrdU 1 hr prior to collecting tissues in
blood dLN ndLN tumor0
5
10
15
20
(%) S
/G2/M
am
ong
CD
8+ C
D44
hi ce
lls
0 103 104 105
0
103
104
105
0 103 104 105
0
103
104
105
9 14100
101
102
103
104
Pm
el-1
cel
ls
Pm
el-1
cel
ls
Day -1 Day 0 Day 5 Day 9 Day 14
Pmel-1 i.v.B16F10 s.c.
_CD4 i.p. FCM analyses(blood, dLN, ndLN,spleen, tumor)BrdU i.p. 1hr pulse
Thy1.1
TCR
V`1
3
BrdU
SS
C
Control _CD4
A
D
F G H
E
I J
CB
Day 14 blood
Days after tumor inoculation
Days after tumor inoculation
dLN tumor
dLN tumor
blood dL
Nnd
LNsp
leen
tumor
101
102
103
104
105
106 Control_CD4
0 103 104 105
0
103
104
105
0 103 104 105
0
103
104
105
CD3+ CD8+ (tumor day 14)
Control _CD4
Pmel1(0)
Pmel-1(0.11)
Control_CD4
Control_CD4
Control_CD4
CD3+ CD8+ Thy1.1+ TCRV`13+
(dLN day 14)
Brd
U+ P
mel
-1 c
ells
Brd
U+ P
mel
-1 c
ells
dLN
tumor
101
102
103
104
105
Day 14
0 103 104 1050
100K
200K
300K
400K
500K
BrdU+
(10.3)
0 103 104 1050
100K
200K
300K
400K
500K
BrdU+
(13.6)
Control _CD4
CD8+ CD44hi (dLN day 8) Control_CD4
(73.1) (8.9)
(16.7)
Fucci-green (mAG)
Fuuc
i-ora
nge
(mK
O2)
9 14102
103
104
105
9 14100
101
102
103
104
105
9 14102
103
104
105
9 14100
101
102
103
**
*
**
G0/G1
(81.5)S/G2/M(4.7)
M/G1(12.5)
Fucci-orange+ (mKO2+)Fucci-green+ (mAG+)
S
M
G2G1
CELLCYCLE
G0
8 10 12 14 16 180
1000
2000
3000
4000
Days after tumor inoculation
Tum
or v
olum
e (m
m3 ) cont
_CD4
order to label proliferating cells. (B) Tumor growth. (C) Flow cytometry plots showing
tumor-infiltrating Pmel-1 CD8+ T cells on day 14. (D and E) Pmel-1+ CD8+ T cell numbers in
different tissue compartments. (F) Proportions of BrdU labeled cells within the Pmel-1 CD8+ T
cell population in the dLN on day 14. (G and H) BrdU+ Pmel-1 CD8+ T cell numbers in the dLN
and tumor. (I and J) Fluorescent ubiquitination-based cell cycle indicator (Fucci) transgenic
mice bearing subcutaneous B16F10 tumors were injected intraperitoneally with anti-CD4 mAb
on day 5 and the cell cycle of CD8+ T cells was analyzed based on the expression of mKO2 and
mAG on day 8. (I) Flow cytometry plots showing mKO2 and mAG expression within CD44hi
CD8+ T cells in the dLN. (J) Proportions of mAG+ (S/G2/M phases) cells among CD8+CD44hi T
cells in different tissue compartments on day 8. Data represent mean and SE of 3–4 mice per
group and are representative of two independent experiments. Numbers in flow cytometry plots
indicates mean frequencies within parental populations. *, P < 0.05; **, P < 0.01 (compared to
control).
Supplementary Figure S8. Anti-CD4 mAb treatment enhances proliferation of
tumor-specific CD8+ T cells in the dLN.
(A) CD45.2+ C57BL/6 mice were inoculated s.c. with B16F10 on day –6 and administrated i.p.
with anti-CD4 mAb on day –1. CD45.1– CD45.2+ CD90.1+ Pmel-1 CD8+ T cells (B16-specific),
CD45.1+ CD45.2– CD90.1– OT-1 CD8+ T cells (ovalbumin-specific) and CD45.1+ CD45.2+
CD90.1– polyclonal CD8+ T cells were purified by negative selection with antibodies against
CD4, CD11b, B220, NK1.1 and TER-119. The cells were mixed equally, labeled with CFSE,
and then adoptively transferred into CD45.1- CD45.2+ CD90.1– recipients on day 0. The
distribution and cell division of donor CD8+ T cell populations were analyzed by flow
cytometry on day 3. (B) Representative plots of IVS-CD45– CD8+ cells in the dLN. (C)
Histograms showing the dilution of CFSE signal in Pmel-1, OT-1 and polyclonal CD8+ T cells
in recipients with various tumor and treatment combinations. Numbers on histograms indicate
cell generation (undivided = generation 0). (D) Proportions of cells within generations 0–1, 2–4
and 5–8 among Pmel-1 CD8+ T cells in the dLN of B16-bearing mice with or without anti-CD4
mAb treatment. (E) Numbers of generation 5–8 Pmel-1 CD8+ T cells in the blood, dLN, ndLN,
spleen and tumor of B16 tumor-bearing mice with or without anti-CD4 mAb treatment. Pmel-1
B
A
D E
C
0 103
104
105
0
100K
200K
300K
400K
500K
0 102
103
104
105
0
102
103
104
105
0 102
103
104
105
0 102
103
104
105
0 102
103
104
105
CD90.1
FS
C
CD45.1
CD
45
.2
CFSE
Pmel-1CD90.1-
CD90.1- Pmel-1 OT-1 polyclonal
dLN IVS CD45- CD8+
(PI- CD11b/CD19/NK1.1-)
B16 _CD4
(-) (-)
(-) (+)
(+) (-)
(+) (+)
B16 sc. or untreatedd -6
Recipient: CD45.1- CD45.2+ CD90.1+
_CD4 mAb ip. or untreatedd -1
CFSE labeled CD8+ T cell iv. transfer
FCM analysis (blood, LN, spleen, tumor)
d 0
d 3
Pmel-1
OT-1
polyclonal
1
2
2
Polyclonal
OT-1
5-8 2-4 0-1
0-1 2-4
generation
5-8
0
20
40
60
80
(%) P
mel-1
***
***
**
**
*
bloo
ddL
N
ndLN
spleen
tum
or
0
2000
4000
6000
8000
G5-8
Pm
el-1
CD
8+ T
cells
congenic markersdonor
CD8*
equal mixture
(4 x 105 CD8+ each)2
1
1/2
CD45CD90
*Enriched CD8+ T cells by depleting lineage (CD4,
CD11b, B220, NK1.1, TER-119, Gr-1) positive cells
Control
_CD4
but not OT-I or polyclonal CD8+ T cells selectively proliferated in the dLN of B16
tumor-bearing mice. These results indicate that CD4 depletion-induced expansion of CD8+ T
cells requires specific tumor antigen, and that the lymphopenic condition is not sufficient to
induce expansion of CD8+ T cells. (D and E) Data represent mean and SE of 5 mice per group.
*, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared to control).
Supplementary Figure S9. Anti-CD4 mAb treatment or combination treatment with
anti-CD4 and anti-PD-1 or PD-L1 mAbs induces anti-tumor gene expression within the
tumor.
Mice bearing B16F10 tumors received anti-CD4 mAb, anti-PD-L1, anti-PD-1 or a combination
of these, according to the treatment schedule shown in Figure 4A. (A and B) Expression of the
anti-tumor cytokine genes Ifng and Tnf, the IFNγ-inducible genes Cxcl10 and Cd274/PD-L1,
and genes for the proapoptotic molecules Fasl, Prf1/perforin, and Gzmb/Granzyme B in whole
tumors on day 14 after tumor inoculation are presented relative to Rps3 expression. Results are
shown for (A) anti-PD-L1 mAb experiments and (B) anti-PD-1 mAb experiments. (C)
Expression of PD-L1 in tumor tissue sections on day 14 after tumor inoculation. Green, CD8;
red, PD-L1; blue, propidium iodide (PI). Scale bar represents 100 µm. Data represent mean ±
SE of 3–4 mice per group and are representative of two independent experiments. *, P < 0.05;
**, P < 0.01; ***, P < 0.001.
Rel
ativ
e to
Rps3
Rel
ativ
e to
Rps3
Control_CD4_PD-1_CD4/_PD-1
Control_CD4_PD-L1_CD4/_PD-L1
A
B
C
Tnf Ifng Cxcl10 Fasl Prf1 Gzmb
Tnf Ifng Cxcl10 Cd274
Cd274
Fasl Prf1 Gzmb10-6
10-5
10-4
10-3
10-2***
*** *** ***
10-6
10-5
10-4
10-3
10-2***
*** *** ***
10-3
10-2
10-1***
*** *** ***
10-6
10-5
10-4
10-3
10-2***
*** *** ***
10-4
10-3
10-2
10-1***
*** *** ***
10
10
10
10
-4
-3
-2
-1
100 ***
*** *** ***
10-6
10-5
10-4
10-3
10-2*
10-6
10-5
10-4
10-3
10-2**
** ** **
10-4
10-3
10-2
10-1**
*** ***
10-6
10-5
10-4
10-3
10-2*
* * *
10-5
10-4
10-3
10-2
10-1*
* * *
10-5
10-4
10-3
10-2
10-1
100**
** * *
10-4
10-3
10-2
10-1
100
10-4
10-3
10-2
10-1
100
Control _CD4
CD8 PD-L1 PI