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