, Nonclinical Research Center

Identiﬁcation of tumor inﬁltrating lymphocyte subsets
using the CT26WT tumor-bearing mouse model○ Hiroyuki Okada, Tomohiro Tanaka, Shinichiro Tsunesumi, Shigenori Enoki, Seiichi Katayama,Naoyuki Hironaka, Katsuhide Nishi

Summary in Japanese

ObjectiveImmune checkpoint inhibitors such as anti-PD -1 antibody andanti-CTLA-4 antibody have recently been approved for the treatment ofmelanoma or non-small cell lung cancer. Further developments oftherapeutic methods using their inhibitors have been advanced in othertypes of cancer. Therefore, it is important to establish an evaluation
method for their inhibitors.
In this study, we examined an evaluation method for mouse tumor
inﬁltrating lymphocytes (TILs) subsets (regulatory T cells, CD8+T cells,
dendritic cells, bone marrow-derived suppressor cells), which is one of
the evaluation items of immune checkpoint inhibitors, using syngeneic
mouse model.

免疫チェックポイント阻害薬（抗 PD-1抗体，抗 CTLA-4 抗体など）は，近年悪性黒色腫や非小細胞性肺癌などのがん治療に認可されている．さらに他のがん種においても免疫チェックポイント阻害薬を用いた治療法の開発が進んでおり，これらの薬剤の評価系の確立は重要である．今回我々はシンジェニックマウ
スモデルを用いて免疫チェックポイント阻害薬の評価項目の1つであるマウス腫瘍浸潤リンパ球（TILs）サブセット（制御性 T 細胞，CD8+T 細胞，樹状細胞，骨髄由来免疫抑制細胞）の解析法について検討した．
その結果，腫瘍中のリンパ球サブセットが検出でき，免疫チェックポイント阻害薬による変化が認められたことから，本解析法は免疫チェックポイント阻害薬の評価系として有用と考えられる．

Results
Tumor growth treated with anti-PD-1 antibody and/or anti-CTLA-4 antibody

Materials and Methods

5000

Mice, BALB/cAnNCrlCrlj, ♀, 7w (At inoculation of cells)【Cell Line】

CT26WT (ATCC, mouse colon carcinoma)
The cell suspension (1×106 cells) was injected subcutaneously into theright abdominal region of each animal.

3000*20000: The points of administration (i.p.): The points of tumor diameter measurement

123478Regulatory T cells (Treg : Foxp3+CD25+CD4+T cells) in TILs

50K

003333【Evaluation】(1) Measurement of tumor diameter
Tumor diameter was measured using a caliper on Day 0, 3, 7, 10 and 14.
Tumor volume was calculated by the following formula.
Estimated tumor volume (mm3) =1/2 × long diameter (mm) × short diameter (mm) × short diameter (mm)(2) Measurement of tumor inﬁltrating lymphocytes
At 8 days after administration, tumors were excised from mice anddispersed using gentleMACS Octo Dissociator (Miltenyi Biotec).
Cells were passed through a 70μm cell strainer, centrifuged at 190×g
and supernatant was removed.
Blocking reagent and CD45 microbeads were added to the cellsuspension and incubated for appropriate time.
Magnetic cell sorting was performed to isolate CD45 positive cells using
QuadroMACS Separator and MACS columns (Miltenyi Biotec).
Cell staining was performed by the antibodies shown in the table below.
Samples were measured by a ﬂow cytometer (FACSLyric, BD Biosciences)
and analyzed using BD FlowJo software (BD Biosciences).
Panel 1: Treg

Panel 2: CD8 T cells+Panel 3: DC and MDSC

AntigenFluorochrome

AntigenFluorochrome

AntigenFluorochrome

CD45
CD3
CD4
CD25
Foxp3
Live/Dead

PerCP-Cy5.5
FITC
PE-Cy7
BV421
AF647
FVS510

CD45
CD3
CD8a
CD69
PD-1
Live/Dead

APC
FITC
APC-H7BB700PEFVS510

CD45
CD3
CD11c
CD11b
Gr-1
MHC II
Live/Dead

PE
FITC
BB700
BV421
APC
PE-Cy7FVS510Conclusion(1) In antitumor test, an antitumor eﬀect was observed in theCTLA-4 group and the PD-1+CTLA-4 group.
(2) Change in distribution of TILs was observed as below.
・ The proportion of Treg in tumor decreased with no signiﬁcantdiﬀerence in the CTLA-4 group and the PD-1+CTLA-4 groupcompared to control group.
・ The proportion of CD69+CD8+T cells (activated CD8+T cells)
in tumor signiﬁcantly increased in the PD-1+CTLA-4 antibody
group compared to control group. On the other hand, theproportion of PD-1+CD8+T cells (exhausted CD8+T cells) intumor signiﬁcantly decreased in the PD-1 group and thePD-1+CTLA-4 group compared to control group.
・ No signiﬁcant diﬀerence in the proportion of DC and MDSC
in tumor was found in any of the test groups compared tocontrol group.
In conclusion, TILs subsets in tumor could be detected and thechanges in the proportion of TILs subsets by immune checkpoint
inhibitors could be observed.
This evaluation method is considered to be useful to evaluate
immune checkpoint inhibitors.

100K

0004
104
10

CD25+CD4+T cells

CD45+CD3+cells
24.5

1100100110 10210310441010

CD3 (FITC)9.0491.02101100 0100110 1010Foxp3+CD25+CD4+T cells

3101100105FSC-A

510CD25+CD4+cells
6.02

21021050K 100K 150K 200K 250K

SSC-A

310310050K 100K 150K 200K 250K

FSC-A
510100K50K50K 100K 150K 200K 250K

510150K50K

2310410105001210 10

10CD4 (PE-Cy7)

10341010510Foxp3 (AF647)

The proportion of Treg in tumor decreased with no signiﬁcant diﬀerence in the CTLA-4 groupand the PD-1+CTLA-4 group compared to control group.
% of Foxp3+CD25+CD4+cells
in CD45+CD3+cells

Control (IgG2a + IgG2b) 200 + 150
PD-1200CTLA-4150
PD-1 + CTLA-4200 + 150

0150K

Each CD8+T cells subsets in TILs
Activated CD8a+T cells

60100

**

50403020100Control

PD-1

CTLA-4

Exhausted CD8a+T cells

806040****1614121086420

Control

200

PD-1+CTLA-4

PD-1

CTLA-4 PD-1+CTLA-4

The columns are the mean and S.E. of 3 animals.
ControlPD-1

CTLA-4PD-1+CTLA-4

The columns are the mean and S.E. of 3 animals.
**: p<0.01; Signiﬁcant diﬀerence from control group. (Studentʼs t-test)

CD69+CD8a+T cells

CD45+CD3+T cells
101010101010(Activated CD8a+T cells)

5104
3CD45+CD3+cells
31.82
101010101010100101102103

CD3 (FITC)

104

PD-1+CD8a+T cells

10(Exhausted CD8a+T cells)

538.8

1039.6410

PD-1 (PE)

6666

Number of(μg/body)animalsDose

510200K

3102101051015.2
1006.52
1011021031041010538.828.615.117.443210510010CD8a (APC-H7)

1102103104105CD8a (APC-H7)

The proportion of CD69+CD8+T cells (activated CD8+T cells) in tumor signiﬁcantly increasedin the PD-1+CTLA-4 group compared to control group. On the other hand, the proportion ofPD-1+CD8+T cells (exhausted CD8+T cells) in tumor signiﬁcantly decreased in the PD-1 group andthe PD-1+CTLA-4 group compared to control group.

DC (Dendritic cells) and MDSC (Myeloid-derived suppressor cells) in TILs
CD45+CD3-cells

DC (MHCⅡ+CD11c+cells)
510510410310210CD45-CD3-cells
54.9

11001001011021031041031021011027.0

1.93110210310CD3 (FITC)

CD11c (BB700)

DCMDSC

1635

141210864

PD-1+CTLA-410Control

PD-1

CTLA-4

50.1

8.71

40.3

0.89

4103102101100
100
10110210310410510No signiﬁcant diﬀerencein the proportion of DC andMDSC in tumor was found in anyof the test groups compared tothe control group.

150510Gr-1 (APC)

200CTLA-4510255PD-1410302
Control59.101051012.0410010

MDSC (Gr-1+CD11b+cells)

CD11b (BV421)

Control (IgG2a + IgG2b) 200 + 150
PD-1200
CTLA-4150
PD-1 + CTLA-4200 + 150

Group

410

CD25 (BV421)

Number of(μg/body)animals

TILs measurement

310% of MDSC
in CD45+CD3-cells

Group conﬁgulation

CD45 (PerCP-Cy5.5)Group allocation

210CD45+CD3+T cells

CD45 (PE)BioXcell
BioXcell
BioXcell
BioXcellCD45 (PE)RMP1-142A3
9D9
MPC-11110FVS510 (BV510)% MHC II+CD11c+cells
in CD45+CD3-cells

PD-1
IgG2a
CTLA-4IgG2bDay 80

10% of CD69+CD8+T cells
in CD45+CD3+cells

Manufacturer

100K

Lymphocytes
49.5

250K

Single Cells
95.2

200K

150K

50K

Antibody
Clone

250K

Single Cells
86.7

Lymphocytes

SSC-A

100K

Antibody

250K

CD25 (BV421)

SSC-A

150K

: The points of administration (i.p.): The points of TILs measurement

Doublets exclusion (SSC)

200K

FSC-H

200K

Experiment Schedule 2 (TILs measurement)

DoseLive
15.6

Doublets exclusion (FSC)SSC-HDay 14

Dead cells exclusion

Day 09 10 11 12 13 14

CD69 (BB700)

Day 10250KGroup6

In antitumor test,an antitumor eﬀect was observedin the CTLA-4 group and the PD-1+CTLA-4 group.

DaysPoints, mean and S.E for 6 animals; vertical bars, S.E.
*: p<0.05, **: p<0.01; Signiﬁcant diﬀerence from control group.
(Studentʼs t-test)

Group allocation

Anti-tumor study

5MHCⅡ (PE-Cy7)

Day 7

0% of PD-1+CD8+T cells
in CD45+CD3+cells

Day 3CTLA-4
PD-1 + CTLA-4**1000Experiment Schedule 1 (Anti-tumor study)

Day 0Control
PD-1

4000

Tumor volume (mm3)【Animal】

PD-1+CTLA-4

The columns are the mean and S.E. of 3 animals.

COI 開示☑ 発表内容に関連し，過去３年間，開示すべきCOI 関係にある企業などはありません

筆頭発表者︓ 岡田 紘幸