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Supplementary Materials for

PD-1 modulates regulatory T cell homeostasis during low-dose IL-2 therapy

Takeru Asano, Yusuke Meguri, Takanori Yoshioka, Yuriko Kishi, Miki Iwamoto, Makoto

Nakamura, Yasuhisa Sando, Hideo Yagita, John Koreth, Haesook T. Kim, Edwin P. Alyea,

Philippe Armand, Corey S. Cutler, Vincent T. Ho, Joseph H. Antin, Robert J Soiffer,

Yoshinobu Maeda, Mitsune Tanimoto, Jerome Ritz and Ken-ichi Matsuoka

This file includes:

Supplemental Figure1

Supplemental Figure2

Supplemental Figure3

Supplemental Table1

Corresponding Author:

Ken-ichi Matsuoka M.D., Ph.D

Department of Hematology and Oncology, Okayama University Graduate School of

Medicine, Dentistry, and Pharmaceutical Sciences, Shikata-cho 2-5-1, Kita-ku, Okayama-city,

Okayama, Japan

Phone +81-86-235-7227

Fax +81-86-232-8226

E-mail: k-matsu@md.okayama-u.ac.jp

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Figure S1. Characteristics of IL-2 treated central memory phenotype Tregs.

Wild type C57BL/6 mice received control vehicle or 5,000 IU human recombinant IL-2 once

daily for 14 days spleen cells were analyzed on day 15. (A) Representative flow cytometry

histograms for identification of Ki-67+ proliferating cells in CD44highCD62Lhigh central

memory phenotype Tregs. Percentage of Ki-67+ cells is shown for each histogram. (B)

Percentage of Ki-67+ proliferating cells in each Treg subset. (C) Representative flow

cytometry histograms detecting expression of CD25, Foxp3, CCR4, CCR7, GITR, CTLA-4,

LAG-3 and Tim-3 on CD44highCD62Lhigh central memory phenotype Tregs. n = 4 mice per

group per experiment. Data are representative of two independent experiments and expressed

as means +/- SEM. ***P<0.001. N : Naïve, CM : Central Memory, EM : Effector Memory.

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Figure S2. Low dose IL-2 increased the expression of PD-ligand 1 on murine Tregs.

Wild type C57BL/6 mice received control vehicle or 5,000 IU human recombinant IL-2 once

daily for 14 days spleen cells were analyzed on day 15. (A) Representative flow cytometry

histograms measuring expression of PD-L1 on CD8 T cells, Tcons and Tregs. (B) Expression

of PD-L1 (MFI) in CD8 T cells, Tcons and Tregs after IL-2 therapy. (C) Representative

histograms used to identify CD11c+ MHCclassⅡ+ dendritic cells and quantify expression of

PD-L1 and PD-L2 on dendritic cells. (D) Expression of PD-L1 (upper) and PD-L2 (lower) on

dendritic cells after ILK-2 therapy. n = 4 mice per group per experiment. Data are

representative of three independent experiments and expressed as means +/- SEM. *P<0.05

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Figure S3. IL-2 expanded PD-1-/- Treg retain suppressive function in vitro.

Tcons labeled with CellTrace Violet from wild type C57BL/6 were cultured at 1:1 ratio with

Tregs isolated from control vehicle or IL-2 treated wild type or PD-1-/- mice in the presence

of CD3/28 stimulation for 3 days. (A) Representative flow cytometry histograms used to

measure Tcon proliferation in vitro. Percentage of divided cells is shown for each histogram.

(B) Suppressive activity PD-1wt and PD-1-/- Tregs. Percentage of divided Tcons was

measured at various Tcon:Treg cell ratios. n = 4 mice per group per experiment. Data are

representative of two independent experiments and expressed as means +/- SEM.

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Supplemental Table1.

Clinical characteristics of patients who were evaluated for clinical response and PD-1

expression on Tregs during IL-2 therapy (n=11)

Characteristic

Clinical

Responders

(n=6)

Clinical

non-responders

(n=5)

p-value

Age, median (range) 47 (44-65) 53 (27-61) > 0.99

M/F 5/1 2/3 0.24

Median time from HSCT

days (range)

752 (420-1575)

1425 (525-2766)

0.25

Median time from oncet of cGVHD

days (range)

476 (117-1127)

1186 (161-2624)

0.42

Concurrent cGVHD therapy

median no of agents (range)

3 (1-3)

3 (2-3)

0.70

Use of concominat agents, no 0.82

Systemic steroids 6 5

MMF 3 2

Calcineurin inhibitor 1 3

Sirolimus 3 4

Previous therapy for cGVHD

median no of agents (range)

0 (0-2)

2 (0-5)

0.09

Discontinued therapies, no > 0.99

Rituximab 1 3

Extracorporeal photopheresis 1 2

MMF 0 2

Calcineurin inhibitor 1 1

Alemutumab 0 1

Sirolimus 0 1

Thalidomide 0 1

Denileukin diftitox 0 1

Area of cGVHD

median no of sites (range)

2 (1-5)

2 (1-4)

0.79

Site of cGVHD, no 0.87

Skin 5 4

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Joint, fascia, muscle 4 1

Liver 1 2

Eye 2 2

Mouth 2 3

Lung 2 1

Peropheral nerves 1 0

Diagnosis 0.08

AML 1 3

CLL 4 0

CML 0 1

HL 1 0

NHL 0 1

Conditioning regimen

Myeloablative 1 4

Nonmyeloablative 5 1

Stem cell source 0.45

PBSCs 6 4

BM and PBSCs 0 1

AML, Acute Myeloid Leukemia; CLL, Chronic Lymphocytic Leukemia; CML, Chronic

Myelogenous Leukemia; HL, Hodgkin Lymphoma; ALL, Acute Lymphoblastic Leukemia;

NHL, Non-Hodgkin Lymphoma; PBSC, Peripheral Blood Stem Cells; BM, Bone Marrow;

MMF, Mycophenolate mofetil

Statistical analysis

In murine experiments, results are presented as means +/- SEM. The Student’s t test was used

to assess statistical significance between two groups and 1-way ANOVA was used to

compare >2 groups. P values < 0.05 were taken to indicate statistically significant. Fisher’s

exact test was used for group comparisons for categorical variables and the Wilcoxon rank

sum test was used for group comparisons for continuous variables in Table S1. In human

subject analyses, the Wilcoxon signed rank test was performed for paired group comparisons.

All tests were two-sided at the significance level of 0.05.