pd-1 modulates regulatory t cell homeostasis during low
TRANSCRIPT
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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: [email protected]
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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.