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DOI: 10.3109/08923973.2015.1038752

RESEARCH ARTICLE

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Immunomodulatory effects of the pentapeptide YGSRS on humanperipheral-blood mononuclear cells
Yurie Nakamura1, Akemi Hayakawa2,3, Hitomi Ishizawa1, Yuzuru Kamei2, Sachiko Tanaka1, Kenji Onda1,
Kentaro Sugiyama1, and Toshihiko Hirano1
1

Department of Clinical Pharmacology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Hachioji, Japan, 2Department of
Plastic and Reconstructive Surgery, Nagoya University Graduate School of Medicine, Nagoya, Japan, and 3Department of Biological Science andTechnology, Faculty of Industrial Science and Technology, Tokyo University of Science, Tokyo, Japan
Abstract

Keywords

Context: The pentapeptide YGSRS is originated from coffee bean, while its pharmacologicalfeatures have little been examined.
Objectives: We investigated the effects of YGSRS on proliferation, cytokine production and CD4+
CD25+ Foxp3+ regulatory T (Treg) cell frequency of human peripheral blood mononuclear cells(PBMCs) activated by T-cell mitogen.
Materials and methods: The effects of YGSRS on T-cell mitogen-activated PBMCs were assessedby WST assay procedures. Concentrations of Th1/Th2/Th17 cytokines in the PBMCs culture
medium were analyzed with beads-array procedures followed by analysis with flow cytometry.
The CD4+ CD25+ Foxp3+ Treg cells in mitogen-activated PBMCs were stained withfluorescence-labeled specific antibodies followed by flow cytometry.
Results: YGSRS at 1–10 000 ng/ml (1.56–15 600 nM) has a tendency to promote the mitogenactivated proliferation of PBMCs, but the effects were not statistically significant. YGSRS affectthe production of tumor necrosis factor (TNF) a, interleukin (IL)-4, IL-6 and IL-10 from theactivated PBMCs, and statistically significant increase in the concentrations of IL-6 and IL-10 in
the medium were observed at 1–1000 ng/ml (1.56–1560 nM) (p50.05). YGSRS has a tendency
to decrease the frequency of Treg cells in the activated PBMCs, but the difference was notstatistically significant.
Discussion and conclusions: The data suggest that the pentapeptide YGSRS affects the
production of several types of cytokines from activated human peripheral T cells, which may
modulate Th2 type immunity.

CD4+ CD25+ Foxp3+ regulatory T cells,
cytokines, peripheral lymphocytes, T-cell
mitogen, YGSRSIntroduction
Immunomodulatory and anti-inflammatory activities of several types of naturally occurring or synthetic oligopeptides
and peptides have been reported1–4. We have also found thatcertain types of peptide antibiotics suppress proliferation andseveral cytokine productions in mitogen-activated humanperipheral blood mononuclear cells (PBMCs)5,6. A wellknown immunosuppressive drug cyclosporine widely used fortreatment of transplant recipients and patients with autoimmune disorders has also a cyclic peptide structure7.
Gramicidin D-containing linear pentadecapeptides wasreported to have immunosuppressive efficacy, which may be
useful as maintenance immunosuppressive therapy for organtransplantations8.

Address for correspondence: Toshihiko Hirano, PhD, Department ofClinical Pharmacology, School of Pharmacy, Tokyo University of
Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 1920392, Japan. Tel: +81 42 676 5794. Fax: +81 42 676 5798. E-mail:hiranot@toyaku.ac.jp

History
Received 2 February 2015
Revised 4 March 2015
Accepted 4 April 2015
Published online 15 May 2015

The pentapeptide YGSRS was isolated from coffee bean
and its chemical structure had been identified, whereas its
biological and pharmacological activities have little been
investigated so far. The observations concerning immunomodulatory activities of several types of peptides, as describedearlier, remind us to examine immunomodulating efficacies
of YGSRS using PBMCs. Increasing evidences support that
regulatory T (Treg) cells contribute to the suppression ofcellular immunity9, and thereby the activation and/or
increased number of Treg cells may result in therapeutic
efficacy against several immunological disorders with progressive autoimmune pathophysiology10,11. Thus, Treg cell
frequency in patients with autoimmune diseases appears to be
critical for the clinical outcome and the efficacy of immunosuppressive therapy in these patients.
The present study was thus conducted to examine theimmunomodulatory efficacy of the pentapeptide YGSRS on
proliferation, cytokine production and CD4+ CD25+ Foxp3+
Treg cell frequency in human PBMCs activated by T-cell
mitogen. Our data suggest that the pentapeptide YGSRS
affects the production of several types of cytokines from

DOI: 10.3109/08923973.2015.1038752

Pentapeptide YGSRS on human peripheral-blood mononuclear cells

T-cell mitogen-activated human peripheral lymphocytes,
which may modulate Th2 type immunity.

Methods

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Materials
RPMI 1640 medium and fetal calf serum were purchased from
Gibco Co., Grand Island, NY. Concanavalin A was obtained
from Seikagaku Kogyo Co., Tokyo, Japan. YGSRS used in thisstudy is a synthetic linear pentapeptide synthesized at Peptide
Institute Inc. (Osaka, Japan) with purity of 99.4%, which was
kindly provided by Dr. Henry Aoki, President at YGSRS Co.,
Acton, MA. The peptide was dissolved in distilled water at aconcentration of 0.5 mg/ml as a stock solution. The stock
solution was diluted to working concentrations by RPIM 1640
medium before use. Cyclosporine A was provided from
Novartis Pharma Co., Tokyo, Japan. The cell proliferation
WST-8 assay kits were obtained from Promega Co. (Madison,
WI). BDÔ cytometric bead array human Th1/Th2/Th17
cytokine kits, Human Foxp3 Stain Kit PE, Human CD4
FITC, Human CD25 allophycocyanin (APC), fluorescein
isothiocyanate (FITC) Mouse IgG1 k Isotype control, APC
Mouse IgG1 k Isotype control and phycoerythrin (PE) Mouse
IgG1 k Isotype control were obtained from BD Biosciences,
San Jose, CA. MEMa medium was from Lonza Japan Co.
(Tokyo, Japan). Prednisolone, Cell Lysis solution and trypan
blue were purchased from Sigma Chemical Co. (St. Louis,
MO). Sterilized plastic plates for cell cultures were obtained
from Iwaki Co. (Tokyo, Japan). All the other reagents were of
the best available grade.
Subjects
The present study was carried out in accordance with The Codeof Ethics of the World Medical Association (Declaration of
Helsinki). The study was approved by the Ethical Committee ofTokyo University of Pharmacy and Life Sciences (Number:
10-01) and written informed consent was obtained from all thehealthy volunteers included in the study. The study includes
totally eight healthy subjects (four male and four female; mean
age of 30.8 years). These subjects had neither a history of
immunological disorders nor a history of taking immunosuppressive drugs including glucocorticoids.
Isolation of PBMCs and evaluation of anti-proliferative
effects in vitro
Twenty milliliters of venous blood were taken from healthy
subjects between 9:30 and 11:00 in the morning andheparinized. This 20 ml sample size was the smallest possible
to examine the effects of the peptide YGSRS, cyclosporineand prednisolone. The heparinized blood was loaded on 3 mlof Ficoll-Hypaque (Nakarai Co., Japan), centrifuged at1300  g for 20 min and PBMCs were separated as described
previously12–14. For evaluation of the effects of the peptide on
PBMCs, the cells were washed and suspended in RPMI 1640
medium containing 10% fetal bovine serum, 100 000 IU/lpenicillin and 100 mg/l streptomycin to a final density of1  106 cells/ml. About 196 ml of this cell suspension were
loaded into wells of a 96-well plate. Concanavalin A, as a
T-cell mitogen, was added to each well to a final

319

concentration of 5.0 mg/ml. Subsequently, 4 ml of an ethanol
solution containing the peptide YGSRS were added to give
final concentrations of 1–10 000 ng/ml (1.56–15 600 nM).
Four microliters of ethanol were added to the control wells.
The plate was incubated for 96 h in 5% CO2/air at 37 C. Afterthe culture, 20 ml of Cell Counting Kit-8 reagent solution wasadded to each well, and the plate was incubated for another2 h. PBMC proliferation was determined by measuring theoptical density at 450 nm absorbance (ref. 650 nm).
MCF-7 cell culture
MCF-7 cells were maintained in MEMa medium containing
10% fetal bovine serum, 100 units/ml penicillin and 100 lg/ml
streptomycin. These cells at densities of 1–5  105 cells/ml
suspended with each medium were incubated in 5% CO2/air at
37 C for 3–4 days in a humidified chamber. The cell numberof the cell suspension was adjusted to 1–5  105 living cells/ml,
by counting living cells with a trypan-blue dye exclusion
test15. Subsequently, 98 ll of this cell suspension were placedin each well of a 96-well flat-bottomed plate, and 2 ml of thesolution containing YGSRS were added to yield final peptide
concentrations of 1–10 000 ng/ml (1.56–15 600 nM), respectively. The cell suspensions were mixed, incubated for 72 h andthen the proliferated cells were analyzed with WST assay
using Cell Counting Kit-8 as described earlier.
Cytokine analysis
Peripheral blood mononuclear cells were incubated for 96 h in
the presence of concanavalin A and the peptide YGSRS as
described earlier, and the culture supernatant was stored at
80 C until measurement of cytokine concentrations. The
concentrations of interferon-g (IFN-g), tumor necrosis factor(TNF) a, interleukin (IL)-2, IL-4, IL-6, IL-10 and IL-17 in
the supernatant of culture medium were measured with beadsarray procedures using Human Th1/Th2/Th17 Cytokine Kit,
followed by flow cytometry16, according to the instructions of
BD Biosciences (San Jose, CA).
CD4+ CD25+ Foxp3+ Treg cell analysis
The analysis was carried out according to the manufacturer’s
instructions (BD Biosciences), which has been reported in ourprevious study17. In brief, PBMCs treated with the peptide
YGSRS in the presence of concanavalin A were washed twicewith phosphate-buffered saline (PBS) and re-suspended with
PBS at a cell density of 1  106 cells/ml. To 1 ml of this cellsuspension, 20 ml of Human CD4 FITC and 20 ml of Human
CD25 APC (BD Biosciences) were added. Twenty microlitersof FITC Mouse IgG1 k Isotype control and 20 ml of APC
Mouse IgG1 k Isotype control (BD Biosciences) were added
to the control cells. These cell suspensions were incubated for
20 min in the dark. After incubation, the cells were washedwith PBS and resuspended with 1 ml of PBS. Then, 1 ml of
10-fold diluted Human Foxp3 Buffer A (BD Biosciences)with distilled water was added to the cell suspension, andincubated for 10 min in the dark. The cells were washed with
PBS, re-suspended with 0.5 ml of Human Foxp3 Buffer B(BD Biosciences) diluted with the 10-fold diluted Human
Foxp3 Buffer A, and incubated for 30 min in the dark.

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Y. Nakamura et al.

After incubation, 1 ml of PBS was added and centrifuged for
5 min. The supernatant was removed and the cells were
washed with PBS. Then, 20 ml of Human Foxp3 PE or 20 ml of
PE Mouse IgG1 k Isotype control for the control wells wereadded, and the cell suspension was incubated for 30 min in thedark. After washing the cells with PBS, the cells were
re-suspended with 0.6 ml of PBS, and then analyzed with flow
cytometry.
The data were analyzed with a FACSCan ÔII (BD
Biosciences), using CellQuest software (Becton Dickinson,
Franklin Lakes, NJ)18. CD4+ cells in the lymphocyte fraction
were gated, and the percentages of CD4+ CD25+ Foxp3+
cells, as Treg cells, in the CD4+ cell fraction were calculated.
Statistics
Differences in the percentages of PBMC-proliferation,amounts of cytokines produced in the culture medium, andpercentages of Treg cells in the CD4+ cell fraction after cell
culture in the presence of serial concentrations of the peptide
YGSRS were analyzed with Bonnferroni tests. The correlation coefficients between the concentrations of any two kindsof cytokines in PBMC culture medium were analyzed with
the Spearman test. These analyses were performed with
GraphPad PRISM 4.0 (GraphPad Software Inc., San Diego,
CA). In each case, two-sided p values 50.05 were considered
to be significant.

Immunopharmacol Immunotoxicol, 2015; 37(3): 318–323

Results
Effects of the pentapeptide YGSRS on proliferationof mitogen-activated PBMCs
We first examined the effects of the pentapeptide YGSRS oncell proliferation of human PBMCs activated with a T-cell
mitogen, concanavalin A, in vitro. The PBMCs were cultured
in the presence of concanavalin A and serial concentrations of
YGSRS for 96 h, and the proliferative response of PBMCs
was estimated by WST assay procedures (Figure 1). YGSRS
appeared to modulate the mitogen-activated proliferation of
PBMCs, but the effects showed wide individual differences
between subjects, and no statistically significant change wasobserved by 1–10 000 ng/ml (1.56–15 600 nM) YGSRS(Figure 1A). Immunosuppressive drugs cyclosporine andprednisolone, used as positive controls, dose-dependently
inhibited the concanavalin A-stimulated proliferation of
PBMCs (Figure 1B).
YGSRS also showed no effects on the in vitro growth of cellsof a human breast cancer cell line MCF-7 at a concentration
range of 1–10 000 ng/ml (1.56–15 600 nM) (Figure 1C).
Effects of YGSRS on cytokine production frommitogen-activated PBMCs
The effects of YGSRS on the production of Th1/Th2/Th17
cytokines from the T-cell mitogen-activated PBMCs were

Figure 1. Effects of YGSRS on proliferation of human PBMCs activated with concanavalin A and cells of a breast cancer cell line MCF-7 in vitro. (A)PBMCs were cultured with concanavalin A and serial concentrations of YGSRS for 96 h, and the cell proliferation was analyzed as escribed in‘‘Materials and methods’’ section. Dose–response curves of the peptide on the proliferation of PBMCs obtained from eight individual healthy subjects
were expressed. Statistically significant changes by YGSRS, as compared to control (0 ng/ml), were not observed. (B) Effects of cyclosporine (opencircle) and prednisolone (closed circle) on the proliferation of human PBMCs activated with concanavalin A. Typical data of each drug were shown. (C)Dose–response curve of MCF-7 cell proliferation in the presence of YGSRS. The mean (SD) of five independent experiments were shown. Statisticallysignificant changes by YGSRS, as compared to control (0 ng/ml), were not observed.

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DOI: 10.3109/08923973.2015.1038752

Pentapeptide YGSRS on human peripheral-blood mononuclear cells

321

Figure 2. Cytokine concentrations in the supernatant of T-cell mitogen-activated PBMCs treated with YGSRS. PBMCs were stimulated withconcanavalin A for 96 h in the absence (0 ng/ml) or presence (1–10 000 ng/ml) (1.56–15 600 nM) of YGSRS. Then, the culture supernatant was
analyzed for concentrations of IFN-g, TNF-a, IL-2, IL-4, IL-6, IL-10 and IL-17 (n ¼ 4 for each) using beads-array procedures followed by flow
cytometry. *p50.05 and **p50.01, as compared to each control.

examined (Figure 2). YGSRS significantly increased theconcentration of IL-6 and IL-10 in the medium of themitogen-activated PBMC culture at 10–1000 ng/ml(15.6–1560 nM) (p50.05 or p50.01). YGSRS also appeared
to affect the production of INFg, TNF-a, IL-4 and IL-17A
from the activated PBMCs, but the changes were notstatistically significant.
The concentrations of IL-6 and IL-10 in the PBMC culture
medium stimulated with concanavalin A in the presence ofseveral concentrations of YGSRS were significantly correlated each other, as can be seen in Figure 3. Whereas, nosignificant correlation was observed between concentrationsof any other two types of cytokines produced in the presenceof YGSRS (r ¼ 0.6167, p ¼ 0.0004).
Effects of YGSRS on the frequency of CD4+ CD25+
Foxp3+ Treg cells in mitogen-activated PBMCs
The percentages of CD4+ CD25+ Foxp3+ Treg cells in
mitogen-activated PBMCs cultured in the presence or absenceof YGSRS for 96 h were analyzed with flow cytometry. CD4+
cells in the lymphocyte fraction of the T-cell mitogenactivated PBMCs were gated, and the percentages of CD25+
Foxp3+ cells in the CD4+ cell fraction (Treg cells) were
calculated (Figure 4). YGSRS at concentrations >1 ng/ml
tended to decrease the percentages of CD4+ CD25+ Foxp3+
Treg cells in CD4+ cells in lymphocytes, but the changes

were not statistically significant (Figure 4). We also examine
the relationship between mitogen-stimulated PBMC proliferation and the ratios of CD4+ CD25+ Foxp3+ Treg
cells treated by YGSRS, but there was no significant
correlation (Figure 5).

Discussion
Several types of naturally occurring or synthetic oligopeptides
and peptides have been reported to show immunomodulatory
and anti-inflammatory activities1–4. Clinically used immunosuppressive drug cyclosporine has a cyclic peptide structure7.
We have also reported that gramicidin D, which contains
linear pentadecapeptides, shows immunosuppressive efficacy
in rat heterotopic heart transplantation model8. The present
study was undertaken to examine the immunomodulatory
efficacies of a linear pantapeptide YGSRS in vitro using
PBMCs obtained from healthy subjects. Although precise
underlying mechanism remains to be elucidated, our datasuggest that YGSRS affects the production of several types ofcytokines from activated human peripheral T cells, which
may modulate Th2 type immunity.
Helper T (Th) cells play critical roles in orchestrating
adaptive immune responses. They exert such functions mainly
through secreting cytokines and chemokines that activate and/or recruit target cells. An imbalance between Th1 and Th2
cytokine production has been shown to play a key role in theImmunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by Tokyo Yakka Daigaku on 06/29/15
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Y. Nakamura et al.

Immunopharmacol Immunotoxicol, 2015; 37(3): 318–323

Figure 3. Correlation between concentrations of IL-6 and IL-10 in the
PBMC culture medium stimulated with concanavalin A in the presenceof several concentrations (1–10 000 ng/ml) (1.56–15 600 nM) of YGSRS.
A statistically significant positive correlation was observed between thetwo indices (r ¼ 0.6167, p ¼ 0.0004).

Figure 5. Relationship between the PBMC proliferative indices (%of control: see Figure 1) and CD4+ CD25+ Foxp3+ cell ratios(Figure 4) under stimulation by concanavalin A in the presence ofseveral concentrations (1–10 000 ng/ml) (1.56–15 600 nM) of YGSRS.
Statistically significant correlation was not observed.

Figure 4. Flow cytometric analysis of CD4+ CD25+ Foxp3+ cells (Tregcells) in concanavalin A-stimulated PBMCs treated with YGSRS. The
mean percentages of CD4+ CD25+ Foxp3+ Treg cells in concanavalin
A-stimulated PBMCs cultured in the presence or absence of serial
concentrations (1–10 000 ng/ml) (1.56–15 600 nM) of YGSRS for 96 h
were shown. The data were indicated as the mean (SD) percentages of
six independent experiments using PBMCs obtained from six individual
healthy subjects. Statistically significant changes by YGSRS, as
compared to control (0 ng/ml), were not observed.

affected function of helper T cells in PBMCs. Cyclosporine
is known to inhibit production of several cytokines including
IL-2 from human PBMCs activated by T-cell mitogen8, andthis agent suppressed the concanavalin A-induced PBMC
proliferation at concentrations 510 ng/ml dose-dependently inthe present assay system (Figure 1B). As described earlier, weobserved a promotive effect of YGSRS on activated PBMC
proliferation, as well as an enhancement of secretion of IFNg, TNF-a, IL-4 and IL-17A, up to 1000 ng/ml (1560 nM).
Although these effects of the peptide were not statisticallysignificant, the data suggest immune enhancing efficacies of
YGSRS. These effects are possibly implicated in anticancer
effects of this peptide by modulating tumor immunity, and arethus worth pursuing of this peptide in future studies.
CD4+ Th cells can be induced to differentiate toward Treg
and Th17 phenotype according to the local cytokine milieu23.
Treg provide a substantial component of the autoimmune
counterbalance, and the thymus plays a role in Treg development and induction of tolerance24. Treg cells are reported
to have a suppressive role on cellular immunity9, andtherefore, Treg cell frequency in patients with autoimmune
or malignant diseases appears to be critical for the clinicaloutcome of these patients. However, YGSRS did not significantly change the relative percentages of Treg cells in CD4+
cells in peripheral lymphocytes (Figure 4). Moreover, therelative percentages of Treg cells in CD4+ cells did notcorrelate with the proliferation indices of mitogen-stimulated
PBMCs in the presence of serial concentrations of YGSRS(Figure 5). We detected Treg cells in the peripheral-blood
samples of healthy subjects as CD4+ CD25+ Foxp3+
lymphocytes in the present study, since Foxp3 has beenreported to define identity and function of Treg in theselymphocytes as described earlier25. Thus, we thought that theCD4+ CD25+ Foxp3+ lymphocytes we detected in thepresent study have typical Treg features and functions. The
increase in the prototypical Treg cytokine IL-10 by treatment
with YGSRS may relate to increased frequency of Treg, but
we did not observe such correlation in the present study as

induction and development of autoimmune diseases19–22. The
pentapeptide YGSRS was isolated from coffee bean and its
chemical structure had been identified, whereas its biologicaland pharmacological activities have little been investigated sofar. The present data suggest that YGSRS promoted, rather
than suppressed, many types of Th1/Th2/Th17 cytokines,
especially IL-6 and IL-10. Although individual differenceswere observed between the PBMC donors in the effects of
YGSRS on the proliferation of T-cell mitogen-activated
PBMCs, the effects of YGSRS appeared to be also promotive,
rather than suppressive, on the PBMC proliferation. Theseobservations suggest that the modulatory effects of YGSRS
on cytokine production are related to the effects of the peptide
on T-cell proliferation. Thus, the data suggest that YGSRSDOI: 10.3109/08923973.2015.1038752

Pentapeptide YGSRS on human peripheral-blood mononuclear cells

described earlier. Thus, we suggest that the immunomodulation by YGSRS is independent of Treg frequency in PBMCs.
Our present study, in summary, raises the possibility thatthe pentapeptide YGSRS affects the production of several
types of cytokines from T-cell mitogen-activated humanperipheral lymphocytes, which may modulate Th2 type
immunity.

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Conclusions
This study was conducted to examine the effects of thepentapeptide YGSRS on the proliferation, cytokine production and CD4+ CD25+ Foxp3+ Treg cell-frequency inhuman PBMCs activated by T-cell mitogen in vitro. YGSRS
did not significantly affect the mitogen-activated proliferationof PBMCs, though the effects were somewhat promotive.
YGSRS at a concentration range of 1–1000 ng/ml (1.56–
1560 nM) tended to promote the production of Th1/Th2/Th17
cytokines from the activated PBMCs, and the effects on IL-6
and IL-10 production were statistically significant (p50.05).
YGSRS did not significantly affect Treg cell-frequency in theactivated PBMCs. These observations suggest that the
pentapeptide YGSRS affects the production of several typesof cytokines from activated human peripheral T cells, which
may modulate Th2 type immunity.

Acknowledgements
We thank Dr Henry Aoki, President at YGSRS Co., Acton,
MA, USA, for providing the pentapeptide YGSRS.

Declaration of interest
The authors declare that there are no conflicts of interest in
this study.

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