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Biological and Pharmaceutical Bulletin Advance Publication by J-STAGE DOI:10.1248/bpb.b15-00331
Ⓒ 2015 The Pharmaceutical Society of Japan
Advance Publication September 4, 2015
Biol. Pharm. Bull. Regular Article
Epigallocatechin-3-gallate (EGCG) suppresses the trafficking of lymphocytes to epidermal
melanocytes via inhibition of JAK2: Its implication for vitiligo treatment
Weixuan Ninga,Suiquan Wang
b,Xiaowu Dongc, Dongyin Liu
b, Lifang Fu
b, Rong Jin
b,
Aie Xub,
*
Affiliation
aDepartment of Dermatology, Guangxing Affiliated Hospital of Zhejiang Chinese Medical University,
Hangzhou, Zhejiang 310007, P.R. China bDepartment of Dermatology, Third People’s Hospital of Hangzhou, Hangzhou 310009, P.R. China
cZJU-ENS Joint Laboratory of Medicinal Chemistry, Zhejiang Province Key Laboratory of Anti-
Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou,
310058, P. R. China
*Correspondence
Dr. Aie Xu, Department of Dermatology, Third People’s Hospital of Hangzhou, Hangzhou 310009,
P.R. China. E-Mail: [email protected]; Tel.: +86 571 87827535; Fax: +86 571 87814481.
Abstract
Vitiligo is an inflammatory skin disorder in which activated T cells play an important role in
its onset and progression. Epigallocatechin-3-gallate (EGCG), the major chemical
constituent of green tea, exhibits remarkable anti-oxidative and anti-inflammatory properties.
EGCG administration has been confirmed to decrease the risk of vitiligo; however, the
underlying mechanism is undetermined. In this study, we proved that EGCG directly
inhibited the kinase activity of Janus kinase 2 (JAK2). In primary cultured human
melanocytes, EGCG pre-treatment attenuated IFN-γ-induced phosphorylation of JAK2 and
its downstream STAT1 and STAT3 in a dose-dependent manner. We further examined the
chemoattractant expression in melanocytes and demonstrated that EGCG significantly
inhibited IFN-γ-induced expression of ICAM-1, CXCL10, and MCP-1 in human
melanocytes. In addition, EGCG reduced the protein levels of the corresponding receptors
including CD11a, CXCR3, and CCR2 in human T lymphocytes. As a consequence, adhesion
of human T cells to melanocytes induced by IFN-γ was effectively suppressed by EGCG.
Taken together, our results provided new evidence for the effectiveness of EGCG in vitiligo
treatment and supported JAK2 as a molecular target for vitiligo medicine development.
Key words
vitiligo; Epigallocatechin-3-gallate; melanocyte; Janus kinase 2; chemoattractant
Biological and Pharmaceutical Bulletin Advance Publication
Introduction
Vitiligo is a depigmentation disorder caused by the destruction of epidermal melanocytes [1]. The exact
pathophysiological mechanism of vitiligo remains elusive. However, several hypotheses, including
autoimmune, oxidant-antioxidant, genetic susceptibility, neural and viral mechanisms, have been
proposed to explain the selective destruction of melanocytes. Currently, clinical and bench findings
suggest the major role of autoimmune factors in the progress of vitiligo. Inflammatory cells, mostly T
lymphocytes, have been identified close to vitiligous skin lesion [2, 3]. Lesional CD8+ T cells specially
induce melanocyte apoptosis in unaffected skin ex vivo, and the frequency of anti-melanocyte CD8+ T
cells in both the blood and skin of vitiligo patients correlates with the severity of disease [4, 5]. Moreover,
immunosuppressive therapies targeting T-cell activation have been shown to be effective for vitiligo
treatment [6, 7]. These evidences all support a direct role for T lymphocytes in melanocyte destruction
in human vitiligo.
EGCG, a major catechin in green tea, is considered beneficial for human health, especially as ananti-
oxidative agent[8]. In addition, EGCG was confirmed to directly inhibit protein kinases by working as
an ATP analog [9]. Previously, we reported the therapeutic effect of EGCG in vitiligo induced by
monobenzone in mice [10]. Our results suggested that it could contribute to the suppression of CD8+ T
cell migration and the inflammatory cytokine expression. However, the mechanism regarding the EGCG
regulation of immune responses in vitiligo is still unclear.
The aim of study is to elucidate the inhibitory effects of EGCG on inflammatory signaling pathways and
to identify the target of EGCG inhibition.
Results and Discussion
EGCG inhibited Jak2 kinase activity in vitro
The JAKs are a family of four non-receptor tyrosine kinases including JAK1, JAK2 JAK3 and TYK2.
Recruitment of stimuli to cell surface receptors activates JAKs which, in turn, phosphorylates and
Biological and Pharmaceutical Bulletin Advance Publication
stimulates latent cytoplasmic STAT proteins to an active dimer, leading to nuclear translocation and
DNA binding and subsequently modulating gene transcription [11]. The JAK/STAT signal pathway
controls a number of important biological responses, including immune functions, cellular growth,
cellular differentiation and hematopoiesis. Dysregulation of JAK signaling has been identified in
multiple autoimmune disorders [12]. Due to their ability to selectively modulate immune function,
targeted JAK inhibitors are attractive candidates for some skin diseases such as psoriasis and atopic
dermatitis [13-15].
Our computer simulation result suggested that EGCG may bind to JAK2 protein (Fig. 1C). To determine
the effect of EGCG binding on JAK2 kinase activity, EGCG and its derivative, peracetylated EGCG
(AcEGCG, Fig. 1A) were tested by Caliper mobility shift assay, along with the pan-kinase inhibitor
Staurosporine (STS) as the positive control. The results showed that EGCG inhibited the kinase activity
of JAK2 with the IC50 at 18.85nM. While, peracetylated EGCG with the all the hydroxyl groups
substituted showed much higher IC50 for JAK2 inhibition (2126nM) (Fig. 1B). These results indicated
that EGCG could bind to JAK2 and directly inhibited its kinase activity.
EGCG inhibited IFN-γ-induced phosphorylation of JAK2, STAT1 and STAT3 in human melanocytes
Various cytokines elicit their biological effects through the activation of JAK-STAT pathways [16, 17].
To characterize the inhibition of JAK2 activity in melanocytes, we examined the cellular
phosphorylation levels of JAK1, JAK2, STAT1 and STAT3 in human melanocytes after treatment with
IFN-γ and EGCG. It was shown that while IFN-γ increased the phosphorylation of JAK2, STAT1 and
STAT3, EGCG suppressed the upregulation of protein phosphorylation in response to IFN-γ in a dose-
dependent manner (Fig. 2). In terms of JAK1, its phosphorylation level wasn’t changed upon IFN-γ
stimulation, but slightly increased after EGCG treatment. It is then suggested that EGCG specifically
targets JAK2 in JAK-STAT pathway.
Various studies have supported a critical role of IFN-γ in vitiligo development [4, 18, 19]. In vitiligo
patients and animal models, IFN-γ level is significantly elevated in lesional skin [4,20]. IFN-γ signaling
regulates the expression of cytokine as well as adhesion molecules facilitating the lymphocyte migration
Biological and Pharmaceutical Bulletin Advance Publication
[16, 24]. So that, local IFN-γ release promotes further recruitment of melanocyte-specific CD8+ T cells
into the vitiligo lesion [21]. Moreover, our previous study proved that IFN-γ directly caused apoptosis
and senescence in melanocytes, and induced melanocyte to secret autoimmune enhancers including IL-
6 and HSP70 [18]. Since IFN-γ mainly engages JAK-STAT pathways to achieve its biological effects,
blocking IFN-γ signaling via the inhibition of JAK2 activity could be a promising therapeutic approach
to prevent vitiligo development.
EGCG inhibited the production of chemoattractants
Increased level of chemoattractants including ICAM-1 and CXCL10 has been found in the skin lesions
of vitiligo patients. [2, 22-24]. To further address the effects of EGCG on the expression of
chemoattractants, we measured the expression levels of ICAM-1 and CXCL10, as well as MCP-1 which
is also implicated in the pathogenesis of certain autoimmune diseases [25, 26]. The results showed that
IFN-γ stimulation significantly increased the mRNA expression and protein secretion of ICAM-1,
CXCL10, and MCP-1. Compared with the IFN-γ stimulation group, EGCG pretreatment significantly
suppressed the IFN-γ-induced production of ICAM-1, CXCL10, and MCP-1 in a dose-dependent
manner. As a positive control, the JAK2-specific kinase inhibitor AG490 also inhibited the induction of
ICAM-1, CXCL10 and MCP-1 by IFN-γ (Fig.3).
Chemoattractants are involved in the attraction and activation of leukocytes during the inflammatory
process. ICAM-1, which can be expressed on the cellular membrane or secreted into extracellular matrix,
is a ligand for leukocyte adhesion protein LFA-1. ICAM-1 promotes the leukocyte trans-endothelial
migration as well as the localization of leukocyte to target cells [24, 23]. MCP-1 was also suggested to
mediate lymphocyte migration [27]. CXCL10 engagement is responsible for the stimulation of
monocytes and the migration of NK and T-lymphocytes. CXCL10 was elevated in both vitiligo patient
skin and serum. Due to the critical roles of these molecules in the skin homing of leukocytes, it is
reasonable to presume them as targets for vitiligo treatment. In fact, application of CXCL10-neutralizing
Biological and Pharmaceutical Bulletin Advance Publication
antibody has been proven to remarkably decrease the development of depigmentation in a mouse model
of vitiligo [24].
It is notable that the expression of T cell chemoattractants is also controlled by other cytokines. For
example, IL-6 could create a MCP-1-gradient, and increase the expression of ICAM-1, functional to
inflammatory cells chemotaxis [28-31]. As we have mentioned above, many cytokines elicit their
biological effects through the activation of JAK-STAT pathways including IL-6 [28]. JAK2 inhibition
by EGCG treatment simultaneously blocks the signaling from various cytokines and maximizes the
effect on the blockage of chemoattractant expression. On the other side, the inhibition of chemoattractant
expression by EGCG was more potent than that by AG490 treatment, suggesting that EGCG may work
via other mechanism besides targeting JAK2 to achieve that effect.
EGCG inhibited the production of chemoattractant receoptors in T lymphocytes
CXCR3 and CCR2 are the receptors to CXCL10 and MCP-1, respectively. CD11a is one of the
molecules that form LFA-1 which is the receptor of ICAM-1. Levels of CXCR3 and CD11a were
elevated on autoreactive lymphocytes in both human vitiligo patients and vitiligo models. In addition,
CXCR3 was demonstrated to be essential for autoreactive T cell accumulation in the skin and subsequent
depigmentation in a mouse model, implicating this chemokine pathway as functionally required for
vitiligo [24, 32]. We then evaluated the effect of EGCG on the expression of CD11a, CXCR3, and CCR2
in activated human T cells. Western blot analysis revealed that EGCG treatment for 24 h decreased the
expression of CD11a, CXCR3, and CCR2 in Jurkat, a CD4+T leukemia cell line, in a dose-dependent
manner (Fig. 4A). In purified CD8+T cells from PBMC, EGCG inhibited the expression of those
receptors as well (Fig. 4B). Comparatively, AG490 also inhibited the expression of CD11a, CXCR3,
and CCR2, suggesting that the inhibiting effect of EGCG on chemoattractant receptor induction was at
least partially through the inhibition of JAK2 activity.
Suppression of T lymphocyte adhesion to melanocytes by EGCG treatment
Biological and Pharmaceutical Bulletin Advance Publication
Given the importance of CXCL10/CCR3 and ICAM-1/LFA-1 for lymphocyte migration and adhesion,
we further examined the effects of EGCG on the adhesion of T lymphocytes to melanocytes. In our study,
treatment of human melanocytes monolayers for 24 h with 200U/mL IFN-γ resulted in a 5.7-fold (Jurkat
cells, Fig. 5A) and 6.3-fold (CD8+T cells, Fig. 5B) increase of T lymphocyte adhesion. In comparison,
IFN-γ-induced T cell adhesion was strongly suppressed when both T cells and melanocytes were pre-
treated with 40 µM EGCG (Fig. 5A and 5B, the far right column). Furthermore, EGCG treatment of T
cells or melanocytes alone also decreased the adhesion between T lymphocytes and melanocytes, which
is consistent with the above results showing that the expression of chemoattractants and their receptors
were all inhibited by EGCG,.
Adhesion to target cells is the initial and necessary step for T lymphocytes to exert their functions.
Adhesion molecules such as ICAM-1 and its receptors LFA-1 are crucial for immune synapse formation,
antigen presentation [33] and lymphocyte killing [34, 35]. It has been reported that adhesion defect in
CD8+T lymphocytes, which is caused by a combination of decreased cell surface levels of adhesion
molecules, deficient LFA-1 activation, and the failure to recruit essential adhesion receptors to the
membrane contact site formed with cognate target cells, weakens their capacity of cytolysis [36].
Therefore, EGCG-caused compromise of interaction between T lymphocytes and melanocytes will
profoundly weaken the T lymphocyte activation and decrease the cytolysis of melanocytes by CD8+CTL.
Collectively, EGCG inhibited the chemoattractant expression in melanocytes and the expression of the
corresponding receptors in T lymphocytes via directly targeting JAK2 kinase, leading to the decrease of
lymphocyte migration/accumulation in vitiligo lesion as well as the weakening of T cell adhesion to
melanocytes. The overall effects will be less activation of auto-reactive T lymphocytes and less
destruction of melanocytes. Our study provides insights into the effects of EGCG on inflammation in
vitiligo and elucidates JAK2 as its novel anti-inflammatory target. Further investigations should be
carried out to further clarify the JAK2 as an independent anti-inflammatory drug target in vitiligo therapy.
Materials and methods
Reagents
Biological and Pharmaceutical Bulletin Advance Publication
EGCG, DMSO, EDTA, Phorbol 12-Myristate 13-Acetate (PMA), Ionomycin, Human recombinant IFN-
γ and IL-2 were obtained from Sigma (St. Louis, USA). F12 medium, RPMI-1640 medium, penicillin,
streptomycin, L-gultamine, and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific
(Carlsbad, CA, USA). Ficoll-Paque was obtained from GE Healthcare (Little Chalfont, UK). Human
CD8+ T Cell Isolation Kit was purchased from MiltenyiBiotec (Cologne, Germany). All antibodies for
Western blotting were purchased from Cell Signaling Technology (Danvers, MA, USA).
Computer analysis of JAK2-EGCG binding
The molecular docking was performed by using LigandFit module embedded in Discovery Studio 2.5.29
At first, the crystal structure of JAK-2 was obtained from PDB bank (entry code: 4GMY), and then was
removed water molecules and charged by CHARMm force field. The binding site was derived from the
volume of co-crystal ligand. For generation of the ligands’ conformations, variable numbers of Monte
Carlo simulations were employed. All the calculations during the docking steps were performed under
the PLP energy grid. A short rigid body minimization was then performed and 50 preferable poses were
saved according to their dock score. Based on the dock score and visual inspection, the most possible
pose was selected for the further analysis.
In vitro kinase assays
Inhibition of EGCG and peracetylated EGCG (AcEGCG) on JAK2 kinase activity was tested in
Shanghai Chempartner Co., LTD (Shanghai, China). EGCG and AcEGCG were tested in duplicate for
10 concentrations. Samples were analyzed by Caliper mobility shift assay using LabChip 3000 (Caliper
Life Sciences, MA, USA). Data was analyzed using HTS Well Analyzer Software from Caliper Life
Sciences to determine IC50.
Cell isolation and culture
Methods for the isolation and cultivation of primary normal melanocytes were described previously [37].
Briefly, melanocytes were isolated from human foreskin specimens obtained after circumcision surgery.
Biological and Pharmaceutical Bulletin Advance Publication
Cells were cultured in Hu16 medium (F12 supplemented with 10% fetal bovine serum (FBS), 20 ng/ml
bFGF and 20 mg/ml IBMX) and cultured at 37 ℃ in an incubator with 5% CO2 and 95% humidity. The
cells were used between passages 2 and 5. Melanocytes were seeded at a density of 1×104 cells per well
into 96-well plates, or at a density of 1×105 cells per well into 24 -well plates, or at a density of 3×105
cells per well into 6-well plates and incubated overnight before experiments.
Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using Ficoll-Paque
density centrifugation. CD8+T cells were then separated from PBMC using Human CD8+ T Cell
Isolation Kit according to the protocol from manufacturer. Both purified CD8+T cells and the human T
lymphoma cell line Jurkat cells were cultured in RPMI-1640 medium supplemented with 100 units/mL
streptomycin, 100 units/mL penicillin, 2 mM gultamine, 1 mM sodium pyruvate, 10 mM nonessential
amino acids, and 10% FBS. CD8+T cells or Jurkat cells were stimulated for 48 h with PMA (25 ng/mL)
and Ionomycin (1 µM) in the presence IL-2 (10U/mL) before each experiment.
RNA Isolation and quantitative PCR
Total RNA was extracted from melanocytes with SV total RNA purification kit (Promega, Shanghai,
China). Reverse transcript reaction was performed using QuantiTect Reverse Transcription Kit (Qiagen,
Germany). Real time PCR was performed using QuantiFast SYBR Green PCR Kit (Qiagen, Germany).
The expression levels of each gene was normalized against β-actin using the comparative Ct method,
and expressed as percentage of IFN-γ group, with the IFN-γ group as 1. primer sequences were as
follows: CXCL10: forward primer 5’-GTGGCATTCAAGGAGTACCTC-3’, reverse primer 5’-
TGATGGCCTTCGATTCTGGATT-3’; ICAM-1: forward primer 5’-TATGGCAACGACTCCTTC T-
3’, reverse primer 5’-CATTCAGCG TCACCTTGG-3’; MCP-1:forward primer 5’-
TGTCCCAAAGAAGCTGTAGTATTTGT-3’, reverse primer 5’-
TTCTGATCTACTTGGTTCTGGTC-3’; β-actin: forward primer 5’-
ATAGCACAGCCTGGATAGCAACGTAC-3’, reverse primer 5’-
CACCTTCTACAATGAGCTGCGTGTG-3’.
Biological and Pharmaceutical Bulletin Advance Publication
Enzyme-linked immunosorbant assay (ELISA)
Melanocytes were plated into six-well plates and incubated in 37℃ in a humidified incubator at an
atmosphere containing 5% CO2. After being washed with warm PBS, melanocytes were pre-treated with
increasing concentrations of EGCG (0, 10, 20, and 40 µM) and 25 µM AG-490 in Hu16 medium for 1
h, followed by IFN-γ (200 U/ml) stimulation for 48 h. The cell supernatants were then collected, and the
expression levels intercellular adhesion molecule-1 (ICAM-1), monocyte chemotactic protein-1 (MCP-
1), and chemokines CXCL10 were analyzed by ELISA kits (R&D Systems, Minneapolis, USA)
according to the manufacturer’s instructions.
Western blot analysis
Cells were collected and lysed in RIPA buffer (10 mM NaPO4, pH 7.4, 300 mMNaCl, 0.1% SDS, 1%
Nonidet P-40, 1% deoxycholic acid and 2 mM EDTA) with protease inhibitors (Pierce, USA). Aliquots
of 20-30μg of protein from each sample (treated as indicated in the legends) were separated by 8-10%
SDS-PAGE and transferred onto a PVDF membrane (Millipore, Bedford, MA). After blocked with 5%
BSA (Beyotime, China) for 1 h, membrane was incubated with specified antibodies overnight at 4°C
followed by incubation with fluorescent dye-labeled secondary antibodies for 1 h at room temperature
in the dark. The protein immuno-complex was visualized by an Odyssey Infrared Imaging System (LI-
COR, USA). Quantification of band intensity was performed using the quantification software of the
digital imaging system (ChemiDoc™ XRS+, Bio-Rad, USA).
Adhesion assay
Melanocytes were grown to confluence in six-well plates and incubated with or without IFN-γ
(200U/mL) for 24 h. Human T cells (Jurkat cells or purified CD8+T cells) were labeled with 5 µM of
DiO, a fluorescent dye, at 37 ℃, for 5 minutes, and subsequently washed twice by PBS. Labeled T cells
(2×106 cells/well) were then incubated with melanocytes in six-well plates at 37 ℃ for 30 min. Non-
Biological and Pharmaceutical Bulletin Advance Publication
adherent T cells were removed, and plates were gently washed twice with PBS. The numbers of adherent
T cells were counted by four fields per 200×high-power field well using fluorescence microscope.
Statistical analysis
All experiments were performed at least three times in triplicate, with one representative experiment
shown. All experimental data obtained from cultured cells were expressed as mean ± SD. One-way
ANOVA, followed by Student-Newman-Keuls multiple comparison tests, was used to analyze the
statistical significance between groups. P values < 0.05 were considered statistically significant.
Biological and Pharmaceutical Bulletin Advance Publication
Acknowledgments
This study was supported by the Natural Science Foundation of Zhejiang, China (Grant no. Y2111310
and no. LY13H110001), the National Natural Science Foundation of China (grant no. 81271758 and no.
81472887), the Key Scientific Innovation Program of Hangzhou (20122513A02), and the National Key
Clinical Specialty Construction Project of China.
Biological and Pharmaceutical Bulletin Advance Publication
Conflicts of Interest
The authors declare no conflict of interest.
Biological and Pharmaceutical Bulletin Advance Publication
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Figure Legends
Fig. 1 Inhibition of EGCG on Jak2 in vitro
(A) Molecular structures of EGCG and its derivative AcEGCG. (B) IC50 profiling results of EGCG,
AcEGCG and pan-kinase inhibitor staurosporine (STS) for JAK2 kinase inhibition. (C) Interaction mode
between EGCG and the binding pocket of JAK2.
Fig. 2 EGCG inhibited IFN-γ-induced phosphorylation of JAK2/STATs in human melanocytes
The primary human melanocytes were exposed to EGCG at the concentration of 0, 10, 20, 40 µM for
1h. Then, IFN-γ was then administered in a concentration of 200 U/ml for 1h. Whole cell lysates were
subjected to SDS-PAGE and analyzed by western blot with corresponding antibodies.
Fig.3 EGCG dose-dependently inhibited ICAM-1, CXCL10, and MCP-1 production in IFN-γ-stimulated
melanocytes
Primary human melanocytes were pre-treated with various concentrations of EGCG (0, 10, 20, and 40
μM) or JAK2 specific inhibitor AG490 (25 μM) for 1 h before incubated with 200U/mL of IFN-γ. Total
RNA was extracted from melanocytes at 24 h after IFN-γ treatment, and qRT-PCR was performed to
measure the mRNA expression of ICAM-1, CXCL10 and MCP-1. The expression level of each gene
was normalized against β-actin using the comparative Ct method. The level of mRNA expression was
expressed as fold-change relative to that in IFN-γ group (IFN-γ group as 1). Supernatants were collected
at 48 h after IFN-γ treatment, ICAM-1, CXCL10, and MCP-1 concentration was measured by ELISA.
The values shown represent the mean±SD of three independent experiments. * P<0.01, ** P<0.01,
*** P<0.01 vs. IFN-γ group.
Fig. 4 Effect of EGCG on CD11a, CXCR3 and CCR2 expression in human T cells
Human T cells were activated for 48 h with PMA (25 ng/mL) and Ionomycin (1 µM) in the presence IL-
2 (10U/mL) before incubation with various concentrations of EGCG or AG-490 for 24 h. Western blot
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analysis was used to detect protein expressions of CD11a, CXCR3 and CCR2. β-actin was probed as
loading control. (A) Effect of EGCG on CD11a, CXCR3 and CCR2 expression in Jurkat cells. (B) Effect
of EGCG on CD11a, CXCR3 and CCR2 expression in purified CD8+T cells from PBMC. Results from
three experiments were quantified by densitometry (bottom panels). * P<0.01, ** P<0.01, vs. control
group.
Fig. 5 EGCG blocks adhesion of human T cells to melanocytes
Primary human melanocytes (MC) were cultured with various combination of IFN-γ (200 U/mL) and
EGCG (40 µM) treatment for 24 h. Human T cells (Jurkat cells (A) or purified CD8+T cells from PBMC
(B)) were incubated without or with 40µM EGCG for 24 h, and then labeled with 5 µM of DiO before
the adhesion assay. The numbers of adherent T cells were counted by four fields per 200×high-power
field well using fluorescence microscope. The values shown represent the mean ± SD of three
independent experiments. *P<0.01 verse control group in which melanocytes (MC) were untreated by
IFN-γ. #P<0.05 verse group in which melanocytes were treated with IFN-γ alone and T cells (Jurkat or
CD8+T cells) were untreated.
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