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Increased IFN-γ+ T cells are responsible for the clinical responses of low-dose
DNA demethylating agent decitabine anti-tumor therapy
Xiang Li1, Yan Zhang
1, Meixia Chen
1, Qian Mei
1, Yang Liu
1, Kaichao Feng
1, Hejin
Jia1, Liang Dong
1, Lu Shi
1, Lin Liu
2, Jing Nie
1, *, Weidong Han
1, *
1Department of Immunology & Biological Therapy, Institute of Basic Medical
Science, PLA General Hospital, Beijing 100853, China
2Department of General Surgery, PLA General Hospital, Beijing 100853, China
* Correspondence to:
Weidong Han, Ph.D., M.D.
Phone: +86-10-6693-7463, Fax: +86-10-6693-7516,
E-mail: [email protected]
Jing Nie, Ph.D.
Phone: +86-10-6693-7917, Fax: +86-10-6693-7516,
E-mail: [email protected]
Running title: Immune-related prognosis for DAC therapy in solid tumors
Key words: decitabine; epigenetic therapy; IFN-γ; anti-tumor immunity
Abbreviations: decitabine (DAC); DNA methyltransferase (DNMT);
5-aza-2’-deoxycitidine (AZA); myelodysplastic syndrome (MDS); acute myeloid
leukemia (AML); peripheral blood mononuclear cells (PBMCs); dendritic cells (DCs);
cytotoxic T lymphocytes (CTLs); regulatory T cells (Treg); tumor draining lymph
node (TDLN); disease control rate (DCR); objective response rate (ORR);
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progression-free survival (PFS); overall survival (OS)
Grant support: The study was supported by National Key Research and
Development Program of China (No. 2016YFC1303501 and 2016YFC1303504 to
WDH), National Natural Science Foundation of China (31671338, 81472838,
81230061, 81650025,) and the Science and Technology Planning Project of Beijing
City (Z151100003915076).
Clinical trial information: NCT01799083
Disclosure of Potential Conflicts of Interest
All authors declare no potential conflict to disclose.
Statement of translational relevance
The DNA demethylating drug decitabine (DAC) has been used clinically for the
treatment of both hematological malignancies and solid tumors. However, the
previous researches mainly focused on its effects on cancer cells; whether and how
low-dose DAC treatment directly influences T cell activation and cytolysis-mediated
anti-tumor responses remain unknown. Moreover, whether the anti-tumor immune
responses modulated by DAC can predict the responses of patients is an interesting
but unclear issue. Faced with questions, we have conducted a phase I/II clinical trial at
the Chinese PLA general hospital, and demonstrated that low-dose DAC therapy
enhanced the activation, proliferation and cytolytic activity of human IFN-γ+ T cells,
which was correlated with improved therapeutic responses and survival of cancer
patients.
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Abstract
Purpose:
Low-dose DNA demethylating agent decitabine therapy is effective in a
subgroup of cancer patients. It remains largely elusive for the biomarker to predict
therapeutic response and the underlying anti-tumor mechanisms, especially the impact
on host anti-tumor immunity.
Experimental design:
The influence of low-dose decitabine on T cells was detected both in vitro and in
vivo. Moreover, a test cohort and a validation cohort of advanced solid tumor patients
with low-dose decitabine-based treatment were involved. The activation, proliferation,
polarization, and cytolysis capacity of CD3+ T cells were analyzed by FACS and
CCK8 assay. Kaplan-Meier and Cox proportional hazard regression analysis were
performed to investigate the prognostic value of enhanced T cell activity following
decitabine epigenetic therapy.
Results:
Low-dose decitabine therapy enhanced the activation and proliferation of human
IFN-γ+ T cells, promoted Th1 polarization and activity of cytotoxic T cells both in
vivo and in vitro, which in turn inhibited cancer progression and augmented the
clinical effects of patients. In clinical trials, increased IFN-γ+ T cells and increased T
cell cytotoxicity predicted improved therapeutic responses and survival in the test
cohort and validation cohort.
Conclusions:
We find that low-dose decitabine therapy promotes anti-tumor T cell responses
by promoting T cell proliferation and the increased IFN-γ+ T cells may act as a
potential prognostic biomarker for the response to decitabine-based anti-tumor
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therapy.
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Introduction
The deregulation of epigenetic modifications is one of the hallmarks of cancer,
especially for DNA methylation (1,2). DNA methyltransferase (DNMT) inhibitors,
such as 5-aza-2’-deoxycitidine (AZA) or decitabine (DAC), have clinical benefits in
both hematological malignancies and solid tumors, especially myelodysplastic
syndrome (MDS) and acute myeloid leukemia (AML) (3,4). Previously, AZA and
DAC were regarded as cytotoxic drugs, and more than 500 mg/m2/cycle high-dose
DAC was used in anti-tumor treatment, showing strong anti-proliferative effects by
inducing DNA strand breaks (5,6). However, intolerable toxicity and primarily
hematologic inhibition have greatly limited the clinical application of DNMT
inhibitors in solid tumors. Owing to knowledge of their DNA demethylation effects, a
lower dose schedule (100-135 mg/m2/cycle) was utilized, and encouraging activity
was determined in myeloid leukemia (3). An even lower dose of DAC (50-90
mg/m2/cycle) was recommended as the “optimal dose” for the treatment of solid
tumors (7-10). Notably, low-dose DAC is more effective than a highly cytotoxic dose
of DAC in both blood neoplasms and solid tumors (10-12). Investigations of
anti-tumor mechanisms of low-dose DAC therapy have previously focused mainly on
its effects on cancer cells, including the upregulation of genes associated with cell
cycle control, cytoskeletal remodeling, apoptosis, cell lineage commitment, anaerobic
glycolysis, antigen exposure, and inflammation (7,13,14). However, as critical roles
in T cell-mediated responses in anti-tumor immunity, whether low-dose DAC
treatment has a direct influence on T cell activation and cytolysis-mediated anti-tumor
responses remains unknown.
Recently, epigenetic therapy has been an important approach to treat cancer
patients. Although low-dose DAC therapy showed beneficial clinical responses in
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approximately 50% of patients with MDS, the overall response rate was 20%~30% in
refractory solid tumor patients (3). Thus, exploring the effective biomarkers to predict
responses to low-dose DAC therapy is of primary importance in clinical epigenetic
therapy for cancer. Numerous studies have sought to illuminate the alterations in a
wide range of genes and pathways following low-dose DAC therapy in both
peripheral blood mononuclear cells (PBMCs) and cancer cells, and found a set of
gene expression changes associated with the cell cycle, DNA damage, and the
immune and inflammatory responses (12,15). Although the number of these genes
may be larger in tumors from clinical responders than non-responders, to date, no
clear evidence has been established for effective biomarkers to predict clinical
responses to low-dose DAC therapy (16). Furthermore, although dose-dependent
DNA demethylation activity of DAC has been observed, the clinical effects were not
positively correlated with the doses of DAC (17). Other mechanisms besides DNA
demethylation may be responsible for the anti-tumor activity of DAC, which was
presumably due to enhancement of the anti-tumor immune response. Therefore,
investigating the immune regulation and elucidating the immune-related biomarkers
for the identification of low-dose DAC therapy responses, especially the biomarkers
in the blood of patients, are an important and promising topic that remains unresolved.
We conducted a phase I/II clinical trial at the Chinese PLA general hospital
(www.clinicaltrials.gov: NCT01799083) and reported that low-dose DAC treatment
prominently prolonged the progression-free survival of patients with advanced
refractory solid tumors (9,18). In our clinical trial, the disease control rate exceeded
70% for advanced ovarian cancer and gastrointestinal cancer, demonstrating a
significant improvement compared with first-line chemotherapy. As the effects of
low-dose DAC on anti-tumor immune responses are unknown, we aimed to examine
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the roles and mechanisms of DAC on the host anti-tumor immune response,
especially T cell activation and function, to suggest effective biomarkers, especially
immune-related biomarkers in the blood, for the identification and prediction of
responses to low-dose DAC therapy.
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Materials and methods
Patients, specimens and DAC-based therapy
This was a single-center, open-label, phase I/II study conducted at Chinese PLA
general hospital (www.clinicaltrials.gov: NCT01799083). This study was undertaken
in accordance with the principles of good clinical practice and was approved by the
appropriate institutional review boards and regulatory agencies. In this paper, there
were two cohorts of patients with solid tumors: the Test Cohort (20 solid tumor
patients) and the Validation Cohort (38 refractory/recurrent ovarian cancer patients)
from Chinese PLA general hospital (Beijing, China), who were enrolled from April 12,
2012 to April 12, 2016. Participants in this study had refractory advanced solid tumors
and included patients with ovarian cancer, lung cancer, and colorectal cancer, among
others. The detailed eligibility criteria were as previously reported in our clinical trials
(9,19). Patients were given 7 mg/m2/day decitabine via intravenous push for five days,
followed by chemotherapy regimen as they previously received, in 28-day cycles.
Baseline patient demographic and clinical characteristics were listed in
Supplementary Table S1 (Test Cohort) and Table S2 (Validation Cohort). All patients
underwent a complete medical interview and a physical examination that included a
blood profile and a CT of the disease lesions. The patients were restaged by CT every
two cycles. The clinical feasibility evaluation was performed using the Response
Evaluation Criteria in Solid Tumors (RECIST 1.1). The primary end points were
disease control rate (percentage of patients with CR+PR+SD), objective response rate
(percentage of patients with CR+PR) and progression-free survival (PFS). Secondary
evaluation included safety assessment, overall survival (OS) and biological research.
A quarterly follow-up evaluated survival until the patient either died or withdrew from
the trial by November 12, 2016, and the median follow-up time was 14 months (for
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test cohort) and 12 months (for validation cohort). Sample size was evaluated by
PASS software (α 0.05, precision 0.15, proportion 0.3).
The peripheral blood was collected before (Day 0) and after DAC treatment (Day
6) during each cycle. Normal peripheral T cells were obtained from healthy donors.
Peripheral blood mononuclear cells were isolated according to standard protocol and
immediately analyzed or frozen for posterior analysis. All donors provided written
informed consent as approved by the ethics committee of the General Hospital of
PLA, Beijing, China.
Reagents
Antibodies against CD3 (ab16669), CD4 (ab25475) and CD8 (ab22378), were
from Abcam. The following antibodies and reagents were obtained from BD
Biosciences: anti-CD3-PerCP (347334, 553067), anti-CD4-APC (340443, 553051),
anti-IFN-γ-FITC/IL-4-PE (340456), anti-IFN-γ-PE (554412), anti-IL-4-PE (554435),
anti-CD25-APC (340939), anti-CD69-APC (555533), anti-CD107a-APC (560664),
anti-Granzyme B-APC (561999), isotype-matched anti-rat IgG1 and the IntraSureTM
Kit (641776). T cell stimulation cocktail (plus protein transport inhibitors)
(00-4975-93) was purchased from eBioscience.
Isolation and treatment of T cells
T cells were isolated from PBLs from healthy donors or cancer patients. CD3+ T
cells were sorted using anti-CD3 antibody by fluorescence activated cell sorter
(FACS). For in vitro treatment, T cells were treated with PBS or 10 nM DAC (or other
doses as indicated) for 3 days in a plate pre-coated with 2 μg/ml anti-CD3 (or the
indicated concentration) and soluble IL-2 (1000 U/ml).
In vivo tumor studies
All animal experiments were undertaken in accordance with the National
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Institute of Health Guide for the Care and Use of Laboratory Animals and with the
approval of the Scientific Investigation Board of PLA General Hospital, Beijing,
China. To evaluate the anti-tumor capacity of low-dose DAC in vivo, 5×106 murine
colon carcinoma CT-26 cells were injected subcutaneously into the back region of
BALB/c mice. After approximately 10 to 12 days when the tumor became palpable,
the animals received a daily intravenous injection of 100 μl PBS or DAC (0.05 mg/kg,
0.20 mg/kg) for 5 days (n=8 per group). The diameter, in two dimensions, was
determined every 2 days. For chemical induction of hepatocarcinogenesis, male mice
at postnatal day 14 were injected intraperitoneally with 20 mg/kg DEN and then
maintained on regular chow food. After approximately 10 months since the initial
injection, the mice were injected intravenously daily with 100 μl PBS or DAC (0.05
mg/kg) for 5 days (n=8 per group). For both mouse models, tumor-infiltrating
lymphocytes (TILs) and T cells in tumor-draining lymph nodes (TDLN) and spleen
were isolated and studied by flow cytometry after 2 or 4 week of treatment. For
intracellular IFN-γ detection, TILs, TDLNs and splenocytes were treated ex vivo with
cell stimulation cocktail and stained with anti-CD3, anti-CD4 and anti-IFN-γ.
Adoptive T cell therapy
CD3+ T cells were sorted from splenocytes of BALB/c mice using anti-CD3
antibody by FACS and treated with 10 nM DAC for 72 h in vitro. PBS or
DAC-treated T cells (5×107) were intravenously transfused into CT26 colon
cancer-bearing BALB/c mice (n=8 per group). Tumor growth was monitored and
recorded. For analysis of the adoptive T cells, both the DAC-treated T cells and
control T cells were labeled with CFSE, and the CFSE-positive T cells in tumor
tissues 48 h post infusion were collected and analyzed by flow cytometry.
Statistical analysis
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Data are presented as the mean±s.d. Statistical comparisons between groups were
analyzed using the unpaired Student’s t-test, and a two-tailed p<0.05 was used to
indicate statistical significance. The median time of progression free survival (PFS)
and overall survival (OS) were calculated from the date of first decitabine treatment.
For PFS, patients who were alive without progressive disease (PD) were censored at
the start of subsequent antitumor therapy. For OS, patients who were alive through
November 12, 2016 data cutoff were censored at the time of last contact. To analyze
the correlation between T cell activity and clinical parameters, the chi-square test and
one-way ANOVA were applied using SPSS 18.0 (Chicago, IL), and the P values were
shown as indicated. Kaplan-Meier survival analysis was performed to compare patient
survival based on the peripheral T cell status, and the log-rank test was used to
generate P values with SPSS 18.0. Hazard ratios (HRs) with 95% CI were estimated
using a Cox regression model.
Detection of T cell proliferation, immunofluorescence analysis, CCK8 cytotoxicity
test for T cells and quantitative real-time PCR (qRT-PCR)
Please see Supplemental Methods
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Results
Low-dose DAC promotes the activation and proliferation of human T cells
Because the direct effects of low-dose DAC therapy on host anti-tumor immune
responses are still unknown, here we intended to investigate the impact of low-dose
DAC on anti-tumor immunity, especially T cell activation and proliferation because of
their crucial roles in anti-tumor responses. First, human peripheral CD3+ T cells from
four healthy donors were sorted and treated with 10 nM DAC because 10-300 nM in
vitro DAC treatment was commonly regarded as a low dose, and the 10 nM very low
doses of DAC produced minimal cytotoxicity in leukemia cells (13,20). First, the
expression levels of CD69 and CD25 (early and later T cell activation markers) were
examined following stimulation with anti-CD3 antibody and low-dose DAC as
indicated. We observed enhanced T cell activation following low-dose DAC treatment,
especially at early stage of T cell activation as observed significant CD69
upregulation (Figures 1A, 1B and S1A). CD69 was induced both in CD4+ and CD8+
T cells, while the alteration was more marked in CD4+ T cells (Figure 1C). The
increased phosphorylation levels of CD3-zeta, LAT and Lck in DAC-treated T cells
further confirmed the increased intracellular T cell signaling activation (Figure 1D).
We further evaluated the cell proliferation and found that low-dose DAC treatment
enhanced T cell viability (Figure 1E and 1F). Moreover, the CFSE dilution assay
analysis validated the increased proliferation of T cells following low-dose DAC
treatment (Figure S1B). In addition, cell cycle progression was also promoted by
low-dose DAC treatment (Figure S1C). These results suggested that low-dose DAC
treatment enhanced T cell activation and proliferation in vitro.
We further investigated the effects of low-dose DAC treatment on T cells in vivo.
As shown in Figure 1G and S1D, low-dose DAC (0.2 mg/kg) treatment (21)
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significantly increased CD3+ T cells in the spleen and peripheral blood of mice. In
agreement with the increased T cell frequency, the absolute number was also
increased (Figures 1H and S1E). Other immune cells were examined and were not
influenced by low-dose DAC, including CD19+ B cells, F4/80+Ly-6C+ macrophages
and CD11b+CD11c+ dendritic cells (DCs) (Figure 1G, 1H and Figure S1D, S1E).
Furthermore, after in vivo low-dose DAC treatment, the activation and viability of T
cells was enhanced, as analyzed by the CCK8 assay and CD69 expression (Figures 1I,
1J and Figures S1F, S1G). These results showed that low-dose DAC treatment
enhanced T cell activation and proliferation in vivo. However, in the human T cell
leukemia cell lines Jurkat and Molt4, 10 nM low-dose DAC treatment showed no
effect on the proliferation of these leukemia cell lines (Figures S1H-S1J), which
differed from the effects on T lymphocytes. Taken together, low-dose DAC promoted
the activation and proliferation of T lymphocytes but not T cell leukemia cells.
Low-dose DAC promotes Th1 polarization and CTL cytolytic activity
Because T cell activation was promoted by low-dose DAC treatment, we further
examined its properties. Cytokine production by these human T cells was examined,
and we observed that IFN-γ production was significantly upregulated by low-dose
DAC treatment, while IL-4 production was significantly inhibited using a real-time
PCR array (Figure 2A). The increased IFN-γ production and decreased IL-4
production were validated by flow cytometry analysis (Figures 2B and 2C). The
intracellular cytokine staining assay confirmed the increase in IFN-γ-producing T
cells following low-dose DAC treatment. Additionally, the number of
IFN-γ-producing CD8+ cytotoxic T lymphocytes (CTLs) was prominently augmented
(Figure 2D). These results indicate that low-dose DAC can promote IFN-γ-producing
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Th1 cells and CTLs. Additionally, the frequency of potential regulatory T (Treg) cells
was detected by intracellular staining of Foxp3 in CD4+CD25+ cells, and both the
frequency and total number of CD4+CD25+Foxp3+ cells were deceased following
low-dose DAC treatment (Figures 2E and 2F). These data suggest that low-dose DAC
treatment promoted Th1 polarization and CTL cytolytic activity but not Treg cell
activation.
The molecular mechanisms responsible for low-dose DAC-induced Th1/CTL
activation were then investigated. Because IFN-γ production played pivotal roles in
the Th1/CTL response and was significantly increased by low-dose DAC treatment,
we examined whether low-dose DAC-induced Th1/CTL responses were dependent on
the production of IFN-γ. As shown in Figure 2G and 2H, low-dose DAC failed to
promote human T cell activation and proliferation when IFN-γ was blocked using
neutralizing antibodies against IFN-γ. In addition, the downstream IFN-γ-stimulated
genes, such as IFI27, IFI44, IFIT3, IFITM1, PSMB9 and IRF1, were all induced by
low-dose DAC treatment in human T cells (Figure 2I). Thus, low-dose DAC-induced
Th1/CTL responses were dependent on the enhanced IFN-γ production.
The enhanced Th1 and CTL activation in response to low-dose DAC treatment
inspired us to investigate the roles of these T cells in anti-tumor immunity. When
co-cultured with human hepatocellular carcinoma SMMC7721 cells, low-dose
DAC-treated human CD3+ T cells exhibited elevated cytotoxicity compared with
control T cells (Figures 2J and S2A). Additionally, the secretion of IFN-γ and TNF-α,
and the percentage of activated CTL cells also increased in low-dose DAC-treated T
cells (Figures 2K-2M). Moreover, the increased T cell cytolytic activity induced by
low-dose DAC treatment was validated in several types of cancer cell lines as targets
(Figure S2B). Thus, low-dose DAC-treated T cells inhibited cancer progression in
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vitro.
Low-dose DAC-treated T cells inhibit cancer progression in vivo
To examine the anti-tumor capacity of low-dose DAC-treated T cells in vivo, we
used a mouse colon cancer CT26 cell tumor-bearing xenograft model. We found that
treatment with low-dose DAC inhibited colon cancer growth and prolonged survival
in mice (Figures 3A and 3B). Strikingly, we observed that both the frequency and
absolute number of CD3+ T cells were prominently increased in the spleen and
peripheral blood following low-dose DAC treatment (Figure 3C and Figures
S3A-S3C). Moreover, the activation of T cells and their infiltration in cancer were
significantly increased by low-dose DAC administration (Figures 3D and 3E).
Intracellular staining analysis suggested that the IFN-γ-producing effector cells in the
tumor draining lymph node (TDLN) and spleen were augmented in the low-dose
DAC-treated group (Figures 3F, S3D and S3E). Thus, low-dose DAC treatment
inhibited cancer progression and promoted anti-tumor T cell responses in vivo.
To mimic human tumor development, we used the diethylnitrosamine
(DEN)-induced hepatocellular carcinoma mouse model. As multiple liver tumors were
developed in control-treated mice, low-dose DAC-treated mice presented liver tumors
that were markedly decreased tumor in number and size (Figure 3G). Moreover,
significantly greater number of T cells had infiltrated the tumor following low-dose
DAC treatment, particularly increased IFN-γ-producing Th1 and CTL cells in tumors,
TDLN and spleen (Figure 3H and S3F). These results suggested that low-dose DAC
treatment increased the anti-tumor properties of T cells and inhibited cancer
progression in vivo.
To further confirm the suppression of cancer development by low-dose DAC
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treatment was dependent on its direct impact on T cells, we performed an adoptive
transfer experiment of in vitro low-dose DAC-treated T cells. Remarkably, in a
BALB/c mouse model carrying CT26 tumors, tumor development was inhibited to a
greater extent by transferring 10 nM DAC-treated T cells compared with control T
cells (Figures 3I and 3J). By labeled with CFSE, we observed more tumor infiltrated
T cells with enhanced activation (analyzed by CD69 expression) and IFN-γ-producing
capacities in mice infusion with DAC-treated T cells, as compared with control T cells
(Figure S3G and S3H). Moreover, to exclude the possibility of cancer immune
activation, we constructed a CT26 or SMMC7721 tumor-bearing nude mouse model
and found that the immunogenic properties of cancer tissues were not significantly
altered by low-dose DAC treatment, including the expression of MAGE tumor
antigens, MHC molecules or co-stimulatory factors (Figure 3K and Figure S3I). Thus,
low-dose DAC-induced activation of T lymphocytes was less likely due to enhanced
cancer immunogenicity but to a direct impact on T lymphocytes.
Improved anti-tumor T cell activity in patients following low-dose DAC therapy
Next we explored whether low-dose DAC therapy could enhance the anti-tumor
activity of T cells in patients with solid tumors. In our clinical trials of low-dose DAC
therapy, DAC was administered at a dose of 7 mg/m2 for five days of each 28-day
treatment cycle (9,19). We collected peripheral blood cells from patients before (Day
0) and after DAC administration (Day 6) in the first cycle and sorted CD3+ T cells. In
patients with a good response to low-dose DAC therapy (UPN1 and UPN2), the
number and viability of T lymphocytes was increased following low-dose DAC
therapy (Figures 4A and 4B). We also analyzed serum cytokine levels before and after
DAC infusion in the first cycle. In UPN1 and UPN2, elevation of the cytokine IFN-γ
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and TNF-α, and reduced production of IL-6 and IL-4, were observed in both patients
(Figure 4C). Moreover, we also detected the enhanced T cell proliferation and Th1
cytokine secretion in clinical benefit patients among the other 18 patients in the test
cohort (Figure S4A-S4C). Consistently, increased numbers of IFN-γ-secreting Th1
and CTL cells were observed (Figure 4D). Next, we evaluated the anti-tumor
cytotoxicity properties of T cells in low-dose DAC-treated patients. Using different
tumor cell lines as target cells, we observed that the cytotoxicity of peripheral CD3+ T
cells from these patients was significantly enhanced following low-dose DAC therapy
(Figure 4E). By comparing the T cell cytotoxicity ability and clinical responses in a
total of 20 patients (Test Cohort), we observed that patients with prominently
enhanced T cell cytotoxicity (n=11) showed beneficial clinical responses to low-dose
therapy (including complete response (CR), partial response (PR) and stable disease
(SD)). Moreover, in 6 patients with a SD clinical response, we did not observe marked
cytotoxicity enhancement of T cells. For the other 3 patients with progressive disease
(PD), there was no improvement in T cell cytolytic ability (Figure 4F and S4D).
Hence, low-dose DAC-activated T cells can inhibit cancer progression in patients. In
addition, the increased frequency of IFN-γ+ T cells may be important for the clinical
effects in solid tumor patients (Figure 4G). These data suggest that the cytotoxicity of
peripheral T cells is enhanced following low-dose DAC-based therapy in solid tumor
patients, which may be important for the response of tumor patients.
An increased frequency of IFN-γ+ T cells and T cell cytotoxicity predict improved
responses to low-dose DAC therapy
Because the increase in IFN-γ+ T cell frequency and T cell cytotoxicity were
determined to be important for the clinical effects of low-dose DAC therapy, we next
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examined the correlation between IFN-γ+ T cell frequency or T cell cytotoxicity and
the clinical response to low-dose DAC therapy in solid tumor patients. In the test
cohort of 20 patients, patients with an increased frequency of IFN-γ+ T cells or T cell
cytotoxicity following low-dose DAC therapy demonstrated significantly a higher
disease control rate (DCR) and objective response rate (ORR) compared with patients
without a significant increase in IFN-γ+ T cells or T cell cytotoxicity (Figure 5A).
Additionally, patients with an increased frequency of IFN-γ+ T cells or T cell
cytotoxicity had both improved progression-free survival (PFS) and overall survival
(OS) following low-dose DAC therapy compared with patients without a significant
increase in IFN-γ+ T cells or cytolytic activity (Figures 5B-5E). These data suggest
that the increase in IFN-γ+ T cells or cytotoxicity in T cells is significantly correlated
with the response to low-dose DAC therapy. We also included a validation cohort of
38 patients with refractory/recurrent ovarian cancer and determined that patients with
an increased frequency of IFN-γ+ T cells or T cell cytotoxicity following low-dose
therapy had significantly higher DCR and ORR (Table S2 and Figure 5F), and
improved PFS and OS compared with patients without a significant increase in
IFN-γ+ T cells or cytotoxicity (Figures 5G-5J). Further statistical analysis showed that
the enhanced T cell function was correlated with the best clinical response to DAC
therapy in both test cohort and validation cohort, while no significant correlation was
observed in other parameters (Table S3 and S4). Thus, these data suggest that the
increased IFN-γ+ T cell frequency or T cell cytotoxicity was positively correlated
with the clinical response to low-dose DAC therapy in solid tumor patients.
Detection of peripheral IFN-γ+ T cell frequency following low-dose DAC
treatment in vitro predicts the clinical response to low-dose DAC therapy in solid
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tumor patients
To present a very predictable pattern for the response to low-dose DAC therapy
in solid tumor patients, we evaluated the effects of in vitro low-dose DAC treatment
on peripheral T cells from solid tumor patients. The peripheral CD3+ T cells from
solid tumor patients before DAC therapy were treated with low-dose DAC in vitro,
and the frequencies of IFN-γ+ T cells were measured. We defined increased IFN-γ+ T
cells as the ΔIFN-γ+ T cell frequency >10%, and found that patients with an
increased frequency of IFN-γ+ T cells following in vitro low-dose DAC treatment had
significantly higher levels of DCR and ORR than patients without a significant
increase in IFN-γ+ T cells in both the Test Cohort and the Validation Cohort (Figure
6A). Additionally, patients with an increased frequency of IFN-γ+ T cells with in vitro
DAC treatment showed improvements in both PFS and OS than patients without a
significant increase in IFN-γ+ T cells in both the Test Cohort and the Validation
Cohort (Figures 6B-6E). Importantly, the changes in IFN-γ+ T cell frequency after in
vivo DAC therapy were positively correlated with those following in vitro DAC
treatment in both cohorts (Figures 6F and 6G). In addition, univariate and multivariate
Cox regression identified that the increased IFN-γ-producing T cell frequency with in
vitro DAC treatment might be associated with longer PFS and OS in both test cohort
and validation cohort (Table S5 and S6). Therefore, pre-detection of the IFN-γ+ T cell
frequency following low-dose DAC treatment in vitro may be a useful approach to
predict and determine the clinical response to low-dose DAC therapy in solid tumor
patients (Figure 6H).
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Discussion
DNMT inhibitors including DAC have been widely studied in anti-tumor
treatment. Currently, low-dose DAC therapy has been used for the treatment of both
blood neoplasms and solid tumors, demonstrating effectiveness and less toxicity
(9-11). Concerning the mechanisms of low-dose DAC therapy, previous reports have
determined that it induces the expression of genes involved in cell cycle arrest,
apoptosis, DNA damage responses, defense responses, antigen exposure and lineage
commitment, leading to decreased tumorigenicity and self-renewal. However, these
previous anti-tumor effects of DAC were mainly focused on its influence on cancer
cells; it is still unknown whether low-dose DAC therapy has a direct effect on T
cell-medicated anti-tumor responses. Here, we found that low-dose DAC therapy
could directly enhance the activation and expansion of IFN-γ-producing Th1 and CTL
cells. Furthermore, increased numbers of IFN-γ-producing T cells following DAC
therapy was suggested to be an effective predictor of improved responses to low-dose
DAC therapy in solid tumor patients. Thus, we presented the important roles of
low-dose DAC therapy on host anti-tumor immune responses in this study.
IFN-γ is a Th1 cytokine and plays critical roles in driving naïve CD4+ T cells
toward a Th1 cell phenotype. Here, we found that low-dose DAC therapy
significantly enhanced the numbers of IFN-γ-producing T cells. Th2 type cytokine
production was suppressed, and CD4+CD25+Foxp3+ Treg cells were also inhibited,
which might be mediated by the enhanced Th1 and CTL activation because IFN-γ
production is known to block the expansion of Th2 and Treg cells (22,23).
Additionally, CD4+ T cells can differentiate into Treg cells in the presence of TGF-β,
and DAC has been reported to suppress TGF-β signaling (16,24). Moreover, although
the frequency and absolute number were not markedly altered, the function of other
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21
immune cells, such as macrophage or DCs might be influenced following low-dose
DAC treatment, probably due to changed profiles of cytokines or tumor antigens.
Thus, the roles for epigenetic modifying agent may be multifaceted, and meaningful
anti-tumor immune responses were induced by low-dose DAC therapy, although
whether and how low-dose DAC therapy alters the immunosuppresive
microenvironment in tumors as well as other immune cells requires further
investigation. Interestingly, we also found that low-dose DAC treatment showed
growth-promoting effects in normal human T cells but not T cell leukemia cell lines.
Because increasing evidence has suggested that DAC-regulated genes are different
depending on the cell types and concentrations (25), the distinct genomic or
epigenomic profiles of normal T cells and T leukemia cells may contribute to the
distinct effects mediated by DNMT inhibitors. For example, we observed that the
expression levels of cell cycle-related proteins, such as p21 and p15, were increased
in Jurkat T leukemia cells following low-dose DAC treatment, but the expression of
these genes was slightly reduced in normal human T cells (Figure S1J). Therefore,
low-dose DAC therapy may favor the enhancement of anti-tumor T cell responses but
not the proliferation of cancer cells.
Low-dose DAC therapy has been determined to have promising therapeutic
effects for some types of solid tumors, such as ovarian cancer, colorectal cancer, and
esophageal cancer (8-10). However, only a subgroup of patients have demonstrated a
good response to low-dose DAC therapy, and thus the identification of biomarkers for
the prediction of responses to low-dose DAC therapy is of prominent importance but
remains unsolved. Substantial research has focused on the changes in DNA
methylation and gene expression in tumor cells or tissues during epigenetic therapy,
or the basal levels of DNA methylation of specific genes in tumors. Although both
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global DNA demethylation and the reactivation of some genes have been determined,
such as p15, p16, MLH1, and BRCA1 (26), the relationship between the
demethylation of specific genes in tumors and the responses to low-dose DAC therapy
was identified to be ineffective or even not determined. Since the doses of DAC and
changes in specific genes in tumors were poorly correlated with the responses of
patients, we focused on the effects of low-dose DAC therapy on anti-tumor immune
responses and attempted to analyze whether the enhance numbers of IFN-γ+ T cells
was correlated with the response. Based on our clinical trials of low-dose DAC
therapy, we found that both an increased frequency of IFN-γ+ T cells and T cell
cytotoxicity determined in the peripheral blood of patients following the first cycle of
DAC therapy were significantly correlated with better clinical responses and
improved survival after low-dose DAC therapy. Moreover, we found that in vitro
low-dose DAC treatment in peripheral T cells from solid tumor patients exhibited
similar effects compared with low-dose DAC therapy in vivo, indicating that the
pre-detection of T cell responses to low-dose DAC treatment in vitro would guide the
therapeutic regimen in solid tumors. These data suggest that the detection of
peripheral T cell alterations in response to low-dose DAC in vitro may be a useful
approach for the prediction of responses to low-dose DAC therapy. However, why T
cells from the other subgroup of patients respond poorly to low-dose DAC therapy
remains unknown. Next, we will focus on the mechanisms underlying this
phenomenon and hope to define new approaches to enhance low-dose DAC-induced
T cell anti-tumor immune activation.
In conclusion, we have examined the effects of low-dose DAC therapy on host
anti-tumor immune responses, including enhanced T cell expansion and activation,
increased IFN-γ-producing Th1 and CTL cells and augmented T cell cytotoxicity,
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23
which were correlated with clinical responses following low-dose DAC therapy in
solid tumor patients.
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24
Acknowledgements
The technical assistance of Chao Jia and Wei Li, are gratefully acknowledged.
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Figure legends
Figure 1 Low-dose DAC promotes the activation and proliferation of T cells
(A) CD3+ T cells were sorted using anti-CD3 antibody by FACS from the peripheral
blood cells of 4 healthy donors. The cells were treated with PBS or 10 nM DAC for
48 h, followed by stimulation with different doses of anti-CD3 antibody for 12 h. Cell
activation was measured by staining with antibody against CD69, gated on CD3
positive T cells. (B) Peripheral CD3+ T cells were treated with PBS (control) or 10
nM DAC for 48 h, followed by stimulation with 2 μg anti-CD3 antibody for the times
indicated. Cell activation was measured by staining with antibody against CD69, both
gated on CD3 positive T cells. (C) CD3+ T cells were treated with different doses of
DAC for 48 h, followed by stimulation with anti-CD3 antibody for 12 h. Cell
activation was assessed in CD4+ and CD8+ T cells by detecting the level of CD69. (D)
CD3+ T cells were treated with PBS or 10 nM DAC for 48 h, followed by stimulation
with anti-CD3 antibody for 2 min, and the cells were analyzed by immunoblotting
using the indicated antibodies. (E) CD3+ T cells were treated with different doses of
DAC in the presence of immobilized anti-CD3 antibody, and the cell viability was
measured at the indicated time points using CCK8 assays. (F) CD3+ T cells were
treated with different doses of DAC in the presence of immobilized anti-CD3
antibody for 3 days, and Ki67 levels were detected by flow cytometry, and the
frequencies of Ki67+CD3+ T cells were shown. (G, H) BALB/c mice were treated
with PBS (Con group) or 0.05 mg/kg DAC (DAC group) for 5 days, and
immunocytes were collected from splenocytes 10 days after DAC treatment using the
indicated antibodies. The frequencies (G) and absolute numbers (H) are shown. mø,
macrophage. (I) BALB/c mice were treated with different doses of DAC (0, 0.05, 0.2
mg/kg) for 5 days; splenic T cells were collected 10 days after DAC treatment. The
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percentage of CD3+CD69+ T cells was measured in response to anti-CD3 stimulation.
(J) Splenic CD3+ T cells as in (H) were isolated, and cell viability was determined in
vitro using the CCK8 assay following stimulation with anti-CD3 antibody.
Data represent the mean±s.d. (n=3) or images of representative experiments. Similar
results were obtained in three independent experiments. *, p<0.05.
Figure 2 Low-dose DAC promotes Th1 polarization and CTL cytolytic activity
(A) CD3+ T cells were sorted using anti-CD3 antibody by FACS from peripheral
blood cells of healthy donors. Quantitative PCR analysis of a series of T/B cell
activation-associated genes (Qiagen, PAHS-053Z RT² Profiler™ PCR Array Human
T-Cell & B-Cell Activation) in 10 nM DAC-treated T cells compared with control
cells was performed, and the relative cytokine expression levels are shown. (B, C)
CD3+ T cells were sorted using anti-CD3 antibody by FACS from peripheral blood
cells of healthy donors. Cells were treated with different doses of DAC in the
presence of immobilized anti-CD3 antibody for 3 days, and the production of Th1
(IFN-γ and TNF-α, B) and Th2 (IL-4, C) cytokines in the culture supernatant was
detected by flow cytometry using the human Th1/Th2 cytokine CBA kit. (D) CD3+ T
cells were treated with DAC for 3 days, followed by re-stimulation with cell
stimulation cocktail (including PMA, ionomycin plus protein transport inhibitors),
and intracellular staining with antibodies against IFN-γ and IL-4. CD3+CD4+ cells
(green dots, CD4 T cells) or CD3+CD4- cells (violet dots, CD8 T cells) were gated,
and the percentage of T cells expressing IFN-γ or IL-4 was analyzed by flow
cytometry and shown. (E, F) The percentage (E) and absolute number (F) of
FoxP3+CD25+CD4+ Treg cells was determined by flow cytometry analysis by gating
on CD4+CD25+ T cells. (G) The 10 nM DAC-primed CD3+ T cells were treated with
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IFN-γ neutralization antibody, and T cell activation was evaluated using anti-CD69
antibody. (H) The 10 nM DAC-primed CD3+ T cells were treated with IFN-γ
neutralization antibody, and T cell viability was assessed by the CCK8 assay. (I)
qRT-PCR analysis of the expression levels of IFN-γ-induced genes is shown as
indicated. (J) The cytotoxicity of different doses of DAC-treated CD3+ T cells against
SMMC7721 cells. (K) Control or DAC-treated CD3+ T cells were co-cultured with
SMMC7721 cells for 24 h. IFN-γ and TNF-α secretion was detected using the
Th1/Th2 cytokine CBA kit by flow cytometry. (L, M) Control or 10 nM DAC-treated
CD3+ T cells were co-cultured and re-stimulated with SMMC7721 for 24 h. The
amounts of Granzyme B-expressed (L) or CD107a-expressed (M) cells were detected
by flow cytometry.
Data represent the mean±s.d. (n=3) or images of representative experiments. Similar
results were obtained in three independent experiments. *, p<0.05. N.S., not
significant.
Figure 3 Low-dose DAC treated T cells inhibit cancer progression in vivo
(A) Subcutaneous growth of CT26 tumors in BALB/c mice treated with 0 mg/kg, 0.05
mg/kg or 0.2 mg/kg DAC as indicated from days 12 to 16 (n=8 per group). The
arrows indicate the DAC treatments. (B) Survival curves of CT26-bearing mice
treated with different doses of DAC as indicated (n=8 per group). (C) CT26-bearing
mice were treated with PBS (Con group) or 0.05 mg/kg DAC (DAC group) for 5 days,
and immunocytes were collected from splenocytes 10 days after DAC treatment and
detected by flow cytometry using the indicated antibodies. The frequencies are shown
as indicated. (D, E) Numbers (D) and immunohistochemical (IHC) analysis (E) of the
tumor infiltration of CD3+ T cells in CT26 colon cancer tissues from PBS or 0.05
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mg/kg DAC-treated mice are shown (n=8 per group). (F) Tumor-draining lymph
nodes (TDLNs) from control or 0.05 mg/kg DAC-treated mice were collected after 2
weeks of treatment with ex vivo cell stimulation cocktail for 4 h. Flow cytometry
analysis was performed to detect CD3-PerCP, CD4-APC, IFN-γ-PE and Granzyme
B-FITC. (G) DAC-treated mice displayed decreased numbers of tumor nodules. The
mice were sacrificed at 4 weeks after the initial DAC treatment, which was
approximately 11.5 months after DEN injection. The results were displayed as dots
representing the numbers of tumor nodules per mouse (n=8). The maximum tumor
nodule diameter per mouse was measured. (H) TILs from DEN mice were collected
as in (G), which were stimulated ex vivo with cell stimulation cocktail for 4 h. Flow
cytometry analysis was performed to detect CD3-PerCP, CD4-APC and IFN-γ-PE. (I,
J) Tumor sizes (I) and survival curves (J) for CT26-bearing mice that received PBS
(Con), control T cell (5×107) transfusions (T) or 10 nM DAC-treated T cell (5×10
7)
transfusions (T+DAC) (n=8 per group). (K) Human hepatocellular carcinoma
SMMC7721 cells were injected subcutaneously into nude mice and treated with
control or 0.05 mg/kg DAC for 5 days. The expression levels of CTAs (MAGE A1/A3,
NY-ESO-1), co-stimulatory molecules (CD80, CD86), and MHC molecule (B2M) in
tumor cells were detected by qRT-PCR at 10 days after DAC treatment.
Data represent the mean±s.d. (n=3) or images of representative experiments. Similar
results were obtained in three independent experiments. *, p<0.05. N.S., not
significant.
Figure 4 Improved anti-tumor T cell activity in patients following low-dose DAC
therapy
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(A) Peripheral CD3+ T cells from solid tumor patients before and after DAC infusion
in the first cycle were sorted by FACS using anti-CD3 antibody, and the numbers of T
cells are shown. These cells were used for the functional analysis as in (B to G). (B) T
cell proliferation in patients was measured using the CCK8 assay before and after
DAC treatment during the first cycle. The representations (UPN1 and UPN2) are
shown. (C) Cytokine production in the sera from patients before and after DAC
treatment during the first cycle was detected using the human Th1/Th2 cytokine CBA
kit by flow cytometry. (D) Intracellular IFN-γ staining was used to identify CD4+ and
CD8+ T cells in representative patients. (E) The cytotoxicity of peripheral T cells
before and after DAC therapy against tumor cells, including WT and drug-resistant
colon cancer tumor cells as indicated. (F) The cytotoxicity of peripheral T cells before
and after DAC therapy during the first cycle against HCT116 tumor cells was
determined in all 20 patients. Δcytolysis = post cytotoxicity - pre cytotoxicity. “+”
refers to a beneficial clinical response (including a complete response, partial
response and stable disease), and “-” refers to progressive disease. Δcytolysis >10%
(the average cytolysis of the 20 patients) refers to enhanced cytolysis. (G) The
percentages of IFN-γ-producing CD8 T and CD4 T cells were detected by flow
cytometry. “+” refers to a beneficial clinical response (including a complete response,
partial response and stable disease), and “-” refers to progressive disease.
Data represent the mean±s.d. (n=3) or images of representative experiments. Similar
results were obtained in three independent experiments. *, p<0.05.
Figure 5 Increased frequency of IFN-γ+ T cells and cytotoxicity of T cells
following five days of in vivo DAC therapy predict a good response to low-dose
DAC therapy in solid tumor patients
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(A) The correlation between clinical responses and peripheral T cell activity in the
Test Cohort (frequency of IFN-γ+ T cells and T cell cytotoxicity). P values were
calculated using the chi-square test in SPSS 18.0. Since the average cytotoxicity in the
Test Cohort was 10%, we defined an increased IFN-γ+ T or cytotoxicity as an IFN-γ+
T cell frequency or cytotoxicity after DAC therapy greater than 10% higher compared
with before DAC therapy. The absence of an increase in IFN-γ or cytotoxicity
indicated that the frequency of IFN-γ+ T cells or cytotoxicity after DAC therapy were
less than 10% higher or lower than the values before DAC therapy. (B, C)
Kaplan-Meier survival curves of progression-free survival (B) and overall survival (C)
based on the frequency alteration of peripheral IFN-γ+ T cells in the Test Cohort. Ten
percentages higher was used as the cutoff, using the log-rank test to evaluate
significance. (D, E) Kaplan-Meier survival curves of progression-free survival (D)
and overall survival (E) based on the cytotoxicity alteration of peripheral T cells in the
Test Cohort, using the same cutoff value and statistical analysis as described in (B, C).
(F) Correlation between clinical responses and peripheral T cell activity in the
Validation Cohort (frequency of IFN-γ+ T cells and T cell cytotoxicity). P values were
calculated using the chi-square test in SPSS 18.0. (G, H) Kaplan-Meier survival
curves of progression-free survival (G) and overall survival (H) based on the
frequency alteration of peripheral IFN-γ+ T cells in the Validation Cohort, using the
same cutoff value and statistical analysis as described in (B, C). (I, J) Kaplan-Meier
survival curves of progression-free survival (I) and overall survival (J) based on the
cytotoxicity alteration of peripheral T cells in the Validation Cohort, using the same
cutoff value and statistical analysis as described in (B, C).
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33
Figure 6 Increased IFN-γ+ T cell frequency following 3 days of in vitro DAC
treatment predicts an improved response to low-dose DAC therapy in solid tumor
patients
(A) The peripheral CD3+ T cells from patients before DAC therapy were collected,
sorted and cultured in the presence of immobilized anti-CD3 antibody with control or
10 nM DAC for 3 days. The frequency of IFN-γ+ T cells was detected by flow
cytometry. The correlation between clinical responses and ΔIFN-γ+ T cell frequency
(“frequency with DAC treatment” - “frequency with control treatment”) in the Test
Cohort and Validation Cohort were determined as indicated. P values were calculated
using the chi-square test in SPSS 18.0. (B, C) Kaplan-Meier survival curves of
progression-free survival (B) and overall survival (C) based on the ΔIFN-γ+ T cell
frequency following a 3 day in vitro DAC treatment in the Test Cohort. Ten
percentages higher was used as the cutoff, using the log-rank test to evaluate
significance. (D, E) Kaplan-Meier survival curves of progression-free survival (D)
and overall survival (E) based on the ΔIFN-γ+ T cell frequency following a 3 day in
vitro DAC treatment in the Validation Cohort, using the same cutoff value and
statistical analysis as described in (C, D). (F) Pearson correlation analysis between the
ΔIFN-γ+ T cell frequency following in vitro DAC treatment and in vivo DAC therapy
in the Test Cohort. (G) Pearson correlation analysis between the ΔIFN-γ+ T cell
frequency following in vitro DAC treatment and in vivo DAC therapy in the Validation
Cohort.
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Published OnlineFirst July 13, 2017.Clin Cancer Res Xiang Li, Yan Zhang, Meixia Chen, et al. anti-tumor therapyresponses of low-dose DNA demethylating agent decitabine
+ T cells are responsible for the clinicalγIncreased IFN-
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