Increased IFN dose DNA demethylating agent decitabine anti … · 2017. 7. 13. · osis, cell...

1

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);

Research. on May 4, 2021. © 2017 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 13, 2017; DOI: 10.1158/1078-0432.CCR-17-1201

http://clincancerres.aacrjournals.org/

2

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.




3

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




4

therapy.




5

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




6

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




7

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.




8

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




9

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




10

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




11

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




12

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)




13

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




14

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




15

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




16

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




17

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




18

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




19

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).




20

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




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




22

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,




23

which were correlated with clinical responses following low-dose DAC therapy in

solid tumor patients.




24

Acknowledgements

The technical assistance of Chao Jia and Wei Li, are gratefully acknowledged.




25

References

1. Gal-Yam EN, Saito Y, Egger G, Jones PA. Cancer epigenetics: modifications, screening, and

therapy. Annual review of medicine 2008;59:267-80 doi

10.1146/annurev.med.59.061606.095816.

2. Brien GL, Valerio DG, Armstrong SA. Exploiting the Epigenome to Control Cancer-Promoting

Gene-Expression Programs. Cancer cell 2016;29(4):464-76 doi 10.1016/j.ccell.2016.03.007.

3. Wijermans P, Lubbert M, Verhoef G, Bosly A, Ravoet C, Andre M, et al. Low-dose

5-aza-2'-deoxycytidine, a DNA hypomethylating agent, for the treatment of high-risk

myelodysplastic syndrome: a multicenter phase II study in elderly patients. Journal of clinical

oncology : official journal of the American Society of Clinical Oncology 2000;18(5):956-62.

4. Silverman LR, Demakos EP, Peterson BL, Kornblith AB, Holland JC, Odchimar-Reissig R, et

al. Randomized controlled trial of azacitidine in patients with the myelodysplastic syndrome: a

study of the cancer and leukemia group B. Journal of clinical oncology : official journal of the

American Society of Clinical Oncology 2002;20(10):2429-40.

5. Willemze R, Suciu S, Archimbaud E, Muus P, Stryckmans P, Louwagie EA, et al. A randomized

phase II study on the effects of 5-Aza-2'-deoxycytidine combined with either amsacrine or

idarubicin in patients with relapsed acute leukemia: an EORTC Leukemia Cooperative Group

phase II study (06893). Leukemia 1997;11 Suppl 1:S24-7.

6. Schwartsmann G, Fernandes MS, Schaan MD, Moschen M, Gerhardt LM, Di Leone L, et al.

Decitabine (5-Aza-2'-deoxycytidine; DAC) plus daunorubicin as a first line treatment in

patients with acute myeloid leukemia: preliminary observations. Leukemia 1997;11 Suppl

1:S28-31.

7. Matei D, Fang F, Shen C, Schilder J, Arnold A, Zeng Y, et al. Epigenetic resensitization to

platinum in ovarian cancer. Cancer research 2012;72(9):2197-205 doi

10.1158/0008-5472.CAN-11-3909.

8. Garrido-Laguna I, McGregor KA, Wade M, Weis J, Gilcrease W, Burr L, et al. A phase I/II

study of decitabine in combination with panitumumab in patients with wild-type (wt) KRAS

metastatic colorectal cancer. Investigational new drugs 2013;31(5):1257-64 doi

10.1007/s10637-013-9947-6.

9. Fan H, Lu X, Wang X, Liu Y, Guo B, Zhang Y, et al. Low-dose decitabine-based

chemoimmunotherapy for patients with refractory advanced solid tumors: a phase I/II report.

Journal of immunology research 2014;2014:371087 doi 10.1155/2014/371087.

10. Stathis A, Hotte SJ, Chen EX, Hirte HW, Oza AM, Moretto P, et al. Phase I study of decitabine

in combination with vorinostat in patients with advanced solid tumors and non-Hodgkin's

lymphomas. Clinical cancer research : an official journal of the American Association for

Cancer Research 2011;17(6):1582-90 doi 10.1158/1078-0432.CCR-10-1893.

11. Issa JP, Garcia-Manero G, Giles FJ, Mannari R, Thomas D, Faderl S, et al. Phase 1 study of

low-dose prolonged exposure schedules of the hypomethylating agent 5-aza-2'-deoxycytidine

(decitabine) in hematopoietic malignancies. Blood 2004;103(5):1635-40 doi

10.1182/blood-2003-03-0687.

12. Nie J, Liu L, Li X, Han W. Decitabine, a new star in epigenetic therapy: the clinical application

and biological mechanism in solid tumors. Cancer letters 2014;354(1):12-20 doi

10.1016/j.canlet.2014.08.010.

13. Tsai HC, Li H, Van Neste L, Cai Y, Robert C, Rassool FV, et al. Transient low doses of




26

DNA-demethylating agents exert durable antitumor effects on hematological and epithelial

tumor cells. Cancer cell 2012;21(3):430-46 doi 10.1016/j.ccr.2011.12.029.

14. Wang L, Zhang Y, Li R, Chen Y, Pan X, Li G, et al. 5-aza-2'-Deoxycytidine enhances the

radiosensitivity of breast cancer cells. Cancer biotherapy & radiopharmaceuticals

2013;28(1):34-44 doi 10.1089/cbr.2012.1170.

15. Li H, Chiappinelli KB, Guzzetta AA, Easwaran H, Yen RW, Vatapalli R, et al. Immune

regulation by low doses of the DNA methyltransferase inhibitor 5-azacitidine in common

human epithelial cancers. Oncotarget 2014;5(3):587-98.

16. Fang F, Zuo Q, Pilrose J, Wang Y, Shen C, Li M, et al. Decitabine reactivated pathways in

platinum resistant ovarian cancer. Oncotarget 2014;5(11):3579-89 doi

10.18632/oncotarget.1961.

17. Yang AS, Yang BJ. The failure of epigenetic combination therapy for cancer and what it might

be telling us about DNA methylation inhibitors. Epigenomics 2016;8(1):9-12 doi

10.2217/epi.15.94.

18. Fu X, Zhang Y, Wang X, Chen M, Wang Y, Nie J, et al. Low Dose Decitabine Combined with

Taxol and Platinum Chemotherapy to Treat Refractory/Recurrent Ovarian Cancer: An

Open-Label, Single-Arm, Phase I/II Study. Current protein & peptide science

2015;16(4):329-36.

19. Mei Q, Chen M, Lu X, Li X, Duan F, Wang M, et al. An open-label, single-arm, phase I/II study

of lower-dose decitabine based therapy in patients with advanced hepatocellular carcinoma.

Oncotarget 2015.

20. Cashen AF, Shah AK, Todt L, Fisher N, DiPersio J. Pharmacokinetics of decitabine

administered as a 3-h infusion to patients with acute myeloid leukemia (AML) or

myelodysplastic syndrome (MDS). Cancer chemotherapy and pharmacology

2008;61(5):759-66 doi 10.1007/s00280-007-0531-7.

21. Peng D, Kryczek I, Nagarsheth N, Zhao L, Wei S, Wang W, et al. Epigenetic silencing of

TH1-type chemokines shapes tumour immunity and immunotherapy. Nature

2015;527(7577):249-53 doi 10.1038/nature15520.

22. Glimcher LH, Murphy KM. Lineage commitment in the immune system: the T helper

lymphocyte grows up. Genes & development 2000;14(14):1693-711.

23. St Rose MC, Taylor RA, Bandyopadhyay S, Qui HZ, Hagymasi AT, Vella AT, et al.

CD134/CD137 dual costimulation-elicited IFN-gamma maximizes effector T-cell function but

limits Treg expansion. Immunology and cell biology 2013;91(2):173-83 doi

10.1038/icb.2012.74.

24. Chen WJ, Jin WW, Hardegen N, Lei KJ, Li L, Marinos N, et al. Conversion of peripheral

CD4(+)CD25(-) naive T cells to CD4(+)CD25(+) regulatory T cells by TGF-beta induction of

transcription factor Foxp3. Journal Of Experimental Medicine 2003;198(12):1875-86 doi

10.1084/jem.20030152.

25. Tsujioka T, Yokoi A, Uesugi M, Kishimoto M, Tochigi A, Suemori S, et al. Effects of DNA

methyltransferase inhibitors (DNMTIs) on MDS-derived cell lines. Experimental hematology

2013;41(2):189-97 doi 10.1016/j.exphem.2012.10.006.

26. Baylin SB. DNA methylation and gene silencing in cancer. Nature clinical practice Oncology

2005;2 Suppl 1:S4-11 doi 10.1038/ncponc0354.




27

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




28

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




29

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.


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




30

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.


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




31

(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.


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




32

(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).




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.




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-

Updated version

10.1158/1078-0432.CCR-17-1201doi:

Access the most recent version of this article at:

Material

Supplementary

http://clincancerres.aacrjournals.org/content/suppl/2017/07/13/1078-0432.CCR-17-1201.DC1

Access the most recent supplemental material at:

Manuscript

Authorbeen edited. Author manuscripts have been peer reviewed and accepted for publication but have not yet

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://clincancerres.aacrjournals.org/content/early/2017/07/13/1078-0432.CCR-17-1201To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/lookup/doi/10.1158/1078-0432.CCR-17-1201

http://clincancerres.aacrjournals.org/content/suppl/2017/07/13/1078-0432.CCR-17-1201.DC1

http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://clincancerres.aacrjournals.org/content/early/2017/07/13/1078-0432.CCR-17-1201


Increased IFN dose DNA demethylating agent decitabine anti … · 2017. 7. 13. · osis, cell...

Documents

Transcript of Increased IFN dose DNA demethylating agent decitabine anti … · 2017. 7. 13. · osis, cell...