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Translational Cancer Mechanisms and Therapy
Activated and Exhausted MAIT Cells Foster
Disease Progression and Indicate Poor Outcome in
Hepatocellular Carcinoma
Meng Duan
1
, Shyamal Goswami
2
, Jie-Yi Shi
1
, Lin-Jie Wu
3
, Xiao-Ying Wang
1
, Jia-Qiang Ma
2
,
Zhao Zhang
1
, Yang Shi
4
, Li-Jie Ma
1
, Shu Zhang
1
, Rui-Bin Xi
3,5
,Ya Cao
6
, Jian Zhou
1,7,8
,
Jia Fan
1,7,8
, Xiao-Ming Zhang
2
, and Qiang Gao
1,8
Abstract
Purpose: Innate immunity is an indispensable arm of
tumor immune surveillance, and the liver is an organ with
a predominance of innate immunity, where mucosal-associ-
ated invariant T (MAIT) cells are enriched. However, little is
known about the phenotype, functions, and immunomodu-
latory role of MAIT cells in hepatocellular carcinoma (HCC).
Experimental Design: The distribution, phenotype, and
function of MAIT cells in patients with HCC were evaluated
by both flow cytometry (FCM) and in vitro bioassays. Tran-
scriptomic analysis of MAIT cells was also performed. Prog-
nostic significance of tumor-infiltrating MAIT cells was vali-
dated in four independent cohorts of patients with HCC.
Results: Despite their fewer densities in HCC tumor
than normal liver, MAIT cells were significantly enriched
in the HCC microenvironment compared with other mucosa-
associated organs. Tumor-derived MAIT cells displayed a
typical CCR7
CD45RA
CD45RO
þ
CD95
þ
effector memory
phenotype with lower costimulatory and effector capabilities.
Tumor-educated MAIT cells significantly upregulated inhibitory
molecules like PD-1, CTLA-4, TIM-3, secreted significantly less
IFNgand IL17, andproduced minimalgranzyme B and perforin
while shifting to produce tumor-promoting cytokines like IL8.
Transcriptome sequencing confirmed that tumor-derived MAIT
cells were reprogrammed toward a tumor-promoting direction
by downregulating genes enriched in pathways of cytokine
secretionand cytolysis effector function like NFKB1and STAT5B
and by upregulating genes like IL8, CXCL12,andHAVCR2
(TIM-3). High infiltration of MAIT cells in HCC significantly
correlated with an unfavorable clinical outcome, revealed by
FCM, qRT-PCR, and multiplex IHC analyses, respectively.
Conclusions: HCC-infiltrating MAIT cells were functionally
impaired and even reprogrammed to shift away from antitu-
mor immunity and toward a tumor-promoting direction.
See related commentary by Carbone, p. 3199
Introduction
Hepatocellular carcinoma (HCC) is the fifth most common
cancer in men and the seventh among women in the world (1).
HCC represents a typicalinflammation/immune-related tumor that
usually develops in an inflamed fibrotic or cirrhotic liver (2). The
recent breakthrough in HCC immunotherapy targeting immune
checkpoint PD-1/PD-L1 has substantially improved the patient
survival (3, 4). A deeper understanding of interactions between
immune cells and cancer cells within the HCC microenvironment
may reveal diverse therapeutic approaches for HCC (5, 6). In
addition to adaptive immune system specialized in recognizing
tumor antigen, innate immunity is another indispensable arm of
immune system, directly or indirectly participating in tumor con-
trol (7, 8). Indeed, the liver is an organ with a predominance of
diverse range of innate lymphocytes, among which mucosal-
associated invariant T (MAIT) cells are of paramount importance.
MAIT cells are a specialized innate-like T-cell subset, accounting
for 10% of circulating CD4
T cells in adults, featured with the
expression of the semiinvariant T-cell antigen receptor (TCR,
va7.2-Ja33) that detects microbial vitamin B metabolites presented
by major histocompatibility complex class I–related protein 1
(MR1; refs. 9, 10). Both the TCR expressed by MAIT cells and the
antigen-presenting molecule MR1 are evolutionarily conserved
among mammals, indicating a strong selective pressure to maintain
1
Department of Liver Surgery and Transplantation, Liver Cancer Institute,
Zhongshan Hospital, and Key Laboratory of Carcinogenesis and Cancer
Invasion (Ministry of Education), Fudan University, Shanghai, China.
2
Key
Laboratory of Molecular Virology & Immunology, Institut Pasteur of
Shanghai, Chinese Academy of Sciences, Shanghai, China.
3
School of
Mathematical Sciences, Peking University, Beijing, China.
4
Peking-Tsinghua
Center for Life Sciences, Academy for Advanced Interdisciplinary Studies,
Peking University, Beijing, China.
5
Center for Statistical Sciences, Peking
University, Beijing, China.
6
Cancer Research Institute, Xiangya School of
Medicine, Central South University, Hunan, China.
7
Institute of Biomedical
Sciences, Fudan University, Shanghai, China.
8
State Key Laboratory of
Genetic Engineering, Fudan University, Shanghai, China.
Note: Supplementary data for this article are available at Clinical Cancer
Research Online (http://clincancerres.aacrjournals.org/).
M. Duan, S. Goswami, J.-Y. Shi contributed equally to this article.
Corresponding Authors: Qiang Gao, Liver Cancer Institute, Zhong Shan
Hospital and Shanghai Medical School, Fudan University, 180 Fenglin
Road, Shanghai 200032, China. Phone: 8621-6403-7181; Fax: 8621-6403-
7181; E-mail: gao.qiang@zs-hospital.sh.cn; Jia Fan, E-mail: fan.jia@zs-
hospital.sh.cn; and Xiao-Ming Zhang, Key Laboratory of Molecular Virology
& Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences.
E-mail: xmzhang@ips.ac.cn
Clin Cancer Res 2019;25:3304–16
doi: 10.1158/1078-0432.CCR-18-3040
2019 American Association for Cancer Research.
Clinical
Cancer
Research
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3304
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immunity mediated by MAIT cells (11). Several subsets of
MAIT cells have been defined, most of which, if not all, are
CD161
high
IL17-secreting CD8
þ
T-cell subset (9, 12). After being acti-
vated by microbial antigen bound with MR1, MAIT cells are licensed
to kill targets by secreting IFNg, granzyme, and perforin (13).
Intriguingly, contrary to the name, MAIT cells are most enriched
in normal human livers, comprising up to 50% of all intrahepatic
T cells (14). It has been reported that hepatic MAIT cells are highly
activated within the liver and may be protective against a range of
bacteria, fungi, and viruses along with the large phagocytic Kupf-
fer cell population (8). Overall, a reduction and dysfunction in
MAIT cells were observed in blood and livers from patients with
chronic hepatitis B virus (HBV) or hepatitis B virus (HCV) infec-
tions and nonvirus chronic liver diseases (11, 15–17). As HCC is
usually complicated with those chronic liver diseases, dysfunction
of MAIT cells in HCC could be expected.
It seems plausible that MAIT cells may have a protective role in
the immune system, considering the highly conserved TCR and
the cytokine secretion pattern. Previous studies investigated MAIT
cells mainly in the infectious or autoimmune diseases, showing
that the infiltration and function of the subset were substantially
impaired (11). The only data regarding the role of MAIT cells in
human cancer comes from the colorectal cancer studies, indicat-
ing that activated MAIT cells accumulated in tumors correlated
with inferior patient survival (18–20). Although liver served as an
immunologic organ where MAIT cells are mostly enriched, the
function and immunomodulatory role of the MAIT cells in HCC
have yet not been fully clarified. In this study, we aimed to
determine the distribution, phenotype, function, and clinical
relevance of MAIT cells in human HCC.
Materials and Methods
Patients and samples
For flow cytometry (FCM) analysis, fresh paired tumor/peritu-
mor tissues and peripheral blood samples were obtained from a
cohort of 50 patients with HCC who underwent surgical resection
between 2014 and 2015. For analyzing TCRva7.2-Ja33 mRNA
expression, fresh frozen tumor/peritumor tissues were obtained
from another cohort of 207 patients with HCC who received
curative operation between 2009 and 2013. For tissue microarrays
(TMA), archival tissues were obtained from two independent
cohorts of 224 and 360 patients with HCC who received curative
surgery in 2007 and in 2006, respectively. Patient information
and clinicopathologic features of all the cohorts are summarized
in Supplementary Table S1. Informed consent was obtained from
each patient prior to receive the sample. This study was conducted
in accordance with ethical principles that have their origin in the
Declaration of Helsinki, and the ethical standards of the Research
Ethics Committee of Zhongshan Hospital (Shanghai, China).
Mononuclear cell isolation and FCM
Mononuclear cells from freshly resected liver tissues and peri-
pheral blood were isolated and stained for FCM as described
previously (ref. 21; details in Supplementary Materials and
Methods and Supplementary Table S2).
Immunofluorescence
Immunofluorescence on frozen section was performed accord-
ing to a two-step way method and scanned by TCS SP5 (Leica
Biosystems). Multiplex IHC on paraffin-embedded TMAs was
performed using Opal Tyramide Signal Amplification (TSA)–
based staining regents (PerkinElmer). Detailed information is
provided in Supplementary Materials and Methods.
MAIT cell coculture and cytokine detection
Information of MAIT cell activation, coculture, and cytokine
detection are described in Supplementary Materials and Methods.
RNA isolation, RT-PCR, and RNA sequencing
Total RNA was extracted using TRIzol Reagent (Invitrogen)
according to the manufacturer's instructions. The TCRva7.2-Ja33
mRNA levels were determined by real-time RT-PCR as described
previously (22). RNA sequencing (RNA-seq) was carried out as
described by Picelli and colleagues with minor modifica-
tions (23). Detailed information is described in Supplementary
Materials and Methods.
Statistical analysis
Statistical analyses were performed using GraphPad Prism 7.03
(GraphPad Software). The experimental data were shown as mean
SD. Cutoff values for patient grouping in all the cohorts were
defined by lowest tertile (33rd percentile) of MAIT cell's frequen-
cy/density. A two-tailed P<0.05 was considered significant.
Detailed information is provided in Supplementary Materials
and Methods.
Results
MAIT cell infiltration is significantly decreased in HCC
We first used FCM analysis to determine MAIT cell distribution
in blood and tissue of patients with HCC and healthy donors.
Consistent with the previous report (9), we confirmed that CD4
cells constituted >95% of total MAIT cells, irrespective of speci-
mens from normal liver, HCC tissues, or peripheral blood (Fig. 1A
and B; Supplementary Fig. S1A).
Even in peripheral blood, the frequency of CD4
MAIT cells
among total CD3
þ
T cells was significantly decreased in patients
Translational Relevance
Hepatocellular carcinoma (HCC) represents a typical
inflammation and immune-related cancer. The mucosal-
associat ed invariant T (MAIT) cell, an innate immune cell subset,
is enriched in the liver where innate immunity is dominant.
Here, we demonstrate that although tumor-infiltrating MAIT
cells display a typical effector memory phenotype, their effector
functions and cytotoxic capability are significantly impaired.
Upregulation of PD-1, CTLA-4, and TIM-3, a common feature
of T-cell exhaustion, is evidenced in HCC-derived MAIT cells.
MAIT cells are found to be activated within tumor milieu and
reprogrammed to produce a significantamountofprotumor
cytokines. A high density of tumor-infiltrating MAIT cells signi-
ficantly and independently correlated with dismal clinical
outcomes in patients with HCC. Thus, strategies to modulate
the functional activities of MAIT cells may provide a new avenue
for antitumor therapy in HCC. Extended understanding of
interactions between innate immunity and the HCC microen-
vironment may further provide new clues for more effective
immune therapy.
MAIT Cells in HCC
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Published OnlineFirst February 5, 2019; DOI: 10.1158/1078-0432.CCR-18-3040
with HCC compared with healthy donors (mean: 0.9% vs. 2.1%,
P<0.01; Fig. 1C). Trend continues in tumor tissues, CD4
MAIT
cell frequency within total CD3
þ
T-cell population was signifi-
cantly decreased in HCC tumor compared with either paired
peritumor (mean: 4.2% vs. 16.5%, P<0.001; n¼50) or normal
liver (mean: 4.2% vs. 21.6%, P<0.001; n¼20; Fig. 1C).
Next, immunofluorescence imaging confirmed our FCM find-
ings (Fig. 1D), reflecting a decreased density of CD161
þ
TCRva7.2
þ
MAIT cells in the tumor center compared with peri-
tumor (mean: 7.2 vs. 17.4 cells/mm
2
,P¼0.014; n¼8; Fig. 1E).
Moreover, using TCRva7.2 and IL18R as the surrogate for MAIT
cell detection, the results also showed a reduction of
TCRva7.2
þ
IL18R
þ
MAIT cells in tumor as compared with peri-
tumor (Supplementary Fig. S1B). These results indicated that
either absolute or relative number of MAIT cells was significantly
lower in the blood and tumor samples of patients with HCC.
We further checked the relationship between MAIT cell infil-
tration and clinical features and identified MAIT cell frequency
was lower in patients with liver cirrhosis in FCM cohort (mean:
3.46% vs. 6.90%, P¼0.053; n¼50; Supplementary Table S1). In
addition, in a larger qRT-PCR cohort (n¼209), TCRva7.2-Ja33
mRNA relative expression level was also decreased in patients with
liver cirrhosis (mean: 0.014 vs. 0.069, P¼0.057), or chronic HBV
infection (mean: 0.061 vs. 0.125, P¼0.075; Supplementary
Table S1). The above results suggest that liver cirrhosis is a
potential factor associated with the decrease of MAIT cell
infiltration.
Tumor-infiltrating MAIT cells display a typical effector memory
phenotype
In patients with HCC and healthy donors, we determined the
phenotypic features of MAIT cells, gated on MR1-5-OP-RU
þ
after
CD3
þ
CD4
CD161
þ
TCRva7.2
þ
, to exclude contaminations
from CD161
int
Va7.2
þ
T CD4
þ
mainstream T cells (24), by
comparing with conventional T-cell–related costimulatory/mem-
ory/activation molecules. Our data indicated that most, if not all,
MAIT cells displayed a CCR7
CD45RA
effector memory phe-
notype that may harbor immediate effector function (Supple-
mentary Fig. S2A and S2B). Likewise, MAIT cells expressed high
levels of CD45RO and CD95 in both patients with HCC and
healthy donors, irrespective of in the liver, tumor, and blood
(Fig. 2A; Supplementary Fig. S2C). Of note, the expression of
Figure 1.
MAIT cell definition, analyzing strategy, and distribution in patients with HCC by FCM and immunofluorescence imaging. A, MAIT cell staining and analyzing
strategy by FCM in peripheral blood and tissues of patients with HCC. B, MAIT cell subset composition in tissues and peripheral blood of patients with HCC and
healthy donors. HD, healthy donor; PBMC, peripheral blood mononuclear cells. C, CD4
MAIT cell frequency in total T cells in peripheral blood and tissues of
patients with HCC and healthy donors detected by FCM analysis (,P<0.01; ,P<0.001 by Mann–Whitney Utest or one-way ANOVA and Tukey multiple
comparison tests). HD, healthy donor; PBMC, peripheral blood mononuclear cells. D, Representative staining for TCRva7.2 and CD161 on frozen sections, scanned
by Leica SP5 under 63 objective. E, Summary of density information of CD161
þ
and TCRva7.2
þ
MAIT cells in paired peritumor and tumor tissues (P¼0.018 by
Mann–Whitney Utest). Lines and error bars are presented as the mean SD.
Duan et al.
Clin Cancer Res; 25(11) June 1, 2019 Clinical Cancer Research3306
on January 10, 2022. © 2019 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from
Published OnlineFirst February 5, 2019; DOI: 10.1158/1078-0432.CCR-18-3040
costimulatory molecules CD28 and CD127 on MAIT cells was
significantly decreased in HCC tissues compared with either
peritumor or normal liver tissues (for CD28 mean: 92.6% vs.
97.5% or 98.3%, P¼0.010; for CD127 mean: 92.8% vs. 98.8% or
98.1%, P¼0.031; Fig. 2B), whereas in peripheral blood, no
noticeable expression difference was found between HCC and
healthy donors (Supplementary Fig. S2D). In addition, MAIT cells
expressed significantly higher level of activating markers CD38
and HLA-DR in tumor compared with its counterpart in peritu-
mor or normal liver tissues (for CD38 mean: 14.6% vs. 8.69% or
4.54%, P¼0.044; for HLA-DR mean: 15.9% vs. 6.98% or 3.44%,
P¼0.014; Fig. 2C), whereas no obvious differences of these two
markers were seen on peripheral MAIT cells between HCC
patients and healthy donors (Supplementary Fig. S2E). Elevated
HLA-DR and CD38 expression could be resulted from chronic
infections that coexists and alternatively indicates a common
exhausted T-cell phenotype (25, 26). Furthermore, we checked
a group of ten T-cell and NK cell–related activating and inhibitory
molecules (27) and found that two effector function–related
molecules CD160 (mean: 39.0% vs. 72.3% or 96.3%, P<
0.001) and KLRG1 (mean: 76.5% vs. 82.7% or 95.7%, P¼
0.019; Fig. 2D; Supplementary Fig. S2G) had significantly lower
expression on HCC-infiltrated MAIT cells, whereas CD160 was
also lower on circulating MAIT cells in patients with HCC (Sup-
plementary Fig. S2F). Collectively, these data showed that tumor
MAIT cells displayed a typical CCR7
CD45RA
CD45RO
þ
CD95
þ
effector memory phenotype with activated status and potentially
decreased effector capabilities.
Figure 2.
Expression of costimulatory and activation receptors on intrahepatic and peripheral blood MAIT cells. A, Representative plots of CD45RO and CD95 expression
on tissue and blood MAIT cells (gated on CD161
þ
TCRva7.2
þ
MR1-tet
þ
). Expression of costimulatory receptors (B), activation receptors (C), and inhibiting
receptors (D) on tissue MAIT cells was also detected by FCM (gated on CD161
þ
TCRva7.2
þ
MR1-tet
þ
). Representative overlays for tumor, peritumor, normal liver,
and isotype control and total summary data are shown (,P<0.05; ,P<0.01; ,P<0.001 by one-way ANOVA and Tukey multiple compari son tests). Lines
and error bars are presented as the mean SD.
MAIT Cells in HCC
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Chemokine receptor expression profile in tumor-infiltrating
MAIT cells
Because MAIT cells are accounting almost half of the intrahe-
patic T cells supposed to be mediated by CCR6 and CXCR6, and
evidenced a decrease expression of gut-homing receptor
CCR9 (9, 15). Herein, to further support MAIT trafficking, we
screened the 18 classic chemokine receptors both in patients with
HCC and healthy donors. Compared with healthy donors, the
expression of all the three receptors, CCR6 (MFI mean: 568.8 vs.
876 or 817.8, P¼0.042), CXCR6 (MFI mean: 104.9 vs. 115.1 or
177.6, P¼0.002), and CCR9 (MFI mean: 80.4 vs. 134.9 or 168.9,
P¼0.016), on MAIT cells was significantly downregulated in the
patients with HCC, particularly in the tumor center (Fig. 3A;
Supplementary Fig. S3A), indicating a possible mechanism relat-
ed to low infiltration of MAIT cells in HCC.
Scenario goes different while comparing CXCR3 expression
with chronic liver diseases, where a positive expression of CXCR3
was detected on MAIT cells (15) and mild expression of CXCR3
was detected in patients with HCC (data not shown). CCR2 is
related to IL17-secreting T cells, and is significantly upregulated in
CD161
þ
T cells (28). In our study, there is a tendency of lower
expression of CCR2 in tumor tissues compared with normal livers
(Fig. 3B; Supplementary Fig. S3B). Previous study indicated
heterogeneous levels of CXCR4 expression on MAIT cells (15),
and here, the same phenomenon is reflected (Fig. 3B). In a normal
liver, MAIT cells show a high expression of CCR5 (15), and we
found that HCC-infiltrating MAIT cells maintained high expres-
sion of this marker (Fig. 3B). Taken together, these results showed
a selective downregulation of certain chemokine receptors may
affect the trafficking and residing capacity of tumor-infiltrating
MAIT cells during the progression of hepatocarcinogenesis.
Apoptosis is unlikely involved in the impaired infiltration of
MAIT cells in HCC
In addition to aberrant chemotaxis, apoptosis could also result
in less immune cells in tumor tissues. Bcl-2 family proteins are
Figure 3.
Expression of chemokine receptors and immune checkpoint molecules on circulating and intrahepatic MAIT cells. Aand B, Expressi on of chemokine receptors on
circulating and intrahepatic MAIT cells detected by FCM (gated on CD161
þ
TCRva7.2
þ
MR1-tet
þ
). Representative overlay plots for HCC peripheral blood
mononuclear cells (PBMC) and healthy donor (HD) PBMCs as well as for tumor, peritumor, normal liver, and isotype control are shown. Cand D, Representative
FCM overlay plots for PD-1
þ
MAIT cells in tumor, peritumor, and normal liver gated on total MAIT cells and its summary data (gated on CD4
CD161
þ
TCRva7.2
þ
).
HD, healthy donor; PBMC, peripheral blood mononuclear cells. E, Summary information for the expression of CTLA-4 and TIM-3 on peritumor and tumor-derived
MAIT cells detected by FCM (gated on CD161
þ
TCRva7.2
þ
MR1-tet
þ
). F, Representative FCM overlay plots show PD-1
þ
MAIT cells (black line) had simultaneously
higher CTLA-4 and TIM-3 expression than their PD-1
counterpart (light gray). Lines and error bars are presen ted as the mean SD (,P<0.05; ,P<0.01 by
Mann–Whitney Utest or one-way ANOVA and Tukey multiple comparison tests).
Duan et al.
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central regulators of cell apoptosis (29). Therefore, we compared
the expression of apoptosis-related Bcl-2 family molecules in
MAIT cells between HCC and normal liver tissues. However,
neither proapoptotic molecules like BAX and BID, nor the anti-
apoptotic molecule Bcl-2 showed any differences between
patients with HCC and healthy donors (Supplementary Fig.
S3C). This indicated that apoptosis may not be involved in
reduction of MAIT cell frequency in the tumor tissues.
HCC-infiltrated MAIT cells express higher levels of immune
checkpoints
In HCC, several mechanisms are involved in maintaining
immunosuppressive microenvironment, including the upregula-
tion of immune checkpoints (30). In this study, significant
upregulation of PD-1 on MAIT cells was detected in tumors
compared with peritumor or normal livers in FCM cohort
(mean: 8.8% vs. 4.3% or 3.6%, P<0.001 and P¼0.015,
respectively; Fig. 3C and D). In peripheral blood, patients with
HCC harbored significantly higher PD-1
þ
MAIT cells than healthy
donors (mean: 1.3% vs. 0.6%, P¼0.044; Fig. 3D). Likewise, MAIT
cell expression of another two immune inhibitory molecules,
CTLA-4 (mean: 6.9% vs. 1.6% or 1.2%, P¼0.006) and TIM-3
(mean: 3.2% vs. 1.3% or 0.23%, P¼0.024), was also significantly
increased in the tumor tissues than normal liver tissues in FCM
cohort, whereas mild expression was detected in their peripheral
blood counterparts (Fig. 3E; Supplementary Fig. S4A and S4B).
Moreover, PD-1
high
MAIT cells had simultaneously higher CTLA-4
and TIM-3 expression (Fig. 3F). To validate these findings, we
cocultured MAIT cells sorted from healthy blood with HCC cell
lines using Transwell system. FCM data showed that after cocul-
ture with HCC cells for 48 hours, significant upregulation of PD-1,
CTLA-4, and TIM-3 on MAIT cells was observed in either contact
(P¼0.0010.014) or noncontact manners (P¼0.0010.009;
Supplementary Fig. S4C–S4F). These results indicated that HCC-
infiltrated MAIT cells were educated by tumor cells to be func-
tionally exhausted.
HCC-infiltrated MAIT cells aberrantly produce tumor-
promoting cytokines
Previous studies have indicated that MAIT cells had the ability
to produce both Th1- and Th17-type cytokines after in vitro
stimulation with phorbol 12-myristate 13-acetate (PMA) and
ionomycin or anti-CD3 and anti-CD28 (9, 15, 31). In our study,
we showed that the intrinsic IFNg- and IL17-secreting ability of
tumor-derived MAIT cells was significantly inhibited compared
with their counterparts in peritumor and normal liver tissues (for
IFNgmean: 47.8% vs. 83.1% or 90.2%, P<0.001; for IL17 mean:
5.88% vs. 9.87% or 14.6%, P¼0.028; Fig. 4A and B), after
stimulating with PMA and ionomycin for 5 hours. Consistently,
MAIT cells from peripheral blood of patients with HCC secreted
significantly less IFNgand IL17 compared with healthy donors
(for IFNgmean: 72.9% vs. 90.2%, P¼0.013; for IL17 mean: 4.3%
vs. 14.6%, P<0.001; Supplementary Fig. S5A). Intriguingly, a
significant upregulation of IL8, which has a crucial role in pro-
moting tumor angiogenesis and progression, was detected in
tumor-derived MAIT cells compared with peritumor and healthy
donors [for tissue mean: 0.73% vs. 0.36% or 0.02%, P¼0.017;
for peripheral blood mononuclear cell (PBMC), 0.79% vs. 0.02%,
P¼0.028; Supplementary Fig. S5B and S5C]. By contrast, IL4,
IL10, and IL22 were variable and no differences were observed
among MAIT cells from different sources (Supplementary
Fig. S5B–S5D). Collectively, these results indicated that HCC
microenvironment inhibited the inherent cytokine-secreting
potential of MAIT cells, and promoted the secretion of IL8 by
MAIT cells.
HCC-derived MAIT cells produce minimal granzyme B and
perforin
Besides Th1/Th17 cytokine secretion, effector function of MAIT
cells depends on degranulation to kill sensitized targets (11).
Previous study has indicated that resting human MAIT cells are
featured by a lack of granzyme B and low perforin expression, but
with high expression of granzyme A and granzyme K (13). In this
study, we first determined the expression of granzymes and
perforin on resting MAIT cells. After recovery from liver tissues,
immune cells were stained with fluorescence dye–conjugated
antibodies following a FOXP3 staining protocol. Under ex vivo
condition, neither granzyme B nor perforin was detected in MAIT
cells (Fig. 4C; Supplementary Fig. S5E). Different from granzyme
B and perforin, the secretion of which is tightly controlled in vivo,
both noncytotoxic molecules granzyme A and granzyme K had
high expression in intrahepatic MAIT cells, with a relatively lower
expression of granzyme K in HCC-derived MAIT cells (Fig. 4C;
Supplementary Fig. S5E).
MAIT cells can be activated in a MR1-dependent manner or
through IL12 and IL18 stimulation in a TCR-independent manner
in vitro (31). MAIT cells can also be efficiently activated after
cocultured with nonprofessional antigen-presenting cells (APC)
pretreated with Escherichia coli stimulation, leading to substantial
secretion of granzyme B and perforin (31). Following this strategy,
MAIT cells were successfully activated to produce granzyme B and
perforin after coculture with E. coli–pretreated THP-1 cells for 5
hours (Fig. 4D). However, the frequencies of MAIT cells expres-
sing granzyme B (mean: 1.56% vs. 11.8% or 16.3%, P<0.001)
and perforin (mean: 36.0% vs. 46.3% or 62.7%, P¼0.002) were
significantly lower in tumor compared with peritumor or normal
liver, where MAIT cells from normal liver produced almost 10-
and 2-fold more granzyme B and perforin than HCC tissue,
respectively (Fig. 4E). Furthermore, significantly increased apo-
ptosis and impaired proliferation of HCC cells were observed after
in vitro coculture with MAIT cells derived from PBMCs of healthy
donors or peritumor liver tissues, with markedly upregulated
secretion of GM-CSF, TNFa, IFNg, and MIP1ain the coculture
supernatant (Supplementary Figs. S6 and S7). However, coculture
supernatant from tumor-infiltrating MAIT cells and HCC cells
significantly promoted proliferation and invasion, and inhibited
apoptosis of HCC cells in vitro (Supplementary Fig. S7). These
results indicated that normal MAIT cells could induce
apoptosis of HCC cells, whereas HCC-infiltrated MAIT cells lose
tumor-killing ability, promoted proliferation, and invasion of
HCC cells.
Molecular characterization of intrahepatic MAIT cells by
RNA-seq
Our above data revealed obvious phonotypic differences
between HCC and liver-derived MAIT cells. We further deter-
mined their global gene expression differences by sorting MAIT
cells from paired tumor and peritumor liver tissues in 5 patients
with HCC, as well as from 5 normal liver of healthy donors, for
RNA-seq. RNA-seq analysis detected more than 6,000 significant-
ly differentially expressed genes in tumor-derived MAIT cells
compared with peritumor or normal liver tissues, suggesting an
MAIT Cells in HCC
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Figure 4.
Cytokine secretion and degranulation profile of intrahepatic and peripheral MAIT cells. A, IFNg- and IL17- secreting profiles of intrahepatic MAIT cells after PMA,
ionomycin, and BFA (PIB) stimulation for 5 hours and (B) its summary data (gated on CD4
CD161
þ
TCRva7.2
þ
). C, Representative FCM overlay plots for perforin
and granzymes secreted by peripheral and intrahepatic MAIT cells under still condition (gated on CD161
þ
TCRva7.2
þ
MR1-tet
þ
). HD, healthy donor. D, Bacterial
stimulation led to degranulation and changes in cytotoxic profile of MAIT cells. Sorted MAIT cells from normal liver, peritumor, and tumor tissues were cocultured
with THP-1 cells, pretreated with or without E. coli, and analyzed by FCM to determine perforin and granzyme B secretion in respective tissues. E, Cumulative
data showing reduced frequency of perforin- and granzyme B–expressin g MAIT cells (gated on CD161
þ
TCRva7.2
þ
MR1-tet
þ
). GrB, granzyme B. Lines and error
bars are presented as the mean SD (,P<0.05; ,P<0.01; ,P<0.001 by one-way ANOVA and Tukey multiple comparison tests).
Duan et al.
Clin Cancer Res; 25(11) June 1, 2019 Clinical Cancer Research3310
on January 10, 2022. © 2019 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from
Published OnlineFirst February 5, 2019; DOI: 10.1158/1078-0432.CCR-18-3040
Figure 5.
Gene expression profile of intrahepatic MAIT cells based on RNA-seq data. A, Visualization of 15 tissues by first two principal components (comp) of principle
component analysis computed on gene expression matrix. B, The Venn diagrams of different ially expressed genes for each pair of groups (P for peritumor,
T for tumor, and HDL for normal liver tissues). DEGs were identified by negative binomial generalized linear model (nbGLM) with |log
2
(fold-change)| >1 and
P<0.01. C, Hierarchical clustering for all DEGs in B.D, Enrichment analysis of up- and downregulated DEGs uniquely altered in tumor MAIT cells. Dot size and
color represent the number of genes and Pvalues, respectively. E, Representative expression plot of differentially expressed genes involving cytokine secretion,
tumor promotion, and metabolism in MAIT cells between tumor an d other tissues. |log
2
(fold-change)| >1, P<0.01, by nbGLM.
MAIT Cells in HCC
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aberrant gene expression profile in MAIT cells within HCC micro-
environment (Fig. 5A–C; Supplementary Table S3).
Next, we aimed to determine the biological pathways uniquely
altered in tumor MAIT cells based on those down- and upregu-
lated genes. The downregulated genes, like NFKB1,STAT5B, and
TGFB1 (32–34), were enriched in pathways of cytokine secreting
and cytolysis effector function, consistent with our findings that
effector function of tumor MAIT cells were severely impaired
(Fig. 5D and E; Supplementary Table S3), whereas significantly
upregulated genes in tumor MAIT cells, like APOE and ALDH1A2,
were enriched in pathways involved in aberrant glucose and
cholesterol metabolism (Fig. 5C–E; Supplementary Table S3).
Moreover, 114 differentially expressed genes were shared when
comparing either of tumor, peritumor, and normal liver tissues,
and Gene Ontology analysis indicated that those genes were
mainly involved in metabolism, supporting the notion that
immune cells may undergo metabolic reprogramming in tumor
milieu (Fig. 5B; Supplementary Fig. S8A and S8B; Supplementary
Table S4; refs. 35, 36).
Genes aberrantly upregulated in tumor MAIT cells, like IL8,
CXCL12, and HAVCR2 (TIM-3), foster HCC development
(37, 38). Specially, IL8, an important proinflammatory and
angiogenic factor (39), was one of the most upregulated genes
in tumor-derived MAIT cells, which was consistent with our
previous cytokine secretion result. On the basis of The Caner
Genome Atlas HCC survival data, we showed that patients with
higher expression of IL8 were significantly correlated with reduced
survival (P¼0.012, by log rank test; Supplementary Fig. S8C).
Thus, RNA-seq results clearly indicated an inhibited cytolytic
effector function and induced tumor-promoting potential of
MAIT cells in the HCC microenvironment.
MAIT infiltration correlates with unfavorable outcomes in HCC
On the basis of the unique cytokine secretion pattern, MAIT
cells were reported to have a protective role in constraining
bacterial infection. Although in the tumor context, different
conclusions have been made (18–20, 40). In this study,
we evaluated the relationship of tumor-infiltrating MAIT cell
density with HCC patient prognosis by FCM, qRT-PCR, and
immunostaining.
The 2-year overall survival (OS) and relapse-free survival (RFS)
rates for FCM cohort (n¼50) were 82.3% and 56.6%. To
determine the relationship between MAIT cell infiltration and
survival, patients were divided into MAIT-high and MAIT-low
groups according to MAIT cell percentage in CD3
þ
T cells in
tumors using lowest tertile of MAIT percentage as the cutoff. Log-
rank test showed that patients with high MAIT cell infiltration had
significantly poor RFS (P¼0.031) and relatively lower OS (P¼
0.118) compared with low-infiltration group (Fig. 6A).
TCRva-Ja33 mRNA expression level was previously used to
define MAIT cell infiltration (22, 41). In our study, qRT-PCR
data showed that TCRva-Ja33 mRNA expression was
significantly decreased in tumor compared with peritumor
tissues in a cohort of 207 patients (0.066 vs. 0.222, P<
0.001; Fig. 6B). When stratifying the patients using lowest
tertile of TCRva7.2 mRNA expression level as cutoff, patients
with high TCRva-Ja33 mRNA level showed significantly
dismal survival (P¼0.036) and increased recurrence (P¼
0.034; Fig. 6C). Furthermore, TCRva7.2-Ja33 mRNA expres-
sion level was revealed to be an independent prognostic
factor for both OS [HR 1.92; 95% confidence interval (CI),
1.16–3.24; P¼0.041] and RFS (HR 1.89; 95% CI, 1.14–2.88;
P¼0.046) in addition to the factors like tumor size,
differentiation, capsule, and vascular invasion (Cox propor-
tional hazards regression; Supplementary Table S5).
We further detected absolute number of MAIT cells by TSA
method on HCC TMA (n¼224). MAIT cells were mainly enriched
in portal area (Fig. 6D), the density of which significantly
decreased in tumor compartment than peritumor tissues (mean
number: 4.1 vs. 5.8 cells/core, P<0.001; Fig. 6E). Similarly, in
tumor, when stratifying the patients using lowest tertile of MAIT
cell density as cutoff (1.5 MAIT cells/core), patients with high
MAIT cell infiltration significantly correlated with unfavorable
outcomes (for OS: P¼0.045; for RFS: P¼0.119; Fig. 6F), and
was confirmed as an independent index for OS (Supplementary
Table S6). Then, in the validation cohort, using same cutoff,
we confirmed that high infiltration of MAIT cells was associated
with poor OS (P¼0.005) and RFS (P¼0.01; Supplementary
Fig. S9; Supplementary Table S6). Collectively, survival analysis
of all four cohorts demonstrated that high density of tumor-
infiltrating MAIT cells indicated dismal clinical outcomes in
patients with HCC.
Discussion
Innate immunity plays an important role in antitumor immune
responses, among which MAIT cells are a population of innate-
type T cells preferentially enriched in human liver, indicating its
pivotal role in liver immunology. Importantly, this may be the
first report of such rigorous evaluation of the distribution, phe-
notype, function, and clinical relevance of circulating and infil-
trating MAIT cells in patients with HCC. We found that overall
significant decrease of MAIT cells in tumor and peripheral blood
of patients with HCC signify a systemic dysregulation occurred in
disease state, which was different from the conventional healthy
donors. Of note, tumor-infiltrating MAIT cells displayed an acti-
vating and exhausted phenotype with impaired effector capabil-
ity, and even shifted to produce tumor-promoting cytokines.
Interestingly, our prime findings further reveal that the high
density of tumor-infiltrating MAIT cells significantly correlated
with unfavorable clinical outcomes, and we could possibly infer
that MAIT cells are reprogrammed within the tumor microenvi-
ronment and may contribute to HCC development.
Reduction of circulating MAIT cell frequency has been reported
in various kinds of bacterial infections and viral infections,
including HBV and HCV, as well as in patients with colon
cancer (16, 18–20, 42). Similar to our FCM analysis, we observed
a significant decrease of circulating MAIT cell frequency in patients
with HCC. This reduction may be attributed to tumor-associated
factors or chronic infectious conditions, considering that majority
of patients were HBV
þ
, which couldn't be ruled out. However,
whether MAIT cell infiltration in tumor tissues was decreased or
increased compared with the nontumor counterparts remains
controversial. An increased MAIT cell infiltration in colon cancer
and a decreased infiltration in colorectal hepatic metastases were
observed, which were compared with normal colon and normal
liver respectively (19, 43). Our flow cytometric, IHC, and qRT-
PCR data collectively demonstrated that MAIT cell infiltration was
significantly lower in HCC tumor than of peritumor tissues. Being
aware of the abundance of intrahepatic MAIT cells, we wondered
to compare the absolute number or frequency of infiltrating MAIT
cells aided with multiplex IHC. Our results showed that the
Duan et al.
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Figure 6.
Higher infiltration of MAIT cells correlates with unfavorable clinical outcomes. A, Kaplan–Meier curves for OS and RFS according to MAIT cell frequency in the
FCM cohort (n¼50). B, Relative expression of TCRva7.2 in tumor and peritumor tissues (n¼209, paired ttest). C, Kaplan–Meier curves for OS and RFS
according to TCRva7.2 mRNA expression level in the qRT-PCR cohort. D, Representative images of MAIT cell distribution in peritumor tissues. The subset was
defined as CD3
þ
MDR-1
þ
IL-18R
þ
cells using the TSA method, and immunofluorescence images were scanned at 20on the Vectra Automated Imaging System.
E, MAIT cell absolute number significantly decreased in tumor compared with peritumor (mean number: 4.1 vs. 5.8 cells/core, P<0.001; n¼224, by paired ttest).
F, Kaplan–Meier curves for OS and RFS according to MAIT cell density in the TMA training cohort. Lines and error bars are presented as the mean SD.
MAIT Cells in HCC
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densities of MAIT cells in HCC and paired liver tissues were 7.2
versus 17.4 cells/mm
2
, whereas MAIT cells in colon cancer and
healthy colon mucosa were reported to be 6.1 versus 2.6 cells/
mm
2
(19). Alternatively, when evaluating MAIT cell frequency
among CD3
þ
T cells, similar trend was observed (4.2% and 3.2%
for HCC and colon cancer, respectively; ref. 18). Obviously, MAIT
cell infiltration in HCC slightly surpassed that in colon cancer.
Hence, we assumed that MAIT cells were relatively accumulated in
HCC although less than the peritumor liver tissues, similar to the
findings that MAIT cells heavily infiltrated colorectal hepatic
metastases, but to a lesser extent than the liver (19, 43). A selective
downregulation of chemokine receptors in HCC-infiltrating MAIT
cells observed in our study could be a possible explanation for the
less infiltration.
In both the patients with HCC and healthy donors, MAIT cells
displayed a typical CCR7
CD45RA
CD45RO
þ
CD95
þ
effector
memory phenotype (9, 44). MAIT cells are known to respond
against external stimuli reflecting with the release of cytokines in
the surroundings and have some important implications in
disease regulation. However, HCC-infiltrating MAIT cells' effector
function was found to be severely impaired, and even could
produce tumor-promoting cytokine like IL8. First, both IFNgand
IL17 secreted by tumor-derived MAIT cells were significantly
lower than the counterparts in peritumor and healthy liver tissues.
Meanwhile, our observations also detect an obvious decline of
CD160 and CD127 expression in tumor-derived MAIT cells.
Generally, MAIT cells are known to express higher level of CD160
and CD127 both in peripheral blood and liver tissues, which are
necessary for Th1 and Th17 cytokine production (14, 45). Thus, it
is possible that suppressed IFNgand IL17 secretion of intratumor
MAIT cells could be ascribed to CD160 and CD127 downregula-
tion. Second, MAIT cells' effector function mainly relied on
granzyme B and perforin, which are key molecules necessary for
the efficient cytotoxic activity (46, 47). Our data demonstrated
that HCC-infiltrating MAIT cells produced significantly less gran-
zyme B and perforin than control, indicating the cytotoxic poten-
tial of intratumor MAIT cells was substantially inhibited, assum-
ing a consistent local microenvironmental impact induced the
transition. Third, we found that PD-1, CTLA-4, and TIM-3 expres-
sion was markedly upregulated in HCC-infiltrating MAIT cells,
together with a high expression of the activating markers, CD38
and HLA-DR, which share common features of exhausted T cells,
was thought to be negative immune regulator in the tumor
milieu (26, 48). Finally, a significant upregulation of IL8, known
for tumor-promoting factor (49), was detected in HCC-derived
MAIT cells. Additional in vitro coculture experiments confirmed
the tumor-promoting function mediated by intratumor MAIT
cells as compared with peritumor or circulating MAIT cells.
Altogether, similar to tumor-associated macrophages and neu-
trophils, we postulated that tumor-infiltrating MAIT cells were
reprogrammed to a tumor-promoting direction. As such, cocul-
ture with HCC cells could lead to a significant upregulation of PD-
1, CTLA-4, and TIM-3 on MAIT cells. RNA-seq analysis of infil-
trating MAIT cells further validated that genes related to cell
activation, cytokine secretion, and metabolism were rerouted to
favor a tumor-promoting function in HCC.
Consistent with their tumor-promoting function, we found
that high levels of tumor-infiltrating MAIT cells significantly and
independently correlated with dismal clinical outcomes as estab-
lished in four independent cohorts of patients with HCC. To date,
the prognostic value of MAIT cells has only been reported in colon
cancer, where high densities of tumor-infiltrating MAIT cells were
also associated with poor survival and serum CEA level positively
correlated with MAIT cell infiltration (18). Nonetheless, our
results were mainly derived from patients with HBV-related HCC.
It will be important to validate the prognostic value of MAIT cells
among patients with HCC with other etiologies like in HCV or
fatty liver.
In summary, our findings showed that HCC-infiltrating
MAIT cells were skewed toward a tumor-promoting direction
and were detrimental to patient prognosis. Soluble factors
derived from HCC cells or direct contact with HCC cells can
activate MAIT cells within the tumor milieu, markedly suppress
their cytotoxic capability, and induce them to produce signif-
icant amount of protumor cytokines. These reprogrammed
MAIT cells shifted away from antitumor toward tumor-
suppressive and proangiogenic pathways. Strategies that mod-
ulate the function of MAIT cells may provide a new avenue for
antitumor therapy in HCC.
Transcript Profiling
The RNA-seq data in this paper have been submitted to the National Center
for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO;
accession GSE117627).
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: M. Duan, S. Goswami, J. Fan, X.-M. Zhang, Q. Gao
Development of methodology: M. Duan, S. Goswami, J.-Y. Shi, Q. Gao
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): M. Duan, S. Goswami, J.-Y. Shi, J.-Q. Ma, L.-J. Ma,
R.-B. Xi, Q. Gao
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
computational analysis): M. Duan, S. Goswami, L.-J. Wu, J.-Q. Ma, Z. Zhang,
Y. Shi, Q. Gao
Writing, review, and/or revision of the manuscript: M. Duan, S. Goswami,
X.-Y. Wang, J. Fan, X.-M. Zhang, Q. Gao
Administrative, technical, or material support (i.e., reporting or organizing
data, constructing databases): M. Duan, J.-Y. Shi, Y. Cao, J. Zhou, J. Fan, Q. Gao
Study supervision: M. Duan, X.-Y. Wang, S. Zhang, Y. Cao, J. Zhou, J. Fan,
X.-M. Zhang, Q. Gao
Acknowledgments
The MR1 tetramer technology was developed jointly by Dr. James
McCluskey, Dr. Jamie Rossjohn, and Dr. David Fairlie, and the material
was produced by the NIH. The authors thank Dr. Xiong Ma from the
Department of Gastroenterology and Hepatology, Renji Hospital, Shanghai
Jiaotong University (Shangai, China) for the MR1 tetramer as a generous gift.
This work was supported by the Strategic Priority Research Program (grant
number XDB29030302), Interdisciplinary Innovation Team, Frontier
Science Key Research Project (grant number QYZDB-SSW-SMC036), Chinese
Academy of Sciences (to X.-M. Zhang), National Natural Science Foundation
of China (grant numbers 81772556 and 81572367 to X.-Y. Wang; grant
numbers 81522036 and 81572292 to Q. Gao; and grant number 81872321
to J.-Y. Shi), and National Program for Special Support of Eminent
Professionals (to Q. Gao).
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
Received September 19, 2018; revised December 25, 2018; accepted February
1, 2019; published first February 5, 2019.
Duan et al.
Clin Cancer Res; 25(11) June 1, 2019 Clinical Cancer Research3314
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References
1. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer
statistics, 2012. CA Cancer J Clin 2015;65:87–108.
2. Bruix J, Reig M, Sherman M. Evidence-based diagnosis, staging, and
treatment of patients with hepatocellular carcinoma. Gastroenterology
2016;150:835–53.
3. El-Khoueiry AB, Sangro B, Yau T, Crocenzi TS, Kudo M, Hsu C, et al.
Nivolumab in patients with advanced hepatocellular carcinoma (Check-
Mate 040): an open-label, non-comparative, phase 1/2 dose escalation and
expansion trial. Lancet 2017;389:2492–502.
4. Beatty GL, Gladney WL. Immune escape mechanisms as a guide for cancer
immunotherapy. Clin Cancer Res 2015;21:687–92.
5. Littman DR. Releasing the brakes on cancer immunotherapy. Cell 2015;
162:1186–90.
6. Sia D, Jiao Y, Martinez-Quetglas I, Kuchuk O, Villacorta-Martin C,
de Moura MC, et al. Identification of an immune-specific class of hepa-
tocellular carcinoma, based on molecular features. Gastroenterology 2017;
153:812–26.
7. Woo SR, Corrales L, Gajewski TF. Innate immune recognition of cancer.
Annu Rev Immunol 2015;33:445–74.
8. Jenne CN, Kubes P. Immune surveillance by the liver. Nat Immunol 2013;
14:996–1006.
9. Dusseaux M, Martin E, Serriari N, Peguillet I, Premel V, Louis D, et al.
Human MAIT cells are xenobiotic-resistant, tissue-targeted, CD161hi IL-
17-secreting T cells. Blood 2011;117:1250–9.
10. Kjer -Nielsen L, Patel O, Corbett AJ, Le Nours J, Meehan B, Liu L, et al. MR1
presents microbial vitamin B metabolites to MAIT cells. Nature 2012;491:
717–23.
11. Kurio kaA, Walker LJ, Klenerman P, Willb ergCB. MAIT cells: new guardi ans
of the liver. Clin Transl Immunology 2016;5:e98.
12. Reantragoon R, Corbett AJ, Sakala IG, Gherardin NA, Furness JB, Chen Z,
et al. Antigen-loaded MR1 tetramers define T cell receptor heterogeneity in
mucosal-associated invariant T cells. J Exp Med 2013;210:2305–20.
13. Kurioka A, Ussher JE, Cosgrove C, Clough C, Fergusson JR, Smith K, et al.
MAIT cells are licensed through granzyme exchange to kill bacterially
sensitized targets. Mucosal Immunol 2015;8:429–40.
14. Tang XZ, Jo J, Tan AT, Sandalova E, Chia A, Tan KC, et al. IL-7 licenses
activation of human liver intrasinusoidal mucosal-associated invariant T
cells. J Immunol 2013;190:3142–52.
15. Jeffery HC, van Wilgenburg B, Kurioka A, Parekh K, Stirling K, Roberts S,
et al. Biliary epithelium and liver B cells exposed to bacteria activate
intrahepatic MAIT cells through MR1. J Hepatol 2016;64:1118–27.
16. Eberhard JM, Kummer S, Hartjen P, H€
ufner A, Diedrich T, Degen O, et al.
Reduced CD161þMAIT cell frequencies in HCV and HIV/HCV co-infec-
tion: is the liver the heart of the matter? J Hepatol 2016;65:1261–3.
17. Walke r LJ, Marrinan E, Muenchhoff M, Ferguson J, Kloverpris H, Cheroutre
H, et al. CD8alphaalpha expression marks terminally differentiated human
CD8þT cells expanded in chronic viral infection. Front Immunol 2013;4:
223.
18. Ling L, Lin Y, Zheng W, Hong S, Tang X, Zhao P, et al. Circulating and
tumor-infiltrating mucosal associated invariant T (MAIT) cells in colorec tal
cancer patients. Sci Rep 2016;6:20358.
19. Zabijak L, Attencourt C, Guignant C, Chatelain D, Marcelo P, Marolleau J-P,
et al. Increased tumor infiltration by mucosal-associated invariant T cells
correlates with poor survival in colorectal cancer patients. Cancer Immunol
Immunother 2015;64:1601–8.
20. Sundstr€
om P, Ahlmanner F, Ak
eus P, Sundquist M, Als
en S, Yrlid U, et al.
Human mucosa-associated invariant T cells accumulate in colon adeno-
carcinomas but produce reduced amounts of IFN-g. J Immunol 2015;195:
3472–81.
21. Shi JY, Gao Q, Wang ZC, Zhou J, Wang XY, Min ZH, et al. Margin-infiltrating
CD20(þ) B cells display an atypical memory phenotype and correlate with
favorable prognosis in hepatocellular carcinoma. Clin Cancer Res 2013;19:
5994–6005.
22. Lepore M, Kalinichenko A, Colone A, Paleja B, Singhal A, Tschumi A, et al.
Parallel T-cell cloning and deep sequencing of human MAIT cells reveal
stable oligoclonal TCRbeta repertoire. Nat Commun 2014;5:3866.
23. Picelli S, Bjorklund AK, Reinius B, Sagasser S, Winberg G, Sandberg R. Tn5
transposase and tagmentation procedures for massively scaled sequencing
projects. Genome Res 2014;24:2033–40.
24. Gherardin NA, Souter MN, Koay HF, Mangas KM, Seemann T, Stinear TP,
et al. Human blood MAIT cell subsets defined using MR1 tetramers.
Immunol Cell Biol 2018;96:507–25.
25. Yong YK, Saeidi A, Tan HY, Rosmawati M, Enstrom PF, Batran RA, et al.
Hyper-expression of PD-1 is associated with the levels of exhausted and
dysfunctional phenotypes of circulating CD161(þþ)TCR iValpha7.2(þ)
mucosal-associated invariant T cells in chronic hepatitis B virus infection.
Front Immunol 2018;9:472.
26. Baitsch L, Baumgaertner P, Devevre E, Raghav SK, Legat A, Barba L, et al.
Exhaustion of tumor-specific CD8(þ) T cells in metastases from melanoma
patients. J Clin Invest 2011;121:2350–60.
27. Henson SM, Akbar AN. KLRG1–more than a marker for T cell senescence.
Age 2009;31:285–91.
28. Billerbeck E, Kang YH, Walker L, Lockstone H, Grafmueller S, Fleming V,
et al. Analysis of CD161 expression on human CD8þT cells defines a
distinct functional subset with tissue-homing properties. Proc Natl Acad Sci
U S A 2010;107:3006–11.
29. Reed JC. Proapoptotic multidomain Bcl-2/Bax-family proteins: mechan-
isms, physiological roles, and therapeutic opportunities. Cell Death Differ
2006;13:1378–86.
30. Hato T, Goyal L, Greten TF, Duda DG, Zhu AX. Immune checkpoint
blockade in hepatocellular carcinoma: current progress and future direc-
tions. Hepatology 2014;60:1776–82.
31. Ussher JE, Bilton M, Attwod E, Shadwell J, Richardson R, de Lara C, et al.
CD161þþ CD8þT cells, including the MAIT cell subset, are specifically
activated by IL-12þIL-18 in a TCR-independent manner. Eur J Immunol
2014;44:195–203.
32. Lougar is V, Patrizi O, Baronio M, Tabellini G, Tampella G, Damiati E, et al.
NFKB1 regulates human NK cell maturation and effector functions.
Clin Immunol 2017;175:99–108.
33. Luedde T, Schwabe RF. NF-kappaB in the liver–linking injury, fibrosis and
hepatocellular carcinoma. Nat Rev Gastroenterol Hepatol 2011;8:108–18.
34. Smith AJ, Humphries SE. Cytokine and cytokine receptor gene polymorp h-
isms and their functionality. Cytokine Growth Factor Rev 2009;20:43–59.
35. Biswas SK. Metabolic reprogramming of immune cells in cancer progres-
sion. Immunity 2015;43:435–49.
36. Wu X, Thi VLD, Liu P, Takacs CN, Xiang K, Andrus L, et al. Pan-genotype
hepatitis E virus replication in stem cell–derived hepatocellular systems.
Gastroenterology 2018;154:663–74. e7.
37. Kim H-D, Song G-W, Park S, Jung MK, Kim MH, Kang HJ, et al. Association
between expression level of PD1 by tumor-infiltrating CD8þT cells and
features of hepatocellular carcinoma. Gastroenterology 2018;155:
1936–50.
38. Liu H, Liu Y, Liu W, Zhang W, Xu J. EZH2-mediated loss of miR-622
determines CXCR4 activation in hepatocellular carcinoma. Nat Commun
2015;6:8494.
39. Park SY, Han J, Kim JB, Yang MG, Kim YJ, Lim HJ, et al. Interleukin-8 is
related to poor chemotherapeutic response and tumourigenicity in hepa-
tocellular carcinoma. Eur J Cancer 2014;50:341–50.
40. Zheng C, Zheng L, Yoo J-K, Guo H, Zhang Y, Guo X, et al. Landscape of
infiltrating T cells in liver cancer revealed by single-cell sequencing. Cell
2017;169:1342–56.
41. Peterfalvi A, Gomori E, Magyarlaki T, Pal J, Banati M, Javorhazy A, et al.
Invariant Va7. 2-Ja33 TCR is expressed in human kidney and brain tumors
indicating infiltration by mucosal-associated invariant T (MAIT) cells.
Int Immunol 2008;20:1517–25.
42. Salou M, Franciszkiewicz K, Lantz O. MAIT cells in infectious diseases.
Curr Opin Immunol 2017;48:7–14.
43. Shaler CR, Tun-Abraham ME, Skaro AI, Khazaie K, Corbett AJ, Mele T, et al.
Mucosa-associated invariant T cells infiltrate hepatic metastases in patients
with colorectal carcinoma but are rendered dysfunctional within and
adjacent to tumor microenvironment. Cancer Immunol Immunother
2017;66:1563–75.
44. Takata H, Takiguchi M. Three memory subsets of human CD8þT cells
differently expressing three cytolytic effector molecules. J Immunol 2006;
177:4330–40.
45. Tu TC, Brown NK, Kim TJ, Wroblewska J, Yang X, Guo X, et al. CD160 is
essential for NK-mediated IFN-gamma production. J Exp Med 2015;212:
415–29.
www.aacrjournals.org Clin Cancer Res; 25(11) June 1, 2019 3315
MAIT Cells in HCC
on January 10, 2022. © 2019 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from
Published OnlineFirst February 5, 2019; DOI: 10.1158/1078-0432.CCR-18-3040
46. Makedonas G, Banerjee PP, Pandey R, Hersperger AR, Sanborn KB, Hardy
GA, et al. Rapid up-regulation and granule-independent transport of
perforin to the immunological synapse define a novel mechanism of
antigen-specific CD8þT cell cytotoxic activity. J Immunol 2009;182:
5560–9.
47. Harari A, Bellutti Enders F, Cellerai C, Bart PA, Pantaleo G. Distinct
profiles of cytotoxic granules in memory CD8 T cells correlate with
function, differentiation stage, and antigen exposure. J Virol 2009;83:
2862–71.
48. Sakuishi K, Apetoh L, Sullivan JM, Blazar BR, Kuchroo VK, Anderson AC.
Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore
anti-tumor immunity. J Exp Med 2010;207:2187–94.
49. Coussens LM, Zitvogel L, Palucka AK. Neutralizing tumor-promoting
chronic inflammation: a magic bullet? Science 2013;339:286–91.
Clin Cancer Res; 25(11) June 1, 2019 Clinical Cancer Research3316
Duan et al.
on January 10, 2022. © 2019 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from
Published OnlineFirst February 5, 2019; DOI: 10.1158/1078-0432.CCR-18-3040
2019;25:3304-3316. Published OnlineFirst February 5, 2019.Clin Cancer Res
Meng Duan, Shyamal Goswami, Jie-Yi Shi, et al.
and Indicate Poor Outcome in Hepatocellular Carcinoma
Activated and Exhausted MAIT Cells Foster Disease Progression
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