Published OnlineFirst August 21, 2018; DOI: 10.1158/2326-6066.

CIR-18-0020

Research Article Cancer

Immunology
Research
TLR2 Promotes Glioma Immune Evasion by
Downregulating MHC Class II Molecules in
Microglia
Jiawen Qian1,2, Feifei Luo2,3, Jiao Yang1,2, Jun Liu1, Ronghua Liu1, Luman Wang1,
Chen Wang1,2, Yuting Deng1,2, Zhou Lu1, Yuedi Wang1,2, Mingfang Lu1,
Ji-Yang Wang1, and Yiwei Chu1,2

Abstract
Gliomas, the most common primary neoplasms in the
brain, are notorious for their ability to evade the immune
response. Despite microglial infiltration in gliomas, expres-
sion of MHC class II molecules in those microglia is
compromised. Here, we report that Toll-like receptor 2
(TLR2) activation downregulated expression of MHC class
II molecules in microglia in an orthotopic murine glioma
model. TLR2-induced microglial impairment hindered the
proliferation and activation of CD4þ T cells, which facili-
tated glioma immune evasion. TLR2-induced downregula-
tion of MHC class II molecules was caused by suppression
of the master regulator of MHC class II molecule transcrip-
tion, Ciita. TLR2 activation triggered downstream MAPK/
ERK1/2 signaling and loss of histone H3 acetylation at
Ciita promoters, which in turn inhibited Ciita expression.
In glioblastoma tissues, various endogenous TLR2 ligands,
including the heat shock proteins that are endogenous TLR2
ligands, were upregulated, a response that correlated with
CIITA inhibition. Thus, TLR2 promotes glioma immune-
system evasion. These results advance our understanding
of microglia as antigen-presenting cells in the context
of glioma. In the glioma tumor microenvironment, TLR2
activation of microglia induces downregulation of micro-
glial MHC class II expression. Impaired MHC class II
expression limits T-cell–dependent antitumor immunity.
Cancer Immunol Res; 6(10); 1220–33.
2018 AACR.

Introduction Normally, microglia scan the environment to detect pathogens

and other disturbances (8). Resting microglia express low
Gliomas are the most common primary intracranial malig-
amounts of the accessory molecules needed for antigen presen-
nancy in humans, and despite clinical intervention, the prog-
tation (8). Under pathologic conditions like multiple sclerosis
nosis for patients with glioma remains dismal (1). Median
and infections, microglia move toward lesions, transform to
overall survival of the patients with IDH1/2 wild-type (WT)
amoeboid-like phagocytic cells, and upregulate expression of
glioblastoma multiforme (GBM) is shorter than 15 months (2).
MHC class II molecules (MHC class II; refs. 6, 9), suggesting their
Fortunately, immunotherapies are being investigated for glio-
participation in antigen presentation. Glioma-associated micro-
mas, including peptide or dendritic cell–based vaccines, chi-
glia (GAM), which show compromised ability to upregulate MHC
meric T-cell receptors, and checkpoint inhibitors (3). Many of
class II expression in a murine model (10), are likely less effective
these strategies are dependent on the T-cell–mediated immune
than they could be at activating T helper cells. In humans,
response, which needs local antigen-presenting cells (APCs) for
expression of MHC class II by microglia is reduced in high-grade
initiation and sustenance (4). Microglia, the major APC subset
gliomas (11). Expression of costimulatory molecules CD80 and
within the central nervous system (CNS; refs. 5, 6), infiltrate
CD86 in glioma-infiltrating microglia (10, 12) is also reduced.
gliomas (7). However, microglia are functionally compromised
Expression of MHC class II, CD80, and CD86 increased after
in the glioma microenvironment, thus limiting the effective-
microglia were isolated from intracranial tumors and cultured
ness of T-cell–dependent tumor eradication.
ex vivo (13). These studies indicate that the tumor microenviron-
ment renders GAM incompetent to induce T-cell activation
and antiglioma immunity. The mechanism by which molecules
1
Department of Immunology, School of Basic Medical Sciences, and Institute of related to antigen-presenting in GAM are downregulated has not
Biomedical Sciences, Fudan University, Shanghai, P.R. China. 2Biotherapy been identified.
Research Center, Fudan University, Shanghai, P.R. China. 3Department of
Toll-like receptors (TLR) are necessary for microglia to ini-
Digestive Diseases, Huashan Hospital, Fudan University, Shanghai, P.R. China.
tiate the innate and adaptive immune responses in the CNS
Note: Supplementary data for this article are available at Cancer Immunology (14). TLRs are activated not only by pathogen-associated
Research Online (http://cancerimmunolres.aacrjournals.org/).
molecular patterns but also by danger-associated molecular
Corresponding Author: Yiwei Chu, Fudan University, No. 138, Yi Xue Yuan Rd., patterns, such as heat shock proteins (HSP) and extracellular
Shanghai 200032, P.R. China. Phone: 8602154237324; Fax: 8602154237324; matrix (ECM) components (15). Activation of TLR signaling
E-mail: yiwei_chu@126.com
pathways leads to the induction of several genes that function
doi: 10.1158/2326-6066.CIR-18-0020 in host defense, including those encoding inflammatory cyto-

2018 American Association for Cancer Research. kines and chemokines (16). In certain types of innate immune

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 Published OnlineFirst August 21, 2018; DOI: 10.1158/2326-6066.CIR-18-0020
cells, such as macrophage and dendritic cells, activation
of TLR signaling is associated with the expression of costimu-
latory molecules and MHC molecules (17, 18). Whether TLR
signaling regulates the antigen-presentation function of GAM
remains unknown.
In the present study, we found that TLR2 expression was
upregulated in GAM. Glioma-derived factors impaired MHC class
II expression of microglia in a TLR2-dependent manner, which
dampened the activation and proliferation of CD4þ T cells.
According to The Cancer Genome Atlas (TCGA) database and
the Human Protein Atlas database, several kinds of endogenous
TLR2 ligands are upregulated in human glioblastoma tissues.
Among these ligands, we identified HSPs as inducers of TLR2
activation in correlation with the MHC class II inhibition in
human GBM samples. Our results clarified the mechanisms
behind glioma-induced impairment of APCs and revealed the
function of TLR in tumor immune evasion.
Materials and Methods
Mice
C57BL/6 mice (6–8 weeks) were purchased from Shanghai Slac
Laboratory Animal Co., Ltd. TLR2-deficient C57BL/6 mice were
purchased from the Model Animal Research Center, Nanjing
University (Nanjing, China). OT II transgenic mice were kindly
provided by Dr. Rui He (Fudan University, Shanghai, China). All
mice were housed under the pathogen-free condition in the
Animal Facility of Fudan University (Shanghai, China). All animal
experiments adhered to the Guidelines for the Care and Use of
Laboratory Animals (No. 55 issued by Ministry of Health, the
People's Republic of China on January 25, 1998) and were
approved by the Institutional Animal Care and Use Committee
of Fudan University.
Cell lines
The murine glioma cell line, GL261, was kindly provided by
Dr. Liangfu Zhou (Huashan Hospital, Shanghai, China) in 2012.
The murine microglial cell line, BV2, was obtained from the
Shanghai Cell Bank of the Chinese Academy of Science (Shanghai,
China) in 2014. After obtaining the cell lines, cells were expanded
to passage 5 and stored in aliquots in liquid nitrogen. All cell lines
were grown in DMEM/F12 (Thermo Fisher) supplemented with
10% heat-inactivated FBS (Thermo Fisher), 2 mmol/L glutamine
(Thermo Fisher), 100 U/mL penicillin (Thermo Fisher), and
100 mg/mL streptomycin (Thermo Fisher). Cell cultures were
maintained in the incubator at 37 C in a humidified atmosphere
of 5% CO2/95% with routine checks for mycoplasma contami-
nation every 3 months. All cells were used in experiments before
they reach passage 15. All cell lines were authenticated using short
tandem repeat profiling in 2015. Glioma-derived conditioned
medium (GCM) was collected after 24 hours from 80% confluent
GL261 cultures, filtered through 0.2 mm filter, and applied to
microglia cultures.
Primary adult microglia culture, cell stimulation, and T-cell
coculture assay
Microglia were prepared from 6- to 8-week-old mice: the
cerebrum was dissected and transferred to cold PBS. The tissue
was triturated mechanically and collected in a 50-mL Falcon
tube and washed with PBS by centrifuging for 7 minutes at
500  g at 4 C. The supernatant was discarded, and pellets
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TLR2 Downregulates Microglial MHC Class II in Glioma
were resuspended and adjusted to 37% Percoll. Percoll gradients
(70%/37%/30%/0%) were set up and centrifuged for 5 minutes at
500  g at 18 C with low acceleration with the brake off.
Mononuclear cells were collected at the 70%/37% interface and
washed with PBS. Microglia were enriched by adhesion to CD11b
microbeads (BD Bioscience) and harvested for purity check and
further tests. For cultivation, microglia were plated onto 24-well
plates at a density of 1  105 cells per well with DMEM/F12
supplemented with 10% heat-inactivated FBS, 2 mmol/L gluta-
mine, 100 U/mL penicillin, 100 mg/mL streptomycin, 5 ng/mL
recombination TGFb1 (Miltenyi), and 10 ng/mL Recombinant
Mouse M-CSF Protein (R&D). Half of the medium was changed
every 3 days, for a total of 10 to 14 days.
In most cases of the study, for microglial TLR2 activation,
primary microglia or BV2 cells were treated with 100 ng/mL
Pam3CSK4 (Invivogen) or GCM (20%, vol/vol) for 24 hours.
For MHC class II induction, primary microglia or BV2 cells were
treated with 20 ng/mL IFNg (PeproTech) for another 24 hours.
Other TLR agonists (Invivogen) used in the study include Poly
(I:C) (10 mg/mL), LPS (100 ng/mL), Pam2CSK4 (100 ng/mL),
and CpG (2.5 mmol/L). The inhibitors in specific experiments
were used by pretreatment of cell cultures for 30 minutes. The
working concentrations of inhibitors are listed as follows: PI3K
inhibitor (CST), Ly294002 (10 mmol/L); ERK1/2 inhibitor
(Selleck), U0126 (10 mmol/L); p38 inhibitor (Selleck), SB203580
(10 mmol/L); JNK inhibitor (Selleck), SP600125 (10 mmol/L);
DNMT inhibitor (Sigma-Aldrich), 5-aza-20 -deoxycytidine
(5-AZA, 1 mmol/L); histone deacetylase (HDAC) inhibitor (MCE),
Trichostatin A (TSA, 0.1 mmol/L); HDAC inhibitor (Selleck),
Nicotinamide (Nico, 10 mmol/L).
For T-cell coculture assays, freshly isolated adult microglia were
plated onto 96-well plates at a density of 1  105 cells per well.
Half of the medium was changed every 3 days, for a total of 7 days.
On day 8, microglia were treated with 100 ng/mL Pam3CSK4 or
20% GCM for 24 hours. Then, microglia were washed with PBS
after stimulation. The CD4þ T Cell Isolation Kit (Miltenyi)
was used for purification of CD4þ T cells from the spleen of
OT II mice. CD4þ T cells were stained with carboxyfluorescein
succinimidyl ester (CFSE) dye (Invitrogen) following the manu-
facturer's instructions. The microglia were washed with PBS for
3 times, and then cocultured with CD4þ T cells (4  105 cells per
well) for 4 days supplied with 0.1 mmol/L OVA323-339 peptides
(Sigma-Aldrich). After coculture, both microglia and T cells were
determined by flow cytometric analysis.
Stereotactic intracranial tumor inoculation and cannula
implantation
GL261 glioma cells were harvested and diluted to a concen-
tration of 2  107 cells/mL. Anesthetized mice were immobi-
lized and mounted onto a stereotactic head holder in the flat-
skull position. The skin of the skull was dissected in the midline
by a scalpel. The skull was carefully drilled with a 20-gauge
needle tip (ML: þ2.0 RC: þ1.0 DV: 3.0 mm). Then a microliter
Hamilton syringe was inserted to a depth of 3 mm and retracted
to a depth of 2.5 mm from the dural surface. Note that 5 mL
(2  104 cells/mL) cell suspension or PBS was slowly injected
over 2 minutes. The needle was then slowly taken out from
the injection canal, and the skin was sutured. For cannula
implantation, a hole was drilled for insertion of the cannula
(ML: þ1.0 RC: –0.4 DV: 2.0 mm), which was used for intracer-
ebroventricular (ICV) injection. In the study, Pam3CSK4 (5 mg)
Cancer Immunol Res; 6(10) October 2018 1221
 Published OnlineFirst August 21, 2018; DOI: 10.1158/2326-6066.CIR-18-0020
Qian et al.
were delivered to the lateral ventricle via cannula on days 6, 12,
and 18 after tumor inoculation, and mice were sacrificed on day
20 for further analysis.
RNA isolation, reverse transcription, and quantitative RT-PCR
RNA was isolated with RNAiso (Takara) following the man-
ufacturer's protocol. RNA samples were reversely transcribed
using the PrimeScriptTM RT reagent Kit with gDNA Eraser
(Perfect Real Time; Takara), and gene expression was detected
using the SYBR Premix Ex TaqTM II (Tli RNaseH Plus) Kit
(Takara). All real-time PCR amplifications were performed in
triplicates in a 20 mL reaction volume with the indicated primer
pairs (Supplementary Table S1). RT-PCRs were performed
using 7500 Fast Real-Time PCR System (Applied Biosystems).
The amount of target mRNA was normalized to the expression
level of b-actin generated from the same sample and subse-
quently to controls. Relative expression was calculated as 2DCt.
Fold induction was calculated as 2DDCt.
Chromatin immunoprecipitation
Primary microglia (contain 5.0  105–1.0  106 cells per
sample) were used for following chromatin immunoprecipitation
(ChIP), performed using the SimpleChIP Enzymatic Chromatin
IP Kit (Cell Signaling Technology) according to the manufac-
turer's protocol. DNA was eluted and purified, followed by qPCR
analysis using primers specific for Ciita promoter (Supplementary
Table S1). All antibodies used for these experiments were listed in
Supplementary Table S1, and normal rabbit IgG (Cell Signaling
Technology) was used as the isotype control. The fold enrichment
method was used for data normalization.
RNA-seq
Total RNA was extracted using TRIZOL Reagent (Thermo
Fisher) following the manufacturer's protocol. Paired-end
libraries were synthesized by using the TruSeq RNA Sample
Preparation Kit (Illumina) following TruSeq RNA Sample
Preparation Guide. The cluster was generated by cBot with the
library diluted to 10 pmol/L and then was sequenced on the
Illumina HiSeq X Ten (Illumina). The P value significance
threshold in multiple tests was set by the FDR. The fold changes
were also estimated according to the fragments per kilobaseof
exon per million fragments mapped (FPKM) in each sample.
The differentially expressed genes were selected using the fol-
lowing filter criteria: FDR  0.05 and fold change  2. R
language was used for Kyoto Encyclopedia of Genes and Gen-
omes (KEGG) pathway analysis and Gene Ontology analysis.
The Gene Expression Omnibus (GEO) accession number for
the RNA-seq data is GSE109297.
Immunofluorescence
For frozen sections, mice were fully anesthetized, transcardially
perfused with 20 mL of PBS, and followed by 20 mL of parafor-
maldehyde (PFA; 4% in PBS). Tissues were incubated in PFA
overnight at 4 C, then transferred to sucrose (20%) for 1 day at
4 C, and finally transferred to sucrose (30%) for another day at
4 C. All tissues were embedded and frozen in Optimal Cutting
Temperature compound, and the brains were mounted on a
freezing microtome (Leica). Sections (20 mm thick) were obtained
and stored at –80 C for all subsequent staining and analysis.
For immunofluorescence, sections were thawed and dried at
room temperature and rinsed in PBS. For cell culture, cells were
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washed with PBS and followed by 4% PFA cell fixation (5 minutes
at room temperature). Samples were permeabilized with 0.25%
Triton-X 100 for 15 minutes at room temperature and blocked in
blocking buffer containing 10% donkey serum for 2 hours at
room temperature or overnight at 4 C. Then, samples were
incubated with indicated primary antibodies (Supplementary
Table S2) overnight at 4 C. Samples were then washed with PBS
and incubated with the appropriate fluorophore-conjugated sec-
ondary antibodies: Alexa Flour-488, 594 (Thermo Fisher), and
Cy3 (Jackson ImmunoResearch Laboratory) at a dilution of 1:500
in 1% BSA for 1 hour at room temperature, and 40 , 6-diamidino-2-
phenylindole (DAPI) were used as a counterstain. Images were
acquired by a fluorescence microscope Olympus IX73 (Olympus)
or a spectral confocal microscope (TCS SP5, TCS SP8; Leica).
Appropriate gain and black level settings were determined by
control tissues stained with secondary antibodies alone. Analyses
of images were performed using ImageJ software (U.S. National
Institutes of Health).
Fluorescence-activated cell sorting analyses
For fluorescence-activated cell sorting (FACS) analysis of brain
tumor–infiltrated immune cells, mice were euthanized at the
defined endpoint or when symptomatic (poor grooming, lethar-
gy, weight loss, or seizures). Brains were harvested with olfactory
bulbs and cerebella removed, dissociated mechanically with
Dounce homogenizers to make homogenates, and then forced
through a filter for single-cell suspension and washed with PBS.
Cell pallets were resuspended in 37% Percoll. Percoll gradients
(70%/37%/30%/0%) were set up and centrifuged for 40 minutes
at 200  g at 18 C with low acceleration with the brake off.
Mononuclear cells were collected at 70%/37% interface and
washed with PBS. Mononuclear cells were stained afterward with
the respective antibodies for FACS analysis.
For flow cytometry, cells were counted and incubated with Fc
block (eBioscience) for 30 minutes, followed by another
30-minute incubation with conjugated antibodies for extracellu-
lar markers. For intracellular cytokine detection, cells were stim-
ulated in vitro with Cell Stimulation Cocktail (eBioscience) for
5 hours at 37 C before FACS analysis. After stimulation, the cells
were stained for surface markers and cytokines with Intracellular
Fixation and Permeabilization Buffer Set (eBioscience). All anti-
bodies used for these experiments were listed in Supplementary
Table S2. Proper isotype controls and compensation controls
were performed in parallel. A BD Biosciences Canto II was used
for flow cytometry, and a Beckman Coulter MoFlo XDP was
used for cell sorting. FlowJo software (Tree Star) was used for
FACS data analysis.
Immunoblotting analysis
The procedure was performed as described previously. Briefly,
proteins were extracted using radio-immunoprecipitation assay
(RIPA) buffer (Cell Signaling Technology) according to the man-
ufacturer's protocol. Equal amounts of protein were resolved by
SDS/PAGE (Beyotime) and then transferred to polyvinylidene
difluoride membranes (Merck Millipore). Membranes were
probed with the primary Abs after blocking with 5% nonfat milk.
All antibodies used for these experiments were listed in Supple-
mentary Table S2. After that, membranes were washed and
incubated with horseradish peroxidase–conjugated secondary
Abs. Specific proteins were detected by enhanced chemilumines-
cence (Thermo Fisher).
Cancer Immunology Research
 Published OnlineFirst August 21, 2018; DOI: 10.1158/2326-6066.CIR-18-0020
Data presentation and statistical analysis
GraphPad Prism 6.0 (GraphPad Software Inc.) was used for all
data analysis. Parametric data are presented as mean  SEM.
Differences between two groups were analyzed using the Student
unpaired t test. ANOVA was used to investigate more than two
groups, and the Pearson correlation coefficient was used to
analyze the correlation among the levels of genes. For survival
curves, the cumulative survival time was calculated using
the Kaplan–Meier method and analyzed using the log-rank test.
P < 0.05 was considered statistically significant.
Results
Microglia accumulate at the tumor site and upregulate TLR2
expression
To investigate the mechanism underlying the downregulation
of molecules related to antigen-presenting in GAM, we used the
GL261 murine glioma model due to its immunogenic features
and predictable tumor progression (19). The GL261 tumor-
bearing WT C57BL/6 mice died around 4 weeks after GL261
inoculation. The histologic hematoxylin and eosin (H&E) stain-
ing showed that tumor size expanded 20 days after of inoculation
(Supplementary Fig. S1A), with significant loss of body weight
(Supplementary Fig. S1B). Weight of spleens of tumor-bearing
mice significantly increased between day 10 and day 20 (Supple-
mentary Fig. S1C), concomitant with increased numbers of
tumor-infiltrating peripheral immune cells (CD45hi cells; Sup-
plementary Fig. S1D and S1E). These results indicate that immune
microenvironment is established along with glioma progression.
Around day 20, the balance tilts in favor of the tumor, resulting in
rapid deterioration of the mouse.
We found that expression of microglial protumor genes, includ-
ing Il6, Il10, Tgfb2, Mmp14 (encoding MT-MMP1), Cd274 (encod-
ing PDL1), and Ccl2 (7), was elevated in the tumor-bearing brain;
expression of some of these genes peaked on day 20 (Supple-
mentary Fig. S1F). Next, we investigated the microglial infiltration
pattern on day 20 after GL261 inoculation. Immunofluorescence
staining showed that Iba1þ microglia accumulated along the
tumor perimeter, where they change morphology from ramified,
characteristic of normal tissue, to the amoeboid form character-
istic of tumor invasion. Within the tumor, microglia appear
as rod-like shapes (Fig. 1A). Based on the Iba1þ cell counts and
Iba1-labeled area, microglial density increased successively from
normal tissue, tumor rim, invasive margin, to intratumoral region
(Fig. 1B).
We then focused on expression of TLRs in GAM. In the normal
brain, according to an RNA-sequencing database of the cerebral
cortex (20), most TLRs are expressed by microglia. Certain TLRs,
namely TLR2, TLR7, and TLR9, are predominantly expressed in
microglia (Fig. 1C). We compared the TLR expression pattern
between microglia isolated from the normal and tumor-bearing
brain by qPCR analysis. The results revealed that Tlr2 and Tlr8
mRNA levels were upregulated in the GAM (Fig. 1D), which is
consistent with the previous study of the GL261 model (21). In
a genetically engineered mouse model of glioma, TLR2 is also the
predominant TLR expressed by GAM (22). Expression of TLR2
in human glioma samples was higher than that in normal tissues
and was associated with higher-grade malignancy (Fig. 1E).
The amount of TLR2 protein in GAM was consistent with the
amount of mRNA (Fig. 1F and G). TLR2 is mainly expressed in
microglia and peripheral macrophages in the tumor-bearing brain
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TLR2 Downregulates Microglial MHC Class II in Glioma
(Supplementary Fig. S2A–S2C). These findings indicate that TLR2
is upregulated in GAM and may modulate GAM function.
TLR2 activation downregulates MHC class II in microglia
To investigate the role of TLR2 in the modification of anti-
gen-presenting function of GAM, we established the GL261
model in Tlr2/ and WT mice. The analysis of cumulative
survival revealed that Tlr2/ mice survived longer than WT
mice (Fig. 2A) with less tumor burden on day 20 according to
the histologic H&E staining (Fig. 2B). We evaluated the expres-
sion of molecules related to antigen-presenting in GAM by flow
cytometry (Supplementary Fig. S3). The brain-resident micro-
glia were gated as CD45loCD11bþCD49d/lo cells according to
the previous report (22). Molecules related to antigen-present-
ing function on Tlr2/ and WT-derived microglia were mea-
sured. Results showed no difference in the expression of costi-
mulatory molecules (CD80 and CD86) or the coinhibitory
molecule (PD-L1) between Tlr2/ and WT mice, whereas
expression of MHC class II was upregulated in microglia from
Tlr2/ mice (Fig. 2C). Histology indicated the MHC class IIþ
microglia were mainly in the tumor invasive margin, and that
microglia in Tlr2/ mice expressed more MHC class II than
WT mice (Fig. 2D).
To clarify that MHC class II downregulation was caused by
activation of TLR2 on microglia rather than on other cells, we
set up a primary adult microglia culture system. Murine adult
microglia were isolated with the purity checked by flow cyto-
metry (Supplementary Fig. S4A) and cultured in the presence of
M-CSF and TGFb1. TGFb1 is essential for maintenance and
survival of microglia (23). The reactivity of the primary micro-
glia was tested (Supplementary Fig. S4B). The expression of
microglia-specific genes (23) was higher in adult microglia
cultured with TGFb1 (Supplementary Fig. S4C). GCM was used
to stimulate the primary cultured microglia. MHC class II
expression could be induced in normal microglia by IFNg, but
less so in GCM-treated microglia (Fig. 2E). Primary microglia
appeared more amoeboid in shape, consistent with the previ-
ous observation in tumor sites. These results indicate that
tumor-derived factors could impair microglial MHC class II
expression.
When WT and Tlr2/ microglia were treated with GCM, and
we observed no morphologic change in Tlr2/ microglia, we
evaluated the MHC class II expression of GCM-treated Tlr2/
microglia by flow cytometry and found that the MHC class II level
remained unchanged (Fig. 2F), indicating that tumor-derived
endogenous TLR2 ligands triggered microglial MHC class II
downregulation. We then applied TLR2 agonist Pam3CSK4 to
mimic the GCM-induced effect. The MHC class II level of
Pam3CSK4-treated cells showed a dose-dependent decrease
compared with control cells (Fig. 2G). We confirmed that
TLR2-mediated downregulation of MHC class II also occurs in
bone marrow–derived macrophage (BMDM) (Supplementary
Fig. S5A and S5B) and CD45þCD11bþ peripheral myeloid cells
(Supplementary Fig. S5C). We tested the TLR2-induced effect
in vivo by ICV injection of Pam3CSK4 in the glioma model. We
buried the cannula in the lateral ventricle ipsilateral to the tumor
inoculation site. Pam3CSK4 or PBS was given via cannula on day 6
after tumor inoculation and given every 6 days for a total of 3
times. All animals were sacrificed on day 20 for further analysis
(Fig. 2H). We found that MHC class II expression in microglia was
lower in the Pam3CSK4-treated group in a time-dependent
Cancer Immunol Res; 6(10) October 2018 1223
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Qian et al.

Figure 1.
Microglia accumulate at tumor sites and upregulate TLR2 expression. A, Representative staining for Iba1 (green) and DAPI (blue) in the tumor-bearing brain
on day 20 after GL261 inoculation. Right plots, different regions of the tumor-bearing brain. N, normal tissue; TR, tumor rim; IM, invasive margin; IT,
intratumoral region. B, Iba1þ cell counts per field and percentage of Iba1 labeled area were calculated. Data were collected from 20 random fields for each
region per mouse, n ¼ 3. C, TLR expression in different cell subsets of the normal brain according to GEO dataset (GSE52564). D, qPCR analysis of TLRs'
expression pattern in FACS-sorted CD45loCD11bþ microglia isolated from glioma model (day 20) and normal brain, each sample represents 5 mice brains
pooled, and expression was normalized to b-actin (n ¼ 3). E, mRNA expression of TLR2 in human glioma samples, data from TCGA LGG/GBM datasets. N,
normal tissue, n ¼ 4; OD, oligodendroglioma, n ¼ 191; OA, oligoastrocytoma, n ¼ 130; AST, astrocytoma, n ¼ 194; GBM, n ¼ 152. F and G, Flow cytometry
analysis of CD45loCD11bþ microglia for TLRs' expression pattern, n ¼ 5. Unpaired Student t test was performed in D and G. One-way ANOVA was performed in E.

, P < 0.05 and   , P < 0.01. All values are shown as mean  SEM.

manner (Fig. 2I). These data suggest that TLR2 induced down-
regulation of MHC class II in microglia.
Downregulation of MHC class II in microglia impedes their
antigen-presenting function
To elucidate whether the TLR2-induced downregulation
of MHC class II affected the antigen-presenting function of
microglia, we assayed OT-II T-cell proliferation (Fig. 3A). After
coculture, microglia in both the Pam3CSK4- and GCM-treated
groups showed impaired MHC class II expression (Fig. 3B). T-cell
proliferation was evaluated by the percentage of divided cells. T
cells in both Pam3CSK4- and GCM-treated groups showed less
proliferation than the OVA group (Fig. 3C). When TLR2/
microglia were tested, T cells in the GCM-treated group showed
consistent proliferation as in the OVA group (Supplementary Fig.
S6A). With CD69 and PD-1 as T-cell activation markers, T cells in
both Pam3CSK4- and GCM-treated groups showed diminished
activation (Fig. 3C). These results demonstrate that TLR2-induced
microglial MHC class II downregulation could diminish the
antigen-presenting function of microglia.
We then analyzed the condition of CD4þ T cells in vivo. CD4þ
T-cell density in the tumor tissue of Tlr2/ mice was higher than
in the tumor tissue of WT mice (Fig. 3D). The MHC class IIþ
microglia accumulated at the invasive margin and colocalized

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TLR2 Downregulates Microglial MHC Class II in Glioma

Figure 2.
TLR2 activation downregulates MHC class II in microglia. A, The Kaplan–Meier survival curves represent the cumulative survival of WT and TLR2/ mice after
GL261 inoculation, n ¼ 8. B, H&E staining for sections of tumor-bearing WT and TLR2/ mice (day 20). Scale bar, 500 mm. Right plot, tumor area was
calculated, n ¼ 5–15. C, Flow cytometry analysis of the glioma model (day 20) derived CD45loCD11bþ microglia for antigen-presenting function-related markers.
Right plot, summary of the data, n ¼ 8–10. Data are representative of three independent experiments. D, Representative staining for Iba1 (green), MHC class II (red),
and DAPI (blue) in tumor-bearing brains on day 20 after GL261 inoculation. Dashed line, tumor boundary; IM, invasive margin; T, tumor; N, normal tissue.
Bottom plots, higher magnification of the regions indicated in the dashed boxes. E, Representative staining for Iba1 (green), MHC class II (red), and DAPI
(blue) in primary microglia. Control, microglia treated with IFNg. GCM, microglia treated with GCM and followed with IFNg. F, Flow cytometry analysis of primary
microglia for MHC class II expression. Right plot, summary of the data, n ¼ 3. G, Flow cytometry analysis of primary microglia (treated with the indicated
dose of Pam3CSK4 and followed with IFNg) for MHC class II expression. Right plot, the summary of the data: mean fluorescence intensity (MFI) values were
normalized against respective control groups, n ¼ 3. Data are representative of three independent experiments. H, Schematic figure of the Pam3CSK4 administration
experiments in vivo. I, Flow cytometry analysis of CD45loCD11bþ microglia that isolated from mice described in H; the MFI values of MHC class II were normalized
against corresponding normal brain samples (N), pooled data from two separate experiments. Ctr, mice treated with PBS; 1st, 2nd, and 3rd, mice treated
with Pam3CSK4 (5 mg) for 1, 2, and 3 times at indicated time points. The log-rank (Mantel–Cox) test was performed in A. One-way ANOVA was performed in G and I.
Two-way ANOVA was performed in F. Unpaired Student t test was performed in C.  , P < 0.05 and   , P < 0.01. All values are shown as mean  SEM.

with CD4þ T cells. CD4þ T cells appeared morphologically
activated in Tlr2/ tumor brain (Fig. 3E). We analyzed the
phenotype of CD4þ T cells isolated from tumor-bearing brain.
Based on expression capacity of IFNg, the function of CD4þ T cells
was more Th1-biased in Tlr2/ mice than in WT mice. However,
TNFa expression and Foxp3þ regulatory T-cell numbers were
comparable between WT and Tlr2/ mice (Fig. 3F). We found
more PD-1, an activated T-cell marker, in Tlr2/ mice (Fig. 3F).
We found that expression of T-cell activation markers CD25 and
CD69 was higher in the CD4þ T cells from TLR2/ mice,
although exhaustion marker Lag-3 was higher as well (Supple-
mentary Fig. S6B). All these results suggest that the CD4þ T cells

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Figure 3.
TLR2-induced downregulation of MHC class II suppresses the antigen-presenting function of microglia. A, Timescales of the microglia-induced OT II CD4þ
T-cell proliferation assay. B, Flow cytometry analysis of the cocultured microglia for MHC class II level. Histogram numbers indicated the mean fluorescence
intensity (MFI) values of MHC class II for each group, representative data of two experiments. C, Flow cytometry analysis of cocultured OT II CD4þ T cells
for proliferation, CD69, and PD-1 expression. Right plots, the summary of the data, n ¼ 3, representative data of three independent experiments. D,
Representative staining for CD4 (green) and DAPI (blue) in the tumor-bearing brain on day 20 after GL261 inoculation; right panel, CD4þ cell counts per
field were calculated. Data were collected from 16 to 20 random fields per mouse, n ¼ 3. Scale bar, 50 mm. E, Representative staining for CD4 (green), Iba1 (red),
MHC class II (white), and DAPI (blue) in the tumor-bearing brain on day 20 after GL261 inoculation. Scale bar, 100 mm. F, Flow cytometry analysis of tumor-infiltrated
CD4þ T cells for IFNg, TNFa, Foxp3, and PD-1. Right plots, the summary of the data, n ¼ 4–5, representative data of two independent experiments. One-way
ANOVA was performed in C. Unpaired Student t test was performed in D and F.  , P < 0.05 and   , P < 0.01. All values are shown as mean  SEM.

have a relatively better response in the tumor site in Tlr2/ mice
compared with WT mice.
MHC class II transactivator, Ciita, is inhibited in TLR2-activated
microglia
To figure out the mechanisms of the TLR2-induced down-
regulation of MHC class II in microglia, we conducted RNA-seq
analysis to generate the transcriptomic profile of TLR2-activated
microglia. The microglia were treated with or without
Pam3CSK4 for 24 hours, followed by stimulation with IFNg
for 24 hours for MHC class II induction. As expected, several
inflammatory cytokines and chemokines, including Ccl3, Ccl5,
and Il1b, were upregulated in Pam3CSK4-treated microglia, so
was Nos2. P2ry12, the signature gene of microglia, was down-
regulated in the Pam3CSK4-treated group (Fig. 4A). P2ry12
expression is robust in the "resting" status but dropped after
microglial activation (24). Some MHC class II–related genes
were downregulated in Pam3CSK4-treated microglia, including
genes encoding mouse MHC class II molecules (H2-A and
H2-E), invariant chain (Ii), and nonclassical MHC class II
molecules (H2-M; Fig. 4A). Expression of Ciita was decreased
in the Pam3CSK4-treated microglia (Fig. 4A and B). CIITA
is the master coactivator for regulation of MHC class II
transcription (25). Inhibition of Ciita in TLR2-activated micro-
glia shuts down these MHC class II–related genes. Pathway
analysis indicated that the TLR, TNF, and NF-kB signaling
pathways were activated (Fig. 4C), as expected (16). Inhibition
of antigen processing– and presentation–related pathways was
also evident (Fig. 4C). We applied Gene Ontology analysis for
all categories of genes that differed in expression between the

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TLR2 Downregulates Microglial MHC Class II in Glioma

Figure 4.
RNA-seq analysis reveals the inhibition of MHC class II–related genes in TLR2-activated microglia. Primary microglia treated with or without Pam3CSK4 and
followed with IFNg. Pam3, microglia with Pam3CSK4 treatment; Control, microglia without Pam3CSK4 treatment. A, Scatterplot of FPKM values for all genes
in both groups. The differentially expressed genes were selected using the following filter criteria: FDR  0.05 and fold change  2. B, Heat map of the most
upregulated and downregulated genes (based on P value) in Pam3 group compared with Control group. Genes in red, MHC class II–related genes. C and D,
KEGG pathway (C) and Gene Ontology enrichment analysis (D) were performed using the differentially expressed genes. The details of the enriched Gene
Ontology terms were listed in Supplementary Table S3.

two groups and listed top related pathways in Fig. 4D. The most
enriched pathways were related to antigen presentation. Most
of the genes in these pathways were downregulated. Other
enriched pathways were related to inflammation, cytokines,
and immune cell chemotaxis. These results indicate that TLR2
activation leads to Ciita shutdown and inhibition of MHC class
II–related genes.
TLR2-induced activation of ERK1/2 signaling pathway inhibits
Ciita in microglia
To clarify the pathway that mediates TLR2-induced
downregulation of MHC class II in microglia, we confirmed
that Pam3CSK4-induced downregulation of MHC class II was
associated with the decrease in MHC class II mRNA (Fig. 5A).
Ciita mRNA expression decreased in the Pam3CSK4-treated
group (Fig. 5B), suggesting that failure of the transcription
factor Ciita causes downregulation of MHC class II, consistent
with data from RNA-seq analysis. Then, we analyzed the effects
of TLRs' agonists in the BV2 microglia cell line. Most TLRs'
agonists caused reduced Ciita expression in microglia, although
TLR3 ligands Poly (I: C) did not, suggesting that the effect is
controlled via the Myd88-dependent pathway, not the TRIF-
dependent pathway (Fig. 5C). Next, we stimulated microglia
with IFNg for 24 hours and then added Pam3CSK4 at time
points from 30 minutes to 24 hours. Ciita mRNA began to
decrease 2 hours after Pam3CSK4 stimulation and continued

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Qian et al.

Figure 5.
TLR2-induced MAPK Erk1/2 signaling pathway inhibits Ciita expression, leading to the downregulation of MHC class II in microglia. qPCR analysis of primary
microglia for MHC class II (H2-Ab1) mRNA (A) and Ciita mRNA (B), n ¼ 3. The data presented represent one of three individual experiments. C, BV2 microglia
were treated with GCM or different TLRs' ligands and IFNg. Ciita mRNA was measured by qPCR, and expression was normalized to b-actin and subsequently
to control group, n ¼ 3. D, BV2 microglia were treated with IFNg and followed with Pam3CSK4 for indicated time points, Ciita mRNA was measured by
qPCR, and expression was normalized to b-actin and subsequently to control group, n ¼ 3. E and F, BV2 microglia were pretreated with the PI3K (E) or MAPK (F)
inhibitors and treated with Pam3CSK4,for 2 hours followed with IFNg for 18 hours. Ciita mRNA was measured by qPCR, and expression was normalized to
b-actin and subsequently to control group, n ¼ 3. Bottom plot in E, phosphorylated Akt of the corresponding samples was analyzed by Western blotting. Bottom plot
in F, phosphorylated p65, phosphorylated ERK1/2, phosphorylated p38, and phosphorylated JNK of the corresponding samples were analyzed by
Western blotting. G and H, Primary microglia were pretreated with U0126 and treated with Pam3CSK4 for 2 hours, followed by IFNg for 18 hours, and MHC
class II was quantified by flow cytometry (G). Summary of the MHC class II quantification (H), n ¼ 3. Bottom plot, phosphorylated ERK1/2, total ERK1/2,
phosphorylated p65, and phosphorylated Akt of the corresponding samples were analyzed by Western blotting. b-Actin was used as an internal control.
All experiments were repeated 3 times. One-way ANOVA was performed in C, D, E, F, and H. Unpaired Student t test was performed in A and B.  , P < 0.05
and   , P < 0.01. All values are shown as mean  SEM.

decreasing over 24 hours (Fig. 5D). These results indicate that
the effect is related to signaling early in the process. We thus
focused on the PI3K and MAPK signaling pathways. We used
inhibitors to block individual signaling pathways (Fig. 5E
and F). In the Erk1/2 inhibitor–treated group, Erk1/2 phos-
phorylation was decreased, and expression of Ciita was
restored. We verified this mechanism in primary microglia
by flow cytometric analysis. Results showed that Erk1/2 inhib-
itor U0126 restored microglial MHC class II amounts in
Pam3CSK4-treated microglia (Fig. 5G and H). These data
suggest that TLR2-induced downregulation of MHC class II
relates to the Erk1/2 signaling pathway.

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TLR2 Downregulates Microglial MHC Class II in Glioma

Figure 6.
Histone acetylation at Ciita promoters
was reduced in TLR2-activated
microglia. A, The modular structure of
the regulatory region of Ciita. Ciita
isoforms 1, 2, and 3 are shown as typical
mRNA isoforms that derive from
promoter pI, pIII, and pIV, respectively.
For gene structure visualization,
SnapGene software was used. B, TLR2
activation impairs the expression of
Ciita mRNA derived from promoters
pI and pIV. Primary microglia treated
with or without Pam3CSK4 and
followed by IFNg, qPCR analysis of the
microglia for the isoforms of Ciita
mRNA. Expression was normalized to
b-actin, n ¼ 3. C, Primary microglia
were pretreated with epigenetic
inhibitors, and then treated with or
without Pam3CSK, followed with IFNg.
qPCR analysis of the microglia for MHC
class II (H2-Ab1), Total Ciita, pI Ciita, and
pIV Ciita mRNA. Expression was
normalized to b-actin, n ¼ 3. D, ChIP
qPCR was applied to quantify H3K9ac
and H3K4me2 in the promoter region of
Ciita. The fold enrichment method was
used for data normalization. Shown is
one of two individual experiments.
One-way ANOVA was performed in C.
Unpaired Student t test was performed
in B and D.  , P < 0.05 and   , P < 0.01. All
values are shown as mean  SEM.

H3K9ac deacetylation contributes to the TLR2-induced Ciita
inhibition
The Ciita gene is transcriptionally regulated by three distinct
promoters, each transcribing a different first exon (ref. 26; Fig. 6A).
Transcription of Ciita from promoter I (pI) is restricted to cells of
the myeloid lineage including conventional dendritic cells and
macrophages (27), whereas promoter III (pIII) is active primarily
in cells of lymphoid lineage, including B and T cells (28, 29), and
promoter IV (pIV) is responsive to IFN in nonhematopoietic cells
(30). However, it remains unclear which promoter is used in adult
microglia. To address this issue, we analyzed amounts of Ciita
mRNA isoforms derived from different promoters (Fig. 6A). We
found that adult microglia use both pI and pIV for Ciita tran-
scription, and Pam3CSK4 stimulation could impair Ciita mRNA
expression derived from both promoters (Fig. 6B).
To understand if there was an epigenetic modulation con-
trolling such Ciita inhibition, a DNMT inhibitor, 5-AZA,
and two HDAC inhibitors, TSA and Nico, were applied. TSA

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Qian et al.
selectively inhibits class I and II HDACs (31), whereas Nico
inhibits class III HDACs (32). The results indicated that TSA
could reverse the Pam3CSK4-induced Ciita inhibition in
both promoters (Fig. 6C), indicating that class I or II HDACs
participate in Pam3CSK4-induced histone deacetylation of
CIITA promoters. Next, to assess H3 acetylation at promoters,
ChIP qPCR was performed. We found that H3K9ac at both
pI and pIV promoters was decreased after Pam3CSK4 stimu-
lation, but H3K4me2 remained unchanged (Fig. 6D). These
data indicate that TLR2 activation in microglia could lead to
the H3 deacetylation of the Ciita promoters, inhibiting Ciita
expression.
Increase of endogenous HSPs is correlated with Ciita inhibition
in patients with glioma
Several endogenous TLR ligands have been reported, such as
HSPs and ECM components (33). According to proteomics data
(34), glioma cell line GL261 secretes several TLR2 ligands, includ-
ing HSPA4, HSPA5, HSPA9, HSPA13, HSPD1, and VCAN. We
searched for TLR2 ligands in the TCGA database that were secreted
by GL261. We used the GlioVis data portal to export the data
describing gene expression from the TCGA GBM datasets (35). We
found that many of these ligands were upregulated in GBM
samples, including HSPA5, HSPD1, BGN, VCAN, and HMGB1
(Fig. 7A). According to the Human Protein Atlas database (36),
TLR2 ligands are upregulated in human GBM samples compared
with normal tissue (Fig. 7B), and some TLR2 ligands with
unchanged mRNA expression, such as HSPA1A and HSPA9, are
also overexpressed as proteins in GBM samples compared with
normal tissues.
We further analyzed expression of genes encoding TLR2
ligands and MHC class II–related molecules in the RNA-seq
database of the Ivy Glioblastoma Atlas Project (Fig. 7C). The
RNA-seq profiles contain GBM samples from different laser-
microdissected structures, including leading edge, infiltrating
tumor, cellular tumor, pseudopalisading cells around necrosis
(PCAN), and microvascular proliferation (MvP), thus facilitat-
ing our research for gene expression patterns at a fine scale.
Based on PTPRC (which encodes CD45) and CD4, the periph-
eral infiltrating CD4þ T cells accumulated mainly at the PCAN
and MvP regions, indicating that these regions are where T cells
recognize antigens. We checked for MHC class II–related genes,
including genes encoding three human MHC class II (HLA-DP,
HLA-DQ, and HLA-DR) and nonclassical MHC class II
(HLA-DM and HLA-DO). These genes were barely expressed
in PCAN region, indicating that the APC in this region had
impaired MHC class II expression, which is similar to the
phenomenon in the murine glioma model. The CD4 expression
pattern was identical to that of the MHC class II–related genes,
consistent with the colocalization of MHC class IIþ microglia
and CD4þ T cells in the murine glioma model.
Intracellular chaperone HSPs signal CNS injury by activating
microglia through TLR- and MyD88-dependent pathways.
HSPs are released from CNS cells undergoing necrotic or
apoptotic cell death and bind to microglia (37). According to
the Ivy GAP database, genes encoding HSPs are more highly
expressed at the necrotic PCAN region than in the MvP region,
which is better oxygenated and has fewer necrotic cells. We
conducted a series of correlation analyses between CIITA and
genes encoding endogenous TLR2 ligands based on the pool of
samples from CD4þ T cells that infiltrated PCAN and MvP
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regions (Fig. 7D). We found that several kinds of HSPs were
negatively correlated with CIITA expression. Meanwhile, no
significant correlation was found between CIITA and ECM
components (BGN and VCAN). The HLA-DPA1 and CD4 levels
were positively correlated with CIITA expression as expected.
These data suggest that the presence of HSPs correlates with
CIITA inhibition in patients with GBM.
Discussion
Although microglia infiltrate gliomas, their expression of MHC
class II is compromised. In the current study, we showed that TLR2
suppresses GAM function. TLR2 is upregulated in GAM, and
several kinds of endogenous TLR2 ligands are enriched in the
glioma microenvironment. TLR2 activation triggers its down-
stream MAPK Erk1/2 signaling pathway and subsequently inhi-
bits CIITA expression by epigenetic modulation, ultimately caus-
ing the downregulation of MHC class II in GAM. Furthermore,
impaired MHC class II expression limits the activation and
maintenance of CD4þ T cells at tumor sites, jeopardizing
T-cell–dependent antitumor immunity.
In the brain parenchyma, only microglia express the immuno-
proteasome, a multicatalytic protease complex involved in the
generation of antigenic peptides presented on MHC (38). Micro-
glia are the predominant APC subset in the GL261 glioma model,
whereas macrophages only contribute to the tumor mass at the
late stage of growth and constitute merely 25% of the myeloid
cells (39). GAM upregulate MHC class II expression somewhat
compared with resting microglia, but this upregulation of MHC
class II is impaired. MHC class II induction after injection of IFNg
is muted in tumor microglia (10). The upregulation of MHC class
II by local APC facilitates tumor eradication by T cells during the
early stages of tumor growth. We have found that the reduced
amounts of microglial MHC class II could lessen the activation
and proliferation of antigen-specific CD4þ T cells in vitro.
IFNg þCD4þ T cells could induce tumor eradication in GL261
murine glioma model (40). The TLR2-induced downregulation of
MHC class II may reflect negative feedback regulation, which
would prevent excessive T-cell–mediated immunity. With TLR2
ligands, the tumor cells have subverted this otherwise protective
mechanism to ensure their own survival.
Various TLR2 ligands, including HSPs and ECM components,
are enriched in human glioma tissues. It remains unclear
whether a specific TLR2 ligand downregulates MHC class II in
microglia and whether nonprotein TLR2 ligands, such as hya-
luronic acid fragments and pyrroles (41, 42), can also down-
regulate MHC class II. The RNA-seq profiles of laser-microdis-
sected structures revealed that necrotic cells might be a source
of TLR2 ligands in gliomas. Necrotic cells induce expression of
genes involved in inflammatory and tissue-repair responses in
macrophages via TLR2 (43). In gliomas, such TLR2-induced
tissue-repair responses may promote tumor angiogenesis and
tumor growth. TLR2 is also associated with the invasive growth
pattern of glioma and protumor phenotype reprogramming of
DCs (44, 45). In the experimental autoimmune encephalomy-
elitis (EAE) model and in multiple sclerosis patients, TLR2
agonists function as inhibitors of neuroinflammation, and TLR
tolerance induces the immune regulatory response to create an
immunosuppressive microenvironment (46, 47). Our study
uncovered the role of TLR2 and its endogenous ligands in
downregulating microglial MHC class II expression and
Cancer Immunology Research
 Published OnlineFirst August 21, 2018; DOI: 10.1158/2326-6066.CIR-18-0020

TLR2 Downregulates Microglial MHC Class II in Glioma

Figure 7.
HSPs are enriched in GBM samples and correlate with CIITA inhibition. A, Expression of endogenous TLR2 ligand mRNAs in human GBM samples, data
from TCGA database. Normal, n ¼ 4; GBM, n ¼ 156. Underlined ligands are detectable in GL261 glioma supernatants. Unpaired Student t test was performed.

, P < 0.01. Values are shown as mean  SEM. B, Immunohistochemistry staining for TLR2 ligands in human GBM samples; image credit, Human Protein Atlas.
Scale bar, 200 mm. The information details (including URL links) of the GBM samples were listed in Supplementary Table S4. C, The RNA-seq profiles of
laser-microdissected structures of GBM samples. Expression from genes encoding TLR2 ligands and MHC class II–related molecules is indicated as heat maps.
The data were derived from the Ivy Glioblastoma Atlas Project; image credit, Allen Institute. D, Correlation analysis between CIITA and genes encoding
endogenous TLR2 ligands was based on the pool of samples from immune cells' infiltrated PCAN and MvP regions. The data were derived from the Ivy
Glioblastoma Atlas Project. Pearson correlation coefficient was calculated to analyze the correlation between gene expression levels.

elucidated the mechanisms that underlie glioma escape from
the attack of infiltrated immune cells.
Activation of TLRs' signaling initiates the immune response and
leads to the increase of MHC class II expression in myeloid cells
like DCs (16). NF-kB activation induced by TLR signaling leads
to assembly of the transcription complex regulating CIITA expres-
sion in DCs (48). However, during DC maturation, TLR signaling
inhibits de novo biosynthesis of MHC class II by reduction of
CIITA mRNA expression (49, 50). TLR signaling–induced CIITA
shutdown limits the presentation of antigens associated with
the maturation stimulus and prohibits the presentation of
antigens experienced by the APCs (49). The mechanisms
drive immune regulation as well as functional accuracy and
efficiency. In gliomas, microglia transiently upregulate MHC
class II in the early stage of tumor growth. However, long-term
exposure to TLR2 ligands results in the shutdown of Ciita
expression and downregulation of MHC class II in microglia.
We believe that the CIITA shutdown by TLR2 ligands is a
protective mechanism by which microglia shield brain tissue
from the attack of immune system and initiate tissue-repairing
process. Unfortunately, such mechanisms allow the glioma to
escape the immune system.

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Qian et al.
In conclusion, our findings highlight the role of TLR2 in GAM.
TLR2 ligands in the tumor microenvironment could facilitate
tumor immune evasion. Similar mechanisms may also occur in
macrophages or dendritic cells in various solid tumors. Because
MHC class II are downregulated in GAM, antagonists for TLR2
or downstream signaling might modify the function of tumor-
infiltrated APCs and improve antiglioma immunity. Our results
might inform the development of more efficient immunothera-
pies for the treatment of glioma.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: J. Qian, Y. Chu
Development of methodology: J. Qian, F. Luo, J. Yang, J. Liu, Z. Lu, Y. Chu
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): J. Qian, F. Luo, J. Yang, R. Liu
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
computational analysis): J. Qian
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Acknowledgments
This work was supported by the National Science Foundation of China
(31570892, 31770992, 81730045, and 91527305) and the Science and Tech-
nology Commission of Shanghai Municipality (15JC1401200).
We thank Dr. Rui He for providing the OT-II transgenic mice, Dr. Liangfu
Zhou for providing GL261 murine glioma cell line, and Dr. Dapeng Yan for the
helpful support and discussions. We thank Dr. Xiaoming Liu and Enyu Huang
for critiquing the article.
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 January 19, 2018; revised June 15, 2018; accepted August 15, 2018;
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Cancer Immunol Res; 6(10) October 2018 1233
 Published OnlineFirst August 21, 2018; DOI: 10.1158/2326-6066.CIR-18-0020

TLR2 Promotes Glioma Immune Evasion by Downregulating MHC

Class II Molecules in Microglia
Jiawen Qian, Feifei Luo, Jiao Yang, et al.

Cancer Immunol Res 2018;6:1220-1233. Published OnlineFirst August 21, 2018.

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