Calnexin Impairs the Antitumor Immunity of CD4+ and CD8+ T Cells

Published OnlineFirst November 6, 2018; DOI: 10.1158/2326-6066.CIR-18-0124

Research Article Cancer Immunology Research Calnexin Impairs the Antitumor Immunity of CD4þ and CD8þ T Cells Yichen Chen1,DaMa1, Xi Wang1, Juan Fang1, Xiangqi Liu1, Jingjing Song1, Xinye Li1, Xianyue Ren1, Qiusheng Li1, Qunxing Li1, Shuqiong Wen1, Liqun Luo2, Juan Xia1, Jun Cui3, Gucheng Zeng2, Lieping Chen4, Bin Cheng1, and Zhi Wang1

Abstract

Elucidation of the mechanisms of T-cell–mediated anti- knockdown of calnexin enhanced the infiltration and effec- tumor responses will provide information for the rational tor functions of T cells in the tumor microenvironment design and development of cancer immunotherapies. Here, and conferred better control of tumor growth, whereas we found that calnexin, an endoplasmic reticulum (ER) treatment with a recombinant calnexin protein impaired chaperone protein, is significantly upregulated in oral squa- the infiltration and effector functions of T cells and pro- mous cell carcinoma (OSCC). Upregulation of its membra- moted tumor growth. We also found that calnexin þ þ nous expression on OSCC cells is associated with inhibited enhanced the expression of PD-1 on CD4 and CD8 T T-cell infiltration in tumor tissues and correlates with poor cells by restraining the DNA methylation status of a CpG survival of patients with OSCC. We found that calnexin island in the PD-1 promoter. Thus, this work uncovers a þ þ inhibits the proliferation of CD4 and CD8 T cells isolated mechanism by which T-cell antitumor responses are regu- from the whole blood of healthy donors and patients with lated by calnexin in tumor cells and suggests that calnexin OSCC and inhibits the secretion of IFNg,TNFa, and IL2 might serve as a potential target for the improvement of from these cells. Furthermore, in a melanoma model, antitumor immunotherapy.

Introduction a reliable predictive biomarker of response to immune checkpoint blockade (4–6). Some new strategies have been developed to Although T-cell immunity plays a critical role in mediating convert immunologically "cold" tumors into "hot" tumors. Exam- antitumor immunity, the molecular mechanisms underlying ples of these strategies include metabolic reprogramming of T cells impaired antitumor T-cell immunity are not fully understood. or modulation of the gut microbiome (7, 8). Still another Immune checkpoint blockade with mAbs directed against the approach needs to be developed based on novel insights into inhibitory immune receptors CTLA-4, PD-1, and PD-L1 has T-cell responses and immune systems. emerged as a successful treatment approach that has shown Endoplasmic reticulum (ER) chaperones, including BiP/ striking antitumor activity in a variety of cancer types (1–3). GRP78, calreticulin, calnexin, GRP94, and ERP57, are a large Currently, there are more than 10 CTLA4, PD-1, and PD-L1 family of proteins that have been discovered to have many antibodies in different stages of clinical trial in tumors. Despite important roles in maintaining ER homeostasis and contributing the durable response rates observed with cancer immunothera- to cancer cell survival and progression. The correlation between pies, the majority of patients do not benefit from the treatment. A ER chaperone expression and tumorigenesis has been extensively "hot" tumor microenvironment, typified by an increased number þ studied in various cancers, and most reports have indicated that of CD8 CTLs and PD-L1–positive cells, has been identified to be these proteins promote the proliferation, migration, and attach- ment of cancer cells (9–13). The ER chaperone calnexin, which is fi 1Guanghua School of Stomatology, Guangdong Provincial Key Laboratory of an ER-speci c type I transmembrane protein, regulates the folding Stomatology, Stomatological Hospital, Sun Yat-Sen University, Guangzhou, P.R. and quality control of newly synthesized glycoproteins (14). China. 2Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, P.R. Calnexin can escape from the ER and be transported to the plasma China. 3Key Laboratory of Gene Engineering of the Ministry of Education, State membrane or released into the extracellular space by interacting Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, – 4 with glycoproteins (15 17) or via its phosphorylation at two Guangzhou, P.R. China. Department of Immunobiology and Yale Comprehen- serine residues (Ser554/564) by protein kinase CK2 (18). Some sive Cancer Center, Yale University, New Haven, Connecticut. studies have found that extracellular calnexin could be involved in Note: Supplementary data for this article are available at Cancer Immunology innate and adaptive immunity in non-mammals (19–21), but the Research Online (http://cancerimmunolres.aacrjournals.org/). impact of surface or secreted calnexin on the human immune Y. Chen, D. Ma, and X. Wang contributed equally to this article. system has not been reported. The results from a lung cancer Corresponding Authors: Zhi Wang, Sun Yat-Sen University, No. 56, Lingyuan- patient cohort provided evidence that calnexin may be a sero- west Road, Guangzhou 510055, Guangdong, China. Phone: 8620-8733-0591; diagnostic marker for lung cancer (15). Fax: 8620-8382-2807; E-mail: [email protected]; and Bin Cheng, In this study, we found that calnexin inhibits the infiltration [email protected] and effector functions of T cells and enhances the expression of doi: 10.1158/2326-6066.CIR-18-0124 PD-1 on T cells. Membrane expression of calnexin was positively 2018 American Association for Cancer Research. correlated with poor prognosis of patients with oral squamous

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cell carcinoma (OSCC). Our work thus uncovers a mechanism by PMA (Sigma-Aldrich) and 5 mg/mL ionomycin (Sigma-Aldrich) in which antitumor responses are regulated by calnexin expressed in the presence of GolgiPlug (BD Biosciences). After 4 hours, cells tumor cells and suggests that calnexin may serve as a target for were stained for dead cells using a FVD dye (eBioscience) and antitumor immunotherapy. Elucidation of these mechanisms will surface markers then fixed prior to intracellular staining for reveal clues as to the next steps that need to be taken to potentially cytokines. Data were analyzed using a FACSVerse flow cytometer overcome resistance to immunotherapy. (BD Biosciences) and using FlowJo software (Tree Star). The following antibodies were used for flow cytometry: anti-mouse CD4 (clone GK1.5); anti-mouse CD8a (clone 53-6.7); anti-mouse Materials and Methods CD3 (clone 17A2); anti-mouse/rat Foxp3 (clone FJK-16s); Patients and tissue samples anti-mouse CD45.2 (clone 104); anti-mouse ki-67 (clone For Western blot analyses, 8 pairs of primary OSCC samples 16A8); anti-mouse TNFa (clone MP6-XT22); and anti-mouse and corresponding normal oral epithelial tissues were obtained IFNg (clone MOB-47). during surgeries at the Hospital of Stomatology, Sun Yat-sen University (Guangzhou, Guangdong, P.R. China). For real-time Xenograft assays in immunodeficient mice PCR analyses, 33 pairs of primary OSCC samples and corres- CNX-knockdown (sh-CNX) cells or control cells (6 106) were ponding normal oral epithelial tissues were obtained during injected subcutaneously into right flank of NCG mice, and 1 107 surgeries at the Hospital of Stomatology, Sun Yat-sen University human peripheral blood mononuclear cells (PBMCs) were (Guangzhou, Guangdong, P.R. China). For IHC and immuno- injected via the tail vein after tumor implantation. The animals fluorescence analyses, the expression of calnexin was investigated were monitored for tumor formation every 2 days and euthanized using a tissue microarray (TMA) containing samples from 357 3 weeks later. Tumor length (L) and width (W) were measured at patients with primary OSCC who were treated at the Hospital of the end of the experiment, and tumor volume was calculated by Stomatology, Sun Yat-sen University (Guangzhou, Guangdong, the formula (L W2)/2. Serum cytokines were analyzed at the þ P.R. China), between January 2007 and January 2009. All speci- indicated time points, and human CD3 T cells were counted after mens were confirmed histologically by hematoxylin and eosin tumor dissociation. staining, and tumor tissue was present in more than 80% of the specimens. The follow-up interval was calculated from the date of Tumor experiment surgery to the date of death or last clinical evaluation. The detailed B16F10 tumor cells were retrovirally transduced with sh-CNX information of these patients is described in Supplementary Table or a control and selected with puromycin (3 mg/mL). For tumor S2. This study protocol was approved by the Institutional Review vaccination, na€ve C57BL/6 mice were immunized with 1 106 Board of the Hospital of Stomatology, Sun Yat-sen University irradiated B16F10 (1 104 rad) cells that were inoculated (Guangzhou, Guangdong, P.R. China) and was conducted in subcutaneously into the left flank. On day 14, the vaccinated agreement with the Helsinki Declaration, and written informed mice were challenged with live transduced tumor cells that were consent was obtained from all study participants. inoculated subcutaneously into the right flank. A CNX-Ig fusion protein or Flag-Ig (200 mg) was injected intraperitoneally (i.p.) Cell lines and reagents into each mouse once a week. Tumor growth was monitored every The HSC-3 cell line was purchased from the cell bank of the 2 days. The mice were euthanized when the tumor size reached Japanese Collection of Research Bioresource (JCRB). SCC15, 15 mm diameter. SCC25, CAL27, B16F10, and MB49 cells were purchased from the ATCC. The normal keratinocyte cell line (NOK-SI) was kindly Isolation of tumor-infiltrating leukocytes from tissues provided by J. Silvio Gutkind (UCSD, San Diego, CA). Cell lines Tumor-infiltrating leukocytes (TILs) from the xenograft used in these experiments were passaged a maximum of four tumors were prepared according to the protocol described times before the experiments. Cells were tested for Mycoplasma previously (22, 23). Briefly, tumors were dissected and homog- contamination and identified by short tandem repeat. enized using a GentleMACS dissociator (Miltenyi Biotec), digested with 0.05% collagenase IV (Sigma-Aldrich), 0.002% Mice DNase I (Roche) at 37C for 1 hour prior to centrifugation on C57BL/6 mice were purchased from the experimental animal Percoll density gradient (40%–80%), and the TILs were washed center of Sun Yat-sen University (Guangzhou, Guangdong, P.R. and resuspended in RPMI. China). NOD-Prkdcem26Cd52Il2rgem26Cd22/Nju (NCG) mice were purchased from Nanjing Biomedical Research Institute of Retroviral constructs and transduction of OSCC cell lines Nanjing University (Nanjing, China). All experiments were HSC3 cells were transfected with CNX shRNA or empty vector approved by the Institutional Animal Care and Use Committee (Genechem) using Polybrene. The cells were trypsinized and of Sun Yat-sen University (Guangzhou, Guangdong, P.R. China) replated in 0.5 mg/mL puromycin 48 hours after transfection. and performed following local rules. Two months later, the puromycin-resistant stable line was established and maintained in medium with 1 mg/mL puromycin. Flow cytometry The transfected cells were incubated for 24 hours and harvested Single-cell preparations were stained with antibodies pur- for real-time PCR and Western blot analysis. chased from eBioscience, BD Biosciences, and BioLegend. Iso- type-matched control mAbs were used. Intracellular staining was In vitro antigen-specific T-cell response assay done using a Foxp3/Transcription factor staining kit or Intracel- OSCC tumor cells were isolated from fresh specimen; single- lular fix & Perm Set according to the manufacturer's instructions cell suspensions were obtained as described above. Human (BD Biosciences). Briefly, cells were stimulated with 50 ng/mL PBMCs from 6 healthy donors and 8 patients with OSCC were

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density-enriched by Ficoll (TBD). Tumor antigens were prepared In vitro plate-bound T-cell activation assay þ as described previously (24). Briefly, 2 107 tumor cells were Human CD3 T cells were isolated from PBMCs of healthy subjected to four freeze (liquid nitrogen) and thaw (37C water donor using pan T-cell isolation kits (Stem Cell Inc.). Then, bath) cycles to obtain a crude lysate as tumor antigen. After labeling was performed by incubating 106 cells/mL at 37C for removal of large particles by centrifugation and sterilization by 15 minutes with 5 mmol/L CFSE in PBS. Carboxyfluorescein filtering (0.22 mm), the protein concentration in the supernatant diacetate succinimidyl ester (CFSE) was quenched by adding was measured (Coomassie blue protein assay kit, Thermo Scien- twice the volume of complete media, followed by three washes tific) and aliquots stored at 80C until use. A total of 1 105 in complete media before stimulation. 96-well flat-bottom plates PBMCs were stimulated for 48 hours in the presence or absence of were coated with 1 mg/mL anti-CD3 (clone OKT3) at 4C over- tumor lysate from the same patient and 1 mg/mL PHA-M (Sigma night. The wells were washed three times with PBS to remove Aldrich). GolgiPlug was added for 5 hours to the cells in culture. unbound antibody and coated with another 5 mg/mL (ratio 1:5) After 5 hours, cells were stained for viability using a FVD dye calnexin-Ig or control-Ig protein in PBS at 4C overnight. (eBioscience), and surface markers were then fixed prior to intra- Wells were washed three times with PBS before adding cells. cellular staining for cytokines using Transcription Factor Buffer Set Replicate cultures (1 105 cells per well) were maintained and Fixation/Permeabilization Solution Kit (BD Biosciences) as in complete RPMI1640 medium supplemented with 10% per the manufacturer's instructions (25). The culture supernatants FBS, 10 mmol/L HEPES, 50 mmol/L b-mercaptoethanol, and were collected for examination by a human Th1/Th2 cytometric penicillin/streptomycin/L-glutamine. The cultures were analyzed bead assay (BD Biosciences) to quantitatively measure IL2, IL4, for CFSE profiles according to a time course as indicated. IL5, IL10, TNFa, and IFNg protein levels. Briefly, test samples (50 mL) and PE detection antibody were incubated with capture Real-time PCR bead reagent for 3 hours in the dark at room temperature. All Total RNA was derived from cultured cells with Rnaiso Reagent unbound antibodies were washed (1.0 mL wash buffer) and (Takara). Quantitative real-time PCR was done using SYBR Green resuspended in 300 mL before acquisition on FACSVerse Flow I Dye (Roche) and according to the protocol of Light Cycle 480 kits Cytometer (BD Biosciences). (Roche). The primer sequences of CNX were 50-CATGATGGA- CATGATGATGACAC-30 (forward) and 50-CTAGAGGCTTGGT IHC and immunofluorescence GTATAC-30. Results were normalized to the expression of GAPDH Tissues were deparaffinized and rehydrated prior to antigen (forward, 50-AACTTTGGCATTG TGGAAGG-30; reverse, 50-ACA- retrieval in citrate buffer. Tissues were stained with anti-calnexin CATTGGGGGTAGGAACA-30). (C5C9, Cell Signaling Technology), anti-CD3 (17A2, eBioscience), anti-CD4 (OKT4, eBioscience), anti-CD8 (HIT8A, Western blot analysis eBioscience). Horseradish peroxidase staining was visualized with Cells and tissues were lysed with radioimmunoprecipitation 3-39 diaminobenzidine (Gene Company), and fluorescence assay (RIPA) buffer containing proteases inhibitor cocktail staining was visualized with Alexa Fluor–conjugated secondary (Sigma Aldrich) and ultrasonication. Protein quantification was antibody (Thermo Scientific). Slides were counterstained, cleared, performed using BCA Protein Assay Reagent (Thermo Fisher Sci- and mounted. entific), and 45 mg protein per sample was loaded into SDS-PAGE and sequentially immunoblotted with calnexin mAb (C5C9, Cell Pathology assessment Signaling Technology). The proteins were using GAPDH antibo- Each TMA staining result was confirmed by a tissue slide dies (D16H11, Cell Signaling Technology) as loading controls. from the same patient. The slides were scanned using an Aperio Scan Scope AT Turbo for digital image analysis. The images CTL killing assay þ were blindly reviewed and scored by two certified anatomic Tumor antigen–specificCD8 human T-cell clones were gener- pathologists. Calnexin expression was defined as cytoplasmic ated from PBMCs from a healthy donor by in vitro stimulation using or membranous based on its immunoreactivity. As previously dendritic cells loaded with corresponding peptide epitopes (irradi- described (26–28), cytoplasmic calnexin staining in tumor cells ated HSC3 cells were used as tumor antigens). CNX-overexpressing was evaluated using the staining–intensity distribution (SID) HSC3 cells or control cells were labeled with CFSE and cocultured score. The staining intensity of positive tumor cells was cate- with CTLs at an effector-to-target ratio (E/T) of 5:1 and 10:1 for 4 gorized into three grades by comparing the intensity with that hours. Then, 0.1 mg of DAPI was added to each sample, and the of internal controls: 0, negative staining; 1 (weak), lighter than samples were immediately analyzed by flow cytometry. CTL killing þ þ þ skeletal muscle; 2 (moderate), equal to skeletal muscle; and 3 (%) ¼ CFSE DAPI cells/total CFSE cells 100%. (strong), more intense than skeletal muscle. The distribution of positive tumor cells was graded as follows: 0, no stained cells; 1, Statistical analysis <25% stained cells; 2, 25%–50% stained cells; and 3, >50% Baseline characteristics were described by mean and SD for stained cells. After multiplying the distribution score by the continuous variables or described by numbers and percentages for intensity score in eight different high-powered image fields, the categorical variables. To compare the baseline characteristics average of the eight fields was the SID score for the sample. between different groups, Student t test was used for continuous Cytoplasmic calnexin expression was categorized into low and variables, whereas x2 tests were used for categorical variables. high expression groups using the median of SID scores of the Overall survival was calculated and described by Kaplan–Meier total patients. Although upon analysis, calnexin membranous method. The difference of survival curves was tested by log-rank immunoreactivity correlated with overall survival, we defined test. Univariate and multivariate Cox proportional models were extensive staining as positive and no staining or sporadic used to analyze the associations between baseline characteristics staining as negative. and overall survival, and the HRs with 95% confidence interval (CI)

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were calculated. All statistical analyses were performed by control normal tissue. In total, 6 pairs of tissue lysates were GraphPad Prism 7.0 and Stata/MP 14.0. All tests were two-sided, analyzed. When the protein patterns of the primary tumor and and a P value of less than 0.05 was considered significant. its corresponding normal tissue were compared, multiple proteins were found to be differentially expressed. Supplementary Table S1 shows 43 proteins that are significantly upregulated Results (>2-fold). Calnexin exhibited a higher expression in cancer Upregulation of membranous calnexin is correlated with tissues (2.7-fold elevation) when compared with the corre- reduced T-cell infiltration sponding normal tissues. We further confirmed its expression iTRAQ-coupled 2D LC-MS/MS technique was used to study by qRT-PCR (Fig. 1A), Western blotting (Fig. 1B), and IHC the protein expression patterns of OSCC tumor tissue and staining (Fig. 1C) in OSCC cells; paired cancer and adjacent

ABP = 0.0121 * **** NOK Cal27HSC3 SCC15 SCC25 10 25 **** CNX 8 20 * 6 15 GAPDH 4 10 1 23 4 5 6 7 8

expression Ca Ca N Ca N Ca 2 5 N Ca N Ca N Ca N Ca N N Relative mRNA CNX/GAPDH Ratio 0 0 CNX r e al 5 nc rm OK SC3 GAPDH N AL27H CC15CC2 Ca No C S S C D

4X OSCC DAPI WGA CNX Merge

Melanoma DAPI WGA CNX Merge 40X 40X OSCC Normal tissue

E 8.0 M 8.0 M F

6.0 M 6.0 M

4.0 M 4.0 M 5 R= −0.5197 3 /g) R − 2.0 M 2.0 M /g) = 0.5080 7 4 P= 0.0189 7 P = 0.0222 0 0 SSC SSC 0 2.0 M 4.0 M 6.0 M 8.0 M 1023 10 10 4 10 5 10 67 10 3 2 FSC PI 2 Cells (x10 Cells (x10 1 + 1 +

7 7 CD4 10 10 0 CD8 0

106 0246 0246 106

105 + 7 + 7 105 CNX Epithelial cells (x10 /g) CNX Epithelial cells (x10 /g)

104

103

103 0

102 CD4/8 CNX

102 103 104 105 106 107 1023 10 10 4 10 5 10 67 10 EpCAM CD3

Figure 1. Calnexin expression is upregulated in OSCC, and its membranous expression is correlated with reduced infiltration of T cells in OSCC tissues. A, qRT-PCR analyses of calnexin (CNX) expression in tumor and adjacent normal tissues derived from the same OSCC patient (n ¼ 33; left); qRT-PCR analyses of CNX expression in OSCC cell lines (right). NOK cells served as control cells. B, Western blotting analysis of CNX expression in the cell lines (top) and paired tissues (bottom). N, normal tissue; Ca, cancer (n ¼ 8). C, IHC analysis of CNX expression in tumor and adjacent normal tissues derived from a typical patient with progressive OSCC. D, Representative immunofluorescence microscopic images of CNX in OSCC tissue and melanoma tissue. In addition to be primarily expressed in the cytosol of OSCC cells, a significant fraction of calnexin colocalized with Wheatgerm agglutinin (WGA) in the cell membrane of tumor cells (scale bar, þ þ 50 mm). E, The gating strategy of CNX epithelial cell and T cells in single-cell suspension of fresh surgical specimen. F, The number of CNX epithelial cells among every gram of OSCC tissue, was correlated with CD4þ and CD8þ T cells in corresponding single-cell suspensions, n ¼ 20. Pearson correlation coefficient was used. Bar graph, mean SEM. , P < 0.05; , P < 0.001; , P < 0.0001. One representative experiment of three is depicted.

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noncancerous tissues were derived from the same patients with Calnexin inhibits T-cell proliferation and antitumor effector OSCC. We found that calnexin was significantly upregulated functions in OSCC tissues. Because upregulation of calnexin in OSCC tumor tissues was þ þ In addition, immunofluorescence analysis suggested that correlated with reduced infiltration of CD4 and CD8 T cells, we although most of the calnexin protein expression was observed hypothesized that calnexin impairs the antitumor immunity of in the cytosol of OSCC cells, a considerable fraction of effector T cells. A calnexin-Ig fusion protein (CNX-Ig) was gener- calnexin colocalized with WGA in the cell membrane of tumor ated to examine the regulatory roles of calnexin in T-cell cells. Membrane localization of calnexin has been found in responses. Indeed, we found that when immobilized on a micro- several tumors, including OSCC and melanoma (Fig. 1D), plate, calnexin-Ig, but not control-Ig, suppressed the proliferation þ þ suggesting that calnexin may exert its biological or immuno- of bulk purified CD4 and CD8 T cells in response to anti-CD3 logic regulation functions as either a secreted or membrane- stimulation (Fig. 3A) and inhibited the production of effector bound form. Because T cells play a critical role in mediating molecules such as IFNg, TNFa, and IL2 (Fig. 3B). Furthermore, þ antitumor immunity, and impaired T-cell infiltration is pos- calnexin inhibited the antitumor cytolytic functions of CD8 itively correlated with poorer prognosis of tumor patients T cells against HSC3 tumor cells (Fig. 3C). These data collectively (29), we first determined whether there was any correlation suggested that calnexin inhibited the proliferation and antitumor þ þ between membranous expression of calnexin and T-cell infil- effector functions of CD4 and CD8 T cells. Although the trationintumortissues.Cellsurfacecalnexinexpressionwas receptor for calnexin is unknown, we speculated that the engage- determined by flow cytometry. We found that higher calnexin ment of calnexin-R on T cells suppresses T-cell receptor (TCR) membranous expression was negatively correlated with the signaling. To test this hypothesis, proximal TCR signaling events þ þ numbers of infiltrated CD4 and CD8 T cells in OSCC tumor were examined using calnexin-Ig. LAT is a proximal signaling tissues (Fig. 1E and F). adaptor that is phosphorylated upon TCR stimulation and forms a complex with multiple signaling molecules, including SH2 Upregulation of membranous calnexin correlated with poor domain containing a leukocyte protein of 76 kDa (SLP76) and clinical prognosis phospholipase C (PLC)-g1 (30). Immobilized calnexin-Ig sub- Given that calnexin membranous expression was upregu- stantially reduced the amount of SLP76 recruited to the CD3 lated in OSCC tissues, we next determined whether calnexin complex, as well as its phosphorylation. When total cell lysates expression in the cytosol and cell membrane was correlated were examined, the phosphorylation of several downstream with tumor prognosis. To address this, IHC staining of calnexin signaling molecules, such as Akt and Erk1/2 was also impaired was performed to assess the expression of calnexin in samples (Fig. 3D). from 357 patients with primary OSCC. Representative images In addition to demonstrating the inhibitory effect of calnexin of the intensity stages are shown in Fig. 2A. Overall, cyto- on peripheral blood T cells from healthy donors, we also evalu- plasmic calnexin expression was categorized into low (178 of ated its role in circulating blood T cells from patients with OSCC. the 357 tumor samples, 49.86%) and high (179 of the 357 Tumor lysates from the same patients were used as tumor antitumor samples, 50.14%) expression groups using the cutoff gens. We found that PBMCs from OSCC patients cocultured with point 5.04 based upon the median of SID scores of the total calnexin-Ig showed inhibitory effects on the proliferation of þ patients. In addition, 71 of the 357 tumor samples (19.89%) CD8 T cells (Fig. 4A) and reduced the number of functional þ showed apparent calnexin expression at the plasma membrane, CD8 T cells producing IFNg by nearly half (Fig. 4B). These whereas 286 of the 357 tumor samples (80.11%) showed findings were confirmed by our cytometric bead assay (CBA) negative staining of calnexin at the plasma membrane (Sup- results, which showed decreased production of IFNg and TNFa plementary Table S2). Representative images are shown but increased production of IL10 by T cells (Fig. 4C). These in Fig. 2A. The expression of calnexin was assessed for associ- changes were more significant in the tumor antigen–experienced ation with a number of clinicopathologic variables (Supple- cells. Because an increase in IL10 production by T cells was mentary Table S2). observed, the induction of Treg was examined. However, calnexin Kaplan-Meier survival curves show, overall patient survival could not promote Treg production in an antigen-specific manner was not significantly different when compared between low (Supplementary Fig. S1). These data collectively suggested that and high cytoplasmic expression of calnexin (P ¼ 0.405), calnexin inhibited the proliferation and antitumor effector func- þ whereas patients with positive calnexin membranous expres- tions of CD8 T cells in patients with OSCC in an antigen- sion had a significantly reduced overall survival than patients dependent manner. with negative expression [3-year OS: 47.89% (35.93%– 58.88%) vs. 66.43% (60.64%–71.58%), P ¼ 0.016; Fig. 2B]. Calnexin promotes OSCC tumor growth in a humanized mouse In the univariate analysis, calnexin membranous expression model (P ¼ 0.018) along with nodal stage (P < 0.001), clinical TNM Because calnexin inhibits the proliferation and effector func- þ þ stage (P ¼ 0.030), and radiotherapy (P ¼ 0.042) were signif- tions of CD4 and CD8 T cells, we next determined whether icantly associated with overall survival. Adjusted for nodal stage calnexin-mediated impairment of antitumor T-cell responses and radiotherapy, patients with positive membranous expres- contributes to tumor growth. Because OSCC tumor cells do not sion of calnexin was significantly associated with reduced grow well in wild-type mice, a humanized mouse model was used overall survival, compared with patients with negative expres- (31, 32). HSC3 tumor cells expressing shRNA-CNX (sh-CNX) or sion (HR, 1.59; 95% CI, 1.10–2.30; P ¼ 0.013; Fig. 2C). There shRNA-control (sh-NEG) were inoculated into NCG mice, and the was no significant association between cytoplasmic expression mice were engrafted with human PBMCs after tumor implanta- of calnexin and overall survival among patients with OSCC tion. The mice were euthanized before experiencing weight loss, a (Supplementary Tables S3 and S4). symptom of graft-versus-host disease (GVHD) that occurs in this

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A CNX Cytoplasmic staining

CNX Intensity (weak) CNX Intensity (moderate) CNX Intensity (strong)

CNX Plasma membrane staining

Positive Negative

B CNX Cytoplasmic expression CNX Membrane expression

100 ) 100

High expression Negative 50 50 Low expression Positive Log-rank test: P = 0.405 Log-rank test: P = 0.016 Overall survival (%) 0 Overall survival (% 0 0 122436486072 0 122436486072 Months to last follow-up Months to last follow-up NO. at risk NO. at risk High expression 179 153 123 114 105 83 3 Negative 286 250 205 190 163 118 6 Low expression 178 154 122 110 92 60 3 Positive 71 57 40 34 34 25 0

C Multivariable risk factor HR (95% CI) p

CNX membranous expression 1.59 (1.1−2.3) 0.013 (Positive vs. Negative) Nodal stage 1.88 (1.36−2.59) <0.001 (N1-N3 vs. N0) Radiotherapy 0.56 (0.36−0.88) 0.012 (Yes vs. No)

0 1 2 3

Figure 2. Upregulation of membranous calnexin is correlated with poorer overall survival rates of patients with OSCC. A, The calnexin (CNX) cytoplasmic expression was determined on the basis of the staining intensity and proportion, and then divided into high and low expression groups using the cutoff point 5.04 based upon the median of SID scores of the total patients. The calnexin plasma membrane expression was determined, and the samples were divided into positive and negative expression groups. Representative images showing strong-, moderate- and weak-intensity cytoplasmic staining and positive/negative membranous staining in tumor tissue samples derived from patients with OSCC. B, Analysis of the associations between the cytoplasmic and membranous expression of calnexin and overall survival among 357 patients with OSCC. Kaplan–Meier survival curves showed that patients with positive membranous expression of calnexin had a significantly reduced overall survival than patients with negative expression (P ¼ 0.016). Calnexin cytoplasmic expression was not significantly associated with overall survival (P ¼ 0.405). The P value was determined by log-rank test. C, Adjusted multivariable risk factor cohort of overall survival. Calnexin membranous expression was significantly associated with overall survival (P ¼ 0.013).

humanized mouse model (Fig. 5A). In this model, the tumor injection in HSC3 tumor model and found that in contrast to the growth in the sh-CNX group was lower than that in the sh-NEG results from humanized mice, calnexin silencing promoted tumor group (P ¼ 0.047; Fig. 5B). We also detected increased frequencies growth in the immunodeﬁcient mice (Fig. 5E and F), indicating þ of multifunctional CD3 T cells producing IFNg in the sh-CNX that calnexin might have another tumor-intrinsic role that is group compared with the sh-NEG group upon PBMC engraftment independent of its function on T cells. These data indicated that (Fig. 5C and D). We also examined control mice without PBMC calnexin suppressed antitumor immunity and promoted OSCC

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CD4 CD8 A α-CD3 B 800 * ** * 1,500 *** α-CD3+CNX-Ig 600 α-CD3+Flag-Ig 1,000

400 (pg/mL) Control (pg/mL) γ 2 N 500 IL

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4 8 2 4 8 2 CFSE 2 4 7 2 4 7 Time (h) Time (h) N.S. N.S. * ** **** 500 * CNX-Ig 100 100 **** **** ** 400 Flag-Ig ** **** %) 80 80 300 pg/mL)

60 60 α( 200

40 40 TNF 100 CFSE-low ( CFSE-low (%) + + 20 20 0 CD4 0 CD8 0 4 8 2 2 4 7 α-CD3 +++ − α-CD3 +++ − Time (h) − −− − −− Flag-Ig + Flag-Ig + D CNX-Ig Flag-Ig CNX-Ig + −−− CNX-Ig + −−− p-SLP76 C CNX Vector 5 5 105 0 7.91 10 0 25.5 10 0 5.68 SLP76

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3 3 103 10 10 p-PLCγ-1

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1 1 101 10 10 0 92.1 0 74.5 0 94.3 PLCγ-1 0 0 100 10 10 DAPI 01 2 3 4 5 1001 10 10 2 10 3 10 4 10 510 01 10 10 2 10 3 10 4 10 5 10 10 10 10 10 10 CFSE p-AKT Effector : Target = 5 : 1 Effector : Target = 0 : 1

50 AKT 5 5 *** *** CNX 10 0 14.3 10 0 38.8 40

%) Vector 4 4 10 10 p-Erk1/2 g( 3 3 30 10 10 illin 2 2 10 10 20

LK Erk1/2 1 1 10 10 10 0 85.7 0 61.2 CT 0 0 10 10 DAPI 0 1 2 3 4 5 0 1 2 3 4 5 10 10 10 10 10 10 10 10 10 10 10 10 0 β-Actin CFSE :1 1 5 0: Effector : Target = 10 : 1 1

Figure 3. Calnexin inhibited the proliferation of CD4þ/CD8þ T cells and the cytotoxicity of CD8þ T cells. Fresh PBMCs were isolated from 6 healthy donors. A, CFSE-labeled, bulk-purified pan T cells were stimulated by plate-bound anti-CD3 together with coabsorbed calnexin-Ig (CNX-Ig) or control-Ig (Flag-Ig) protein. Top, representative CFSE dilution profiles. Bottom, the percentage of CFSE-low cells was quantified. B, Culture supernatants in A were collected at the þ indicated times. The concentrations of IL2, IFNg, and TNFa were analyzed by ELISA. C, Tumor antigen–specific CD8 human T-cell clones (CTL) were generated from PBMCs of a healthy donor by in vitro stimulation using dendritic cells loaded with irradiated HSC3 cells. Calnexin-overexpressing HSC3 cells (CNX) or control cells (Vector) were labeled with CFSE and cocultured with CTLs at an effector-to-target ratio (E/T) of 5:1 and 10:1 for 4 hours. D, Engagement of calnexin during TCR activation maximally suppresses proximal adaptor signaling. Na€ve pan-T cells purified from human PBMCs were incubated on ice for 30 minutes with anti-CD3/CD28 and CNX-Ig or Flag-Ig. Then, cell lysates were prepared, and the phosphorylation status of SLP76, PLC-g1, AKT, and Erk1/2 was examined by immunoblotting. Bar graph is shown as the mean SEM (n ¼ 6); N.S., not significant. , P < 0.05; , P < 0.01; , P < 0.001; , P < 0.0001. One representative experiment of three is depicted. tumor growth via inhibiting the proliferation and effector cells expressing shRNA targeting calnexin (sh-CNX) or control þ þ functions of CD4 and CD8 T cells. shRNA (sh-NEG) and monitored tumor growth. To generate protective immunity, na€ve mice were vaccinated with irradiated Calnexin deficiency promotes antitumor immunity and B16F10 tumor cells in advance (Fig. 6A). We found that knock- controls tumor growth down of calnexin in melanoma tumor cells significantly inhibited We next developed a mouse melanoma model to determine melanoma growth in mice, whereas administration of calnexin-Ig whether calnexin-mediated impairment of T cells contributes to enhanced melanoma growth (Fig. 6B). Furthermore, knockdown tumor growth. We injected mice subcutaneously with B16F10 of calnexin in melanoma tumor cells increased the infiltration of

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A 40 N.S. 5 5 5 5 * 10 10 10 10 17.7 13.4 37.2 21.9

4 4 4 4 30 10 10 10 10

3 3 3 3 10 10 10 10 20

2 2 2 2 10 10 10 10 among CD8 (%) 10

0 0 0 0 + Ki67 2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5 010 10 10 10 010 10 10 10 010 10 10 10 010 10 10 10

Ki67 0 CD8 Flag-Ig CNX-Ig Flag-Ig CNX-Ig Tumor antigen −− ++ Medium Tumor antigen Flag-Ig ++−− CNX-Ig− + − + B 40 N.S. ****

among CD810 (%) +

IFNγ 0 IFNγ CD8 Tumor antigen −− ++ Flag-Ig CNX-Ig Flag-Ig CNX-Ig Flag-Ig ++−− Medium Tumor antigen CNX-Ig− + − + C

150 500 ** * 200 N.S. * N.S. * 400 150 100 300 100 200 50

IFNγ (pg/mL) IFNγ 50 IL10 (pg/mL) IL10

TNFα (pg/mL) TNFα 100 0 0 0

Tumor antigen -- ++ Tumor antigen − − ++ Tumor antigen − − ++ Flag-Ig ++- - Flag-Ig ++− − Flag-Ig ++− − CNX-Ig + - + - CNX-Ig + − + − CNX-Ig + − + −

Figure 4. þ Calnexin inhibited tumor antigen–specific Ki67 and IFNg expression on CD8 T cells during progressive OSCC. Fresh PBMCs isolated from 8 patients with progressive OSCC were restimulated by tumor lysate in the presence of calnexin (CNX)-Ig or Flag-Ig for 48 hours. The PBMCs were then subjected to flow cytometric analyses of Ki67 and IFNg expression, and the culture supernatants were subjected to cytometric bead assay (CBA) analysis of Th1/Th2 cytokine concentrations. A, Representative flow cytometric data and bar graph show the percentages of Ki67þCD8þ T cells in the presence of OSCC tumor lysate þ þ with calnexin-Ig or Flag-Ig. B, Representative flow cytometric data and bar graph data show the percentages of IFNg CD8 T cells in the presence of OSCC tumor lysate with calnexin-Ig or Flag-Ig. C, Concentrations of IFNg, TNFa, and IL10 in the culture supernatants. Bar graph, mean SEM (n ¼ 8). N.S., not significant; , P < 0.05; , P < 0.01; , P < 0.0001. One representative experiment of two is depicted.

þ þ þ CD3 , CD4 , and CD8 T cells in melanoma tumors (Fig. 6C and No differences in Tregs and MDSC frequencies among TILs were þ þ D) and enhanced the expression of Ki67 in CD4 and CD8 found (Supplementary Fig. S2). There were no significant þ T cells (Fig. 6E). In addition, treatment with calnexin-Ig inhibited differences in the proliferation and effector functions of CD4 þ this infiltration in melanoma tumors (Fig. 6C and D) and the and CD8 T cells located in the spleen, lymph nodes and expression of Ki67 in these T cells (Fig. 6E). Moreover, knockdown PBMCs between the groups (Supplementary Fig. S3). To con- of calnexin in melanoma tumor cells enhanced the expression firm that there is no intrinsic enhancement of tumor growth in þ of the antitumor effector molecules IFNg and TNFa by CD4 the absence of T-cell–mediated antitumor immunity, tumors þ and CD8 T cells in melanoma tumors, and this effect was were inoculated in T-cell–deficient nude mice. As shown in significantly reversed by treatment with calnexin-Ig (Fig. 6F). Supplementary Fig. S4, administration of calnexin-Ig no longer

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A B Blood withdrawal

) 800 NCG mice 3 sh-CNX Day7 Day14 600 sh-NEG Day1 Day21 sh-NEG 400 * 1×107 human-PBMC i.v. Tumor isolated 200 + sh-CNX 6×106 sh-CNX or sh-NEG HSC3 s.c. Tumor volume (mm 0 0 5 10 15 20 25 C D Days 600 * 50 N.S. * sh-CNX 40

Tcellin 400 sh-NEG + 30 200 20 10 each mg of tumor

0 Human-IFNγ (pg/mL) Human-CD3 0

y7 a h-CNX D ay14 s sh-NEG D E F

) 800 NCG mice 3 sh-CNX sh-NEG sh-NEG 600 Day1 Day22 ** 400 sh-CNX 6×106 sh-CNX Tumor isolated 200 or

sh-NEG HSC3 s.c. Tumor volume (mm 0 0 5 10 15 20 25 Days

Figure 5. Calnexin promotes OSCC tumor growth in humanized NCG mice. A, Schematic diagram showed the experiment protocol used to determine the role of calnexin (CNX) in OSCC tumor growth in immune-integrity environment. On day 1, mouse was transplanted with HSC3 cells transduced with sh-CNX or sh-NEG, 1 107 human PBMCs were injected intraperitoneally. On days 7 and 14, peripheral blood samples were taken. B, Representative in situ images of OSCC tumors and tumor volume kinetics were measured and calculated using the following formula: V ¼ L W2/2. C and D, The bar graph shows the increased þ frequencies of CD3 T cells and functional T cells producing IFNg in the sh-calnexin group compared with the sh-NEG group after PBMC engraftment. E, Schematic diagram shows the experiment protocol used to determine the role of calnexin in OSCC tumor growth in immune-deﬁcient environment. HSC3 cells transduced with shRNA targeting calnexin (sh-CNX) or control shRNA (sh-NEG) were injected subcutaneously at indicated time. F, Representative in situ images of OSCC tumors in NCG mice and tumor volume kinetics were measured and calculated using the formula described in B. Bar graph, mean SEM (n ¼ 5); , P < 0.05. One representative experiment of two is depicted.

enhanced melanoma growth upon T-cell deficiency. Calnexin- the expression of PD-1, but not TIGIT, CTLA-4, PD-1H, or LAG-3, þ þ silenced B16F10 tumors grew more rapidly than control in CD4 and CD8 T cells derived from melanoma tumor tumors, which is consistent with our previous observation samples (Fig. 7A). In contrast, calnexin-Ig treatment partly þ þ (Fig. 5E and F) that tumor-intrinsic calnexin itself suppressed reversed the decrease in PD-1 expression on CD4 and CD8 tumor growth. Together, these data indicated that calnexin T cells conferred by knockdown of calnexin in melanoma tumors deficiency promotes antitumor immunity and controls tumor (Fig. 7A). Similar results were found in an MB49 tumor model growth in a T-cell–dependent manner. with calnexin-Ig treatment (Fig. 7B). In addition, calnexin-Ig þ enhanced the expression of PD-1 on CD8 T cells in PBMCs Calnexin enhances the expression of PD-1 by repressing PD-1 derived from patients with progressive OSCC, and this enhance- promoter methylation ment was more significant in tumor antigen–experienced T cells, Given that T-cell surface receptors such as TIGIT, CTLA-4, PD-1, as shown in Fig. 7C. Thus, these data suggested that calnexin þ þ and LAG-3 play critical roles in inhibiting T-cell responses, we next enhanced the expression of PD-1 on CD4 and CD8 T cells in determined whether upregulation of calnexin might enhance the OSCC in an antigen-dependent manner. expression of these molecules and therefore induce impairment of We then determined the mechanism by which calnexin þ þ the proliferation and effector functions of CD4 and CD8 T cells enhanced the expression of PD-1 on T cells in tumors. Because in tumors. To address this, we analyzed the expression of TIGIT, PD-1 promoter CpG island methylation status plays a central role þ þ CTLA-4, PD-1H, PD-1, and LAG-3 on CD4 and CD8 T cells in mediating PD-1 expression (33, 34), we analyzed the methyl- derived from melanoma tumor samples. We found that knock- ation of this region using bisulfite sequencing in T cells from down of calnexin in melanoma tumor cells significantly decreased OSCC patients' PBMCs (Fig. 7D). In contrast to control-Ig, T cells

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Figure 6. Calnexin expressed in tumor cells inhibits the antitumor protective immunity of CD4þ and CD8þ T cells in a mouse melanoma model. A, Schematic diagram shows the protocol used to determine the effect of calnexin (CNX) on melanoma tumor growth in mice. B, Kinetics of tumor volumes in mice as indicated (n ¼ 4–5). C, Flow cytometry analysis of the number of CD4þ and CD8þ T cells infiltrated in tumors derived from mice with the indicated treatments. Note that knockdown of calnexin (CNX) significantly increased the number of infiltrated CD4þ or CD8þ T cells in tumors. However, treatment with calnexin-Ig decreased the number of tumor-infiltrated CD4þ or CD8þ T cells (n ¼ 4–5). D, IHC analysis of tumors derived from mice with indicated treatments suggested that significantly larger numbers of infiltrated CD3þ T cells in tumors were observed in the calnexin-deficient group. E, Flow cytometry analysis of the Ki67 expression on tumor infiltrated CD4þ and CD8þ Tcells.F, Representative flow cytometric analysis of expression of IFNg and TNFa in CD4þ or CD8þ T cells isolated from tumors derived from mice with the indicated treatments; note that knockdown of calnexin in melanoma tumor cells enhanced expression of þ þ the antitumor effector molecules IFNg and TNFa produced by CD4 and CD8 T cells, and this enhancement of effector functions was significantly reversed by treatment with the calnexin-Ig. Bar graph, mean SEM (n ¼ 4–5); , P < 0.05; , P < 0.01; , P < 0.001; , P < 0.0001; N.S., not significant. One representative experiment of two is depicted.

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Figure 7. Calnexin promotes the expression of PD-1 on CD4þ/CD8þ T cells in tumor by restraining the DNA methylation status of a CpG region in the PD-1 (PDCD1) promoter. A, Representative flow cytometric analysis and dot plot data show the expression of PD-1, TIGIT, CTLA-4, PD-1H, and LAG-3 on CD4þ or CD8þ T cells isolated from mice in Fig. 6. The data showed that knockdown of calnexin (CNX) in B16F10 tumor cells significantly reduced the expression of PD-1, but not TIGIT, CTLA-4, PD-1H, or LAG-3, on CD4þ or CD8þ T cells (n ¼ 4–5). B, Representative flow cytometric analysis and dot plot data from an MB49 tumor model showed that treatment with calnexin-Ig, but not Flag-Ig protein, significantly enhanced the expression of PD-1 on CD4þ and CD8þ T cells (n ¼ 4–5). One representative experiment of two is depicted. C, Representative flow cytometric data and bar graph data showed that calnexin-Ig increased the expression of PD-1 among CD8þ T cells in the presence of OSCC tumor antigen (n ¼ 8). D, Schematic of the CpG island and bisulfite pyrosequencing region in the PDCD1 promoter. TSS, transcription start site; red letters, CG sites for bisulfite pyrosequencing. Bisulfite pyrosequencing was used to detect the methylation of PD-1 promoter CpG island. E, The average methylation in the calnexin-Ig and Flag-Ig group was calculated. Note that recombinant calnexin-Ig significantly suppressed PD-1 promoter CpG island methylation in T cells. The data are representative of three independent experiments. Bar graph is shown as mean SEM; , P < 0.05; , P < 0.01; , P < 0.001; N.S., not significant.

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treated with calnexin-Ig significantly repressed the methylation of of antitumor responses during PD-1 blockade should be deter- the PD-1 promoter CpG island (Fig. 7E). mined in further studies. Although we found that calnexin expressed in tumor cells þ þ Discussion limited the infiltration and effector functions of CD4 and CD8 T cells in tumors and therefore promoted tumor cell growth, the In this study, we discovered that the ER chaperone protein þ þ specific receptor expressed on CD4 and CD8 T cells that calnexin was highly upregulated in OSCC tumor tissues and interacts with calnexin remains unknown. In addition, the obser- multiple tumors. Upregulation of membranous calnexin was vations that only membranous calnexin expressed in tumor cells positively correlated with poor prognosis of patients with OSCC. was associated with poorer survival of patients with OSCC indi- We found that calnexin played a central role in inhibiting the cate that direct contact with PBMCs is required for calnexin to exert infiltration and effector functions of T cells and promoting the þ þ its regulatory function. Identification of the receptor that interacts expression of PD-1 on CD4 and CD8 T cells in tumors, which with calnexin expressed in tumor cells will allow us to better therefore enhanced tumor growth, demonstrating the potential of understand the mechanism by which calnexin impairs the infil- calnexin as a new antitumor immunotherapy target. þ þ tration and effector functions of CD4 and CD8 T cells in the Calnexin has been reported to play a role in the folding and tumor microenvironment. Although low concentrations of cal- quality control of newly synthesized glycoproteins (14, 35). A nexin were isolated from lung cancer patients' peripheral blood wide variety of important cellular and viral glycoproteins are serum (15), the interaction between calnexin and T cells may known substrates of calnexin, including HIV gp120 and gp160, primarily occur in the tumor site. Thus, the interaction between class I MHC heavy chain, and TCR subunits (36, 37). Although calnexin and T cells in the circulation and lymphoid tissue may be several reports have shown that calnexin expression may be not sufficient for inhibition of the proliferation and effector associated with the progression of breast cancer, lung cancer, and functions of T cells. Previous studies from other groups suggest colorectal cancer, most previous studies of calnexin focused on the that calnexin could be transported to the plasma membrane to relationship between the expression of calnexin and clinical interact with glycoproteins such as clonotype-independent CD3 outcome (38–40). Whether calnexin regulates the T-cell response complexes (16, 45). Further studies are required to determine during tumor development is unknown. Here, we first identified what protein interacts with calnexin. that upregulation of calnexin in tumor cells could inhibit the infiltration of T cells in tumors and the proliferation and effector þ þ Disclosure of Potential Conflicts of Interest functions of CD4 and CD8 T cells. As increasing evidence has fi fi L. Chen is a scienti c founder of and has ownership interest in NextCure and suggested that the in ltration and effector functions of T cells in TAYU Biotech, reports receiving commercial research grant funding NextCure, tumors are critical for antitumor immunity, this finding therefore andisa consultant/advisory board member for Pfizer, Vcanbio, andGenomiCare. reveals a mechanism responsible for poor survival of tumor No potential conflicts of interest were disclosed by the other authors. patients. A finding of this study is the establishment of an immunologic Authors' Contributions link between calnexin and PD-1 on T cells. We found that Conception and design: Y. Chen, J. Cui, G. Zeng, L. Chen, B. Cheng, Z. Wang knockdown of calnexin in melanoma tumor cells significantly Development of methodology: D. Ma, X. Wang, J. Song decreased the expression of PD-1. In addition, calnexin-Ig treat- Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Y. Chen, D. Ma, X. Wang, J. Fang, Q. Li, Q. Li, S. Wen ment partly reversed the decrease of PD-1 expression on T cells. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, Calnexin-Ig also enhanced the expression of PD-1 on T cells in computational analysis): Y. Chen, D. Ma, X. Li, X. Ren, L. Chen PBMCs derived from patients with progressive OSCC by inhibit- Writing, review, and/or revision of the manuscript: J. Cui, G. Zeng, L. Chen, ing the PD-1 promoter CpG island methylation. PD-1 is upregu- Z. Wang lated on activated T cells. The binding of PD-1 and PD-L1 induces Administrative, technical, or material support (i.e., reporting or organizing T-cell anergy and cell death (5, 41). This study provides evidence data, constructing databases): X. Wang, X. Liu, L. Luo, J. Xia Study supervision: B. Cheng, Z. Wang suggesting that PD-1 expression on T cells can be influenced by the expression of calnexins. The detailed mechanism by which cal- Acknowledgments nexin mediates PD-1 expression during tumor development We thank Luisa A. DiPietro (College of Dentistry, UIC) for the suggestions on should be investigated in future studies. There are substantial this manuscript. This project was supported by the National Natural Science efforts underway to identify reliable predictive biomarkers of Foundations of China (grant nos. 81772896, 81472524, 81630025, 81602383, response and resistance to immune checkpoint blockade, includ- and 81602384). ing total tumor mutational load (42, 43), as well as markers of an effective immune infiltrate within a tumor signifying a "hot" or The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "cold" tumor microenvironment (44). This study provides a advertisement – in accordance with 18 U.S.C. Section 1734 solely to indicate potential target for the improvement of responses to anti PD-1 this fact. immunotherapy. Because the density or distribution of T cells and PD-1/PD-L1 axis activation could affect the differential responses Received March 7, 2018; revised August 6, 2018; accepted November 2, 2018; to checkpoint blockade, the effect of calnexin on the enhancement published first November 6, 2018.

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Calnexin Impairs the Antitumor Immunity of CD4+ and CD8+ T Cells

Yichen Chen, Da Ma, Xi Wang, et al.

Cancer Immunol Res Published OnlineFirst November 6, 2018.

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