Mechanisms by Which Dendritic Cells

Published OnlineFirst July 17, 2018; DOI: 10.1158/2326-6066.CIR-17-0716

Research Article Cancer Immunology Research Mechanisms by Which Dendritic Cells Present Tumor Microparticle Antigens to CD8þ T Cells Jingwei Ma1,2, Keke Wei2, Huafeng Zhang2, Ke Tang2, Fei Li1, Tianzhen Zhang3, Junwei Liu4, Pingwei Xu2, Yuandong Yu2, Weiwei Sun2, LiYan Zhu2, Jie Chen2, Li Zhou2, Xiaoyu Liang3, Jiadi Lv3, Roland Fiskesund3, Yuying Liu3, and Bo Huang1,2,3

Abstract

Tumor cell–derived microparticles (T-MP) contain tumor MHC class I–tumor antigen peptide complexes. Concurrent- antigen profiles as well as innate signals, endowing them ly, endocytosis of T-MPs results in the upregulation of with vaccine potential; however, the precise mechanism CD80 and CD86. T-MP–increased ROS activate lysosomal þ þ by which DCs present T-MP antigens to T cells remains Ca2 channel Mcoln2, leading to Ca2 release. Released þ unclear. Here, we show that T-MPs activate a lysosomal Ca2 activates transcription factor EB (TFEB), a lysosomal pathway that is required for DCs presenting tumor antigens master regulator that directly binds to CD80 and CD86 of T-MPs. DCs endocytose T-MPs to lysosomes, where T- promoters, promoting gene expression. These findings elu- MPs increase lysosomal pH from 5.0 to a peak of 8.5 cidate a pathway through which DCs efficiently present þ via NOX2-catalyzed reactive oxygen species (ROS) produc- tumor antigen from T-MPs to CD8 T cells, potentiating tion. This increased pH, coupled with T-MP–driven lyso- T-MPsasanoveltumorcell–free vaccine with clinical somal centripetal migration, promotes the formation of applications. Cancer Immunol Res; 6(9); 1057–68. 2018 AACR.

Introduction that present tumor antigens. Tumor cell–derived cytokines (e.g., VEGF, IL10, and TGFb) and biologic factors (e.g., galectin-1, Whole tumor cells may be a promising source of tumor indoleamine 2,3-dioxygenase, and lipid droplets) have been antigens for the use in construction of cancer vaccines for several shown to suppress DC maturation and T-cell activation, likely reasons. They contain the complete repertoire of mutated neoan- limiting the efficacy and efficiency of whole cell–based vaccina- tigens and tumor-associated antigens, which will likely reduce tion (8, 9). Therefore, a vital consideration for developing tumor the possibility of immune escape and development of resistance cell–based vaccines is providing DCs with appropriate innate (1, 2). Also, the manipulation of tumor cell vaccination is very signals apart from the tumor antigens. simple and convenient by means of culture and irradiation (3). Tumor cells are capable of generating and releasing microve- The immunogenicity of whole tumor-cell vaccines can be aug- sicles of various sizes into the extracellular space (10). These mented by the addition of adjuvants, including, but not limited subcellular structures not only contain tumor antigens, but also to, Freund's incomplete adjuvant, BCG, GM-CSF, and Toll-like include information that is unique from the parental cells (11, receptor agonists (4–6). Despite the potential success of these 12), suggesting the potential for tumor cell–derived microvesicles vaccines, previous reports of autologous and allogeneic whole to be developed as tumor vaccines. In response to stimuli or tumor cell–based vaccines in clinical trials in patients with apoptotic signals, cells may alter their cytoskeleton to encapsulate various tumor types demonstrate limited efficacy and efficiency cytosolic contents within the cellular membrane to form subcel- of these vaccines (7). The antitumor immune response is mainly þ lular vesicles. These 0.1–1 mm vesicles, termed microparticles, are conveyed by tumor-lytic CD8 T cells via dendritic cells (DC) subsequently released into extracellular spaces (13–16). We have previously shown that tumor cell–derived microparticles (T-MP) can be readily taken up by DCs, leading to maturation and 1Department of Immunology, Tongji Medical College, Huazhong University of presentation of multiple antigens. T-MPs may represent an effec- Science and Technology, Wuhan, China. 2Department of Biochemistry and tive vaccination platform to trigger antitumor T-cell immune Molecular Biology, Tongji Medical College, Huazhong University of Science and response (17); however, the underlying molecular mechanisms 3 Technology, Wuhan, China. Department of Immunology and National Key by which DCs mature and present tumor antigens to tumor-lytic Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, þ CD8 T cells remain unclear. Chinese Academy of Medical Sciences, Beijing, China. 4Cardiovascular Surgery, Union Hospital, Huazhong University of Science and Technology, Wuhan, China. Lysosomes are the sites at which class I peptides are generated for DCs to load them onto MHC class I, forming a complex for the Note: Supplementary data for this article are available at Cancer Immunology Research Online (http://cancerimmunolres.aacrjournals.org/). cross-presentation of T-MP antigens; thus, we speculate that, following endocytosis by DCs, T-MPs may alter the function of J. Ma and K. Wei contributed equally to this article. lysosomes. At least two key issues regarding antigen cross-pre- Corresponding Author: Bo Huang, Chinese Academy of Medical Sciences, sentation are dependent on lysosomal alterations. First, to gen- 5 Dong Dan San Tiao, Beijing 100005, China. Phone: 86-10-69156447; Fax: erate the 8 to 11 amino acid–length antigenic peptide, a transient 86-10-65229258; E-mail: [email protected] increase of lysosomal pH is necessary to avoid the degradation of doi: 10.1158/2326-6066.CIR-17-0716 tumor antigens into smaller peptides or single amino acids. 2018 American Association for Cancer Research. Second, lysosomal alteration might be required to regulate the

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expression of costimulatory molecules, such as CD80 and Preparation of bone marrow–derived DCs, splenic-derived þ þ CD86, for effective presentation of tumor antigens to cytolytic CD8a DCs, and CD8 T cells þ CD8 T cells. In this present study, we demonstrate that B16- Bone marrow cells were harvested from femurs of mice and OVA melanoma tumor cell–derived microparticles (OVA-MP) cultured in RPMI 1640 supplemented with 10% FBS, 100 U/mL can readily enter DC lysosomes, increasing lysosomal pH and penicillin, and 100 mg/mL streptomycin. The cells were cultured upregulating CD80 and CD86 expression via a NOX2–ROS– in 6-well plates with 20 ng/mL GM-CSF (PeproTech) and 20 ng/ TFEB signaling pathway, leading to subsequent activation of mL IL4 (PeproTech), and cytokines were replenished on days 3 þ tumor-lytic CD8 Tcells.Thesefindings highlight tumor cell– and 5; nonadherent cells were harvested for experiments. We also derived microparticles as cell-free vaccines with potential clin- used Flt3L (200 ng/mL) to treat mouse bone marrow cells for þ ical applications. 10 days to induce Flt3L-BMDCs. Murine splenic CD8a DCs from pooled spleens (from at least 3 spleens) were purified first by density-gradient centrifugation and then by magnetic-activated Materials and Methods þ cell separation. OT-I and pmel-1 mice–derived splenic CD8 Cell lines and animals T cells were purified by magnetic-activated cell separation (purity Murine melanoma cell lines B16-OVA and B16 were > 90%, Miltenyi Biotec). obtained from China Center for Type Culture Collection in 2014 and maintained in a rigid dish with 1640 cell culture T-cell proliferation assay medium (Invitrogen) supplemented with 10% FBS (Gibco) at þ fi Splenic CD8 T cells were puri ed through negative selection 37 Cwith5%CO2. Cells were determined to be mycoplasma- (Miltenyi Biotec) from OT-I or pmel-1 mice, then fluorescently free, free of interspecies cross-contamination and authenticated labeled with 5 mmol/L CFSE (Sigma-Aldrich). DCs were incubated by isoenzyme and short tandem repeat analyses in Cell with OVA-MPs for 12 hours to obtain OVA-MP–loaded DCs. Resource Centre of Peking Union Medical College before the CFSE-labeled T cells were incubated with OVA-MP–loaded DCs or study. Cell lines used in the experiments were within 20 empty DCs for 2 to 3 days before flow-cytometric analysis. passages. Female wild-type C57BL/6J (6–8 weeks old) were purchased from the Centre of Medical Experimental Animals of In vitro cytokine secretion assay þ Hubei Province (Wuhan, China). OT-I TCR-transgenic mice Splenic CD8 T cells purified from OT-1 mice were incubated (C57BL/6-Tg (TcraTcrb) 1100Mjb/J) were a gift from Dr. Hui with empty DCs or OVA-MP–loaded DCs. IFNg in the super- þ Zhang (Sun Yat-Sen University, Guangdong, China). Pmel-1 natants of CD8 T cells was assessed by the mouse mini ELISA kit transgenic mice were presented by Dr. Ying Wan (Third Military (PeproTech) according to the manufacturer's protocol. Medical University, Chongqing, China). All mice were bred in fi speci c pathogen-free conditions. All animal experiments were Lysosomal staining and pH measurement performed in accordance with the National Institute of Health Lysosomal staining was performed using LysoTracker, a lyso- GuidefortheCareandUseofLaboratoryAnimalsalongwith somotropic probe (Invitrogen). The treated cells were incubated fi approval from the Scienti c Investigation Board of the Tongji for 30 minutes at 37 C with 1 mmol/L of LysoTracker. The cells Medical College, Wuhan, China. were examined using a confocal microscope. LysoSensor Green DND-189 is commonly used to qualitatively measure the pH of Reagents and antibodies acidic organelles, such as lysosomes, which become more fluo- Amiloride hydrochloride, DPI, NAC, ryanodine, CGP37157, rescent in acidic environments and less fluorescent in alkaline cyclosporin A (CsA), sodium vanadate (Na3VO4), PKH26 Red, environments. DCs were loaded with 0.1 mmol/L LysoSensor and PKH67 Green Fluorescent Cell Linker Kit were purchased Green DND-189 in prewarmed RPMI 1640 medium for 30 from Sigma. ER-, Mito-, LysoTracker Green/Red, LysoSensor minutes at 37C. The cells were then washed twice with PBS Green, LysoSensor Yellow/Blue, and CellLight Golgi-GFP were and immediately analyzed by fluorescence microscope. Quanti- purchased from Invitrogen. The following primary antibodies fication of lysosomal pH was performed using a ratiometric were purchased from Abcam: anti-LAMP1 (ab13523), anti-gp91 lysosomal pH dye LysoSensor Yellow/Blue DND-160. The pH (ab80508), anti-Rab7 (ab137029), anti-dynein (ab157468), calibration curve was generated according to the manufacturer's anti-histone H3 (ab8284), and anti-TFEB (ab2636, for ChIP). protocol. DCs were trypsinized and labeled with 2 mmol/L Lyso- Anti-b-actin (ANT009) was purchased from Ant Gene. The fol- Sensor Yellow/Blue DND-160 for 30 minutes at 37C in RPMI lowing secondary antibodies were purchased from Abcam: goat 1640 medium, and excess dye was washed away using PBS. The anti-mouse IgG FITC, donkey anti-rabbit IgG Alexa Fluor488, and labeled cells were treated for 10 minutes with 10 mmol/L mon- goat anti-rabbit IgG Dylight594. ensin and 10 mmol/L nigericin in 25 mmol/L MES calibration buffer, pH 4.5–7.5, containing 5 mmol/L NaCl, 115 mmol/L Generation and isolation of microparticles KCl, and 1.2 mmol/L MgSO4. Quantitative comparisons were Tumor cells were exposed to ultraviolet irradiation (300 J/m2) performed in a 96-well plate, and the fluorescence was measured for 1.5 hours, and 18 hours later, supernatants were used for with a microplate reader at 37C. microparticle isolation as described previously (17). Briefly, supernatants were centrifuged at 1,000 g for 10 minutes to Detection of reactive oxygen species remove whole cells and then centrifuged for 2 minutes at 14,000 Analysis of intracellular reactive oxygen species (ROS) produc- g to remove debris. The supernatant was further centrifuged for tion was conducted according to the manufacturer's protocol. 60 minutes at 14,000 g to pellet microparticles. The pellets were Briefly, DCs with indicated treatments were incubated with washed 3 times and resuspended in culture medium for the 2.5 mmol/L CellROX Green at 37C for 30 minutes and analyzed subsequent experiments. by flow cytometry.

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Plasmid constructs and transfection ATCTCAGCAAAAGGTGG-30 (sense) and 50-GTACTGTCCCAC- Recombinant vectors encoding murine TFEB were constructed CTCCATCTTG-30 (antisense); TAP1, 50-CAGCGGCAACCTTGTCT- by PCR-based amplification from cDNA of DCs and then were CAT-30 (sense) and 50-TTCCAGGATGCAGGGTGAAC-30 (anti- subcloned into the pcDNA3.1–3 Flag eukaryotic expression sense); TAP2, 50-TATGGCCTGAGGGACTGTGA-30 (sense) and 50- vectors. All constructs were confirmed by DNA sequencing in BGI TCCAGTTCTGTAGGGCCTGT-30 (antisense); Mcoln1, 50-ACCATC- (Shenzhen, China). Sequencing primers: forward, 50-CGCAAA- TCGGGGACTGTCAT-30 (sense) and 50-CAGGTAGCGAATGA- TGGGCGGTAGGCGTG-30 and reverse, 50-TAGAAGGCACAGTC- CACCGA-30 (antisense); Mcoln2, 50-GCATTCTGGTGTGGCTG- GAGG-30. Plasmids were transiently transfected into BMDCs with TTC-30 (sense) and 50-GGTGTGGTAAGAGTCGGTG A-30 (anti- Lipofectamine RNAiMAX Transfection Reagent (Invitrogen). sense); Tpcn1, 50-GGACGGCGCGTACCTTA-30 (sense) and 50- CGGTCCTCAGGATACAACGG-30 (antisense); Tpcn2, 50-GCCTT- Gene silencing experiments CCTGGTTGACCTCTC-30 (sense) and 50-CGAAACGATCCAGTC- siRNAs targeting mouse gp91 (siRNA#1: GCTGAATGTC- CACCA-30 (antisense); b-actin, 50-CATTGCTGACAGGATGCA- TTCCTCTTT; siRNA#2: CCATGGAGCTGAACGAATT; siRNA#3: GAAGG-30 (sense) and 50-TGCTGGAAGGTGGACAGTGAGG-30 GCACCATGATGAGGAGAAA), mouse dynein (siRNA#1: GAA- (antisense). ATCAACTTGCCCGATA; siRNA#2: CCACGTGCCTGTTGTATAT; siRNA#3: GCAGGCAGATGAGCAGTTT), mouse Rab7 (siRNA#1: Flow-cytometric analysis GGAAGAAAGTGTTGCTGAA; siRNA#2: CCATCAAACTGGAC- For phenotypic analysis of DCs, cells were stained with surface AAGAA; siRNA#3: GTACAAAGCCACAATAGGA), mouse Mcoln2 antibodies: anti-CD11c (clone N418), anti-CD80 (clone 16- (siRNA#1: GCAGTTCATTCCCGAGAGA; siRNA#2: GCTGAG- 10A1), anti-CD86 (clone GL1), anti-MHC I (clone M5/ GAAGAGATTTCTA; siRNA#3: GCTTGAAGGTCTGTAAGCA), 114.15.2), anti-MHC II (clone M5/114.15.2), anti-CCR7 (clone mouse TFEB (siRNA#1: GCAGGCTGTCATGCATTAT; siRNA#2: 4B12), anti-CD40 (clone 1C10), anti-OX40L (clone RM134L), CCAAGAAGGATCTGGACTT; siRNA#3: CCATGGCCATGCTA- and anti-PDL1 (clone 10F.9G2). Rat IgG2a, k, Rat IgG2b, k and CATAT) and negative control siRNAs (NC) were purchased from Armenian Hamster IgG were used as isotype controls. All anti- RiboBio. siRNA (50 nmol/L) was transfected into DCs using bodies were purchased from eBioscience or BioLegend, and flow- lipofectamine RNAiMax (Invitrogen) according to the manufac- cytometric analysis was performed with Accuri C6 (BD). turer's instruction. Two-photon confocal microscopy Real-time PCR Isolated OVA-MPs were labeled with a green fluorescent cell Real-time PCR analyses were performed with 2 mg of cDNA as a linker (PKH67; Sigma-Aldrich), according to the manufacturer's template, using an SYBR Green mix (Applied Biosciences) and an protocol. Labeled OVA-MPs were incubated with DCs at 37C for Agilent Technologies Stratagene Mx3500P real-time PCR system. 6 hours before staining with PE-CD11c antibody (eBioscience), Relative quantitative RNA was normalized using the housekeep- according to the manufacturer's protocol and visualized by two- ing gene GAPDH. Analysis of the results was performed using Bio- photon fluorescent microscopy. For intracellular staining, DCs Rad CFX Manager and relative quantification was performed. The were fixed in 2% paraformaldehyde for 10 minutes at room entire procedure was repeated in at least 3 biologically indepen- temperature, permeabilized with 100 mmol/L digitonin and dent samples. The primer sequences are shown as follows: blocked with 1% BSA for 1 hour at 25C. Samples were incubated LAMP1, 50-ACAGGGATATATGGGCAGGGA-30 (sense) and 50- with primary antibody (in PBS with 1% BSA and 0.1% Tween-20) AGCCAGGACACCCTTACCTC-30 (antisense); LAPM2, 50-AGGA- overnight at 4C. Following overnight incubation, cells were ATGTGCTGCTGACTCTG-30 (sense) and 50-AATGGAAGCACGA- washed 3 times in PBS and incubated with secondary antibodies GACTGGC-30 (antisense); TFEB, 50-CCACCCCAGCCATCAA- for 1 hour at room temperature. Nuclei were stained in DAPI CAC-30 (sense) and 50-CAGACAGATACTCCCGAACCTT-30 (anti- solution (1 mg/mL). Merge figure shows the bright field image and sense); ATP6V0A1, 50-CCGAGGACGAAGTGTTTGACT-30 (sense) fluorescent image observed under a two-photon fluorescent and 50-ATCAGCAGGATAGCCACGGTAA-30 (antisense); ATP6- microscope. V0A2, 50-TGGTGCAGTTCCGAGACCT-30 (sense) and 50-GCAG- GGGAATATCAGCTCTGG-30 (antisense); ATP6V0C, 50-ACTTAT- Western blot analysis CGCTAACTCCCTGACT-30 (sense) and 50-ACACCAGCATCTCC- Whole cell lysates were prepared from DCs and separated by GACGA-30 (antisense); ATP6V0E, 50-GCATACCACGGCCTT- SDS–PAGE at 100 V for 1 hour. Separated proteins were then ACTGT-30 (sense) and 50-TGATAACTCCCCGGTTAGGAC-30 transferred to nitrocellulose membranes (Millipore). The mem- (antisense); ATP6V1A, 50-ACAGAGGAAGCGTGACTTACA-30 branes were blocked in 5% BSA in TBS containing 0.1% Tween-20 (sense) and 50-CACTTGGACCATGCTGAACTT-30 (antisense); for 1.5 hours at room temperature. Then, the membranes were ATP6V1B2, 50-ATGCGGGGAATCGTGAACG-30 (sense) and 50- incubated with anti-TFEB, anti-Histone H3, or anti b-actin over- AGGCTGGGATAGGTAGTTCCG-30 (antisense); ATP6V1C1, 50- night at 4C. The membranes were washed 5 times and incubated ACTGAGTTCTGGCTCATATCTGC-30 (sense) and 50-TGGAAGA- with HRP-conjugated secondary antibodies for 1.5 hours at room GACGGCAAGATTATTG-30 (antisense); ATP6V1E1, 50-GAAT- temperature. Proteins were visualized by ECL Western blotting CAAGCAAGGCTCAAAGTCC-30 (sense) and 50-CGGGTCG TATC- substrate (Thermo Scientific Pierce). TTTTACCACC-30 (antisense); ATP6V1F, 50-GCGGGCAGAGG- þ TAAGCTAAT C-30 (sense) and 50-TTAGGGTGGCGGTTCTTGTTT- Intracellular Ca2 measurement 30 (antisense); ATP6V1G1, 50-CCCAGGCTGAAATTGAACAGT-30 DCs were cultured in 24-well plates at the density of 5 104 þ (sense) and 50-TTCTGGAGGACGGTCATCTTC-30 (antisense); cells/well in RMPI 1640 medium overnight. Before Ca2 mea- ATP6V1H, 50-GGATGCTGCTGTCCCAACTAA-30 (sense) and 50- surement, cells were washed with PBS for 3 times and incubated TCTCTTGCTTGTCCTCGGAAC-30 (antisense); gp91, 50-TGGCG- for 60 minutes in Hanks' balanced salt solution containing

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4 mmol/L Fluo-4AM in the dark at room temperature. The cells DCs (Fig. 1F). Thus, the uptake of OVA-MPs by DCs was necessary were then washed with Hanks' balanced salt solution 3 times and for subsequent tumor-specific T-cell activation. B16 melanoma incubated at room temperature for another 10 minutes. Then cells commonly express tumor-associated antigen gp100 (18). 200 nmol/L ionomycin (iono) was applied extracellularly at Consistent with this, bone marrow–derived DCs also presented 30 seconds, and the cytosolic calcium release was recorded by the MHC class I–gp100 peptide complexes to the surface, follow- two-photon confocal microscope. Images were collected every ing the B16-MP treatment, concomitant with the upregulation of 2 seconds and analyzed by ImageJ software (NIH). CD80, CD86, CCR7, CD40, and OX40L and IFNg production þ (Supplementary Fig. S1C–S1E). As a result, gp100-specific CD8 Chromatin immunoprecipitation T cells, isolated from pmel-1 T-cell receptor transgenic mice, were A chromatin immunoprecipitation (ChIP) assay kit (Active stimulated to proliferate effectively by the DCs (Supplementary Motif) was utilized to examine the binding of TFEB to the CD80 Fig. S1F). To dissect the uptake process, we stained OVA-MPs and and CD86 promoter. Untreated and MP-treated DCs were fixed BMDC organelles. We did not observe any colocalization of OVA- with 1% formaldehyde on ice to cross link the proteins bound to MPs with mitochondria, endoplasmic reticulum (ER), or Golgi the chromatin DNA. After washing, the chromatin DNA was apparatus (Fig. 1G). However, OVA-MPs were found to be colo- þ þ sheared by enzymatic force to produce DNA fragments of around calized with Rab5 early endosomes, Rab7 late endosomes, and 200 to 1,000 bp. The same amounts of sheared DNA were used for lysosomes (Fig. 1H), suggesting that OVA-MPs are taken up via immunoprecipitation with a TFEB antibody or an equal amount endocytosis and trafficked to the lysosomes of DCs. To further of preimmune IgG. The immunoprecipitate then was incubated verify this, we added the endocytosis inhibitor amiloride hydro- with protein G Magnetic Beads, and the antibody–protein G chloride to block BMDCs from taking up OVA-MPs. As a result, Magnetic Beads complex was collected for subsequent reverse BMDCs were not able to induce OT-I T-cell activation (Fig. 1I). We þ cross-linking. The same amount of sheared DNA without anti- additionally analyzed the Flt3L-induced DCs and CD8a DCs body precipitation was processed for reverse cross-linking and isolated from the spleen (19–22). These DCs induced OT-1 T-cell served as input control. DNA recovered from reverse cross-linking proliferation and IFNg production after OVA-MPs pulsing (Sup- was used for PCR. PCR was performed with primers for the CD80 plementary Fig. S2A–S2D) and upregulated the expression of and CD86 promoter flanking the TFEB binding site at 59C for 36 CD80, CD86, CCR7, CD40, and OX40L (Supplementary Fig. cycles (CD80 primers, forward, 50-CGCTCTGGATAACCTGCACT- S2E and S2F). The ability of DCs to cross-present T-MPs was also 30 and reverse, 50-ACAGCGGTGTGTAAGCTGTC-30; CD86 pri- confirmed in vivo. We injected OVA-MPs into the footpads of mers, 50-GTGAGACTGGGACACCAACA-30 and reverse, 50-GCTC- C57BL/6 mice (8 105, once per day) for 4 days, followed by the TGCCGCTATCTAGCTT-30). adoptive transfer of OT-I T cells. We found that OVA-MP treatment resulted in DCs in popliteal LNs presenting MHC I–OVA Statistical analysis peptide complexes and upregulating CD80, CD86, CCR7, CD40, All experiments were performed at least 3 times. Results were and OX40L (Supplementary Fig. S3A and S3B). Also, OT-I T-cell expressed as mean SEM and analyzed by an unpaired two-tailed proliferation was induced in the spleen and draining lymph nodes Student t test. P values of <0.05 were considered statistically (Supplementary Fig. S3C). Together, these data suggest that DCs significant. The analysis was conducted using the GraphPad Prism endocytose T-MPs to lysosomes, leading to tumor antigen pre- þ 6.0 software. sentation and tumor-specific CD8 T-cell activation.

Results Endocytosed OVA-MPs increase DC lysosomal pH and number þ DCs endocytose T-MPs, leading to tumor-specific CD8 T-cell Next, we examined whether and how endocytosed OVA-MPs activation affect the lysosomes of DCs. A fundamental function of lysosomes Previously, we showed that the immunogenicity of T-MPs is degrading biomolecules, a process that strictly relies on the required the uptake of DCs (17). In this study, we investigated acidic microenvironment (pH 4.5–5.0) in lysosomal lumen. the underlying mechanisms. Using B16 melanoma cells expres- Staining OVA-MP–treated BMDCs with LysoSensor Green, we sing the model tumor antigen ovalbumin (OVA) and OVA-spe- found that the normalized fluorescence intensity decreased by þ cific CD8 T cells derived from OT-I T-cell receptor transgenic 75%, indicating that lysosomal pH was elevated (Fig. 2A). The mice, we found that bone marrow–derived DCs (BMDC), which lysosomal pH increased to a peak of 8.5 within 24 hours and then were generated by GM-CSF/IL4 stimulation and pretreated with decreased to 6.9 at 48 hours(Fig. 2B). Lysosomal pH alteration is B16-OVA cell–derived microparticles (OVA-MP), stimulated commonly associated with lysosome biogenesis (23). In line with þ OVA-specific CD8 T-cell proliferation as well as IFNg production increased pH, the normalized fluorescence intensity of Lyso- (Fig. 1A and B), suggesting that BMDCs effectively process and Tracker Red increased about 2.5 times, indicating that lysosome present OVA antigen to T cells. Consistently, we observed that number increased in OVA-MP–treated BMDCs (Fig. 2C). Consis- almost 100% of the BMDCs efficiently took up OVA-MPs by tent with this observation, 2 lysosomal genes, LAMP1 and LAMP2, fluorescent microscopy (Fig. 1C). The uptake rate of OVA-MPs were upregulated in the treated BMDCs (Fig. 2D). Transcription was detected by flow cytometry (Supplementary Fig. S1A and factor EB (TFEB) is known to be critical for lysosomal biogenesis S1B). The MHC class I–OVA peptide complexes were confirmed to upon its translocation into the nucleus (24). Both TFEB expres- be expressed on BMDC membrane surface in a dose- and time- sion and its entry into the nucleus were found to be upregulated in dependent manner (Fig. 1D and E). In addition, DC maturation the OVA-MP–treated BMDCs, compared with untreated BMDCs surface markers, including CD80, CD86, CCR7, MHC class I/II, (Fig. 2E and F). Together, these data suggest that endocytosed CD40, and OX40L, were all upregulated upon 24-hour incuba- OVA-MPs induce an increase of both lysosomal pH and lyso- tion with OVA-MPs, but PD-L1 expression was not upregulated on somal number in DCs.

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Figure 1. DCs endocytosed T-MPs for tumor-specific CD8þ T-cell activation. A and B, Splenic CD8þ T cells purified from OT-1 mice were incubated with untreated DCs or OVA- þ MP–loaded DCs. T-cell proliferation was examined by CFSE dilution assay (A) and IFNg in supernatants of splenic CD8 T cells was measured by ELISA (B) after a 2-day coculture. C, DCs were incubated with PKH26-labeled MPs for 24 hours, and the uptake of microparticles was observed by fluorescent microscopy. Scale bar, 10 mm. D and E, DCs were incubated with different ratio of OVA-MPs for 24, 48, and 72 hours, the expression (mean fluorescence intensity, MFI) of H-2Kb-OVA (257–264) was detected by flow cytometry. F, DC maturation was analyzed for expression of CD80, CD86, CCR7, MHC I/II, CD40, OX40L, and PD-L1 by flow cytometry after coculture with OVA-MPs for 48 hours. G and H, DCs were incubated with PKH26/67-labeled MPs for 24 hours and then analyzed with mitochondria, ER, Golgi, and lysosome Green/Red Trackers under two-photon confocal microscope. Scale bar, 10 mm. I, DCs were treated with OVA-MPs in the þ presence or absence of endocytosis inhibitor amiloride hydrochloride (75 and 100 mmol/L) for 24 hours and then incubated with OT-1 mice–derived CD8 T cells. T- cell proliferation was examined by CFSE dilution assay. Data shown are representative of 3 independent experiments, and error bars represent mean SEM; , P <0.01; , P < 0.001; , P < 0.0001.

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Figure 2. Endocytosed OVA-MPs increase lysosomal pH value and the number in DCs. A and B, DCs were treated with OVA-MPs at different time points and then incubated with 0.1 mmol/L LysoSensor for 30 minutes, the DCs were observed by ﬂuorescence microscope (A) and the pH value of DCs was detected by microplate reader (B). Scale bars, 10 mm. C, DCs were treated with OVA-MPs for 24 hours or untreated, and then the number of DC lysosomes was observed by ﬂuorescence microscopy. Scale bars, 10 mm. D, DCs were treated with OVA-MPs for 48 hours, or untreated, and then the expression of LAMP1 and LAMP2 was analyzed by real-time PCR. E, DCs were treated with OVA-MPs for 6, 12, 24, and 48 hours, or untreated, and the expression of TFEB was analyzed by real-time PCR. F, DCs were treated with OVA-MPs for 3, 6, 12, 24, and 48 hours, or untreated, and the expression of TFEB was analyzed by Western blot. Data shown are representative of 3 independent experiments, and error bars represent mean SEM; , P < 0.01; , P < 0.001; , P < 0.0001.

Endocytosed T-MPs increase lysosomal pH via NOX2-mediated BMDCs with CellROX Green, we found that the fluorescence ROS production intensity of ROS increased about 2 times in OVA-MP–treated þ Low lysosomal pH is maintained by the vacuolar-type H - BMDCs, as measured by fluorescence microscopy and flow cyto- ATPase (V-ATPase)-mediated pumping of protons into the metry (Fig. 3D and E). Superoxide anion in lysosomes can be lumen. V-ATPase consists of V0 and V1 domains, each with quickly reduced to hydrogen peroxide by reacting with protons, multiple subunits (25). The real-time PCR result did not show thus consuming protons and increasing pH (27). To examine this, differential expression of subunit members of V-ATPase between we used NADPH oxidase inhibitor diphenylene iodonium (DPI) MP-treated and untreated BMDCs (Fig. 3A). The enzyme NADPH or gp91phox siRNA to block NOX2 activity. We found that the oxidase 2 (NOX2, previously known as gp91phox) is also ubiqui- above ROS and lysosomal pH decreased to the levels of untreated tously integrated into lysosomal membrane of phagocytes in a BMDCs (Fig. 3F and G), suggesting that endocytosed T-MPs heterodimer form with p22phox. During phagocytosis or upon increase lysosomal pH via NOX2-mediated ROS production. We stimulation, cytosolic regulatory proteins p47phox, p67phox, further speculated that the increase of lysosomal pH by T-MPs was p40phox, and GTP-binding Rac are recruited to the membrane, required for DCs to present tumor antigens, because low pH where they assemble with NOX2-p22phox to form an active oxidase confers the ability of lysosomal enzymes to degrade tumor anti- complex, leading to transfer of electron from NADPH to molec- gens into small peptides or single amino acids. To test this, we ular oxygen and the production of superoxide anion (26). Here, treated OVA-MP–phagocytosed BMDCs with either DPI or we found that endocytosed T-MPs could upregulate the expres- gp91phox siRNA and cultured the cells with OT-I T cells. We found sion of gp91phox (Fig. 3B), and that gp91phox was effectively that the DPI or siRNA treatment significantly inhibited T-cell recruited to lysosomes in MP-treated BMDCs (Fig. 3C). Staining proliferation (Fig. 3H). Moreover, we found that the expression

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Figure 3. Endocytosed T-MPs increase lysosomal pH via a pathway of NOX2-mediated ROS production. A, DCs were treated with OVA-MPs for 36 hours, and then the expression of V-ATPase subunits was analyzed by real-time PCR. B, DCs were treated with OVA-MPs for 12, 24, 36, and 48 hours, and then the expression of gp91phox was analyzed by real-time PCR. C, DCs were treated with PKH26-labeled OVA-MPs for 24 hours or untreated and stained with anti-LAMP-1 (green) and anti-gp91phox (purple). DAPI was used to stain the cell nuclei (blue). Then, the cells were observed under two-photon confocal microscope. Scale bars, 10 mm. D and E, DCs were treated with OVA-MPs at different time points, or untreated, and the ROS production (mean fluorescence intensity, MFI) of DCs was detected by confocal microscope (D)andflow cytometry (E). F and G, DCs were transfected with gp91phox siRNAs or treated with DPI, respectively, and ROS production and lysosomal pH of DCs were analyzed by flow cytometry (F) and fluorescence microscope (G). H and I, DCs were transfected with gp91phox siRNAs or treated with DPI, respectively, and then the T-cell proliferation was examined by CFSE dilution assay (H) and the expression of H-2Kb-OVA (257–264) was detected by flow cytometry (I). Data shown are representative of 3 independent experiments, and error bars represent mean SEM; , P < 0.01; , P < 0.001; , P < 0.0001.

of MHC class I–OVA peptide complexes on the surface of those (Fig. 4A). This result suggested that increased lysosomal pH DCs was significantly reduced by DPI or gp91phox siRNA (Fig. 3I). directly causes the productionofclassIpeptideswiththe8–11 Together, these data suggest that endocytosed T-MPs increase amino acid length. Notwithstanding the generation of class I lysosomal pH via the pathway of NOX2-mediated ROS produc- peptide in lysosomes of T-MP–phagocytosed DCs, the mech- tion, leading to generation of MHC–tumor antigenic peptide anism by which class I peptide contacts and complexes with complexes. the MHC class I molecule needed further clarification. TAP1 and TAP2 mediate the entry of cytosolic class I peptide into ER T-MPs promote lysosomal migration and tumor antigen cross- (28). Here, we found that T-MP–treated BMDCs upregulated presentation the expression of TAP1 and TAP2 (Fig.4B),andknockdownof Increased lysosomal pH likely results in long peptide (>11 TAP1/2 resulted in abrogation of BMDCs presenting OVA-MP amino acids) production due to decreased enzymatic activity. tumor antigen to OT-I T cells (Fig. 4C). This result implies that Large peptides cannot be accommodated by the peptide-bind- class I peptide is released from lysosomes and translocated to ing cleft of MHC class I for DC cross-presentation. We hypoth- ER in this way. In addition to digestion, lysosomes are also esized that large peptides exited lysosomes and entered able to transport molecules. Lysosomes are capable of bidi- theproteasomedegradationpathwaytogenerate8–11-amino rectional migration along microtubule tracks, upon recruiting acid MHC class I peptides. However, when we treated OVA- specific regulatory molecules to their membranes. For example, MP–endocytosed BMDCs with the pan-proteasome inhibitor centripetal (inward) movement is guided by the small GTPase MG-132, we found that this blockade did not affect the ability Rab7, which recruits the minus end-directed microtubule þ of DCs to express and present OVA peptide to CD8 T cells motor dynein to lysosomes (29). By contrast, centrifugal

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Figure 4. T-MPs promote lysosomal centripetal migration to facilitate tumor antigen cross-presentation. A, OVA-MP–loaded DCs were treated with MG132 (5 mmol/L) for 12 hours or untreated; the expression (mean fluorescence intensity, MFI) of H-2Kb-OVA (257–264) was detected by flow cytometry. B, DCs were treated with OVA-MPs for 12, 24, 36, and 48 hours; the expression of TAP1 and TAP2 was analyzed by real-time PCR. C, TAP siRNAs or control siRNAs were transfected into DCs. Twenty-four hours later, DCs were treated with OVA-MPs and the expression of H-2Kb-OVA (257–264) was detected by flow cytometry. D, DCs were treated with or without OVA-MPs for 24 hours and stained with anti-LAMP-1 (red), anti-Rab7 (green), and anti-dynein (blue) antibodies. DAPI was used to stain the cell nuclei (purple). Then DCs were observed by two-photon confocal microscopy. Scale bars, 10 mm. E, Dynein siRNAs, Rab7 siRNAs, or control siRNAs were transfected into DCs. Twenty-four hours later, DCs were treated with OVA-MPs for 72 hours, and then the expression of H-2Kb-OVA (257–264) was detected by flow cytometry. F, Dynein siRNAs, Rab7 siRNAs, or control siRNAs were transfected into DCs. Twenty-four hours later, DCs were pretreated with OVA-MPs and then incubated with OT-1 mice–derived splenic CD8þT cells. T-cell proliferation was examined by CFSE dilution assay after a 2-day coculture. Data shown are representative of 3 independent experiments, and error bars represent mean SEM; , P < 0.05; , P < 0.01; , P < 0.001; , P < 0.0001.

(outward) movement is directed by another small GTPase expression of MHC class I–OVA peptide complex in the treated Arl8, which links lysosomes to the plus end-directed microtu- BMDCs (Fig. 4E) and a subsequent reduction in OT-I T-cell bule motor kinesin (30). Lysosomal migration can proliferation (Fig. 4F). Together, these data suggest that endo- be regulated by pH alteration (31). We previously reported cytosed T-MPs facilitate tumor antigen cross-presentation by that T-MPs facilitate the inward migration of lysosomes in promoting centripetal migration of lysosomes via a Rab7/ tumor cells (32). Here, we further hypothesized that lysosomes dynein-mediated pathway. facilitate tumor antigenic peptides to access the ER through a centripetal transport pathway. In line with the hypothesis, we Endocytosed T-MPs upregulate CD80/CD86 expression via found that Rab7 and dynein were recruited to the lysosomal ROS production membrane in OVA-MP–endocytosed BMDCs (Fig. 4D). More- The above data indicate that DCs efﬁciently process tumor over, either dynein or Rab7 siRNA led to downregulation of the antigens of T-MPs and present them as MHC class I-peptide

Figure 5. Endocytosed T-MPs upregulates the expression of CD80 and CD86 in a ROS-dependent manner. A, DCs were pretreated with DPI (2.5 mmol/L) for 1 hour and then treated with OVA-MP for 24 hours; the expression (mean fluorescence intensity, MFI) of CD80 and CD86 was detected by flow cytometry. B and C, DCs were pretreated with NAC (20 mmol/L) for 1 hour, and then treated with OVA-MP for 24/48 hours. The ROS production and CD80/CD86 expression were detected by flow cytometry. D, Splenic CD8þ T cells purified from OT-1 mice were incubated with untreated DCs, OVA-MP–loaded DCs, and OVA-MPþNAC– treated DCs, respectively, for 48 hours. Then, the T-cell proliferation was examined by CFSE dilution assay after a 2-day coculture. Data shown are representative of 3 independent experiments, and error bars represent mean SEM; , P < 0.01; , P < 0.001; , P < 0.0001.

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complexes to the cellular membrane, thus providing the first channels (TPC1/2 and Mcoln1/2) have been reported to mediate signal for tumor-specific T-cell activation. However, the second lysosomal calcium release (36). Here, we found that endocytosed signal by CD80/CD86 is also required for T-cell activation. T-MPs only upregulated the expression of Mcoln2 in BMDCs Twenty-eight hours after incubation of BMDCs with T-MPs, we (Fig. 6B). Knockdown of Mcoln2 with the siRNA led to inhibition þ determined the expression of CD80 and CD86 by flow cytometry. of T-MP–induced Ca2 release (Fig. 6C) as well as CD80 and We found that the expression of CD80 and CD86 was upregulated CD86 upregulation in BMDCs (Fig. 6D), concomitant with the þ (Fig. 5A), which was abrogated by the addition of DPI (Fig. 5A). impaired CD8 T-cell proliferation (Fig. 6E). In addition, treat- þ Given that T-MPs augmented ROS production in DCs via acti- ment with cyclosporin A (CsA), a Ca2 signaling inhibitor (37), vating the NOX2 pathway, we then used ROS scavenger NAC to also blocked the effect of T-MPs on CD80 and CD86 upregulation treat OVA-MP–loaded BMDCs. The result showed that the inhi- (Fig. 6F). Given that T-MP caused ROS production and pH bition of ROS production downregulated the expression of CD80 increase in lysosomes, we assumed that the ROS production and þ and CD86 (Fig. 5B and C), concomitant with decreased OT-I T-cell pH alteration resulted in Mcoln2-mediated Ca2 release. To proliferation (Fig. 5D). Together, these data suggest that NOX2- clarify this, we used either NAC or DPI to block the ROS/pH mediated ROS production upregulates the expression of CD80 pathway. As a result, both DPI and NAC downregulated the þ and CD86 in T-MP–endocytosed DCs. expression of Mcoln2 and inhibited lysosomal Ca2 release in T-MP–treated BMDCs (Fig. 6G and H). Together, these data ROS-triggered lysosomal calcium signaling upregulates CD80/ suggest that ROS-triggered lysosomal calcium signaling upregu- CD86 expression lates the expression of CD80 and CD86 in T-MP–loaded DCs. Next, we investigated the mechanism by which ROS regulates þ the expression of CD86 and CD80. Although activation of TLR Released lysosomal Ca2 activates TFEB to increase CD80 and signaling is a common pathway for CD80 and CD86 upregulation CD86 expression þ (33), Ca2 signaling is also able to effectively upregulate their Finally, we investigated the mechanism by which lysosomal þ expression (34). Coincidently, lysosomes are organelles for cal- Ca2 signaling regulates expression of CD86 and CD80. TFEB is cium storage (35). Here, we report that the intracellular calcium not only a master transcriptional regulator for lysosomal biogen- levels were elevated in BMDCs upon uptake of T-MPs (Fig. 6A). esis but also regulates immune-related genes (38). Phosphory- Using ryanodine or CGP37157 to block calcium release from the lated TFEB, which is localized in the cytosol, translocates to the þ þ ER or mitochondria did not affect the T-MP–mediated Ca2 nucleus upon dephosphorylation by Ca2 release-activated cal- þ increase in DCs (Fig. 6A), suggesting that lysosomes, rather than cineurin (39). We hypothesized that lysosomal Ca2 signaling- þ þ ER or mitochondria, release Ca2 to the cytosol. Several Ca2 activated TFEB upregulates CD80 and CD86 expression.

Figure 6. ROS-triggered lysosomal calcium signaling upregulates the expression of CD80 and CD86. A, OVA-MP–loaded DCs were pretreated with ER or mitochondria calcium release inhibitor ryanodine or CGP37157 for 1 hour and stained with Fluo-4AM for 1 hour or untreated. Then, 200 nmol/L ionomycin (iono) was applied extracellularly at 30 seconds, and the cytosolic calcium release was recorded by two-photon confocal microscope (mean fluorescence intensity, MFI). B, DCs were treated with OVA-MPs for 12, 24, 36, and 48 hours; the expression of Mcoln1/2 and Tpcn1/2 was analyzed by real-time PCR. C and D, Mcoln2 siRNAs or control siRNAs were transfected into DCs. Twenty-four hours later, DCs were treated with OVA-MPs and the cytosolic calcium release was recorded by two-photon confocal microscope (C), and CD80/CD86 expression were detected by flow cytometry (D). E, Mcoln2 siRNAs or control siRNAs were transfected into DCs. Twenty-four hours later, DCs were pretreated with OVA-MPs and then incubated with OT-1 mice–derived splenic CD8þT cells. T-cell proliferation was examined by CFSE dilution assay after a 2-day coculture. F, DCs were pretreated with CsA (5 nmol/L and 10 nmol/L) for 1 hour, and then treated with OVA-MPs for 24 hours; the expression of CD80 and CD86 was then detected by flow cytometry. G, NAC-pretreated DCs were treated with OVA-MPs for 4, 8, 12, and 24 hours, and the expression of Mcoln2 was analyzed by real-time PCR. H, Untreated DCs, OVA-MP–loaded DCs, or MPþNAC–treated DCs were stained with Fluo-4, and then the cytosolic calcium release was recorded by two-photon confocal microscope. Data shown are representative of 3 independent experiments, and error bars represent mean SEM; , P < 0.05; , P < 0.01; , P < 0.001; ****, P < 0.0001.

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In Fig. 2E, the real-time PCR result showed that TFEB was upre- Tumor cells are capable of releasing different types of micro- gulated in T-MP–loaded BMDCs. Here, immunostaining and vesicles, the roles of which in tumor immunity are controversial. Western blot showed that abundant TFEB was translocated into Exosomes are small, endosome-derived extracellular microvesi- the nucleus in T-MP–treated DCs, but not in untreated BMDCs cles (30–100 nm), delivering contents such as proteins, messenger (Fig. 7A and B). To clarify whether TFEB regulates CD80 and CD86 RNAs, and microRNAs to recipient cells. Although tumor exo- expression, we knocked down TFEB by siRNA (Fig. 7C), which led somes contain tumor antigens, studies have shown that tumor to downregulation of CD80 and CD86 expression (Fig. 7D). exosomes actually mediate tumor immunosuppression as well as Na3VO4, a serine/threonine phosphatase inhibitor that promotes metastasis (41). However, subcutaneous inoculation or oral TFEB phosphorylation, also downregulated the expression of administration of T-MPs results in the generation of antitumor CD80 and CD86 (Fig. 7E). In contrast, when we forced over- T-cell immunity (17, 42, 43). The opposite consequence of expression of TFEB (TFEB-OE) in DCs, the expression of CD80 treatment with T-MPs, compared with exosomes, may be ascribed and CD86 was upregulated (Fig. 7F). We performed chromatin to the different contents within these two vesicles. In particular, T- immunoprecipitation (ChIP)-qPCR assay and found that TFEB MPs contain genomic and mitochondrial DNA fragments, but indeed bound the promoters of CD80 and CD86 genes (Fig. 7G). exosomes may not contain these (17). Such DNA fragments, Together, these data suggest that activation TFEB transcriptional which are capable of stimulating DCs to release IFNb via the þ activity by T-MP–triggered lysosomal Ca2 signaling directly cGAS–STING pathway, play an important role in polarizing upregulates the expression of CD80 and CD86. macrophages toward an M2 phenotype, which promotes tumor growth (44). The molecular mechanism through which T-MPs have differential effects on DCs and macrophages remains Discussion unclear. One observation is that T-MPs increase the lysosomal The antitumor immune response is mainly mediated by tumor- pH of DCs but decrease the lysosomal pH of macrophages. Such þ specific CD8 T cells. DCs are crucial in the generation of these an unexpected result warrants further investigation. þ CD8 T cells, as they are required not only to present tumor Endocytosis is the initial step in cross-presentation of exoge- þ antigen peptides, but also to provide costimulating signals to nous tumor antigens by antigen-presenting cells to CD8 T cells. þ CD8 T cells. Thus, an ideal tumor vaccine ought to simulta- Endocytosed antigens are then transited to endolysosomes where neously possess abundant tumor antigens as well as suitable a delicate degradation of tumor antigens occurs: too much deg- innate signals (40). We have already demonstrated that tumor radation may destroy potential T-cell epitopes, but some sufficient cell–derived microparticles (T-MP) have these dual advantages degradation is required to generate antigenic peptides. The com- and represent an effective vaccination platform to trigger antitu- patible antigenic peptides are then translocated into the ER, mor T-cell immunity (17). In this study, we demonstrate the where they bind to MHC class I molecules (45). Lysosomal unusual action pathway of T-MPs in DCs through which T-MPs proteases that mediate protein degradation normally require an induce DCs to become highly efficient in presenting tumor anti- acidic pH (5.5–6.5) to exert their optimal function, and two þ gens to tumor-lytic CD8 T cells. systems of V-ATPase and NOX2 regulate the lysosomal pH in

Figure 7. Activation of TFEB transcriptional activity by released lysosomal calcium signaling upregulates CD80 and CD86 expression. A and B, DCs were treated with OVA-MPs for 12, 24, 36, and 48 hours, the expression and location of TFEB were analyzed by two-photon confocal microscope (A) and Western blot (B). C, TFEB siRNAs or control siRNAs (NC) were transfected into DCs. Twenty-four hours later, the expression of TFEB was analyzed by real-time PCR. D, TFEB siRNAs or control siRNAs (NC) were transfected into DCs. Twenty-four hours later, the expression (mean ﬂuorescence intensity, MFI) of CD80 and CD86 was analyzed by ﬂow

cytometry. E, DCs were pretreated with Na3VO4 for 1 hour, and then treated with OVA-MP for 24 hours; the expression of CD80 and CD86 was evaluated by ﬂow cytometry. F, TFEB-overexpressing (TFEB-OE) or mock plasmid was transfected into DCs. Twenty-four hours later, DCs were treated with OVA-MPs for 36 hours, and the expression of CD80 and CD86 was detected by ﬂow cytometry. G, Chromatin immunoprecipitation (ChIP) with anti-TFEB followed by PCR with CD80 and CD86 promoter primers in DCs treated with OVA-MPs or untreated. IgG ChIP was used as the negative control, total genomic DNA as INPUT. H, A proposed model of dendritic cell activation and presentation of tumor microparticle–derived antigens to CD8þ T cells. Data shown are representative of 3independent experiments, and error bars represent mean SEM; P < 0.01, P < 0.001.

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DCs (46–48). V-ATPase pumps protons from the cytosol to aureus, implying that TFEB may directly transcriptionally regulate lysosomal lumen, thus acidifying lysosomes. In contrast, NOX2 immune genes (53). In the current study, we found that TFEB transfers electrons from NADPH to molecular oxygen, and the binds to and transcriptionally activates CD80 and CD86 promo- generated superoxide anion causes hydrogen peroxide formation ters. Although we verified the pathway of T-MP–induced ROS by consuming protons, leading to alkalizing lysosomes. In this upregulation of CD80 and CD86, we have not yet elucidated the study, we report that endocytosed T-MPs influenced antigen mechanism through which T-MPs activate NOX2 and subse- degradation in lysosomes of DCs. T-MPs seemed not to alter the quently increase ROS production in DCs. This work is ongoing. V-ATPase system, but activated the NOX2 system, and subse- In summary, the work presented here demonstrates that T-MPs, quently increase lysosomal pH. As a result, tumor antigens from T- by activating lysosomal ROS, induce initiation of two concom- MPs were effectively degraded into antigenic peptides in the itant pathways. T-MPs induce production and translocation of lysosomes of DCs. Consistent with the current study, previous tumor antigenic peptides to the ER via T-MP–increased lysosomal work showed that excessive antigen degradation occurs in NOX2- pH and T-MP–triggered dynein–microtubule transportation in þ deficient DCs, resulting in a defect in cross-presentation to CD8 T DCs in one pathway, and upregulation of CD80 and CD86 þ cells (27). Incomplete lysosomal degradation might cause greater expression by TFEB activated by ROS-triggered Ca2 signaling on length antigenic peptides (>8–11 amino acids), which are the other (Fig. 7H). These findings reveal the molecular pathways required to be transferred to proteasomes for the secondary by which T-MP vaccines prime DCs to efficiently present T-MP þ degradation. However, when we used a proteasome inhibitor to tumor antigens within CD8 T cells, thus opening a new avenue block this process, DC presentation of T-MP tumor antigens was for cancer immunotherapy. not affected, suggesting that DCs effectively generate 8–11 amino acids antigenic peptides in lysosomes, upon uptake of T-MPs. T- Disclosure of Potential Conflicts of Interest MPs are capable of promoting centripetal migration of lysosomes No potential conflicts of interest were disclosed. toward ER through recruiting small G protein Rab7 to the lysosomal membrane, leading to activating dynein–microtubule Authors' Contributions transportation system (32). Thus, the T-MP–triggered pathway Conception and design: J. Ma, Y. Yu, B. Huang induces efficient translocation of antigenic peptides to the ER. Development of methodology: J. Ma, K. Wei, J. Chen, B. Huang Acquisition of data (provided animals, acquired and managed patients, One finding of this study is that DCs mobilize T-MP–induced provided facilities, etc.): K. Wei, H. Zhang, K. Tang, T. Zhang, P. Xu, J. Chen, ROS to enhance presentation of tumor antigen. The generation of L. Zhou, X. Liang, R. Fiskesund ROS by T-MPs not only increases pH for proper degradation of Analysis and interpretation of data (e.g., statistical analysis, biostatistics, tumor antigen into antigenic peptide, but also induces the upre- computational analysis): J. Ma, H. Zhang, F. Li, P. Xu, Y. Yu, W. Sun, gulation of CD80 and CD86 expression, key costimulatory mole- R. Fiskesund cules for T-cell activation. Although pathogen-associated molec- Writing, review, and/or revision of the manuscript: J. Ma, R. Fiskesund, B. Huang ular patterns, such as LPS, or damage-associated molecular pro- Administrative, technical, or material support (i.e., reporting or organizing teins, such as HMGB1, readily upregulate CD80 and CD86 data, constructing databases): J. Ma, K. Wei, J. Liu, Y. Liu expression through the NF-kB and MAPK pathways (33, 49), Study supervision: K. Wei, Y. Liu, B. Huang þ here, we find that lysosomal Ca2 signaling triggers CD80 and CD86 upregulation via activation of TFEB, the master lysosomal Acknowledgments þ regulator. Conventionally, ER is thought to be the Ca2 storing This work was supported by CAMS Initiative for Innovative Medicine (2017- þ organelle (50); however, lysosomes also store Ca2 (51). In this I2M-1-001) and the National Natural Science Foundation of China (81601447, þ study, we find that T-MP–induced ROS activates lysosomal Ca2 81788101, 91742112, 81661128007, and 81530080). þ þ channel, leading to lysosomal Ca2 release. Such Ca2 signaling The costs of publication of this article were defrayed in part by the may activate the phosphatase calcineurin that dephosphorylates payment of page charges. This article must therefore be hereby marked TFEB. Subsequently, TFEB enters the nucleus and stimulates advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate transcription (38, 39, 52). Although TFEB regulates genes this fact. involved in autophagy and lysosome biogenesis, some immune molecules such as IL1b, IL6, TNFa, and CCL5 are downregulated Received December 11, 2017; revised May 17, 2018; accepted July 11, 2018; in TFEB-knockdown macrophages infected with Staphylococcus published first July 17, 2018.

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1068 Cancer Immunol Res; 6(9) September 2018 Cancer Immunology Research

Mechanisms by Which Dendritic Cells Present Tumor Microparticle Antigens to CD8 + T Cells

Jingwei Ma, Keke Wei, Huafeng Zhang, et al.

Cancer Immunol Res 2018;6:1057-1068. Published OnlineFirst July 17, 2018.

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