Dendritic Cells Recruit Exosomes via Exosomal LFA-1 Leading to Inhibition of CD8+ CTL Responses through Downregulation of Peptide/MHC Class I and This information is current as -Mediated Cytotoxicity of September 25, 2021. Yufeng Xie, Haifeng Zhang, Wei Li, Yulin Deng, Manjunatha Ankathatti Munegowda, Rajni Chibbar, Mabood Qureshi and Jim Xiang J Immunol 2010; 185:5268-5278; Prepublished online 29 Downloaded from September 2010; doi: 10.4049/jimmunol.1000386 http://www.jimmunol.org/content/185/9/5268 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Dendritic Cells Recruit T Cell Exosomes via Exosomal LFA-1 Leading to Inhibition of CD8+ CTL Responses through Downregulation of Peptide/MHC Class I and Fas Ligand-Mediated Cytotoxicity

Yufeng Xie,*,†,‡,1 Haifeng Zhang,*,†,‡,1 Wei Li,*,†,‡ Yulin Deng,*,†,‡ Manjunatha Ankathatti Munegowda,*,†,‡ Rajni Chibbar,x Mabood Qureshi,x and Jim Xiang*,†,‡

Active T cells release bioactive exosomes (EXOs). However, its potential modulation in immune responses is elusive. In this study, we + + in vitro generated active OVA-specific CD8 T cells by cultivation of OVA-pulsed dendritic cells (DCOVA) with naive CD8 T cells Downloaded from derived from OVA-specific TCR transgenic OTI mice and purified EXOs from CD8+ T cell culture supernatant by differential + ultracentrifugation. We then investigated the suppressive effect of T cell EXOs on DCOVA-mediated CD8 CTL responses and antitumor immunity. We found that DCOVA uptake OTI T cell EXOs expressing OVA-specific TCRs and Fas ligand via peptide/ MHC Ag I–TCR and CD54–LFA-1 interactions leading to downregulation of peptide/MHC Ag I expression and induction of of DCOVA via Fas/Fas ligand pathway. We demonstrated that OVA-specific OTI T cell EXOs, but not lymphocytic

+ + http://www.jimmunol.org/ choriomeningitis virus-specific TCR transgenic mouse CD8 T cell EXOs, can inhibit DCOVA-stimulated CD8 CTL responses and antitumor immunity against OVA-expressing B16 melanoma. In addition, these T cell EXOs can also inhibit DCOVA-mediated CD8+ CTL-induced diabetes in transgenic rat insulin promoter-mOVA mice. Interestingly, the anti–LFA-1 Ab treatment signifi- cantly reduces T cell EXO-induced inhibition of CD8+ CTL responses in both antitumor immunity and autoimmunity. EXOs released from T cell hybridoma RF3370 cells expressing OTI CD8+ TCRs have a similar inhibitory effect as T cell EXOs in + DCOVA-stimulated CTL responses and antitumor immunity. Therefore, our data indicate that Ag-specific CD8 T cells can modulate immune responses via T cell-released EXOs, and T cell EXOs may be useful for treatment of autoimmune dis- eases. The Journal of Immunology, 2010, 185: 5268–5278. by guest on September 25, 2021 endritic cells (DCs) are the most potent APCs in immune involved in Ag presentation via MHC class I (MHC I) and responses (1). One general characteristic of tumor cells MHC class II, costimulatory (CD80 and CD86), and tetraspan D is releasing or shedding membrane vesicular bodies, (CD63 and CD82) molecules (4). Ag-specific DC-derived EXOs nowadays, called as exosomes (EXOs), which was initially de- can induce tumor-specific CTL responses via either indirectly or scribed by Taylor et al. (2) 25 y ago. EXOs are small (∼100 nm in directly stimulating Ag-specific T cells (5-7) or transferring Ags to diameter) membrane-bound vesicles of the endocytic pathway that the host DC leading to stimulation of CD8+ CTL responses (8, 9). are externalized by a variety of cell types. They are formed by These EXOs have been used for antitumor vaccines (10, 11). the fusion of multivesicular bodies with the plasma membrane, Active CD8+ T cells have been shown to release lysosomes followed by exocytosis (3). Such EXOs display a discrete set of containing cytolytic granzyme and perforin as well as TCRs, CD3, and CD8 (12), which play an important role in specific delivery of cytolytic effect to target cells. In addition, these active T cells also *Research Unit, Division of Health Research, Saskatchewan Cancer Agency; and †Department of Oncology, ‡Department of Immunology, and xDepartment of Pathol- secrete bioactive EXOs, which express TCRs and Fas ligand ogy, University of Saskatchewan, Saskatoon, Canada (FasL) (13, 14) and can be taken up by APCs or B cells via direct 1Y.X. and H.Z. contributed equally to this work. cultivation (15). It has also been demonstrated that these FasL- Received for publication February 3, 2010. Accepted for publication August 29, expressing T cell EXOs induced apoptosis formation of the by- 2010. stander T cells with unknown mechanism (16). However, the This work was supported by research grants from the Canadian Institutes of Health potential effect of these active T cell EXOs on modulation of Research (MOP 79415 and MOP 89713). DC-induced immune responses is still largely unknown. Address correspondence and reprint requests to Dr. Jim Xiang, Saskatoon Cancer In this study, we generated OVA-specific CD8+ T cells by Center, 20 Campus Drive, Saskatoon, Saskatchewan S7N 4H4, Canada. E-mail ad- + dress: [email protected] cultivation of OVA-pulsed DCs (DCOVA) with naive CD8 T cells derived from OVA-specific TCR transgenic OTI mice (17) and Abbreviations used in this paper: BL6-10OVA, OVA-transfected BL6-10; CFSE-EXO, + + + CFSE-labeled OTI CD8 T cell-released exosome; DC, ; DCMut1,Mut1 purified CD8 T cell EXOs from OVA-specific CD8 T cell cul- peptide-pulsed dendritic cell; DCOVA, OVA-pulsed dendritic cell; ECD, energe-coupled high ture supernatant by differential ultracentrifugation (18). We then dye; EXO, exosome; EXOcont, control exosome; FasL, Fas ligand; H, CFSE ; i.d., + intradermally; L, CFSElow; LCMV, lymphocytic choriomeningitis virus; LCMV-TCR, investigated the suppressive effect of CD8 T cell-released EXOs + lymphocytic choriomeningitis virus gp33–41-specific TCR transgenic; MFI, mean fluo- in in vitro and in vivo CD8 T cell proliferation assays and in rescence intensity; MHC I, MHC class I; pMHC, peptide/MHC Ag; RIP, rat insulin OVA-specific animal tumor model and in transgenic rat insulin promoter; Tg-GFP, GFP-transgenic. promoter (RIP)-mOVA mice. We found that T cell EXOs ex- Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 pressing OVA-specificTCRs and FasL bound to DCOVA via peptide/ www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000386 The Journal of Immunology 5269

MHC Ag (pMHC) I/TCR and CD54–LFA-1 interactions leading to used them as a control EXO (EXOcont). The yields of EXOs, EXOcont, m 3 6 downregulation of pMHC I expression and induction of apoptosis and EXORF were estimated to be 2.3, 3.5, and 3.8 g/1 10 cells/24 h, respectively. of DCOVA through Fas/FasL pathway. For the first time, to our + knowledge, we have demonstrated that OVA-specific OTI CD8 Electron microscopic analysis T cell EXOs can inhibit both in vitro and in vivo DCOVA-mediated CD8+ CTL responses and in vivo antitumor immunity against OVA- EXOs were fixed in 4% paraformaldehyde. The pellets were then loaded onto carbon-coated formvar grids. After incubation in a moist atmosphere expressing B16 melanoma OVA-transfected BL6-10 (BL6-10OVA). for 20 min, the samples were washed twice in PBS and then fixed for 5 min In addition, these T cell EXOs can also inhibit DCOVA-induced in 1% glutaraldehyde. After washes for three times, the EXO sample-loaded CD8+ CTL-mediated diabetes in transgenic RIP-mOVA mice. grids were stained for 10 min with saturated aqueous uranyl. EXO samples Interestingly, the anti–LFA-1 Ab treatment significantly reduces were then examined with a JEOL 1200EX electron microscope at 60 kV (20). + + CD8 T cell EXO-induced inhibition of CD8 CTL responses in Western blot analysis both antitumor immunity and autoimmunity. EXO samples (10 mg/lane) were loaded onto 12% acrylamide gels, sub- jected to NaDodSO4-PAGE (SDS-PAGE), and subsequently transferred Materials and Methods onto nitrocellulose membrane (Millipore, Bedford, MA). The membrane Reagents, cell lines, and animals was blocked by incubation for 2 h at room temperature with ODYSSEY blocking buffer (LI-COR, Lincoln, NE) and immunoblotted with a panel of The FITC- or biotin-labeled Abs specific for Vb5.1,5.2 TCR, H-2Kb,Iab, Abs specific for CD54, LFA-1, Vb5.1,5.2 TCR, and calnexin at 4˚C for CD11c, CD80, CD28, CD54, LFA-1, Fas and FasL, calnexin Ab, and overnight. After washes for three times with PBS containing 0.05% (v/v) FITC-conjugated avidin Ab were all obtained from BD Biosciences Tween 20, the membrane was further incubated with goat anti-rat/mouse b Downloaded from (Mississauga, Ontario, Canada). The anti–H-2K /OVA257–264 (OVAI) IRDyeR800CW and scanned using ODYSSEY instrument according to the (SIINFEKL) (pMHC I) Ab was obtained from Dr. R. Germain (National manufacturer’s instruction (LI-COR). Institutes of Health, Bethesda, MD). The PE-labeled H-2Kb/OVAI tetramer and FITC-labeled anti-mouse CD8 Ab were obtained from Beckman Phenotypic analysis by flow cytometry b Coulter (San Diego, CA). The MHC I (H-2K )–restricted OVAI peptide + For the phenotypic analysis, DCOVA, active CD8 T, and RF3370 cells were and irrelevant Mut1 peptide (FEQNTAQP) were synthesized by Multiple b b Peptide Systems (San Diego, CA). The recombinant mouse IL-2, IL-4, and stained with a panel of Abs specific for H-2K ,Ia, CD11c, CD80, CD8, CD28, CD54, LFA-1, Vb5.1,5.2 TCR, pMHC I, Fas, and FasL, re- GM-CSF were obtained from Endogen (Woburn, MA). The mouse T cell http://www.jimmunol.org/ hybridoma cell line RF3370 expressing TCRs specific for H-2Kb/OVAI spectively, and analyzed by flow cytometry as described previously (20). EXO samples (10 mg/100 ml PBS) were mixed with FITC Abs (2 ml, 1 mg/ complex was obtained from Dr. K. Rock (University of Massachusetts b Medical Center, Worcester, MA). The highly lung metastatic B16 mouse ml) specific for CD8, CD28, CD54, LFA-1, V 5.1,5.2 TCR, FasL, and CD11c, kept on ice for 30 min, and then analyzed by flow cytometry (13, melanoma BL6-10 and BL6-10OVA cell lines were generated in our own b 20). Briefly, EXOs were first gated using calibrated polystyrene latex laboratory (17). The female wild-type C57BL/6 (H-2K ) mice were ob- m tained from Charles River Laboratories (St. Laurent, Quebec, Canada). microbeads (3.8 m) bound to fluorescent dye (Sigma-Aldrich, St. Louis, MO) and analyzed for expression of the above molecules by flow cy- The OVA257–264-specific Vb5.1,5.2 TCR transgenic (OTI), OVA323–339- specific TCR-transgenic (OTII), lymphocytic choriomeningitis virus tometry. To determine the potential mechanism associated with EXO- mediated inhibition of CD8+ T cell proliferation, the pMHC I expression (LCMV) gp33–41-specific TCR transgenic (LCMV-TCR), and UBC-GFP- + 2/2 on CD8 T cell EXO-treated DCOVA was analyzed by flow cytometry. transgenic (Tg-GFP) mice, and Fas gene knockout (KO) (Fas ) mice on + Briefly, DCOVA were incubated with CD8 T cell EXOs or EXOcont (10 mg/ by guest on September 25, 2021 a C57BL/6 background were obtained from The Jackson Laboratory (Bar 3 6 Harbor, ME). The homozygous OTI/LFA-12/2 mice were generated by 1 10 DCOVA) in serum-free AIM-V medium at 37˚C for 4 h. After backcrossing the LFA-1 gene KO mice onto the OTI background for three treatment, DCOVA were harvested, washed with PBS, stained with anti- pMHC I Ab, and analyzed by flow cytometry. To further investigate their generations, and homozygosity was confirmed by PCR according to The 3 6 Jackson Laboratory’s protocol. The transgenic RIP-mOVA mice on a in vivo effect on modulation of pMHC I expression, DCOVA (5 10 /mouse) C57BL/6 background were obtained from Dr. W. Heath (Walter and Eliza derived from Tg-GFP mice were intradermally (i.d.) injected into the flank and scruff of neck of C57BL/6 mice with i.v. injection of CD8+ T cell EXOs Hall Institute of Medical Research, Melbourne, Victoria, Australia) (19). All m mice were housed in the animal facility at the Saskatoon Cancer Center or EXOcont (30 g/mouse). At various time points (12, 24, and 36 h) after (Saskatoon, Saskatchewan, Canada), with all animal experiments carried out i.d. DCOVA injection, axillary, brachial, and inguinal lymph nodes were in accordance to the Canadian Council for Animal Care guidelines. isolated and subjected to collagenase treatment to release DCs (22). After collagenase treatment, the cells were resuspended in AIM-V medium with 5 Purification of CD8+ T cell-released exosomes mM EDTA to prevent clumping of the DCs. These recovered cells were then stained with PE-conjugated anti-Iab, anti-CD11c, and anti-B220 Abs and 2/2 C57BL/6, Tg-GFP, and Fas mouse bone marrow-derived DCs (DCOVA, analyzed by flow cytometry. These recovered cells were also stained with 2/2 [Tg-GFP]DCOVA and [Fas ]DCOVA) were generated in presence of PE-anti–CD11c and energe-coupled dye (ECD)-anti–pMHC I Abs, and the GM-CSF/IL-4 (20 ng/ml) and pulsed with OVA (0.1 mg/ml) for pMHC I expression on injected Tg-GFP DCOVA was analyzed by gating on overnight as described previously (17). To generate OVA-specific CD8+ GFP+CD11c+ events by flow cytometry. T cells, naive CD8+ T cells (4 3 105 cells/ml) from OTI or OTI/LFA-12/2 5 mice were stimulated for 72 h with irradiated (4000 rad) DCOVA (1 3 10 Uptake of EXOs by DCs cells/ml) in presence of IL-2 (20 U/ml) (17). The active CD8+ T cells were For assessment of exosomal uptake, DC were incubated with CFSE- further purified using MACS anti-CD8 beads (Miltenyi Biotec, Auburn, OVA EXO (10 mg/1 3 106 DC ) in serum-free AIM-V medium at 37˚C for CA). These purified OVA-specific active CD8+ T cells with or without 0.5 OVA 4 h, washed with PBS, and then analyzed for CFSE expression by flow mM CFSE staining were then cultured in serum-free AIM-V medium cytometry (20). To examine the molecular mechanism involved in EXO (Invitrogen, Carlsbad, CA) with IL-2 (20 U/ml) for 24 h. EXOs were uptake, DC were incubated with a panel of Abs specific for H-2Kb, purified from the above T cell culture supernatants by differential ultra- OVA CD28, and LFA-1 and anti–H-2Kb plus anti–LFA-1 Abs, respectively, on centrifugation and quantified by protein content using a Bradford assay + ice before and during coculture with CFSE-EXO in AIM-V medium at (20). DCOVA-activated CD8 T cell EXOs derived from OTI and OTI/LFA- 2 2 2 2 37˚C for 4 h and washed with PBS, and the CFSE-positive cells were then 1 / mice were termed EXOs and EXO(LFA-1 / ), respectively. EXOs detected and counted by flow cytometry and confocal fluorescence mi- derived from CFSE-stained CD8+ T cells were termed CFSE-labeled OTI croscopy. CD8+ T cell-released EXOs (CFSE-EXO). EXOs were incubated with anti–LFA-1 or anti-CD28 Ab (30 mg EXO/3 mg Ab) on ice for 30 min and T cell proliferation assays termed anti–LFA-1 Ab-treated EXOs and control Ab-treated EXOs, re- spectively. EXOs derived from a T cell hybridoma cell line RF3370 In in vitro T cell proliferation assay, purified naive OTI CD8+ and OTII + + 5 expressing OTI CD8 TCRs were termed EXORF. We previously demon- CD4 T cells (0.4 3 10 cells/well) were incubated with irradiated (4000 5 strated that in vitro Con A-stimulated T cells had similar phenotype and rad) DCOVA (0.1 3 10 cells/well) and its 2-fold dilutions in absence or profile as in vitro DC-activated ones (21). We purified EXOs from presence of different amount (1, 5, and 20 mg/ml) of EXOs or EXOcont. culture supernatants of Con A-stimulated LCMV-TCR transgenic mouse After incubation for 48 h, OTI CD8+ and OTII CD4+ T cell proliferation CD8+ T cells expressing monoclonal TCRs not for OVA but for LCMV and was measured by adding 1 mCi [3H]thymidine (1 mCi/ml; GE Healthcare, 5270 CD8+ T CELL EXOSOMES EXERT IMMUNE-SUPPRESSIVE EFFECT

Waukesha, WI) to each well. After incubation for overnight, [3H]thymi- mouse using Prism software (GraphPad Software, San Diego, CA) (8). A dine incorporation was determined by liquid scintillation counting. In p value ,0.05 was considered significant. in vivo CD8+ T cell proliferation assay, C57BL/6 mice (eight mice per 6 group) were i.v. immunized with DCOVA (1 3 10 cells/mouse) alone or + 2/2 Results together with CD8 T cell EXOs, EXOcont, (LFA-1 )EXO, EXORF, and + + anti–LFA-1 Ab- or anti–CD28 (control) Ab-treated EXOs (30 mg/mouse), CD8 T cell EXO express CD8 T cell molecules such as CD8, respectively. In another set of in vivo experiments, RIP-mOVA transgenic LFA-1, TCR, and FasL + 3 mice (10 mice/group) were first i.v. injected with OTI CD8 T cells (0.5 + 6 The in vitro-irradiated DCOVA-stimulated CD8 T cells derived 10 /mouse). One day later, the mice were then i.v. immunized with DCOVA (1 3 106 cells/mouse) alone or together with CD8+ T cell EXOs, EXOcont, from OTI mice were purified using MACS anti-CD8 beads. The and anti–LFA-1 Ab- or anti–CD28 (control) Ab-treated EXOs (30 mg/ highly purified CD8+ T cell preparation did not have any con- mouse), respectively. Six days later, the immunized mouse tail blood sam- b tamination of irradiated DCOVA (Fig. 1A). They expressed T cell ples were stained with PE-H-2K /OVAI tetramer and FITC-anti–CD8 Ab surface molecules such as CD8, CD28, CD54, Vb5.1,5.2 TCR, according to the company’s protocol and analyzed by flow cytometry. LFA-1, and FasL but no DC marker CD11c (Fig. 1B). These pu- Cytotoxicity assays rified CD8+ T cells were further cultured in presence of IL-2 for

+ production of EXOs. EXOs released from the above purified In in vitro cytotoxicity assay, DCOVA were incubated with CD8 T cell + 2/2 EXOs (10 mg/1 3 106 DC ) in presence or absence of different amounts monoclonal CD8 T cells derived from OTI and OTI/LFA-1 OVA + (0.1, 1, and 10 mg/ml) of anti-FasL blocking or control Ab at 37˚C for mice and EXOs released from Con A-stimulated monoclonal CD8 FITC 8 or 16 h. The EXO-treated DCOVA were stained with Annexin V T cells derived from LCMV-TCR transgenic mice were purified (BD Biosciences, Mississauga, Ontario, Canada) and analyzed by flow from T cell culture supernatants by differential ultracentrifugation, cytometry or subjected to analysis with TUNEL assay using In Situ Cell and termed EXOs, (LFA-12/2)EXO, and EXOcont, respectively. Downloaded from Death Detection (Roche Applied Science, Laval, Quebec, Canada) according to Roche TUNEL Staining Kit User Manual. Briefly, the above These EXOs were then subjected to electron microscopic, flow EXO-treated DCOVA were first fixed with fixing solution (4% para- cytometric, and Western blot analysis. As shown in Fig. 1C, EXOs formaldehyde in PBS), permeabilized with permeabilization solution and EXOcont had a typical exosomal characteristic of “saucer” (0.1% Triton X-100 in 0.1% sodium citrate), and then stained with TUNEL or round shape with a diameter between 50 and 90 nm (20). reaction mixture in the dark at 37˚C for 1 h. After three washes with PBS, + 2/2 2/2 the cells were analyzed by flow cytometry. In in vivo cytotoxicity assay, OTI CD8 (LFA-1 ) T cell-released (LFA-1 )EXO had a

2/2 6 http://www.jimmunol.org/ (Tg-GFP)DCOVA or CFSE (3.0 mM)-labeled (Fas )DCOVA (5 3 10 / similar morphological and biochemical characteristics as OTI mouse) were i.d. injected into the flank and scruff of neck of C57BL/6 CD8+ T cell-released EXOs (data not shown). OTI CD8+ T cell + mice with i.v. injection of CD8 T cell EXOs or EXOcont (30 mg/mouse) EXOs were then stained with a panel of Abs and analyzed by flow to assess EXO-mediated cytotoxicity to DCOVA. The draining lymph nodes cytometry calibrated with microbeads (3.8 mm in diameter) (Fig. were removed at different time points (24 and 48 h) postinjection and treated with collagenase to release DCs. The cells were stained with PE- 1D). Sorted EXOs displayed expression of molecules (CD8, CD28, anti–CD11c Ab, and the number of GFP+CD11c+ or CFSE+CD11c+ cells CD54, Vb5.1,5.2 TCR, LFA-1, and FasL) but to a much less ex- was then determined by flow cytometry. In another set of in vivo cyto- tent than OTI CD8+ T cells (Fig. 1D). In addition, they did not + toxicity assay for assessment of killing activity of DCOVA-induced CD8 b 3 6 express CD11c. Except for LFA-1 and V 5.1,5.2 TCR expression, CTLs, C57BL/6 mice were i.v. immunized with DCOVA (1 10 cells/ 2/2 + 2/2 (LFA-1 )EXO and EXOcont also had a similar phenotype as

mouse) alone or together with CD8 T cell EXOs, EXOcont, (LFA-1 ) by guest on September 25, 2021 + EXO, and anti–LFA-1 Ab- or anti-CD28 (control) Ab-treated EXOs OTI CD8 T cell-released EXOs, respectively (data not shown). (30 mg/mouse), respectively. The C57BL/6 mouse spleen cells pulsed with EXOs also displayed EXO-associated proteins, such as CD54, high OVAI peptide were strongly labeled with CFSE (3.0 mM, CFSE ) and LFA-1, and Vb5.1,5.2 TCR, but not apoptotic body marker calnexin served as OVA-specific target cells, whereas spleen cells pulsed with ir- low (4) by Western blot analysis (Fig. 1E), indicating that apoptotic relevant Mut1 peptide were weakly labeled with CFSE (0.6 mM, CFSE ) and served as nonspecific control target cells. Six days following the above vesicles are absent in the EXO and EXOcont samples. However, immunization, the immunized mice were then i.v. injected with a 1:1 EXOcont did not express Vb5.1,5.2 TCR because monoclonal (CFSEhigh:CFSElow) mixture of splenocytes targets. Sixteen hours after CD8+ T cells derived from LCMV-TCR transgenic mice express target cell delivery, spleens of the recipient mice were removed, and the Vb8.1,2.4 TCRs (23). relative proportions of CFSEhigh and CFSElow target cells remaining in the spleens were analyzed by flow cytometry (18). T cell EXOs downregulate DC pMHC I expression and inhibit DC-stimulated CD8+ T cell proliferation in vitro Animal studies We previously established a protocol for in vitro generation of For evaluation of antitumor immunity, C57BL/6 mice (eight mice per 6 mature DCs by culturing mouse bone marrow cells in presence of group) were i.v. vaccinated with DCOVA (1 3 10 cells/mouse) alone or 6 + GM-CSF (20 ng/ml) and IL-4 (20 ng/ml) (17) and demonstrated DCOVA (1 3 10 cells/mouse) together with i.v. injection of CD8 T 2/2 cell EXOs, EXOcont, (LFA-1 )EXO, EXORF, anti–LFA-1 Ab-treated that these DCs can uptake DC-released EXOs (8). To assess the m EXOs, and anti-CD28 (control) Ab-treated EXOs (30 g/mouse), re- phenotype of bone marrow-derived mature DCOVA, we performed spectively. Eight days after immunization, mice were challenged by s.c. 3 6 flow cytometric analysis. As shown in Fig. 2A,DCOVA expressed injection of 0.3 10 BL6-10OVA tumor cells. Animal tumor growth was b monitored daily for 40–60 d; for ethical reasons, all mice with tumors that CD11c, CD80, and Ia , indicating that they are mature DCs. In achieved a size of 1.5 cm in diameter were sacrificed. To assess the in- addition, they also expressed pMHC I complexes and Fas, whereas duction of diabetes, RIP-mOVA transgenic mice (10 mice/group) were i.v. Mut1 peptide-pulsed DCs did not display OVA-specific pMHC I injected with OTI CD8+ T cells (0.5 3 106 cells/mouse), followed by i.v. 6 6 detected by anti-pMHC I Ab. The downregulation of pMHC I immunization of DCOVA (1 3 10 cells/mouse) alone or DCOVA (1 3 10 + complexes on DCOVA has been reported when DCOVA interact with cells/mouse) together with CD8 T cell EXOs, EXOcont, anti–LFA-1 + Ab-treated EXOs, and anti-CD28 (control) Ab-treated EXOs (30 mg/ active CD8 T cells via TCR-mediated internalization of TCR- mouse), respectively. The above mice were monitored for 10 d after im- pMHC I complexes into DCOVA (24). We incubated DCOVA with munization for diabetes by urine glucose testing (19). Animals were CD8+ T cell EXOs or EXOcont for 4 h and then characterized $ considered to be diabetic after 2 consecutive days with readings of 55 EXO-treated DC by flow cytometry. We found that pMHC I mmol/L glucose. OVA expression on EXO-treated DCOVA but not on EXOcont-treated Statistical analysis DCOVA was downregulated, possibly because of TCR-mediated internalization (Fig. 2A). However, expression of other mole- Statistical analysis were carried out to perform Student t test for comparison b b of variables from different groups in both in vitro and in vivo experiments, cules, such as H-2K ,Ia, CD11c, CD80, and Fas on EXO-treated and log- test for comparison of animal survival in different groups of DCOVA, was not affected. The fact that EXO-treated DCOVA dis- The Journal of Immunology 5271

b played similar amount of H-2K expression as DCOVA, although they displayed reduced amounts of pMHC I, may result from a b compensation via uptake of H-2K –expressing EXOs by DCOVA. Interestingly, EXO-treated DCOVA with pMHC I downregulation, but not EXOcont-treated DCOVA, also failed in efficient stimula- tion of CD8+ T cell proliferation in an EXO–dose-dependent fashion (Fig. 2B), indicating that OTI CD8+ T cell EXOs inhibit + in vitro DCOVA-mediated CD8 T cell proliferation. However, the + stimulation of CD4 T cell proliferation by EXO-treated DCOVA was not affected (Fig. 2C). DC uptake T cell EXO via pMHC I–TCR and CD54–LFA-1 interactions It has been demonstrated that DCs can uptake T cell EXOs (15). To assess T cell EXO uptake by DCs, DCOVA were incubated with CFSE-EXO for 4 h and then analyzed by flow cytometry and confocal fluorescence microscopy. As shown in Fig. 3A,DCOVA displayed CFSE expression after incubation with CFSE-EXO, in-

dicating that DCOVA uptake T cell EXOs. However, either DCOVA Downloaded from incubated with CFSE-EXOcont or DCs incubated with CFSE- EXO displayed reduced amount of CFSE expression (Fig. 3A), indicating that cognate pMHC I–TCR interactions may play an important role in DCs’ uptake of T cell EXOs. To assess the molecular mechanism for uptake of T cell EXOs by DCOVA,we added blocking reagents to the above mixture of DCOVA and http://www.jimmunol.org/ CFSE-EXO. We found that both anti–H-2Kb and anti–LFA-1 Abs, but not anti-CD28 Ab, significantly inhibited the uptake of CFSE- EXO by DCOVA (p , 0.05) (Fig. 3B,3C), indicating that both pMHC I/TCR and CD54–LFA-1 interactions mediate DCOVA’s absorption of CD8+ T cell EXOs. T cell EXOs downregulate pMHC I on DCs in vivo To assess whether T cell EXOs also downregulate pMHC I on DCs in vivo, we then generated DCOVA derived from Tg-GFP mice. by guest on September 25, 2021 High amounts of GFP were expressed by all these DCOVA, which allowed an easy identification of these cells after they were transferred into other mice. To assess in vivo downregulatory ef- fect, CD8+ T cell EXOs were i.v. injected into C57BL/6 mice with i.d. immunization of Tg-GFP DCOVA derived from Tg-GFP mice. The cells derived from draining lymph nodes were har- vested, stained with PE-conjugated anti-Iab, anti-CD11c, and anti- B220 Abs, and analyzed by flow cytometry. We found that 96% of GFP+ cells recovered from the mouse draining lymph nodes were Iab+CD11c+B2202 (data not shown), indicating that the recovered GFP+ cells are predominantly DCs. The cells recovered from the draining lymph nodes were also stained with PE-anti–CD11c and ECD-anti–pMHC I Abs, and the pMHC I expression on injected + + DCOVA was then analyzed by gating on GFP CD11c events by flow cytometry. We found that expression of pMHC I complexes on injected Tg-GFP DCOVA decreased only minimally over time in immunized mice with i.v. injection of EXOcont, whereas its ex- pression fell rapidly and was barely detectable 36 h after transfer + FIGURE 1. Phenotypic analysis of DCOVA-activated CD8 T cells and into mice with i.v. injection of EXOs (Fig. 3D), indicating that CD8+ T cell- or RF3370-released EXOs. A,DC and purified DC - + OVA OVA CD8 T cell EXOs also downregulate pMHC I on DCOVA in vivo. activated OTI CD8+ T cells were stained with FITC-anti–CD8 Ab (FITC- CD8) and ECD-anti–CD11c Ab (CD11c-ECD) and analyzed by flow T cell EXOs induce cytotoxicity to DCs via Fas/FasL pathway + cytometry. B,DCOVA-activated OTI CD8 T cells were stained with It has been demonstrated that FasL expressing tumor cell-released a panel of Abs (solid lines) or isotype-matched irrelevant Abs (dotted EXOs induced CD8+ T cell apoptosis (25). To assess the potential lines) and then analyzed by flow cytometry. C, Electron micrograph of killing effect of FasL-expressing EXOs to Fas-expressing DCs, we EXOs. Scale bar, 100 nm. D, Flow cytometric analysis of DC -activated OVA stained EXO-treated DC with Annexin VFITC and then ana- OTI CD8+ T cell-released EXOs and microbeads (3.8 mm in diameter). OVA CD8+ T cell EXOs were sorted (in circle) for analysis of expression of lyzed them by flow cytometry. As shown in Fig. 3E, 78% of EXO- surface molecules using FITC-conjugated Abs against immunological treated DCOVA displayed expression of annexin V (early apoptosis molecules (solid lines) or isotype-matched FITC-conjugated irrelevant Abs marker), compared with only 14% of DCOVA with spontaneous + (dotted lines). E, Western blot analysis of EXOs, EXORF, and EXOcont apoptosis, indicating that CD8 T cell EXOs can induce apoptosis using a panel of Abs. One representative experiment of two is shown. to DCs. Interestingly, the anti-FasL Ab, but not the isotype- 5272 CD8+ T CELL EXOSOMES EXERT IMMUNE-SUPPRESSIVE EFFECT

FIGURE 2. T cell EXOs downregulate pMHC I + expression of DCOVA and inhibit DC-induced CD8 T cell proliferation in vitro. A,DCOVA, Mut1 peptide- pulsed DCs (DCMut1), and EXO- or EXOcont-treated DCOVA were stained with a panel of Abs (solid lines) or isotype-matched irrelevant Abs (dotted lines) and then analyzed by flow cytometry. Naive OTI CD8+ T cells (B) or naive OTII CD4+ T cells (C) were in- cubated with irradiated DCOVA in absence or pres- ence of different amounts of EXOs or EXOcont in a Downloaded from [3H]thymidine uptake assay. pp , 0.05 versus cohorts of DCOVA or DCOVA plus EXOcont (Student t test). One representative experiment of three is shown. http://www.jimmunol.org/

+ by guest on September 25, 2021 matched irrelevant control Ab, almost completely blocked EXO- DCOVA together with CD8 T cell EXOs. We found that DCOVA + + induced apoptosis to DCOVA in a dose-dependent manner, in- were able to induce 1.76% tetramer CD8 T cells in the total dicating that CD8+ T cell EXOs are cytotoxic to DCs via Fas/FasL mouse CD8+ T cell population at day 6 after immunization (Fig. + pathway. To further confirm the above results, we performed 4A). However, immunization of DCOVA with OTI CD8 T cell + TUNEL assay. Similarly, we found that EXO-treated DCOVA,but EXOs, but not LCMV-TCR transgenic mouse CD8 Tcell + + not EXO-treated DCOVA in the presence of anti-FasL Ab, dis- EXOcont, significantly reduced stimulation of tetramer CD8 played increased cell apoptosis (Fig. 3F). To assess the potential T cell response from the original 1.76–0.34% in the total CD8+ in vivo cytotoxicity, CD8+ T cell EXOs were i.v. injected into T cell population (p , 0.05), indicating that OTI CD8+ T cell C57BL/6 mice with i.d. immunization of Tg-GFP DCOVA. At dif- EXOs expressing OVA-specific TCR inhibit DCOVA-stimulated ferent time points after injection, the cells in the draining lymph OVA-specific CD8+ T cell responses. To assess a CD8+ T cell nodes were harvested, stained with PE-anti–CD11c Ab, and then effector’s function, we adoptively transferred the OVAI peptide- analyzed by flow cytometry as described above. We found that the pulsed splenocytes that had been strongly labeled with CFSE + + high number of GFP CD11c DCOVA mildly decreased in mice over (CFSE ) as well as the Mut1 control peptide-pulsed splenocytes the time course of the experiments (Fig. 3G). However, the that had been weakly labeled with CFSE (CFSElow) into the re- + + number of injected GFP CD11c DCOVA significantly decreased cipient mice that had been vaccinated with DCOVA and DCOVA + + in mice with injection of GFP CD11c DCOVA and EXOs, but not plus EXOs, respectively. We then assessed loss of OVAI-specific EXOcont (p , 0.05), compared with mice with injection of GFP+ CFSEhigh target cells in the recipient mice, which represent the + + CD11c DCOVA alone (Fig. 3G), indicating that T cell EXOs ex- killing activity of OVA-specific effector CD8 CTLs in the re- pressing FasL may also induce in vivo apoptosis of Fas-expres- cipient mice. As shown in Fig. 4B, no CFSEhigh target cell loss was sing DCs leading to reduced DC survival. To further confirm it, we observed in mice immunized with PBS. As expected, there was 2/2 high used CFSE-labeled DCOVA derived from Fas mice (CFSE- a substantial loss (83.5%) of OVA-specific target (CFSE )cellsin 2/2 [Fas ]DCOVA). We found that there was no significant differ- mice immunized with DCOVA, indicating that DCOVA can stimulate 2/2 + ence between the numbers of CFSE-(Fas )DCOVA in the CD8 T cell differentiation into CTL effectors in vivo. Compared draining lymph nodes of C57BL/6 mice with and without EXO with DCOVA-immunized group, DCOVA plus EXOcont immuniza- treatment (p . 0.05) (Fig. 3H). tion similarly resulted in a substantial loss (83.1%) of OVA-specific high CFSE cells. However, DCOVA plus EXO immunization killed only 38.8% of OVA-specific CFSEhigh cells (p , 0.05), indicating that T cell EXOs inhibit in vivo OVA-specific CD8+ CTL responses OVA-specific, TCR-specific T cell EXOs can inhibit DCOVA-stimu- and antitumor immunity lated effector CD8+ CTL responses. To assess whether EXOs inhibit + To assess whether CD8 T cell EXOs have an in vivo-suppressive antitumor immunity, C57BL/6 mice were i.v. immunized with DCOVA effect, we i.v. immunized C57BL/6 mice with DCOVA alone or or DCOVA plus EXOs. Eight days after the immunization, the im- The Journal of Immunology 5273

FIGURE 3. T cell EXO downregulate pMHC I expression of DCOVA in vivo and induce cytotox- icity to DCs. A,DCOVA and DCs were cocultured with (solid line) or without (dotted line) CFSE- EXO and CFSE-EXOcont, respectively, and then analyzed by flow cytometry. DCOVA were incubated with CFSE-EXO in presence of a panel of Abs.

CFSE-positive DCOVA were then detected and counted by flow cytometry (B) and confocal fluo- rescence microscopy (C), respectively. pp , 0.05 versus cohorts of EXOs or EXOs plus anti-CD28 Ab (Student t test). One representative experiment of three is shown. D, CD8+ T cell EXO down- regulate pMHC I of DCOVA in vivo. Tg-GFP DCOVA were i.d. injected into C57BL/6 mice with or without i.v. injection of CD8+ T cell EXOs or

EXOcont. At various time points after i.d. DCOVA transfer, the cells recovered from draining lymph Downloaded from nodes were stained with PE-CD11c and ECD- pMHC I Abs, and the pMHC I expression on injected Tg-GFP DCOVA was then analyzed by gating on GFP+CD11c+ events by flow cytometry. Points in the curve, mean fluorescence intensity (MFI) of pMHC I expression on GFP+CD11c+ http://www.jimmunol.org/ cells; bars, SD. pp , 0.05 versus cohorts of DCOVA or DCOVA plus EXOcont (Student t test). One representative of six experiments is shown. E and F,

In vitro cytotoxicity assay. DCOVA were incubated with CD8+ T cell EXO in presence or absence of anti-FasL blocking or control Ab for 8 and

16 h. The EXO-treated DCOVA were stained with Annexin VFITC (E) and assessed in TUNEL assay (F), respectively, and then analyzed by flow cy-

tometry. In TUNEL assay, we assessed DCOVA, by guest on September 25, 2021 DCOVA plus EXO with or without anti-FasL, or control Ab (solid lines) and used positive (solid line) and negative (dotted lines) controls provided in the In Situ Cell Death Detection Kit. pp , 0.05 versus cohorts of DCOVA; ppp , 0.05 versus co- horts of DCOVA plus EXO or DCOVA plus EXO and control Ab (Student t test). One representative experiment of three is shown. G and H,Invivo cytotoxicity assay. Tg-GFP DCOVA (G) or CFSE- labeled DCOVA (H) derived from Fas gene KO mice were i.d. injected into C57BL/6 mice with i.v. in- jection of CD8+ T cell EXOs or EXOcont. The draining lymph nodes were removed at different time points after injection and treated with colla- genase to release DCs. The percentage of GFP+ CD11c+ or CFSE+CD11c+ cells was determined by flow cytometry. The absolute number of GFP+ CD11c+ or CFSE+CD11c+ cells was then counted by multiplying this percentage by the total cell number. pp , 0.05 versus cohorts of DCOVA or DCOVA plus EXOcont (Student t test). One repre- sentative of six experiments is shown.

+ munized mice were s.c. challenged with BL6-10OVA tumor cells. that OTI CD8 T cell EXOs can also inhibit OVA-specific anti- As shown in Fig. 4C, all the mice injected with PBS died of tumor tumor immunity. within 21 d after tumor cell challenge. DCOVA vaccine protected T cell EXOs also inhibit DC-stimulated diabetes in transgenic eight of eight (100%) mice from tumor growth. DCOVA plus EXOs RIP-mOVA mice or EXO-pulsed DCOVA, but not DCOVA plus EXOcont vaccine, had significant inhibition on DCOVA-mediated immune protection RIP-mOVA transgenic mice with moderate expression of self-OVA + against OVA-expressing BL6-10OVA tumors (p , 0.05), indicating exhibited deletional tolerance mediated by autoreactive CD8 5274 CD8+ T CELL EXOSOMES EXERT IMMUNE-SUPPRESSIVE EFFECT Downloaded from http://www.jimmunol.org/

FIGURE 4. T cell EXO suppress CD8+ CTL responses and antitumor immunity. A, In vivo CD8+ T cell proliferation assay. Six days after immunization + 2/2 of C57BL/6 mice with DCOVA alone, DCOVA plus CD8 T cell EXOs, EXOcont, (LFA-1 )EXO, anti–LFA-1 Ab-treated EXOs, and anti-CD28 (control) Ab-treated EXOs, respectively, the immunized mouse tail blood samples were stained with PE-H-2Kb/OVAI tetramer and FITC-anti–CD8 Ab and then analyzed by flow cytomery. The value in each panel represents the percentage of tetramer+CD8+ T cells versus the total CD8+ T cell population. The value in parenthesis represents the SD. pp , 0.05 versus cohorts of DCOVA or DCOVA plus EXOcont; ppp , 0.05 versus cohorts of DCOVA plus EXOs or DCOVA plus EXOs and control Ab (Student t test). B, In vivo cytotoxicity assay. Six days after immunization, the immunized mice were i.v. injected with 2 3 106 high low

cells containing a 1:1 mixture of CFSE - and CFSE -labeled splenocytes that had been pulsed with OVAI or Mut1 peptides, respectively. After 16 h, the by guest on September 25, 2021 spleens of immunized mice were removed, and the percentages of the residual CFSEhigh (H) and CFSElow (L) target cells remaining in the recipients’ spleens were analyzed by flow cytometry. The value in each panel represents the percentage of CFSEhigh versus CFSElow target cells remaining in the spleen. The value in parenthesis represents the SD. pp , 0.05 versus cohorts of DCOVA or DCOVA plus EXOcont; ppp , 0.05 versus cohorts of DCOVA plus EXOs or DCOVA plus EXOs and control Ab (Student t test). C, Animal studies. C57BL/6 mice were i.v. vaccinated with DCOVA or EXO-pulsed DCOVA + 2/2 alone, DCOVA plus CD8 T cell EXOs, EXOcont, (LFA-1 )EXOs, anti–LFA-1 Ab-treated EXOs, and control Ab-treated EXOs, respectively. Eight days after immunization, the immunized mice were s.c. inoculated with BL6-10OVA tumor cells. Animal mortality and tumor growth were monitored daily for up to 40–60 d. pp , 0.05 versus cohorts of DCOVA or DCOVA plus EXOcont; ppp , 0.05 versus cohorts of DCOVA plus EXOs or DCOVA plus EXOs and control Ab (log-rank test). One representative experiment of two is shown.

+ T cells (26), which can be overcome by more OVA-specific CD8 of 10 RIP-mOVA mice, whereas DCOVA injection together with T precursors (27). To assess the CD8+ T cell EXO-mediated in- EXOs only induced mild diabetes in 4 of 10 RIP-mOVA mice. hibitory effect on DCOVA-induced diabetes in transgenic RIP- In addition, we found the destruction of pancreatic islet tissues mOVA mice, these mice were given i.v. injection of OTI CD8+ with lymphocyte and infiltration in these diabetic mice T cells and then i.v. immunization with DCOVA or DCOVA plus (Fig. 5C). EXOs or EXOcont. Six days after immunization, mouse tail blood b Exosomal LFA-1 plays an important role in T cell samples were stained with PE-H-2K /OVAI tetramer and FITC- + anti–CD8 Ab for assessment of OVA-specific CD8+ T cell re- EXO-mediated inhibition of in vivo DC-stimulated CD8 CTL responses in antitumor immunity and autoimmunity sponses by flow cytometry. As shown in Fig. 5A,DCOVA were unable to stimulate OVA-specific CD8+ CTL responses in trans- To assess the potential role of LFA-1 expression in CD8+ T cell genic RIP-mOVA mice with OVA-specific self- EXO-mediated suppression, we repeated the above in vivo ex- (26). However, when these mice were given OTI CD8+ T cells, periments by using anti–LFA-1 Ab-treated EXOs. We found that + DCOVA became capable of stimulating OVA-specific CD8 CTL injection of anti–LFA-1 Ab-treated EXOs, but not the control responses (accounting for 2.26% in the total mouse CD8+ T cell Ab-treated EXOs, significantly resumed OTI CD8+ T cell EXO- + + population). Interestingly, treatment of mice with CD8 T cell induced inhibition of DCOVA-stimulated CD8 T cell responses in + EXOs but not EXOcont significantly inhibit DCOVA-mediated re- C57BL/6 mice (Fig. 4A), CD8 T cell effector function (Fig. 4B), + sponses of CD8 CTL responses (accounting for 0.48% in the and antitumor immunity against BL6-10OVA tumor cell challenge total CD8+ T cell population) (p , 0.05). Furthermore, RIP-mOVA (p , 0.05) (Fig. 4C), indicating that LFA-1 adhesion plays an + mice immunized with DCOVA plus EXO treatment were monitored important role in CD8 T cell EXO-mediated inhibition of in vivo for 10 d for diabetes by urine glucose testing. As shown in Fig. 5B, OVA-specific CD8+ CTL responses and antitumor immunity. To 2/2 + DCOVA injection alone or together with EXOcont into RIP-mOVA confirm it, we further used (LFA-1 )EXO released from CD8 mice with transfer of OTI CD8+ T cells induced diabetes in all 10 T cells derived from OTI/LFA-12/2 mice in the above experiments. The Journal of Immunology 5275 Downloaded from http://www.jimmunol.org/

FIGURE 5. T cell EXOs inhibit CD8+ CTL responses and diabetes in transgenic RIP-mOVA mice. A, In vivo CD8+ T cell proliferation assay. RIP-mOVA + + transgenic mice were i.v. immunized with DCOVA or i.v. injected with OTI CD8 T cells, then i.v. immunized with DCOVA alone or DCOVA plus CD8 T cell EXO, EXOcont, anti–LFA-1 Ab-treated EXOs, and anti-CD28 (control) Ab-treated EXOs, respectively. Six days later, the immunized mouse tail blood samples were stained with PE-H-2Kb/OVAI tetramer and FITC-anti–CD8 Ab and then analyzed by flow cytometry. The value in each panel represents the + + + percentage of tetramer CD8 T cells versus the total CD8 T cell population. The value in parenthesis represents the SD. pp , 0.05 versus cohorts of DCOVA by guest on September 25, 2021 or DCOVA plus EXOcont; ppp , 0.05 versus cohorts of DCOVA plus EXOs or DCOVA plus EXOs and control Ab (Student t test). B, T cell EXOs inhibit DCOVA-stimulated diabetes in RIP-mOVA mice. The above immunized RIP-mOVA mice were monitored for 10 d after immunization for diabetes by urine glucose testing. The SDs in the glucose curves were calculated from the data derived from diabetic mice only. pp , 0.05 versus cohorts of DCOVA or DCOVA plus EXOcont; ppp , 0.05 versus cohorts of DCOVA plus EXOs or DCOVA plus EXOs and control Ab (Student t test). C, H&E staining of pancreas tissues. The pancreas tissues of the immunized transgenic RIP-mOVA mice were fixed in 10% neutral-buffered formalin and embedded in paraffin. Tissue sections were stained with H&E and examined by microscopy. The destruction of pancreatic islet tissues with lymphocyte and monocyte infiltration in immunized RIP-mOVA mice was marked with arrows. One representative experiment of two is shown. Original magnification 3150.

We found that LFA-1–deficient EXOs did not show any inhibition associated proteins including LFA-1 and Vb5.1,5.2 TCRs were + + on DCOVA-induced CD8 CTL responses (Fig. 4A), CD8 Teffector abundant by Western blot analysis (Fig. 1E). EXORF displayed function (Fig. 4B), and antitumor immunity (Fig. 4C), thus con- expression of molecules (Vb5.1,5.2, TCR LFA-1, and FasL) but to firming that T cell EXO-induced inhibition of in vivo OVA-specific a much less extent than RF3370 hybridoma cells (Fig. 6A). Both + CD8 CTL responses and antitumor immunity is mediated by RF3370 cells and EXORF did not express CD54. To assess its exosomal LFA-1. Similarly, injection of anti–LFA-1 Ab-treated in vivo inhibitory effect, we further used EXORF released from EXOs, but not the control Ab-treated EXOs, significantly resumed RF3370 hybridoma cells in experiments as described in the above + DCOVA-stimulated CD8 T cell responses in RIP-mOVA mice sections. We found that RF3370 T cell hybridoma-derived EXORF (p , 0.05) (Fig. 5A), leading to diabetes in all 9 of 10 RIP-mOVA expressing OVA-specific TCRs and LFA-1 also inhibited DCOVA- mice with the anti–LFA-1 Ab-treated EXO injection (Fig. 5B), induced OVA-specific CD8+ CTL responses (Fig. 6B) and anti- thus confirming that exosomal LFA-1 plays an important role in tumor immunity against OVA-expressing BL6-10OVA tumor cell + T cell EXO-mediated inhibition of in vivo OVA-specific CD8 challenge (Fig. 6C), indicating that EXORF have a similar in- + CTL responses and autoimmunity. hibitory effect as CD8 T cell EXOs on in vivo DCOVA-stimulated OVA-specific CD8+ CTL responses and antitumor immunity. T cell hybridoma-released EXOs similarly inhibit + DC-stimulated CD8 CTL responses and antitumor immunity Discussion EXOs derived from a T cell hybridoma cell line RF3370 expressing Intercellular trogocytosis, a phenomenon of intercellular molecule OTI CD8+ TCRs were purified from its culture supernatant by transfer, which plays an important role in modulation of immune differential ultracentrifugation and termed EXORF. These EXORF responses, has been attracting more and more attention (28). were then subjected to electron microscopic, Western blot, and Surface molecule transfer has been observed for various cell types flow cytometric analyses. As shown in Fig. 1C, EXORF had a but has been predominantly reported for transfer of DC surface typical exosomal characteristic of “saucer” or round shape with a molecules to T cells in a unidirectional way (29, 30). As a result, diameter between 50 and 90 nm. We also confirmed that EXO- T cells with acquired DC molecules have been shown to be either 5276 CD8+ T CELL EXOSOMES EXERT IMMUNE-SUPPRESSIVE EFFECT

sibility to the following Ag-specific T cells (36). In this study, we showed that OVA-specific CD8+ T cell-released EXOs can induce in vitro and in vivo OVA-specific pMHC I downregulation + on DCOVA, leading to loss of DCs’ stimulatory effect on CD8 T cell proliferation. Cytotoxic CD8+ T cells kill target cells through two distinct cytolytic pathways, the perforin-dependent granule exocytosis and the Fas/FasL pathway (38). It has been demonstrated that FasL-expressing T cells mediated effect- ive killing of prostate cancer cells (39) and induced apoptosis to bystander T cells with unknown mechanism (16). It has also been shown that FasL-expressing tumor cell-released EXOs or pregnancy-associated EXOs induce CD8+ T cell apoptosis via Fas/ FasL pathway or exhibit suppression of T cell CD3d and JAK3 (25, 40-42), and diavylglycerol kinase regulates the secretion of lethal EXOs bearing FasL during activation-induced cell death of T lymphocytes (43, 44). In this study, we further demonstrated that T cell EXOs expressing FasL induce DC apoptosis in vitro in a dose-dependent manner and reduce DC survival in vivo.

Therefore, our data, for the first time to our knowledge, indicate Downloaded from that T cell EXOs are able to induce both pMHC I downregulation and cytotoxicity to DCs, leading to inhibition of both in vitro and in vivo OVA-specific DC-mediated CD8+ CTL responses as well as antitumor immunity. Thus, in addition to the previously re- ported mechanism of CD8+ CTL-mediated killing of DCs (17), + FIGURE 6. T cell hybridoma-derived EXORF similarly inhibit CD8 our study provides another mechanism of how DC-induced im- http://www.jimmunol.org/ CTL responses and antitumor immunity. A, Phenotypic analysis of RF3370 mune responses can be regulated by CTL-released EXOs, espe- T cell hybridoma and RF3370 cell-released EXOs by flow cytometry. cially when CTL responses are too strong. RF3370 cells and RF3370-released EXOs were stained with a panel of Abs Type I diabetes is a chronically progressive, T cell-mediated (solid lines) or isotype-matched irrelevant Abs (dotted lines) and then that affects and inbred strains of ro- + analyzed by flow cytometry. B, In vivo CD8 T cell proliferation assay. Six dents such as the NOD mice. It has been demonstrated that CD8+ days after immunization of C57BL/6 mice with DCOVA alone or DCOVA T cells, which recognize Ags in context of MHC I molecules, are plus EXO or EXOcont, the immunized mouse tail blood samples were RF the effector cells that kill the b cells of the islets in a Fas- or stained with PE-H-2Kb/OVAI tetramer and FITC-anti–CD8 Ab and then analyzed by flow cytomery. The value in each panel represents the per- perforin-dependent manner (45, 46). In this study, we demon-

+ by guest on September 25, 2021 centage of tetramer+CD8+ T cells versus the total CD8+ T cell population. strated that Ag-specific OTI mouse CD8 T cell EXOs can also + The value in parenthesis represents the SD. pp , 0.05 versus cohorts of inhibit OVA-specific DC-mediated CD8 CTL responses and di-

DCOVA or DCOVA plus EXOcont (Student t test). C, Animal studies. abetes in transgenic RIP-mOVA mice. Interestingly, EXOs derived C57BL/6 mice were i.v. vaccinated with DCOVA alone or DCOVA plus from an immortal T cell hybridoma cell line RF3370 generated + EXORF or EXOcont. Eight days after immunization, the immunized mice by fusion of OTI CD8 T cells with T cell leukemia cells + were s.c. inoculated with BL6-10OVA tumor cells. Animal mortality and have a similar inhibitory effect as OTI CD8 T cell EXOs on p , tumor growth were monitored daily for up to 40–60 d. p 0.05 versus DC-mediated CD8+ CTL responses and antitumor immunity. cohorts of DCOVA or DCOVA plus EXOcont (log-rank test). One repre- Therefore, our data indicate that Ag-specific T cell EXOs, espe- sentative experiment of two is shown. cially when they are derived from immortalized T cell hybrid- omas, may be useful in treatment of autoimmune diseases. immunogenic (21, 31) or tolerogenic (32, 33) in modulation of It has been shown that immature DCs take up EXOs via milk fat immune responses. In addition, T cells with uptake of DC-released globule-E8, CD11a, CD54, CD9, and CD81 on EXOs and aVb4 EXOs can be used as T cell vaccine for stimulation of antitumor integrin, CD11a, and CD54 on DCs (47). EXOs were either in- immunity (18, 34). Recently, however, we have shown that molec- ternalized by or fused with DCs and then processed by DCs for ular transfer between DCs and T cells is bidirectional, and DCs can presentation of exosomal Ag to CD4+ T cells (48). It has also been also acquire T cell molecules via a dissociation-associated pathway demonstrated that naive T cells absorbed DC EXOs via either (35). Busch et al. (36) have also demonstrated that transfer of T cell TCRs or CD28 (49) and LFA-1 (6). We previously demonstrated molecules onto DCs occurred, when DCs activated CD4+ T cells, via that active CD4+ T cells can uptake DC EXOs via TCR–MHC and two distinct mechanisms including an early independency and a late LFA-1–CD54 interactions (18). Activation of specific G protein dependency on Ag-specific T cell activation. Similar to DCs, active receptors, cytokine stimulation motility, and TCR-mediated T cells can also secrete bioactive EXOs (13, 14), which can be signals all induce a transient conformational change in LFA- further taken up by APCs or B cells via cultivation (15). However, 1, thereby highly increasing T cell LFA-1 avidity for CD54 the potential effect of these T cell EXOs on modulation of DC- (50). This process is called “inside-out” signaling (51). Recently, stimulated immune responses is elusive. it has been further demonstrated that active T cells recruit DC It has been shown that pMHC I expression of DCs could EXOs by LFA-1 (52), indicating that CD54–LFA-1 interaction be downregulated after in vitro and in vivo interactions with Ag- plays an important role in not only intercellular cell–cell inter- specific CD8+ T cells via TCR-mediated internalization pathway actions but also T cell EXO absorption by DC or DC EXO uptake (24, 37). It has also been demonstrated that DCs that received by T cells. In this study, we showed that DCs can absorb EXOs TCR molecules were less efficient in priming T cell responses, expressing OVA-specific TCRs derived from transgenic OTI CD8+ possibly because the transferred TCR molecules might mask T cells via MHC–TCR and CD54–LFA-1 interactions because the Ag-bearing MHC complexes, thereby reducing their acces- addition of blocking reagents such as anti–H-2Kb and anti–LFA-1 The Journal of Immunology 5277

Ab to the mixture of DCs and T cell EXOs inhibited in vitro EXO inducing ligand-carrying microvesicles during activation-induced death of hu- + man T cells. J. Immunol. 167: 6736–6744. uptake by DCs. OTI CD8 T cell EXOs expressing OVA-specific 17. Xia, D., S. Hao, and J. Xiang. 2006. CD8+ cytotoxic T-APC stimulate central TCRs, but not EXOs without OVA-specific TCR expression, which memory CD8+ T cell responses via acquired peptide-MHC class I complexes and were derived from LCMV-TCR transgenic mouse monoclonal CD80 costimulation, and IL-2 secretion. J. Immunol. 177: 2976–2984. + + 18. Hao, S., Y. Liu, J. Yuan, X. Zhang, T. He, X. Wu, Y. Wei, D. Sun, and J. Xiang. CD8 T cells, can inhibit in vivo DCOVA-induced CD8 CTL 2007. Novel exosome-targeted CD4+ T cell vaccine counteracting CD4+25+ responses and antitumor immunity. Furthermore, we also demon- -mediated immune suppression and stimulating efficient central strated that the anti–LFA-1 Ab treatment completely blocks OTI memory CD8+ CTL responses. J. Immunol. 179: 2731–2740. + + 19. Behrens, G. M., M. Li, G. M. Davey, J. Allison, R. A. Flavell, F. R. Carbone, and CD8 T cell EXO-mediated inhibition of CD8 CTL responses, W. R. Heath. 2004. Helper requirements for generation of effector CTL to islet antitumor immunity, and autoimmunity. Our data thus indicate that antigens. J. Immunol. 172: 5420–5426. DCs recruit T cell exosomes in vivo via exosomal LFA-1. 20. Xie,Y.,O.Bai,H.Zhang,J.Yuan,S.Zong,R.Chibbar,K.Slattery,M.Qureshi,Y.Wei, + Y. Deng, and J. Xiang. 2009. Membrane-bound HSP70-engineered myeloma cell- Taken together, our study demonstrates that OVA-specific CD8 derived exosomes stimulate more efficient CD8+ CTL- and NK-mediated antitumor T cell EXOs expressing OVA-specific TCRs and FasL can inhibit immunity than exosomes released from heat-shocked tumor cells expressing cyto- DC-mediated antitumor and autoimmune CD8+ CTL responses. plasmic HSP70. J.Cell.Mol.Med. 21. Umeshappa, C. S., H. Huang, Y. Xie, Y. Wei, S. J. Mulligan, Y. Deng, and Their inhibitory effect is via downregulation of pMHC I and in- J. Xiang. 2009. CD4+ Th-APC with acquired peptide/MHC class I and II duction of apoptosis of DCs through Fas/FasL pathway. Therefore, complexes stimulate type 1 helper CD4+ and central memory CD8+ Tcell our data indicate that Ag-specific CD8+ T cells can modulate im- responses. J. Immunol. 182: 193–206. 22. Zhang, X., H. Huang, J. Yuan, D. Sun, W. S. Hou, J. Gordon, and J. Xiang. 2005. mune responses via T cell-released EXOs, and T cell EXOs may CD4282 dendritic cells prime CD4+ T regulatory 1 cells to suppress antitumor be useful for treatment of autoimmune diseases. immunity. J. Immunol. 175: 2931–2937.

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