Autophagic Compartments Gain Access to the MHC Class II Compartments in Thymic Epithelium

This information is current as Michiyuki Kasai, Isei Tanida, Takashi Ueno, Eiki of September 29, 2021. Kominami, Sachiko Seki, Tohru Ikeda and Toshiaki Mizuochi J Immunol published online 13 November 2009 http://www.jimmunol.org/content/early/2009/11/13/jimmuno

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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 © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published November 13, 2009, doi:10.4049/jimmunol.0804087 The Journal of Immunology

Autophagic Compartments Gain Access to the MHC Class II Compartments in Thymic Epithelium

Michiyuki Kasai,1* Isei Tanida,† Takashi Ueno,‡ Eiki Kominami,‡ Sachiko Seki,§ Tohru Ikeda,¶ and Toshiaki Mizuochi*

The presentation of self-peptides in the context of MHC molecules by thymic epithelial cells (TECs) is essential for repertoire selection in the . However, the underlying mechanisms of this process have not been fully elucidated. To address whether autophagy, a catabolic process involving the degradation of a cell’s components through the lysosomal machinery, intersects the MHC class II-restricted Ag presentation pathway in TECs, we investigated the colocalization of LC3, a peculiar autophagy marker molecule, with MHC class II compartments in in vitro-established TEC lines by immunofluorescence microscopy and Western blotting analyses. We found that in both cortical and medullary TEC lines, LC3 was colocalized with the H2-DM-positive lyso-

somal compartments, in which MHC class II plus class II-associated invariant chain peptides complexes are formed. Furthermore, Downloaded from our analysis of thymic cryosections from 1-day-old mice revealed that LC3 colocalizes with the H2-DM-positive compartments in TECs. These results strongly suggest that the cytoplasmic self-Ags gain access to the H2-DM-positive compartments via the autophagic process in the thymus. The Journal of Immunology, 2009, 183: 7278–7285.

he recognition of self-peptides in the context of MHC are delivered into the MHC class II compartments through the 2 molecules on cortical thymic epithelial cells (TECs) is trans-Golgi network (4); and the autophagic pathway, in which http://www.jimmunol.org/ T critical for double-positive naive T cells to acquire MHC- cytoplasmic and nuclear Ags are sequestered in autophagosomes restriction by positive selection, whereas the recognition of such and delivered into the MHC class II compartments (5–7). After the Ϫ complexes on medullary TECs is crucial for CD4 or CD8-single Ags are digested into peptides, complexes of MHC class II ␣␤ positive T cells to acquire by negative selection heterodimers and the antigenic peptides (referred to as MHC class (1). The formation of the complexes composed of nominal anti- II plus peptide complexes) are formed in the MHC class II genic peptides and MHC molecules in professional APCs, such as compartments. macrophages and dendritic cells, has been extensively studied (2, In in vitro-established TEC lines, endocytosed Ags are delivered 3). However, the mechanisms that underlie the formation of the into the MHC class II compartments via the endocytic pathway by guest on September 29, 2021 complexes composed of self-peptides and MHC molecules in both and endogenous Ags in the endoplasmic reticulum are delivered cortical and medullary TECs are poorly understood. into the MHC class II compartments via the constitutive secretory Concerning the MHC class II-restricted Ag presentation, pro- pathway (8–12). In addition, autophagy in TECs is critically in- fessional APCs mainly use three types of intracellular delivery volved in shaping the CD4-single positive T cell repertoire and pathways for nominal Ags into the MHC class II compartments. establishing central tolerance (13). However, the intersection of These pathways are the endocytic pathway, in which endocytosed autophagy with the MHC class II-restricted Ag-presentation path- Ags are delivered from endosomes or phagosomes into the MHC way in TECs has not been verified. In general, autophagy is the class II compartments (4); the constitutive secretory pathway, in bulk degradation process of cytoplasmic components, including which Ags endogenously synthesized in endoplasmic reticulum organelles, to generate recycled amino acids during stress and star- vation (14). However, thymic autophagy constitutively occurs ir- respective of nutrient conditions (14). In the autophagy process, *Department of Safety Research on Blood and Biological Products, National Institute of Infectious Diseases, Musashimurayama-shi, Tokyo, Japan; †Department of Bio- some of the cytoplasmic components are sequestrated in autopha- chemistry and Cell Biology, National Institute of Infectious Diseases, Shinjuku-ku, gosomes and then delivered into autolysosomes, which are formed Tokyo, Japan; ‡Department of Biochemistry, Juntendo University School of Medi- after the fusion of autophagosomes with lysosomes. Therefore, it cine, Bunkyo-ku, Tokyo, Japan; §Department of Pathology and Immunology, Grad- uate School, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan; and appears that thymic autophagy may be extensively involved in ¶Department of Oral Pathology and Bone Metabolism, Nagasaki University School of lysosomal delivery and the degradation of cytoplasmic Ags in thy- Dentistry, Nagasaki-shi, Japan mic stromal cells, and may intersect the MHC class II presentation Received for publication December 10, 2008. Accepted for publication October 4, 2009. pathway, in which the complexes of thymic peptides with MHC class II molecules are formed. To investigate this possibility, we The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance examined the expression and the localization profiles of LC3 by with 18 U.S.C. Section 1734 solely to indicate this fact. Western blot analysis and confocal microscopy in the in vitro- 1 Address correspondence and reprint requests to Dr. Michiyuki Kasai, Department of established TEC lines, in which both the autophagy process and Safety Research on Blood and Biological Products, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama-shi, Tokyo 208-0011, Japan. E-mail ad- the formation of MHC class II plus peptide complexes were ar- dress: [email protected] rested by pepstatin A, an aspartyl protease inhibitor, and E64d, a 2 Abbreviations used in this paper: TEC, thymic epithelial cell; CLIP, class II-asso- cysteine protease inhibitor. In the presence of these protease in- ciated invariant chain peptide; cTEC, cortical TEC; mTEC, medullary TEC; PNS, hibitors, LC3 was detected in cell lysates from TEC lines and was postnuclear supernatant. colocalized in compartments that contained LAMP-1, H2-DM, and Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 MHC class II plus class II-associated invariant chain peptides

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0804087 The Journal of Immunology 7279

(CLIP) complexes. To address whether the autophagic process had streptavidin for3hatroom temperature. After washing three times with access to the MHC class II presentation pathway in thymus, we TBS, the slides were sealed with PBS(Ϫ)-glycerol (for fluorescence mi- examined the expression and the localization profiles of LC3 on croscopy; Merck) solution (1:9). Thymuses from 1-day-old mice were removed, embedded in optimal thymic cryosections of 1-day-old mice by confocal microscopy. cutting temperature (O.C.T) compound, and frozen in liquid nitrogen. To We found that LC3 colocalized with the compartments in which stain with Abs against cytokeratins, the 5-␮m cryosections from an O.T.C- MHC class II plus CLIP complexes as well as H2-DM molecules embedded thymus were fixed with cold acetone (kept at 4oC) for 10 min resided. These results suggest that the autophagic process may and washed three times with TBS. The cryosections were incubated over- night with 3 ␮g/ml Alexa Fluor 546-conjugated primary Ab in 1% BSA- intersect the MHC class II presentation pathway not only in the in TBS in a refrigerator. After washing three times with TBS, the samples vitro established TEC lines but also in TECs in situ. We discuss the were incubated overnight in a refrigerator with 3 ␮g/ml Alexa Fluor 488- role of the autophagic process in the MHC class II-restricted pre- conjugated secondary Ab or 1 ␮g of secondary Ab labeled by Zenon Alexa sentation of thymic self-peptides in light of the thymic selection of Fluor 488 rabbit IgG labeling reagent in 1% BSA-TBS. After the slides Ϫ T cell repertoires. were washed three times with TBS, they were sealed with PBS( )-glycerin solution (1:9). The sealed samples were imaged with a Zeiss LSM 510 confocal mi- Materials and Methods croscope equipped with a ϫ64 1.4 NA plan-apochromat oil-immersion lens Cell culture or a ϫ40 1.2 NA C- apochromat water-immersion lens (Carl Zeiss). A 488-nm Ar laser line and a 545-nm He-Ne laser line were used for exci- Cortical and medullary TEC lines were established and cultured as previ- tation of Alexa Fluor 488 and Alexa Fluor 543, respectively. Emission ously described (15). wavelengths were separated by band pass (505–530 nm) and long pass Mice (560 nm) filters, respectively. Downloaded from We obtained C57BL/6 mice from Japan SLC and performed all animal Quantification of immunofluorescent pictures experiments according to the guidelines of the Institutional Animal Care Pictures were taken with the LSM510 confocal software and were assem- and Use Committee of Nagasaki University. bled into RGB images with Photoshop (Adobe Systems). Quantification of Abs and reagents red, green, or yellow fluorescing dots in the pictures was performed with the image analysis software MetaMorph (Molecular Devices). The following Abs were purchased and used in this study: rabbit anti-pan cytokeratin Ab (A0575; DakoCytomation), rabbit anti-mouse cytokeratin 5 Isolation of H2-DM-positive vesicles from TEC lines http://www.jimmunol.org/ Ab (Covance), mouse anti-cytokeratin 8 mAb (PROGEN Biotecnik), Magnetic beads (M-450 Dynabeads conjugated with anti-rabbit IgG Ab; mouse mAb to LC3 (clone 4E12; MBL International), and FITC-conju- Dynal Biotech) were conjugated with rabbit anti-H2-DM Ab according to gated rat anti-mouse CD107a (LAMP-1) mAb (BD Pharmingen). Rabbit the manufacturer’s protocol. The TEC lines were cultured in 100-mm antisera against LC3 were raised against thioredoxin-human LC3 and pu- dishes in DMEM with 10% FCS and then incubated in DMEM with 7.5% rified by affinity chromatography on a glutathione-S-transferase-immobi- FCS and without IFN-␥ or with 103 IU/ml IFN-␥ (PeproTech) for 72 h to lized human LC3-Sepharose column (16). We obtained Y3P mouse mAb express MHC class II complexes and their related molecules. The culture against mouse I-Ab (17) from Dr. Saizawa (Nihon Medical College, Tokyo, b,d medium was replaced by fresh medium every 24 h. The TEC lines were Japan), M5–114 rat mAb against mouse I-A (18) from Dr. Uchida (Na- incubated in 7.5% FCS-DMEM or 7.5% FCS-DMEM containing 6 ␮g/ml tional Institute of Infectious Diseases, Tokyo, Japan), In-1.1 mAb against E64d and 6 ␮g/ml pepstatin A for 4 h. After incubation, these TEC lines mouse Ii (19) from Dr. G. J. Ha¨mmerling (German Cancer Research Cen- b were rinsed once with 5 ml of PBS and once with 2.0 ml of 0.25M sucrose by guest on September 29, 2021 ter, Heidelberg, Germany), and 30-2 mAb, which reacts with the I-A solution containing 0.02% EDTA, 1 mM PMSF, and 0.1 mM tosylphenyl- molecules associated with CLIP (20), from Dr. A. Rudensky (Howard alanyl chloromethyl ketone (TPCK). Then the cells were scraped with 1.0 Hughes Medical Institute, University of Washington School of Medicine, ␤ ml of 0.25M sucrose solution containing 0.02% EDTA and protease inhibitors Seattle, WA). Rabbit antisera to H2-DM 2 chain was raised against a (1 mM PMSF, 0.1 mM TPCK, 10 ␮g/ml aprotinin, and 0.1 mM leupeptin). keyhole limpet hemocyanin-coupled synthetic peptide derived from the ␤ The cell suspension was placed into a 15-ml plastic tube and homogenized by cytoplasmic tail of the H2-DM 2 chain (RKSHSSSYTPLPGSTYPEGRH) passing the 0.25M sucrose solution 20 times through a 23-gauge needle at- (21). We purified the antisera with the synthetic peptide-conjugated, ep- tached to a disposable 1-ml plastic syringe. The postnuclear supernatant (PNS) oxy-activated Sepharose 6B and designated it as anti-H2-DM Ab after was obtained by centrifugation at 1000 ϫ g for 10 min. Rabbit anti-H2-DM purification. Abs were labeled with biotin or Alexa Fluor according to the Ab-conjugated Dynabeads M450 (2 ϫ 107 beads) were added to 1 ml of PNS. manufacture’s instructions (Molecular Probes). The mixture was rotated gently for 24 h in a refrigerator. The magnetic bead Staining and immunofluorescence fraction was washed twice with PBS with the aid of a magnet. The fraction was stored at Ϫ80°C until used for SDS-PAGE. TEC lines were cultured on eight-hole heavy Teflon-printed glass slides (Thermo Fisher Scientific) in 10% FCS-DMEM and then incubated in SDS-PAGE and Western blotting DMEM containing 10% FCS and 103 IU/ml IFN-␥ (PeproTech) for 72 h to To analyze the H2-DM-positive vesicles bound to the magnet beads by express MHC class II molecules and their related molecules. The culture SDS-PAGE and Western blotting, 50 ␮l of SDS-sample buffer without medium was replaced with fresh medium every 24 h. After incubation with 2-ME was added to the magnetic bead-fraction, and the proteins included IFN-␥, the TEC lines were incubated with DMEM containing 10% FCS or in the H2-DM-positive vesicles were eluted. We diluted the eluate 1/4 with DMEM containing 10% FCS and 6 ␮g/ml E64d and 6 ␮g/ml pepstatin A H O and determined its protein concentration by the Bradford method for 4 h. The TEC lines were incubated for 10 min in serum-free medium 2 (Bio-Rad) according to the manufacturer’s protocol. We added an equal at room temperature and then fixed with PBS containing 4% paraformal- volume of sample buffer containing 10% (v/v) of 2-ME to each sample. dehyde (pH 7.0) for 10 min at room temperature. Slides were treated with Proteins (1 ␮g), with or without boiling, were separated on a 12.5% poly- 0.1 M glycine (adjusted to pH 7.0 with 1 M Tris) to block the remaining acrylamide gel. For Western blotting, proteins were transferred from poly- paraformaldehyde activity and then washed three times with TBS for 10 acrylamide gels to polyvinylidene difluoride membranes (Immobilon-P min. To stain the TEC lines with Abs against intracellular compartments, transfer membrane; Millipore). The membranes were blocked with TBS the cells were permeabilized with TBS containing 0.05% saponin for 10 containing 5% BSA and probed with 1–2 ␮g/ml the primary Ab in TBS min after fixation and then washed three times with TBS. The TEC lines containing 1% BSA and 0.02% NaN . The membranes were washed once were incubated overnight with 1–3 ␮g/ml of the primary Ab in 1% BSA- 3 with TBS containing 0.02% Tween 20 and twice with TBS. Then primary TBS in a refrigerator. After washing three times with TBS, the samples Abs were probed with HRP-conjugated anti-Ig Ab in TBS containing 1% were incubated with 1 ␮g/ml Alexa Fluor 546-conjugated anti-IgG Ab in BSA, followed by washing once with TBS containing 0.02% Tween 20 and 1% BSA-TBS for3hatroom temperature. After washing three times with twice with TBS. Signals were detected by using super signal chemilumi- TBS, the samples were incubated overnight in a refrigerator with 1–3 nescent substrate (Pierce). ␮g/ml biotin-conjugated secondary Ab in 1% BSA-TBS or 3 ␮g Alexa Fluor 488-conjugated secondary Ab or 1 ␮g of secondary Ab labeled by Statistics Zenon Alexa Fluor 488 IgG labeling reagent (Molecular Probes). After incubation with biotin-conjugated Ab, the samples were washed with TBS Values of p were calculated by the Student’s t test (Microsoft Excel software) three times and then incubated with 1 ␮g/ml Alexa Fluor 488-conjugated with two-tailed distribution and two-sample unequal variance parameters. 7280 THYMIC AUTOPHAGY IN THE MHC CLASS II-ANTIGEN PRESENTATION Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 1. Detection of LC3 in the lysates from TEC lines and colocalization of LC3 with the H2-DM-positive lysosomal compartments in TEC lines. A, Cellular lysates were prepared from both TEC lines (cTEC and mTEC) cultured in DMEM containing 7.5% FCS and 103 IU/ml IFN-␥ for 3 days and then analyzed by Western blotting and SDS-PAGE. On the Western blot, the rabbit anti-LC3 Ab detected a membrane-bound form of LC3, i.e., LC3-II, which is ϳ16 kDa in molecular mass. The amounts of LC3-II were increased upon incubation of both TEC lines in the culture medium containing 6 ␮g/ml E64d and 6 ␮g/ml pepstatin A (ϩPI, with protease inhibitors). B, The cTEC line (a–c) and the mTEC line (d–f) were cultured in DMEM containing 7.5% FCS and 103 IU/ml IFN-␥ for 3 days. They were then incubated for4hinthemedium alone (ϪPI, without protease inhibitors; b and e) or with 6 ␮g/ml E64d and 6 ␮g/ml pepstatin A (ϩPI; c and f). They were fixed and stained with rabbit anti-LC3 Ab followed by Alexa Fluor 543-conjugated anti-rabbit IgGs. Negative controls (a and d) indicate TEC lines incubated with protease inhibitors and fixed and stained with normal rabbit serum followed by Alexa Fluor 543-conjugated anti-rabbit IgGs. White bars are 10 ␮m. C, After the culture of the both TEC lines in the above condition, TEC lines were fixed and stained with rabbit anti-LC3 Ab followed by Alexa Fluor 543-conjugated anti-rabbit IgGs. The amounts of red-color compartments in the immunofluo- rescent pictures were quantified by the image analysis software MetaMorph (Molecular Devices). The average intensity indicates the amount of red-color compartments in the immunofluorescent picture. Data are representative of five independent immunofluorescent experiments. Error bars indicate SDs. Values of p were obtained by Student’s t test. D–I, The TEC lines were cultured in DMEM containing 7.5% FCS and 103 IU/ml IFN-␥ for 3 days and then incubated for4hinthemedium containing 6 ␮g/ml E64d and 6 ␮g/ml pepstatin A. Both the cTEC line (D, F, and H) and the mTEC line (E, G, and I) were fixed and stained with rabbit anti-LC3 Ab followed by Alexa Fluor 543-conjugated anti-rabbit IgGs (LC3; D–I). After the staining with the primary Ab, both TEC lines were further stained with FITC-conjugated anti-LAMP-1 mAb (LAMP-1; D and E), with a biotin-conjugated rabbit anti-H2-DM Ab (H2-DM; F and G) or a biotin-conjugated Y3P mAb (MHC class II; H and I). The biotin-conjugated Abs were followed by Alexa Fluor 488-conjugated streptavidin. White bars are 10 ␮m.

Results ized in H2-DM-positive compartments and on the plasma mem- Colocalization of LC3, MHC class II, and MHC class II-related brane. Ii chains and the MHC class II plus CLIP complexes are molecules in TEC lines also localized in the H2-DM-compartments (10–12). To determine Both the cortical TEC (cTEC) and medullary TEC (mTEC) lines whether the autophagic process in both TEC lines is involved in express MHC class II as well as MHC class II-related molecules, the MHC class II-Ag presentation pathway, we examined the ex- such as Ii chains and H2-DM molecules, upon stimulation with pression levels and localization profiles of LC3 by Western blot IFN-␥ (15). In both TEC lines, MHC class II molecules are local- analysis and immunofluorescence staining. In the cell lysates from The Journal of Immunology 7281

Table I. Quantification of the colocalization of LC3-positive cytoplasm of the treated cells (16). Therefore, the increased levels compartments with LAMP-1, H2-DM, or MHC class II molecules in of LC3-II in lysates of both TEC lines may be due to the accu- a TEC lines mulation of the autophagic compartments in the cytoplasm. Immunostaining analyses revealed that the intracellular com- cTEC mTEC (yellow:red (%) Ϯ SD) (yellow:red (%) Ϯ SD) partments bound with LC3-II (referred to as LC3-positve compart- ments) were clearly probed with the anti-LC3 Ab in the cytoplasm LC3 colocalized with: of both TEC lines stimulated with IFN-␥ (Fig. 1B, b and e). Fur- Ϯ Ϯ LAMP-1 49.7 5.82 31.7 3.71 thermore, when both TEC lines were precultured in medium con- H2-DM 38.9 Ϯ 16.2 39.9 Ϯ 8.62 MHC class II 42.1 Ϯ 12.7 43.4 Ϯ 11.8 taining the protease inhibitors, pepstatin A and E64d (referred to as the protease inhibitors hereafter), the amounts of LC3-positve a Colocalization was quantified according to Materials and Methods. Results are representative of at least five independent experiments. compartments were increased in the cytoplasm of both the cTEC and the mTEC lines (Fig. 1B, c and f). When the amounts of LC3- positve compartments shown in Fig. 1B were quantified, they were both TEC lines stimulated with IFN-␥, the rabbit anti-LC3 Ab increased with statistical significance in the cytoplasm of the both detected LC3 as a membrane-bound form, i.e., LC3-II, which is TEC lines after incubation with the protease inhibitors (Fig. 1C). ϳ16 kDa in molecular mass (Fig. 1A) (16). This Ab hardly de- These results suggested that, in the presence of protease inhib- tected a cytosolic form of LC3, i.e., LC3-I, which is ϳ18 kDa in itors, the increased amount of LC3-II in the cell lysates is attrib- molecular mass. In fact, this Ab may be more likely to react to uted to the accumulation of the LC3-positive compartments in the LC3-II than to LC3-I in HeLa cell and A431, which are originated cytoplasm of both TEC lines. Downloaded from from epithelium; however, this Ab reacts to LC3-I as well as In the same culture condition, ϳ50% of the LC3-positive com- LC3-II in HEK 293 cell (16). The expression level of LC3-II was partments in the cTEC line and 32% of the LC3-positive compart- apparently increased when the TEC lines were precultured in the ments in the mTEC line were costained with anti-LAMP-1 Ab presence of both 6 ␮g/ml pepstatin A (an aspartyl protease inhib- (Table I and Fig. 1, D–E), suggesting that the autophagic process itor) and 6 ␮g/ml E64d for 4 h. The combination of pepstatin A was arrested at the autolysosome stage. Furthermore, ϳ40% of the and E64d arrests the autophagic process at the stage of autopha- LC3-positive compartments in both TEC lines were costained with http://www.jimmunol.org/ gosomes or autolysosomes; thus, these vesicles accumulate in the both anti-H2-DM Ab (Table I and Fig. 1, F–G) and with anti-MHC by guest on September 29, 2021

FIGURE 2. Colocalization of LC3 with the compartments in which MHC class II plus CLIP complexes reside in TEC lines. A–D, The cTEC lines and the mTEC lines were stimulated with 103 IU/ml IFN-␥ for 3 days. The TEC lines were incubated for4hinthemedium alone (A, cTEC, without protease inhibitor (ϪPI) and C, mTEC, ϪPI) or with 6 ␮g/ml E64d and 6 ␮g/ml pepstatin A (B, cTEC, with protease inhibitor (ϩPI) and D, mTEC, ϩPI) and then fixed. The TEC lines were first stained with rabbit anti-LC3 Ab followed by Alexa Fluor 543-conjugated anti-rabbit IgGs (red, LC3) and next stained with a biotin-conjugated 30-2 mAb against MHC class II plus CLIP complexes followed by Alexa Fluor 488-conjugated streptavidin (green, 30-2). The merged profiles (yellow, merge) are set in the right side of each figure. White bars are 10 ␮m. E, The amount of red, green, or yellow color-compartments in the immunofluorescent pictures was respectively quantified by the image analysis software, MetaMorph, as described in Materials and Methods. The average intensity indicates the amount of each color compartment in the immunofluorescent picture. Data are representative of three independent immunofluorescent experiments. Error bars indicate SDs. Values of p were obtained by Student’s t test. F, In the presence (ϩPI) or the absence (ϪPI) of the protease inhibitors, the amounts of LC3-positive compartments (shown by red columns in E) and those of the colocalization of 30-2-positive compartments with LC3-positive compartments (shown by yellow columns in E) were quantified according to Materials and Methods. The ratios of the yellow compartment to the red compartment are shown. Data are representative of three independent immunofluorescent experiences. Error bars indicate SDs. Values of p were obtained by Student’s t test. 7282 THYMIC AUTOPHAGY IN THE MHC CLASS II-ANTIGEN PRESENTATION class II Ab, respectively (Table I and Fig. 1, H–I). Taken together, in both TEC lines, LC3-II seemed to be colocalized in the lyso- somal compartments that contain both MHC class II and H2-DM molecules. However, there was a difference in the ratio of the colocalization of LC3-positive compartments with LAMP-1 be- tween in the cTEC line and the mTEC line, suggesting that the nature of the autolysosome in the cTEC line may be subtly differ- ent from that in the mTEC line (10–12). The combination of pepstatin A and E64d not only arrests the autophagic process by accumulation of autophagosomes/autoly- sosomes in the cytoplasm, but also affects both the generation of CLIP from Ii chains and the dissociation of CLIP from MHC class II ␣␤ heterodimers (22–24). Thus, the MHC class II plus CLIP complexes accumulate in the H2-DM-positive compart- ments. In the next experiment, TEC lines were cultured with or without the protease inhibitors and then stained with anti-LC3 FIGURE 3. Detection of LC3 in the H2-DM-positive vesicles isolated Ab followed by staining with 30-2 mAb that reacts to the MHC from TEC lines. The TEC lines were stimulated with 103 IU/ml IFN-␥ for class II plus CLIP complexes (20). In the cultures without the 3 days and then incubated for4hinthepresence of 6 ␮g/ml E64d and 6 Downloaded from protease inhibitors, some of the LC3-positive and the 30-2-pos- ␮g/ml pepstatin A. The H2-DM-positive vesicles were isolated from the itive compartments were detected in the cytoplasm of both TEC PNS of both TEC lines by using magnetic beads coated with anti-H2-DM lines (Figure 2, A and C); however, the distribution of the 30- Ab. Proteins eluted from the beads with SDS-sample buffer were analyzed 2-positive compartments were uneven. Both TEC lines have with Western blotting followed by SDS-PAGE in the boiling (A, C, and D) been established via several cloning procedures, but when both or non-boiling (B) conditions. A, Blots were probed with rabbit anti- H2-DM Ab. Arrowhead indicates H2-DM molecules (DM). B, Blots were

TEC lines are placed on slide glass, they may adhere to the probed with rat M5–114 mAb. One of the arrowheads indicates the com- http://www.jimmunol.org/ glass and start growing in its own manner. Then the cell cycle plexes composed of MHC class II heterodimers and partially digested of both the TEC lines may not synchronize. In addition, when forms of Ii chains (p70), ϳ10 kDa. The other arrowhead indicates het- the TEC lines are incubated in the medium containing IFN-␥, erodimer complexes (HD). C, Blots were probed with rat anti-Ii chain the expression of MHC class II and H2-DM molecules in mAb, In-1.1. Two arrowheads indicate 35 (Ii-35) and 30 (Ii-30) kDa of Ii both the TEC lines may not synchronize. The above un-syn- chain-isoforms, respectively. D, Blots were probed with rabbit anti-LC3 chronizing cell cycle and expression may cause the uneven dis- Ab. Arrowhead indicates LC3-II, i.e., membrane-bound form of LC3. m, medullary TEC; c, cortical TEC. tribution of the 30-2-positive compartments. When the staining profiles with anti-LC3 Ab were merged to those with 30-2 mAb, a few of the yellow compartments, in which the LC3-positive by guest on September 29, 2021 compartments were colocalized with the 30-2-positive compart- Western blot analysis of the H2-DM vesicles isolated from ments, were found in the cytoplasm of both TEC lines (Fig. 2, TEC lines A and C). Approximately 10% of LC3-positive compartments in the cTEC line and 7% of those in the mTEC line were colocal- To confirm that LC3-positive compartments were docked to the ized with the 30-2-positive compartments (Fig. 2F). In the cul- compartments containing H2-DM molecules, we isolated H2-DM- tures with the protease inhibitors, the amounts of the 30-2- and positive vesicles from the PNS of both TEC lines after incubation LC3-positive compartments were increased in the cytoplasm of with the protease inhibitors. The isolated vesicles were then sub- jected to SDS-PAGE and Western blotting analyses. We detected both TEC lines (Fig. 2, B and D), although the distribution of H2-DM molecules in the isolated vesicles from both TEC lines the 30-2-positive compartments was uneven. After the addition (Fig. 3A). LAMP-1, a typical lysosomal marker, and a series of of the protease inhibitors, quantification analysis of red and soluble endosomal/lysosomal proteases, i.e., cathepsins-B, -D, -L, green color-compartments in the pictures of both the TEC lines and -S, were detected in the same manner as in our previous re- clearly showed that not only the amount of LC3-positive com- ports (Ref. 15 and data not shown). Both MHC class II het- partments but also the amount of the 30-2-positive compart- erodimers and P70 complexes that were composed of the het- ments were increased in the cytoplasm of both TEC lines after erodimers and partially digested Ii chains (10–12) were probed culture with the protease inhibitors (Fig. 2E). When the staining with M5–114 (Fig. 3B), and two intact isoforms of Ii chains (35 profiles with 30-2 mAb were merged to those with anti-LC3 Ab, kDa and 31 kDa) were probed with anti-Ii chain mAb (In-1.1; Fig. it was observed that the amount of the yellow compartments 3C). It is noteworthy that LC3-II was detected on this blotting (Fig. increased in the cytoplasm of both TEC lines (Fig. 2, B and D). 3D). These results, together with the results in Figs. 1 and 2, may When the amounts of yellow color-compartments in the imply that the combined protease inhibitors not only affect the merged-pictures were quantified, the amount of yellow color- degradation of Ii chains but also accumulate the LC3-II molecules compartments increased (Fig. 2E). In this condition, ϳ20% of in the H2-DM-positive vesicles. LC3-positive compartments were colocalized with the 30-2- positive compartments in the cTEC lines as well as in mTEC Colocalization of LC3-II molecules and MHC class II plus CLIP lines (Fig. 2F). These results indicated that the considerable complexes in thymus amounts of the 30-2-positive compartments in both TEC lines To determine whether the autophagic process participates in the pre- were colocalized with the LC3-positive compartments. These sentation of cytoplasmic self-Ags not only in the in vitro-established results also suggested that the LC3-positive compartments TEC lines but also in the thymus in situ, we analyzed mouse thymic could fuse to the H2-DM-positive compartments in which MHC cryosections that were immunostained. Thymic autophagy occurs class II complexes were formed. even without starvation. In addition, the autophagy is immediately The Journal of Immunology 7283 Downloaded from

FIGURE 4. Colocalization of LC3 with the H2-DM-positive compartments in which MHC class II plus CLIP complexes reside in thymus in situ.

Cryosections prepared from the 1-day-old thymuses were fixed with cold acetone (4°C) and then stained with mouse anti-LC3 mAb (4E12) conjugated with http://www.jimmunol.org/ Alexa Fluor 543. A, After staining with anti-LC3 mAb conjugated with Alexa Fluor 543, the cryosections were stained with rabbit anti-pan cytokeratin conjugated by Zenon Alexa Fluor 488 rabbit IgG labeling reagent. B, These cryosections were single-stained with mouse anti-LC3 mAb conjugated with Alexa Fluor 543. C–H, After staining with anti-LC3 mAb conjugated with Alexa Fluor 543, the cryosections were probed with one of the following Abs: a rabbit anti-cytokeratin-5 Ab conjugated by Zenon Alexa Fluor 488 rabbit IgG labeling reagent (C), a mouse anti-cytokeratin-8 mAb conjugated by Zenon Alexa Fluor 488 mouse IgG labeling reagent (D), FITC-conjugated rat anti-mouse LAMP-1 mAb (E), a rabbit anti-H2-DM Ab conjugated with Alexa Fluor 488 (F), an Alexa Fluor 488-conjugated mouse Y3P mAb against MHC class II molecules (G), or an Alexa Fluor 488-conjugated mouse 30-2 mAb against MHC class II plus CLIP complexes (H). White bars are 20 ␮minA, C, and D; and 10 ␮minB and E–H.

induced in various tissues after birth and is then involved in the deg- would be autolysosomes. The LC3-positive compartments were by guest on September 29, 2021 radation of self-proteins and the production of amino acids (14). Thus, costained with anti-MHC class II Ab (Fig. 4G); however, the it is plausible that thymic autophagy might occur with a greater fre- ratio of colocalization with the LC3 was around 20%. The anti- quency shortly after birth than in the constitutive condition. We there- MHC class II Ab used in this staining reacts to MHC class II fore analyzed thymuses taken from 1-day-old mice. plus peptide complexes generated from MHC class II plus Ii LC3-II was detected across the cryosections of the 1-day-old complexes. Therefore, the lower colocalization ratio suggests thymus and colocalized with pan-cytokeratin-positive stromal that autophagosomes fused with compartments transporting cells (Fig. 4A) and in a vesicle-like manner in the cytoplasm of MHC class II plus Ii complexes would progress via autolyso- the stromal cells (Fig. 4B). The LC3-positive compartments in somes to the LC3-positive compartments colocalized with thymic cryosections resided in some of the medullary epithelial MHC class II plus peptide complexes. In contrast, ϳ60% of the cells that were stained with anti-cytokeratin 5 Ab (Fig. 4C) and LC3-positive compartments were stained with 30-2 mAb (Fig. in some of the cortical epithelial cells that were stained with 4H and Table II), suggesting that the LC3-positive compart- anti-cytokeratin 8 Ab (Fig. 4D). Quantification analyses (Table ments colocalized with MHC class II plus CLIP complexes II) indicated that ϳ40% of the LC3-positive compartments in would be generated immediately after the fusion of autophago- the thymic cryosections were costained with anti-LAMP-1 Ab somes with compartments transporting MHC class II plus Ii (Fig. 4E) and anti-H2-DM Ab (Fig. 4F), suggesting that, similar complexes. Taken together, these results indicate that the to the TEC lines as shown above, most of these compartments autophagic compartments in thymus in situ could access the H2-DM positive compartments in which the MHC class II plus peptide complexes are formed. Table II. Quantification of the colocalization of LC3-positive compartments with LAMP-1, H2-DM, MHC class II, or MHC class II Discussion plus CLIP molecules in thymic cryosectionsa The presentation of cytoplasmic self-Ags by TECs to developing/ immature T cells is critical for the generation of T cell reper- Thymic cryosections (yellow:red (%) Ϯ SD) toires (1). Although the presentation process of exogenous Ags to mature T cells performed by professional APCs has been LC3 colocalized with: extensively investigated, mechanisms underlying the presenta- LAMP-1 37.0 Ϯ 9.33 H2-DM 38.7 Ϯ 3.83 tion of endogenous Ags, including self-derived cytoplasmic MHC class II 21.6 Ϯ 3.16 Ags, by TECs are almost unknown. The present study intro- MHC class II plus CLIP 59.6 Ϯ 17.9 duces the intriguing possibility that the autophagic process con- a Colocalization was quantified according to Materials and Methods. Results are tributes to the MHC class II presentation pathway for cytoplas- representative of eight independent experiments. mic self-Ags in TECs. 7284 THYMIC AUTOPHAGY IN THE MHC CLASS II-ANTIGEN PRESENTATION

We demonstrated that LC3-II, a membrane marker of autopha- the thymic self-Ags promiscuously expressed by autoimmune reg- gosomes, is localized in the H2-DM-positive compartments in ulator and transport them into the MHC class II compartments in which MHC class II plus CLIP complexes are formed (Fig. 1G). which lysosomal enzymes may degrade the thymic self-Ags and The combination of two lysosomal protease inhibitors, pepstatin A generate the effective repertoire of thymic self-peptides for nega- and E64d, arrests not only the autophagic process at the stage of tive selection. In any case, it is suggested that the autophagic pro- autolysosomes (16), but also the formation of MHC class II plus cess in the thymus may participate in producing the various spectra peptide complexes at the formative stage of the MHC class II plus of self-peptides for thymic selection. CLIP complex (21–23). E64d, a potent cysteine protease, may af- In conclusion, we have shown that LC3-II molecules are in fact fect the proteolytic activity of cathepsins L and S to cleave Ii colocalized with the H2-DM-positive compartments in which chains (4, 22–24). Therefore, the use of these protease inhibitors MHC class II complexes are formed, not only in the in vitro- facilitates the intersection of the autophagic process in the MHC established TEC lines but also in thymic cryosections. This finding class II Ag-presentation pathway. implies that autophagy could intersect the MHC class II presenta- To determine whether the autophagic process could access the tion pathway and consequently play an important role in present- MHC class II compartments in the thymus in situ, we used immu- ing self-Ags to developing/immature T cells in the thymus. Our nofluorescent probes to analyze cryosections of thymuses from conclusion is concordant with the report by Nedjic et al. in which 1-day-old mice. In newborn mice, autophagy is actively induced they addressed the role of autophagy in T cell repertoire selection and helps to rescue neonates from severe starvation (25). There- in the thymus by using Atg5-deficeint mice that are deprived of an fore, thymic autophagy may also occur with an increased fre- essential component of autophagosome formation (13). In the quency during this period. As expected, LC3-II was expressed present study, the intracellular localization profiles of the autoph- Downloaded from across the cryosection of the neonate thymus (Fig. 4A), suggesting agic marker, i.e., LC3-II, in in vitro-established TECs as well as in that the autophagic process occurs in both the cortical region and the thymus in situ, confirms the involvement of autophagy in the the medullary region. In fact, some LC3-II molecules were colo- presentation of the thymic self-Ags. The degradation of the thymic calized with the thymic stromal cells that are positive for cytoker- self-Ags via the proteosome processing system and the autophagy- atin 5 (Fig. 4C) as well as cytokeratin 8 (Fig. 4D). Moreover, in mediated lysosomal processing system produced a variety of com-

some thymic stromal cells, LC3-II was clearly colocalized with the plexes composed of various thymic self-peptides and MHC mol- http://www.jimmunol.org/ compartments containing LAMP-1, H2-DM molecules, MHC ecules. These complexes may lead to efficient positive selection of class II molecules, and MHC class II plus CLIP complexes (Figs. double-positive T cells in the thymic cortex as well as negative 4, E–H). These results indicate that the autophagic process in the selection of self-reactive, single-positive T cells in the thymic me- thymus may access the endosomal/lysosomal compartments in dulla. The autophagic process may play a supporting role or even which MHC class II plus peptide complexes are formed. a crucial role in the presentation of self-Ags in the thymus to shape We previously demonstrated that cTEC lines mainly present in- the T cell repertoires. tracellular Ags via the constitutive secretory pathway but do not present the extracellular Ags via the endocytic pathway, whereas

Acknowledgments by guest on September 29, 2021 mTEC lines present the extracellular Ags via the endocytic path- We thank Drs. Y. Takahama and M. Matsumoto for valuable suggestions way and the intracellular Ags via the constitutive secretory path- and for reviewing the manuscript. way (8–12). In the present study, we found that the autophagic process could intersect the MHC class II presentation pathway in both in vitro-established TEC lines and the thymus in situ. There- Disclosures fore, we speculate that the thymic self-Ags in cTECs could be The authors have no financial conflict of interest. delivered into the MHC class II compartments via the autophagic pathway as well as the constitutive secretory pathway. In the References mTECs, thymic self-Ags could be delivered into the MHC class II 1. Starr, T. K., S. C. Jameson, and K. A. Hogquist. 2003. Positive and negative selection of T cells. Annu. Rev. Immunol. 21: 139–176. compartments via the following three pathways: the autophagic 2. Cresswell, P., A. L. Ackerman, A. Giodini, D. R. Peaper, and P. A. Wearsch. pathway, the endocytic pathway, and the constitutive secretory 2005. Mechanisms of MHC class I-restricted antigen processing and cross-pre- pathway. sentation. Immunol. Rev. 207: 145–157. 3. Villadangos, J. A., P. Schnorrer, and N. S. Wilson. 2005. Control of MHC class Takahama et al. suggested that the unique characteristics of pro- II antigen presentation in dendritic cells: a balance between creative and destruc- tein degradation in the cortical thymus are critical for positive tive forces. Immunol. Rev. 207: 191–205. selection (26). Nakagawa et al. demonstrated that cathepsin L, a 4. Trombetta, E. S., and I. Mellman. 2005. Cell biology of antigen processing in vitro and in vivo. Annu. Rev. Immunol. 23: 975–1028. lysosomal cysteine protease expressed by cTECs, is required for 5. Dengjel, J., O. Schoor, R. Fischer, M. Reich, M. Kraus, M. Mu¨ller, the generation of CD4-single positive T cells in the context of K. Kreymborg, F. Altenberend, J. Brandenburg, H. Kalbacher, et al. 2005. Au- positive selection involved with MHC class II (22). We previously tophagy promotes MHC class II presentation of peptides from intracellular source proteins. Proc. Natl. Acad. Sci. USA 102: 7922–7927. verified that cathepsin L is produced both in vitro in the cTEC line 6. Paludan, C., D. Schmid, M. Landthaler, M. Vockerodt, D. Kube, T. Tuschl, and and in situ in the thymic cortex (27). Therefore, in cTECs, the C. Mu¨nz. 2005. Endogenous MHC class II processing of a viral nuclear antigen autophagic process, in conjunction with the constitutive secretory after autophagy. Science 307: 593–596. 7. Schmid, D., M. Pypaert, and C. Mu¨nz. 2007. Antigen-loading compartments for pathway, could contribute to the efficient transport of thymic self- major histocompatibility complex class II molecules continuously receive input Ags into the MHC class II compartments in which cathepsin L from autophagosomes. Immunity 26: 1–14. could be involved in degradation of the thymic self-Ags and gen- 8. Mizuochi, T., M. Kasai, M. T. Kokuho, T. Kakiuchi, and K. Hirokawa. 1992. Medullary but not cortical thymic epithelial cells present soluble antigens to eration of the thymic self-peptides with the unique characteristics helper T cells. J. Exp. Med. 175: 1601–1605. for positive selection. In contrast, promiscuous expression of the 9. Oukka, M., P. Andre, P. Turmel, N. Besnard, V. Angevin, L. Karlsson, P. L. Trans, D. Charron, B. Bihain, K. Kosmatopoulos, and V. Lotteau. 1997. thymic self-Ags by activity in mTECs may Selectivity of the major histocompatibility complex class II presentation pathway be essential for negatively selecting autoreactive single-positive T of cortical thymic epithelial cell lines. Eur. J. Immunol. 27: 855–859. cells in the medulla (28). Therefore, in mTECs, the autophagic 10. Kasai, M., K. Hirokawa, K. Kajino, K. Ogasawara, M. Tatsumi, E. Hermal, J. J. Monaco, and T. Mizuochi. 1996. Difference in antigen presentation pathways pathway, in conjunction with the endocytic and constitutive secre- between cortical and medullary thymic epithelial cells. Eur. J. Immunol. 26: tory pathways, may contribute to the comprehensive collection of 2101–2107. The Journal of Immunology 7285

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