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Functional Early Endosomes Are Required for Maturation of Major Histocompatibility Complex Class II Molecules in Human B Lymphoblastoid Cells

1999; Elsevier BV; Volume: 274; Issue: 25 Linguagem: Inglês

10.1074/jbc.274.25.18049

ISSN

1083-351X

Autores

L Pond, Colin Watts,

Tópico(s)

RNA Interference and Gene Delivery

Resumo

Major histocompatibility complex (MHC) class II molecules are targeted together with their invariant chain (Ii) chaperone from the secretory pathway to the endocytic pathway. Within the endosome/lysosome system, Ii must be degraded to enable peptide capture by MHC class II molecules. It remains controversial exactly which route or routes MHC class II/Ii complexes take to reach the sites of Ii processing and peptide loading. We have asked whether early endosomes are required for successful maturation of MHC class II molecules by using an in situ peroxidase/diaminobenzidine compartment ablation technique. Cells whose early endosomes were selectively ablated using transferrin-horseradish peroxidase conjugates fail to mature their newly synthesized MHC class II molecules. We show that whereas transport of secretory Ig through the secretory pathway is virtually normal in the ablated cells, newly synthesized MHC class II/Ii complexes never reach compartments capable of processing Ii. These results strongly suggest that the transport of the bulk of newly synthesized MHC class II molecules through early endosomes is obligatory and that direct input into later endosomes/lysosomes does not take place. Major histocompatibility complex (MHC) class II molecules are targeted together with their invariant chain (Ii) chaperone from the secretory pathway to the endocytic pathway. Within the endosome/lysosome system, Ii must be degraded to enable peptide capture by MHC class II molecules. It remains controversial exactly which route or routes MHC class II/Ii complexes take to reach the sites of Ii processing and peptide loading. We have asked whether early endosomes are required for successful maturation of MHC class II molecules by using an in situ peroxidase/diaminobenzidine compartment ablation technique. Cells whose early endosomes were selectively ablated using transferrin-horseradish peroxidase conjugates fail to mature their newly synthesized MHC class II molecules. We show that whereas transport of secretory Ig through the secretory pathway is virtually normal in the ablated cells, newly synthesized MHC class II/Ii complexes never reach compartments capable of processing Ii. These results strongly suggest that the transport of the bulk of newly synthesized MHC class II molecules through early endosomes is obligatory and that direct input into later endosomes/lysosomes does not take place. MHC 1The abbreviations used are: MHC, major histocompatibility complex; EBV, Epstein-Barr virus; DHB, Dulbecco's modified Eagle's medium, 25 mm Hepes, pH 7.5, and 5 mg/ml bovine serum albumin; Tf-HRP, transferrin-horseradish peroxidase; HRP, horseradish peroxidase; DAB, diaminobenzidine; mAb, monoclonal antibody. class II molecules are peptide-binding glycoproteins that display a highly diverse set of peptides on the surface of B lymphocytes, dendritic cells, and macrophages. Specific peptide/MHC class II complexes are recognized by CD4+ T cells, for example, to trigger protective immune responses. Extensive studies on the biosynthesis of MHC class II molecules have established that unlike MHC class I molecules, they intersect the endocytic pathway before their appearance on the surface (Refs. 1Cresswell P. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 8188-8192Crossref PubMed Scopus (124) Google Scholar and 2Neefjes J.J. Stollorz V. Peters P.J. Geuze H.J. Ploegh H.L. Cell. 1990; 61: 171-183Abstract Full Text PDF PubMed Scopus (369) Google Scholar; reviewed in Refs. 3Germain R.N. Margulies D.H. Annu. Rev. Immunol. 1993; 11: 403-450Crossref PubMed Scopus (954) Google Scholar, 4Wolf P.R. Ploegh H.L. Annu. Rev. Cell Dev. Biol. 1995; 11: 267-306Crossref PubMed Scopus (252) Google Scholar, 5Watts C. Annu. Rev. Immunol. 1997; 15: 821-850Crossref PubMed Scopus (660) Google Scholar). This diversion is essential (a) to enable proteolytic removal of the invariant chain (Ii, a specialized chaperone), and (b) to ensure admixing of MHC class II molecules with processed peptide material from endocytosed exogenous antigens. It is well established that targeting motifs within Ii are responsible for the delivery of MHC class II/Ii complexes to the endocytic pathway (6Bakke O. Dobberstein B. Cell. 1990; 63: 707-716Abstract Full Text PDF PubMed Scopus (508) Google Scholar, 7Lotteau V. Teyton L. Peleraux A. Nilsson T. Karlsson L. Schmid S.L. Quaranta V. Peterson P.A. Nature. 1990; 348: 600-605Crossref PubMed Scopus (447) Google Scholar, 8Odorizzi C.G. Trowbridge I.S. Xue L. Hopkins C.R. Davis C.D. Collawn J.F. J. Cell Biol. 1994; 126: 317-330Crossref PubMed Scopus (160) Google Scholar, 9Pond L. Kuhn L.A. Teyton L. Schutze M.P. Tainer J.A. Jackson M.R. Peterson P.A. J. Biol. Chem. 1995; 270: 19989-19997Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 10Pieters J. Bakke O. Dobberstein B. J. Cell Sci. 1993; 106: 831-846Crossref PubMed Google Scholar), but it has been more difficult to resolve the precise route taken from the Golgi apparatus to the endosome system. Much of the data on this pathway has been obtained by studying human B lymphoblastoid cells that are potent antigen-presenting cells and express high levels of MHC class II molecules. Earlier studies in these cells have shown that most MHC class II molecules that have assembled with peptide are found in late endosomes or lysosomes before their expression on the cell surface (11Peters P.J. Neefjes J.J. Oorschot V. Ploegh H.L. Geuze H.J. Nature. 1991; 349: 669-676Crossref PubMed Scopus (555) Google Scholar, 12West M.A. Lucocq J.M. Watts C. Nature. 1994; 369: 147-151Crossref PubMed Scopus (322) Google Scholar, 13Peters P.J. Raposo G. Neefjes J.J. Oorschot V. Leijendekker R.L. Geuze H.J. Ploegh H.L. J. Exp. Med. 1995; 182: 325-334Crossref PubMed Scopus (117) Google Scholar). This observation suggested that transport might occur directly from the Golgi network to the late endosomes/MHC class II compartments, and several studies appear to support this possibility (14Benaroch P. Yilla M. Raposo G. Ito K. Miwa K. Geuze H.J. Ploegh H.L. EMBO J. 1995; 14: 37-49Crossref PubMed Scopus (153) Google Scholar, 15Morton P.A. Zacheis M.L. Giacoletto K.S. Manning J.A. Schwartz B.D. J. Immunol. 1995; 154: 137-149PubMed Google Scholar, 16Glickman J.N. Morton P.A. Slot J.W. Kornfeld S. Geuze H.J. J. Cell Biol. 1996; 132: 769-785Crossref PubMed Scopus (59) Google Scholar). However, reports that some Ii could be detected on the surface of antigen-presenting cells (17Wraight C.J. van Endert P. Moller P. Lipp J. Ling N.R. MacLennan I.C. Koch N. Moldenhauer G. J. Biol. Chem. 1990; 265: 5787-5792Abstract Full Text PDF PubMed Google Scholar) were followed by a direct biochemical demonstration of the transient appearance of newly synthesized class II/Ii complexes on the cell surface (18Koch N. Moldenhauer G. Hofmann W.J. Moller P. J. Immunol. 1991; 147: 2643-2651PubMed Google Scholar, 19Roche P.A. Teletski C.L. Stang E. Bakke O. Long E.O. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8581-8585Crossref PubMed Scopus (189) Google Scholar, 20Warmerdam P.A. Long E.O. Roche P.A. J. Cell Biol. 1996; 133: 281-291Crossref PubMed Scopus (63) Google Scholar). In addition, a recent reappraisal of the steady-state distribution of Ii, MHC class II molecules, and other markers in human B lymphoblastoid cells indicates the presence of class II/Ii complexes in early endosomes (21Kleijmeer M.J. Morkowski S. Griffith J.M. Rudensky A.Y. Geuze H.J. J. Cell Biol. 1997; 139: 639-649Crossref PubMed Scopus (198) Google Scholar). In murine B cells, there is also evidence that newly synthesized MHC class II molecules are found in early endosomes (22Castellino F. Germain R.N. Immunity. 1995; 2: 73-88Abstract Full Text PDF PubMed Scopus (188) Google Scholar, 23Brachet V. Raposo G. Amigorena S. Mellman I. J. Cell Biol. 1997; 137: 51-65Crossref PubMed Scopus (81) Google Scholar) as well as in later compartments (24Rudensky A.Y. Maric M. Eastman S. Shoemaker L. De Roos P.C. Blum J.S. Immunity. 1994; 1: 585-594Abstract Full Text PDF PubMed Scopus (112) Google Scholar, 25Qiu Y. Xu X. Wandinger Ness A. Dalke D.P. Pierce S.K. J. Cell Biol. 1994; 125: 595-605Crossref PubMed Scopus (209) Google Scholar). Thus, morphological and biochemical evidence indicates that in human B lymphoblastoid cells, at least a proportion of newly synthesized MHC class II/Ii complexes are found on the cell surface or in early endosomes before Ii processing and peptide loading. However, using these techniques, it is difficult to determine what fraction of molecules travel by this route. Morphology can only reveal steady-state MHC class II/Ii levels that may be low for rapidly traversed sectors of the pathway. Similarly, asychronous kinetics of MHC class II molecule transport through the secretory and endocytic pathway make it difficult to establish exactly what proportion of molecules must pass through a particular sector. One way of resolving this issue would be to selectively block the early part of the endocytic pathway and to assess the ability of MHC class II molecules to mature in these cells. If traffic through the cell surface or early endosomes is obligatory, maturation should be blocked under these conditions. On the other hand, if MHC class II molecules can target from the Golgi to both early and late endocytic compartments, the ablation of the early part of the pathway should not disrupt trafficking to later elements, and successful MHC class II molecule maturation should occur. To date, this type of approach has given rise to somewhat contradictory results. Using concanamycin B to block transport from early to late endosomes, Benaroch et al. (14Benaroch P. Yilla M. Raposo G. Ito K. Miwa K. Geuze H.J. Ploegh H.L. EMBO J. 1995; 14: 37-49Crossref PubMed Scopus (153) Google Scholar) showed that newly synthesized MHC class II molecules appeared only slowly on the cell surface and were not accessible to endocytosed neuraminidase. They concluded that the major route was direct transport from the Golgi to late endosomes and/or MHC class II compartments (14Benaroch P. Yilla M. Raposo G. Ito K. Miwa K. Geuze H.J. Ploegh H.L. EMBO J. 1995; 14: 37-49Crossref PubMed Scopus (153) Google Scholar). In contrast, Wang et al. (26Wang K. Peterson P.A. Karlsson L. J. Biol. Chem. 1997; 272: 17055-17060Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) recently analyzed MHC class II molecule maturation in transfected HeLa cells expressing a dominant negative mutant of the GTPase dynamin, which is required for endocytosis through clathrin-coated pits. In these cells, MHC class II molecules failed to mature, indicating that transport via the cell surface was the major route followed by newly synthesized MHC class II molecules (26Wang K. Peterson P.A. Karlsson L. J. Biol. Chem. 1997; 272: 17055-17060Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Because of these contradictory results, and because of the possibility that trafficking of ectopically expressed MHC class II molecules in cells such as HeLa might be different from trafficking in bona fide antigen-presenting cells, we have reassessed the importance of the early endosome system in MHC class II maturation in human B lymphoblastoid cells. To do this, we have used a peroxidase-ablation technique that allows selected domains of the endocytic pathway to be inactivated in intact cells. Here we show that ablation of early endosome function in human EBV-B cells prevents detectable maturation of MHC class II molecules as measured by Ii processing and peptide binding. These results are consistent with a model that involves obligatory passage of MHC class II molecules through the early endosome system before transport to the sites of Ii processing and peptide loading. Human EBV-transformed cell lines FC4 and EDR have been described previously (12West M.A. Lucocq J.M. Watts C. Nature. 1994; 369: 147-151Crossref PubMed Scopus (322) Google Scholar). Pala cells were a kind gift from P. Cresswell. All cells were maintained in RPMI 1640 medium or in Iscove's modified Dulbecco's medium (Life Technologies, Inc.) supplemented as described previously (27Pond L. Watts C. J. Immunol. 1997; 159: 543-553PubMed Google Scholar). Cells were pre-incubated in methionine and cysteine-free medium (MEM; Sigma) for 15–30 min at 37 °C before labeling. 35S-Translabel (Amersham Pharmacia Biotech) was added to a final concentration of 0.5–1.0 mCi/ml for 15–30 min as indicated, and then the cells were washed in DHB. The cells were resuspended in DHB containing 2 mm methionine and, where indicated, 10 μg/ml transferrin-horseradish peroxidase (Tf-HRP) conjugate at 37 °C. In some experiments, leupeptin (Sigma) was included at a final concentration of 1 mm. After Tf-HRP loading, the cells were collected by centrifugation and resuspended in DHB for peroxidase-mediated ablation. Human transferrin was conjugated to HRP exactly as described previously (27Pond L. Watts C. J. Immunol. 1997; 159: 543-553PubMed Google Scholar). The Tf-HRP conjugate was used at 10 μg/ml and loaded at 37 °C in DHB medium for 20 min or for the times indicated. Peroxide and DAB-mediated ablation was performed essentially as described previously (27Pond L. Watts C. J. Immunol. 1997; 159: 543-553PubMed Google Scholar), except that the removal of surface-bound Tf-HRP by acid treatment was made unnecessary by the use of ascorbic acid to quench any extracellular peroxidase activity (28Stoorvogel W. Oorschot V. Geuze H.J. J. Cell Biol. 1996; 132: 21-34Crossref PubMed Scopus (320) Google Scholar). Cells (107 cells/ml) were resuspended in 85 mm NaCl, 50 mm ascorbic acid, 20 mm Hepes, pH 7.4, and 0.01% H2O2, with or without 1.5 mg/ml DAB. The two aliquots were incubated in the dark for 30 min at 0 °C. After peroxidase-mediated ablation, the cells were washed extensively and either prepared for immunoprecipitation (see below) or, in some experiments, subjected to an additional chase at 37 °C in complete Iscove's medium supplemented with 2 mm methionine. The viability of the cells after the peroxidase-mediated ablation and subsequent chase steps was ∼90%. For precipitation of MHC class II complexes, cells were lysed at 2 × 107 cells/ml in lysis buffer as described previously (12West M.A. Lucocq J.M. Watts C. Nature. 1994; 369: 147-151Crossref PubMed Scopus (322) Google Scholar). After centrifugation (14,000 rpm, 2 min, 4 °C) to remove nuclei and cell debris, the supernatants were subjected to pre-clearing with Pansorbin (Calbiochem). In some experiments, a second pre-clearing step was performed with protein G-Sepharose (Amersham Pharmacia Biotech). The pre-cleared lysates were incubated with mAb DA6.231 (29Guy K. van Heyningen V. Cohen B.B. Deane D.L. Steel C.M. Eur. J. Immunol. 1982; 12: 942-948Crossref PubMed Scopus (144) Google Scholar) for 1 h at 0 °C followed by protein G-Sepharose for 1 h. The washed immunoprecipitates were eluted in SDS sample buffer at 25 °C or at 95 °C, as indicated, and analyzed by SDS-polyacrylamide gel electrophoresis as described previously (27Pond L. Watts C. J. Immunol. 1997; 159: 543-553PubMed Google Scholar). For reprecipitation of MHC class II-associated Ii molecules, washed DA6.231 immunoprecipitates were resuspended in 50 μl of phosphate-buffered saline/1% SDS and eluted at 95 °C for 5 min. The eluted proteins were transferred to 950 μl of lysis buffer, and Ii molecules were immunoprecipitated using mAb VIC-Y1 (30Quaranta V. Majdic O. Stingl G. Liszda K. Honigsmann H. Knapp W. J. Immunol. 1984; 132: 1900-1905PubMed Google Scholar). To capture Ig secreted during chase incubations, cells were removed by centrifugation, and the supernatants (∼1.0 ml) were incubated at 4 °C for 1 h with protein A-Sepharose or fixedStaphylococcus aureus cells (Pansorbin; Calbiochem). The beads or bacteria were washed several times in Tris-buffered saline containing 1% Triton X-100 and once in Tris-buffered saline before the elution of immunoglobulin into SDS sample buffer. Fluorescence microscopy of peroxidase-ablated cells was performed essentially as described previously (27Pond L. Watts C. J. Immunol. 1997; 159: 543-553PubMed Google Scholar). After peroxidase-mediated ablation as described above, the cells (5 × 104 cells in 0.5 ml) were resuspended at 106cells/ml, and aliquots (0.05 ml) were spun onto a microscope slide in a Cytospin centrifuge (800 rpm, 4 min; Shandon Scientific, Runcorn, United Kingdom). The cells were fixed in 3.7% HCHO, quenched, and permeabilized and pre-blocked with 0.2% saponin and 0.2% fish skin gelatin as described previously (27Pond L. Watts C. J. Immunol. 1997; 159: 543-553PubMed Google Scholar). Cells were incubated with primary antibodies diluted in saponin/fish skin gelatin/phosphate-buffered saline for 30 min and then washed extensively before incubation with fluorescein isothiocyanate-conjugated secondary antibodies. Antibodies were as follows: (a) transferrin receptor, mAb OKT9; (b) MHC class II, mAb DA6.231; and (c) TGN-46, sheep antiserum to human TGN-46 (final concentration, 10 μg/ml; a kind gift of S.Ponnambalam). Secondary antibodies were fluorescein isothiocyanate-coupled donkey anti-mouse or anti-sheep Ig (Jackson Laboratories). The cells were mounted in Citifluor and viewed on a Zeiss Axioplan microscope. Images were recorded using a Kodak DCS 420 digital camera and processed using Adobe Photoshop software. To assess whether or not functional early endosomes are required for newly synthesized MHC class II molecules to reach the sites of Ii processing and peptide loading, we used a peroxidase-mediated compartment ablation technique. As described previously, this technique allows selective ablation within intact cells of those compartments to which HRP has been targeted (12West M.A. Lucocq J.M. Watts C. Nature. 1994; 369: 147-151Crossref PubMed Scopus (322) Google Scholar,27Pond L. Watts C. J. Immunol. 1997; 159: 543-553PubMed Google Scholar, 31Stoorvogel W. Schwartz A.L. Strous G.J. Fallon R.J. J. Biol. Chem. 1991; 266: 5438-5444Abstract Full Text PDF PubMed Google Scholar, 32Ajioka R.S. Kaplan J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6445-6449Crossref PubMed Scopus (58) Google Scholar). For example, Tf-HRP conjugates selectively ablate early endosomes and recycling endosomes. The DAB polymerization reaction is controlled in all experiments by the omission or inclusion of DAB monomer. We asked first whether newly synthesized MHC class II molecules could mature to yield SDS-stable αβ dimers in cells lacking functional early endosomes. The human EBV-transformed B cell line FC4 was pulse-labeled with [35S]methionine/cysteine and then loaded with Tf-HRP for 30 min. Cell aliquots were exposed to DAB alone or to DAB plus H2O2 at 0 °C and then chased at 37 °C for different times. As shown in Fig.1, cells chased in the presence of H2O2 alone showed a band of approximately 60 kDa that increased in intensity during the chase and disappeared upon heating the sample to 95 °C. However, in cells exposed to H2O2 and DAB to allow polymer formation, SDS-stable dimers failed to appear during the chase. This result suggested that functional early endosomes are required for successful MHC class II molecule maturation. To confirm that compartment ablation had been selective in these cells, we assessed the integrity of different compartments by fluorescence microscopy. Cells loaded with Tf-HRP and exposed to H2O2 alone showed staining for intracellular transferrin receptor, the trans-Golgi marker TGN-46, and MHC class II molecules (Fig. Figure 2, Figure 3, a,c, and e). Cell surface staining for transferrin receptors and MHC class II molecules was also observed. In contrast, in cells incubated with H2O2 and DAB, intracellular staining of transferrin receptors could no longer be detected (Fig. 2 b). Importantly, both intracellular MHC class II and TGN-46 labeling were unaffected by Tf-HRP-catalyzed DAB polymerization, demonstrating selective ablation of a defined sector of the endocytic pathway (Fig. 2, d and f) and making it unlikely that the failure of MHC class II molecules to mature was simply due to the ablation of the Golgi apparatus by low levels of transferrin receptor trafficking through this compartment (33Stoorvogel W. Geuze H.J. Griffith J.M. Strous G.J. J. Cell Biol. 1988; 106: 1821-1829Crossref PubMed Scopus (97) Google Scholar). Quantitative immunoblotting for TGN-46 confirmed that there was no loss of this marker in ablated cells (data not shown).Figure 3The leupeptin-induced Ii peptide (LIP) does not appear in cells lacking functional early endosomes. EBV-B cells (clone EDR) were labeled with35S-Translabel in the presence of 1 mmleupeptin. After 15 min, the cells were washed and resuspended in Tf-HRP plus leupeptin. After a 30-min loading at 37 °C, the cells were divided and incubated at 4 °C in H2O2with or without DAB. After further incubation at 37 °C for the times shown, MHC class II molecules and associated Ii fragments were immunoprecipitated with the DA6.231 mAb.View Large Image Figure ViewerDownload (PPT) A requirement for early endosomes in MHC class II molecule maturation could be explained either by a specific requirement for MHC class II molecule traffic through these compartments or by a requirement for the delivery of some other factor, for example, processed peptide material, to downstream sites of MHC class II molecule maturation. In the latter case, early endosomes would still be required even if the targeting of newly synthesized MHC class II molecules themselves was direct. To analyze the transport and maturation of MHC class II molecules in a way that did not depend on peptide availability and SDS-stable dimer formation, we asked whether Ii processing was taking place in cells lacking functional early endosomes. We took advantage of the fact that defined intermediates in Ii processing can be amplified in cells treated with leupeptin. In particular, an Ii-processing intermediate (leupeptin-induced peptide) of around 22 kDa accumulates in leupeptin-treated cells (34Blum J.S. Cresswell P. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 3975-3979Crossref PubMed Scopus (320) Google Scholar). As before, pulse-labeled EBV-B cells were loaded with Tf-HRP and then incubated with H2O2 in the presence or absence of DAB. To amplify Ii-processing intermediates, the cells were additionally incubated in the presence of leupeptin. As expected, under these conditions, we could not detect the accumulation of SDS-stable dimers (data not shown). Instead, after 60 min of chase, we observed the accumulation of the Ii-derived leupeptin-induced peptide fragment in Tf-HRP-loaded cells that had been treated with H2O2 alone after Tf-HRP loading but not in cells treated with DAB and H2O2. Thus, in cells lacking functional early endosomes, class II/Ii complexes cannot access the sites of Ii processing. To substantiate the above finding (and to rule out the possibility that leupeptin failed to reach the relevant compartments in the ablated cells), we analyzed the processing of Ii/MHC class II complexes in Pala cells in which Ii-processing intermediates can be readily detected even in the absence of protease inhibitors (for example, see Ref. 35Riberdy J.M. Avva R.R. Geuze H.J. Cresswell P. J. Cell Biol. 1994; 125: 1225-1237Crossref PubMed Scopus (75) Google Scholar). As shown in Fig.4 a, a particularly prominent fragment migrating with an apparent size of ∼14 kDa was readily observed, was most prominent after 150 min of chase, and declined thereafter. To confirm that this fragment arose from the N terminus of Ii, we treated immunoprecipitates of MHC class II molecules at 95 °C and reprecipitated them with VIC-Y1 antibody, which is specific for the N-terminal cytosolic domain of Ii (30Quaranta V. Majdic O. Stingl G. Liszda K. Honigsmann H. Knapp W. J. Immunol. 1984; 132: 1900-1905PubMed Google Scholar). As shown in Fig. 4 b, the p14 fragment (as well as larger forms of Ii) was precipitated under these conditions, confirming that it is an N-terminal fragment of the Ii. We next established the location of the MHC class II molecules associated with this p14 Ii fragment with respect to the early endosome system. Cells were pulse-labeled, chased for different times in the presence of Tf-HRP, and then incubated at 0 °C in the presence of H2O2 with or without DAB. As shown in Fig.4 a, ablation of early endosomes using this protocol did not result in any significant loss of the p14 Ii fragment, indicating that it was accumulating downstream of early endosomes, as defined by the itinerary of the recycling Tf-HRP conjugate (Fig. 4 a). This is fully consistent with our earlier data that showed that the majority of both biochemically detectable SDS-stable MHC class II dimers and peptide/class II complexes detectable by T cells are found in late endosomes/lysosomes before expression on the cell surface (12West M.A. Lucocq J.M. Watts C. Nature. 1994; 369: 147-151Crossref PubMed Scopus (322) Google Scholar, 27Pond L. Watts C. J. Immunol. 1997; 159: 543-553PubMed Google Scholar). However, when early endosomes were ablated after pulse labeling but before the chase (pulse-ablate-chase protocol), the appearance of both the Ii p14 fragment and an Ii fragment around 22 kDa was completely abolished (Fig. 5 a). Quantitation of a second experiment clearly showed that neither of these Ii fragments is produced in cells lacking functional early endosomes (Fig. 5 b). However, transport of class II/Ii complexes through the Golgi complex still occurred, as indicated by the conversion of oligosaccharides on the MHC class II β chain from their immature to mature state (Fig. 5, b and c). To demonstrate unequivocally that selective ablation of early endosomes accounted for the failure of MHC class II molecules to mature, we asked whether secretory protein traffic could still occur in the HRP-ablated cells. After first establishing that Pala secretes immunoglobulin into the medium like other EBV-transformed cell lines, we pulse-labeled cells with [35S]methionine/cysteine, loaded them with Tf-HRP, and ablated the Tf-HRP-positive compartments as described above. After reincubation at 37 °C for different times, both the cells and the reincubation medium were retained for separate analysis. Any Ig secreted into the medium during the chase was captured on protein A and analyzed on a 10% SDS gel. As shown in Fig.6 a, radiolabeled Ig heavy and light chains appeared in the medium during the chase from cells incubated in H2O2 alone but also, importantly, from cells incubated in H2O2 and DAB. This demonstrates that compartment ablation under our conditions of Tf-HRP loading is confined to early endosomes and does not extend into the secretory pathway. Ablation of early endosomes in this experiment was successful because in the same cells, we observed, as before, a virtually complete blockade in Ii processing (Fig. 6 b). Quantitation of Ig secretion in several experiments indicated that there was a lag in the ablated cells relative to the non-ablated cells (Fig. 6 b). We do not know the reason for this, but one possibility is that the ablation of the early endosome system interferes with the recycling of soluble or membrane-bound proteins that may be needed for optimal secretory pathway traffic. In any case, it is clear that the secretory pathway is still functional in the ablated cells, as judged by both β chain maturation and by Ig secretion. Taken together, these data demonstrate that a functional early endosome system is required for the successful maturation of newly synthesized MHC class II molecules. This is true despite the fact that the products of maturation (Ii-processing intermediates and peptide/αβ dimers) accumulate downstream of these compartments. Several previous studies have investigated how newly synthesized MHC class II/Ii complexes are delivered to the endocytic pathway. As outlined under "Introduction," class II/Ii complexes have been detected not only in later endosomal and lysosomal compartments, but also at much lower levels in early endosomes and, indeed, on the cell surface (17Wraight C.J. van Endert P. Moller P. Lipp J. Ling N.R. MacLennan I.C. Koch N. Moldenhauer G. J. Biol. Chem. 1990; 265: 5787-5792Abstract Full Text PDF PubMed Google Scholar, 18Koch N. Moldenhauer G. Hofmann W.J. Moller P. J. Immunol. 1991; 147: 2643-2651PubMed Google Scholar, 19Roche P.A. Teletski C.L. Stang E. Bakke O. Long E.O. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8581-8585Crossref PubMed Scopus (189) Google Scholar, 22Castellino F. Germain R.N. Immunity. 1995; 2: 73-88Abstract Full Text PDF PubMed Scopus (188) Google Scholar, 36Romagnoli P. Layet C. Yewdell J. Bakke O. Germain R.N. J. Exp. Med. 1993; 177: 583-596Crossref PubMed Scopus (124) Google Scholar, 37Warmerdam P.A.M. Long E.O. Roche P.A. J. Cell Biol. 1996; 133: 281-291Crossref PubMed Scopus (64) Google Scholar). This has led to a model whereby at least some MHC class II/Ii complexes leaving the Golgi apparatus are transported initially to the earliest parts of the endocytic pathway and/or the cell surface. However, other evidence indicates that MHC class II/Ii complexes are directly targeted to later endosomal and lysosomal compartments (13Peters P.J. Raposo G. Neefjes J.J. Oorschot V. Leijendekker R.L. Geuze H.J. Ploegh H.L. J. Exp. Med. 1995; 182: 325-334Crossref PubMed Scopus (117) Google Scholar, 14Benaroch P. Yilla M. Raposo G. Ito K. Miwa K. Geuze H.J. Ploegh H.L. EMBO J. 1995; 14: 37-49Crossref PubMed Scopus (153) Google Scholar, 15Morton P.A. Zacheis M.L. Giacoletto K.S. Manning J.A. Schwartz B.D. J. Immunol. 1995; 154: 137-149PubMed Google Scholar, 16Glickman J.N. Morton P.A. Slot J.W. Kornfeld S. Geuze H.J. J. Cell Biol. 1996; 132: 769-785Crossref PubMed Scopus (59) Google Scholar). Here, we have used a new approach to address this important question. Selective inactivation or ablation of intracellular compartments can be achieved by targeting HRP to those compartments. This has been achieved on the secretory pathway by expression of a secreted form of HRP (38Connolly C.N. Futter C.E. Gibson A. Hopkins C.R. Cutler D.F. J. Cell Biol. 1994; 127: 641-652Crossref PubMed Scopus (130) Google Scholar) and in the endocytic pathway by endocytosis of HRP conjugated to transferrin or other endocytosed ligands including antigens (12West M.A. Lucocq J.M. Watts C. Nature. 1994; 369: 147-151Crossref PubMed Scopus (322) Google Scholar, 27Pond L. Watts C. J. Immunol. 1997; 159: 543-553PubMed Google Scholar, 31Stoorvogel W. Schwartz A.L. Strous G.J. Fallon R.J. J. Biol. Chem. 1991; 266: 5438-5444Abstract Full Text PDF PubMed Google Scholar, 32Ajioka R.S. Kaplan J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6445-6449Crossref PubMed Scopus (58) Google Scholar). By functionally inactivating the earliest compartments of the endocytic pathway in this way, we have been able to ask whether or not it is required for successful MHC class II/Ii maturation. We find that the invariant chain is not processed and that SDS-stable class II/peptide dimers do not assemble in cells whose early endosomes have been inactivated. The possibility that our internalized Tf-HRP conjugate functionally inactivated later endosomes or lysosomes is rendered very unlikely by our earlier demonstration that this conjugate recycles efficiently (27Pond L. Watts C. J. Immunol. 1997; 159: 543-553PubMed Google Scholar), does not ablate the major cellular MHC class II compartments (Ref. 12West M.A. Lucocq J.M. Watts C. Nature. 1994; 369: 147-151Crossref PubMed Scopus (322) Google Scholar and Fig. 2), and does not affect compartments in which processed fragments of Ii accumulate (Fig. 4 a). Moreover, whereas Tf-HRP can access the Golgi complex in some cell types (33Stoorvogel W. Geuze H.J. Griffith J.M. Strous G.J. J. Cell Biol. 1988; 106: 1821-1829Crossref PubMed Scopus (97) Google Scholar), this did not occur under the conditions of loading we used because in Tf-HRP-loaded and ablated cells, immunodetection of the trans-Golgi marker TGN-46 was still observed, oligosaccharide maturation on MHC class II β chains still occurred, and immunoglobulin was still secreted into the culture medium. Taken together with our own earlier data (12West M.A. Lucocq J.M. Watts C. Nature. 1994; 369: 147-151Crossref PubMed Scopus (322) Google Scholar, 27Pond L. Watts C. J. Immunol. 1997; 159: 543-553PubMed Google Scholar), this result suggests a model for MHC class II maturation in human B cells (Fig.7). Three principal points can be made. First, as shown previously (12West M.A. Lucocq J.M. Watts C. Nature. 1994; 369: 147-151Crossref PubMed Scopus (322) Google Scholar) and in this study (Fig. 4 a), the products of MHC class II maturation, namely, invariant chain processing intermediates and SDS-stable αβ dimers, accumulate, for the most part, downstream of transferrin-positive early endosomes, as judged by their resistance to Tf-HRP-mediated cross-linking. Second, these early endosomes were not necessary for the transport of assembled peptide/αβ complexes to the cell surface, as measured by the biologically relevant assay of T-cell stimulation (27Pond L. Watts C. J. Immunol. 1997; 159: 543-553PubMed Google Scholar). Third, despite this lack of involvement of early endosomes in the later stages of MHC class II/peptide assembly and surface transport, they are nonetheless an essential gateway through which the majority of newly synthesized MHC class II molecules must pass to reach the sites of Ii processing and peptide loading. We therefore propose that the points of entry and exit of newly synthesized MHC class II molecules along the endocytic pathway are different. Newly synthesized MHC class II/Ii complexes are delivered primarily into early transferrin receptor-positive domains but leave, once peptide-loaded, primarily from later transferrin receptor-negative domains (Fig. 7). As also documented by others, transport of MHC class II molecules between these domains is accompanied by Ii processing and peptide loading (18Koch N. Moldenhauer G. Hofmann W.J. Moller P. J. Immunol. 1991; 147: 2643-2651PubMed Google Scholar, 19Roche P.A. Teletski C.L. Stang E. Bakke O. Long E.O. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8581-8585Crossref PubMed Scopus (189) Google Scholar, 20Warmerdam P.A. Long E.O. Roche P.A. J. Cell Biol. 1996; 133: 281-291Crossref PubMed Scopus (63) Google Scholar, 21Kleijmeer M.J. Morkowski S. Griffith J.M. Rudensky A.Y. Geuze H.J. J. Cell Biol. 1997; 139: 639-649Crossref PubMed Scopus (198) Google Scholar, 22Castellino F. Germain R.N. Immunity. 1995; 2: 73-88Abstract Full Text PDF PubMed Scopus (188) Google Scholar, 23Brachet V. Raposo G. Amigorena S. Mellman I. J. Cell Biol. 1997; 137: 51-65Crossref PubMed Scopus (81) Google Scholar, 24Rudensky A.Y. Maric M. Eastman S. Shoemaker L. De Roos P.C. Blum J.S. Immunity. 1994; 1: 585-594Abstract Full Text PDF PubMed Scopus (112) Google Scholar, 25Qiu Y. Xu X. Wandinger Ness A. Dalke D.P. Pierce S.K. J. Cell Biol. 1994; 125: 595-605Crossref PubMed Scopus (209) Google Scholar). Recently, Kleijmeer et al. (21Kleijmeer M.J. Morkowski S. Griffith J.M. Rudensky A.Y. Geuze H.J. J. Cell Biol. 1997; 139: 639-649Crossref PubMed Scopus (198) Google Scholar) have reassessed the steady-state distribution of MHC class II molecules, Ii, and other markers of the endocytic pathway in both human and murine B-cell lines. In a human EBV-transformed B-cell line similar to those used in our study, they defined up to six different types of structures involved in MHC class II maturation. Their analysis revealed Ii, presumably associated with MHC class II, in the so called "early" MHC class II compartments and, to some extent, in conventional early endosomes (21Kleijmeer M.J. Morkowski S. Griffith J.M. Rudensky A.Y. Geuze H.J. J. Cell Biol. 1997; 139: 639-649Crossref PubMed Scopus (198) Google Scholar). They proposed that MHC class II/Ii complexes leaving the Golgi apparatus target to a variety of endosomal structures. Our results are consistent with this study and with other studies that reported the presence of newly synthesized MHC class II molecules in low-density, early endosomes and/or on the cell surface (19Roche P.A. Teletski C.L. Stang E. Bakke O. Long E.O. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8581-8585Crossref PubMed Scopus (189) Google Scholar, 20Warmerdam P.A. Long E.O. Roche P.A. J. Cell Biol. 1996; 133: 281-291Crossref PubMed Scopus (63) Google Scholar, 21Kleijmeer M.J. Morkowski S. Griffith J.M. Rudensky A.Y. Geuze H.J. J. Cell Biol. 1997; 139: 639-649Crossref PubMed Scopus (198) Google Scholar, 22Castellino F. Germain R.N. Immunity. 1995; 2: 73-88Abstract Full Text PDF PubMed Scopus (188) Google Scholar, 23Brachet V. Raposo G. Amigorena S. Mellman I. J. Cell Biol. 1997; 137: 51-65Crossref PubMed Scopus (81) Google Scholar, 37Warmerdam P.A.M. Long E.O. Roche P.A. J. Cell Biol. 1996; 133: 281-291Crossref PubMed Scopus (64) Google Scholar). The requirement that we observe for functional transferrin receptor-positive endosomes now suggests that the input of MHC class II/Ii does not occur later than type 3 structures as defined by Kleijmeer et al. (21Kleijmeer M.J. Morkowski S. Griffith J.M. Rudensky A.Y. Geuze H.J. J. Cell Biol. 1997; 139: 639-649Crossref PubMed Scopus (198) Google Scholar). MHC/Ii complexes found in later compartments must therefore traffic there from earlier transferrin-positive endosomes. Our results are more difficult to reconcile with studies that indicate that direct transport of MHC class II/Ii complexes to later endocytic compartments occurs. In part, this might be explained by the difficulties noted above of detecting rapid passage through compartments in which MHC class II molecules do not accumulate. When traffic from early to late endosomes was disrupted by the vacuolar H+-ATPase inhibitor concanamycin B, MHC class II molecules did not appear to accumulate in early endosomes, as might have been expected if early endosomes are the principal target of post-Golgi vesicles carrying class II molecules (14Benaroch P. Yilla M. Raposo G. Ito K. Miwa K. Geuze H.J. Ploegh H.L. EMBO J. 1995; 14: 37-49Crossref PubMed Scopus (153) Google Scholar). Conceivably, concanamycin B interferes not only with normal early to late endosome traffic, but also with normal fusion of Golgi-derived vesicles with early endosomes. This could explain why Ii degradation was strongly inhibited in concanamycin B-treated cells and why the accumulated MHC class II/Ii complexes were not accessible to endocytosed tracers (14Benaroch P. Yilla M. Raposo G. Ito K. Miwa K. Geuze H.J. Ploegh H.L. EMBO J. 1995; 14: 37-49Crossref PubMed Scopus (153) Google Scholar). The compartment ablation technique we have used cannot easily determine what proportion of newly synthesized complexes reach early endosomes via the cell surface or, instead, directly from the Golgi apparatus. Using a mutant of dynamin that blocks clathrin-coated pit function, Wang et al. (26Wang K. Peterson P.A. Karlsson L. J. Biol. Chem. 1997; 272: 17055-17060Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) recently provided convincing evidence that a majority of complexes in transfected HeLa cells reach early endosomes via the cell surface. It remains to be established whether this is also the case in bona fide antigen-presenting cells such as B lymphocytes or whether both routes are used to direct newly synthesized MHC class II molecules to early endosomes, as indicated by other studies (37Warmerdam P.A.M. Long E.O. Roche P.A. J. Cell Biol. 1996; 133: 281-291Crossref PubMed Scopus (64) Google Scholar, 41Saudrais C. Spehner D. de la Salle H. Bohbot A. Cazenave J.P. Goud B. Hanau D. Salamero J. J. Immunol. 1998; 160: 2597-2607PubMed Google Scholar). Passage through the early endosome system via the cell surface or by direct transport from the Golgi apparatus to early endosomes should ensure exposure of the maturing population of MHC class II molecules to the full range of processed antigenic material. The fact that lysosomal hydrolases also appear to be targeted initially to the earliest parts of the endocytic pathway (39Ludwig T. Griffiths G. Hoflack B. J. Cell Biol. 1991; 115: 1561-1572Crossref PubMed Scopus (127) Google Scholar, 40Rijnboutt S. Stoorvogel W. Geuze H.J. Strous G.J. J. Biol. Chem. 1992; 267: 15665-15672Abstract Full Text PDF PubMed Google Scholar) should facilitate such a scenario. We thank S. Blackwood for technical assistance, S. Ponnambalam for the gift of anti-TGN-46 antisera, W. Knapp for mAb VIC-Y1, A. Lanzavecchia and P. Cresswell for cell lines, and M. A.West for comments on the manuscript.

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