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• W1982944034 abstract "Major histocompatibility complex (MHC) class I and class II molecules have been shown to present peptides of different origin to αβ T cells. Most peptides presented by class I molecules are derived from endogenously synthesized proteins, whereas most peptides presented by class II molecules are from exogenous sources. This functional dichotomy can largely be achieved by the preferential intracellular association of the invariant chain (Ii) with MHC class II molecules, which may inhibit binding of endogenous peptides to class II molecules and direct them to endocytic compartments where extracellularly derived peptides can be sampled. Here, we show that Ii also can associate with a subset of MHC class I molecules and direct them to endocytic compartments. Ii was coprecipitated with class I molecules after lysis of human lymphocytes in mild detergent such as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid or digitonin, and the association was more clearly visualized by the use of dithiobis[succinimidylpropionate], a homobifunctional chemical cross-linker. The class I•Ii complex was reconstituted in Ii negative cells by transfection of corresponding cDNA clones and was found to be transported through the Golgi to acidic endocytic compartments. These observations may explain how some exogenous antigens can be presented by MHC class I molecules and how MHC class II molecules can bind self peptides derived from MHC class I molecules in endocytic compartments. Major histocompatibility complex (MHC) class I and class II molecules have been shown to present peptides of different origin to αβ T cells. Most peptides presented by class I molecules are derived from endogenously synthesized proteins, whereas most peptides presented by class II molecules are from exogenous sources. This functional dichotomy can largely be achieved by the preferential intracellular association of the invariant chain (Ii) with MHC class II molecules, which may inhibit binding of endogenous peptides to class II molecules and direct them to endocytic compartments where extracellularly derived peptides can be sampled. Here, we show that Ii also can associate with a subset of MHC class I molecules and direct them to endocytic compartments. Ii was coprecipitated with class I molecules after lysis of human lymphocytes in mild detergent such as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid or digitonin, and the association was more clearly visualized by the use of dithiobis[succinimidylpropionate], a homobifunctional chemical cross-linker. The class I•Ii complex was reconstituted in Ii negative cells by transfection of corresponding cDNA clones and was found to be transported through the Golgi to acidic endocytic compartments. These observations may explain how some exogenous antigens can be presented by MHC class I molecules and how MHC class II molecules can bind self peptides derived from MHC class I molecules in endocytic compartments. Major histocompatibility complex (MHC) 1The abbreviations used are: MHCmajor histocompatibility complexIiinvariant chainERendoplasmic reticulumβ2mβ2-microglobulinmAbmonoclonal antibodyHLAhuman leukocyte antigenDSPdithiobis[succinimidylpropionate]PAGEpolyacrylamide gel electrophoresisendo Hendoglycosidase HFITCfluorescein isothiocyanateCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. class I and class II molecules are integral membrane glycoproteins whose primary function is to present bound antigenic peptides to T cells expressing αβ T cell receptor (1Germain R.N. Margulies D.H. Annu. Rev. Immunol. 1993; 11: 403-450Crossref PubMed Scopus (952) Google Scholar). MHC class I molecules bind peptides of intracellular origin, such as those derived from endogenously synthesized viral proteins, and present them to CD8+ T cells, whereas MHC class II molecules bind exogenous antigens and present them to CD4+ T cells(2Van Bleek G.M. Nathenson S.G. Nature. 1990; 348: 213-216Crossref PubMed Scopus (586) Google Scholar, 3Davidson H.W. Reid P.A. Lanzavecchia A. Watts C. Cell. 1991; 67: 105-116Abstract Full Text PDF PubMed Scopus (168) Google Scholar). This functional difference is believed to be explained by the capability of class II molecules to bind intracellularly the invariant chain (Ii), which directs them to endocytic vesicles where they encounter exogenously derived peptides(4Teyton L. O'Sullivan D. Dickson P.W. Lotteau V. Sette A. Fink P. Peterson P.A. Nature. 1990; 348: 39-44Crossref PubMed Scopus (258) Google Scholar, 5Lotteau V. Teyton L. Peleraux A. Nilsson T. Karlsson L. Schmid S.L. Quaranta V. Peterson P.A. Nature. 1990; 348: 600-605Crossref PubMed Scopus (444) Google Scholar, 6Lamb C.A. Yewdell J.W. Bennink J.R. Cresswell P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5998-6002Crossref PubMed Scopus (129) Google Scholar). major histocompatibility complex invariant chain endoplasmic reticulum β2-microglobulin monoclonal antibody human leukocyte antigen dithiobis[succinimidylpropionate] polyacrylamide gel electrophoresis endoglycosidase H fluorescein isothiocyanate 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. Ii is a nonpolymorphic type II transmembrane glycoprotein that exists in humans in four forms, designated according to their molecular sizes as p31, p33, p41, and p43 (note that p31 and p33 are referred to as p33 and p35, respectively, by some authors)(7Claesson L. Larhammar D. Rask L. Peterson P.A. Proc. Natl. Acac. Sci. U. S. A. 1983; 80: 7395-7399Crossref PubMed Scopus (107) Google Scholar, 8Strubin M. Mach B. Long E.O. EMBO J. 1984; 3: 869-872Crossref PubMed Scopus (78) Google Scholar). These different forms of Ii are generated by the use of alternative translation initiation codons and alternative mRNA splicing(9Strubin M. Berte C. Mach B. EMBO J. 1986; 5: 3483-3488Crossref PubMed Scopus (130) Google Scholar). Amino acid residues 12-15 (measured from the Ii p31 N-terminal cytoplasmic tail), which are shared among all the four forms of Ii, have been identified as critical residues for endosomal targeting(10Bakke O. Dobberstein B. Cell. 1990; 63: 707-716Abstract Full Text PDF PubMed Scopus (506) Google Scholar). Assembly of MHC class II α and β chains with Ii occurs in the endoplasmic reticulum (ER). Newly synthesized Ii associates as a trimer with calnexin(11Anderson K.S. Cresswell P. EMBO J. 1994; 13: 675-682Crossref PubMed Scopus (107) Google Scholar), an ER resident molecular chaperone(12Hochstenbach F. David V. Watkins S. Brenner M.B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4734-4738Crossref PubMed Scopus (213) Google Scholar, 13David V. Hochstenbach F. Rajagopalan S. Brenner M.B. J. Biol. Chem. 1993; 268: 9585-9592Abstract Full Text PDF PubMed Google Scholar, 14Bergeron J.J.M. Brenner M.B. Thomas D.Y. Williams D.B. Trends Biochem. Sci. 1994; 19: 124-128Abstract Full Text PDF PubMed Scopus (455) Google Scholar). Newly synthesized class II α and β chains also bind to calnexin and dissociate when a complete nonamer complex, containing three αβ dimers and an Ii trimer, is formed(11Anderson K.S. Cresswell P. EMBO J. 1994; 13: 675-682Crossref PubMed Scopus (107) Google Scholar). Subsequently, the class II•Ii complex exits the ER and is transported through the Golgi to a specialized endocytic compartment, distinct from early/late endosomes or dense lysosomes, where Ii is proteolytically cleaved and class II molecules acquire antigenic peptides of exogenous origin(15Peters P.J. Neefjes J.J. Oorschot V. Ploegh H.L. Geuze H.J. Nature. 1991; 349: 669-676Crossref PubMed Scopus (553) Google Scholar, 16Amigorena S. Drake J.R. Webster P. Mellman I. Nature. 1994; 369: 113-120Crossref PubMed Scopus (396) Google Scholar, 17Tulp A. Verwoerd D. Dobberstein B. Ploegh H.L. Pieters J. Nature. 1994; 369: 120-126Crossref PubMed Scopus (380) Google Scholar, 18West M.A. Lucocq J.M. Watts C. Nature. 1994; 369: 147-151Crossref PubMed Scopus (322) Google Scholar, 19Qiu Y. Xu X. Wandinger-Ness A. Dalke D.P. Pierce S.K. J. Cell Biol. 1994; 125: 595-605Crossref PubMed Scopus (209) Google Scholar). Following dissociation of Ii and binding of peptides, class II molecules are transported to the cell surface for recognition by CD4+ T cells(20Germain R.N. Hendrix L.R. Nature. 1991; 353: 134-139Crossref PubMed Scopus (361) Google Scholar). Newly synthesized class I heavy chains also bind to calnexin during their biosynthesis and assembly(21Degen E. Cohen-Doyle M.F. Williams D.B. J. Exp. Med. 1992; 175: 1653-1661Crossref PubMed Scopus (151) Google Scholar, 22Sugita M. Brenner M.B. J. Exp. Med. 1994; 180: 2163-2171Crossref PubMed Scopus (80) Google Scholar). We recently demonstrated in human cells that, following association with β2m, class I heavy chains dissociate from calnexin and subsequently bind peptides of endogenous origin(22Sugita M. Brenner M.B. J. Exp. Med. 1994; 180: 2163-2171Crossref PubMed Scopus (80) Google Scholar). These peptides are transported from the cytoplasm by TAP molecules, and their association with nascent class I chains in the ER might be facilitated by physical association of the class I heavy chain•β2m heterodimer with TAP molecules (23Ortmann B. Androlewicz M.J. Cresswell P. Nature. 1994; 368: 864-867Crossref PubMed Scopus (325) Google Scholar). Fully assembled class I complexes leave the ER and are transported through the Golgi to the cell surface without intersecting the endocytic route(24Neefjes J.J. Stollorz V. Peters P.J. Geuze H.J. Ploegh H.L. Cell. 1990; 61: 171-183Abstract Full Text PDF PubMed Scopus (367) Google Scholar). Segregation of class I and class II takes place in the trans-Golgi reticulum, where class I molecules traffic to the plasma membrane while class II molecules are sorted to endocytic compartments(15Peters P.J. Neefjes J.J. Oorschot V. Ploegh H.L. Geuze H.J. Nature. 1991; 349: 669-676Crossref PubMed Scopus (553) Google Scholar). Thus, the different origin of peptides presented by class I and class II molecules may reflect different intracellular pathways through which these molecules traffic in the cell, which apparently result from the ability of Ii to direct class II molecules away from the default secretory pathway by virtue of endosomal targeting/retention signals(25Hämmerling G.J. Moreno J. Immunol. Today. 1990; 11: 337-340Abstract Full Text PDF PubMed Google Scholar, 26Neefjes J.J. Ploegh H.L. Immunol. Today. 1992; 13: 179-184Abstract Full Text PDF PubMed Scopus (177) Google Scholar, 27Cresswell P. Annu. Rev. Immunol. 1994; 12: 259-293Crossref PubMed Google Scholar). Accumulating evidence has shown that the dichotomy in presentation of antigen from endogenous and exogenous origin is not absolute. Some endogenous antigens can be presented by class II molecules(28Nuchtern J.G. Biddison W.E. Klausner R.D. Nature. 1990; 343: 74-76Crossref PubMed Scopus (247) Google Scholar, 29Chen B.P. Madrigal A. Parham P. J. Exp. Med. 1990; 172: 779-788Crossref PubMed Scopus (105) Google Scholar, 30Malnati M.S. Marti M. LaVaute T. Jaraquemada D. Biddison W. DeMars R. Long E.O. Nature. 1992; 357: 702-704Crossref PubMed Scopus (187) Google Scholar), whereas exogenous antigens are in some cases presented by class I molecules(31Carbone F.R. Bevan M.J. J. Exp. Med. 1990; 171: 377-387Crossref PubMed Scopus (354) Google Scholar, 32Rock K.L. Gamble S. Rothstein L. Science. 1990; 249: 918-921Crossref PubMed Scopus (274) Google Scholar, 33Pfeifer J.D. Wick M.J. Roberts R.L. Findlay K. Normark S.J. Harding C.V. Nature. 1993; 361: 359-362Crossref PubMed Scopus (547) Google Scholar, 34Kovacsovics-Bankowski M. Clark K. Benacerraf B. Rock K.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4942-4946Crossref PubMed Scopus (528) Google Scholar). The exact mechanisms accounting for these observations are not well understood. In this paper, we show that Ii can associate with a subset of class I molecules and direct them into acidic endosomal compartments. This observation suggests a mechanism that could explain how exogenous antigens can be presented by class I molecules and how self class I-derived peptides gain access to endosomal compartments for binding to class II molecules. The human T × B hybrid T1 and T2 cell lines (35Riberdy J.M. Cresswell P. J. Immunol. 1992; 148: 2586-2590PubMed Google Scholar) were cultured in RPMI 1640 (Life Technologies, Inc.) supplemented with 10% bovine calf serum (Hyclone, Logan, UT). The human epithelial cell line, HeLa(5Lotteau V. Teyton L. Peleraux A. Nilsson T. Karlsson L. Schmid S.L. Quaranta V. Peterson P.A. Nature. 1990; 348: 600-605Crossref PubMed Scopus (444) Google Scholar), was grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% bovine calf serum. Monoclonal antibodies (mAbs) W6/32 (specific for β2m-associated HLA class I heavy chain)(36Brodsky F.M. Parham P. J. Immunol. 1982; 128: 129-135PubMed Google Scholar), ME.1 (specific for HLA-B27)(37Ellis S.A. Taylor C. McMichael A. Hum. Immunol. 1982; 5: 49-59Crossref PubMed Scopus (197) Google Scholar), BBM.1 (specific for human β2m)(38Brodsky F.M. Bodmer W.F. Parham P. Eur. J. Immunol. 1979; 9: 536-545Crossref PubMed Scopus (248) Google Scholar), L243 (specific for HLA-DR)(39Lampson L.A. Levy R. J. Immunol. 1980; 125: 293-299PubMed Google Scholar), and P3 (negative control) (40Kohler G. Milstein C. Nature. 1975; 256: 495-497Crossref PubMed Scopus (12503) Google Scholar) were obtained from the American Type Culture Collection. mAb HC10, which recognizes β2m-unassociated HLA class I heavy chain(41Stam N.J. Spits H. Ploegh H.L. J. Immunol. 1986; 137: 2299-2306PubMed Google Scholar), was provided by Dr. Hidde Ploegh (Massachusetts Institute of Technology, Cambridge, MA). Anti-human Ii mAb PIN.1 (42Lamb C.A. Cresswell P. J. Immunol. 1992; 148: 3478-3482PubMed Google Scholar) was a generous gift from Dr. Peter Cresswell (Yale University, New Haven, CT). Anti-human calnexin antibody, AF8, was generated in our laboratory(12Hochstenbach F. David V. Watkins S. Brenner M.B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4734-4738Crossref PubMed Scopus (213) Google Scholar). mAb UPC10, a negative control antibody, was purchased from Sigma. Human Ii p31 cDNA in expression vector pCMU4 (5Lotteau V. Teyton L. Peleraux A. Nilsson T. Karlsson L. Schmid S.L. Quaranta V. Peterson P.A. Nature. 1990; 348: 600-605Crossref PubMed Scopus (444) Google Scholar) was a kind gift from Dr. Per Peterson (The Scripps Research Institute, La Jolla, CA). HLA-B2705 cDNA as a 1.1-kilobase SalI-HindIII insert in phagemid pT7T3-18H was provided by Dr. William Biddison (National Institutes of Health, Bethesda, MD). A SalI-HindIII fragment of HLA-B27 cDNA was first inserted into pBluescript SK to generate a SalI-XbaI fragment, which was then subcloned into pSRα-neo(43Takebe Y. Seiki M. Fujisawa J.-I. Hoy P. Yokota K. Arai K.-I. Yoshida M. Arai N. Mol. Cell. Biol. 1988; 8: 466-472Crossref PubMed Google Scholar). Following transfection of HeLa cells with HLA-B27 cDNA in pSRα-neo either with pCMU4 vector or with Ii p31 cDNA in pCMU4 by the calcium phosphate precipitation method(44Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4799) Google Scholar), HLA-B27-positive cells with or without Ii p31 expression were cloned from the G418 (0.5 mg/ml, Sigma)-resistant cell population. Metabolic labeling of cells with [35S]methionine was performed as described(22Sugita M. Brenner M.B. J. Exp. Med. 1994; 180: 2163-2171Crossref PubMed Scopus (80) Google Scholar). Radiolabeled cells were lysed in 0.3% CHAPS in phosphate-buffered saline, pH 8, either in the presence or absence of 0.1 mM dithiobis[succinimidylpropionate] (DSP) (Pierce) and incubated on ice for 30 min. Chemical cross-linking was quenched by the addition of 10 mM glycine. The nuclei were removed by centrifugation, and the lysates were precleared overnight at 4°C with Staphylococcusaureus Cowan strain I (Pansorbin, Calbiochem, La Jolla, CA). Immunoprecipitation was performed with indicated mAbs, followed by incubation with protein A-Sepharose CL-4B (Pharmacia Biotech, Inc.). Pellets were washed 5 times with 0.3% CHAPS in Tris-buffered saline, resuspended in 1 × sample buffer containing 1.5% SDS and 5% 2-mercaptoethanol, and boiled for 5 min to cleave DSP. The samples were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) under reducing conditions. The gels were fluorographed using Me2SO/2,5-diphenyloxazole, dried, and exposed to Kodak XAR film. For peptide mapping, the gel slices containing Ii p31 after separation on an SDS-PAGE gel were overlaid with 0.5 μg of V8 protease (Promega, Madison, WI) and were electrophoresed on a 15% SDS-PAGE gel. Endoglycosidase H (endo H) digestion was performed as described(45Rajagopalan S. Brenner M.B. J. Exp. Med. 1994; 180: 407-412Crossref PubMed Scopus (84) Google Scholar). HeLa transfectant cells grown on glass coverslips were fixed in 3.7% paraformaldehyde in phosphate-buffered saline for 10 min, permeabilized in 0.1% digitonin (Aldrich), and incubated with mAb ME.1 followed by rhodamine B-conjugated goat anti-mouse antibodies (Tago, Burlingame, CA). After blocking free antigen binding sites of the goat antibodies with isotype-matched mouse control antibodies, cells were incubated with FITC-conjugated PIN.1 antibody. In some experiments, transfectant cells, incubated for 2 h with 1 mg/ml of Texas red-conjugated ovalbumin (Molecular Probes, Inc., Eugene, OR), were labeled with mAb ME.1 followed by detection with FITC-conjugated goat anti-mouse antibodies (Tago). The cells were viewed and photographed using a Nikon Optiphot-2 fluorescence microscope (Melville, NY) with FITC and rhodamine filter sets. T2 is a mutant cell line with a homozygous deletion in the MHC on chromosome 6, including all functional class II genes and the TAP-1 and TAP-2 peptide transporter genes. Thus, T2 cells completely lack the expression of MHC class II molecules and have impaired assembly of class I molecules due to restricted availability of peptides in the ER resulting in reduced class I expression on the cell surface. Class I heavy chains that are assembled with β2m were detected either by immunoprecipitation with conformation-dependent antibody, W6/32, or by coimmunoprecipitation with β2m-specific BBM.1 antibody (Fig. 1, lanes6 and 4, respectively). We observed that mAb BBM.1 immunoprecipitated 12-kDa β2m and 43-kDa class I heavy chain and coprecipitated a scant amount of 33-kDa protein barely detected on this gel exposure (see arrow, Fig. 1, lane4). This association was more clearly visualized by the use of the thiol-cleavable, bifunctional chemical cross-linker, DSP (Fig. 1, lane10). Radiolabeled protein that comigrated exactly with the 33-kDa protein was also detected by coimmunoprecipitation with the anti-calnexin AF8 antibody and with mAb HC10, which recognizes β2m-unassociated class I heavy chains (see arrow, Fig. 1, lanes8 and 11, respectively). mAb PIN.1 is an anti-Ii antibody that was generated by immunization with a synthetic peptide corresponding to a sequence located in the cytoplasmic domain of all forms of Ii (residues 12-28, measured from the Ii p31 N terminus)(42Lamb C.A. Cresswell P. J. Immunol. 1992; 148: 3478-3482PubMed Google Scholar). Ii p31 is the most abundant form, and it was visualized prominently by immunoprecipitation with mAb PIN.1 (Fig. 2A, lane4). Importantly, the 33-kDa protein associated with class I (Fig. 2A, lanes2 and 3) or calnexin (lane1) comigrated with Ii p31. The identity of this 33-kDa protein with Ii p31 was confirmed by peptide mapping with V8 protease. The peptide map of either class I-associated or calnexin-associated 33-kDa protein was exactly the same as that of Ii p31 (Fig. 2B). Thus, we concluded that Ii p31 was associated with class I molecules in T2 cells. To rule out the possibility that this novel association might be achieved only in class II negative, mutant T2 cells with impaired class I assembly, we examined wild type T1 cells. Unlike T2 cells, T1 cells have normal class I assembly, which was manifested by the appearance of abundant assembled class I heavy chains detected with mAb W6/32 (Fig. 3, lane4) and fewer β2m-unassociated heavy chains detected with mAb HC10 (lane3). Importantly, mAb W6/32 coimmunoprecipitated a protein comigrating with Ii p31 (Fig. 3, lane4, shown with an arrow). The identity of this class I-associated protein to Ii p31 was confirmed by resolution on nonequilibrium pH gradient electrophoresis/SDS-PAGE two-dimensional gels (not shown). This association was also observed in a human B cell line, JY (data not shown). Thus, we concluded that the association of Ii p31 with class I molecules was not attributable to the abnormal class I assembly in T2 cells but was a more general phenomenon that also occurred in cells with normal class I assembly and class II expression. In order to confirm the association of Ii p31 with class I molecules and to determine the intracellular transport and distribution of the complex, we reconstituted the association in HeLa cells, an Ii-negative cervical epithelial carcinoma cell line. HeLa cells were transfected with an HLA-B27 cDNA with or without an Ii p31 cDNA. After selection with G418, stable transfectant clones expressing either HLA-B27 alone (HeLaB27 mock) or both HLA-B27 and Ii p31 (HeLaB27Iip31) were obtained. No PIN.1-reactive material was immunoprecipitated from radiolabeled HeLaB27 mock cells (Fig. 4, lane5), and class I-associated 33-kDa protein was not detected in these cells (lanes2-4). In contrast, transfected Ii p31 was abundantly expressed in HeLaB27Iip31 cells (Fig. 4, lane10, shown at an arrow), and the association of Ii p31 with class I molecules could be demonstrated in these cells (lanes7-9, see arrow). The intracellular transport of the class I•Ii p31 complex was analyzed by pulse-chase experiments in which the susceptibility of the Ii to endo H digestion was examined. These experiments were performed either in the presence or absence of concanamycin B, a vacuolar proton ATPase inhibitor(46Yilla M. Tan A. Ito K. Miwa K. Ploegh H.L. J. Biol. Chem. 1993; 268: 19092-19100Abstract Full Text PDF PubMed Google Scholar). Following a 10-min [35S]methionine pulse labeling of HeLaB27Iip31 cells, endo H-sensitive (S) radiolabeled Ii p31 decreased during the chase period, and endo H-resistant (R) Ii p31 appeared after a chase of 60 min (Fig. 5A, upperpanel). This endo H-resistant form of Ii p31 disappeared after a chase of 240 min in the absence of concanamycin B, while in its presence, persistence of the endo H resistant species was observed (Fig. 5A, lowerpanel). This could be explained by concanamycin B-mediated, impaired acidification of vacuolar organelles such as endosomes(47Mellman I. Fuchs R. Helenius A. Annu. Rev. Biochem. 1986; 55: 663-700Crossref PubMed Google Scholar), where Ii is proteolytically degraded by proteases that require an acidic environment for their activity(48Blum J.S. Cresswell P. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 3975-3979Crossref PubMed Scopus (320) Google Scholar, 49Roche P.A. Cresswell P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3150-3154Crossref PubMed Scopus (205) Google Scholar, 50Maric M.A. Taylor M.D. Blum J.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2171-2175Crossref PubMed Scopus (169) Google Scholar). Thus, we reasoned that if the class I•Ii p31 complex exits the ER and is transported to acidic compartments, class I molecules might remain associated with Ii p31 that acquires resistance to endo H, and the association should be prolonged in the presence of concanamycin B. To examine this possibility, immunoprecipitation with mAb W6/32, specific for assembled class I molecules, was performed at each chase time (Fig. 5B). This experiment revealed that class I molecules associated with an endo H-sensitive form of Ii p31 (approximately 33 kDa) during earlier chase periods, and this became an endo H-resistant form of Ii p31 (35 kDa) after a 1-h chase (Fig. 5B, lanes1-4). Moreover, prolonged association of class I molecules with the endo H-resistant species occurred in the presence of concanamycin B (Fig. 5B, lanes5-8). Thus, we concluded that, following assembly of class I molecules with Ii in the ER, the complex left the ER and was transported through the Golgi to acidic compartments in which acidification depended on the function of vacuolar proton ATPases. Immunofluorescence microscopy was carried out to determine the subcellular localization of class I•Ii p31 complex. Staining of permeabilized HeLa transfectant cells expressing both HLA-B27 and Ii p31 (HeLaB27Iip31) with an HLA-B27-specific antibody revealed the presence of HLA-B27 in large vesicles as well as on the cell surface (Fig. 6A, left). In double-label experiments, the Ii-specific PIN.1 antibody also stained these vesicles (Fig. 6A, right), which indicated that HLA-B27 and Ii were colocalized in these intracellular compartments. These vesicles observed when Ii p31 is expressed in Ii-negative cells have been shown to correspond to endosomal compartments(5Lotteau V. Teyton L. Peleraux A. Nilsson T. Karlsson L. Schmid S.L. Quaranta V. Peterson P.A. Nature. 1990; 348: 600-605Crossref PubMed Scopus (444) Google Scholar, 51Romagnoli P. Layet C. Yewdell J. Bakke O. Germain R.N. J. Exp. Med. 1993; 177: 583-596Crossref PubMed Scopus (123) Google Scholar). We examined if these vesicular structures belonged to the endocytic system by incubating HeLaB27Iip31 cells in media containing Texas red-conjugated ovalbumin. Exogenously added ovalbumin is endocytosed into the cell and transported intracellularly via endocytic pathways so that endocytic compartments are labeled with Texas red(52Swanson J. Methods Cell Biol. 1989; 29: 137-151Crossref PubMed Scopus (71) Google Scholar). With this method, the large vesicles were clearly visualized (Fig. 6B, right) and, importantly, were costained with the HLA-B27-specific antibody (Fig. 6B, left). Such a striking colocalization of HLA-B27 with endocytosed ovalbumin was not observed in Ii-negative HeLa cells (data not shown). Thus, we concluded that class I•Ii p31 complexes were transported into endocytic compartments in these transfectant cells. Using direct biochemical analyses of human lymphocytes (Figure 1:, Figure 2:, Figure 3:) and reconstitution by transfection in HeLa cells (Fig. 4), we have demonstrated an association between class I molecules and Ii. The association of class II molecules with Ii is fairly strong and is maintained in detergents such as 1% Triton X-100(11Anderson K.S. Cresswell P. EMBO J. 1994; 13: 675-682Crossref PubMed Scopus (107) Google Scholar). In contrast, the association of class I molecules with Ii was too weak at least in vitro to be maintained in 0.5% Triton X-100 (data not shown). We observed that a small amount of Ii was coimmunoprecipitated with class I molecules in milder detergents such as 0.3% CHAPS (Fig. 1) and 1% digitonin (not shown), and we succeeded in visualizing the association more clearly in a reproducible way by the use of low concentrations of the chemical cross-linker DSP (Fig. 1). Previously, mouse class I heavy chains were expressed artificially in human cells, and an association with human Ii was reported(53Cerundolo V. Elliott T. Elvin J. Bastin J. Townsend A. Eur. J. Immunol. 1992; 22: 2243-2248Crossref PubMed Scopus (26) Google Scholar). Here, we demonstrate the presence of class I•Ii complexes under physiological conditions in untransfected human cell lines. Moreover, we show that the class I•Ii complex remained assembled in vivo during transport from the ER, through the Golgi, to endocytic compartments, which was evidenced by pulse-chase experiments (Fig. 5) and immunofluorescence microscopy analysis (Fig. 6). Thus, the association of Ii with class I molecules is stable in vivo and results in sorting of this pool of class I molecules out of the direct secretory pathway. What is the biological significance of this association? The colocalization of class I•Ii complex with endocytosed ovalbumin leads one to consider the possible involvement of this complex in presentation of exogenous antigens by class I molecules. Several reports have shown that exogenous antigens can be presented by class I molecules to T cells both in vivo and in vitro(31Carbone F.R. Bevan M.J. J. Exp. Med. 1990; 171: 377-387Crossref PubMed Scopus (354) Google Scholar, 32Rock K.L. Gamble S. Rothstein L. Science. 1990; 249: 918-921Crossref PubMed Scopus (274) Google Scholar, 33Pfeifer J.D. Wick M.J. Roberts R.L. Findlay K. Normark S.J. Harding C.V. Nature. 1993; 361: 359-362Crossref PubMed Scopus (547) Google Scholar, 34Kovacsovics-Bankowski M. Clark K. Benacerraf B. Rock K.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4942-4946Crossref PubMed Scopus (528) Google Scholar). Recent evidence has shown that cells capable of this presentation are phagocytic macrophages, and indeed phagocytosis by these cells of exogenous antigens, such as bacteria or ovalbumin linked to beads, and transport of these antigens to endosomal compartments are critical for antigen presentation(33Pfeifer J.D. Wick M.J. Roberts R.L. Findlay K. Normark S.J. Harding C.V. Nature. 1993; 361: 359-362Crossref PubMed Scopus (547) Google Scholar, 34Kovacsovics-Bankowski M. Clark K. Benacerraf B. Rock K.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4942-4946Crossref PubMed Scopus (528) Google Scholar). We speculate that Ii may transport a subset of class I molecules, as well as class II molecules, to endocytic compartments, where Ii is cleaved and class I molecules acquire peptides derived from endocytosed exogenous antigens. Although a recently identified compartment where peptide loading onto class II molecules takes place does not contain class I molecules, a small amount of class I molecules are found in endosomes in a human B cell line, JY(15Peters P.J. Neefjes J.J. Oorschot V. Ploegh H.L. Geuze H.J. Nature. 1991; 349: 669-676Crossref PubMed Scopus (553) Google Scholar), in which no internalization from the cell surface is detected for class I molecules(24Neefjes J.J. Stollorz V. Peters P.J. Geuze H.J. Ploegh H.L. Cell. 1990; 61: 171-183Abstract Full Text PDF PubMed Scopus (367) Google Scholar). Further studies are required to determine definitively the extent to which class I molecules (colocalized with Ii and with class II molecules) are present in endocytic compartments. Another possible function of class I•Ii complexes is to provide the source of self class I-derived peptides for binding to class II molecules. Analysis of naturally processed peptides bound to HLA-DR1 shows that self HLA-A2 derived peptides as well as those derived from Ii are found in association with DR1 molecules(54Chicz R.M. Urban R.G. Lane W.S. Gorga J.C. Stern L.J. Vignali D.A.A. Strominger J.L. Nature. 1992; 358: 764-768Crossref PubMed Scopus (669) Google Scholar), and indeed MHC class II molecules have been shown to present endogenous class I-derived peptides to cytotoxic T cells in a Brefeldin A-sensitive manner(29Chen B.P. Madrigal A. Parham P. J. Exp. Med. 1990; 172: 779-788Crossref PubMed Scopus (105) Google Scholar). Given the limited internalization of class I molecules from the cell surface, Ii-mediated transport of class I molecules to endocytic compartments might be crucial to the supply of certain self antigens (such as MHC class I proteins) for presentation by class II molecules. Finally, based on these results and previous studies from our laboratory(22Sugita M. Brenner M.B. J. Exp. Med. 1994; 180: 2163-2171Crossref PubMed Scopus (80) Google Scholar), we believe that calnexin may act as a molecular chaperone in the assembly of class I•Ii complexes. Calnexin has been shown to play an important role in the assembly of both class I and class II molecules(11Anderson K.S. Cresswell P. EMBO J. 1994; 13: 675-682Crossref PubMed Scopus (107) Google Scholar, 21Degen E. Cohen-Doyle M.F. Williams D.B. J. Exp. Med. 1992; 175: 1653-1661Crossref PubMed Scopus (151) Google Scholar, 22Sugita M. Brenner M.B. J. Exp. Med. 1994; 180: 2163-2171Crossref PubMed Scopus (80) Google Scholar, 55Schreiber K.L. Bell M.P. Huntoon C.J. Rajagopalan S. Brenner M.B. McKean D.J. Int. Immunol. 1994; 6: 101-111Crossref PubMed Scopus (43) Google Scholar). In the case of class II molecules, calnexin binds newly synthesized α, β and Ii chains and remains associated with assembling intermediate complexes until the complete nonamer, containing three αβ dimers and the Ii trimer, is formed (11Anderson K.S. Cresswell P. EMBO J. 1994; 13: 675-682Crossref PubMed Scopus (107) Google Scholar). In the case of class I assembly, we recently showed that newly synthesized class I heavy chains bind to calnexin and that association with β2m triggers heavy chains to dissociate from calnexin(22Sugita M. Brenner M.B. J. Exp. Med. 1994; 180: 2163-2171Crossref PubMed Scopus (80) Google Scholar). Since the interaction between class I heavy chains and Ii was also observed in β2m deficient Daudi cells (data not shown), we speculate that calnexin may mediate association of a subset of class I heavy chains with Ii, and that, following association with β2m, class I heavy chain•β2m•Ii complex may dissociate from calnexin. These findings potentially indicate stronger parallels between the assembly of class II molecules and a subset of class I molecules than previously have been recognized. We thank Dr. Per Peterson for Ii p31 cDNA; Dr. William Biddison for HLA-B27 cDNA; Dr. Peter Cresswell for mAb PIN.1, T1, and T2 cells; and Dr. Hidde Ploegh for mAb HC10 and concanamycin B. We also thank Dr. Steven Porcelli for critically reading the manuscript." @default.
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• W1982944034 title "Association of the Invariant Chain with Major Histocompatibility Complex Class I Molecules Directs Trafficking to Endocytic Compartments" @default.
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