0022-1767/90/ zyxwvutsrqponm 1448-2915S02.00/0 zyxwvutsrqpon Copyrlght zyxwvutsrqponmlkji G 1990 by The American Assoclation of lrnrnunologlsts THE JOURNAL OF I~MUNOLOGY Vol. 144. 2915-2924. No. zyx 8. April 15. 1990 zy Printed in U.S.A. THEEXTRACELLULAR DOMAINS OF MHC CLASS I1 MOLECULES DETERMINE THEIR PROCESSING REQUIREMENTS FOR ANTIGEN PRESENTATION' LARS KJER-NIELSEN," JEANNE D. PERERA," LISA F. BOYD,t DAVID H. MARGULIES,t AND JAMES MCCLUSKEY~" zyxwv From the *Department of Pathology and Immunology. Monash Medical School, Commercial Road, Melbourne, Australia: and 'Laboratory zyxwvutsrq of Immunology. National Institute of Allergy and Infectious Disease, National Institutes of Health. " " Bethesda We have evaluated the relative contributions of the extracellular and cytoplasmic domains of MHC class I1 molecules in determining the Ag-processing requirements for class 11-restricted Ag presentation to T cells. Hybrid genes were constructed to encode a heterodimeric I-A" molecule in which the extra- cellular portion of the molecule resembled wild type I-A" but where the connecting stalk, transmem- brane and cytoplasmic domains of both the a- and &chain were derived from theclass I molecule H-2Dd. Mutant I-Ak molecules were expressed a s het- erodimeric membrane glycoproteins reactive with mAb specific for wild type I-A". Fibroblast and B lymphoma cells expressing either wild type or mu- tant I-A" molecules were able to process and present hen egg lysozyme (HEL) and conalbumin to Ag-spe- cific, I-A"-restricted, T cell hybridomas or clones. The mutant-expressing cells presented native and peptide Ag less efficiently than the wild type-ex- pressing cells, suggesting that the disparity in pres- entation efficiency was not due to a difference in Ag processing. CD4 interaction was intact on the mu- tant I-Ak molecules. Presentation of native Ag by mutant and wild type-I-Ak-expressing cells was abol- ished by preincubation with chloroquine, or after paraformaldehyde fixation. After transfection of a cDNA encoding the gene for HEL,3 neither mutant nor wild type-I-Ak-expressing cells presented endog- enously synthesized HEL to a specific T hybrid. Newly synthesized mutant I-Ak molecules were as- sociated with invariant chain. These data demon- strate the ability of hybrid class I1 molecules to associate intracellularly with invariant chain and degraded foreign Ag in a conventional class II-re- stricted processing pathway indicating that the ex- tracellular domains of class I1 molecules play a dom- inant role in controlling these Ag-processing re- quirements. Received for publication October 26, 1989. Accepted for publication January 17, 1990 The costs of publication of this article were defrayed in part by the advertisement in accordance with 18 U.S.C. Section 1734 solely to indi- payment of page charges. This article must therefore be hereby marked cate this fact. thritisFoundation, National Health and Medical Research Council of ' This work was supported in part by grants from the American Ar- Australia and Monash University Special Grants, L.K.-N. is supported by a Commonwealth Postgraduate Scholarship. McCluskey. Department of Pathology and Immunology, Monash Medical 'Address for correspondence and reprint requests to Dr. James School. Commercial Road, Melbourne, Australia 31 81. Abbreviations used in this paper: HEL. hen egg lysozyme; la. immune associated or class I1 molecules; Ii. invariant chain: tk. thymidine kinase. MD 20892 MHC class I molecules generally restrict the cytolytic T cell recognition of viral and minor Ag endogenously ex- pressed by APC (1 -4). Processing of these Ag for recog- nition by class I-restricted T cells probably takes place in a nonendosomal compartment, most likelythe cytosol (2, 5). Class I-restricted presentation of exogenous Ag is apparently unusual although this has been reported for hepatitis B surface Ag (6) and has also been demonstrated after in vitro manipulations of the mode of Ag delivery to APC (7-9). which presumably result in direct cytosolic delivery of antigen. In contrast, MHC class I1 molecules usually restrict the recognition of exogenous soluble Ag (1, 3) and although presentation of certain endogenous antigens has been described (6. 10, 11) in some cases this may involveinternalization of surface or shed Ag via an endosomal pathway (3, 4, 6). Thus Ag processing for class I1 restricted, but not class I-restricted presentation, is sensitive to lysosomotropic agents and probably occurs in an acidified endosomal compartment (4). By contrast, Ag presentation for class I-restricted, but not class II- restricted recognition, is inhibited by Brefeldin A, an antifungal agent that prevents the egress of newly syn- thesized proteins from the endoplasmic reticulum (12, 13). In addition, recent evidence implies that the associ- ation of endogenouspeptideswithnewlysynthesized class I proteins may be a prerequisite for their proper assembly in the endoplasmic reticulum (14, 15), consist- ent with Brefeldin A-sensitivity of class I-restricted pres- entation: This apparent dichotomy in processing require- ments for MHC class I- and class 11-restricted presenta- tion, although not absolute, is believed to reflect general differences in the site of Ag processing and/or the cellular pathway and mechanisms by which processed foreign Ag is assembled with MHC class I and class I1 molecules (1, 16). This notion suggests that some integral structural feature of class I and I1 molecules may stronglyinfluence the choice of Ag-processing pathway. We have evaluated the relative contributionsof the MHC class I1 ectodomain (external domains) and endodomain (transmembrane and cytoplasmic domains) in determining the Ag processing and presentation pathway used to generate Ia/Ag com- plexes. We show that hybrid class II/class I MHC mole- cules comprising a class I1 ectodomain and class I trans- membrane and cytoplasmic domains associate with in- variant chain and are able to present foreign Ag with the same qualitative processing requirements as conven- tional class I1 MHC molecules indicating that the external domains of the class I1 a- and P-chains determine the class I1 Ag presentation pathway. 2915