MHC Class II Expression in Double Mutant Mice Lacking Invariant Chain and DM Functions George Kenty, W. David Martin, Luc Van Kaer and Elizabeth K. Bikoff This information is current as of September 28, 2021. J Immunol 1998; 160:606-614; ; http://www.jimmunol.org/content/160/2/606 References This article cites 84 articles, 37 of which you can access for free at: Downloaded from http://www.jimmunol.org/content/160/2/606.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: by guest on September 28, 2021 http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. MHC Class II Expression in Double Mutant Mice Lacking Invariant Chain and DM Functions1 George Kenty,* W. David Martin,2† Luc Van Kaer,3† and Elizabeth K. Bikoff4* Invariant (Ii) chain and DM functions are required at distinct stages during class II maturation to promote occupancy by diverse peptide ligands. The class II molecules expressed by mutant mouse strains lacking Ii chain or DM activities display discrete structural and functional abnormalities. The present report describes the cellular and biochemical characteristics of Ii2DM2 doubly deficient mice. As for Ii chain mutants, their mature AabAbb dimers similarly exhibit reduced mobilities in SDS-PAGE, and in functional assays these molecules behave as if empty or occupied by an easily displaced peptide. Additionally, the present experiments demonstrate that the production of floppy AabAbb dimers is TAP independent. In comparison with Ii chain mutants, Ii2DM2 doubly deficient cell populations exhibit increased peptide binding activities and consistently greater pre- sentation abilities in T cell stimulation assays. These functional differences appear to reflect higher class II surface expression Downloaded from associated with their increased representation of B lymphocytes. We also observe defective B cell maturation in mice lacking Ii chain or DM expression, and interestingly, B cell development appears more severely compromised in Ii2DM2 double mutants. These mutant mice lacking both Ii chain and DM activities should prove useful for analyzing nonconventional class II Ag presentation under normal physiological conditions in the intact animal. The Journal of Immunology, 1998, 160: 606–614. onsiderable progress has been made over recent years later inside the endocytic compartment(s) to cause dissociation of http://www.jimmunol.org/ toward understanding the complex cellular and biochem- a relatively short proteolytic product of the Ii chain corresponding C ical events necessary for guiding MHC class II peptide to the so-called CLIP region, in exchange for tightly bound peptide acquisition. Important contributions are made by both the invariant ligand(s) (14, 15). Recent studies demonstrate the DM also asso- (Ii)5 chain and DM to facilitate surface expression of diverse pep- ciates with empty class II molecules (16–18) and acting in this tide ligands, but these activities are required at distinct stages of manner may function as a peptide editor, serving to increase the class II maturation (1). MHC class II a and b subunits are found overall affinities of peptide/class II complexes (19, 20). DM con- coassembled with the Ii chain shortly after synthesis in the ER (2). tains its own endosomal targeting signal(s) located in the b chain In the exceptional case of AabAbb molecules, the Ii chain is es- cytoplasmic tail (21, 22), but there is also evidence suggesting that by guest on September 28, 2021 sential for production or maintenance of ab dimers (3). This early DM transport is mediated via an association with the Ii chain (21). class II association with the Ii chain probably prevents irreversible In contrast to ab-Ii complexes formed early during biosynthesis, misfolding or aggregation of the subunits and protects the empty far less is known about transient associations among class II ab peptide groove from association with molecular chaperones such dimers, Ii chain degradation products, and DM molecules during as BiP and calnexin that are responsible for ER quality control peptide acquisition in the endocytic compartment(s). (4–8). The Ii chain facilitates export of correctly folded ab dimers The specific defects described for Ii chain (23–25) and DM (26– past the Golgi complex (9, 10) and directs their delivery to the 28) mutant mouse strains created using embryonic stem cell tech- endocytic compartment(s) (11, 12). Selective Ii chain degradation nology partially overlap. Both these mutations disrupt class II mat- subsequently permits occupancy of the class II peptide uration, Ag presentation, and CD41 T cell development. However, groove (13). DM mutant spleen cells efficiently express surface AabAbb/CLIP The nonconventional class II product DM, originally described complexes at levels equivalent to those in wild-type AabAbb mol- as a facilitator of Ag presentation in mutant human cell lines, acts ecules (26–28). In contrast, the loss of Ii chain function leads to markedly reduced AabAbb surface expression, largely due to de- *Department of Molecular and Cellular Biology, The Biological Laboratories, creased rates of export (23–25). Thus in the absence of Ii chain, the Harvard University, Cambridge, MA 02138; and †Howard Hughes Medical In- vast majority of class II ab dimers fail to acquire endoglycosidase stitute, Department of Microbiology and Immunology, Vanderbilt University H-resistant glycans and are rapidly degraded. The few mature School of Medicine, Nashville, TN 37232 AabAbb dimers produced by Ii chain mutants exhibit reduced mo- Received for publication July 16, 1997. Accepted for publication September 25, 1997. bilities in SDS gels, and in functional assays these molecules be- The costs of publication of this article were defrayed in part by the payment of have as if empty or occupied by an easily displaced peptide. The page charges. This article must therefore be hereby marked advertisement in structural basis for the exceptional abilities of these floppy accordance with 18 U.S.C. Section 1734 solely to indicate this fact. AabAbb dimers to escape ER quality control has yet to be 1 This work was supported by Grant AI19047 from the National Institutes of determined. Health (to E.K.B.). In the present study we examine class II expression in double 2 Present address: Purdue University, West Lafayette, IN 47907 mutant mice lacking both Ii chain and DM functions. For the most 3 Assistant Investigator with the Howard Hughes Medical Institute. part, their cellular and biochemical defects closely parallel those 4 Address correspondence and reprint requests to Dr. Elizabeth K. Bikoff, De- observed for Ii chain mutants. Thus we found that Ii2DM2 double partment of Molecular and Cellular Biology, The Biological Laboratories, Har- vard University, 16 Divinity Ave., Cambridge, MA 02138. mutants display markedly reduced class II surface expression, pep- 1 5 Abbreviations used in this paper: Ii, invariant; ER, endoplasmic reticulum; tide occupancy, and CD4 T cell development. The loss of DM CLIP, class II-associated Ii chain peptide; PE, phycoerythrin. function has no noticeable effect on the production of floppy Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 The Journal of Immunology 607 AabAbb dimers. Additionally, our experiments demonstrate that Immunofluorescence analysis expression of floppy AabAbb dimers is TAP independent. Com- 2 2 For class II analysis, spleen cell suspensions depleted of erythrocytes by pared with Ii chain mutants, Ii DM double-mutant spleen cells ammonium chloride-Tris treatment were incubated on ice with saturating consistently display increased peptide binding activities and en- amounts of biotin-conjugated Abs followed by FITC-labeled avidin D. hanced presentation abilities in T cell stimulation assays. This Fluorescence was analyzed using a FACScan flow cytometer (Becton probably reflects higher levels of AabAbb surface molecules as- Dickinson Co., Mountain View, CA), and the data are displayed as cell number vs log fluorescence. For double-staining experiments, spleen or sociated with an increased representation of B lymphocytes. B cell 9 lymph node cells were incubated with PE-conjugated goat F(ab )2 anti- maturation is partially defective in the absence of Ii chain or DM mouse IgM (m) (catalogue no. M31604, Caltag, San Francisco, CA) or expression and, interestingly, appears more severely compromised PE-conjugated rat anti-mouse CD45R/B220 (catalogue no. 01125A, in Ii2DM2 double mutants. These mutant mice lacking both Ii PharMingen, San Diego, CA) as a pan-B cell marker used in combination with FITC-labeled Abs directed against the IgE Fc receptor CD23 (Phar chain and DM functions should prove useful for studying class II Mingen catalogue no. 01234D) or surface IgD (PharMingen catalogue