Cell, Vol. 64, 767-776,

February

22, 1991, Copyright

0 1991 by Cell Press

MHC Class II-Restricted of Intracellular Antigen

Presentation

Siegfried Weiss’t* and Bjarne Bogent l Base1 Institute for Immunology Grenzacherstrasse 487 Ch-4005 Base1 Switzerland tlnstitute for immunology and Rheumatology Fr. Qvamsgt. 1 N-0172 Oslo Norway

Summary An endogenously produced immunogiobuiin light chain (h2315) is processed and presented to T cells in association with major histocompatibiiity complex (MHC) class ii molecules. Using transfectants producing variant forms of h2315 that am neither expressed on the ceil surface nor secreted, we demonstrate that intracellular h2315, which has never been exported outside of the ceil, is the source of processed k2315 idiotype. This challenges the currently accepted paradigm that endogenous antigens are only presented by MHC class I molecules. Variants of XZ315 protein that are retained in the endoplasmic rectlcuium (ER) are also presented. Variants that are expressed in the cytosoi as well as those that an? transported into the nucleus rather than the ER are not presented. Thus, the ER is likely to be the processing compartment. Introduction The receptor of conventional T cells recognizes short peptides derived from the nominal antigen by proteolysis, on antigen presenting cells in association with major histocompatibility complex (MHC) molecules (Davis and Bjorkman, 1989; Babbit et al., 1985). It is still an enigma what determines whether processed peptides derived from a given antigen will be presented on class I or on class II MHC molecules. The currently accepted paradigm is that peptides from endogenous proteins are presented on class I MHC molecules while peptides derived from exogenous proteins are presented on class II molecules (Morrison et al., 1988; Germain, 1986; Bevan, 1987; Moore et al., 1988; Sweetser et al., 1989). Peptides processed from proteins are thought to complex with class I molecules in a pre-Golgi compartment (Townsend et al., 1989; Nuchtern et al., 1989; Yewdell and Bennick, 1989). Endocytosed proteins are processed in endosomes and their peptides are thought to become associated in post-Golgi vesicles with newly synthesized (Neefjes et al., 1990; Guagliardi et al., 1990) or recycling (Harding et al., 1989) class II molecules. However, exogenous hepatitis B antigen (Jin et al., 1988; Barnaba et al., 1990) HIV-1 envelope *Present address: GBF, Mascheroder Federal Republic of Germany.

Weg 1, D-3300

Braunschweig,

protein in ISCOMs (Takahashi et al., 1990) ovalbumin on T cells (Dick and Fleske-Kunz, 1989) and exogenous cell-associated antigen in vivo (Staerz et al., 1987; Carbone and Bevan, 1990) are all presented on class I MHC molecules. Furthermore, endogenously produced immunoglobulin (Weiss and Bogen, 1989; Bikoff and Eckhardt, 1989; Yurin et al., 1989), measle virus matrix protein (Jacobson et al., 1989) and influenza A virus matrix protein (Nuchtern et al., 1990) are presented by class Ii molecules. Thus, the endogenous-class I, exogenous-class II rule may need to be reappraised. Recently anti-idiotypic T cell clones (a/8+, CD4+) were isolated that recognize an idiotypic determinant of the h2315 immunoglobulin light chain bound to the class Ii molecule I-Ed (Bogen et al., 1986a). The idiotypic determinant resides on a synthetic peptide covering amino acids 91-101 of h2315 (Bogen and Lambris, 1989), residues PheQ4 Arga5 Asng6 (FRN) being essential (Bogen et al., 1986b). B lymphoma ceils transfected with the h2 light chain gene of MOPC 315 constitutively process and present the I.2315 idiotypic determinant to class Il-restricted T cells (Weiss and Bogen, 1989). Such transfectants express h2315 as the light chain of immunogiobulin (slg) on the cell surface, and a small amount is secreted or shed into the medium. Thus, the above finding could be reconciled with the endogenous-class I, exogenous-class II rule if the idiotypic determinant were processed and presented only when recycled from the membrane or taken up from the medium. The main thrust of the paper is to demonstrate that this is not the case. Using molecular engineering techniques, we construct mutants of h2315, which when transfected into B lymphoma cells express variant proteins that cannot be expressed on the cell surface or secreted into the medium. As we show below such variants are also processed and presented to T cells in association with class Ii MHC molecules. Results Presentation of Mutant h2315 Lacking Surface Expression In previous transfection experiments (Weiss and Bogen, 1989) we optimized h2315 expression by using a h2315 gene construct containing the mouse immunoglobulin heavy chain enhancer within the major intron (F9, Figure 1). The enhancer, however, is not essential; F14 transfected with the normal h2315 gene presents the idiotypic determinant, although at a lower level (Figures l-5). Transfectants described below were therefore established with constructs containing the enhancer, except where it was found to have little effect (F70) or to be deleterious (F59) (Figure 1). We initially tested whether the macromolecular context of the idiotypic sequence would influence processing and presentation. This does not seem to be the case. No influence was found in an exon shuffling experiment; a chimeric

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(1) A 20/46 is a BALB/c-derived B lymphoma cell line. (2) CA36.2.1 is an L cell transfectant expressing the class II molecule EakEpd, which functions like I-Ed. When presenting X2315, this cell line is an extremly sensitive target for the anti-idiotypic T cell clones (Bogen et al., 1966a). (3) Cell-associated Us’5 was estimated by Western blot using anti-12 R/A and serial dilutions of M315 in F55 lysate as standard. (4) nd = not determined. (5) The Pros0 seems to interfere with an antigenic epitope. F45 and F70 were not detected by the 9A3 monoclonal antibody (antiVH/2). (6) lmmunoprecipitation from surface iodinated F70 cells revealed a band with a similar electrophoretic mobility as theexpected band. As the truncated light chain does not bind the endogenous heavy chain (Figure 6) it is unlikely to be expressed on the cell surface. Therefore, the observed band is most likely an artifact of the iodination. (7) “Not found” indicates that the most sensitive sandwich ELIBA did not reveal any k2sq5 protein in the supematant (