Class II-restricted presentation of an endogenously derived immunodominant T-cell determinant of hen egg lysozyme.

Major histocompatibility complex (MHC) class II molecules bind antigenic peptides for display to T lymphocytes. Although the enzymes involved remain to be identified, it is commonly believed that class II associated peptides are released from intact antigens through a series of proteolytic steps carried out inside antigen presenting cells. We have examined the effect of amino acid substitutions on proteolytic processing of the model antigen hen-egg lysozyme (HEL). Altered HEL molecules, engineered by site-directed mutagenesis of a HEL cDNA, were expressed as separate stable transfectants in a B cell lymphoma line. Each transfectant processed a different mutant HEL protein for presentation on MHC class II. We purified the resulting class II-associated peptides and analyzed them by mass spectrometry. Our results strongly support the hypothesis that antigen processing continues after peptide binding to the MHC class II molecule and are most consistent with a scenario in which long peptides first bind to MHC class II and are then trimmed by exopeptidase.

Section: Resultssupporting

confidence: 92%

Amino-terminal trimming of peptides for presentation on major histocompatibility complex class II molecules

Nelson

Vidavsky

Viner

et al. 1997

“…Based upon the location of the antigenic peptide, cDNA constructs were prepared ( Fig. 2) (36,37). Efficient Presentation of Endogenous Antigens from IiAntigen Fusion Proteins in the Absence of Wild-Type Ii.…”

Section: Resultsmentioning

confidence: 99%

Expression of endogenous peptide-major histocompatibility complex class II complexes derived from invariant chain-antigen fusion proteins.

Sanderson

Frauwirth

Shastri

1995

CD4+ T cells recognize major histocompatibility complex (MHC) class II-bound peptides that are primarily obtained from extracellular sources. Endogenously synthesized proteins that readily enter the MHC class I presentation pathway are generally excluded from the MHC class II presentation pathway. We show here that endogenously synthesized ovalbumin or hen egg lysozyme can be efficiently presented as peptide-MHC class II complexes when they are expressed as fusion proteins with the invariant chain (Ii). Similar to the wild-type Ii, the Ii-antigen fusion proteins were associated intracellularly with MHC molecules.Most efficient expression of endogenous peptide-MHC complex was obtained with fusion proteins that contained the endosomal targeting signal within the N-terminal cytoplasmic Ii residues but did not require the luminal residues of Ii that are known to bind MHC molecules. These results suggest that signals within the Ii can allow endogenously synthesized proteins to efficiently enter the MHC class II presentation pathway. They also suggest a strategy for identifying unknown antigens presented by MHC class II molecules.T cells recognize antigens as peptides bound to major histocompatibility complex (MHC) molecules on the surface of antigenpresenting cells (APCs). CD4+ and CD8+ T cells recognize antigens bound to MHC class II and MHC class I, respectively. In addition to providing immunity against foreign pathogens, both CD4+ and CD8+ T cells are implicated in the destruction of self tissues that leads to autoimmune disorders (1). Identification of these peptides and their donor proteins that elicit T-cell immunity is essential for development of vaccines and understanding the role of these antigens in autoimmunity.Antigens that stimulate T cells can in principle be identified by sequencing the purified MHC-bound antigenic peptide. This biochemical approach is, however, made difficult by the fact that many different peptides are bound to MHC on the APC surface, and individual peptides represent only a very small fraction of the total (2-4). We have proposed (5) an alternative genetic strategy for identifying T-cell-stimulating antigens that does not depend upon biochemical purification of peptides but instead, relies upon expression of peptide-MHC complexes in APCs transfected with the antigen genes. The generation of peptide-MHC class I complexes from transiently expressed genes and the ability to detect rare APCs with the single-cell lacZ T-cell assay suggested that these methods could serve as an expression cloning strategy to identify antigens recognized by CD8+ T cells (5,6). Indeed this strategy has allowed the identification of several antigens that are presented by MHC class I molecules (ref. 7 and S. Malarkannan, M. Afkarian, and N.S., unpublished data).The efficiency of peptide-MHC expression after gene transfer is central to expression cloning strategies. Not only should the genes transferred cause expression of peptide-MHC complexes, but this should occur in a large fraction of APCs to pe...

“…Because of the requirement for all three plasmids in the DNA immunization, it is most likely that the cells expressing CD1 from the injected DNA also are expressing Ova and are presenting it. For class II molecules, there are ample precedents for the presentation of peptides derived from endogenously expressed, secreted proteins (34), and, therefore, it is not so surprising that peptides derived from such proteins also reach endosomes containing CD1.…”

Section: Discussionmentioning

confidence: 99%

Presentation of peptide antigens by mouse CD1 requires endosomal localization and protein antigen processing

Tangri

Brossay

Burdin

et al. 1998

Mouse CD1(mCD1) molecules have been reported to present two types of antigens: peptides or proteins and the glycolipid ␣-galactosylceramide. Here, we demonstrate that a protein antigen, chicken ovalbumin (Ova), must be processed to generate peptides presented by mCD1 to CD8 ؉ T cells. The processing and mCD1-mediated presentation of chicken Ova depend on endosomal localization because inhibitors of endosomal acidification and endosomal recycling pathways block T cell reactivity. Furthermore, a cytoplasmic tail mutant of mCD1, which disrupts endosomal localization, has a greatly reduced capacity to present Ova to mCD1 restricted cells. Newly synthesized mCD1 molecules, however, are not required for Ova presentation, suggesting that molecules recycling from the cell surface are needed. Because of these data showing that mCD1 trafficks to endosomes, where it can bind peptides derived from exogenous proteins, we conclude that peptide antigen presentation by mCD1 is likely to be a naturally occurring phenomenon. In competition assays, ␣-galactosylceramide did not inhibit Ova presentation, and presentation of the glycolipid was not inhibited by excess Ova or the peptide epitope derived from it. This suggests that, although both lipid and peptide presentation may occur naturally, mCD1 may interact differently with these two types of antigens.