SummaryRecently several cell lines have been identified with mutations in a major histocompatibility complex (MHC)-linked protein that lead to defects in class II-restricted antigen presentation and a defect in the formation of class II SDS-stable dimers. The defect in these calls has recently been shown to result from the inability to express the MHC-encoded nonclassical class II molecule called DM. To further examine the role of DM in class II-restricted antigen presentation, we asked if this defect would equally affect different allelic and species variants of class II molecules. To investigate this, we transfected the parent call lines T1 and 8.1.6 and their respective antigen presentation mutants T2 and 9.5.3 with the genes encoding I-A d and examined the derived transfectants for their ability to present antigen, the conformation of I-A a at the cell surface, association of I-A a with invariant chain (Ii), and the ability to form I-A d SDS-stable dimers. The lack of functional DM expression did not affect any of the anti-I-A d monoclonal antibody (mAb) epitopes tested or the ability of I-A d to associate and dissociate with Ii. Surprisingly, these studies also revealed that the antigen presentation defect observed for DR. in the 9.5.3 cells did not compromise I-Ad-restricted antigen presentation. In addition, we found that the level of SDS-stable dimer formation did not correlate with antigen presentation capacity for I-A d and that the amount of SDS-stable I-A d dimer depends on the cellular context in which the class II molecule is expressed. Our results suggest that the ability to form SDS-stable dimer is not strictly correlated with dass II-restricted antigen presentation. Finally, when two allelic forms of murine class II molecules were compared in the defective T2 cell line, it was found that I-A k but not I-A d forms SDS-stable dimers equivalent to that seen in the parental cell lines. Overall, our results suggest that DM may modulate rather than play a requisite role in I-A drestricted antigen presentation and SDS-stable dimer formation and that dependency on DM may be allele or species specific. M HC class II molecules have the ability to present a wide . array of antigens derived from both exogenous and endogenous sources. Studies involving the use of lysosomotropic reagents (1), those that have examined the sites of class II localization within the APC (2-6), the biochemistry of invariant chain (Ii) 1 (reviewed in 7), and class II conformation (8-12) have led to a general model of class II-restricted antigen,presentation. In this model, class II molecules bind Ii in the endoplasmic reticulum (ER), an event which blocks the loading of most endogenous peptides (13,14). The class Abbreviations used in this paFr: ADH, alcohol dehydrogenase; c, chicken; CLIP, class lI-assodated invariant chain peptide; ER, endophsmic reticulum; GAM, goat anti-mouse; HEL, hen egg lysosome; Ii, invariant chain; MYO, myoglobin; PAS, protein A-Sepharose; PINS, pork insulin; t, turkey.II/Ii complex then traffics out of th...

Although reported examples of endogenous antigen (Ag) presentation by major histocompatibility complex (MHC) class II molecules have increased, the mechanisms governing this process remain poorly defined. In this communication, we describe an experimental system designed to examine the mechanisms governing class II presentation of internal Ag. Our target peptide is processed from a transmembrane protein constitutively expressed by a variety of nucleated cells (MHC class I, H-2Ld), is naturally displayed by MHC class II molecules in vivo, and is recognized by a class II-restricted, CD4+ T cell hybridoma. Our results indicate that presentation of the Ld target Ag is independent of its plasma membrane expression, may not involve endosomal proteolysis, and thus may be distinct from the classically defined class II presentation pathway. In addition, the observations that Ld presentation does not require a functional TAP-1 complex, is not blocked by invariant chain, and cannot utilize cytoplasmic forms of H-2Ld, suggest that a classical class I pathway is not involved in this presentation event. Finally, our data suggest that different cofactors participate in MHC class II presentation of exogenous and endogenous Ag, and that disparate Ag presenting cells, such as B, T, and pancreatic islet cells, may differentially express these two class II pathways of Ag presentation.

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