The requirement for DM in class II-restricted antigen presentation and SDS-stable dimer formation is allele and species dependent.

Abstract

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...

Relaxed DM Requirements During Class II Peptide Loading and CD4+ T Cell Maturation in BALB/c Mice

“…To further explore allele-specific DM and Ii chain requirements under physiological conditions, mutant spleen cells were tested for their abilities to stimulate a diverse panel of A d -and E d -restricted T cell hybridomas (Table I). Unlike DM-independent activity observed for transfected cell lines (19,20), in this study we found presentation of intact protein Ags to A d -restricted T cell clones strictly requires DM coexpression (Fig. 5a).…”

“…The class II chain specificities (36,37) and Ii chain influences affecting expression of these conformational epitopes (10,34) have been extensively discussed. Rabbit antisera specific for determinants located in the cytoplasmic tails of the ␣-and ␤-chains were generously provided by Ronald N. Germain (YFMYSKLRVQKSTWERG), bacteriophage repressor cI peptide P [12][13][14][15][16][17][18][19][20][21][22][23][24][25][26] (LEDARRLKAIYEKKK), SWM 129 -153 GAMNKALELFRKDI-AAKYKELGYQG, the truncated variant of moth cytochrome 88 -103 previously described as DASP KKANELIAYLKQATK, the Ii chain 85-101 KPVSQMRMATPLLMRPM peptides, and biotin-conjugated variants were purchased from Quality Controlled Biochemicals (Hopkinton, MA).…”

DM Loss in k Haplotype Mice Reveals Isotype-Specific Chaperone Requirements

“…Class II polymorphism also has a significant impact on DM interactions with empty class II, as judged by differential abilities to enhance peptide exchange at acidic pH (8). Allele-specific DM requirements during class II maturation and Ag presentation have been previously investigated, but inconsistencies in the literature make it difficult to evaluate these observations (22)(23)(24)(25)(26). Considerable evidence suggests that the extent of Ii chain and DM functional contributions to class II presentation depends on the particular epitope, its sensitivity to proteases, and its availability inside various peptide-loading compartment(s) (27)(28)(29)(30)(31)(32)(33)(34).…”

Regulation of the Class II MHC Pathway in Primary Human Monocytes by Granulocyte-Macrophage Colony-Stimulating Factor

“…Monocytes from the remaining four experiments did not appreciably change or expressed higher relative CLIP class II levels after treatment with GM-CSF, suggesting that there is variability between donors. This is likely due to allelic MHC differences that affect binding to CLIP and dependence on HLA-DM (23,24). The finding that GM-CSF decreases the relative level of CLIP-loaded class II molecules on the surface of primary human monocytes suggests that HLA-DM expression and/or function may be enhanced, leading to the generation of more stable peptide/MHC complexes on the cell surface.…”