Invariant chain association with MHC class I: preference for HLA class I/beta 2-microglobulin heterodimers, specificity, and influence of the MHC peptide-binding groove.

MHC class I molecules require the assembly of heavy chain with beta2-microglobulin (beta 2m) and peptide in order to present Ag on the cell surface. Endoplasmic reticulum resident proteins associate with class I molecules and aid assembly. Free class I heavy chains associate with calnexin, which may facilitate association with beta 2m. Invariant chain (Ii) also associates with MHC class I molecules, but its role in class I assembly is not clear. We report here that Ii strongly associates with HLA class I/beta 2m heterodimers, but weakly with free class I heavy chains in HLA-B7-transfected T2 cells. Ii/HLA class I complexes persist stably within the endoplasmic reticulum/cis-Golgi compartment in peptide-processing deficient cells, but are much less prominent in normally processing cells. Furthermore, Ii differentially associates with variant HLA-B7 molecules that have peptide-binding groove mutations, and the degree of association correlates with HLA-B7 variant cell surface expression. Ii also shows HLA class I molecule specificity, associating to a greater degree with HLA-B7 than HLA-A2. Together these observations suggest that Ii stabilizes particular HLA class I/beta 2m heterodimers until peptide is loaded, and that this association may enhance class I cell surface expression.

Probing HLA-B7 conformational shifts induced by peptide-binding groove mutations and bound peptide with anti-HLA monoclonal antibodies.

To determine the influence of peptide-binding groove residues and MHC-bound peptide on HLA-B7 conformation, we investigated the binding sites of nine locus- or allele-specific mAbs using a panel of 82 HLA-B7 variants. The functional mAb epitopes encircle the HLA-B7 peptide-binding groove. Three mAbs are affected by mutations at solvent-accessible peptide-binding groove mutations. Mutations in peptide-binding groove residues 45, 63, and 150 affect multiple nonoverlapping mAb epitopes, probably by interaction with other MHC residues or bound peptide. However, 18 of 24 peptide-binding groove mutations do not affect mAb binding, indicating that the conformation of solvent-accessible HLA-B7 structures is largely dissociated from changes in the peptide-binding groove. To test whether bound peptides alter HLA-B7 conformation, we loaded HLA-B7 heavy chains on acid-stripped cells with beta2-microglobulin and 20 individual synthetic peptides. Two of eight mAbs are sensitive to HLA-B7-bound peptides. A likely interpretation of these data is that the conformational flexibility of HLA-B7 is due to peptide-induced conformational shifts in MHC side chains, rather than major shifts in the MHC main chain. These results suggest that HLA-B7 conformation is largely maintained in the context of different bound peptides and different peptide-binding grooves.

Induction of ornithine decarboxylase activity in mouse B lymphocytes.

We have examined the role in B cell activation of ornithine decarboxylase (ODC), the labile rate-limiting enzyme in the synthesis of polyamines thought to be required for S phase entry in all cells. When small resting mouse splenic B cells were stimulated with the mitogenic agents phorbol myristate acetate (PMA) plus ionomycin (lo), LPS or the B cell specific agent F(ab')2 anti-lg, ODC activity was greatly increased. ODC activity in small dense B cells remained near baseline levels for the first 6 h after treatment with LPS, but then increased approximately 150-fold in the next 18 h. When purified B cells were not separated by cell density, ODC activity was 30-fold greater at baseline and rose earlier after LPS stimulation, reaching a level about three times that of LPS-stimulated small, dense B cells at 24 h, implying that large (preactivated) B cells have much greater ODC responses than small, dense B cells. ODC activity, like S phase entry, could also be induced in small, dense B cells by PMA and lo but failed to respond to either agent alone. ODC levels rose transiently by approximately 40-fold between 2 and 6 h following stimulation of small B cells with F(ab')2 anti-lg, then declined to baseline. Whole anti-lg did not stimulate ODC activity and also blocked the F(ab')2 anti-lg mediated increase in ODC activity, just as it produced the expected inhibition of thymidine incorporation and cellular progression into S phase.(ABSTRACT TRUNCATED AT 250 WORDS)