Endogenous and exogenous factors contributing to the surface expression of HLA B27 on mutant APC.
We have examined the expression of HLA B*2705 in the mutant cell line 721.220, which lacks endogenous HLA A and B alleles and expresses a defective tapasin molecule. Several peptide sensitive mAbs distinguish between HLA B*2705 expressed on the surface of 721.220 cells (B27.220) and 721.220 cells co-transfected with human tapasin (B27.220.hTsn). This differential staining defines subtle differences in the conformation of HLA B27, which most likely reflect changes in the repertoire of antigenic peptides bound to B27 in the presence and absence of wild type tapasin. HLA B27 molecules expressed on the surface of 721.220 display increased levels of "free" B27 heavy chain (HC-10 staining), an epitope that is dependent on TAP-translocated peptides. The conformation and stability of B27 molecules was examined by investigating the integrity of mAb epitopes and the half-lives of these complexes on cells cultured with and without serum. The decay of surface B27 epitopes occurred more rapidly in B27.220 and this effect was exaggerated in serum free media. Importantly, the decay of surface B27 molecules in B27.220.hTsn cells was characterized by an early increase in HC-10 staining when the cells were grown in serum free media. This decay of B27 molecules via HC-10 reactive intermediates was not observed in B27.220 cells, implying molecules on these cells may already have passed through this stage prior to surface expression. Taken together these observations indicate that tapasin has a significant contribution to the composition and stability of the B27-bound peptide repertoire.
Purcell
A W
AW
Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia. a.purcell@microbiology.unimelb.edu.au
Kelly
A J
AJ
Peh
C A
CA
Dudek
N L
NL
McCluskey
J
J
D000938:Antigen-Presenting Cells / Q000187:drug effects / Q000276:immunology*; D017920:Antiporters / Q000494:pharmacology; D020126:Brefeldin A / Q000494:pharmacology; D002478:Cells, Cultured; D003470:Culture Media; D016895:Culture Media, Serum-Free; D005434:Flow Cytometry; D015395:Histocompatibility Antigens Class I / Q000494:pharmacology; D006801:Humans; D007136:Immunoglobulins / Q000494:pharmacology; D026901:Membrane Transport Proteins; D009154:Mutation
D017920:Antiporters; D003470:Culture Media; D016895:Culture Media, Serum-Free; D015395:Histocompatibility Antigens Class I; D007136:Immunoglobulins; D026901:Membrane Transport Proteins; C107762:tapasin; D020126:Brefeldin A