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Endogenous and exogenous factors contributing to the surface expression of HLA B27 on mutant APC
A W Purcell1, A J Kelly, C A Peh
1Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia. a.purcell@microbiology.unimelb.edu.au
Abstract:
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.
Insights
Tapasin significantly influences the peptide repertoire and stability of HLA B*2705 molecules. Its presence alters HLA B27 conformation and surface expression dynamics, impacting immune recognition.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- The human leukocyte antigen (HLA) B*2705 plays a crucial role in immune responses.
- Tapasin is an MHC class I-associated molecule essential for proper peptide loading.
- Understanding the role of tapasin in HLA B27 expression is vital for immunology.
Purpose of the Study:
- To investigate the impact of tapasin on the conformation and peptide repertoire of HLA B*2705.
- To analyze the stability and surface expression of HLA B*2705 in the presence and absence of tapasin.
Main Methods:
- Utilized the 721.220 mutant cell line lacking endogenous HLA A and B alleles and expressing defective tapasin.
- Employed peptide-sensitive monoclonal antibodies (mAbs) to differentiate HLA B*2705 conformations.
- Assessed surface expression stability by examining mAb epitope integrity and molecular half-lives.
- Investigated "free" B27 heavy chain levels using HC-10 staining.
Main Results:
- Differential mAb staining revealed conformational changes in HLA B*2705 dependent on tapasin presence.
- HLA B*2705 on tapasin-deficient cells showed increased "free" B27 heavy chain (HC-10 epitope).
- Surface HLA B*2705 decayed more rapidly in tapasin-deficient cells, especially in serum-free media.
- Tapasin-deficient cells exhibited unique HC-10 staining patterns during decay, suggesting altered peptide loading.
Conclusions:
- Tapasin significantly contributes to shaping the peptide repertoire bound by HLA B*2705.
- The presence of tapasin enhances the stability of HLA B*2705 on the cell surface.
- Tapasin influences the conformational state of HLA B*2705, affecting its interaction with peptides and immune recognition.