Modification of membrane cholesterol level affects expression and clustering of class I HLA molecules at the surface of JY human lymphoblasts.
Recently we have found that class I HLA molecules, key elements of the antigen presentation system for CD8 + effector cells, show a clustered lateral distribution (homoassociation) at the surface of activated human T- and B-lymphocytes as well as virus-transformed T- and B-lymphoblasts, in contrast to a disperse distribution on resting human PBLs (Matk6 et al. (1994) J. Immunol. 152, 3353; Bene et al. (1994) Eur. J. Immunol. 24, 2115). Expression of beta2m-free HLA heavy chains and exogenous beta2m have been shown as potential regulation factors of HLA-I clustering, which in turn may affect cytotoxic activity of CD8+ effector cells. Here we report a study on the effect of plasma membrane-modification (by exogenous cholesterol and phosphatidylcholine) on the expression of free HLA heavy chains and beta2m-bound HLA-I molecules on JY human B-lymphoblasts. The modulating effect of these two treatments on the lipid fluidity of cells was demonstrated by fluorescence anisotropy of DPH lipid probe. The lateral clustering (association) of HLA-I molecules was detected by flow cytometric fluorescence resonance energy transfer (FCET) and digital imaging microscopic photobleaching energy transfer (pbFRET) methods, using flourescein-isothiocyanate (FITC) (donor)- and tetramethyl-rhodamine-isothiocyanate (TRITC) (acceptor)-labeled W6/32 or KE2 antibodies directed against intact HLA-I molecules. Cholesterol enrichment of the plasma membrane increased membrane fluidity and reduced the expression of heavy- and light-chain determinants of HLA-I molecules and free heavy chains (FHCs). This was accompanied with a higher degree of HLA-I clustering as shown by the enhanced intermolecular energy transfer efficiency. In contrast, cholesterol depletion resulted in membrane fluidization and increased expression of HLA-I epitopes. Our results suggest that both cholesterol level and lipid structure/fluidity of the plasma membrane in lymphoblastoid cells may also potentially regulate lateral organization and consequently the presentation efficiency of HLA-I molecules.
Bodnár
A
A
Department of Biophysics, Medical University School of Debrecen, Hungary.
Jenei
A
A
Bene
L
L
Damjanovich
S
S
Matkó
J
J
D002461:Cell Line, Transformed; D002462:Cell Membrane; D002784:Cholesterol / Q000378:metabolism*; D000939:Epitopes / Q000276:immunology; D015395:Histocompatibility Antigens Class I / Q000096:biosynthesis*; D006801:Humans; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000166:cytology / Q000276:immunology*
D000939:Epitopes; D015395:Histocompatibility Antigens Class I; D002784:Cholesterol