The full length HLA-G1 and no other alternative form of HLA-G is expressed at the cell surface of transfected cells

In contrast to HLA class Ia, the HLA-G class Ib transcripts can be alternativeley spliced to yield several isoforms including four potentially membrane-bound variants, namely HLA-G1, -G2, -G3 and G4. Ir is so far unclear whether each of these splice variants lacking one or two external domains is properly translated and expressed at the cell surface. We used targeted Enhanced Green Fluorescence Protein (EGFP)-HLA-G fusion cDNA to track HLA-G isoform expression in living murine (L-human beta 2m) and human (JAR) transiently transfected cells. It was demonstrated that he four MLA-G1, -G2, -G3, and -G4 isoforms were translated in these transfectants Ly the means of(i) Western blotting analysis, using an anti-EGFP mAb; (ii) intracellular double labeling flow cytometry analysis, using the EGFP natural fluorescence and phycoerythrin-labeled HCA2 anti-HLA-G mAb; and (iii) immunocytochemistry on isolated acetone fixed transfectants with the use of different anti-HLA-G mAbs. Cell surface flow cytometry analysis using the HCA2 mAb revealed that only the HLA-G1 isoform was expressed as a membrane-bound protein Two color confocal microscopy performed on fixed, permeabilized cells further showed that the EGFP green fluorescence co-localized with anti-calnexin rhodamine florescence in the four HLA-G isoform transfectants but only in HLA-G1 transfectant was the green EGFP fluorescence also detectable at cite outer parr of the cells, suggesting that the HLA-G2, -G3, and G4 were retained in the endoplasm ic reticulum. Such intracellular retention of the three shorter forms of HLA-G suggest chat they may play a role in regulating cell surface expression either of the full length HLA-G1 form or of HLA-E. (C) American Society for Histocompatibility and Immunogenetics, 2000. Published by Elsevier Science Inc.