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

In contrast to HLA class Ia, the HLA-G class Ib transcripts can be alternat iveley spliced to yield several isoforms including four potentially membran e-bound variants, namely HLA-G1, -G2, -G3 and G4. Ir is so far unclear whet her each of these splice variants lacking one or two external domains is pr operly translated and expressed at the cell surface. We used targeted Enhan ced Green Fluorescence Protein (EGFP)-HLA-G fusion cDNA to track HLA-G isof orm expression in living murine (L-human beta 2m) and human (JAR) transient ly transfected cells. It was demonstrated that he four MLA-G1, -G2, -G3, an d -G4 isoforms were translated in these transfectants Ly the means of(i) We stern blotting analysis, using an anti-EGFP mAb; (ii) intracellular double labeling flow cytometry analysis, using the EGFP natural fluorescence and p hycoerythrin-labeled HCA2 anti-HLA-G mAb; and (iii) immunocytochemistry on isolated acetone fixed transfectants with the use of different anti-HLA-G m Abs. 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 tra nsfectant was the green EGFP fluorescence also detectable at cite outer par r of the cells, suggesting that the HLA-G2, -G3, and G4 were retained in th e endoplasm ic reticulum. Such intracellular retention of the three shorter forms of HLA-G suggest chat they may play a role in regulating cell surfac e expression either of the full length HLA-G1 form or of HLA-E. (C) America n Society for Histocompatibility and Immunogenetics, 2000. Published by Els evier Science Inc.