Cellular co-localization of intron-4 containing mRNA and HLA-G soluble protein in melanoma analyzed by fluorescence in situ hybridization.
HLA-G5, -G6, and -G7 soluble isoforms of the immunosuppressive HLA-G molecule are produced from the splice variants of the primary HLA-G mRNA transcript containing intron-4 that encodes a specific 21 amino acids tail. In particular, HLA-G5 interacts with the inhibitory ILT2/4 and KIR2DL4 receptors that are expressed on immune cells. Acquisition of soluble HLA-G in the microenvironment may turn a HLA-G non-expressing cell into a HLA-G-bearing one. To address the question of how to distinguish cells that express soluble HLA-G generated by alternative splicing from those that have acquired HLA-G, we have developed a method capable of detecting intron-4 containing mRNA and protein in situ simultaneously. M8 melanoma cell line either transfected or not with HLA-G5 cDNA was analyzed by indirect immunofluorescence confocal microscopy using double staining with a HLA-G intron-4 digoxygenin labeled probe along with a monoclonal antibody directed against the 21 amino acid tail. The combined fluorescence in situ hybridization was also used on the HLA-G-positive choricarcinoma cell line JEG-3. This method would be helpful to follow-up bona fide HLA-G expression in a heterogeneous cell population and to elucidate the mechanisms underlying soluble HLA-G mediated immune modulation in physiological conditions such as pregnancy and pathophysiological situations such as cancer.
Sangrouber
Déborah
D
Service de Recherches en Hémato-Immunologie, Commissariat à L'Energie Atomique- DSV- I(2)BM, Institut Universitaire d'Hématologie, Hôpital Saint-Louis, Paris, France.
Marcou
Céline
C
Le Discorde
Magali
M
Chang
Chien-Chung
CC
Carosella
Edgardo D
ED
Moreau
Philippe
P
D045744:Cell Line, Tumor; D015342:DNA Probes / Q000138:chemical synthesis / Q000378:metabolism; D006680:HLA Antigens / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D059951:HLA-G Antigens; D015395:Histocompatibility Antigens Class I / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007438:Introns / Q000235:genetics*; D008545:Melanoma / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism*; D012995:Solubility; D014162:Transfection
D015342:DNA Probes; D006680:HLA Antigens; D059951:HLA-G Antigens; D015395:Histocompatibility Antigens Class I; D012333:RNA, Messenger