Isolation of two CD50 (ICAM-3)-negative Jurkat T-cell clones and their application for analysis of CD50 function.
The leukocyte differentiation antigen CD50 (intercellular adhesion molecule-3, ICAM-3), mediates cell-cell adhesion through its ligand LFA-1 and is a transducting receptor molecule during T-cell activation. Since CD50 homologues in other species have not yet been identified, the role of this molecule can only be analyzed in human cell models. Thus, to better study CD50 function in T cells, we have obtained two CD50-negative T-cell clones, named CAMY.1 and CAMY.2. These clones were derived from the Jurkat T-cell variant PPL.1. Data from analysis of protein expression, specific mRNA content and calcium mobilization assays have confirmed the absence of functional CD50 molecules on these two clones. Thus, CAMY.1 and CAMY.2 show no CD50 expression by phenotypical and immunoprecipitation analysis. CD50-specific mRNA content is undetectable by Northern blot analysis in these clones and, only, when RT-PCR was performed could specific mRNA be detected. Additionally, CD50 cross-linking on theses clones shows no increase in intracellular calcium. Transfection of CD50 cDNA on CAMY cells restores not only CD50 surface expression, but its functional ability to induce calcium mobilization, CD69 upregulation and cell morphological changes. The CAMY.1 and CAMY.2 clones provide useful model systems to analyze CD50 function in T cells.
Vilardell
C
C
Servei d'Immunologia, Hospital Clinic, Barcelona, Spain.
Juan
M
M
Miralles
A
A
Barceló
J J
JJ
Esparza
J
J
Palou
E
E
Vilella
R
R
Places
L
L
Lozano
F
F
Alberola-Ila
J
J
Gayà
A
A
Yagüe
J
J
D000818:Animals; D015703:Antigens, CD; D000943:Antigens, Differentiation; D015152:Blotting, Northern; D017252:CD3 Complex / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000502:physiology*; D002469:Cell Separation; D002999:Clone Cells; D003432:Cross-Linking Reagents; D006801:Humans; D019169:Jurkat Cells; D051379:Mice; D011233:Precipitin Tests; D012333:RNA, Messenger; D013601:T-Lymphocytes / Q000378:metabolism / Q000502:physiology*; D014162:Transfection
D015703:Antigens, CD; D000943:Antigens, Differentiation; D017252:CD3 Complex; D015815:Cell Adhesion Molecules; D003432:Cross-Linking Reagents; C483616:ICAM3 protein, human; D012333:RNA, Messenger; D002118:Calcium