IDENTIFICATION OF HYBRIDOMAS DERIVED FROM MOUSE CD5(-LYMPHOCYTES BY FLUORESCENT STAINING FOR CYTOPLASMIC CD5 EXPRESSION() B)

The production of hybridomas derived from CD5(+) B lymphocytes is impo rtant for studying the immunoglobulin gene repertoire and antibody spe cificity of this B-lymphocyte subpopulation. However, hybridomas deriv ed from these lymphocytes invariably lose surface membrane expression of CD5 following hybridization. This impedes the unequivocal assignmen t of the generated hybridomas to the CD5(+) B-lymphocyte subpopulation from unsorted, or partially sorted, cells. Recent studies have shown that mRNA transcripts of the CD5 gene and the protein product can be d etected in the cytoplasm of some mouse hybridomas, indicating that alt hough surface expression has been lost, they may have been generated f rom CD5(+) B lymphocytes. These studies, however, have been unable to discount completely the possibility that aberrant cytoplasmic expressi on of the CD5 gene occurred as a direct consequence of the hybridizati on process. To confirm that cytoplasmic CD5 expression in the hybridom as is in fact directly related to the B-lymphocyte origin we have gene rated hybridomas from FACS-sorted CD5(+) and CD5(-) murine splenic B l ymphocytes, and determined their surface and cytoplasmic CD5 expressio n by fluorescence-activated cell sorting. Our findings reveal that all hybridomas derived from CD5(+) B lymphocytes (nine hybridomas) were n egative for surface but positive for cytoplasmic CD5 expression, where as all hybridomas derived from CD5(-) B lymphocytes (nine hybridomas) were negative for both surface and cytoplasmic CD5 expression. This fi nding shows that staining for cytoplasmic CD5 expression provides an a ccurate method of determining the cellular origin of murine B-lymphocy te hybridomas.