Cloning non-transformed sheep B cells

• Published in: Journal of immunological methods 2000-04, Vol.237 (1), p.19-28
• Main Authors: Griebel, Philip J, Beskorwayne, Terry, Godson, Dale L, Popowych, Yurij, Hein, Wayne
• Format: Article
• Language: English
• Subjects: Animals; Antibody secretion; B-Lymphocytes - cytology; B-Lymphocytes - immunology; Base Sequence; CD40 ligand (CD154); Cell Culture Techniques - methods; Clone Cells; DNA Primers - genetics; DNA, Complementary - genetics; Flow Cytometry; Gamma-chain common cytokines; Genes, Immunoglobulin; Humans; Immunoglobulin G - metabolism; Immunoglobulin genes; Immunoglobulin M - metabolism; Mice; Molecular Sequence Data; Peyer's Patches - cytology; Peyer's Patches - immunology; Peyer’s patches; Receptors, Antigen, B-Cell - metabolism; Sheep - genetics; Sheep - immunology
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/S0022-1759(99)00247-1

The capacity to clone B cells and establish permanent B cell lines has greatly facilitated a wide variety of studies characterising the growth, differentiation, and gene expression of murine and human B cells. Similar investigations of B cell biology for other species have been severely restricted by an inability to culture or clone B cells. This is the first report of a method to clone non-transformed sheep B cells using a culture system based on murine CD154 and a combination of human gamma chain-common cytokines. Sheep Peyer’s patch B cells were cultured for 120 days and then cloned by limiting dilution culture. The parental B cell culture contained both surface immunoglobulin (sIg)M + and sIgG1 + B cells and both types of B cell were cloned. Clonality was confirmed by PCR analysis of Ig heavy chain (HC) and light chain (LC) expression and DNA sequencing of HC V genes. There was agreement between the PCR and flow cytometric analyses of HC isotype expression on the B cell clones but the available monoclonal antibodies specific for sheep λ and κ LC did not react with all clones. Soluble Ig was detected in the culture supernatant of sIgG1 + clones but not sIgM + clones. The B cell clones remained dependent upon CD154 and gamma chain-common cytokine co-stimulation for sustained growth and maintained stable Ig expression. The cloning of non-transformed sheep B cells should provide a valuable tool for studying sheep B cell biology, establishing Ig HC- and LC-specific monoclonal antibodies, analysing the B cell Ig repertoire, and may be used to produce sheep monoclonal antibodies.