Anti-CD3-stimulated T cells induce the production of multiple Ig H chain isotypes by individual human peripheral B lymphocytes.
Polyclonal activation of human B cells is achieved by coculture with T cells stimulated by mAb to the CD3 molecular complex. By formal limiting dilution analysis, approximately 60% of human peripheral blood B cells were found to produce Ig in this system. When individual B cells were cultured in microtiter wells with anti-CD3-activated T cells, more than one-third of cultures producing Ig contained multiple Ig H chain isotypes. Similar results were observed when individual IgM-expressing B cells, selected and dispersed by FACS were cultured with anti-CD3-activated T cells. The clonality of the B cells producing multiple Ig isotypes was supported by L chain analysis of the secreted Ig. Of the wells containing more than one H chain isotype, nearly 85% contained only a single L chain type. Clonality was further examined by polymerase chain reaction amplification of cDNA harvested from cultures originally seeded with individual B cells. In general, only a single VH gene family could be amplified from cultures producing more than one Ig isotype. Three separate VH regions were cloned and sequenced. One, a VHIV-mu was nearly identical to a previously described VH gene VH71.4; as second, a VHIV-gamma was very similar to a previously described VH gene segment V-79, whereas a third, a VHIII-gamma differed by 14% in nucleotide sequence from its closest germline counterpart VH3005. These results indicate that anti-CD3-activated T cells not only stimulate the majority of B cells to secrete Ig, but also induce individual B cells to produce multiple Ig H chain isotypes. Additionally, the procedure described provides a reliable method to sample a large proportion of the human peripheral B cell repertoire.
Kelly
P J
PJ
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235.
Pascual
V
V
Capra
J D
JD
Lipsky
P E
PE
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000945:Antigens, Differentiation, T-Lymphocyte / Q000276:immunology*; D001402:B-Lymphocytes / Q000276:immunology*; D001483:Base Sequence; D017252:CD3 Complex; D002478:Cells, Cultured; D005803:Genes, Immunoglobulin; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis*; D007132:Immunoglobulin Isotypes / Q000096:biosynthesis*; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011948:Receptors, Antigen, T-Cell / Q000276:immunology*; D013601:T-Lymphocytes / Q000502:physiology*
D000911:Antibodies, Monoclonal; D000945:Antigens, Differentiation, T-Lymphocyte; D017252:CD3 Complex; D007143:Immunoglobulin Heavy Chains; D007132:Immunoglobulin Isotypes; D007147:Immunoglobulin Light Chains; D011948:Receptors, Antigen, T-Cell