Efficient isolation of mutant antigen presenting cell lines by functional selection using T cell clones.
An efficient method for the isolation of mutant antigen-presenting cell (APC) lines is described. When mixtures of transfectant APC lines TA beta z (that express A beta z/A alpha d MHC class II molecules) and hypothetical variant APC lines TA beta d (that express A beta d/A alpha d class II molecules) were cultured with and selected by autoreactive A beta z/A alpha d-restricted T cell clones, the percentage of TA beta d APC lines increased from less than 1% of the original APC mixtures to almost 100% after several cycles of selection. This increase of hypothetical variant was shown to be due to the formation of aggregates of wild-type TA beta z APC lines with A beta z/A alpha d-restricted autoreactive T cell clones that results in the inhibition of proliferation and probably killing of TA beta z APC lines. Based on this, ethyl methane sulfonate (EMS)-treated TA beta z APC lines or B-B hybridoma APC lines MW4 (that express A beta z/A alpha d and A beta z/A alpha z class II molecules) were cultured with and selected by A beta z/A alpha d-restricted autoreactive T cell clones to obtain mutant APC lines that escaped the recognition by T cell clones. After cloning, about 43% of clones examined lost the ability to stimulate T cell clones with concomitant loss of class II molecule expression. Less than 1% showed loss of stimulatory activity against T cell clones in spite of the expression of normal amounts of class II molecules. Initial analysis revealed that they include APC mutant lines with (1) altered MHC class II sequences, (2) loss of adhesion molecule expression and (3) possible impairment of the peptide loading. The method described here may provide a variety of mutant APC lines that are useful for the analysis of antigen processing and presentation pathways as well as of class II structure for T cell stimulation.
Koarada
S
S
Department of Immunology, Saga Medical School, Japan.
Kubota
E
E
Tokushima
M
M
Naitoh
K
K
Miyake
K
K
Kimoto
M
M
D000818:Animals; D000911:Antibodies, Monoclonal; D000938:Antigen-Presenting Cells / Q000166:cytology / Q000276:immunology*; D002450:Cell Communication / Q000276:immunology; D002460:Cell Line; D002469:Cell Separation / Q000379:methods*; D002999:Clone Cells; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D014644:Genetic Variation; D000949:Histocompatibility Antigens Class II / Q000235:genetics; D006825:Hybridomas / Q000276:immunology; D007158:Immunologic Techniques; D008297:Male; D051379:Mice; D008814:Mice, Inbred NZB; D009154:Mutation; D013601:T-Lymphocytes / Q000276:immunology*; D014162:Transfection
D000911:Antibodies, Monoclonal; D000949:Histocompatibility Antigens Class II