Limit-dilution assay and clonal expansion of all T cells capable of proliferation.
A limit-dilution microculture system is presented in which almost all mature T cells, cultured at a level of about 1 cell/well, grow and expand to clones averaging 60,000 cells over an 8-9 day period. Cloning efficiency is 70-100%, so the set of the expanded clones is representative of the starting T-cell population. T cells of all Lyt phenotypes form clones of progeny cells. The system involves culture in flat-bottom microtitre trays, in the presence of concanavalin A as the initiating stimulus, together with appropriately irradiated spleen filler cells and a supplementary source of soluble T cell growth factors. The resultant clones may be screened for cytolytic function, as described in the accompanying paper. The system may be used to assay the level of T cells capable of expansion or precursor function (PTL-p) by using [3H]TdR uptake as a readout for the presence or absence of proliferating clones. Analysis of the frequency of positive cultures shows a good fit to the expected Poisson distribution, with no evidence of complicating suppressor or helper effects.
D000818:Animals; D000950:Antigens, Ly / Q000235:genetics; D000961:Antilymphocyte Serum / Q000494:pharmacology; D002999:Clone Cells / Q000166:cytology / Q000276:immunology; D003208:Concanavalin A / Q000494:pharmacology; D003601:Cytotoxicity Tests, Immunologic / Q000379:methods*; D007376:Interleukin-2 / Q000096:biosynthesis; D007700:Kinetics; D008213:Lymphocyte Activation / Q000528:radiation effects; D051379:Mice; D013154:Spleen / Q000166:cytology / Q000276:immunology / Q000528:radiation effects; D013601:T-Lymphocytes / Q000145:classification / Q000166:cytology* / Q000276:immunology
D000950:Antigens, Ly; D000961:Antilymphocyte Serum; D007376:Interleukin-2; D003208:Concanavalin A