title: Quantitative studies on the precursors of cytotoxic lymphocytes. I. Characterization of a clonal assay and determination of the size of clones derived from single precursors.
abstract: A microculture system for estimating the frequency of cytotoxic lymphocyte precursors (CLP) to alloantigens is described. Cytotoxic T lymphocytes (CL) were generated by culturing limiting numbers of RNC (H-2k)-nu/+lymph node (LN) cells with irradiated C3D2F1 (H-2k/d) spleen cells in the presence of RNC-nu/nu spleen cells for 7 days in microtiter trays. At the end of the culture period, individual wells were assayed for cytotoxic effector cells directed against 51CrP815 (H-2d) target cells. The effector cells generated under these conditions are sensitive to killing by rabbit anti-mouse brain serum and guinea pig complement. The process of differentiation from precursors to CL is radiation sensitive (Do approximately 180 rads). The frequency of precursors for H-2d was calculated by fitting the proportion of nonresponding cultures to the zero order term of the Poisson distribution, Po = e-vn, where Po = per cent nonresponders, v = precursor frequency, and N = number of LN cells cultured per well. The frequency of precursors in the RNC-nu/+LN population responding to the H-2d haplotype was found to be 1 in 776. Under conditions where no backstimulation is possible, the frequency of precursors responding to the H-2d haplotype is 1 in 885 for B6-nu/+LN and 1 in 2550 for B6-nu/+ spleen, respectively. In combination with a visual assay for individual CL, it was found that the average clone size per precursor after 7 days in culture is about 1040. Our assay could detect clones with as few as 40 CL/well. Since the average clone size in 26 times the detection limit, we conclude that the assay for CLP is highly sensitive and does not represent a minimum estimate.
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D002999:Clone Cells; D003601:Cytotoxicity Tests, Immunologic; D007519:Isoantigens; D008213:Lymphocyte Activation; D007959:Lymphocyte Culture Test, Mixed; D051379:Mice; D008819:Mice, Nude; D013154:Spleen / Q000276:immunology; D013601:T-Lymphocytes / Q000166:cytology* / Q000276:immunology
chemical_list: D007519:Isoantigens