QUANTITATIVE STUDIES ON PRECURSORS OF CYTOTOXIC LYMPHOCYTES .1. CHARACTERIZATION OF A CLONAL ASSAY AND DETERMINATION OF SIZE OF CLONES DERIVED FROM SINGLE PRECURSORS

  • 1 January 1977
    • journal article
    • research article
    • Vol. 118  (3) , 1049-1056
Abstract
A microculture system for estimating the frequency of cytotoxic [mouse] lymphocyte precursors (CLP) to alloantigens is described. Cytotoxic T [thymus-derived] 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 51Cr-P815 [mastocytoma] (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 [lethal dose] .apprx. 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-.nu.N where Po = % nonresponders, .nu. = 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 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, the average clone size per precursor after 7 days in culture is about 1040. This assay could detect clones with as few as 40 CL/well. Since the average clone size in 26 times the detection limit, the assay for CLP is highly sensitive and does not represent a minimum estimate.