Limit-dilution assay and clonal expansion of all T cells capable of proliferation

J Immunol Methods. 1982 Aug 13;52(3):307-22. doi: 10.1016/0022-1759(82)90003-5.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Antigens, Ly / genetics
  • Antilymphocyte Serum / pharmacology
  • Clone Cells / cytology
  • Clone Cells / immunology
  • Concanavalin A / pharmacology
  • Cytotoxicity Tests, Immunologic / methods*
  • Interleukin-2 / biosynthesis
  • Kinetics
  • Lymphocyte Activation* / radiation effects
  • Mice
  • Spleen / cytology
  • Spleen / immunology
  • Spleen / radiation effects
  • T-Lymphocytes / classification
  • T-Lymphocytes / cytology*
  • T-Lymphocytes / immunology

Substances

  • Antigens, Ly
  • Antilymphocyte Serum
  • Interleukin-2
  • Concanavalin A