Cloning of functional human T lymphocytes by limiting dilution: impact of filler cells and interleukin 2 sources on cloning efficiencies.
Conditions for the cloning of alloactivated human lymphocytes by limiting dilution in the presence of interleukin 2 (IL 2) and filler cells have been investigated. Cloning efficiencies were extremely high (at least 40%) when IL 2 derived from pooled peripheral blood mononuclear cells (PBM), stimulated with phytohaemagglutinin in the presence of irradiated Epstein-Barr virus-transformed lymphoid cell lines (LCL), was used in combination with irradiated pooled PBM as filler cells. Cloning efficiencies were reduced almost twofold using autologous filler cells and were further reduced dramatically using autologous IL2, although some clones could still be obtained. In addition, cloning efficiencies were acceptable (over 30%) when IL 2 produced spontaneously from the leukaemic cell Jurkat (M-N) was used. In contrast, sheep erythrocytes and LCL as filler cells failed to allow successful cloning, although LCL were beneficial in the expansion of cloned populations. Other factors being equal, the use of pooled PBM as filler cells and highly active preparations of IL 2 will be the method of choice for cloning functional human T lymphocytes.
D002455:Cell Division; D002460:Cell Line; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D002999:Clone Cells / Q000166:cytology*; D046508:Culture Techniques / Q000379:methods*; D004854:Herpesvirus 4, Human; D006801:Humans; D007376:Interleukin-2 / Q000494:pharmacology*; D008222:Lymphokines / Q000494:pharmacology*; D013601:T-Lymphocytes / Q000166:cytology*
D007376:Interleukin-2; D008222:Lymphokines