CLONING OF FUNCTIONAL HUMAN LYMPHOCYTE-T BY LIMITING DILUTION - IMPACT OF FILLER CELLS AND INTERLEUKIN-2 SOURCES ON CLONING EFFICIENCIES

Abstract

Conditions for the cloning of alloactivated human lymphocytes by limiting dilution in the presence of interleukin 2 (IL 2) and filler cells was investigated. Cloning efficiencies were extremely high (at least 40%) when IL 2 derived from pooled peripheral blood mononuclear cells (PBM), stimulated with phytohemagglutinin 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 2-fold using autologous filler cells were further reduced dramatically using autologous IL 2, although some clones could still be obtained. Cloning efficiencies were acceptable (> 30%) when IL 2 produced spontaneously from the leukemic cell Jurkat (M-N) was used. 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.