One-step procedure for human T-lymphocyte colony growth and its regulation by T and B cells, and monocytes

Previous systems for cloning human T-cells have been either one step procedures with small colony size (40 less than 40 cells) or two-step procedures with agglutination problems. These systems also require erythrocytes, thiols and human serum. We have regularly grown more than 10(3) colonies (more than 40 cells)/5 X 10(5) cells from purified T-cells or phagocytic and adherent cell depleted mononuclear cells in agar using phytohemagglutinin (PHA) as the mitogenic stimulus. The reason for the poor colony size in other single step procedures using agar may be related to cell-to-cell interaction. We found that both adherent (Ad) and phagocytic cells were suppressive of T-cell colony growth (TCCG) in donors with low or absent TCCG from whole mononuclear cells. This effect was reproduced by adding Ad cells to T-cell concentrated fractions. Both irradiated T and unirradiated enriched B-cells increased TCCG of T fractions. TCCG is possible in simple in vitro systems from both adherent and phagocytic cell depleted fractions and concentrated T-cell fractions. This allows for the examination of factor regulation of TCCG and has enabled the identification of a possible B-cell released T-cell growth factor. Previously described complex growth requirements may be related to cell interactions.