Cloning of human lymphocytes using limiting dilution

A simple technique for cloning lymphocytes at limiting dilution in microwells has been developed using phytohaemagglutinin, irradiated cells and conditioned medium containing interleukin-2. For freshly isolated peripheral blood lymphocytes, the mean frequency of clone forming cells (CFC) was 29.4% and for continuously cultured lymphocytes it was 25.5%. Quantitative considerations, mixing experiments involving HLA-A2 positive and negative cells and replating experiments indicated that growth in most of the positive microwells originated from a single cell which had a very high self-renewal capacity and underwent approximately 15 divisions during the 14 days of culture. The high plating efficiency and sustained growth observed with this technique suggests that it is the method of choice for enumerating clone forming cells or for isolating clones.