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A simple whole blood limiting dilution technique was developed to clone human lymphocytes non-specifically. The geometric mean frequency of clone forming cells in 13 normal individuals was found to be 31.1%. Compared with measurement of proliferation in mass suspension culture, cloning provides a quantitative and easily interpretable endpoint for a variety of lymphocyte studies and its use in measurement of radiation sensitivity is presented as an example.

Detailed methods are presented for measurement and study of in vivo mutations and in vitro mutagenesis in human lymphocytes. The methods described include preparation of conditioned medium containing interleukin-2, enumeration of mutant clones, in vitro mutagenesis, and expansion of mutant clones for further study.

The genetic stability of normal and neoplastic lymphocytes was compared by using base-line mutation frequency and mutation rate/cell generation. Mutations at the hypoxanthine-guanine phosphoribosyltransferase locus were studied by enumerating thioguanine-resistant cells in a clonogenic assay. The base-line ("spontaneous") mutation frequency was 1.52 X 10(-6), 6.38 X 10(-6), and 1.06 X 10(-6) for normal cells from three individuals and was 1.16 X 10(-3), 6.08 X 10(-5), and 3.06 X 10(-5) for the three malignant cell lines, Jurkat (JM), HRIK, FMC-Hu1B, respectively. The mutation cell/generation rate was 24.6 X 10(-8), 15 X 10(-8), and 5.5 X 10(-8) for lymphocytes from the three normal individuals, and 666.4 X 10(-8), 52.8 X 10(-8), and 131 X 10(-8) for the three malignant cell lines. The results suggest that neoplastic lymphocytes are more genetically unstable than normal lymphocytes.

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.

Human lymphocytes mutated at the HLA-A2 or HLA-A3 alleles were enumerated and studied by primary selection using antibody and complement, followed by limiting dilution cloning and secondary selection using immunofluorescence or antibody and complement. The geometric mean frequency of in vivo mutant lymphocytes was 3.08 X 10(-5) for the HLA-A2 allele and 4.68 X 10(-6) for the HLA-A3 allele. Mutagenesis by X-radiation or mitomycin produced a dose-related increase in mutant frequency. HLA-B phenotyping and Southern Analysis of the HLA-A gene suggested that mutation was frequently due to gene deletion, which was often substantial.

Several theories of ageing predict that somatic mutations should increase with age. This prediction was tested for human lymphocytes using a recently developed clonal technique for enumeration of mutations, and an increase of 1.6% per year in mutations with age was detected.

The capacity of lymphocytes from 23 human subjects to metabolize the model carcinogen 2-acetylaminofluorene (AAF) was assessed. These cells metabolized AAF to its 1-, 7- and N-hydroxylated metabolites. Alpha-naphthoflavone and metyrapone exhibited IC50's for all pathways of 0.02 microM and 1 mM, respectively. These data indicate that the same form of cytochrome P450 or forms with similar IC50's are mediating these reactions. No significant difference was observed between lymphocytes from smokers versus non-smokers in the N-hydroxylation of AAF, the initial step in the metabolic activation of this carcinogen. However, lymphocytes from smokers were faster detoxifiers of AAF as seen in their increased capacity to 1- and 7-hydroxylate AAF compared to lymphocytes from non-smokers.

The sensitivity of human peripheral blood lymphocytes to bleomycin and mitomycin-C was assessed by measuring the inhibition of phytohemagglutinin stimulated proliferation. The sensitivity to bleomycin, and not to mitomycin-C, increased with the age.