ANTIGEN-SPECIFIC HUMAN T-CELL COLONY FORMATION IN A LIQUID CULTURE SYSTEM

We have developed a limiting dilution microculture T-cell cloning tech nique to generate antigen-specific T-cell clones. Cultures contained p eripheral blood T-cells, irradiated autologous mononuclear cells (feed ers), human AB serum and recombinant IL-2. Colonies were enumerated at 14 days. Under these conditions the cloning efficiency expressed as t he frequency of proliferating cells in response to PHA, ranged from 1/ 1.07 to 1/3.35. In response to tetanus toroid, the frequency of prolif erating cells ranged from 1/415 to 1/5655. Average colony size ranged from 0.7 x 10(5) to 6.2 x 10(5) cells with a mean T-cell recovery of 2 .5 x 10(5) cells per microculture. Restimulation of 14-day tetanus tor oid T-cell colonies revealed marked proliferation to PHA and tetanus t oroid but only background responses to PPD. These studies document the development of a rapid, antigen-specific liquid culture T-cell clonin g system which is likely to detect T-cell clones that are representati ve of the original T-cell population. The clones are of sufficient siz e to be useful in studies of antigen cross-reactivity.