Antigen-specific human T-cell colony formation in a liquid culture system

Abstract

Keystone E, Poplonski L, Snow KM, Paige C, Miller RG. Antigen-specific human T-cell colony formation in a liquid culture system. Scand J Clin Lab Invest 1993; 53: 783-788.

We have developed a limiting dilution microculture T-cell cloning technique to generate antigen-specific T-cell clones. Cultures contained peripheral blood T-cells, irradiated autologous mononuclear cells (feeders), human AB serum and recombinant IL-2. Colonies were enumerated at 14 days. Under these conditions the cloning efficiency expressed as the frequency of proliferating cells in response to PHA, ranged from 1/1.07 to 1/3.35. In response to tetanus toxoid, the frequency of proliferating cells ranged from 1/415 to 1/5655. Average colony size ranged from 0.7 × 10⁵ to 6.2 × 10⁵ cells with a mean T-cell recovery of 2.5 × 10⁵ cells per microculture. Restimulation of 14-day tetanus toxoid T-cell colonies revealed marked proliferation to PHA and tetanus toxoid but only background responses to PPD. These studies document the development of a rapid, antigen-specific liquid culture T-cell cloning system which is likely to detect T-cell clones that are representative of the original T-cell population. The clones are of sufficient size to be useful in studies of antigen cross-reactivity.

Key Words:

• antigen driven
• limiting dilution
• PHA
• T-cell cloning