Colony formation by subpopulations of human T lymphocytes. I. Effects of phytohaemagglutinin and lymphocytosis-promoting factor from Bordetella pertussis

Abstract

A method is described for the growth in semi-solid agar medium of human lymphocyte colonies in response to stimulation by lymphocytosis-promoting factor (LPF) derived from Bordetella pertussis.

Colony formation was dependent on (a) a liquid pre-culture step with LPF prior to agar seeding, (b) presence of LPF in the agar medium, (c) a cell density in the agar culture of more than 5000 cells per ml.

Optimal colony formation was obtained with 30 μl LPF preparation in the liquid pre-culture step and 20 μl LPF preparation in the agar medium. Moreover, colony development improved after addition of 5 × 10⁻⁵M 2-mercaptoethanol and 0·6% human AB serum. The frequency of LPF-induced T-lymphocyte colonies in ten normal adult donors was 5450±1800 per 10⁶ mononuclear blood cells. In comparison, the frequency of phytohaemagglutinin (PHA)-induced colonies was 20,000±2500 per 10⁶ mononuclear cells plated directly in the agar medium. Lymphocytes seeded in agar medium with LPF started to divide within 2–3 days of culture and formed colonies of 30 to 200 cells on day 7 of culture. Cultures became moribund at day 8. The colony cells were negative for surface immunoglobulin and approximately 75% formed rosettes with sheep red blood cells (SRBC). No synergistic effect between LPF and PHA on colony formation was observed when PHA and LPF were used in the first and second step of culture respectively. Similarly, addition of LPF did not influence the growth of PHA-induced colonies.

When mononuclear cells were depleted of monocytes prior to agar culture addition of supernate factor(s) from cultures of adherent blood mononuclear cells (AC-CM) was necessary to ensure optimal development of colonies.

Mononuclear cells were separated by various rosette-depletion techniques. Colony-forming cells were recovered in the sheep red blood cell-rosetting fraction (E-RFC) of human blood lymphocytes indicating that these precursor cells are themselves T lymphocytes. E-RFCs were separated into Fc(γ)-receptor-positive and negative subpopulations. The frequency of colony-forming cells in the Fc(γ)-receptor-negative T-lymphocyte population was two to ten times higher than that of the Fc(γ)-positive T-cell population. Co-culture of equal numbers of Fc-positive and Fc-negative T lymphocytes reduced the frequency of T colonies by 20 to 95%.

Mononuclear cells were separated by passage through Ig-anti-Ig-coated columns. Attempts to grow LPF-stimulated colonies from passaged lymphocytes failed, suggesting that the colony-forming cells or cells necessary for colony formation were trapped in the column. In contrast, PHA-induced colony formation was enriched in cells which had passed through Ig-anti-Ig-coated columns.