COLONY FORMATION BY SUB-POPULATIONS OF HUMAN LYMPHOCYTES-T .1. EFFECTS OF PHYTOHEMAGGLUTININ AND LYMPHOCYTOSIS-PROMOTING FACTOR FROM BORDETELLA-PERTUSSIS

  • 1 January 1978
    • journal article
    • research article
    • Vol. 34  (3) , 364-373
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 B. pertussis. Colony formation was dependent on a liquid pre-culture step with LPF prior to agar seeding, presence of LPF in the agar medium and a cell density in the agar culture of more than 5000 cells per ml. Optimal colony formation was obtained with 30 .mu.l LPF preparation in the liquid pre-culture step and 20 .mu.l LPF preparation in the agar medium. Colony development improved after the addition of 5 .times. 10-5 M 2-mercaptoethanol and 0.6% human AB serum. The frequency of LPF-induced T[thymus-derived]-lymphocyte colonies in 10 normal adult donors was 5450 .+-. 1800 per 106 mononuclear blood cells. The frequency of phytohemagglutinin (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 30-200 cells on day 7 of culture. Cultures became moribund at day 8. The colony cells were negative for surface immunoglobulin (Ig) 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 1st and 2nd step of culture, respectively. 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 [CFC] were recovered in the SRBC-rosetting fraction (E-RFC) of human blood lymphocytes indicating that these precursor cells are themselves T lymphocytes. E-RFC were separated into Fc(.gamma.)-receptor-positive and negative subpopulations. The frequency of CFC in the Fc(.gamma.)-receptor-negative T lymphocyte population was 2-10 times higher than that of the Fc(.gamma.)-negative T lymphocytes reduced the frequency of T colonies by 20-95%. Mononuclear cells were separated by passage through Ig-anti-Ig-coated columns. Attempts to grow LPF-were trapped in the column. PHA-induced colony formation was enriched in cells which had passed through Ig-anti-Ig-coated columns.