Phytohemagglutinin-Induced Transformation and Aggregation of Guinea Pig Lymphocytes 1

Mitogenic agents which cause a high percentage of blast transformation when added to short-term lymphocyte cultures also cause marked cell aggregation. Conditions which promote cell-to-cell contact may increase the blastogenic reaction as evidenced in antigen-stimulated lymphocyte cultures (1, 2). Removal of those cells capable of adhering to glass eliminates the transformation seen in antigen-stimulated cultures (1–3). Despite the evidence that cell contact is an important aspect of response to specific antigens, the relationship of marked aggregation seen with phytohemagglutinin (PHA) and other nonspecific mitogens to blastogenesis is less clear. Lymphocytes purified by the glass bead method still transform when treated with PHA (1, 2). Coulson (4) has observed PHA-induced transformation even when cells are embedded in agar prior to treatment so that cell contact is eliminated. In the agar system it would seem difficult to measure the magnitude of this response. In the present work, the response of guinea pig lymph node lymphocytes to PHA is related to the aggregation characteristics of the treated cells.

Materials and Methods. Hartley strain inbred guinea pigs (Camm Research Institute, Inc., Wayne, N.J.) 300–350 g males were used in all experiments. These animals were immunized by subcutaneous injection into each hind footpad, 0.2 ml of alumprecipitated DPT antigen (Tri-Solgen, Eli Lily & Co.) at 10-day intervals. Ten to 20 days after the second immunization, the animals were sacrificed. Under aseptic conditions, popliteal lymph nodes were collected in TC 199 (Difco) with 20% fetal calf serum, gently minced, and expressed through a 100-gauge stainless steel wire mesh. This yielded a 98% single cell suspension which was washed twice and adjusted to a concentration of 4 × 10⁶ cells/ml. A 0.5-ml portion of this suspension was then added to 1.5 ml of TC 199, with or without PHA (control), and cultured in vertical, tightly stoppered 15-ml round bottom Kimax tubes at 37° in 5% CO₂-air. Cells were routinely harvested at 1, 48, and 96 hr.

The PHA used in all experiments was from a lot prepared in this laboratory by the method of Börjeson et al. (5), contained 2.8 mg of protein/ml, and was stored frozen at —20°. A new vial was opened for each experiment. The optimum dose of PHA for a culture containing 1 × 10⁶ cells/ml was 0.001 ml (2.8 μg of protein) /ml of culture.