Effect of Phytohemagglutinin on Mouse Peripheral Lymphocytes.

Using a short-term culture technique it has been possible to cultivate mouse peripheral lymphocytes from various strains for as long as 96 hours. With this technique a study was made to determine whether mouse peripheral lymphocytes could be stimulated by PHA « in vitro ». Swiss (non-inbred) and CBA (inbred) mice of both sexes, 3–12 months old, were used throughout this study. Blood was obtained from the retro-orbital sinus of ether-anesthetized animals, collected in heparinized tubes and pooled according to sex and strain. Leukocyte separation was effected by sedimentation in Plasmagel for 15'. The supernatant was transferred to an equal volume of Hanks Balanced Salt Solution (HBSS) and spun down for 20’ at 250–300 × g. The resulting cell sediment was washed twice with HBSS and resuspended in 2 ml Trowell T8 medium which contained 30 - 40 % calf serum which had been pre-heated for 30’ in a 56 °C water bath. After a differential cell count, the cell suspension was further diluted to achieve 106 lymphocytes/ml. Red blood cell contamination corresponded to 30 rbc/1 wbc with granulocyte population varying 10–40 %. 3 × 106 lymphocytes were placed in glass culture tubes prepared in advance by adding 2 ml of 1.5 % agar in HBSS and allowing it to solidify in the bottom of upright tubes. 0.1 ml PHA-M was added to each tube and the cultures were kept upright in a controlled gas phase incubator for 24–96 hours at 37 °C. At the end of the incubation period, the cultures were transferred to centrifuge tubes and spun down for 10’ as above and smears were made directly from the dense sediment after the supernatant was discarded. These smears were fixed, stained and read for the presence of transformed cells scoring at least 1000 cells/culture. Some cultures received 3 μg colchicine 2–6 hours before harvesting and chromosome preparations were made. Mitotic indexes were estimated by scoring 1000 cells/culture. Swiss cultures were also employed for radioautography by adding 3H-thymidine 6 hours before harvesting. Cells were then washed once in HBSS, subjected to short hypotonic treatment, rapidly fixed, pipetted on to slides and allowed to dry. Slides were coated with Kodak NTB2 emulsion, exposed for 9 days, processed and stained through the film and read for labelled cells scoring 500 cells/slide. Microscopic examination revealed a prompt Mastoid transformation of the small lymphocytes which presented a deeply basophilic cytoplasm surrounding a large nucleus with reticular chromatin and one or more prominent nucleoli. Quantitation of the blastoid response at 24-hour intervals throughout the 96 hours of culture disclosed significant blast formation within the first 24 hours for both mouse strains. Differences in transformation between the 2 strains were statistically significant at 24 and 48 hours. 3H-thymidine uptake paralleled blastogenesis by increasing progressively. Differences in uptake between PHA cultures and corresponding controls were found to be statistically significant for all except the 96 - hour cultures. The results obtained suggest that mouse peripheral lymphocytes will respond to PHA stimulation provided culture conditions are adequate. There was clear evidence of stimulation by PHA in both morphology and radioautography studies. However, the day-to-day variation in values of transformation and 3H-thymidine uptake necessitate further studies to improve culture conditions. Higher variability in Swiss (non-inbred) cultures indicates that cellular factors may also influence response « in vitro ». It is concluded that mouse peripheral lymphocytes react « in vitro » to PHA in much the same way as human and other mammalian lymphocytes.