Production of human T-lymphocyte clones. I. Monoclonal culture and functional cytotoxic maturation

Abstract

Well-defined clones of human T-lymphocytes were produced amd monoclonal T-cell cultures were maintained for long periods of time. Single T-lymphocytes were isolated with the help of a micropipette from PBL cultures prior to any cellular stimulation (MLC), collected separately at the bottom of a 200 μl tissue culture microwell under the control of stereomicroscopic observation, and cultured with irradiated lymphoid cells in the presence of TCGF. After 12 days, 20–50% of the seeded wells exhibited clones of 3 × 10 4 − 10 5 T-lymphocytes, which were transferred to largeer tissue culture wells (2 ml, LINBRO) for long-term culture. Recloning of the growing cloned cells under the same conditions as the primary culture was carried out successfully. In the preliminary cytotoxic assays performed in 11 clones (a) a marked activity directed against lectin-coated targets was observed in many clones; (b) an important NK-like activity was exhibited by the clone 45B9 (65% of the tested cells lysed K562 cell targets); (c) 3 clones did not demonstrate cytotoxic activity against either PHA-coated L-1210 cells or K562 cell targets. These results could be explained hypothetically by the difference of functional maturation of T-cells within each clone.