Cloned primed-lymphocyte-test reagents in the dissection of HLA-D

Human T lymphocytes obtained as blasts on day 4 from a primary mixed leukocyte culture (MLC) were cloned in the presence of T cell growth factor (TCGF) and feeder cells. Parameters important in producing higher-specific-activity TCGF were evaluated; irradiation of the responding cells as well as removal of adherent cells or inclusion of indomethacin in the culture was important. In addition, the presence of an irradiated lymphoblastoid cell line (LCL) cell in the TCGF-producing system enhanced activity in the supernate. The long-term maintenance of progeny from clones was achieved by utilizing the LCL autologous with either the responding or sensitizing cells from the initial MLC as feeder cells. Under those conditions, clones could be expanded for 7 or more wk with the maintenance of PLT reactivity. Had all the cells in each clone been maintained for the full 7 wk, more than 1 X 10(10) cells could have been developed in each clone. The cloned reagents provide a higher degree of antigen-specific reactivity than do normal PLT cells. It is to be anticipated that as the requirements for cloning are made more stringent, including the recloning of the cells, these reagents will aid greatly in the dissection of the complexity attendant to HLA-D.