Graft reaction in tissue culture. II. Quantification of the lytic action on mouse fibroblasts by rat lymphocytes sensitized on mouse embryo monolayers.

A method for assaying the lytic action of large pyroninophilic cells (LPC) in cultures of rat lymphocytes on target fibroblasts in mouse embryo monolayers has been described. The basis of the assay was the labelling of fibroblasts with Cr. The rate of Cr release to the medium was found to be proportional to the number of LPC and expressed precisely the actual number of lysed fibroblasts. The lytic activity in LPC suspension was expressed by the lysis index which is the number of LPC per fibroblast lysed at 50 per cent lysis. A suspension of LPC collected 5–6 days after exposure of rat lymphocytes to the mouse monolayers showed a lysis index ranging from 0.7 to 1.3 after 16–48 hours of incubation. The lytic power of a culture was expressed by a value which was obtained by dividing the total number of LPC in a culture by the index. This determined the total number of fibroblasts lysed if the whole culture content were distributed among plates of test monolayer in such a way as to produce 50 per cent lysis in all the plates. Cultures started with 25 × 10 to 30 × 10 lymphocytes produced on the 5th and 6th day a lytic power of as high as 6 × 10 to 12 × 10 fibroblasts. The study has indicated that rats injected with horse serum and boosted 2 days prior to culturing were completely unable to produce graft reaction cultures. Lymphoid cells from non-boosted rats did generate LPC with lytic ability. A quantitative study of the specificity of the lytic reaction has indicated that LPC originated on C57BL/6 monolayers lysed much more strongly monolayers isologous to originator than C3H monolayers, and .