Quantitative aspects of a cell-mediated response to alloantigens

Abstract

1. Cytotoxic lymphocytes were generated by transferring lymphoid cells from parental strain (CBA) mice to irradiated F1 (CBA/DBA) recipients. 2. A sensitive colony inhibition assay was developed for the measurement of cytotoxic lymphocytes. The colony inhibition method was compared with the standard 51Cr release assay for cytotoxicity. The two assays appeared to be measuring the same effector T cells. 3. The capacity of spleen and thymus cells to generate cytotoxic lymphocytes was investigated using the colony inhibition assay for cytotoxicity determinations. For a given number of cells, spleen cells generated more cytotoxic activity than thymus cells. There was not a linear relationship between the amount of cytotoxic activity and the dose of donor spleen cells injected. The contribution of possible cellular interactions to this type of dose-response relationship have been discussed. 4. Foci of cytotoxic lymphocytes were detected by assaying for cytotoxic activity in fragments of recipient spleens. One focus was generated by 2.3 x 10 5 spleen cells 2.7 x 10 6 thymus cells and 2.8 x 10 4 lymph node cells. 5. The ability of lymphoid cells in a population able to generate cytotoxic lymphocytes can be expressed in terms of focus forming cells. The seeding efficiency of parental spleen cells in F1 spleens was established (19%). With this correction the frequency of focus forming cells in a population of spleen cells was 1 per 4.4 x 10 4 or 2.3 x 10 -5. 6. To avoid some of the problems encountered with the in vivo segregation of cytotoxic foci, a culture method was devised to measure the frequency of CL precursors in vitro. Small numbers of parental (CBA) spleen cells were cultured with F1 (CBA/DBA) stimulator spleen cells and cytotoxic foci were segregated in the dimpled surface of a polyacrylamide culture vessel. The frequency of CL precursors specific for DBA alloantigens on P 815-Y mastocytoma cells was calculated to be 1 in 1700 spleen cells. 7. “Spontaneous” cytotoxic foci were detected when spleen cells of CBA, DBA or the F1 hybrid cells were cultured in the absence of stimulator cells. 8. The use of these quantitative techniques to study problems of specificity and cellular interactions in T cell-mediated cytotoxicity is discussed.