The intrathymic injection of donor spleen cells into antilymphocyte serum 
(ALS)-treated mice induces significant prolongation of donor skin grafts. The 
intrathymic route of antigen presentation in this model is superior to the 
intravenous route in achieving unresponsiveness. To elucidate possible 
mechanisms involved in the induction of unresponsiveness in ALS-treated mice 
after intrathymic injection of donor spleen cells, we have analysed the 
reactivity of lymphoid cells from unresponsive mice in various ways. Deletion of 
donor reactive cells has been studied using the Mls antigen system. Functional 
inactivation was analysed by a sequential study of the frequency of donor 
reactive cells. Suppressor cell activity was studied using an adoptive transfer 
assay. Deletion of donor reactive cells was partial and occurred largely in the 
spleen in both early and late stages of unresponsiveness. The frequency of donor 
reactive cytotoxic cells was suppressed in both spleen and lymph nodes from day 
+/- 13 until grafts were rejected, with the exception of a rebound period at day 
+/- 22. In contrast, donor reactive cells were not deleted or inactivated in the 
thymus. Suppressor cell activity could only be detected in mice bearing 
long-term grafts. These results suggest that donor reactive cytotoxic cells are 
functionally inactivated in the spleen and nodes in the early and late stages of 
unresponsiveness after intrathymic injection of antigen. In contrast, donor 
reactive cells in the thymus do not appear to be affected.