Alloreactive and nonalloreactive donor T cells can be distinguished by their phenotype and proliferation kinetics. (a and b) Lethally irradiated (1,300 cGy) C3FeB6F1 (allogeneic, Allo) or B6 (syngeneic, Syn) recipients were transplanted with B6 BM (5 × 106) and CFSE-labeled B6 (Ly5.1+) purified T cells (2 × 107). At various timepoints after BMT, recipients were sacrificed and splenocytes were stained with anti-CD8, -CD25, -CD44, -CD62L, and -CD122 antibodies. Flow cytometric analysis is shown for expression of these antibodies on donor CD8+ T cells 64 hours after BMT (a).CD69 expression on donor CD4 and CD8 T cells in the spleen was determined 16 hours, 40 hours, and 64 hours after BMT (b). (c) Lethally irradiated (750 cGy) B6D2F1/J recipients were transplanted with CFSE-labeled B6 purified T cells (2 × 107). After 64 hours, mice were sacrificed, splenocytes were stained with anti-CD4 and -CD8 antibodies, and FSC intensity was determined. A: nondividing cells; B: slow proliferating cells; C:fast proliferating cells. FSC, forward scatter; % of max, % of maximum number of the cells per group. (d) Lethally irradiated (750 cGy) C3FeB6F1 recipients were transplanted with CFSE-labeled B6 purified T cells (2 × 107). After 64 hours, mice were sacrificed and splenocytes were sorted according to CFSE intensity, shown in a histogram plot as fast- and slow-proliferative cells. Cells were incubated with irradiated C3FeB6F1 splenocytes as stimulators for 5 days. IL-2 (20 IU/ml) was added after 72 hours, and proliferation was determined by 3H-thymidine incorporation.