Generation of 2C/B6 syngeneic chimeras. (A) B6 mice received 3 Gy TBI and 5 × 10⁶ syngeneic 2C BMCs. Seven weeks later, the presence of 2C cells among peripheral blood CD8 cells of the recipient mice was analyzed by FCM analysis using anticlonotypic mAb 1B2. Histograms show 1B2 staining on gated CD8⁺ WBCs. These 2C/B6 syngeneic chimeras then received 3 Gy TBI on day − 1 and allo-BM transplant on day 0. 2C/B6 mice received a transplant of either L^d+ (B10.A) or L^d− (A.SW or B10.S) BM and were injected with anti-CD154 on day 0 or received L^d+ (B10.A) BM without anti-CD154 (MR1). (B) Normalized mean (± SEM) percentage of 2C cells among splenic CD8 cells at 4 and 7 days after allogeneic BMT. A value of 100% was given to the percentage of 2C CD8⁺ T cells in the blood 1 week prior to allogeneic BMT, and the percentage of 2C CD8⁺ T cells in the spleen on days 4 and 7 was normalized to this value. Statistical analyses were performed with a Mann-Whitney U test to compare the “L^d+ group” with the “L^d− group” or the “L^d+ group” with the “L^d+ no MR1 group”: ** indicates P < .005; NS, not significant. Two experiments are shown for the day-4 analysis (n = 2-5 animals/group/experiment) and 3 experiments are shown for the day-7 analysis (n = 3-5 animals/group/experiment). (C) Time course of 2C deletion in peripheral WBCs. A value of 1 was given to the percentage of 2C CD8⁺ T cells in the blood 1 week prior to allogeneic BMT. The percentage of 2C⁺CD8⁺ cells among WBC CD8 cells was then analyzed every 2 weeks and normalized to the value before BMT. The mean (± SEM) is presented. □ indicates recipients of L^d+ (B10.A); ○, recipients of L^d− (B10.S); and ▵, recipients of L^d+ (B10.A) BMCs without anti-CD154.