Administration of anti–CTLA-4 on day 0 impairs the establishment of mixed chimerism in mice receiving CD8 depletion, 3 Gy TBI, anti-CD154 mAb, and BM transplant. The mean percentage of donor cells among CD4, CD8, B-cell, monocyte, and granulocyte lineages in WBCs was determined by FCM at various time points after BMT. The percentage of donor-derived cells within the granulocyte population is illustrated as a representative lineage, and results for individual mice within each group are shown (A-C). Five of 5 control mice achieved and maintained multilineage chimerism more than 26 weeks after BMT (A), in contrast to only 1 of 5 recipients that received anti–CTLA-4 on day 0 (B). Four of 5 mice that received anti–CTLA-4 on day +2 established long-term mixed chimerism (C). (D-F) In mice that received BM transplant with anti-CD154 on day 0 and anti–CTLA-4 on days 0, +2, +4, and +6, long-term mixed chimerism was not achieved (E; 0 of 10 mice). Depletion of peripheral CD4⁺ cells (9 of 10) reversed the effect of the anti–CTLA-4 (F), and permitted levels of chimerism similar to those in mice that received only anti-CD154 with alloBMT (D; 4 of 5).