Both CTLA-4^−/− CD80/86^−/− and CD80/86^−/− reconstituted recipients are resistant to the induction of mixed chimerism in mice treated with anti-CD154. (A) Lethally irradiated B6 (CD45.2, Ly9.2) mice reconstituted with BM from B6-wild type (CD45.1, Ly9.2), 129-TKO (CTLA-4^−/− CD80/86^−/−; CD45.2, Ly9.1), or 129-DKO (CD80/86^−/−; CD45.2, Ly9.1) mice were treated with anti-CD8, 3 Gy TBI, anti-CD154, and allogeneic BMT 6 weeks after reconstitution. Among recipients reconstituted with WT BMCs, 100% (5 of 5) developed and maintained long-term mixed chimerism. In contrast, all recipients of TKO-derived BMCs (5 of 5) and most recipients of DKO-derived BMCs (3 of 4) failed to establish mixed chimerism at 4 weeks after allogeneic BMT. (B) The mean percentage of donor WBCs in WT recipients of BM transplant with anti-CD154 among CD4, CD8, B-cell, monocyte, and granulocyte lineages was determined by FCM at various time points after BMT. Results are shown as mean plus or minus SEM for each lineage. Similar results were obtained in the single chimeric DKO recipient (data not shown).