Lack of a T cell–intrinsic requirement for CD28 for the tolerization of CD4 T cells. (A) Peripheral levels of CD28^−/−-derived WBCs in mixed CD28^± mice. In B6-CD45.1 mice treated with 3 Gy TBI, the administration of increasing doses of syngeneic B6 CD28^−/− (CD45.2) BMCs (1 × 10⁶, 5 × 10⁶, and 25 × 10⁶) led to increasing numbers of peripheral CD45.2⁺ cells in the CD4, CD8, B-cell, monocyte, and granulocyte lineages. Mean levels of CD45.2⁺ cells in each lineage 5 weeks after syngeneic BMT are shown. (B) Mixed CD28^± mice develop long-term mixed chimerism in most instances after treatment with anti-CD8, 3 Gy TBI, anti-CD154 mAb, and allogeneic BMT. WT B6 (CD45.1), B6-CD28^−/− (CD45.2), and CD28^± mice all received B10.A BM transplant with anti-CD8, anti-CD154, and 3 Gy TBI. Although none of the CD28^−/− recipients developed chimerism (0 of 5), all remaining groups, regardless of level of CD28^−./− CD4⁺ T cells in the periphery, developed chimerism at similar rates (9 of 10 for WT; 8 of 8 for CD28^± created with 1 × 10⁶, 7 of 9 for CD28^± created with 5 × 10⁶, and 9 of 10 for CD28^± created with 25 × 10⁶ CD28^−/− BMCs). Chimerism was detected in CD4, CD8, B-cell, monocyte, and granulocyte lineages by FCM analysis for more than 25 weeks after BMT.