In independent trials, two groups of BALB/c (H2^d) mice (n = 5 in each group) were injected with 50 × 10⁶ C57BL/6 (H2^b) bone marrow cells on day 0. All mice were treated with a DST consisting of 10⁷ C57BL/6 spleen cells on day –7 relative to bone marrow transplantation. They also received anti-CD154 mAb at a dose of 0.5 mg intraperitoneally on days –7, –4, 0, and +3 relative to bone marrow transplantation. The percentage of donor-origin PBMCs was measured by flow microfluorometry in all mice 4 weeks, 8 weeks, 14 or 21 weeks, and 30 weeks after bone marrow transplantation as described in Methods.

Intrathymic deletion of host alloreactive DES⁺CD8⁺CD4^– thymocytes. KB5 CBA synchimeras were randomized into two groups. Group 1 (upper panels) was left untreated. Group 2 (lower panels) was injected with a C57BL/6 DST on day –7 and anti-CD154 mAb on days –7, –4, 0, and +3 relative to injection of 50 × 10⁶ C57BL/6 bone marrow cells on day 0. Thymi were recovered 35 weeks after bone marrow transplantation and analyzed by flow microfluorometry for the percentage of DES⁺CD8⁺CD4^– thymocytes as described in Methods. Shown in the left column are representative dot plots; the percentage of cells expressing CD4 and CD8 is indicated in each quadrant. The right column presents histograms; the horizontal bars depict the gates used to determine the number of DES⁺ cells in the CD8⁺CD4^– quadrant. Representative data are shown; the complete data set is given in Table 4.

Deletion of peripheral host alloreactive CD8⁺ T cells. KB5 CBA synchimeras were randomized into four cohorts. Group 1 was untreated. Group 2 was injected with four doses of anti-CD154 mAb on days 0, +3, +7, and +10 (short arrows), and with 50 × 10⁶ C57BL/6 bone marrow cells on day +7 (long arrow). Group 3 received four doses of 0.5 mg of anti-CD154 mAb at the same intervals (short arrows) plus a transfusion of C57BL/6 spleen cells on day 0. Group 4 received a DST of C57BL/6 spleen cells on day 0 and anti-CD154 mAb on days 0, +3, +7, and +10, in addition to an injection of 50 × 10⁶ C57BL/6 bone marrow cells on day +7. The percentage of DES⁺CD8⁺ cells in the blood was determined on day 0 (before any treatment) and then at the indicated times. Within 2 weeks of treatment, the percentage of DES⁺CD8⁺ peripheral blood cells was significantly lower in all treatment groups compared with controls (P < 0.001). Thereafter, the percentage of DES⁺CD8⁺ peripheral blood cells in groups 2 and 3 tended to rise toward that observed in controls, but even at week 15 the percentage remained less than in controls (P < 0.001). In contrast, the percentage of DES⁺CD8⁺ peripheral blood cells in group 4 remained extremely low throughout the course of the experiment and, at week 15, was significantly lower than in all other groups (P ≤ 0.001 for each comparison). With respect to chimerism, defined as at least 0.5% donor-origin (H2-K^b+) PBMCs 9 weeks after transplantation, all mice in group 4 were chimeric and none in groups 2 or 3 was chimeric.