Critical role for donor CD11c-expressing cells in the persistence of donor T_regs in vivo. (A) B6D2 recipients were lethally irradiated on day −1 and then administered 3 × 10⁶ TCD BM cells from either WT Thy1.2⁺ B6 or CD11c-DTR/eGFP Thy1.2⁺ B6 mice plus 1 × 10⁶ CD4⁺CD25⁺ WT Thy1.1⁺ B6 T_regs on day 0. Then 4 × 10⁶ CD25-depleted Thy1.2⁺ T_cons from WT B6 donors were administered on day +2. Recipient mice were treated with DT via intraperitoneal injection at a dose of 4 ng/g body weight on transplant days +6, +8, and +10. On transplant day +12, recipient mice were euthanized, and their spleens disrupted. Donor T_reg numbers were then quantified by flow cytometry using a Thy1.1⁺Foxp3⁺ gate; n = 6 WT BM and 7 CD11c-DTR/eGFP BM recipients. Absolute donor T_reg numbers within the recipient spleen are depicted. *P = .0014 by Student t test. (B) B6D2 recipients were lethally irradiated on day −1 and then administered 3 × 10⁶ TCD BM cells from either WT B6 or CD11c-DTR/eGFP B6 mice plus 1 × 10⁶ CD4⁺CD25⁺ CCR8^−/− eGFP⁺ B6 T_regs on day 0. Then 4 × 10⁶ CD25-depleted T_cons from WT B6 donors were administered on day +2. Recipient mice were treated with DT via intraperitoneal injection on transplant days +6, +8, and +10. On transplant day +12, recipient mice were euthanized, and their spleens disrupted. Donor T_reg numbers were then quantified by flow cytometry using an eGFP⁺Foxp3⁺ gate; n = 6 WT BM and 7 CD11c-DTR/eGFP BM recipients. P = .213. (C-D) “WT” BALB/c and “CD11c.DTR” BALB/c BM chimeras were generated and then used as secondary transplant recipients 8 to 12 weeks later. For these second transplants, WT and CD11c.DTR BALB/c BM chimeric mice were irradiated to 800 rads on transplant day −1. Recipient mice were administered 5 × 10⁶ TCD BM cells from Thy1.1⁺ WT B6 mice and 5 × 10⁵ Thy1.2⁺ WT B6 T_regs on transplant day 0. These cells were administered with (C) and without (D) 2.5 × 10⁵ whole T_cons on day +2. All mice received DT by intraperitoneal injection on transplant days −3, −1, and +1. In the figure label, the “BM Group” refers to the BM product used to generate the 2 varieties of chimeric mice. Both groups of recipients received identical marrow products for transplant 2. On transplant day +7, recipients were euthanized, and their spleens disrupted. Donor T_reg numbers within the recipient spleen were then quantified by flow cytometry using a Thy1.1^–K^d–Foxp3⁺ gate. (C) n = 6 WT BM chimera mice and 7 CD11c.DTR BM chimeras. P = .381 by Student t test. (D) n = 7 WT BM chimera mice and 5 CD11c.DTR BM chimeras. *P = .041. (E-F) “WT” BALB/c and “CD11c.DTR” BALB/c BM chimeras were generated and then used as secondary transplant recipients 12 weeks later. For these second transplants, WT and CD11c.DTR BALB/c BM chimeric mice were irradiated to 800 rads on transplant day −1 and administered 5 × 10⁶ TCD BM cells from K^b+ WT B6 mice without supplementary T_cons or T_regs on transplant day 0. All mice received DT by intraperitoneal injection on transplant days −3, −1, and +1. On transplant day +7, recipients were euthanized, and their spleens disrupted. (D) Donor neutrophil numbers within the recipient spleen were quantified by flow cytometry using a K^d–CD11b⁺Ly6G⁺ gate; n = 3 mice per group. *P = .014 by Student t test. (E) Donor CD11c⁺ APC numbers within the recipient spleen were quantified by flow cytometry using a K^d–CD11c⁺ gate; n = 3 mice per group. *P = .001.