Expansion of luciferase-labeled Treg cells is reduced when CD153 is blocked early after transplantation. (A) Distribution and expansion of luc⁺ donor Treg cells in Balb/c mice (H-2k^d) receiving Tconv cells and Treg cells (both H-2k^q) and isotype IgG Ab or Tconv cells and Treg cells along with anti-CD153 blocking Ab as shown for representative animals on day 7 after BMT. Expansion of luc⁺ Treg cells (first animal) is reduced when the CD30/CD153 interaction is blocked in the early (middle animal) but not in the late (right animal) phase after adoptive transfer. Early blockade represents days −2, 0, 2, and 4, and late phase represents days 4, 6, 8, and 10 after BMT. (B) The number of donor-derived Treg cells (CD4⁺FoxP3⁺H-2Kq⁺) per high-power field was determined in 5 representative areas and plotted for the respective secondary lymphoid organ recovered from Balb/c recipients (n = 5) on day 10 after adoptive transfer of TCD-BM and Tconv cells plus Treg cells. *P < .05, animals receiving anti-CD153 Ab (blue bars) or isotype IgG (green bars) both in the early phase. (C) Whole-body photons derived from luc⁺ Treg cells expanding in Balb/c recipients at serial time points. TCD-BM (▵, n = 10), with Tconv cells and Treg cells and isotype IgG (□, n = 10), with Tconv cells and Treg cells plus early anti-CD153 blocking Ab (○, n = 10), and with Tconv cells and Treg cells plus late anti-CD153 blocking Ab (•, n = 10). Expansion of luc⁺ Treg cells is significantly reduced in the presence of the anti-CD153 blocking Ab during the early expansion phase compared with isotype IgG (○ versus □, P = .007) or late blockade (○ versus •, P = .008). Data are pooled from 3 independent experiments. (D) Treg cells (CD4⁺CD25^highH-2k^b+) from either wild-type (open bars) or CD30^−/− (solid black bars) C57B/6 mice were incubated with CFSE-labeled CD4⁺CD25⁻ T cells (H-2k^b, Thy-1.1⁺) and γ-irradiated (30 Gy) APCs (CD11c⁺H-2k^d+). To measure T-cell proliferation, Thy-1.1⁺ cells were analyzed by FACS after 72 hours. The bars represent the percentage of proliferating CD4⁺Thy 1.1⁺CFSE⁺ cells. One representative experiment of 3 is presented. (E) On days 5 and 7 after BMT, lymphoid organs were removed from recipients of wt or CD30^−/− Treg cells and total cells per organ were counted in a single-cell suspension. Subsequent analysis of the resulting cell suspensions by FACS provided the percentages based on which the absolute cell numbers were calculated. *P < .05. Data are pooled from 2 independent experiments with 6 animals per group and time point. Error bars indicate SD from the mean.