Surface expression of CD30 increases upon allogeneic stimulation in vitro and in vivo. CD30 deficiency affects Treg-mediated GvHD protection. (A) CD4⁺CD25^high FoxP3⁺ cells (H-2^b) were coincubated with CD11c⁺ APCs derived from syngeneic C57B/6 mice (gray open histogram, MFI: 67.1) or allogeneic Balb/c mice (black solid histogram, MFI: 209.2) for 96 hours. The gray solid histogram represents cells stained with isotype control Ab. Cells were gated on CD4⁺FoxP3⁺. (B) eGFP-labeled Treg cells (CD4⁺CD25^highH-2^b) were isolated from secondary lymphoid organs on day 4 after transfer into C57B/6 mice (syngeneic, light gray histogram) or Balb/c mice (allogeneic, black solid histogram). Recipients were given 5 × 10⁶ TCD-BM (H-2^b) after lethal irradiation with 800 cGy. Additionally, 2.5 × 10⁵ eGFP⁺ Treg cells (day 0) plus 5 × 10⁵ CD4⁺/CD8⁺ (1:4) T cells (day +2) were given (both H-2^b). Histograms represent the mean fluorescence intensity for cells that are gated on eGFP⁺ cells. The gray open histogram and the dark gray filled histogram represent naive Treg cells (unmanipulated from 6-week-old mice) and isotype control–stained Treg cells, respectively. CD30 expression is increased after allogeneic compared with syngeneic antigen exposure (MFI black solid versus light gray histogram, P = .003). (C) Relative Foxp3 mRNA expression level was determined by quantitative real-time PCR in eGFP-positive versus -negative cells. The eGFP⁺ population represents the transferred Treg cells after syngeneic or allogeneic transplantation as indicated below the bar pairs. Expression was normalized to GAPDH. The negative controls are donor derived (CD4H-2^b) eGFP⁻ cells (gray bars). Error bars indicate SD from the mean. (D) Survival of recipients given 5 × 10⁶ TCD-BM cells (H-2^b) after lethal irradiation with 800 cGy. Additionally, 2.5 × 10⁵ wt or CD30^−/− Treg cells (day 0) plus 5 × 10⁵ CD4⁺/CD8⁺ (1:4) T cells (day +2) were given (both H-2^b). Survival of mice receiving TCD-BM (▵, n = 15), with Tconv cells (□, n = 15), with Tconv cells and wt Treg cells (▪, n = 14), and with Tconv cells and CD30^−/− Treg cells (○, n = 14). Survival of Balb/c recipients is significantly lower when Treg cells lack CD30 compared with wt Treg cells (○ versus ▪, P = .002). (E) Proliferation of luc⁺ Tconv cells (left column) is reduced by cotransfer of Treg cells (middle column) and to a lesser extent by Treg cells derived from CD30^−/− donors (right column) as depicted for day 7 after BMT. (F) Signal intensity of luc⁺ Tconv cells as quantified in photons per mouse per second over total body area for the indicated group. TCD-BM (▵, n = 15), with Tconv cells (□, n = 15), with Tconv cells and wt Treg cells (▪, n = 14), and with Tconv cells and CD30^−/− Treg cells (○, n = 14). Signal intensity is significantly higher in animals receiving CD30^−/− Treg cells compared with wt Treg cells (○ versus ▪, P = .007, days 6-25). Data are pooled from 2 representative experiments.