Figure 2. Effect of 3B3 anti–Tim-1 mAb on alloreactive Tregs in vitro.

(A) CFSE-labeled CD4⁺CD25⁺ Tregs were stimulated with mature allogeneic DCs in the presence of anti–Tim-1 mAb or IgG2a isotype control mAb. Proliferation of alloreactive CD4⁺CD25⁺ Tregs was assessed by flow cytometry after 6 days of culture through analysis of the CFSE profile of CD4⁺ T cells. (B) Next, proliferating CFSE^dim CD4⁺CD25⁺ Tregs were isolated by flow cytometry for RNA extraction, and relative expression of Treg-associated transcripts was determined by quantitative real-time PCR. (C) FACS-sorted GFP(Foxp3)⁺ Tregs from Foxp3-GFP knock-in mice were cocultured with mature allogeneic DCs in the presence of anti–Tim-1 mAb or IgG2a isotype control mAb. Foxp3 gene expression was assessed by quantitative real-time PCR on days 2, 4 and 6. (D) Neutralizing anti–IL-6 mAb or isotype control mAb was added to the culture of GFP(Foxp3)⁺ Tregs with mature allogeneic DCs. After 6 days of culture, Foxp3 expression was analyzed by quantitative real-time PCR. (E) CFSE-labeled CD45.1⁺ Teffs (CD4⁺CD25^–) were stimulated by plate-bound anti-CD3 and soluble anti-CD28 mAb for 4 days (Teff alone), or cocultured with alloreactive CD45.2⁺ Tregs, previously stimulated either in the presence of IgG2a isotype control mAb (Isotype control mAb proliferating Treg/Teff) or in the presence of anti–Tim-1 mAb (Anti-Tim 1 mAb proliferating Treg/Teff). Proliferation of Teffs was assessed by flow cytometry, based on the CFSE profile of CD45.1⁺ cells. Data are representative of 4 (A and E) independent experiments or are presented as the mean ± SEM of 4 different independent experiments (B–D).