(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).

Diabetic C57BL/6 mice transplanted with DBA/2 pancreatic islet were treated with an anti-CD154 mAb in combination with either anti–Tim-1 mAb (circles; n = 9), or the IgG2a isotype control mAb (triangles; n = 14).

CFSE-labeled C57BL/6 CD4⁺CD25^– and CD8⁺CD25^– cells were stimulated with highly mature allogeneic DBA/2 DCs in the presence of anti–Tim-1 mAb or IgG2a isotype control mAb. Analysis of the CFSE profile (A) and enumeration of the absolute number of CD3⁺CD4⁺ or CD8⁺ cells (B) were used to assess the proliferation and expansion of alloreactive T cells after 2 (A) and 4 (A and B) days of culture. (C) The effect of anti–Tim-1 mAb on the proportion of cells programmed for apoptosis (annexin V⁺ cells) in the proliferating cell population was assessed after 4 days of culture. (D) To gain further insight, we analyzed Bcl-2 transcript expression in alloreactive CD4⁺ or CD8⁺ cells after 2 days of culture. (E) Finally, secretion of IFN-γ (Th1) and IL-17 (Th17) by alloreactive Teffs after 4 days of culture was assessed by intracellular staining. Data represent 1 of 8 independent experiments (A and E) or are presented as the mean ± SEM of at least 5 independent experiments (B–D).

CD4⁺GFP(Foxp3)^– T cells isolated from naive C57BL/6 Foxp3-GFP knock-in mice were cultured with highly mature syngeneic DCs plus anti-CD3 mAb in the presence of IgG2a mAb or anti–Tim-1 mAb and in the presence or absence of TGF-β. Induction of Foxp3 expression was assessed 3 days later by quantitative real-time PCR (A) or by intracellular staining of Foxp3 (B). Quantification of conversion of CD4⁺GFP(Foxp3)^– to CD4⁺Foxp3⁺ Tregs at the level of protein expression was determined by gating onto the CD4⁺ fraction. Differentiation of Th17 cells was assessed by intracellular staining of IL-17 and quantified at the level of protein expression by gating onto the CD4⁺ fraction. Data are presented as the mean ± SEM of 3 independent experiments (A) or are representative of 3 independent experiments (B).