ILT3 is dispensable for induction of CD4⁺Foxp3⁺ regulatory T cells by 1,25(OH)₂D₃-treated DCs. The read-out system to test for suppressive activity was composed of CFSE-labeled CD4⁺CD25-cells from donor A PBMCs cultured with 1:10 LPS-matured monocyte-derived DCs from donor B in the presence of 1 μg/mL anti–human CD3 mAb. To evaluate the induction of Ts, CD4⁺ T cells from donor C were generated by 3 rounds of restimulation with allogeneic immature monocyte-derived DCs from donor D, which were cultured for the last 48 hours with or without 10 nM 1,25(OH)₂D₃. The coculture was carried out with or without anti-ILT3 mAb (20 μg/mL). After 96 hours of culture, cells were analyzed by flow cytometry. Expression of Foxp3 transcripts, evaluated by real time RT-PCR, is shown as arbitrary units (AU) normalized to GAPDH signals. (A) Proliferation of CFSE-labeled CD4⁺CD25^– cells (5 × 10⁴/well). Basal levels of Foxp3 transcripts are expressed as AU. (B) Addition of blood CD4⁺CD25⁺ cells (6.5 × 10³/well) inhibits CD4⁺CD25^– cell proliferation. Foxp3 expression of blood CD4⁺CD25⁺ cells is indicated. (C) CD4⁺ cells (6.5 × 10³/well) stimulated for 3 rounds with allogeneic immature monocyte-derived DCs (CD4-iDC) show enhanced Foxp3 expression and inhibit proliferation of CFSE-labeled CD4⁺CD25^– cells. (D) CD4⁺ cells stimulated for 3 rounds with allogeneic immature monocyte-derived DCs in the presence of 10 nM 1,25(OH)₂D₃ (CD4-iDC-D₃) show enhanced Foxp3 expression and inhibit proliferation of CFSE-labeled CD4⁺CD25^– cells. (E) Addition of anti-ILT3 mAb to CD4⁺ cells stimulated for 3 rounds with allogeneic immature monocyte-derived DCs (CD4-iDC-αILT3) show basal levels of Foxp3 expression and fail to inhibit proliferation of CFSE-labeled CD4⁺CD25^– cells. (F) Addition of anti-ILT3 mAb to CD4⁺ cells stimulated for 3 rounds with allogeneic immature monocyte-derived DCs in the presence of 10 nM 1,25(OH)₂D₃ (CD4-iDC-D₃-αILT3) show enhanced Foxp3 expression and inhibit proliferation of CFSE-labeled CD4⁺CD25^– cells. A representative experiment out of 2 performed is shown.