Migratory DCs are responsible for iTreg cell conversion. (A) In vitro differentiation of CD25⁺Foxp3⁺ 2a T cells induced by migratory (MHC class II^highCD11c⁺, ssmDCs), CLN resident (MHC class II^lowCD11c⁺, CLN res DCs), or spleen resident (MHC class II^int/highCD11c⁺, spleen DCs) DCs from wild-type BALB/c mice loaded with the Bpep. Where indicated, the RA receptor inhibitor LE540 and TGF-β have been added to the cocultures. The experiment has been repeated 3 times with similar results. (B) Inhibition of CD25⁺Foxp3⁺ 2a T-cell generation in vivo by the LE540. DC-tg Rag2-deficient animals were adoptively transferred with naive 2a T cells and treated every second day with the inhibitor for 3 weeks. The percentage of CD25⁺Foxp3⁺ 2a T cells on the total 2a T-cell population was determined by cytofluorimetric analysis 3 weeks after transfer. Data are mean ± SEM of 4 mice analyzed in 2 independent experiments. (C) Flow cytometry of the CCR4 expression by the indicated lymph node populations of 2a T cells 2 weeks after transfer into DC-tg Rag2-deficient mice. The experiment was repeated 3 times with similar results. (D) Reduction in the numbers of CD25⁺Foxp3⁺ 2a T cells in the lymph nodes of transferred mice after treatment with the CCR4 inhibitor, AF399/42018025. DC-tg Rag2-deficient mice were transferred with naive 2a T cells and treated with the CCR4 inhibitor. Absolute numbers of CD25⁺Foxp3⁺ 2a T cells were then assessed by flow cytometry. NT indicates nontreated mice. Data are mean ± SEM of 4 mice analyzed in 2 independent experiments. (E) Flow cytometric analysis of CCR4 expression by CD25⁺Foxp3⁺ 2a T cells generated in vitro as described in panel A. CCR4 mean fluorescence intensity (MFI) on CD25⁺Foxp3⁺ and CD25⁻Foxp3⁻ 2a T cells after coculture with ssmDCs from BALB/c mice in presence of the Bpep. The experiment was repeated twice with similar results. *P < .05; **P < .01.