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. 2005 Sep 6;102(38):13562–13567. doi: 10.1073/pnas.0504484102

Fig. 2.

Fig. 2.

Murine DCs differentiated in the presence of VIP induce regulatory Tr1 cells and tolerance in vivo. DCs were generated from mouse BM cells in the absence (DCcontrol) or presence (DCVIP) of VIP and activated with LPS to induce DC maturation. (a)DCcontrol or DCVIP was added to allogeneic CD4 T cells (5 × 105), and the proliferative response was determined. DCcontrol or DCVIP without T cells did not proliferate. Each result is the mean ± SD of three experiments performed in duplicate. (b) Purified CD4 T cells were exposed to allogeneic DCcontrol or DCVIP and activated with phorbol 12-myristate 13-acetate plus ionomycin. Intracellular cytokines were determined in CD4-gated cells by flow cytometry. Numbers represent percentage of positive cells (n = 5). (c) Purified CD4 T cells were stimulated for 1 week with allogeneic DCcontrol or DCVIP. The resulting regulatory CD4 T cells (Tr) were incubated with syngeneic responder CD4 T cells (rCD4) in the presence of allogeneic mDCs, and the proliferative response was determined (n = 4). (d) Isolated CD4 T cells were cocultured with syngeneic TrVIP and allogeneic mDCs in the presence or absence of blocking anti-IL10 and/or anti-TGFβ. Additionally, CD4+mDCs were separated from TrVIP+mDC in a Transwell system. The proliferative response of responder CD4 T cells was determined (n = 4). (e) Sorted CD4 T cells generated with DCcontrol or DCVIP were analyzed for neuropilin 1 and Foxp3 mRNA expression by real-time RT-PCR and for surface CD103 and glucocorticoid-induced TNF receptor (GITR) expression by flow cytometry. Open histograms and dashed lines represent isotype controls. One representative experiment of two is shown. (f) Mice were injected s.c. with increasing numbers (from 50 to 5 × 105 cells) of Ag-pulsed DCcontrol or DCVIP 1 week before priming with Ag. Five days later, mice were tested for DLN Ag-specific T cell proliferation, serum antibody levels, and DTH responses. Mice injected with Ag alone (None) were used as controls. Results are the mean ± SD for each group (n = 4) tested separately and are representative of three experiments.