(2 106) were cultured with complete medium (RPMI 1640 supplemented with 100 U/mL penicillin–streptomycin, 2 mM L-glutamine, 50 M 2-mercaptoethanol, and 10% heat-inactivated fetal calf serum) containing GM-CSF (800 U/mL PeproTech, Rocky Hill, NJ) and IL-4 (500 U/mL PeproTech) in the absence (DCcontrols) or the presence of VIP (DCVIPs 10 8 M Calbiochem, San Diego, CA). After 6 days, nonadherent cells were collected and subjected to negative selection with anti-CD2 and anti-CD19 mAbs conjugated with immunomagnetic beads (Miltenyi Biotec, Auburn, CA). Resultant cells were cultured for 48 hours with LPS (1 g/mL) or human TNF (10 ng/mL) to induce activation/maturation. Human naive CD4 and CD8 T cells were purified from peripheral blood mononuclear cells (PBMCs) obtained from different donors with use of the CD4/CD45RA and CD8 Multisort kit (Miltenyi Biotec) according to the manufacturer’s recommendations and were typically more than 99% pure, as indicated by flow cytometry analysis (CD4 CD45RO CD62L or CD8 CD45RO CD62L , respectively). Human TH1 cells were generated from naive CD4 T cells, as de- scribed.15 To generate human tetanus toxin (TT)–specific CD4 T cells and allogeneic fibroblast-specific CD8 T cells, PBMCs (107) were primed with TT (1 g/mL) or necrotic allogeneic fibroblasts (106) for 3 weeks in medium containing IL-2 (100 U/mL), and CD4 or CD8 T cells were negatively selected, as described.16 Resultant cells (greater than 95% CD3 CD4 cells or greater than 95% CD3 CD8 cells) were cultured in medium with IL-2 (10 U/mL) for 5 days and were used for subsequent experiments. For isolation of different T-cell populations (CD4 , CD4 CD25 , CD4 CD25 ), cells were labeled with PE-anti-CD25 and PerCP-anti-CD4 antibodies, as described, and the different populations were gated and sorted using a FACSCalibur flow cytometer (Becton Dickinson, San Diego, CA). Flow cytometry Cells were incubated with various PerCP-, FITC-, and PE-labeled mAbs (BD PharMingen, San Diego, CA) diluted at optimal concentration for immunostaining, fixed in 1% paraformaldehyde, and analyzed on a FACSCalibur flow cytometer (Becton Dickinson). We used isotype- matched antibodies as controls and IgG block (Sigma) to avoid nonspecific binding to Fc receptors. For analysis of intracellular CTLA4, cells were stained first for surface CD4 with PerCP-anti-CD4 mAb, fixed with Cytofix/Cytoperm solution (BD PharMingen), incubated with PE-anti- CTLA4 mAb diluted in 0.5% saponin, and analyzed by flow cytometry. Cytokine assays Cytokine contents in the supernatants of DC cultures or DC–CD4 T cell cocultures were determined by specific sandwich ELISA using capture/ biotinylated detection antibodies (BD PharMingen). Endocytosis assay Mannose receptor–mediated endocytosis was measured as the cellular uptake of FITC-dextran (Sigma) and was quantified by flow cytometry, as described.16 Mixed leukocyte reaction Naive CD4 T cells (2 105) were cultured with allogeneic DCcontrols or DCVIPs at various T/DC ratios in the presence or absence of IL-2 (100 U/mL) for 3 days. Cell proliferation in primary cultures was evaluated by using a cell proliferation assay (BrdU) from Roche Diagnostics GmbH (Mannheim, Germany). TT-primed CD4 T cells (5 105) were cultured with unprimed, OVA-primed, or TT-primed DCcontrols or DCVIPs at various T/DC ratios. In some experiments, DCs (105) were cultured with purified syngeneic or allogeneic unprimed or primed CD4 T cells (5 105). After 3 days of culture, T cells were recovered by immunodepletion of CD11c DCs, rested for 3 days in complete medium supplemented with IL-2 (20 U/mL), and restimulated (5 105) with different numbers of allogeneic LPS-matured DCs or syngeneic TT-pulsed DCs (105). Human naive CD8 T cells (5 105) were cocultured with allogeneic DCcontrols or DCVIPs at various T/DC ratios for 5 days, and CD8 T cells were negatively selected by the depletion of DCs. Subsequently, CD8 CD28 and CD8 CD28 T cells were selected using anti–human CD28 mAb (BD PharMingen) plus goat anti–mouse IgG mAb–conjugated immunomagnetic beads, cocultured with allogeneic LPS-matured DCs (104), and assayed for proliferation as described. Analysis of Treg cell function Purified naive CD4 or CD8 T cells were exposed to allogeneic DCcontrols or DCVIPs as described. Five days later, CD4 or CD8 T cells were recovered by immunodepletion of DCs and were cultured with syngeneic TH1 cells (5 105) in the presence of allogeneic mDCs (105). Some cultures were performed in the presence of blocking anti–IL-10 (10 g/mL), anti-TGF 1 (40 g/mL), or anti-CTLA4 (10 g/mL) mAbs or of IL-2 (100 U/mL). To determine the cell-contact dependence of the regulatory response, we placed TH1 cells (5 105) with allogeneic mDCs (105) in the bottom well of a Transwell system (Millipore, Auburn, CA) and the recovered CD4 or CD8 T cells (2 105) with allogeneic mDCs (105) in the upper Transwell chamber. After 72 hours, we measured the proliferative response of the bystander reactive TH1 cells in the bottom well. mRNA analysis mRNA was isolated from different CD4 T cell populations, and Foxp3 and Nrp1 mRNAexpression were determined by real-time reverse transcription– polymerase chain reaction (RT-PCR), as described, and expressed as relative units.17,18 Cytotoxicity assay In vivo–primed CD8 T cells and allogeneic fibroblast-specific CD8 T cells were cultured with Na251CrO4 (100 Ci (3.7 MBq)/106 cells NEN Life Science Products, Boston, MA)–labeled human allogeneic fibroblasts (104) for 4 hours at various effector-to-target cell ratios (E/T ratios). The radioactivity released in the supernatants was measured, and the percentage of specific lysis was calculated.7 Results VIP interferes with the differentiation of human DCs and their subsequent maturation To determine whether exposure to VIP during human DC differen- tiation results in DC phenotypic and functional changes, we compared human monocyte–derived DCs generated in the presence or absence of VIP in terms of surface markers, phagocytic capacity, and cytokine production. As previously described, human mono- cytes cultured with GM-CSF plus IL-4 for 6 days differentiated into iDCs (Figure 1A). With LPS stimulation, iDCs matured to DCs expressing high levels of DC maturation markers (CD83), MHC molecules (classes 1 and 2), and costimulatory molecules (CD40, CD80, CD86) (Figure 1A DCcontrols). However, DCs generated in the presence of VIP (DCVIPs) were resistant to the LPS-induced up-regulation of the costimulatory molecules (CD40, CD80, CD86) and the maturation-associated changes (CD83) (Figure 1A). We observed similar results by quantitative real-time PCR and after stimulation of DCVIPs with TNF , anti-CD40 antibodies, or an inflammatory cocktail containing TNF /IL-6/IL-1 /PGE2 (not shown). Because iDCs and mDCs differ in their phagocytic capacity, we assessed the endocytic ability of DCVIPs by measuring the FITC-dextran uptake through mannose receptors. DCVIPs exhibit higher phagocytic activity than DCcontrols do (Figure 1B). With Toll-like receptor activation, iDCs matured into cells capable of producing high levels of inflammatory cytokines. In contrast to DCcontrols, which produce TNF, IL-12, IL-6, IL-8, and low levels of IL-10, DCVIPs produce very low levels of proinflammatory cyto- kines (TNF, IL-12, IL-6) but release significant levels of the anti-inflammatory cytokine IL-10 (Figure 1C). Taken together, VIP-GENERATED DCs INDUCE Treg CELLS 3633 BLOOD, 1 MAY 2006 VOLUME 107, NUMBER 9 For personal use only. at FMRP on January 19, 2011. www.bloodjournal.org From

these results indicate that the DCs generated in the presence of VIP differ from those generated with GM-CSF/IL-4 alone in terms of costimulatory molecule expression, phagocytic capacity, and cyto- kine profile. The characteristics expressed by DCVIPs are similar to those reported for tolerogenic DCs.1-3,6,8,19 VIP differentiates tolerogenic DCs that induce IL-10/TGF –producing anergic CD4 T cells Tolerogenic DCs are poor stimulators of T-cell proliferation and cytokine production.3,7,8,15,18,19 To examine the capacity of the DCVIPs to prime and differentiate CD4 T cells, we cocultured DCcontrols or DCVIPs with alloreactive CD4 T cells. Priming with DCcontrols results in a strong proliferation of allogeneic CD4 T cells, whereas DCVIPs induce only weak proliferation (Figure 2A pri- mary culture). To determine whether the CD4 T cells exposed to DCVIPs are indeed anergic, we restimulated CD4 T cells primed with DCcontrols or DCVIPs with fresh LPS-matured DCs (mDCs) generated from the same donor as the DCcontrols/DCVIPs. T cells exposed initially to DCcontrols proliferated and produced a typical TH1 cytokine profile (large amounts of IFN and IL-2 but no IL-4, IL-5, or IL-10). In contrast, T cells primed with DCVIPs did not proliferate after successive restimulation, and they exhibited a cytokine profile characteristic of regulatory Treg1 cells—that is, production of high amounts of IL-10 and TGF and no or negligible synthesis of IFN , IL-2, IL-4, and IL-5 (Figure 2A, restimulation). We next examined the effectiveness of DCVIPs for the presentation of processed antigen to antigen-specific T cells. In a first coculture, TT-pulsed DCcontrols, but not unpulsed and ovalbumin-pulsed DCcontrols, induced antigen-specific proliferation and IFN production in TT-specific CD4 T cells. After restimula- tion with syngeneic TT-pulsed mDCs, the CD4 T cells responded strongly in terms of proliferation and IFN production (Figure 2B). In contrast, TT-pulsed DCVIPs elicited weak responses (Figure 2B), suggesting that DCVIPs pulsed with soluble proteins induce a state of tolerance in antigen-specific CD4 T cells. DCVIPs induce functional human CD4 Treg cells in vitro After TCR stimulation, Treg cells suppressed the proliferation and IL-2 production of antigen-specific effector T cells. To determine whether T cells exposed to DCVIPs become functional CD4 Treg cells, we restimulated alloreactive TH1 cells with mDCs in the presence of CD4 T cells derived from the same donor and previously primed with allogeneic DCcontrols (CD4Treg controls) or DCVIPs (CD4Treg VIPs). Treg VIPs inhibited the proliferation of synge- neic TH1 cells in response to allogeneic mDCs in a dose-dependent manner, whereas CD4Treg controls were not suppressive (Figure 3A). Similar results were obtained with respect to IL-2 production (Figure 3A). CD4Treg VIP did not suppress the costimulatory capacity of allogeneic mDCs because mDCs pretreated with CD4Treg VIPs for 48 hours had the same stimulatory capacity as untreated mDCs (not shown). We next investigated whether CD4Treg VIPs and effector TH1 cells have to interact with target structures expressed by the same mDCs. We generated TH1 and CD4Treg VIPs from the same donor (donor C) by priming with Figure 1. VIP interferes with the differentiation of human DCs and their subsequent maturation. Human DCs generated in the absence (DCcontrols) or presence (DCVIPs) of VIP were stimulated with LPS to induce DC activation/maturation. (A) Cell surface, maturation, and costimulatory markers were analyzed by flow cytometry. Dashed lines in top histograms represent the staining profile with isotype-matched control antibodies. Numbers represent the mean channel fluorescence intensity (MFI) for each phenotypic marker. Histograms are representa- tive of 4 independent experiments. (B) DCs differentiated in the presence of VIP show augmented antigen uptake, evaluated by endocytosis, of FITC-dextran at different times. Results are expressed as fluorescence intensity and are the mean SD of 3 experiments performed in duplicate. (C) Differentiation of DCs with VIP modifies their cytokine production after activation. Results are the mean SD of 4 experiments performed in duplicate. Figure 2. VIP generates human tolerogenic DCs, which induce IL-10–producing anergic CD4 T cells. Human DCs generated in the absence (DCcontrols, f) or presence (DCVIPs, ) of VIP were stimulated with LPS to induce DC activation/ maturation. (A) Purified naive CD4 T cells were cultured with graded doses of allogeneic DCcontrols (f) or DCVIPs ( ) in primary cultures (panel i). CD4 T cells from primary cultures were recovered, rested, and restimulated with LPS-matured allogeneic DCs (panel ii). Cultures of DCcontrols or DCVIPs without T cells did not proliferate. Cytokine production was determined in secondary cultures at DC/T cell ratios of 1:10 (panel iii). Each result is the mean SD of 3 experiments performed in duplicate. (B) TT-primed TH1 cells were cocul- tured with TT-pulsed syngeneic DCcontrols (f) or DCVIPs ( ) in primary cultures (panel i). After 3 days of culture, TH1 cells were rescued, rested, and restimulated with TT-pulsed, LPS-matured syngeneic DCs, and proliferation (panel ii) and IFN production (panel iii) were determined. Unpulsed or ovalbumin-pulsed DCcontrols or DCVIPs did not induce the proliferation ofTT-primedTH1 cells. Each result is the mean SD of 3 experiments performed in duplicate. 3634 GONZALEZ-REY et al BLOOD, 1 MAY 2006 VOLUME 107, NUMBER 9 For personal use only. at FMRP on January 19, 2011. www.bloodjournal.org From