The neuropeptide vasoactive intestinal peptide generates tolerogenic dendritic cells

The Neuropeptide Vasoactive Intestinal Peptide GeneratesTolerogenic Dendritic Cells1

Mario Delgado,*† Elena Gonzalez-Rey,* and Doina Ganea2†

Tolerogenic dendritic cells (DCs) play an important role in maintaining peripheral tolerance through the induction/activation ofregulatory T cells (Treg). Endogenous factors contribute to the functional development of tolerogenic DCs. In this report, wepresent evidence that two known immunosuppressive neuropeptides, the vasoactive intestinal peptide (VIP) and the pituitaryadenylate cyclase-activating polypeptide (PACAP), contribute to the development of bone marrow-derived tolerogenic DCs invitro and in vivo. The VIP/PACAP-generated DCs are CD11clowCD45RBhigh, do not up-regulate CD80, CD86, and CD40 followingLPS stimulation, and secrete high amounts of IL-10. The induction of tolerogenic DCs is mediated through the VPAC1 receptorand protein kinase A, and correlates with the inhibition of I�B phosphorylation and of NF-�Bp65 nuclear translocation. TheVIP/PACAP-generated DCs induce functional Treg in vitro and in vivo. The VIP/DC-induced Treg resemble the previouslydescribed Tr1 in terms of phenotype and cytokine profile, suppress primarily Th1 responses including delayed-type hypersensi-tivity, and transfer suppression to naive hosts. The effect of VIP/PACAP on the DC-Treg axis represents an additional mechanismfor their general anti-inflammatory role, particularly in anatomical sites which exhibit immune deviation or privilege. TheJournal of Immunology, 2005, 175: 7311–7324.

D endritic cells (DC)3 are a heterogenous population ofAPCs that contribute to innate immunity and initiateadaptive immune responses to Ags associated with in-

fection and inflammation (1). The successful initiation of the adap-tive immune response requires DC maturation, following signalingthrough the toll-like receptors and CD40. However, in addition totheir proinflammatory role, DC also play an important role in im-mune homeostasis, by inducing and maintaining tolerance (2).

In addition to the elimination of self-reactive T cells in the thy-mus, tolerance is maintained in the periphery through clonal de-letion, induction of anergy, and differentiation of regulatory T cells(Treg). Several populations of CD4 Treg have been described andcharacterized, including the naturally occurring CD4�CD25�

Treg (nTreg) and the induced CD4�CD25� peripheral Treg(iTreg), consisting of IL-10-producing Tr1 and TGF-�-secretingTh3/Tr2 (3–6).

In contrast to the nTreg generated in the thymus, iTreg are gen-erated in the periphery, and DCs appear to play an essential role intheir differentiation. Several recent reports indicate that immature

or semimature DCs contribute to the induction of iTreg. Repeatedexposure to immature monocyte-derived human DCs secretingIL-10 has been shown to induce IL-10 and TGF-�-secreting CD4Tr1 (7). Tolerogenic DCs have been generated in the presence of1,25(OH)2D3, the active form of vitamin D3, and of vitamin D3

analogues from both human blood monocytes and murine bonemarrow and shown to induce Treg (8–10). In addition, DCs witha semimature status were shown to be tolerogenic and induce Tr1differentiation (11). Although most reports focused on the matu-ration stage as directly connected to the tolerogenic capacity, arecent study by Wakkach et al. (12) suggests the existence of adistinct subset of tolerogenic DCs characterized by the stable phe-notype CD11clowCD45Rbhigh. These DCs can be derived frombone marrow cells in the presence of GM-CSF, TNF, and IL-10,secrete high levels of IL-10 following activation, and induce Tr1cells in vivo and in vitro. CD11clowCD45RBhigh tolerogenic DCsare present in the spleen and lymph nodes of normal mice, andsplenic stromal cells were shown to direct their differentiation(12, 13).

The neuropeptides vasoactive intestinal peptide (VIP) and pitu-itary adenylate cyclase-activating polypeptide (PACAP) are potentimmunosuppressive agents, affecting both innate and adaptive im-munity (14–16). Recently, we have shown that VIP/PACAP affectbone marrow-derived DC (BM-DC) differently, depending on theDC maturation state (17). Immature DC treated with VIP orPACAP up-regulate CD86 expression, stimulate T cell prolifera-tion and promote Th2-type responses while inhibiting the Th1-typeproinflammatory response (17). In contrast, VIP and PACAPdown-regulate CD80 and CD86 expression of LPS-matured DCsand inhibit their capacity to activate allogeneic or syngeneic Tcells in vivo and in vitro (17). In the present study, we addressedthe question whether VIP/PACAP induce tolerogenic BM-DCs.We report that VIP/PACAP induce CD11clowCD45Rbhigh DC thatdo not up-regulate CD40, CD80, or CD86, and secrete highamounts of IL-10 upon LPS stimulation. The VIP/PACAP-gener-ated DCs induce Ag-specific Tr1-like Treg in vitro and in vivo, andthese Treg are capable of transferring suppression to naive hosts.

*Department of Biological Sciences, Rutgers University, Newark, NJ 07102; and†Instituto de Parasitologia y Biomedicina, Instituto del Consejo Superior de Investi-gaciones Cientı́ficas, Granada, Spain

Received for publication June 3, 2005. Accepted for publication September 19, 2005.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by National Institutes of Health Grants AI52306 andAI47325 (to D.G.), Johnson & Johnson Neuroimmunology Fellowships (to M.D.),and the Spanish Ministry of Health PI04/0674 (to M.D.).2 Address correspondence and reprint requests to Doina Ganea, Department of Phys-iology, Temple University School of Medicine, 2nd Floor, Old Medical School,3400 North Broad Street, Philadelphia, PA 19140, E-mail address: [email protected] Abbreviations used in this paper: DC, dendritic cell; BM-DC, bone marrow-derivedDC; DLN, draining lymph nodes; iDC, immature DC; mDC, mature DC; DTH, de-layed-type hypersensitivity; GITR, glucocorticoid-induced TNFR; MCF, mean chan-nel fluorescence; Nrp1, neuropilin 1; PACAP, pituitary adenylate cyclase-activatingpolypeptide; PCCF, pigeon cytochrome c fragment; PKA, protein kinase A; Tg, trans-genic; Treg, regulatory T cell; nTreg, naturally occurring CD4�CD25� Treg; iTreg,induced CD4�CD25� peripheral Treg; VIP, vasoactive intestinal peptide.

The Journal of Immunology

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00

Materials and MethodsReagents

Synthetic VIP, PACAP38, and PACAP6-38 were purchased from Calbio-chem-Novabiochem. Capture and biotinylated Abs against murine IL-4,IL-5, IL-2, TNF-�, IL-6, IL-12, TGF-�1, and IFN-�, mAbs to CTLA-4,CD40, CD4, V�3, CD11c, I-Ek, CD80, CD86, and CD103, and recombi-nant murine (rm)IFN-� and rmIL-4 were purchased from BD Pharmingen.Capture and biotinylated Abs against murine IL-10 and rmGM-CSF andrmIL-2 were purchased from PreproTech. Rat anti-glucocorticoid-inducedTNFR (GITR) was purchased from R&D Systems. Pigeon cytochrome cfragment (PCCF) was synthesized and purified by Research Genetics. H89was obtained from ICN Pharmaceuticals. LPS (from Escherichia coli 055:B5), OVA, avidin-peroxidase, PMA, calphostin C, ionomycin, and monen-sin were purchased from Sigma-Aldrich. The VPAC1-agonist[K15,R16,L27]VIP1–7-GRF8–27 and the VPAC1-antagonist [Ac-His1,D-Phe2,K15,R16,L27] VIP3–7-GRF8–27 were previously described (18, 19).

Animals

B10.A (I-Ek), C57BL/6 (H-2b), and TCR-Cyt-5CC7-I/Rag1 transgenic (Tg,I-Ek) mice were obtained from The Jackson Laboratory and TaconicFarms. All mice used were between 7 and 12 wk of age. All animal pro-cedures were approved by the Rutgers University Institutional Animal Careand Use Committee, on accordance with American Association for theAccreditation of Laboratory Animal Care guidelines.

Cell isolation and cultures

BM-DCs were generated from B10.A mice with the exception of the de-layed-type hypersensitivity (DTH) experiments where DC were generatedfrom C57BL/6 mice as previously described (17) in medium containing200 U/ml (20 ng/ml) rmGM-CSF (5 � 106 U/mg) in the presence or ab-sence of VIP or PACAP38 (from 10�12 to 10�6 M). Alternatively, VIP orPACAP (10�8 M) were added at different times after the initiation of cul-ture. On day 6 or 7, nonadherent cells were collected by gently pipettingand centrifuged at 1200 rpm for 5 min. Cells were directly analyzed byflow cytometry or were seeded in flat-bottom 48-well microtiter plates(Corning Glass at 5 � 105 cells per well in a final volume of 500 �l andmatured for 48 h with LPS (1 �g/ml) or CD40L-transfected Chinese ham-ster ovary cells (1 � 105 cells). In some experiments CD11c� DCs werepurified by immunomagnetic methods using anti-CD11c-conjugated mag-netic beads and the AutoMACS system (Miltenyi Biotec).

Purified CD4 T cells from Tg mice were isolated by positive immuno-magnetic selection with anti-CD4-conjugated magnetic beads (MiltenyiBiotec). The purified T cells were �98% CD4� by FACS analysis. EffectorTh1 and Th2 cells were generated as previously described (17). Briefly,purified naive CD4� T cells (3 � 105 cells/ml) from Tg-PCCF mice werecultured with APCs (105 cells/ml) and PCCF (5 �g/ml) plus IL-2 (50U/ml). Th2 polarization was performed in the presence of IL-4 (200 U/ml)plus anti-IFN-� Ab (10 �g/ml), and Th1 polarization was performed in thepresence of IFN-� (1000 U/ml) and anti-IL-4 Abs (10 �g/ml). After 4 days,Th1 and Th2 effectors were characterized by intracellular cytokine profile(IFN-� and IL-4, respectively).

APCs were prepared by T cell depletion of B10.A (I-Ek) spleen cellswith a mixture of anti-CD8- and anti-CD4-conjugated magnetic beads andtreated with 50 �g/ml mitomycin C (Sigma-Aldrich) for 20 min at 37°C.

DCs were also purified from spleen and lymph nodes as previouslydescribed with some modifications (12). In brief, spleen and mesentericlymph nodes were cut into small fragments and digested with collagenaseD (1 mg/ml). Resulting cells were layered over a Percoll gradient, and Tand B cells were depleted by treating the recovered low-density cells witha mixture of mAbs (anti-CD3 and anti-B220) coupled to magnetic beads.

FACS analysis

BM-DCs (1 � 106 cells/ml) were incubated with various mAbs (FITC-anti-CD80, FITC-anti-CD86, FITC-anti-CD40, FITC-anti-I-Ek, FITC-anti-CD45RB, PE-anti-CD11c, 2.5 �g/ml final concentration) at 4oC for 1 h.Isotype-matched Abs were used as controls, and IgG block (Sigma-Al-drich) was used to block nonspecific binding to Fc receptors. After exten-sive washing, the cells were fixed in 1% paraformaldehyde. Stained DC,gated according to forward- and side-scatter characteristics, were analyzedon a FACSCalibur flow cytometer (BD Biosciences). Fluorescence datawere expressed as mean channel fluorescence (MCF), and as percentage(%) of positive cells after subtraction of background isotype-matchedvalues.

For analysis of intracellular CTLA-4, T cells (1 � 106 cells/ml) werestained with PerCP-anti-CD4 mAb for 30 min at 4oC, fixed with Cytofix/Cytoperm solution (BD Pharmingen) and incubated for 45 min at 4oC with

PE-anti-CTLA-4 mAb diluted in PBS � 1% BSA � 0.5% saponin. Afterextensive washing, cells were analyzed on a FACSCalibur flow cytometer.Surface GITR and CD103 expression was determined by FACS using PE-anti-CD103 and PE-anti-GITR Abs.

Endocytosis

Mannose receptor-mediated endocytosis was measured as the cellular up-take of FITC-dextran (Sigma-Aldrich) and the fluid phase endocytosisthrough membrane ruffling was measured as the cellular uptake of LuciferYellow dipotassium salt (Sigma-Aldrich), and both were quantified by flowcytometry. Briefly, DCs (2 � 105 cells/sample) were incubated in mediumcontaining FITC-dextran (1 mg/ml; m.w. 40,000) or with Lucifer Yellow(1 mg/ml) for 0, 60, and 120 min. Afterward incubation cells were washedtwice in wash buffer, fixed in cold 1% paraformaldehyde, and analyzed byflow cytometry.

Assay of DC costimulatory activity

The costimulatory activity for syngeneic Ag-specific CD4 T cells was per-formed by using a TCR Tg model. In brief, various numbers of B10.ABM-DCs differentiated in the absence or presence of VIP/PACAP wereadded to purified PCCF-specific Tg CD4 T cells (5 � 105 cells/well) in thepresence of PCCF (5 �M). Proliferation was determined by measuringBrdU incorporation as recommended by the manufacturer (Roche AppliedScience). Cells cultured with an irrelevant Ag (OVA, 10 �g/ml) were usedas control.

T cell anergy was determined after a secondary restimulation of T cellswith mature DCs. In brief, B10.A BM-DCs (1 � 105 cells/well) differen-tiated in the absence or presence of VIP/PACAP were added to purifiedsyngeneic PCCF-specific Tg CD4 T cells (5 � 105 cells/well) in the pres-ence of PCCF (5 �M). After 3 days of culture, T cells were recovered byFicoll gradient and DC depletion with anti-CD11c microbeads. T cellswere rested for 2 to 4 days in complete medium supplemented with 2 U/mlIL-2, and restimulated (5 � 105 cells/well) with different numbers of LPS-matured B10.A DCs, generated in the absence of VIP/PACAP, and pulsedwith PCCF (5 �M). Proliferation was determined by measuring the BrdUincorporation. Cell-free culture supernatants were harvested and used forcytokine determination by ELISA.

Cytokine Assays

The cytokine content in DCs or DCs-CD4 T cell cultures was determinedby sandwich ELISAs. The Ab pairs used were as follows, listed by capture/biotinylated detection Abs (BD Pharmingen): IL-4, BVD4–1D11/BVD6–24G2; IFN-�, R4–6A2/XMG1.2; IL-5, TRFK5/TRFK4; IL-2, JES6–1A12/JES6–5H4; IL-12p40, C15.6/C17.8; TNF, MP6-XT22/MP6-XT3;IL6, MP5–20F3/MP5–32C11; IL-10, JES5–2A5/JES5–16E3.

For the intracellular cytokine analysis of restimulated CD4 T cells, 106

cells/ml were collected and stimulated with PMA (1 ng/ml) plus ionomycin(20 ng/ml) for 8 h. Monensin (1.33 �mol/ml) was added for the last 4 h ofculture. Cells were stained with PerCP-anti-CD4 mAbs for 30 min at 4oC,washed, fixed/saponin permeabilized with Cytofix/Cytoperm, and stainedwith 0.5 �g/sample of FITC- and PE-conjugated anti-IFN-�-, anti-IL-4-, oranti-IL-10-specific mAbs for 45 min at 4oC. Cells were analyzed by flowcytometry, using FITC/PE-conjugated isotypic mAbs as controls.

Real-time RT-PCR

Total RNA was isolated from CD4 T cells or sorted CD4�CD25� (106

cells) using the Ultraspec RNA reagent (Biotecx). Two micrograms of totalRNA was reverse transcribed. Quantitative real-time RT-PCR was per-formed in an ABI PRISM cycler (Applied Biosystems) using a SYBRGreen PCR kit from Applied Biosystems. A threshold was set in the linearpart of the amplification curve, and the number of cycles needed to reachthe threshold was calculated for every gene. Relative mRNA levels weredetermined by using standard curves for each individual gene and furthernormalization to hypoxanthine phosphoribosyltransferase (HPRT). Melt-ing curves established the purity of the amplified band. Primer sequencesare: neuropilin 1 (Nrp1) (5�-GCCTGCTTTCTTCTCTTGGTTTCA-3�, 5�-GCTCTGGGCACTGGGCTACA-3�); Foxp3 (5�-CTGGCGAAGGGCTCGGTAGTCCT-3�, 5�-CTCCCAGAGCCCATGGCA GAAGT-3�);HPRT (5�-TGGAAAGAATGTCTTGATTGTTGAA-3�, 5�-AGCTTGCAACCTTAACCATTTTG-3�).

In vitro T regulatory activity

B10.A BM-DCs differentiated in the absence or presence of VIP orPACAP were extensively washed, cultured with LPS (1 �g/ml) for 48 h at37oC to induce DC activation/maturation, and added (5 � 104 cells/well)to purified syngeneic PCCF-specific Tg CD4 T cells (5 � 105 cells/well)

7312 VIP AND TOLEROGENIC DC

in the presence of PCCF (5 �M). One week later, T cells were separatedby Ficoll-Hypaque gradient, and DCs were removed by anti-CD11c mi-crobeads (Miltenyi Biotec). Different numbers of purified T cells (Treg)were incubated with syngeneic PCCF-specific Tg CD4 T cells (5 � 105

cells/well) (responder T) in the presence of mitomycin C-treated B10.Aspleen APCs (2 � 104 cells/well) and PCCF. The proliferation of responderCD4 T cells was determined by BrdU incorporation. In some experiments,cocultures were performed in the presence of blocking mouse anti-IL-10(10 �g/ml), anti-TGF-�1 (40 �g/ml) and/or anti-CTLA4 (10 �g/ml)mAbs, or of rmIL-2 (100 U/ml). The anti-IL-10 and anti-TGF-�1 Abs werepreviously titrated. Concentrations leading to undetectable levels of IL-10or TGF-�1 following Ab addition were used in the blocking experiments.Culture supernatants were assayed for IL-2 production by sandwich ELISA(BD Pharmingen).

Transwell experiments were done in 24-well plates (Millicell, 0.4 �m;Millipore). Responder PCCF-specific Tg CD4 T cells (5 � 105 cells) to-gether with mitomycin C-treated B10.A spleen APCs (2 � 104 cells) andPCCF (5 �M) were placed in the bottom wells. T cells (5 � 105 cells)generated in the presence of VIP-DCs were placed together with mitomy-cin C-treated B10.A spleen APCs (2 � 104 cells) and PCCF (5 �M) in theupper wells. Three days later, the basket was removed, and the proliferationof the responder T cells was measured. IL-2 levels in culture supernatantswere determined by ELISA.

Determination of Ab responses

Specific Ab responses in PCCF or OVA-immunized mice were determinedby ELISA. Serum was obtained by cardiac puncture. Maxisorb plates (Mil-lipore) were coated overnight at 4°C with 100 �l of soluble PCCF or OVA(10 �g/ml). The plates were incubated with serial dilutions of serum for 2 hat 37°C. Biotinylated anti-IgG, anti-IgG1, or anti-IgG2a (2.5 �g/ml) (Se-rotec) were added for 1 h at 37°C, followed by streptavidin-HRP. Thebound enzyme was detected with the TMB substrate, and quantitated atOD450 in an ELISA reader (Bio-Tek Intrument). A standard curve wasconstructed for each assay by coating wells with an isotype-specific anti-mouse Ig followed by addition of known concentrations of the correspond-ing mouse Ig isotype.

DTH

Control- or VIP-DC were generated from C57BL/6 mice, pulsed with OVA(5 �M) for 2 h, and cultured with LPS (1 �g/ml) for 24 h to induce DCactivation/maturation. To trace the injected cells in vivo, control-DC andVIP-DC were labeled with 1 ml of PBS, 0.1% BSA, 5 �M CFSE (Mo-lecular Probes) for 10 min at 37oC, and washed three times in RPMI 1640and 10% FBS. Control-DC, VIP-DC (3 � 105 cells) or saline (none) wereinjected s.c. into C57BL/6 mice previously immunized with OVA/CFA(100 �g, s.c.) (groups of nine). Four days after DC transfer, the mice werechallenged with 50 �g OVA in 20 �l of PBS in the left ear with 20 �l ofPBS in the right ear. The presence of the transferred CFSE-stained DCs inthe draining lymph node (DLN) were determined by flow cytometry. Earthickness was measured 24 h after challenge, and the thickness of the earexposed to PBS was subtracted from the thickness of the ear exposedto OVA.

Determination of NF-�Bp65 nuclear translocation and IkBphosphorylation

Nuclear and cytoplasmic protein extracts were isolated from DCs as pre-viously described (20), and the content of NF-�Bp65 and phosphorylated-IkB were determined by using a specific ELISA kit (Imgenex).

ResultsCharacterization of VIP/PACAP-DC

To determine whether exposure to VIP/PACAP during DC differ-entiation results in DC phenotypical and functional changes, wecompared DC generated from B10.A mice in the presence or ab-sence of VIP/PACAP (VIP/PACAP-DC) in terms of surface mark-ers, endocytic capacity, and cytokine production. Bone marrowcell cultures were initiated in medium containing GM-CSF andVIP or PACAP. We harvested the nonadherent cells 6 days laterand determined the levels of surface MHC class II, CD40, CD80,and CD86 expression in the CD11c� cell population (immatureDC (iDC)). The DCs generated in the presence of VIP/PACAPexpress lower levels of CD11c, CD40, CD80, and CD86, and thereduction in costimulatory molecule expression by VIP/PACAP is

dose-dependent (Fig. 1A). Expression of MHC class II and of thecostimulatory molecules CD40, CD80, and CD86 increases uponLPS stimulation of DC (mature DC (mDC)). However, similar toDC generated in the presence of 1,25(OH)2D3 or in the presence ofTNF and IL-10, the VIP/PACAP-DC are resistant to LPS stimu-lation in terms of expression of CD40, CD80, and CD86 (Fig. 1B).

To establish the requirements for exposure to VIP during DCdifferentiation, we compared the effects of VIP added at differenttimes during DC generation. VIP added at day 0 or 1 results inreduction in CD40 and CD80 expression, and VIP added as late asday 3 still affects CD86 (Fig. 1C). In contrast, later additions (day5 or 7) do not affect CD40 and CD80, while inducing CD86 iniDC, and down-regulate CD80 and CD86 in LPS-mDC (Fig. 1C).These results are in agreement with the previously reported effectsof VIP/PACAP added at the end of DC differentiation (day 7) (17).

Because iDC and mDC differ in their phagocytic capacity, weassessed the endocytic ability of VIP/PACAP-DC by using FITC-dextran or Lucifer Yellow. DC generated in the presence of VIP/PACAP exhibit a higher endocytic activity compared with controlDC (Fig. 2A). Upon TLR receptor activation, iDC mature into cellscapable of producing high levels of TNF, IL-12, and IL-6. Wedetermined the cytokine profile for VIP/PACAP-DC stimulatedwith LPS. In contrast to control DC which produce TNF, IL-12p40, and IL-6, and low levels of IL-10, the VIP/PACAP-DCproduce extremely low levels of proinflammatory cytokines (TNF,IL-12, and IL-6), and high levels of IL-10 (Fig. 2B). IL-10 pro-ducing tolerogenic DC, generated in the presence of TNF andIL-10 and expressing low levels of costimulatory molecules, havebeen previously described as being CD11clowCD45RBhigh. TheVIP/PACAP-DC consist of cells with a similar phenotype, i.e.,CD11clowCD45RBhigh (Fig. 2C).

Taken together, these results indicate that the DCs generated inthe presence of the neuropeptides differ from those generated withGM-CSF alone, in terms of costimulatory molecule expression,endocytic capacity, cytokine profile, and CD11cCD45RB pheno-type. The characteristics expressed by the VIP/PACAP-DC arequite similar to those reported for tolerogenic DC generated in thepresence of TNF and IL-10 (12).

VIP/PACAP-DC induce IL-10/TGF-�-producing T cells with lowproliferative capacity

Tolerogenic DC are poor stimulators of T cell proliferation andcytokine production. To examine the capacity of the VIP/PACAP-DC to stimulate T cells, we treated the VIP/PACAP-DCwith LPS or CD40L-transfected cells and cocultured them withsyngeneic PCCF-specific T cells (Tg PCCF-T) in the presence ofPCCF. The T cells exposed to the VIP/PACAP-DC did not pro-liferate (Fig. 2D). Because LPS-stimulated VIP-DCs secrete largeamounts of IL-10 (Fig. 2B), we assessed the role of IL-10 in theinduction of low proliferative T cells by adding anti-IL-10 Abs tothe VIP-DCs/T cell cultures. The Ab dose necessary to neutralizethe secreted IL-10 from LPS-stimulated VIP-DCs was determinedin preliminary experiments. The neutralizing anti-IL-10 Abs didnot affect T cell proliferation induced by control DCs (2.32 � 0.31in the absence of anti-IL-10 Abs and 2.37 � 0.22 in the presenceof anti-IL-10 Abs), but partially reversed the inhibitory effect ofVIP-DCs on T cell proliferation (from 0.51 � 0.12 in the absenceof anti-IL-10 to 0.97 � 0.08 in the presence of anti-IL-10 Abs).

To determine whether the T cells exposed to VIP/PACAP-DCare indeed anergic, we exposed transgenic PCCF-specific T cells(Tg PCCF-T) to control or VIP/PACAP-DC in the presence ofPCCF, followed by re-exposure to fresh LPS-stimulated DC

7313The Journal of Immunology

FIGURE 1. VIP and PACAP induce a stable immature phenotype in BM-DCs. A and B, BM cells were grown in the absence (control-iDC) or presence(VIP-iDC or PACAP-iDC) of different concentrations of VIP or PACAP (10�8 M for histograms). The nonadherent cells were harvested, double stainedwith anti-CD11c and with anti-CD40, I-Ek (MHCII), CD80 or CD86 mAbs, and analyzed by flow cytometry (A), or extensively washed, cultured for 48 hwith LPS (1 �g/ml), and double stained as above (B). Right panels, Dose-response effects for each phenotypic marker in the CD11c� population.Histograms are representative of three independent experiments. Results in right panels are the mean � SD of three experiments performed in duplicate.C, Effect of VIP added at different time points during DC differentiation. VIP (10�8 M) was added at different times after the initiation of the culture. Thecells were double stained with anti-CD11c and with anti-CD40, I-Ek (MHCII), CD80 or CD86 mAbs, and analyzed by flow cytometry. Results show theMCF intensity for each phenotypic marker in the CD11c� population. Data represent the mean � SD of three experiments performed in duplicate.


pulsed with PCCF. T cells exposed initially to control DC prolif-erated and produced IFN-�, whereas the T cells exposed to VIP/PACAP-DC did not proliferate and did not produce IFN-� uponre-exposure to PCCF-loaded DC (Fig. 2E). This suggests that VIP/PACAP-DC induce anergic T cells and/or Treg.

Although neither type of T cells proliferate in vitro in responseto Ag, Treg can release anti-inflammatory cytokines such as IL-10

and TGF-�. Therefore, we assessed the cytokine profile of T cellscocultured with VIP/PACAP-DC. VIP/PACAP-DC were stimu-lated with LPS and added together with PCCF to Tg PCCF-T cells.The T cells were purified 1 week later, restimulated with mitomy-cin C-treated splenic APC plus PCCF, and the cytokine profile wasdetermined by ELISA. Control DC induced a predominant Th1cytokine profile, with high levels of IFN-� and IL-2. In contrast, T

FIGURE 2. VIP/PACAP affectDC endocytosis, cytokine profile, andphenotype and induce T cell hypore-sponsiveness. A, Control-iDC andVIP-iDC or PACAP-iDC were as-sessed for endocytosis using flow cy-tometry. Results are expressed as flu-orescence intensity (MCF) and arethe mean � SD of three experimentsperformed in duplicate. B, CD11c�

control- and VIP-DC were culturedwith LPS (1 �g/ml). Supernatantswere harvested 48 h later and assayedfor TNF, IL-12p40, IL-6, and IL-10cytokines by ELISA. Results are themean � SD of four experiments per-formed in duplicate. C, VIP/PACAPinduce the generation of CD11clow

CD45Rbhigh DCs. Control- and VIP/PACAP-DCs were double stained forCD11c and CD45RB, and analyzedby flow cytometry. Values refer tothe percentage of CD11clow

CD45RBhigh DC cells (rectangle).Dot plots represent the results of onerepresentative experiment of three.D, Control- and VIP/PACAP-DCcells were cultured for 48 h with me-dium, LPS (1 �g/ml), or CD40L-transfected cells. Various numbers ofDCs were added to purified synge-neic PCCF Tg CD4 T cells (5 � 105

cells/well) and PCCF (5 �M), andproliferation was determined. Cul-tures of control-iDC or VIP-iDCwithout T cells did not proliferate.Each result is the mean � SD of threeexperiments performed in duplicate.E, DC were cultured with syngeneicPCCF Tg T cells for 3 days. The re-purified T cells were rested for 2 to 4days in the presence of IL-2, and re-stimulated (5 � 105 cells/well) withdifferent numbers of LPS-maturedDCs and PCCF. T cell proliferationwas determined. IFN-� levels in theculture supernatants were determinedby ELISA. Cells cultured with an ir-relevant Ag (OVA, 10 �g/ml) did notproliferate (A450 � 0.123) or produceIFN-� (�0.1 ng/ml). Each result isthe mean � SD of three experimentsperformed in duplicate.


cells exposed to VIP/PACAP-DC produced IL-10, low levels ofTGF-�, little IFN-�, and no IL-2 (Fig. 3A). T cells exposed re-peatedly to LPS-stimulated VIP/PACAP-DC and PCCF (threeweekly rounds), followed by purification and restimulation withPCCF/splenic APC, produced significantly higher levels of IL-10and TGF-�, but no IFN-�, IL-2, IL-5, and very little if any IL-4(Fig. 3B). Intracellular cytokine staining confirmed the high ex-pression of IL-10 and to a much lesser degree of IL-4, and thereduction in IFN-� in T cells exposed to VIP/PACAP-DC(Fig. 3C).

Induction of tolerogenic DC by VIP is mediated through VPAC1receptors and protein kinase A (PKA)

The biological effects of VIP/PACAP are mediated through threetypes of receptors, i.e., VPAC1, VPAC2, and PAC1 (21). We have

previously reported that BM-DCs express VPAC1 and VPAC2(17). To determine the VIP receptor involved in the generation oftolerogenic DCs, we used a VPAC1 agonist and VPAC1 andVPAC2/PAC1 antagonists. DC generated in the presence of theVPAC1 agonist exhibit a phenotype similar to VIP-DCs(CD11clowCD45RBhighCD40lowCD80lowCD86low) (Fig. 4A). Therole of VPAC1 was confirmed by the fact that the VPAC1 antag-onist, but not the VPAC2/PAC1 antagonist, reversed the effect ofVIP (Fig. 4A). Because VPAC1 activates adenylate cyclase induc-ing intracellular cAMP, we assessed the role of PKA, one of themajor cAMP targets. The PKA inhibitor H89, but not the proteinkinase C inhibitor calphostin C, reversed the effect of VIP(Fig. 4A).

The involvement of VPAC1 and PKA were further confirmed inexperiments assessing the tolerogenic capacity of VIP-DCs. DCs

FIGURE 3. VIP/PACAP-DCs in-duce IL-10-producing CD4 T cells. Aand B, Control- and VIP/PACAP-DCswere cultured with LPS and added(5 � 104 cells/well) to purified synge-neic PCCF-specific Tg CD4 T cells(5 � 105 cells/well) in the presence ofPCCF. One week later (first restimula-tion, A) or after weekly repetitive stim-ulations (for 3 weeks) (third restimula-tion, B), T cells (2 � 105 cells/well)were isolated and restimulated withspleen APCs (2 � 104 cells/well) andPCCF; 48 h later the cytokines weredetermined by ELISA. Cells culturedwith an irrelevant Ag (OVA, 10 �g/ml)did not induce any cytokines: �0.1 ngof IFN-� per milliliter, �30 pg of IL-4per milliliter, �50 pg of TGF-�1 permilliliter, �0.1 ng of IL-10 per millili-ter, �0.1 ng/ml IL-2, and �50 pg ofIL-5 per milliliter. Each result is themean � SD of three experiments per-formed in duplicate. C, Intracellularcytokine staining of activated T cells.Purified syngeneic PCCF Tg CD4 Tcells (5 � 105 cells/well) were stimu-lated as in B, harvested and restimu-lated with PMA (1 ng/ml) plus iono-mycin (20 ng/ml) for 8 h. Monensin(1.33 �mol/ml) was added for the last4 h of culture. Cells were fixed andstained for detection of intracellular cy-tokines using FITC- or PE-conjugatedspecific mAbs. Values indicate the per-centage of positive cells in each quad-rant. Dot plots show PerCyP-stainedCD4 T gated cells. One of four exper-iments with similar results is shown.


generated in various conditions were stimulated with LPS for 24 h,followed by coculture with Tg PCCF T cells and PCCF. Thepurified T cells were restimulated 7 days later with mitomycinC-treated splenic APCs and PCCF and tested for proliferation andcytokine profile. T cells cultured with VIP-DCs or VPAC1-DCsdid not proliferate and produced very low levels of IFN-�, no IL-2,and high levels of IL-10 and TGF-� (Fig. 4, B and C). The effectof VIP was reversed by the VPAC1 antagonist and H89, but not bythe VPAC2/PAC1 antagonist or calphostin C (Fig. 4, B and C).

LPS-induced NF-�B activation is prevented in VIP-DCs

Recently, inhibition of NF-�B has been reported to result in theinduction of tolerogenic DCs (22). Therefore, we determined theeffects of VIP on NF-�Bp65 nuclear translocation and on I�B

phosphorylation. VIP-DCs were stimulated with LPS, and the lev-els of NF-�Bp65 and P-I�B were determined in nuclear and cy-toplasmic extracts. In the absence of LPS stimulation, p65 waslocalized in the cytoplasm, and I�B was not phosphorylated (Fig.4D). Following LPS stimulation, p65 translocated to the nucleus incontrol DCs but not in VIP-DCs, and I�B was phosphorylated incontrol but not VIP-DCs (Fig. 4D). The effect of VIP was reversedby the VPAC1 antagonist and by H89, suggesting that the VIP-induced inhibition of NF-�B activation is mediated by VPAC1and PKA.

VIP/PACAP-DC induce functional Treg in vitro

When stimulated through the TCR, Treg suppress the proliferationand IL-2 production of Ag-specific effector T cells. To determine

FIGURE 4. VIP-induced generation of tolerogenic DCs is VPAC1- and cAMP-dependent, and involves the inhibition of NF-�B nuclear translocation.BM-DC cells were generated in the absence (control) or presence of VIP (10�8 M), VPAC1 agonist (10�8 M), VIP plus VPAC1 antagonist (10�6 M), VIPplus VPAC2/PAC1-antagonist (10�6 M), VIP plus H89 (a PKA inhibitor, 100 nM), or VIP plus calphostin C (a protein kinase C inhibitor, 20 ng/ml).Nonadherent cells were harvested 6 days later, stimulated with LPS (1 �g/ml), double stained for CD11c and CD45RB, CD40, CD80, or CD86, andanalyzed by flow cytometry (A). CD11c� DCs were added (5 � 104 cells/well) to purified PCCF-specific Tg CD4 T cells (5 � 105 cells/well) in thepresence of PCCF. One week later, T cells (2 � 105 cells/well) were isolated, restimulated with spleen APCs (2 � 104 cells/well), and PCCF, and thecytokines were measured 48 h later by ELISA (B), or the purified T cells were added to syngeneic PCCF Tg T cells (5 � 105 cells/well) in the presenceof spleen APCs (2 � 104 cells/well) and PCCF; T cell proliferation was determined (C). Each result is the mean � SD of three experiments performedin duplicate. D, BM-DCs generated as described above were incubated with medium or LPS. After 1 h of culture, the nuclear and cytoplasmic extracts wereisolated, and the content of NF-�B and phosphorylated IkB was determined by ELISA. Each result is the mean � SD of four experiments performed induplicate.


whether T cells exposed to VIP/PACAP-DC become functionalTreg, we cultured Tg-PCCF CD4 T cells for 7 days with PCCF-pulsed DC generated from B10.A mice either in the presence orabsence of VIP and matured with LPS. Tr(control) from culturescontaining DC generated in the absence of the neuropeptide, andTr(VIP) from cultures containing DC generated in the presence ofVIP were purified and cocultured with fresh Tg-PCCF CD4 T cellsin the presence of splenic APC pulsed with PCCF. Tr(VIP) inhib-ited the proliferation of fresh Tg-PCCF T cells, whereas the Tr-(control) did not (Fig. 5A). Similar results were obtained in respectto IL-2 production (Fig. 5A, bottom panel). The inhibition of Tg-PCCF T cell proliferation by Tr(VIP) is dose-dependent (Fig. 5A,central panel).

Next we addressed the question whether Tr(VIP) inhibit effectorT cells through direct cellular contact and/or soluble factors. WhenTr(VIP) and effector CD4 T cells are separated in Transwell ex-periments, the proliferation of effector T cells is still inhibited, butto a lesser degree, indicating that both direct contact and solublefactors mediate the inhibitory effect. In regular cocultures, additionof saturating amounts of anti-TGF-�, anti-IL-10, or anti-CTLA-4Abs reversed inhibition only modestly. However, addition of bothanti-IL-10 and anti-TGF-� has a more pronounced effect, and ad-dition of all three Abs (anti-IL-10, anti-TGF-�, anti-CTLA-4) re-verses the inhibitory effect completely (Fig. 5B).

Because Treg were reported to express high levels of CTLA-4,and our experiments suggest that CTLA-4 plays a role in the in-hibitory activity of Tr(VIP), we measured the levels of CTLA-4 inTr(VIP) and Tr(control). Tr(VIP) express higher levels ofCTLA-4, and the percentage of intracellular IL-10�CTLA-4�

cells is significantly higher in the Tr(VIP) population (Fig. 5C).CD4�CD25� Treg have been reported to express markers such asCD103, GITR, Nrp1, and Foxp3, although different Treg popula-tions vary in the expression of these markers. Tr(VIP) and Tr(con-trol) were restimulated with splenic APCs and PCCF, and stainedwith anti-CD4, anti-CD103 and anti-GITR Abs. In addition, sortedCD4� T cells were analyzed for Foxp3 and Nrp1 expression byRT-PCR. Freshly isolated CD4�CD25� nTregs expressing highlevels of Foxp3, Nrp1, GITR, and CD103 were used as control. Incontrast to nTreg, Tr(VIP) show little if any surface CD103 andGITR and no Nrp1 expression. There is a slight increase in Foxp3expression in Tr(VIP) compared with Tr(control) but much lessthan in naturally occurring Treg (Fig. 5C).

Tr(VIP) inhibit preferentially the response of Th1 effectors

Because VIP/PACAP were reported to favor Th2 over Th1 immu-nity in vivo (20), we compared the ability of Tr(VIP) to inhibit Th1and Th2 effectors. Tr(VIP) and Tr(control) were cocultured withPCCF-specific Th1 or Th2 effectors, followed by measurements ofproliferation and cytokine profile. Tr(VIP) inhibit Th1, and to amuch lesser degree Th2 cell proliferation (Fig. 5D), and reduce theproduction of Th1 cytokines (IFN-� and IL-2), without signifi-cantly affecting the Th2 cytokines (IL-5 and IL-4) (Fig. 5E).

VIP-DC induce Treg in vivo

DCs generated from B10.A mice in the presence or absence of VIPwere stimulated with LPS, loaded with PCCF and injected intoB10.A hosts that received 24 h previously Tg-PCCF CD4 T cells.Splenic CD4 T cells isolated 7 days later were restimulated in vitrowith splenic APC/PCCF. T cells obtained from mice injected withcontrol DC (generated without VIP) proliferate and secrete highlevels of IFN-� and IL-2 (Fig. 6A). In contrast, T cells from miceinjected with VIP-DC exhibit low proliferation, reduced levels ofIFN-� and IL-2, and high levels of IL-10 (Fig. 6A). The suppres-sive activity of the T cells obtained from mice injected with

VIP-DC was tested in cocultures with fresh Tg-PCCF CD4 T cells(responder T cells) in the presence of splenic APC/PCCF. Incontrast to T cells from mice inoculated with control-DC, T cellsfrom VIP-DC inoculated mice inhibit the proliferation of re-sponder T cells (Fig. 6B) and express higher levels of Foxp3 (Fig.6C). Therefore, the T cells developing in vivo in the presence ofDC generated with VIP exhibit Treg characteristics and suppres-sive function, similar to the Tr(VIP) generated in vitro.

VIP-DC induce Ag-specific tolerance in vivo

DC generated from B10.A mice in the presence (VIP-DC) or ab-sence of VIP (control-DC) were stimulated with LPS, loaded withPCCF, stained with CFSE, and injected i.v. into B10.A hosts thatreceived 24 h previously Tg-PCCF CD4 T cells. The animals wereimmunized 1 week later with PCCF/CFA, followed by a boost 2weeks later. The spleens were harvested 2 weeks after the secondimmunization. The presence of the inoculated DC was ascertainedby analyzing spleen cells for CD11c and CFSE expression. Wedetected both control- and VIP-DC in spleen (Fig. 7A).

Next, we assessed the proliferative capacity and the cytokineprofile of splenic T cells following ex vivo restimulation withPCCF. Splenic T cells from mice that received control DC prolif-erate and produce high levels of IL-2 and IFN-� and moderateamounts of IL-4 (Fig. 7, B and C). In contrast, splenic T cells frommice that received VIP-DC exhibit Treg characteristics such asreduced proliferation, reduced IL-2 and IFN-� production, andhigh levels of IL-10 release (Fig. 7, B and C).

The mice injected with control-DC have both serum IgG1 andIgG2a anti-PCCF Abs, and in agreement with the previously de-scribed dominant inhibitory effect of Tr(VIP) on Th1 effectors, theanti-PCCF Abs in mice that received VIP-DC were almost exclu-sively of the IgG1 isotype (Fig. 7D).

To determine whether the in vivo tolerance induced by VIP-DCis Ag-specific, we inoculated VIP-DC stimulated with LPS andloaded with PCCF in B10.A hosts, followed by immunization andT cell restimulation with OVA instead of PCCF. There were nodifferences in terms of proliferation and levels of anti-OVA IgG2aAbs between hosts that received VIP-DC (PCCF) or medium (Fig.7, B and D). These results suggest that the tolerance induced byVIP-DC in vivo is restricted to the Ag presented by theinoculated DC.

VIP-DC inhibit DTH responses in vivo

Because VIP-DC seem to have a predominant effect on Th1 cells,we used an in vivo model of DTH. Control- and VIP-DC generatedfrom C57BL/6 mice were pulsed with OVA, stimulated with LPS,stained with CFSE, and injected s.c. in OVA-immunized C57BL/6mice. The mice were challenged with OVA in the left ear pinna 4days later, and the DTH reaction was assessed by measuring earthickness in comparison to the right ear control (PBS). The pres-ence of control- and VIP-DC in the draining lymph nodes wasassessed by analyzing the lymph node cells for CFSE and CD11cstaining. CFSE-labeled CD11c� cells were detected in the drain-ing lymph nodes, with VIP-DC seemingly having undergone fewerdivisions (Fig. 8A).

Mice that received control-DC developed DTH reactions higherthan controls (no DC), whereas those receiving VIP-DC exhibitreduced DTH reactivity (Fig. 8B). In addition, we also assessed theproliferation of T cells isolated from the draining lymph nodesfollowing ex vivo restimulation with OVA, and the levels of anti-OVA Abs in serum. T cells from mice inoculated with control-DCproliferate at higher levels than controls (no DC) and, again, in-oculation of VIP-DC results in a substantial reduction in T cell


FIGURE 5. VIP/PACAP-DCs induce Treg in vitro that differentially regulate Th1 and Th2 effectors. Control- and VIP-DC were cultured with LPS andadded (5 � 104 cells/well) to purified syngeneic PCCF Tg CD4 T cells (5 � 105 cells/well) in the presence of PCCF. A, One week later, the T cells werepurified (Tr) and added (left panels, 2.5 � 105 cells/well; right panel, different numbers) to syngeneic PCCF Tg T cells (5 � 105 cells/well) in the presenceof spleen APCs (2 � 104 cells/well) and PCCF (5 �M). Responder T cell proliferation was determined. IL-2 levels in the culture supernatants weredetermined by ELISA (left bottom panel). Cells cultured with an irrelevant Ag (OVA, 10 �g/ml) did not proliferate or produce IL-2. B, Treg (Tr) (2.5 �105 cells/well) generated in the presence of control-DC or VIP-DC were added to responder PCCF-specific Tg T cells (5 � 105 cells/well) in the presenceof spleen APCs (2 � 104 cells/well) and PCCF (5 �M). Coculture experiments were also performed in the presence of saturating blocking anti-IL-10 (10�g/ml), anti-TGF-�1 (40 �g/ml), and/or anti-CTLA-4 (10 �g/ml) mAbs, or of rmIL-2 (100 U/ml). The addition of isotype control (IgG) did not affect theeffect of Tr (VIP-DC) on the proliferation of responder T cells. Responder PCCF-specific Tg T cells (5 � 105 cells) together with spleen APCs (2 � 104

cells) and PCCF (5 �M) were added to the bottom wells of a Transwell system, with Tr (5 � 105 cells) generated in the presence of VIP-DC, spleen APCs(2 � 104 cells) and PCCF (5 �M) added to the upper well. Three days later, the basket was removed and responder T cell proliferation was determined.Results represent mean � SD of triplicates from one representative experiment of three. C, Treg (Tr) (5 � 105 cells/well) generated with control-DC orVIP-DC were restimulated in the presence of spleen APCs (2 � 104 cells/well) and PCCF, were stained for CD4 and intracellular CTLA-4 and IL-10 andanalyzed by flow cytometry. Left panel, CTLA-4 fluorescence intensity (MCF); middle panel, percentage of CD4� CTLA-4� cells; right panel, doublestaining for IL-10 and CTLA-4 expression. Values refer to percentage of positive cells in each quadrant. The data are from a representative experiment oftwo. CD4 T cells generated with control-DCs or VIP-DCs were analyzed for neuropilin 1 and Foxp3 mRNA expression by real-time RT-PCR, and for


proliferation for all OVA concentrations tested (Fig. 8C). Simi-larly, mice inoculated with control-DC produce high levels of anti-OVA Abs, whereas those inoculated with VIP-DC have a level ofanti-OVA Abs below the controls (no DC) (Fig. 8D).

Transfer of tolerance with CD4 T cells from VIP-DC inoculateddonors

Ag-specific tolerance can be adoptively transferred with Treg. Weinvestigated the potential of Treg generated by VIP-DC to transfertolerance to naive hosts. DCs generated from B10.A mice in thepresence or absence of VIP were stimulated with LPS, loaded withPCCF, and injected i.v. into B10.A hosts that received 24 h previouslyTg-PCCF CD4 T cells. Splenic CD4 T cells harvested 7 days laterwere transferred i.v. into naive B10.A mice, which were immunizeda week later with PCCF/CFA and received a boost 2 weeks later.Splenic T cells were analyzed 2 weeks after the boost in terms ofproliferation and cytokine profile following ex vivo restimulation, andserum anti-PCCF levels were determined. T cells from donors inoc-ulated with VIP-DC are capable of transferring the suppressive activ-ity into B10.A naive hosts, as determined by the reduction in prolif-eration, IL-2 and IFN-� production, and increased IL-10 productionby host splenic T cells (Fig. 9, A and B). In agreement with the sub-stantial reduction in Th1 cytokines, the levels of anti-PCCF IgG2aAbs in hosts receiving T cells from the VIP-DC inoculated donorswere significantly reduced (Fig. 9C).

In vivo induction of tolerogenic DC by VIP

In the experiments described above, the VIP-induced tolerogenic DCwere generated in vitro. Next, we addressed the question whether VIPcan induce tolerogenic DC in vivo, by administering VIP togetherwith PCCF (Ag) to Tg PCCF mice. Controls were injected with Agalone. Eight days later, spleen and mesenteric lymph node cells de-pleted of T and B cells consisted of approximately the same percent-age of CD11c� (DC) in both controls and VIP-injected mice (26–29%) (Fig. 10A). However, the CD45RBhigh population increasedfrom 7.2% (Ag control) to 17.3% (VIP�Ag), and these cells ex-pressed lower levels of CD11c, similar to the profile of the VIP-DCgenerated in vitro (Fig. 2C).

Also, similar to the in vitro generated VIP-DCs, the CD11c�

cells generated in vivo following VIP administration expressedlower levels of CD40, CD80, and CD86 following LPS stimulation(Fig. 10B). To determine whether the VIP-DCs generated in vivofunction as tolerogenic DCs, we stimulated T- and B-depletedspleen and lymph node cells with LPS, isolated the CD11c� DCsand cultured them with purified Tg PCCF T cells in the presenceof PCCF. Four days later, we measured proliferation and cytokinerelease. The cultures containing the in vivo generated VIP-DCs didnot proliferate, produced much less IFN-� and IL-2, increased lev-els of TGF-�, and significantly higher amounts of IL-10 (Fig.10C). This suggests that, similar to the in vitro VIP-DCs, the invivo VIP-DCs induce IL-10/TGF-�-producing T cells with lowproliferative capacity.

To verify that the IL-10/TGF-� producing T cells induced incocultures with the in vivo generated VIP-DCs (Ag�VIP) func-tion as Treg (Tr), we added different numbers of Tr cells to syn-geneic PCCF Tg T cells (responder T cells) in the presence ofsplenic APCs and PCCF. The proliferation of the responder T cells

surface CD103 and GITR expression by flow cytometry, and compared with freshly isolated splenic CD4�CD25� T cells. Open histograms and dashed linesrepresent isotype controls. One representative experiment of two is shown. D, Control- and VIP-DC were cultured with LPS and added (5 � 104 cells/well) topurified syngeneic PCCF Tg T cells (5 � 105 cells/well) in the presence of PCCF. One week later, the T cells were repurified and added (2.5 � 105 cells/well)to syngeneic PCCF-specific Th1 or Th2 effectors (5 � 105 cells/well) together with spleen APCs (2 � 104 cells/well) and PCCF (5 �M). The proliferation ofresponder Th1 or Th2 cells was determined. E, The cytokine content in the culture supernatants was determined by ELISA. Th1 and Th2 cells alone cultured withPCCF in the absence of APCs did not proliferate or produce cytokines. Each result is the mean � SD of three experiments performed in duplicate.

FIGURE 6. VIP-DC induce Treg in vivo. Control- and VIP-DCs wereloaded with PCCF (5 �M) and cultured with LPS. DCs (3 � 105 cells)were injected i.v. into B10.A mice (groups of nine) transferred 24 h pre-viously with PCCF Tg CD4 T cells (2 � 106 cells). One week later, spleenCD4 T cells (5 � 105 cells/well) were isolated and stimulated with PCCFand spleen APCs (2 � 104 cells/well). A, The proliferation and cytokineproduction were determined. Controls transferred with PCCF Tg CD4 Tcells without DCs were included (None). Cells cultured with an irrelevantAg (OVA, 10 �g/ml) did not proliferate or produce cytokines. Resultsrepresent mean � SD of triplicates from one representative experiment ofthree. B, Purified spleen CD4 T cells from mice injected with control-DCor VIP-DC (2.5 � 105 cells/well) were added to responder PCCF Tg Tcells (5 � 105 cells/well) in the presence of spleen APCs (2 � 104 cells/well) and PCCF. Proliferation was determined as above. C, Purified CD4splenic T cells from mice injected with control-DC or VIP-DC were ana-lyzed for Foxp3 expression by real-time RT-PCR.


was inhibited in a dose-dependent manner (Fig. 10D). These re-sults indicate that in vivo administration of VIP results in the in-duction of tolerogenic DCs, which in turn can induceIL-10/TGF-�-producing Treg.

DiscussionVIP and PACAP are potent immunosuppressive agents that affectboth innate and adaptive immunity (14–16). Until now, the mech-anisms described for their immunosuppressive activity includedmacrophage/dendritic cell/microglia deactivation, and support forTh2 effector differentiation and survival. In this study, we report onthe induction of Treg through the VIP/PACAP generation oftolerogenic DCs.

We reported previously that VIP/PACAP have different effects onimmature and LPS-matured DCs (17). The VIP/PACAP treatment ofimmature DCs led to the up-regulation of CD86, and increased ca-pacity to stimulate CD4 T cells and promote Th2-type responses. Incontrast, the VIP/PACAP treatment of LPS-matured DCs preventedthe expression of CD80 and CD86, reduced the stimulatory activityfor CD4 T cell proliferation and cytokine production, and differen-tially affecting Th1- and Th-2 chemoattractants (23, 24). These resultssupport the previously reported effect of VIP/PACAP on the immuneresponse, i.e., the inhibition of the proinflammatory, Th1-dependentresponse, with a concomitant bias toward Th2 responses.

In this study, we generated BM-DCs in the presence of VIP/PACAP. The addition of the neuropeptides early during DC differ-

entiation (up to day 3) results in DCs phenotypically resembling theTr1-inducing DCs described by Wakkach et al. (12). Similar to DCsgenerated in the presence of GM-CSF, TNF, and IL-10 (12), the DCsgenerated with VIP/PACAP are CD11clowCD45Rbhigh, do not up-regulate the expression of CD80 and CD86, and secrete IL-10 but notIL-12, following LPS treatment. Similar CD11clowCD45Rbhigh

tolerogenic DC were also generated in vivo upon administration ofVIP and Ag. These results suggest that VIP and PACAP provide amaturation signal similar to IL-10 (12) or to the active form of vitaminD3 1,25(OH)2D3 (8, 9, 25–27) leading to the differentiation of tolero-genic “semimature” or “quiescent” DCs (28–30).

The effect of VIP in generating tolerogenic DCs is mediatedthrough the VPAC1 receptor and the cAMP/PKA signaling path-way. VPAC1 has been previously identified as the major func-tional receptor in dendritic cells, macrophages, and bone marrowcells (14, 17, 31). Previously VIP has been reported to inhibitNF-�Bp65 nuclear translocation, DNA binding and transactivatingactivity in macrophages (20). Here, we report that both NF-�Bp65nuclear translocation and I�B phosphorylation are inhibited inVIP-DCs. The connection between NF-�B transactivating activity,CD40 expression, and DC function has been established recently. Theassociation between tolerogenic DCs and lack of CD40 expression orsignaling has been demonstrated both in vivo and in vitro (32, 33).Expression of CD40 depends on NF-�B, primarily p65 (32, 33). Arecent report by Yang et al. (22) demonstrates that inhibition ofNF-�B in DCs leads to failure of CD40, CD80, and CD86 expression

FIGURE 7. VIP-DCs induce Ag-specific tolerance in vivo. Control- and VIP-DCs were loaded with PCCF, cultured with LPS, and stained with CFSE.Control-DC, VIP-DC (3 � 105 cells) or saline (none) were injected i.v. into B10.A mice (groups of nine) transferred 24 h earlier with PCCF Tg CD4 Tcells (2 � 106 cells). One week later, the mice were immunized s.c. with PCCF/CFA (200 �g), followed 2 weeks later by a boost (s.c. 200 �g). The micewere sacrificed 2 weeks later. Mice immunized s.c. with an irrelevant Ag (OVA/CFA, 200 �g) were used as controls. A, The presence of the transferredCFSE-stained DCs in the spleen was determined by flow cytometry 2 weeks after transfer. Results represent mean � SD of triplicates from one repre-sentative experiment of three. B and C, Splenic CD4 T cells harvested from mice immunized with PCCF (left panel) or OVA (right panel) and injectedwith saline (None), control-DC plus PCCF, or VIP-DC plus PCCF, were restimulated in vitro with splenic APCs and PCCF or OVA and tested forproliferation or cytokine production. D, Sera from each group of mice were collected and PCCF-specific IgG1 and IgG2a levels were measured by ELISA.


upon LPS stimulation, and to the generation of tolerogenic DCs.Therefore, we propose that the mechanism by which VIP/PACAPinduce tolerogenic DCs involves the VPAC1 3 cAMP 3 PKA-mediated inhibition of I�B phosphorylation and NF-�Bp65 nucleartranslocation, leading to lack of CD40 expression.

Dendritic cells play an important role in the differentiation of pe-ripherally induced Tr1 and Th3/Tr2 Treg (5, 28, 36, 37). The gener-ation of Treg by VIP-DCs is partially mediated through the release ofVIP-DCs-derived IL-10, as shown by the effect of neutralizing anti-IL-10 Abs. However, because saturating concentrations of anti-IL-10could not restore �50% of T cell proliferation, other VIP-DCs relatedfactors must play a role. The best candidate is the reduced CD40expression on VIP-DCs, because blockade of CD40 signaling hasbeen shown to promote tolerance in immature DCs (38–40).

CD4� T cells exposed to VIP/PACAP-generated DCs do notproliferate and secrete large amounts of IL-10, some TGF-�, andno IL-2 or IFN-�, following re-exposure to stimulatory Ag-pulsedDCs. The cytokine profile resembles best the previously describedTr1 cells (12). In addition, CD4 T cells exposed to VIP/PACAP-generated DCs function as Treg, inhibiting the proliferation andIL-2 production of responder T cells. These cells express highlevels of CTLA-4, but low levels of other Treg markers such asFoxp3, CD103, GITR, and neuropilin 1. The suppressive functionappears to be mediated through both soluble factors (IL-10 andTGF-�) and direct cellular contact. CTLA-4 may play a role insuppression through direct cellular contact, because the combina-tion of anti-IL-10, anti-TGF-�, and anti-CTLA-4 Abs completelyreverses suppression. Although CTLA-4 is expressed at high levels

FIGURE 9. CD4 T cells from mice tolerized with VIP-DCs transfer tolerance. Control- and VIP-DCs were loaded with PCCF, cultured with LPS, andinjected i.v. into B10.A mice transferred 24 h earlier with PCCF Tg CD4 T cells (2 � 106 cells). One week later, splenic-purified CD4 T cells (2 � 106

cells) were adoptively transferred (i.v.) into naive B10.A mice (groups of six). One week later, the hosts were immunized s.c. with PCCF/CFA (200 �g),followed 2 weeks later by a boost (s.c., 200 �g). The mice were sacrificed 2 weeks later. A, Spleen cells were stimulated in vitro for 72 h with differentconcentrations of PCCF, and the proliferation was determined as described above. Cells cultured with an irrelevant Ag (OVA, 10 �g/ml) did not proliferate.B, Spleen cells were stimulated with PCCF and the cytokine content in the culture supernatants were measured by ELISA. Cells cultured with an irrelevantAg (OVA, 10 �g/ml) did induce any cytokines. C, Serum PCCF-specific IgG2a levels were determined by ELISA. Results represent mean � SD oftriplicates from one representative experiment of two.

FIGURE 8. VIP-DC inhibit DTH. Control- or VIP-DC were pulsed with OVA for 2 h, cultured with LPS and stained with CFSE. Control-DC, VIP-DC(3 � 105 cells) or saline (none) were injected s.c. into C57BL/6 mice previously immunized with OVA/CFA (100 �g, s.c.) (groups of nine). Four daysafter DC transfer, the mice were challenged with 50 �g of OVA in 20 �l of PBS in the left ear and with 20 �l of PBS in the right ear. A, The presenceof the transferred CFSE-stained DCs in the DLN were determined by flow cytometry. B, Ear thickness was measured 24 h after challenge. � ear thickness thickness of left ear � thickness of right ear. C, Isolated inguinal lymph node cells (4 � 105 cells/well) were incubated with different concentrations ofOVA, and proliferation was determined as described above. D, Serum was collected and assayed for OVA-specific IgG Abs by ELISA. Results representmean � SD of triplicates from one representative experiment of three.


in Treg, its role in the development and/or function of Treg is notclear. This is partly because CTLA-4-deficient mice have normalTreg development and homeostasis. However, these mice exhibitincreased levels of IL-10 and TGF-�, which suggests the existenceof compensatory mechanisms (41). In support for a CTLA-4 rolein Treg suppression, CTLA-4 binding to B7 has been shown toinduce IDO in DCs, leading to tryptophan depletion and inductionof proapoptotic molecules (42).

CD4�CD25� nTreg have been characterized by high expressionof Foxp3 and GITR, and recently of neuropilin 1 (43–46). In con-trast, the Tr1 cells generated with VIP/PACAP-DCs express lowlevels of Foxp3, and very little, if any, Nrp1 and GITR. In agree-ment with our results, Tr1 cells generated by repetitive stimulationwith IL-10-secreting iDCs have been shown to express low levelsof CD25 and Foxp3 (7).

Although all DC subsets are generated from bone marrow pro-genitors, recent studies indicate that with the exception of plasma-cytoid DCs, all the other DC subsets differentiate at their periph-eral homing site (47). Blood monocytes differentiate intomacrophages or DCs upon migration into the tissues, with cyto-kines and environmental factors playing an important role in shift-ing the balance between these cell types (48, 49). In physiologicalconditions, VIP could affect DCs differentiation in the bone mar-row as well as in the periphery. Indeed, VIP-ergic nerve fibershave been identified in the bone marrow, as well as skin,gastrointestinal tract, and secondary lymphoid organs (50, 51). Inaddition, activated Th2 effectors have been shown to release func-tional VIP (52). Therefore, in steady-state conditions, VIP releasedfrom the innervation or secreted by immune cells could act as anendogenous maturation signal driving the differentiation of tolero-genic DCs loaded with self- or commonly encountered Ags.

In addition, the VIP-induced generation of tolerogenic DCs invitro could lead to new therapeutic developments in autoimmunediseases and in allogeneic transplantation. We showed that in vitropulsing of tolerogenic VIP-DCs with Ag followed by administra-tion in vivo leads to Ag-specific tolerance mediated by Treg. Wewould like to propose that the in vitro generation of VIP-DCsfollowed by loading with specific Ags might provide a more tar-geted approach for the induction of endogenous Ag-specific Tregin the treatment of autoimmune diseases.

DisclosuresThe authors have no financial conflict of interest.

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The neuropeptide vasoactive intestinal peptide generates tolerogenic dendritic cells

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