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Published online before print October 4, 2005

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(Journal of Leukocyte Biology. 2005;78:1327-1338.)
© 2005 by Society for Leukocyte Biology

Vasoactive intestinal peptide generates CD4⁺CD25⁺ regulatory T cells in vivo

Mario Delgado^*,, Alejo Chorny, Elena Gonzalez-Rey and Doina Ganea^*,1

^* Department of Biological Sciences, Rutgers University, Newark, New Jersey; and
Instituto de Parasitologia y Biomedicina, Instituto del Consejo Superior de Investigaciones Científicas, Granada, Spain

¹ Correspondence: Rutgers University, Department of Biological Sciences, 101 Warren St., Newark, NJ 07102. E-mail: dganea{at}andromeda.rutgers.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
CD4⁺CD25⁺ regulatory T (Treg) cells control the immune response to a variety of antigens, including self-antigens, and several models support the idea of the peripheral expansion of CD4⁺CD25⁺ Treg cells. Although hormones such as estrogen and -melanocyte-stimulating hormone have been recently reported to expand the CD4⁺CD25⁺ Foxp3-expressing Treg cell compartment, little is known about the endogenous factors and mechanisms controlling the peripheral expansion of CD4⁺CD25⁺ Treg cells. In this study, we report on the capacity of the vasoactive intestinal peptide (VIP), an immunosuppressive neuropeptide, to induce functional Treg cells in vivo. The administration of VIP together with specific antigen to T cell receptor (TCR)-transgenic (Tg) mice results in the expansion of the CD4⁺CD25⁺, Foxp-3/neuropilin 1-expressing T cells, which inhibit responder T cell proliferation through direct cellular contact. In addition to the increase in the number of CD4⁺CD25⁺ Treg cells, VIP induces more efficient suppressors on a per-cell basis. The VIP-generated CD4⁺CD25⁺ Treg cells transfer suppression, inhibit delayed-type hypersensitivity in TCR-Tg hosts, and prevent graft-versus-host disease in irradiated hosts reconstituted with allogeneic bone marrow.

Key Words: neuropeptides • tolerance • collagen-induced arthritis • graft-versus-host disease

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The immune system mounts a protective response against pathogen-derived antigens and maintains tolerance for self-antigens. Various mechanisms, including T cell apoptosis, anergy, changes in the balance of effector T cells, and the generation and activation of regulatory T (Treg) cells, contribute to the control of ongoing immune responses and tolerance.

Two major types of Treg cells have been characterized in the CD4⁺ population, i.e., the naturally occurring, thymus-generated Treg cells and the peripherally induced, interleukin (IL)-10 or transforming growth factor-ß (TGF-ß)-secreting Treg cells [^{1 2 3} ]. The CD4⁺CD25⁺ Foxp3-expressing, naturally occurring Treg cells generated in the thymus migrate and are maintained in the periphery. The signals for their thymic generation and maintenance in the periphery are not defined entirely, although CD28 stimulation and IL-2 appear to be required [⁴ , ⁵ ]. Several experimental models support the idea of peripheral generation of CD4⁺CD25⁺ Treg cells from CD4⁺CD25^– T cells [⁶ ]. However, in contrast to the Tr1/T helper cell type 3 (Th3; Tr2) Treg cells induced by IL-10, TGF-ß, or through infectious tolerance [^{7 8 9} ], the CD4⁺CD25⁺ Treg cells developing de novo in the periphery exert their suppressive function in a cell contact-dependent manner [¹⁰ ]. The endogenous factors and mechanisms controlling the peripheral expansion of CD4⁺CD25⁺ Treg cells are mostly unknown. Recently, Polanczyk et al. [¹¹ ] reported that estrogen promotes tolerance by expanding the CD4⁺CD25⁺ Foxp3⁺-expressing regulatory compartment.

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are two neuropeptides with profound immunoregulatory activities. We have shown previously that VIP/PACAP inhibit macrophage/dendritic cell/microglia activation and promote Th2-type responses at the expense of proinflammatory Th1 immunity [^{12 13 14} ]. As Treg cells are major players in limiting the immune response, we investigated the possibility that VIP and PACAP promote Treg cell development and/or activation. In this study, we characterized the functional Treg cells generated in vivo following VIP administration. The major Treg cell population induced in vivo following VIP and antigen administration is CD4⁺CD25⁺, Foxp-3^hi, neuropilin (Nrp)^hi and inhibits responder T cell proliferation through direct cellular contact. These cells exhibit increased suppressive activity compared with Treg cells generated in response to antigen alone. The in vivo VIP-generated Treg cells transfer suppression, inhibit delayed-type hypersensitivity (DTH) in T cell receptor (TCR)-transgenic (Tg) hosts and prevent graft-versus-host disease (GVHD) in irradiated hosts reconstituted with allogeneic bone marrow (BM).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Reagents
Synthetic VIP was purchased from Calbiochem-Novabiochem (Laufelfingen, Switzerland). Capture and biotinylated antibodies against murine IL-4, IL-5, IL-2, TGF-ß1, and interferon- (IFN-); monoclonal antibodies (mAb) to cytotoxic T-lymphocyte antigen (CTLA)-4, CD4, Vß3 (clone KJ25), I-E^k, I-E^d [major histocompatibility complex type II (MHC-II)], CD25, CD69, B220, CD103, CD62 ligand (CD62L), and CD45RB; and recombinant murine (rm)IFN- and rmIL-4 were purchased from BD PharMingen (San Diego, CA). Capture and biotinylated antibodies against murine IL-10 and rmIL-2 were purchased from PreproTech Inc. (Rocky Hill, NJ). Pigeon cytochrome c fragment (PCCF) was synthesized and purified by Research Genetics (Huntsville, AL). Ovalbumin (OVA) and avidin-peroxidase were purchased from Sigma Chemical Co. (St. Louis, MO). Rat anti-glucocorticoid-induced tumor necrosis factor receptor (GITR) antibody was purchased from R&D Systems (Minneapolis, MN).

Animals
B10.A (I-E^k), C57Bl/6 (H-2^b), BALB/c (H-2^d), and TCR-Cyt-5CC7-I/Rag1 Tg (I-E^k) mice were obtained from Jackson Laboratory (Bar Harbor, ME) and Taconic Farms (Germantown, NY). All mice used were between 7 and 12 weeks of age.

Cell isolation and cultures
Purified, naïve CD4 T cells from Tg mice were isolated by positive immunomagnetic selection by treating spleen cells with magnetic beads conjugated with anti-CD4 mAb [magnetic cell sorter (MACS), Miltenyi Biotec, Auburn, CA]. The purified T cells were >98% CD4⁺ by fluorescein-activated cell sorter (FACS) analysis.

Antigen-presenting cells (APC) were prepared by T cell depletion of B10.A (I-E^k) spleen cells by treating the cells with a mixture of magnetic beads conjugated with anti-CD8 and anti-CD4 mAb (MACS) at 15°C for 20 min, followed by magnetic separation, and treatment with 50 µg/ml mitomycin C (mitC; Sigma Chemical Co.) for 20 min at 37°C.

CD4⁺CD25⁺ and CD4⁺CD25^– cell populations were isolated with an AutoMACS magnetic cell sorter (Miltenyi Biotec). Briefly, PCCF TCR-Tg spleen and draining lymph node (LN; popliteal, inguinal, and mesenteric LN) cells were depleted of red blood cells, of adherent cells, and CD8⁺ T cells by incubation for 2 h at 37°C in Petri dishes followed by negative magnetic selection with anti-CD8 microbeads (Miltenyi Biotec). Resulting cells were labeled with biotinylated anti-CD25 mAb for 45 min at 4°C, washed, and incubated with a 1:10 dilution of streptavidin-conjugated magnetic beads (Miltenyi Biotec) at a concentration of 10⁸ cells/ml in phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin (BSA) and 1 mM EDTA for 20 min at 8°C. The cells were washed once, passed through a nytex filter to remove clumps, pelleted, and resuspended at a concentration of 2 x 10⁸ cells/ml. CD25⁺ T cells were isolated with the AutoMACS, and CD4⁺ T cells were isolated from the CD25^–fraction with anti-CD4 microbeads (Miltenyi Biotec). Contaminating MHC-II⁺ cells were removed from the CD25⁺ and CD4⁺CD25^– fractions by panning with anti-I-E^k-coated plates for 12 h at 37°C. The CD25⁺ populations were >95% CD4⁺CD25⁺, and the CD4⁺CD25^– populations were 98% pure, as determined by flow cytometry.

In some of the in vivo experiments, the cells were labeled with 1 ml PBS-0.1% BSA-5 µM carboxyfluorescein succinimidyl ester (CFSE) for 10 min at 37°C, washed three times in RPMI-10% fetal bovine serum, and injected intravenously (i.v.; 100 µl) as described below.

To determine T cell anergy, isolated CD4 T cells (5x10⁴ cells/well) were stimulated with mitC-treated B10.A spleen APC (10⁵ cells/well) and varying concentrations of PCCF. Proliferation was determined following pulsing with bromodeoxyuridine (BrdU) for the last 16 h of a 4-day culture period. BrdU incorporation was measured by using a peroxidase-labeled anti-BrdU mAb detected with tetramethylenbenzidine (TMB) substrate and quantified at optical density (OD)₄₅₀ in an enzyme-linked immunosorbent assay (ELISA) reader, according to the manufacturer’s (Roche Applied Science, Mannheim, Germany) protocol. Results are expressed as arbitrary units of OD₄₅₀. To determine the in vitro Treg cell function, different numbers of CD4⁺, CD4⁺CD25⁺, or CD4⁺CD25^– T cells isolated from naïve mice or mice inoculated with PCCF or PCCF plus VIP were cocultured with responder CD4⁺ T cells (rCD4; 5x10⁴ cells/well), isolated from naïve PCCF-Tg mice and stimulated in the presence of mitC-treated B10.A spleen APC (10⁵ cells/well) and PCCF (5 µM). Proliferation was determined as described above. Culture supernatants were collected and assayed for cytokine determination as described below. In some experiments, cocultures were performed in the presence of blocking mouse anti-IL-10 (10 µg/ml), anti-TGF-ß1 (40 µg/ml), and/or anti-CTLA-4 (10 µg/ml) mAb or of rmIL-2 (100 U/ml). Transwell experiments were done in 24-well plates (Millicell, 0.4 µm, Millipore, Bedford, MA). Responder PCCF-specific-Tg CD4 T cells (5x10⁵ cells), together with mitC-treated B10.A spleen APC (2x10⁴ cells) and PCCF (5 µM), were placed in the bottom wells. CD4⁺ T cells or CD4⁺CD25⁺ T cells (5x10⁵ cells) isolated from PCCF- or PCCF + VIP-treated Tg mice were placed together with mitC-treated B10.A spleen APC (2x10⁴ cells) and PCCF (5 µM) in the upper wells. Three days later, the basket was removed, and the proliferation of the responder T cells was measured.

FACS analysis
Spleen and LN (popliteal, inguinal, and mesenteric LN) cells (10⁶ cells/ml) were harvested in ice-cold RPMI complete medium and washed twice with PBS containing 0.1% sodium azide plus 2% heat-inactivated fetal calf serum (wash buffer). Cells were incubated with various mAb [phycoerythrin (PE)-anti-CD25, fluorescein isothiocyanate (FITC)-anti-CD62L, FITC-anti-CD69, FITC-anti-CD103, FITC-anti-Vß3, FITC-anti-CD45RB, FITC-anti-GITR, peridinin chlorophyll protein (PerCP)-anti-CD4, 2.5 µg/ml final concentration] at 4°C for 1 h. Isotype-matched antibodies were used as controls, and immunogloublin G (IgG) block (Sigma Chemical Co.) was used to block the nonspecific binding to Fc receptors. After extensive washing, the cells were fixed in 1% paraformaldehyde. Stained T cells, gated accordingly to CD4 staining, were analyzed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA). Samples with isotype-matched antibodies instead of specific antibodies were used to determine the proper region or window setting. Fluorescence data were expressed as mean channel fluorescence and as percentage of positive cells after subtraction of background, isotype-matched values.

For analysis of intracellular CTLA-4, spleen and LN T cells (10⁶ cells/ml) were harvested in ice-cold RPMI complete medium, washed twice with wash buffer, and stained with PerCP-anti-CD4 and FITC-anti-CD25 mAb for 30 min at 4°C, followed by fixation with Cytofix/Cytoperm solution (BD PharMingen) and incubation for 45 min at 4°C with PE-anti-CTLA-4 mAb diluted in PBS + 1% BSA + 0.5% saponin. After extensive washing, cells were analyzed using a FACSCalibur flow cytometer.

Cytokine assays
Cytokine contents in the supernatants of T cell cultures or APC-CD4⁺ T cell cocultures were determined by specific sandwich ELISAs. The cytokine amount in each supernatant was extrapolated from the standard curve. The antibody pairs used were as follows, listed by capture/biotinylated detection antibodies (BD PharMingen): IL-4, BVD4-1D11/BVD6-24G2; IFN-, R4-6A2/XMG1.2; IL-5, TRFK5/TRFK4; IL-2, JES6-1A12/JES6-5H4.

Determination of antibody responses
Specific antibody responses in the PCCF or OVA-immunized mice were determined by ELISA. Serum was obtained by cardiac puncture. Maxisorb plates (Millipore) were coated overnight at 4°C with 100 µl soluble PCCF or OVA (10 µg/ml). After washing with PBS containing 0.05% v/v Tween-20, the plates were blocked with 3% BSA and incubated with serial dilutions of serum for 2 h at 37°C. After washing, biotinylated anti-IgG, anti-IgG1, or anti-IgG2a (2.5 µg/ml; Serotec, Oxford, UK) was added for 1 h at 37°C. The plates were washed, followed by incubation with streptavidin-horseradish peroxidase. The bound enzyme was detected with the TMB substrate and quantified at OD₄₅₀in an ELISA reader (Bio-Tek Instrument, Inc., Winooski, VT). Standard curves were constructed for each assay by coating wells with an isotype-specific anti-mouse Ig, followed by addition of known concentrations of the mouse Ig isotype.

Real-time reverse transcriptase-polymerase chain reaction (RT-PCR)
Total RNA was isolated from CD4⁺ T cells or sorted CD4⁺CD25⁺(10⁶ cells) using the Ultraspec RNA reagent (Biotecx, Houston, TX). Total RNA (2 µg) was reverse-transcribed with oligo-dT primers and Moloney murine leukemia virus-RT polymerase (Invitrogen, Carlsbad, CA). Quantitative real-time RT-PCR was performed in an ABI PRISM cycler (Applied Biosystems, Foster City, CA) using a SYBR Green PCR kit from Applied Biosystems. A threshold was set in the linear part of the amplification curve, and the number of cycles needed to reach the threshold was calculated for each gene. Relative mRNA levels were determined by using standard curves for each individual gene and further normalization to hypoxanthine guanine phosphoribosyl transferase (HPRT). Melting curves established the purity of the amplified band. Primer sequences are: Nrp1 (5'-GCCTGCTTTCTTCTCTTGGTTTCA-3', 5'-GCTCTGGGCACTGGGCTACA-3'); Foxp3 (5'-CTGGCGAAGGGCTCGGTAGTCCT-3', 5'-CTCCCAGAGCCCATGGCAGAAGT-3'); HPRT (5'-TGGAAAGAATGTCTTGATTGTTGAA-3', 5'-AGCTTGCAACCTTAACCATTTTG-3').

Acute GVHD and graft-versus-leukemia (GVL)
GVHD
BALB/c mice (H-2^d) were irradiated lethally (8Gy TBI with a 200-Kv X-ray source) and reconstituted i.v. with 100 µl T cell-depleted (TCD), allogeneic BM cells (5x10⁶ cells) from B10.A mice (H-2^k) plus CD4 T cells (10⁶ cells) and CD4⁺CD25^– or CD4⁺CD25⁺ cells (0.5x10⁶ cells), isolated from naïve PCCF-Tg mice or from mice immunized with PCCF or PCCF plus VIP as described above. The survival and appearance of the BALB/c hosts were monitored daily, and body weight was measured weekly.

GVL
Irradiated BALB/c mice were reconstituted with TCD allogeneic BM cells (B10.A, 10⁶ cells). The recipients also received a single i.v. injection of CD4 T cells (10⁶ cells) isolated from PCCF-Tg mice immunized with PCCF or PCCF plus VIP and A20 leukemic cells (derived from BALB/c mice, 10⁴ cells). The recipients were monitored once every day from the day of transplantation until they succumbed naturally to GVHD and/or tumor burden. Tumor growth/elimination was assessed by the presence of A20 cells in blood and detected based on coexpression of B220 and H-2K^d and size by flow cytometry.

Data analysis
All values are expressed as mean ± SD of mice/experiment. The differences between groups were analyzed by Mann-Whitney U test and if appropriate, by Kruskal-Wallis ANOVA test.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In vivo administration of VIP increases the number of CD4⁺CD25⁺ T cells
Previous reports from our laboratory indicated that in the presence of immunogenic doses of antigen, VIP promotes Th2-type immune responses in vivo and in vitro. In vivo inoculation of VIP into PCCF-TCR-Tg mice results in long-term survival of Th2 effectors and predominant development of Th2 memory cells. As antigen-specific tolerance can be induced with low doses of antigen, we assessed the ability of VIP to facilitate CD4⁺CD25⁺ Treg cell induction in vivo in the presence of low doses of antigen. PCCF-TCR-Tg mice were inoculated with low doses of antigen (50 µg PCCF) on Days 0 and +2, with or without VIP (5 nmol, 750 µg/kg/day). Eight days later, FACS determined the numbers of CD4⁺CD25⁺ T cells from spleen and mesenteric LN. Administration of antigen plus VIP results in the doubling of CD4⁺CD25⁺ cells in spleen and LN (Fig. 1A ). Similar numbers were observed 4 weeks after immunization, indicating a long-term effect of VIP on the expansion of the CD4⁺CD25⁺ T cell population (Fig. 1B) . The VIP-induced increase was observed only in the antigen-specific T cell population, as determined by triple staining for CD4, CD25, and the TCR/Vß3 (Fig. 1C) . The VIP-induced increase in CD25⁺Vß3⁺ T cells was observed only in the presence of low doses of antigen (Fig. 1D , left and middle panels), and VIP did not increase the number of CD25^–Vß3⁺ T cells, which presumably represent effector T cells whose CD25 expression is transient (Fig. 1D , right panel).

View larger version (41K): [in this window] [in a new window]   Figure 1. VIP expands CD25+ CD4 T cells following peripheral tolerance induction with peptide antigen. (A–D) PCCF-Tg mice were injected intraperitoneally (i.p.) on Days 0 and +2 with medium (Naïve) or with antigen (Ag; PCCF, 50 µg) and with or without VIP (5 nmol; 750 µg/kg/day). (A) Eight days after initial antigen stimulation, spleen and LN cells (inguinal, mesenteric, and popliteal) were isolated and analyzed for CD4 and CD25. Values indicate the percentage of positive cells in each quadrant. The results are representative of three experiments, three mice per group. (B) Four weeks after initial antigen stimulation, mesenteric, inguinal, and popliteal LN cells were isolated and analyzed for CD4 and CD25. The results are representative of two experiments with three mice per group. (C) Eight days after initial antigen stimulation, spleen and LN cells were isolated and analyzed for CD4, CD25, and the Tg TCR using the clonotypic mAb KJ25 (Vß3). Each result is the mean ± SD of three independent experiments with three mice per group. *, P< 0.001, versus mice treated with antigen alone. (D) PCCF-Tg mice were injected i.p. on Days 0 and +2 with antigen (PCCF, 500 µg or 50 µg), with or without VIP. Spleen cells were collected on the indicated days after initial antigen stimulation and analyzed for CD4, CD25, and the Tg TCR using the clonotypic mAb KJ25. Percentage and total numbers of spleen CD25+ Tg+ and CD25– Tg+ T cells were determined. Each result is the mean ± SD of two or three independent experiments with three mice per group.

View larger version (40K): [in this window] [in a new window]   Figure 2. VIP induces the in vivo emergence of CD4+ T cells with a regulatory phenotype. PCCF-Tg mice were injected i.p. with medium (Naïve) and antigen (PCCF), with or without VIP. Eight days after initial antigen stimulation, spleen CD4 and CD4+CD25+ and CD4+CD25– T cells were isolated. Cells from naïve PCCF-Tg mice were used as control. Foxp3 and Nrp1 mRNA expression was analyzed by real-time RT-PCR and normalized using HPRT expression. GITR, CD103, CD45RB, CD62L, and CD69 expression was determined by flow cytometry. Results are representative of three independent experiments. *, P < 0.001, versus mice treated with antigen alone.

View larger version (26K): [in this window] [in a new window]   Figure 3. VIP induces the in vivo emergence of functional Treg CD4+ cells. PCCF-Tg mice were injected i.p. on Days 0 and +2 with medium (Naïve) or antigen (PCCF), with or without VIP. Eight days after initial antigen stimulation, spleen CD4+ T cells were isolated. Spleen cells from naïve PCCF-Tg mice were used as control. (A) VIP induces CD4+ T cell hyporesponsiveness. Isolated CD4+ T cells (5x104 cells/well) were stimulated with mitC-treated B10.A spleen APC (1x105 cells/well) and varying concentrations of PCCF. BrdU was added for the last 16–18 h of a 4-day culture, and T cell proliferation was assayed as described in Materials and Methods. Each result is the mean ± SD of three experiments performed in duplicate. A450, Absorbance at 450 nm. (B) VIP induces IL-10/TGF-ß1-producing T cells. Isolated CD4+ T cells (2x105 cells/well) were stimulated with mitC-treated B10.A spleen APC (5x105 cells/well) and PCCF. After 48 h culture, cytokine secretion was determined by ELISA. Cells cultured with an irrelevant antigen (OVA, 10 µg/ml) did not secrete cytokines. Each result is the mean ± SD of three experiments performed in duplicate. (C and D) In vivo treatment with VIP induces functional Treg cells. Different numbers (5x104 cells/well in C) of CD4+ T cells isolated from mice inoculated with PCCF [CD4(Ag)] or PCCF plus VIP [CD4(Ag+VIP)] were cocultured with responder CD4+ T cells (rCD4+, 5x104 cells/well), isolated from naïve PCCF-Tg mice and stimulated in the presence of mitC-treated B10.A spleen APC (105 cells/well) and PCCF (5 µM). Proliferation was determined by using BrdU. Secreted IL-2 was quantified by ELISA. Each result is the mean ± SD of three experiments performed in duplicate. *, P < 0.001, versus mice treated with antigen alone.

To assess the suppressive activity of the T cells induced by antigen + VIP inoculation, we cocultured CD4⁺ T cells from mice injected with antigen alone [CD4⁺(antigen)] or from mice injected with VIP plus antigen [CD4⁺(antigen+VIP)] with CD4⁺ T cells from nontreated PCCF-Tg mice (rCD4) in the presence of APC/PCCF. CD4⁺(antigen) T cells proliferate in response to antigen and did not suppress the proliferation of responder T cells. In contrast, CD4⁺(antigen+VIP) proliferated less and suppressed the proliferation of the responder T cells (Fig. 3C) . The suppression increased with the number of CD4⁺(antigen+VIP) cells (Fig. 3D) .

VIP enhances the efficiency of pre-existing CD25⁺ Treg cells
To evaluate the suppressive activity of VIP-induced CD25⁺ Treg cells, we purified CD4⁺CD25⁺ T cells from mice inoculated with medium (naïve), with antigen alone, or with VIP plus antigen and compared them in terms of cytokine production and suppressive activity. Upon ex vivo stimulation with APC and various concentrations of antigen, the CD4⁺CD25^– T cells from naïve, antigen, and antigen + VIP-inoculated mice proliferate, and the CD4⁺CD25⁺ T cells from all three groups show a similar lack of proliferation (Fig. 4A ). However, differences become apparent when equal numbers of T cells from antigen and antigen + VIP-inoculated mice are compared in terms of cytokine production. The CD4⁺CD25^–(antigen+VIP) T cells secrete less IFN- and more IL-10 and TGF-ß than the CD4⁺CD25^–(antigen) or the CD4⁺CD25^–(naïve; Fig. 4B ), which probably reflects the presence of developing Tr1/Th3-like Treg cells within the CD25^– population. The difference is even greater within the three CD4⁺CD25⁺ populations. Although CD4⁺CD25⁺(antigen+VIP), CD4⁺CD25⁺(antigen), and CD4⁺CD25⁺(naïve) do not secrete IL-2, the CD4⁺CD25⁺(antigen+VIP) cells produce much less IFN-, more IL-10, and much more TGF-ß than the CD4⁺CD25⁺(antigen) or the CD4⁺CD25⁺(naïve) populations (Fig. 4B) .

View larger version (46K): [in this window] [in a new window]   Figure 4. VIP induces highly efficient CD4+CD25+ Treg cells. PCCF-Tg mice were injected i.p. with medium (Naïve) or with PCCF (Ag), with or without VIP (5 nmol; 750 µg/kg). Eight days after initial antigen stimulation, spleen CD4+CD25+ and CD4+CD25– T cells were isolated. (A) Isolated CD4+CD25+ and CD4+CD25– T cells (5x104 cells/well) were stimulated with mitC-treated B10.A spleen APC (105 cells/well) and varying concentrations of PCCF. Proliferation was determined by using BrdU. Each result is the mean ± SD of three experiments performed in duplicate. (B) Isolated CD4+CD25+ and CD4+CD25– T cells (2x105 cells/well) were stimulated with mitC-treated B10.A spleen APC and PCCF. After 48 h culture, cytokine secretion was determined by ELISA. Cells cultured with an irrelevant antigen (OVA, 10 µg/ml) did not secrete cytokines. Each result is the mean ± SD of three experiments performed in duplicate. (C) rCD4+ T cells (5x104 cells/well) isolated from naïve PCCF-Tg mice were cocultured with different numbers of CD4+CD25+ and CD4+CD25– T cells (ratios of Treg/Tresp from 1:2 to 1:10) in the presence of mitC-treated B10.A spleen APC plus PCCF or soluble anti-CD3 antibody (5 µg/ml). Proliferation was determined by using BrdU. Each result is the mean ± SD of three experiments performed in duplicate. *, P < 0.001, versus mice treated with antigen alone. (D) Functional CD4+CD25+ Treg cells generated by VIP require TCR stimulation. CD4+CD25+ T cells (105 cells/well) from PCCF-Tg mice treated with PCCF plus VIP for 8 days were cocultured with CD4+CD25– T cells (5x105 cells/well) from OVA-immunized B10.A mice in the presence of mitC-treated B10.A spleen APC and antigen (5 µM PCCF and/or 200 µg/ml OVA) or soluble anti-CD3 antibody (5 µg/ml). Proliferation was determined by using BrdU. Each result is the mean ± SD of two experiments performed in duplicate.

It has been reported previously that Treg cells need TCR activation to function as suppressors. To assess the requirement for TCR activation in the CD4⁺CD25⁺(antigen+VIP) population, we isolated CD4⁺CD25⁺(antigen+VIP) from PCCF-immunized mice and cocultured them with CD4⁺CD25^– T cells from OVA-immunized MHC II-compatible mice in the presence of OVA, OVA plus PCCF, or anti-CD3. Suppression occurred only in the cultures containing PCCF or anti-CD3 (Fig. 4D) , indicating that the CD4⁺CD25⁺(antigen+VIP) Treg cells need to be activated through the TCR to function as suppressors.

The in vivo VIP-induced Treg cells act primarily through cell-cell contact
Naturally occurring CD4⁺CD25⁺ Treg cells exert their suppressive activity primarily through direct cellular contact, whereas Tr1/Tr2(Th3) suppressors act primarily through cytokines. To evaluate the requirement for direct cellular contact in the suppressive activity of CD4⁺CD25⁺(antigen+VIP), we generated CD4⁺(antigen+VIP; containing CD25⁺ and CD25^– T cells) and purified CD4⁺CD25⁺(antigen+VIP) and cocultured them with rCD4⁺ T cells in the presence of APC/PCCF. For the cultures containing CD4⁺(antigen+VIP), addition of saturating amounts of anti-IL-10 or anti-TGF-ß-neutralizing antibodies slightly reversed the inhibitory effect on responder T cell proliferation, and a combination of anti-IL-10, anti-TGF-ß, and anti-CTLA-4 antibodies completely reversed suppression (Fig. 5A , left panel). Also, when CD4⁺(antigen+VIP) and responder T cells were separated in Transwell experiments, there was a slight reduction in proliferation (P<0.05 for Transwell vs. rCD4+APC samples; Fig. 5A , left panel). This suggests that the CD4⁺(antigen+VIP) cells consist of two Treg cell populations: a major, contact-dependent, cytokine-independent population and a minor population, exerting suppression through soluble cytokines. In contrast, the suppressive activity of the CD4⁺CD25⁺(antigen+VIP) was entirely contact-dependent, with no reversal upon anti-IL-10 or anti-TGF-ß administration, complete reversal in the presence of anti-CTLA-4 antibodies, and no inhibition in the Transwell experiments (Fig. 5A , right panel). As expected, exogenous IL-2 reversed the suppressive effect for CD4⁺(antigen+VIP) and CD4⁺CD25⁺(antigen+VIP) Treg cells.

View larger version (41K): [in this window] [in a new window]   Figure 5. In vivo VIP-induced Treg cells act through direct cell contact. PCCF-Tg mice were injected i.p. with medium (Naive) or PCCF with or without VIP. Eight days later, spleen CD4+, CD4+CD25+, and CD4+CD25– T cells were isolated. (A) Total CD4+ T cells [CD4(Ag+VIP)] or sorted CD4+CD25+ T cells [CD25(Ag+VIP); 2.5x105 cells/well] were cocultured with rCD4+ T cells from naïve-Tg mice (5x104 cells/well) in the presence of mitC-treated B10.A spleen APC and PCCF. Coculture experiments were also performed in the presence of blocking mouse anti-IL-10 (10 µg/ml), anti-TGF-ß1 (40 µg/ml), and/or anti-CTLA-4 (10 µg/ml) mAb or of rmIL-2 (100 U/ml). Proliferation was determined by using BrdU. In addition, rCD4+ T cells (5x104 cells) were placed with mitC-treated B10.A spleen APC and PCCF in the bottom wells of a Transwell system, and total CD4+ or sorted CD4+CD25+ Treg cells (2x104 cells) from PCCF + VIP-treated mice were placed with mitC-treated B10.A spleen APC and PCCF in the upper wells. Three days later, the basket was removed, and the proliferative response of the bystander rCD4+ T cells was measured after a pulse with BrdU during the final 16 h of the 72-h culture. Results represent mean ± SD of triplicates from one representative experiment out of three. *, P < 0.001, versus untreated samples (None). (B) Spleen cells from mice treated with medium (Naïve), PCCF, or PCCF plus VIP were analyzed for CD4, CD25, and intracellular CTLA-4 by flow cytometry. (Left panels) Double-staining for CD25 and CTLA-4 expression of gated CD4 T cells. Values refer to percentage of positive cells in each quadrant. (Right panels) Intracellular CTLA-4 expression was analyzed for the CD4+CD25+ and CD4+CD25– populations. Values refer to the percentage of CTLA-4+ cells. One representative experiment out of three is shown.

Transfer of antigen-specific, suppressive activity with VIP-generated Treg cells
Treg cells retain their suppressive activity upon adoptive transfer. To assess the capacity of the in vivo VIP-generated Treg cells, we purified splenic CD4⁺ and CD4⁺CD25⁺ T cells from PCCF-Tg mice injected with PCCF (antigen) or with PCCF plus VIP (antigen+VIP). CD4⁺ T cells were injected i.v. into PCCF-Tg naïve hosts, followed 24 h later by immunization with PCCF/complete Freund’s adjuvant (CFA) in the footpads. The spleen, draining LN (DLN), and peripheral nondraining LN (PLN) were obtained. The spleen and DLN from mice transferred with CD4⁺(antigen) were significantly larger than the counterparts from animals transferred with CD4⁺(antigen+VIP; Fig. 6A ). This is presumably because of the Treg cells present in the CD4⁺(antigen+VIP) population, which upon transfer, suppress the host’s response to PCCF.

View larger version (42K): [in this window] [in a new window]   Figure 6. In vivo VIP-induced Treg cells are capable of transferring tolerance. PCCF-Tg mice were injected i.p. with PCCF (Ag) or with PCCF plus VIP (Ag+VIP). Eight days later, spleen CD4+ or CD4+CD25+ T cells were isolated. (A) Total CD4+ T cells (1x106 cells; 100 µl) were injected i.v. into PCCF-Tg mice. Twenty-four hours later, mice were immunized subcutaneously (s.c.) with 100 µg PCCF/CFA in the footpads. Two weeks after immunization, spleen, PLN, and DLN were isolated. Results are representative of three experiments. (B) Total CD4+ or sorted CD4+CD25+ T cells (106 cells) were injected i.v. into PCCF-Tg mice. Mice injected with medium instead of cells were used as controls. Twenty-four hours later, mice were immunized with 100 µg PCCF/CFA in the footpads (s.c.). Two weeks after immunization, the mice were challenged intradermally with 50 µg PCCF in 20 µl PBS in the left ear or with 20 µl PBS in the right ear. Ear thickness was measured 24 h later. ear thickness = thickness of left ear – thickness of right ear. In addition, 2 weeks after immunization, mice were analyzed individually for splenic T cell proliferation in response to antigen and serum antibody responses. Spleen cells (4x105 cells/well) were incubated with PCCF (5 µM), and the proliferative response was determined by BrdU incorporation. Serum was collected and assayed for PCCF-specific IgG antibodies by ELISA. Results represent mean ± SD of triplicates from one representative experiment out of three. *, P < 0.001, versus mice treated with antigen alone. (C) The relative presence and the proliferative state of the transferred CFSE-stained CD4+ or CD25+ cells in the spleen were determined by flow cytometry. One representative experiment out of three is shown.

The presence and proliferative state of the transferred T cells were ascertained by FACS analysis for CFSE, CD4, and CD25. CD25^– and CD25⁺ CFSE-labeled cells from antigen and antigen + VIP-injected donors were present in the spleen of recipients 2 weeks after inoculation (Fig. 6C) . Although both populations proliferated, CD25⁺ cells proliferated less than CD25^– T cells.

Effects of VIP-generated Treg cells in GVHD and GVL
The treatment of choice for hematological malignancies consists of allogeneic BM transplantation (BMT) following host irradiation or chemotherapy. The donor T cells responsible for the elimination of the surviving malignant cells are also the reason for the major complication, i.e., GVHD. Recently, it has been reported that Treg cells play an essential role in establishing tolerance to alloantigens and that Treg cells can be used to control GVHD. The question remains whether Treg cells, whereas limiting GVHD, still allow a graft-versus-tumor (GVT) response. We assessed first the potential use of VIP-induced Treg cells in a model of GVHD. Irradiated BALB/c recipients (H-2^d) were reconstituted with allogeneic (H-2^k) TCD BM cells together with CD4⁺ splenic T cells obtained from Tg-PCCF mice (H-2^k), previously inoculated with PCCF [CD4⁺(antigen)] or with PCCF + VIP [CD4⁺(antigen+VIP)] or from naïve mice (control). Mice that received CD4⁺(antigen) developed severe GVHD, including weight loss, reduced mobility, hunched posture, diarrhea, and ruffled fur and died within 30 days. In contrast, mice that received CD4⁺(antigen+VIP) were protected, and more than 60% survived for more than 75 days (Fig. 7A ). Control mice that received CD4⁺ T cells from naïve Tg PCCF mice developed GVHD similar to those receiving CD4⁺(antigen; results not shown). When the CD4⁺ T cells were separated into CD25^– and CD25⁺ subpopulations, the CD25⁺(antigen+VIP) cells were more protective than the CD25⁺(antigen) cells (Fig. 7B) . Next, we determined the effect of CD4⁺(antigen) and CD4⁺(antigen+VIP) cells on anti-tumor activity. Host-irradiated BALB/c mice (H-2^d) were reconstituted with TCD allogeneic BM cells (H-2^k) plus A20 leukemic cells (H-2^d) and CD4⁺(antigen) or CD4⁺(antigen+VIP) T cells (H-2^k). Blood A20 leukemic cells were identified as B220⁺H-2^d by FACS analysis. Mice receiving only BM cells and A20 died between Days 20 and 30, and A20 cells were present in high numbers in the blood. Mice receiving BM and CD4⁺(antigen) T cells died of GVHD between Days 5 and 24, although the A20 tumor cells had been eliminated. In contrast, 80% of the mice that received BM and CD4⁺(antigen+VIP) T cells survived, and no A20 cells were present in blood (Fig. 7C) . These results suggest that the CD4⁺(antigen+VIP) T cells consist of a population that eliminates the allogeneic tumor cells and a second population that prevents GVHD. The anti-tumor T cells resided within the CD4⁺CD25^– population, as purified CD4⁺CD25⁺ T cells from the antigen + VIP-injected mice did not eliminate the tumor cells (Fig. 7C) .

View larger version (39K): [in this window] [in a new window]   Figure 7. In vivo VIP-induced Treg cells inhibit acute lethal GVHD. PCCF-Tg mice were injected i.p. with PCCF (Ag) or with PCCF plus VIP (Ag+VIP). Eight days later, spleen CD4+, CD4+CD25–, and CD4+CD25+ T cells were isolated. (A) BALB/c mice (groups of 10, H-2d) were irradiated and injected i.v. with TCD BM cells (5x106 cells), isolated from B10.A mice (H-2k), with or without CD4+ T cells (106 cells) from PCCF-Tg mice inoculated with PCCF or PCCF plus VIP as described above. Survival and appearance were monitored daily, and body weight was measured weekly after BMT. (B) BALB/c hosts (groups of 10) were irradiated and injected i.v. with B10.A TCD BM (5x106 cells) and B10.A spleen CD4+ T cells (0.5x106 cells), plus CD4+CD25– or CD4+CD25+ cells (0.5x106 cells) from PCCF-Tg mice inoculated with PCCF (Ag) or PCCF plus VIP (Ag+VIP) as described above. (C) Irradiated BALB/c hosts (groups of eight) were inoculated with BMT (106 cells) and A20 leukemic cells (1x104 cells). CD4+ T cells or CD4+CD25+ cells (106 cells) from PCCF-Tg mice treated with PCCF (Ag) or PCCF plus VIP (Ag+VIP) were also injected. Survival was monitored, and tumor growth/elimination was assessed by the presence of A20 cells in blood detected by size and coexpression of B220 and H-2Kd. (D) GVT is maintained in the presence of VIP-induced CD25+ Treg cells. Irradiated BALB/c hosts (groups of 10) were inoculated with BMT (B10.A TCD BM cells, 106 cells) and A20 leukemic cells (derived from BALB/c mice, 1x104 cells). B10.A splenic CD4+ T cells (0.5x106 cells) and CD4+CD25+ cells (0.5x106 cells) from PCCF-Tg mice treated with PCCF (Ag) or PCCF plus VIP (Ag+VIP) were injected. Survival and tumor growth/elimination were assessed as in C. *, P < 0.001, versus mice treated with antigen alone.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
VIP and PACAP are potent, immunosuppressive agents that proved to be protective in models of autoimmune diseases such as collagen-induced arthritis and inflammatory bowel disease [^{15 16 17} ]. Until now, the VIP/PACAP were described as deactivators of macrophages, dendritic cells, and microglia and promoters of Th2 effector differentiation and survival [^{12 13 14} ]. This is the first report about the in vivo effect of VIP on CD4⁺CD25⁺ Treg cell activity.

We compared the effects of antigen and antigen + VIP administration in TCR-Tg mice and concluded that VIP contributes primarily to the expansion of the CD4⁺CD25⁺ Treg cell population. The increase in the numbers of CD4⁺CD25⁺ cells occurred only in the antigen-specific T cells for low doses of antigen and was maintained long-term. CD4⁺CD25⁺ Treg cells have been characterized by high expression of the transcription repressor Foxp3, high surface expression of GITR, Nrp1, CD103, CD62L, and CD69, and low expression of CD45RB [^{18 19 20 21 22} ]. The CD4 population from antigen + VIP-inoculated mice showed a decrease in CD45RB and increases in the expression of all the other markers, as compared with the CD4⁺ T cells from antigen-inoculated mice. However, the purified CD4⁺CD25⁺ T cells from antigen- or antigen + VIP-inoculated mice expressed similar levels of the markers characteristic for CD4⁺CD25⁺ Treg cells. These results suggest that VIP expands the CD25⁺ population without inducing increases in Foxp3 or Nrp1 (or other markers) on a per-cell basis. In addition to expanding the CD4⁺CD25⁺ population, VIP also induced more efficient Treg cells in terms of suppressive activity. On a per-cell basis, the VIP-induced CD4⁺CD25⁺ Treg cells are stronger suppressors of responder T cell proliferation, particularly at low Treg/Tresp cell ratios.

Several subsets of Treg cells with different phenotypes and suppressive mechanisms have been described [⁷ , ²³ ]. In contrast to Tr1/Tr2(Th3), which suppress mostly through cytokine release (IL-10 and TGF-ß), CD4⁺CD25⁺ naturally occurring Treg cells suppress through direct cellular contact. The in vivo VIP-generated CD4⁺ Treg cells appear to consist of two populations: a major CD4⁺CD25⁺ population, whose suppressive mechanism is mediated through direct cellular contact and does not involve IL-10 or TGF-ß, and a minor CD4⁺ population, which produces and uses IL-10 and/or TGF-ß as suppressive molecules. The major population of Treg cells induced by VIP indeed resembles the recently reported CD25⁺ cytokine-independent suppressors recruited from the peripheral CD25^– population by CD4⁺CD25⁺ T cells stimulated with IL-2 and TGF-ß [²⁴ ].

Whether VIP acts directly on T cells inducing the generation or expansion of CD4⁺CD25⁺ Treg cells remains to be established. Another neuropeptide, the melanocyte-stimulating hormone, has been shown to induce CD4⁺CD25⁺ Treg cells in vitro in conjunction with antigen stimulation or TGF-ß [²⁵ , ²⁶ ]. The role of TGF-ß in the expansion of CD4⁺CD25⁺ Treg cells has been demonstrated in vivo and in vitro [²⁷ , ²⁸ ]. Experiments are in progress to determine whether the in vitro treatment with VIP and TGF-ß converts antigen-primed T cells to CD25⁺Foxp3⁺ Treg cells.

The in vivo VIP-induced CD4⁺CD25⁺ Treg cells transfer antigen-specific suppression to naïve hosts, inhibiting DTH, a Th1-dependent immune reaction, and antibody formation, a Th2-dependent response. The in vivo induction of Treg by VIP is physiologically significant from at least two points of view. First, our results suggest that VIP is one of the endogenous factors controlling the peripheral expansion of the CD4⁺CD25⁺ Treg cells. This is important, as little is presently known about the factors and mechanisms controlling Treg cell expansion. Second, the induction of Treg cells adds an additional dimension to the general anti-inflammatory role of VIP, which was reported previously to occur through macrophage/microglia deactivation and bias toward Th2 immunity.

The possible therapeutic use of the VIP-induced Treg cells has been assessed in a model of allogeneic BMT. Allogeneic BMT is a treatment of choice in many hematopoietic malignancies. Following high-dose chemotherapy or irradiation, the host is reconstituted with BM cells, and the donor T cells are responsible for the GVT effects, which eliminate the remaining malignant cells in the host. However, the same donor T cells initiate a GVHD reaction, which represents the major complication following allogeneic stem cell transplantation. Therefore, a desirable therapy will eliminate GVHD without affecting the GVT response. In mice, Treg cells have been shown to prevent lethal GVHD in lethally irradiated hosts reconstituted with allogeneic BM [²⁹ , ³⁰ ]. Recent studies have also demonstrated that although controlling GVHD, CD4⁺CD25⁺ Treg cells maintain the GVL response [³¹ ]. Similar to these reports, the VIP-induced CD4⁺CD25⁺ Treg cells prevented GVHD and maintained GVT in a murine model for allogeneic BMT. These studies suggest the possible use of VIP-induced Treg cells in the survival after hematopoietic stem cell transplantation.

	ACKNOWLEDGEMENTS

 
This work was supported by Grants AI52306 and AI47325 (D. G.), Johnson and Johnson Neuroimmunology Fellowships (M. D.), the Spanish Ministry of Health PI04/0674, and Ramon Areces Foundation (M. D.). The authors have no conflicting financial interests.

Received June 3, 2005; revised July 29, 2005; accepted August 1, 2005.

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