Vasoactive intestinal peptide (VIP) is a potent anti-inflammatory neuropeptide that, by inhibiting Th1-driven responses and inducing the emergence of regulatory T cells (Treg), has been proven successful in the induction of tolerance in various experimental models of autoimmune disorders. of the CDK inhibitor p27kip1 and impairment of phosphatidylinositol 3-kinase (PI3K)-Akt signaling. Inhibition of interleukin 2 (IL-2) transcription and downregulation of signaling through NFAT, AP-1, and Ras-Raf paralleled the VIP-induced cell cycle arrest. Noteworthy from a functional point of view is the fact that VIP-treated T cells show a regulatory phenotype characterized by high expression of CD25, cytotoxic-T-lymphocyte-associated protein 4 (CTLA4), and Forkhead box protein 3 (FoxP3) and potent suppressive activities against effector T cells. CTLA4 appears to be critically involved in the generation and suppressive activities of VIP-induced Treg. Finally, cyclic AMP (cAMP) and protein kinase A (PKA) activation seems to mediate the VIP-induced cell cycle arrest and Treg generation. Regulatory T cells (Treg) have emerged as a unique population of suppressor T cells orchestrating peripheral immune tolerance (54). Two major populations of Treg, with complementary and overlapping functions in the control of immune response Treg function or infusing Treg isolated and manipulated/expanded ex vivo. Several approaches have been used to expand naturally occurring human CD4+ CD25+ Treg, mainly by T-cell receptor (TCR)-CD28 stimulation in combination with interleukin 2 (IL-2) and/or IL-15 (36, 47). An alternative approach consists of the conversion of CD4+ CD25+ Treg from conventional CD4 T cells with inducible factors. Whereas a large body of literature has been dedicated to describing how Treg control ongoing immune responses and tolerance, especially regarding their phenotype, ontogeny, and mechanisms of suppression (38, 54), the endogenous molecules controlling the peripheral expansion or generation of Treg remain largely unknown. For example, the suppressive cytokine transforming growth factor 1 buy 900515-16-4 (TGF-1) buy 900515-16-4 or immunosuppressive drugs, such as FK778, generate CD4+ CD25+ Treg from the CD4+ CD25? T-cell compartment (14, 19, 35, 53, 66, 67). The identification of additional Treg-inducing factors should extend the applicability of immunotherapy based on Treg in human patients. Vasoactive intestinal peptide (VIP) is an immunosuppressive neuropeptide with potent anti-inflammatory effects (16). VIP is produced by Th2 cells upon antigenic stimulation and mediates regulatory actions on both innate and adaptive immunity (16). Indeed, VIP-based therapy has been proven successful in the treatment of various experimental models of inflammatory and autoimmune disorders (25). Beside its inhibitory effect on inflammatory and Th1-driven responses, VIP induces the emergence of Treg in animals with experimental autoimmune encephalomyelitis and arthritis (20, 26). The VIP-induced Treg seem to consist of two populations: a major population of FoxP3+ CD4+ NR4A3 CD25+ Treg, the suppressive mechanism for which is mediated through direct cellular contact that is mainly dependent on cytotoxic-T-lymphocyte-associated protein 4 (CTLA4), and a minor CD4+ T-cell population, which uses IL-10 and/or TGF-1 as a suppressive molecule (29). We have recently reported the potential of VIP to promote immune tolerance for alloantigens by generating Treg displaying suppressive functions against allospecific effector T cells and protecting against acute graft-versus-host disease in a mouse model of allogeneic bone marrow transplantation (48). The aim of this study was to investigate the molecular mechanisms involved in the immunosuppressive activity of VIP on human T cells and on the generation of human Treg. MATERIALS AND METHODS Antibodies and reagents. VIP, secretin, pituitary adenylate cyclase (AC)-activating polypeptide (PACAP), VIP10-28, VIP1-12, okadaic acid, forskolin, 8-Br-cyclic AMP (cAMP), H-89 {kinase assays. Proteins from whole-cell, nuclear, and cytoplasmic extracts were prepared as described previously (15), separated (70 g/lane) by SDS-polyacrylamide gel electrophoresis (PAGE) (12%; 8% for phosphorylated Rb [pRb]), and blotted onto polyvinylidene difluoride membranes (Millipore) using a semidry system. The membranes buy 900515-16-4 were blocked (Tris-buffered saline-Tween 20-3% nonfat dry milk; 1 h; 22C), probed with the indicated primary antibody overnight at 4C, immunodetected with horseradish peroxidase-conjugated secondary antibodies, and visualized by enhanced chemiluminescence (ECL) (Amersham Pharmacia). Equal protein loading was controlled by reprobing with anti-actin, anti-Zap70 (zeta chain-associated protein kinase), buy 900515-16-4 anti–tubulin, or anti-laminin B antibody. The band intensity was quantitatively determined with ImageJ software (National Institutes of Health) and expressed as densitometric units normalized for the expression of actin, Zap70, -tubulin, or laminin B in each sample. The levels of phosphorylated proteins were normalized to the total expression of the corresponding protein. To study the association between cyclin/cyclin-dependent kinase (CDK) complexes and p27kip1, endogenous cdk4, cdk2, cyclin D2, cyclin E, and cyclin D3 were immunoprecipitated from whole-cell lysates (250 g/sample) by incubation with the corresponding antibody (5 buy 900515-16-4 g; 2.