Two days after adoptive transference, comparable numbers of SarSP-DCs and control DCs were detected in DLNs, indicating that both DC subsets displayed equivalent DLN-homing capacities (Figure 5A). DC survival via PI3K-Akt signaling cascade. In adoptive transfer experiments, NK1R-signaled BMDCs loaded with Ag exhibit increased longevity in draining lymph nodes, resulting in enhanced and prolonged effector cellular immunity. Our results contribute to the understanding of the interactions between the immune and nervous systems that control DC function and present a novel approach for ex vivogeneration of potent immune-stimulatory DCs. == Introduction == Because of their crucial role in initiation and control of innate and adaptive CFTR-Inhibitor-II immunity, myeloid dendritic cells (DCs) are the favored target Ag-presenting cells for positive cellular vaccination protocols.1During the past decade, ex vivogenerated DCs have been used in immunization approaches for prevention and treatment of cancer and infectious diseases.2Current DC-based vaccines focus on the adjuvant effect of proinflammatory mediators, conferring DCs the capability to initiate and bias T-cell immune responses.35However, despite initial promising results, DC-based vaccines do not usually elicit potent T-cell immunity.6 Generation of efficient T-cell immunity using ex vivogenerated DCs requires a critical number of adoptively transferred DCs capable of surviving apoptosis.7Indeed, DCs used as cellular vaccines are exposed to proapoptotic stimuli at the injection sites and in tissue-draining lymph nodes (DLNs).7,8Accordingly, exposure of DCs to proapoptotic stimuli brought on by lytic infections, ultraviolet B (UVB) irradiation, tumor mediators, and cytotoxic cells results in immune suppression.9In addition, interaction of DCs with cells in early apoptosis down-regulates the T cellstimulatory ability CFTR-Inhibitor-II of DCs and induces immunological tolerance.1013Conversely, proinflammatory mediators and growth factors promoting DC survival, including granulocyte macrophagecolony stimulating factor (GM-CSF),14prostaglandin E2(PG-E2),15lipopolysaccharide (LPS),16and CCR717and CD4018ligands, correlate with enhanced T-cell immunity. The intracellular signaling involved in DC survival is currently being elucidated. Activation through CD40 promotes DC survival by favoring a positive balance of nuclear factor kappa B (NF-B) versus activator protein-1 (AP-1) pathway,19whereas GM-CSF, LPS, and PG-E2prevent DC apoptosis by signaling via phosphatidylinositol 3-kinase (PI3K) and protein kinase B (referred as Akt).14,15,20In contrast, the immune-suppressive drug rapamycin induces DC death by antagonizing GM-CSF signaling via inhibition of the PI3K-Akt signaling cascade.14,21 Recently, it has become evident that the outcome of the immune response is highly regulated by neuropeptides. The balance between anti-inflammatory and proinflammatory neuropeptides is crucial to maintain the immune privilege of the central nervous system (CNS) and the steady-state condition in peripheral tissues, and altering this delicate balance plays a relevant role in the pathogenesis of chronic inflammatory and autoimmune diseases.2226Proinflammatory neuropeptides such as substance P (SP) favor CD4+T helper (Th)1 bias and cellular immunity, whereas vasoactive intestinal peptide promotes CD4+Th2 bias.2734Conversely, calcitonin gene-related peptide and the anti-inflammatory products of proopiomelanocortin cleavage, -melanocyte-stimulating hormone and adrenocorticotropin, are potent suppressors of cellular immunity.23 The proinflammatory tachykinins SP and hemokinin-1 (HK-1) exert their immune-stimulatory functions by binding with high affinity the neurokinin 1 receptor (NK1R), a 7 transmembrane domain G protein-coupled receptor.35SP and HK-1 induce interferon- secretion and proliferation of T cells.25,26,3638Our laboratory has recently described that skin DCs express functional NK1R and elicit in vivo CD4+Th1 and CD8+cytotoxic T lymphocyte (CTL) immunity in response to NK1R agonists.30Nevertheless, because the NK1R is usually expressed by several skin cells, the potential of NK1R agonists to modulate directly the immune-stimulatory function of DCs remains unknown. In the present study, we examined the expression of functional NK1R by bone marrowderived DCs (BMDCs) and the ability of NK1R agonists SP, HK-1, and the synthetic NK1R ligand [Sar9Met(O2)11]-SP (SarSP) to rescue BMDCs from apoptosis induced by deprivation of GM-CSF and IL-4. We also CFTR-Inhibitor-II investigated in vivo the immunological relevance of signaling BMDCs with these proinflammatory neuropeptides. == Methods == == Mice == Eight- to 12-week-old wild-type C57BL/6 mice (B6) (The Jackson Laboratory, Bar Harbor, ME) and B6 NK1R/KOmice (kindly provided by Dr Christopher Paige, HPTA University of Toronto, Toronto, ON) were housed in the pathogen-free animal facility of the University of Pittsburgh and used according to institutional guidelines with approval of the University of Pittsburgh Institutional Animal Care and Use Committee. == Generation of BMDCs == BMDCs were generated by culturing mouse BM-precursors for 6 days in RPMI 1640 medium supplemented with 10% fetal bovine serum (Gemini, West Sacramento, CA; complete medium) and GM-CSF and IL-4 cytokines (both at 1000 models/mL; R&D Systems, Minneapolis, MN), as described previously.39,40Total CD11c+BMDCs were purified by.