Treatment with ex vivo-generated regulatory T cells (T-reg) has been regarded as a potentially attractive therapeutic approach for autoimmune diseases. However, the dynamics and function of T-reg in autoimmunity are not well understood. Thus, we developed Foxp3gfp knock-in (Foxp3gfp.KI) mice and myelin oligodendrocyte glycoprotein (MOG) /IAb (MHC class II) tetramers to track autoantigen-specific effector T cells (T-eff) and T-reg in vivo during experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. MOG tetramer-reactive, Foxp3 + T-reg expanded in the peripheral lymphoid compartment and readily accumulated in the central nervous system (CNS), but did not prevent the onset of disease. Foxp3 + T cells isolated from the CNS were effective in suppressing naive MOG-specific T cells, but failed to control CNS-derived encephalitogenic T-eff that secreted interleukin (IL)-6 and tumor necrosis factor (TNF). Our data suggest that in order for CD4 + Foxp3 + T-reg to effectively control autoimmune reactions in the target organ, it may also be necessary to control tissue inflammation.Thymus-derived (natural) T-reg are crucial for preventing generalized multiorgan autoimmunity throughout the lifespan of an individual 1,2 . The importance of antigenspecific T-reg in conferring genetic resistance to organ-specific autoimmunity 3 COMPETING INTERESTS STATEMENTThe authors declare no competing financial interests.Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions NIH Public Access 10,11 .In vitro, natural T-reg are anergic and suppress T-eff responses in a cell contact-dependent manner 12 . In vivo, T-reg can proliferate and their mechanism of suppression remains largely unknown 13,14 . It was suggested that the encounter with autoantigens was required for their maintenance in vivo 15 . However, it is unclear whether expansion of antigen-specific T-reg can actually be triggered during an inflammatory autoimmune response. This is a fundamental question as T-reg that bear transgenic autoreactive T-cell receptors (TcRs) seem to suppress autoimmune diabetes better than a polyclonal T-reg population does 16 . Furthermore, it has been shown that naive T cells develop reciprocally into T-reg or pathogenic T-helper type 17 (T H 17) cells depending on the absence or presence of IL-6 in the local cytokine milieu [17][18][19] . Thus, the origin and population dynamics of T-reg in autoimmune tissue inflammation are unclear. It has been proposed that in EAE, T-reg may not even reach the target organ but may prevent the trafficking of autopathogenic T-eff into the CNS (ref. The transcription factor Foxp3 has been identified as crucial for the commitment of thymocytes to the T-reg lineage [21][22][23] . We and others have generated Foxp3gfp.KI mice in order to reliably monitor T-reg in vivo based on the linked expression of green fluorescent protein (GFP) in Foxp3 + T cells 19,24 . Using Foxp3gfp.KI mice in combination with a MOG 35-55 /IAb-specifi...

Distinct dendritic cell (DC) subsets differ with respect to pathways of Ag uptake and intracellular routing to MHC class I or MHC class II molecules. Murine studies suggest a specialized role for CD8α+ DC in cross-presentation, where exogenous Ags are presented on MHC class I molecules to CD8+ T cells, while CD8α− DC are more likely to present extracellular Ags on MHC class II molecules to CD4+ T cells. As a proportion of CD8α+ DC have been shown to express langerin (CD207), we investigated the role of langerin+CD8α+ DC in presenting Ag and priming T cell responses to soluble Ags. When splenic DC populations were sorted from animals administered protein i.v., the ability to cross-present Ag was restricted to the langerin+ compartment of the CD8α+ DC population. The langerin+CD8α+ DC population was also susceptible to depletion following administration of cytochrome c, which is known to trigger apoptosis if diverted to the cytosol. Cross-priming of CTL in the presence of the adjuvant activity of the TLR2 ligand N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-Cys-[S]-Serl-[S]-Lys4-trihydrochloride or the invariant NKT cell ligand α-galactosylceramide was severely impaired in animals selectively depleted of langerin+ cells in vivo. The production of IL-12p40 in response to these systemic activation stimuli was restricted to langerin+CD8α+ DC, and the release of IL-12p70 into the serum following invariant NKT cell activation was ablated in the absence of langerin+ cells. These data suggest a critical role for the langerin+ compartment of the CD8α+ DC population in cross-priming and IL-12 production.

Purpose: The prognosis for patients with glioblastoma multiforme (GBM) remains extremely poor despite recent treatment advances. There is an urgent need to develop novel therapies for this disease.Experimental Design: We used the implantable GL261 murine glioma model to investigate the therapeutic potential of a vaccine consisting of intravenous injection of irradiated whole tumor cells pulsed with the immuno-adjuvant a-galactosylceramide (a-GalCer).Results: Vaccine treatment alone was highly effective in a prophylactic setting. In a more stringent therapeutic setting, administration of one dose of vaccine combined with depletion of regulatory T cells (Treg) resulted in 43% long-term survival and the disappearance of mass lesions detected by MRI. Mechanistically, the a-GalCer component was shown to act by stimulating "invariant" natural killer-like T cells (iNKT cells) in a CD1d-restricted manner, which in turn supported the development of a CD4 þ T-cell-mediated adaptive immune response. Pulsing a-GalCer onto tumor cells avoided the profound iNKT cell anergy induced by free a-GalCer. To investigate the potential for clinical application of this vaccine, the number and function of iNKT cells was assessed in patients with GBM and shown to be similar to agematched healthy volunteers. Furthermore, irradiated GBM tumor cells pulsed with a-GalCer were able to stimulate iNKT cells and augment a T-cell response in vitro.Conclusions: Injection of irradiated tumor cells loaded with a-GalCer is a simple procedure that could provide effective immunotherapy for patients with high-grade glioma.