Neuron-mediated generation of regulatory T cells fromencephalitogenic T cells suppresses EAEYawei Liu1, Ingrid Teige1, Bryndis Birnir2& Shohreh Issazadeh-Navikas1Neurons have been neglected as cells with a major immune-regulatory function because they do not express majorhistocompatibility complex class II. Our data show that neurons are highly immune regulatory, having a crucial role in governingT-cell response and central nervous system (CNS) inflammation. Neurons induce the proliferation of activated CD4+T cells throughB7-CD28 and transforming growth factor (TGF)-b1–TGF-b receptor signaling pathways, resulting in amplification of T-cell receptorsignaling through phosphorylated ZAP-70, interleukin (IL)-2 and IL-9. The interaction between neurons and T cells results in theconversion of encephalitogenic T cells to CD25+TGF-b1+CTLA-4+FoxP3+T regulatory (Treg) cells that suppress encephalitogenicT cells and inhibit experimental autoimmune encephalomyelitis. Suppression is dependent on cytotoxic T lymphocyte antigen(CTLA)-4 but not TGF-b1. Autocrine action of TGF-b1, however, is important for the proliferative arrest of Tregcells. Blockingthe B7 and TGF-b pathways prevents the CNS-specific generation of Tregcells. These findings show that generation of neuron-dependent Tregcells in the CNS is instrumental in regulating CNS inflammation.The role of neurons in the regulation of CNS inflammation has beenaddressed in only a few earlier studies—mainly through their inter-action with residual antigen-presenting cells (APCs), namely glial cells,in the CNS1,2. Collectively, these reports contribute to the notion thatneurons might indirectly function as T-cell regulators through controlof CNS-specific APCs. Even less is known about direct communica-tion between T cells and neurons, but cells from the immune systemhave been implicated in protecting neurons from degeneration3–5.These studies suggested the existence of an extensive cross-talkbetween the immune system and the CNS.Neurons can induce T-cell apoptosis6, and T cells have been shownto bind to neurons through leukocyte function-associated antigen 1(LFA-1; on T cells) and intercellular adhesion molecule 5 (ICAM-5; onneurons)7,8. Further supporting an immune-regulatory function ofneurons, we reported that neurons produce the anti-inflammatorycytokine TGF-b1, which is associated with resistance to experimentalautoimmune encephalomyelitis (EAE)9–12. In addition, recovery fromEAE in mice was correlated with upregulation of B7.1 and TGF-b1inneurons—both of which are vital to the regulation of the immuneresponse12. Together, these data suggest a role for neurons in theregulation of CNS inflammation.TGF-b1 is a crucial cytokine in the regulation of T cell–mediatedimmune responses and in the induction of immune tolerance13,14.When TGF-b1 signaling was abolished in T cells15,16, mice deve-loped unchecked T-cell proliferation as well as inflammatory andautoimmune-like diseases. The suppressive role of TGF-b hasalso been established in EAE, in which in vivo blocking of TGF-bat early stages was reported to accelerate and aggravate thedisease17. These reports show the importance of TGF-b–dependentsignaling in T-cell activation and tolerance in vivo. The role of theB7 family (B7.1 and B7.2) as costimulatory molecules is welldocumented and is important in the activation and termination ofthe T-cell response through binding to CD28 or CTLA-4, respectively,thus inducing a secondary signal after engagement of the T-cellreceptor (TCR)18.Here, we investigated how neurons interact with T cells andexamined the potential role of the neuronal B7 interaction withligands on T cells in the absence of major histocompatibility complex(MHC) class II–TCR signaling. Further, we investigated the role ofneuronal TGF-b1 in the outcome of the T-cell response. To studythe interaction of neurons and encephalitogenic T cells, we used anin vitro coculturing system and extended the findings in vivo usingactive and adoptive transfer EAE. Our data show that neurons have acrucial role in the regulation of the T-cell response.RESULTST cells induce upregulation of TGF-b1 and B7 in neuronsWe first investigated the circumstances under which neurons upregu-late TGF-b1 and B7 and asked whether these molecules influenceT-cell regulation in the CNS. Cultured neurons expressed Tgfb1(which encodes TGF-b1) and Cd80 (which encodes B7.1) mRNAand corresponding proteins (Fig. 1a–d). Neuronal production ofTGF-b1 was visualized by confocal microscopy in vitro and byimmunohistochemistry in vivo in the CNS of mice with EAE at day 7after immunization (Fig. 1e,f). In addition, neurons in culturesecreted a substantial amount of TGF-b1 into the culture medium.When neurons were cocultured with T cells, however, TGF-b1 levels inthe culture medium dropped, suggesting that neuronal TGF-b1isReceived 27 June 2005; accepted 4 April 2006; published online 23 April 2006; doi:10.1038/nm14021Neuroinflammation Unit, Institute for Experimental Medical Science, University of Lund, BMC, I13, 221 84 Lund, Sweden.2Department of Clinical Sciences, ClinicalResearch Center, University of Lund, 91-11-59, 205 02, Malmo¨, Sweden. Correspondence should be addressed to S.I.-N. ([email protected]).518 VOLUME 12[NUMBER 5[MAY 2006 NATURE ME DICI NEARTICLES© 2006 Nature Publishing Group http://www.nature.com/naturemedicinetaken up by T cells. Blocking the TGF-b receptor (TGF-bR) onT cells before coculture restored TGF-b1 levels in the culturemedium (Fig. 1g).Neuron–T cell interaction resulted in upregulation of Cd80, Cd86,Icam1, Tgfb1 and Tgfbr2 mRNA (encoding B7.1, B7.2, ICAM-1, TGF-b1and TGF-bR, respectively) in neurons (Fig. 2a,b). In addition, thepercentage of neurons expressing B7.1, B7.2, TGF-b1, TGF-bRandICAM-1 was also significantly increased upon coculture with T cells(Fig. 2c). Blocking production of interferon (IFN)-g and tumornecrosis factor (TNF)-a in T cells before coculture inhibited upregula-tion of these molecules on neurons (Fig. 2c). When recombinantIFN-g and TNF-a were added to the neurons in culture, neuronalexpression of B7.1, B7.2, TGF-b1, TGF-bR and ICAM-1 was induced(Fig. 2d). Together, these data suggest that production of IFN-g andTNF-a by encephalitogenic T cells triggers induction of these mole-cules on neurons. Furthermore, T-cell interaction with neurons resultsin significantly higher neuron survival, suggesting that this interactionis protective for neurons (Fig.