CD3-specific antibody–induced immune tolerance involves transforming growth factor-b from phagocytes digesting apoptotic T cells Sylvain Perruche 1 , Pin Zhang 1 , Yongzhong Liu 1 , Philippe Saas 2 , Jeffrey A Bluestone 3 & WanJun Chen 1 Intact CD3-specific antibody transiently depletes large numbers of T cells and subsequently induces long-term immune tolerance. The underlying mechanisms for the systemic tolerance, however, remain unclear. We show here that treatment of normal mice with intact antibody to CD3 increases systemic transforming growth factor-b (TGF-b) produced by phagocytes exposed to apoptotic T cells. Among the phagocytes, macrophages and immature dendritic cells (iDCs) secrete TGF-b upon ingestion of apoptotic T cells, which induces CD4 + Foxp3 + regulatory T cells in culture and contributes to immune tolerance mediated by CD3-specific antibody in vivo. In accordance with these results, depletion of macrophages and iDCs not only abrogates CD3-specific antibody–mediated prevention of myelin oligodendrocyte glycoprotein–induced acute experimental autoimmune encephalomyelitis (EAE), but also reverses the therapeutic effects of antibody to CD3 on established disease in a model of relapsing-remitting EAE. Thus, CD3-specific antibody–induced immune tolerance is associated with TGF-b production in phagocytes involved in clearing apoptotic T cells, which suggests that apoptosis is linked to active suppression in immune tolerance. Because CD3-specific antibody induces immune tolerance, it is used to enhance the efficacy of transplantation and to treat autoimmune diseases 1–5 . Treatment of nonobese diabetic (NOD) or EAE mice with either mitogenic conventional monoclonal antibodies or nonmito- genic antibody to CD3 results in T cell anergy 6 , CD4 + CD25 + T regulatory (T reg ) cell production of TGF-b 7,8 and remission with long-term immune tolerance 9,10 . This suggests that an immuno- regulatory environment is established by treatment with intact CD3-specific antibody, but how it is created remains elusive. Here we present a new model that links T cell depletion to long- term immune tolerance through TGF-b production by phagocytic macrophages 11–13 and iDCs 14,15 . The TGF-b production is triggered by the apoptotic T cells induced by intact CD3-specific antibody. This apoptotic T cell–phagocyte–mediated immunoregulation is also involved in the prevention and suppression of EAE by CD3-specific antibody treatment. RESULTS CD3-specific antibody treatment increases systemic TGF-b Consistently with previous reports 9,16 , injection of intact mitogenic CD3-specific antibody (2C11) into normal mice resulted in the rapid disappearance of the majority of T cells in the circulation within 2 h, during which time a substantial proportion (450%) of the total T cells in the body were eliminated. The effect lasted for several days (Fig. 1a). CD3-specific antibody injection resulted in a transient (1–3-h) increase in inflammatory cytokines in the circulation, includ- ing interleukin-2 (IL-2), interferon-g (IFN-g) and tumor necrosis factor (TNF), followed by rapid clearance by 6 h 17,18 . The amount of total TGF-b (in plasma treated with 1 N HCl) was upregulated at about 1.5 h but was slightly decreased at 6 h after CD3-antibody injection (Fig. 1b). Notably, however, TGF-b abundance was signifi- cantly increased by 24 h after antibody treatment and was sustained at higher amounts than in untreated controls for several days (Fig. 1b). The bioactive TGF-b (without acid treatment) content in plasma was only marginally elevated by 24 h (PBS-treated, 507 ± 10 pg/ml, n ¼ 7; antibody to CD3–treated, 536 ± 71 pg/ml; n ¼ 4, P ¼ 0.6). Thus, injection of intact CD3-specific antibody increases systemic TGF-b, which is sustained at high levels for several days. Upregulation of TGF-b requires macrophages and DCs in vivo We then studied the cellular source of the TGF-b increase that resulted from the CD3-specific antibody treatment. Because intact CD3- specific antibody induces apoptosis in a large number of T cells, and these apoptotic T cells can be efficiently engulfed and digested by professional phagocytes in vivo, we reasoned that phagocytes in the treated mice might be responsible for TGF-b production. To test this, we injected clodronate-loaded liposomes 19 into mice, which eradi- cated most macrophages and a considerable proportion of DCs compared to PBS-loaded liposomes in spleens (Fig. 1c) and peripheral blood (data not shown). Clodronate-loaded liposomes selectively Received 12 October 2007; accepted 7 March 2008; published online 27 April 2008; doi:10.1038/nm1749 1 Mucosal Immunology Unit, Oral Infection and Immunity Branch, National Institute of Dental and Craniofacial Research (NIDCR), US National Institutes of Health (NIH), 30 Convent Drive, Bethesda, Maryland 20892, USA. 2 Institut National de la Sante ´ et de la Recherche Me ´ dicale, UMR645, University of Franche-Comte ´, IFR133, Etablissement Franc ¸ ais du Sang de Bourgogne Franche-Comte ´ , 25020 Besanc ¸ on, France. 3 University of California–San Francisco Diabetes Center, 513 Parnassus Avenue, San Francisco, California 94143, USA. Correspondence should be addressed to W.C. (wchen@mail.nih.gov). 528 VOLUME 14 [ NUMBER 5 [ MAY 2008 NATURE MEDICINE ARTICLES