TRAF6 is a T cell–intrinsic negative regulator required for the maintenance of immune homeostasis Carolyn G King 1 , Takashi Kobayashi 1,3 , Pedro J Cejas 1 , Taesoo Kim 1 , Kwiyeom Yoon 1,3 , Gregory K Kim 1 , Elise Chiffoleau 2,3 , Somia P Hickman 2 , Patrick T Walsh 2 , Laurence A Turka 2 & Yongwon Choi 1 TRAF6 has a key role in the regulation of innate immune responses by mediating signals from both TNF receptor and interleukin-1 receptor/Toll-like receptor superfamilies. Here we show that T cell–specific deletion of TRAF6 unexpectedly results in multiorgan inflammatory disease. TRAF6-deficient T cells exhibit hyperactivation of the phosphatidylinositol 3- kinase (PI3K)-Akt pathway compared with wild-type T cells and, as a result, become resistant to suppression by CD4 + CD25 + regulatory T cells. These data identify a previously unrecognized role for TRAF6 in the maintenance of peripheral tolerance, and suggest the presence of a T cell–intrinsic control mechanism to render responder T cells susceptible to tolerizing signals. Tumor necrosis factor receptor–associated factors (TRAFs) are impor- tant signaling adaptors downstream from tumor necrosis factor receptor (TNFR) superfamily members, and are known to activate transcription factors including NF-kB, NFAT, Akt and MAP kinases 1 . TRAF6, unique among TRAF proteins, is able to transduce signals from both TNFR and interleukin (IL)-1/Toll-like receptor superfami- lies, thereby playing an essential role in innate immune responses 2–8 . In addition, TRAF6 has a crucial role in central tolerance by regulating thymic epithelial cell development and organization 9 . A physiologic role for TRAF6 in T cells has not been previously described, however. TRAF6 has been shown to act as an E3 ubiquitin ligase and several other E3 ubiquitin ligases have reported roles in T cells, including Cbl-b and Itch. Deletion of the genes encoding either of these proteins results in the development of autoimmune disease 10,11 . Unlike Cbl-b and Itch, however, TRAF6-mediated ubiquitination is associated with protein activation independent of proteasomal degradation 12 . Recently, we found that Traf6 –/– fetal liver chimeras and Traf6 –/– mice created by Rag2 –/– blastocyst complementation developed a progressive inflammatory disease characterized by hyperactivation of CD4 + T cells 13 . In addition, activated T cells rapidly upregulate Traf6 mRNA and protein expression (Fig. 1a). Together, these data sug- gested the possibility that TRAF6 had a T cell–intrinsic role in the maintenance of immune homeostasis. To test this possibility, we generated mice with TRAF6 deletion specific to T cells (Traf6-DT) by crossing floxed Traf6 mice with CD4-Cre transgenic mice (Supple- mentary Fig. 1 online). Traf6-DT mice were born at normal mendelian ratios and appeared healthy and viable at birth. By 10–12 weeks, however, a majority of Traf6-DT mice exhibited splenomegaly and lymphadenopathy (11 of 14 versus 0 of 10 littermate controls, P o 0.0001; Fig. 1b). Analysis of Traf6-DT peripheral lymphoid organs showed an approximate twofold increase in splenic and lymph node B-cell numbers (Fig. 1c) and a significant decrease in the percentage of CD8 + T cells (17.5 ± 6.1% Traf6-DT lymph node CD8 T cells versus 35.9% ± 5.8 control, P o 0.0001). This difference was accompanied by an increase in CD4 + T-cell numbers and percentage of activated (CD69 + ) and effector-memory (CD44 hi , CD62L lo ) CD4 + T cells (Fig. 1c,d). Sub- sequent histopathological examination showed signs of systemic inflammatory disease, including mononuclear cell infiltrates in intes- tine, liver, lung and kidney (Fig. 1e). Infiltrates primarily consisted of B220 + cells and, to a lesser extent, CD4 + cells (Supplementary Fig. 2 online). IL-4 and IL-10 could be detected in the infiltrated organs, suggesting abnormal T helper type 2 (T H 2) cytokine production in vivo (Supplementary Fig. 2) and, consistent with these observations, Traf6-DT T cells produced higher levels of IL-4 and IL-5 upon in vitro stimulation (Fig. 1f ). In addition, Traf6-DT mice showed signs of hyperactive humoral immunity including increased serum levels of IgG1, IgE and IgM, and DNA autoantibodies (Fig. 1g), and Traf6-DT CD4 + T cells proliferated more strongly in syngeneic mixed lympho- cyte reactions, indicating the presence of autoreactive T cells (Fig. 1h). Floxed TRAF6 mice crossed with Lck-Cre transgenic mice 14 exhibited similar perturbations in peripheral lymphoid organs, confirming that T cell–specific loss of TRAF6 was responsible for the observed phenotype (Supplementary Fig. 3 online). These findings define a previously unrecognized role for T-cell expression of TRAF6 in the regulation of immune cell homeostasis. Autoimmune disease generally occurs as a result of defects in either central or peripheral tolerance. However, no considerable differences in thymic cellularity, percentages of CD4 + and CD8 + thymocytes or alterations of the peripheral Vb repertoire were observed (Supple- mentary Fig. 4 online). Traf6-DT thymocytes showed normal Received 27 January; accepted 16 June; published online 20 August 2006; doi:10.1038/nm1449 1 Department of Pathology and Laboratory Medicine, Abramson Family Cancer Research Institute and 2 Department of Medicine, University of Pennsylvania School of Medicine, 421 Curie Boulevard, Philadelphia, Pennsylvania 19104, USA. 3 Current addresses: Division of Molecular and Cellular Immunology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi Higashi-ku, Fukuoka 812-8582 Japan (T.K.). Division of Molecular Life Sciences and Center for Cell Signaling Research, Ewha Womans University, Seoul 120-750, Republic of Korea (K.Y.). INSERM Unite ´ 437 and Institut de Transplantation et de Recherche en Transplantation, Nantes, F-44035, France (E.C.). Correspondence should be addressed to Y.C. (ychoi3@mail.med.upenn.edu). 1088 VOLUME 12 [ NUMBER 9 [ SEPTEMBER 2006 NATURE MEDICINE LETTERS © 2006 Nature Publishing Group http://www.nature.com/naturemedicine