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© 2007 Nature Publishing Group http://www.nature.com/natureimmunology ARTICLES Critical regulation of CD4+ T cell survival and autoimmunity by b-arrestin 1 Yufeng Shi1, Yan Feng2, Jiuhong Kang1, Chang Liu1, Zhenxin Li3, Dangsheng Li4, Wei Cao2, Ju Qiu2, Zhengliang Guo5, Enguang Bi1, Lei Zang1, Chuanzhen Lu3, Jingwu Z Zhang2,5,6 & Gang Pei1 CD4+ T cells are important in adaptive immunity, but their dysregulation can cause autoimmunity. Here we demonstrate that the multifunctional adaptor protein b-arrestin 1 positively regulated naive and activated CD4+ T cell survival. We found enhanced expression of the proto-oncogene Bcl2 through b-arrestin 1–dependent regulation of acetylation of histone H4 at the Bcl2 promoter. Mice deficient in the gene encoding b-arrestin 1 (Arrb1) were much more resistant to experimental autoimmune encephalomyelitis, whereas overexpression of Arrb1 increased susceptibility to this disease. CD4+ T cells from patients with multiple sclerosis had much higher Arrb1 expression, and ‘knockdown’ of Arrb1 by RNA-mediated interference in those cells increased apoptosis induced by cytokine withdrawal. Our data demonstrate that b-arrestin 1 is critical for CD4+ T cell survival and is a factor in susceptibility to autoimmunity. Apoptosis of T cells is tightly controlled to maintain proper immune homeostasis. Selected by means of T cell receptor signaling in the thymus, most T cells die, and those remaining enter the peripheral lymphoid organs and form the peripheral T cell repertoire. Peripheral CD4+ T cells are maintained in a homeostatic balance between their production and their elimination1,2. After antigen recognition, naive CD4+ T cells proliferate and differentiate into effector T cells to regulate immune responses. When the immune response is reduced, several distinct pathways, including cell inactivation, activationinduced cell death and activated T cell–autonomous death, are used to quell the number and activity of effector T cells. In all of these processes, failure of CD4+ T cell death caused by incorrectly expressed death-signaling regulators may either prevent the apoptosis of potentially autoreactive CD4+ T cells or prolong CD4+ T cell immune responses, both of which can result in disturbed T cell homeostasis and immune diseases such as autoimmunity and leukemogenesis3–6. In mammals, CD4+ T cell death is mediated mainly by two distinct signaling pathways1,6. One pathway is induced by cell surface receptors such as TNF, TRAIL receptor and Fas, which are death factors for activated CD4+ T cells; signaling through these receptors directly activates apoptosis through the recruitment and activation of caspase enzymes6. In addition to the death-receptor pathway, the Bcl-2 protein family regulates T cell apoptosis through a separate mitochondriamediated pathway. The Bcl-2 protein family is composed of pro- and antiapoptotic members that regulate apoptosis by controlling the integrity of mitochondrial membranes and the release of proapoptotic molecules that reside in the mitochondria7,8. Proteins of the Bcl-2 family are critical regulators of cytokine withdrawal–induced and stress-induced T cell apoptosis, as well as autonomous death of activated T cells1,9–12. Bcl-2 is the prototypic member of this large protein family. In Bcl-2-deficient mice, T cells are much more susceptible to death, and leukopenia occurs as these mice grow old13. Conversely, in Bcl2-transgenic mice, T cells are more resistant to many apoptotic stimuli, immune responses10,11 are prolonged, and some autoimmune symptoms have been reported14. The two b-arrestins, b-arrestin 1 and b-arrestin 2, are multifunctional signaling molecules15 with well established functions in the desensitization and endocytosis of diverse cell surface receptors16–19. They have been proposed to regulate the activities and/or subcellular distributions of various signaling molecules, including the kinase Akt (also called protein kinase B), Src family kinases and certain components of the mitogen-activated protein kinase family, such as Erk15,20,21. In addition to the pivotal functions of b-arrestins in signaling regulation in the cytoplasm, b-arrestin 1 has a function in the nucleus, where it binds and recruits histone acetylase p300 to specific gene promoters and increases local acetylation of histone H4 and gene transcription22. The b-arrestins are universally expressed, but the neural and immune systems have much higher expression of b-arrestin proteins23,24. It has been shown that b-arrestin 2 negatively modulates Toll-like receptor signaling in innate immunity, but it has 1Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, China. 2Joint Immunology Laboratory of Institute of Health Sciences and Shanghai Institute of Immunology, Shanghai JiaoTong University School of Medicine and Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200025, China. 3Institute of Neurology, Huashan Hospital, Shanghai Medical College of Fudan University, Shanghai 200040, China. 4Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China. 5Department of Neurology, Ruijin Hospital, Shanghai JiaoTong University School of Medicine, Shanghai 200025, China. 6E-Institutes of Shanghai Universities, Shanghai 200240, China. Correspondence should be addressed to G.P. ([email protected]) or J.Z. ([email protected]). Received 29 March; accepted 15 June; published online 8 July 2007; doi:10.1038/ni1489 NATURE IMMUNOLOGY ADVANCE ONLINE PUBLICATION 1 ARTICLES 102 * Total CD4 CD8 0 24 48 72 50 * 25 0 ** ** 0 24 48 72 Arr1tg WT Arr1 –/– * ** Total CD4 75 50 25 0 0 24 48 72 Time after culture (h) 100 CD8 cells (‘fold expansion’) LN 100 75 50 25 0 0 24 48 72 CD8 e Figure 1 Involvement of b-arrestin 1 in the homeostasis and survival of CD4+ T cells. (a) Flow cytometry of the expression of CD4 and CD8 in lymph node cells (LN) and splenocytes (Spleen) from Arrb1–/– mice, Arrb1tg mice and wild-type mice (WT) at 13–18 weeks of age. Numbers in quadrants indicate percent CD4+CD8– cells (top left) or CD4–CD8+ cells (bottom right). Below, cell numbers for various populations of splenocytes (left) and lymph node cells (right). Data are representative of four experiments with more than ten mice per group. (b,c) Survival assays of splenic CD4+ and CD8+ T cells from Arrb1–/–, Arrb1tg and wild-type mice left untreated (Naive; b) or activated with anti-CD3 and anti-CD28 (c). Viable cells were identified by propidium iodide and annexin V exclusion. Data are representative of three experiments. (d,e) Flow cytometry of total peripheral blood mononuclear cells from Arrb1–/–, Arrb1tg and wild-type mice (five mice per group) immunized intraperitoneally with 100 mg staphylococcal enterotoxin B (SEB) for the population expansion of T cell receptor Vb8.1,2+ (d) or Vb6+ (e) CD4+ and CD8+ T cells. Values are relative to those at time 0. Data are pooled from three independent experiments. *, P o 0.05, and **, P o 0.01, versus the wild-type control. also been reported that b-arrestin 2 regulates the development of allergic asthma25,26. The function of b-arrestin 1 in immune cells, however, remains mostly uncharacterized. Here we report that b-arrestin 1 has a positive regulatory function in CD4+ T cell survival and homeostasis. This function of b-arrestin 1 in T cell biology seems to be mediated mainly by its nuclear function, as it promoted acetylation of histone H4 at the Bcl2 locus and Bcl2 expression. The physiological relevance of CD4+ T cell regulation by b-arrestin 1 was strongly supported by our findings that in mice ‘programmed’ to develop the autoimmune demyelinating disease experimental autoimmune encephalomyelitis (EAE), the disease phenotype was alleviated or aggravated considerably in Arrb1-knockout or Arrb1-transgenic mice, respectively, and that higher expression of Arrb1 and Bcl2 in autoreactive CD4+ T cells from multiple sclerosis patients contributed to the survival of these cells. Our results collectively identify b-arrestin 1–dependent mechanisms critical for the regulation of CD4+ T cell physiology and link b-arrestin 1 to the pathogenesis of autoimmunity. RESULTS Regulation of CD4+ T cell survival and homeostasis Analysis of the immune system, in which the expression of b-arrestins was relatively high (Supplementary Fig. 1a online), showed that adult Arrb1–/– mice had fewer peripheral CD4+ cells, but not CD8+ T cells and B cells, than wild-type mice had (Fig. 1a and Supplementary Fig. 1b). Homeostasis of peripheral CD4+ T cells is maintained between their production and their elimination. As our analysis of thymocyte populations of Arrb1–/– and wild-type mice by flow cytometry showed no significant differences (Supplementary Fig. 1c), we investigated the effects of b-arrestin 1 on the survival of peripheral CD4+ T cells. We purified CD4+ T cells from the spleens of 2 Arr1tg ** ** 1.5 1.0 * 0.5 0 Arr1–/– * 0 5 10 15 20 0 5 10 15 20 2.0 1.5 1.0 0.5 0 Time after SEB injection (d) Time after culture (h) CD4 cells (‘fold expansion’) * 6.0 5.0 4.0 3.0 2.0 1.0 0 ** CD8 cells (‘fold expansion’) Cells (× 106) © 2007 Nature Publishing Group http://www.nature.com/natureimmunology CD8 Spleen 125 * 75 2.0 17.3 20.2 20.1 100 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 50 40 30 20 10 0 Vβ8.1,2 WT 100 CD4 cells (‘fold expansion’) 17.8 125 100 75 50 25 0 Live CD4 T cells (%) 8.1 30.4 101 d Activated T cells Live CD8 T cells (%) Spleen CD4 10.3 10.7 101 c Naive T cells 13.0 102 100 104 31.9 103 LN b Arr1 –/– WT 20.4 Live CD4 T cells (%) Arr1tg 104 21.5 103 Live CD8 T cells (%) a Vβ6 1.5 1.0 0.5 0 0 5 10 15 20 1.5 1.0 0.5 0 0 5 10 15 20 Time after SEB injection (d) Arrb1–/– mice, Arrb1-transgenic (Arrb1tg) mice and wild-type mice and activated some with antibody to CD3 (anti-CD3) and anti-CD28, followed by culture in medium without additional cytokines. After activation, CD4+ T cells from Arrb1tg mice showed increased survival, whereas both naive and activated CD4+ T cells from Arrb1–/– mice were more prone to apoptosis than those from the wild-type mice (Fig. 1b,c). These data indicated that b-arrestin 1 promotes the survival of naive and activated CD4+ T cells in vitro. To examine the function of b-arrestin 1 in vivo, we challenged Arrb1–/–, Arrb1tg and wild-type mice with staphylococcal enterotoxin B and monitored the responding variable b-chain (Vb)8.1,2 and nonresponding Vb6 T cells at various intervals11. Vb8.1,2 T cell populations in mice of the various genotypes expanded similarly on day 3 after injection of staphylococcal enterotoxin B, but their viability decreased thereafter (Fig. 1d). During the subsequent observation period, it was evident that Arrb1tg mice had more circulating CD4+ Vb8.1,2 T cells than wild-type control mice had, whereas Arrb1–/– mice had fewer such cells than wild-type control mice had (Fig. 1d), suggesting that b-arrestin 1 confers substantial protection to the responding CD4+ T cells from staphylococcal enterotoxin B–induced cell death in vivo. The protective effect seemed to be specific to CD4+ T cells, as attrition of the corresponding CD8+ T cells was similar in all three groups of mice (Fig. 1d). In contrast, the populations of nonresponding Vb6 T cells (CD8+ or CD4+) were not affected in these mice throughout the observation period (Fig. 1e). It is well known that the cytokine interleukin 2 (IL-2) is essential for CD4+ T cell survival27–29 and that after activation in vitro, CD4+ T cells make large amounts of IL-2, which prevents apoptosis1. We found, however, that IL-2 production was slightly higher in CD4+ T cells from Arrb1–/– mice than in cells from wild-type mice after activation with anti-CD3 and anti-CD28 (Supplementary Fig. 2a ADVANCE ONLINE PUBLICATION NATURE IMMUNOLOGY ARTICLES a Time (h) 0 48 96 168 0 12 24 36 48 60 72 84 40 44 48 52 56 60 64 Actin Arrb1 Arrb2 Bcl-2 b Relative mRNA Bax Bcl2 Bax 150 100 50 ** Relative mRNA 24 48 72 24 48 72 Bax WT Time (h) 0 24 48 72 Arr1tg ** Arrb1 Arrb2 ** Bcl-2 0 24 48 72 Bax Arr1tg 150 Time (h) 0 Actin 100 0 Time (h) 0 0 Arrb1 Arrb2 Actin 100 WT Relative mRNA © 2007 Nature Publishing Group http://www.nature.com/natureimmunology ** ** 150 50 24 48 72 Arr1–/– 200 0 Time (h) 0 24 48 72 d 0 Bcl-2 0 WT 50 24 48 72 Actin ** * * 0 Time (h) 0 24 48 72 c Arr1–/– WT Time (h) 0 Figure 2 Promotion of Bcl2 expression by b-arrestin 1 in CD4+ T cells. (a) Immunoblot of the expression of b-arrestin 1 (Arrb1), b-arrestin 2 (Arrb2), Bcl-2 and Bax in splenic CD4+ T cells activated with anti-CD3 and anti-CD28 (time, above lanes). b-actin (Actin), loading control. (b–d) Quantitative RT-PCR analysis of Bcl2 and Bax mRNA (left) and immunoblot analysis of b-arrestin 1, Bcl-2 and Bax (right) in wild-type, Arrb1–/– (b), Arrb1tg (c) and Arrb2–/– (d) CD4+ T splenocytes activated with anti-CD3 and anti-CD28 (time, below graphs and above lanes). Results for quantitative RT-PCR are normalized to those obtained for the gene encoding mouse hypoxanthine phosphoribosyl transferase and are presented relative to those obtained at time 0, set as 100%. b-actin, protein loading control (immunoblot). **, P o 0.01, versus the corresponding control. Quantitative RT-PCR data are pooled from three independent experiments; all immunoblot analyses are representative of at least three independent experiments. ** –/– 0 Arr2 24 48 72 Arrb1 Arrb2 ** 24 48 72 WT WT 24 48 72 Bcl-2 0 24 48 72 Arr2 Bax –/– online). Thus, the lower survival of CD4+ T cells from Arrb1–/– mice did not seem to be due to lower production of IL-2. Epigenetic regulation of Bcl2 in CD4+ T cells We further investigated the molecular mechanisms underlying b-arrestin 1–induced survival of CD4+ T cells. Given the known functions of the Akt30 and Erk31 signaling pathways in T cell survival and the positive effect of b-arrestin 1 on the activation of Akt and Erk15, we investigated whether b-arrestin 1 enhanced CD4+ T cell survival by increasing Akt and Erk activity. We activated CD4+ T cells purified from Arrb1–/– and wild-type mice as described above or left them untreated and assessed activation of Akt and Erk by monitoring their phosphorylation status. We found that b-arrestin 1 had no effect on Erk phosphorylation in naive or activated CD4+ T cells and that Akt activation was slightly lower in Arrb1–/– CD4+ T cells only after their activation (Supplementary Fig. 2b). Given that b-arrestin 1 promoted CD4+ T cell survival in both naive and activated states (Fig. 1) and that b-arrestin 1 did not seem to affect the activation of Akt and Erk in naive cells, we reasoned that there must be additional mechanisms by which b-arrestin 1 regulates T cell survival. We thus did microarray analyses with Affymetrix gene chips and found that inhibition of Arrb1 expression mediated by Arrb1-specific small interfering RNA (siRNA) downregulated transcription of the antiapoptotic gene Bcl2 but did not affect other Bcl2 family members, such as Bax, Bcl2l11 (encoding Bim) and Bcl2l1 (encoding Bcl-xL; data not shown). We confirmed this finding in the Jurkat cell line by reverse transcription and quantitative real-time PCR and further found that it was nuclear b-arrestin 1 that upregulated the transcription of Bcl2, as both b-arrestin 2 and mutant b-arrestin 1 with a Q394L substitution, located exclusively in the cytoplasm32,33, had no such effect (Supplementary Fig. 3b online). Given that Bcl-2 is critically involved in regulating the apoptosis of both naive and activated CD4+ T cells1,11 and given the potential regulation of its expression by b-arrestin 1, we examined the expression NATURE IMMUNOLOGY ADVANCE ONLINE PUBLICATION of Arrb1 and Bcl2 in CD4+ T cells after activation. We found that in the first 2 d after CD4+ T cell activation, b-arrestin 1 and Bcl-2 protein gradually decreased but then mostly recovered by day 3 and remained high thereafter (Fig. 2a). To analyze the changes of Arrb1 and Bcl2 expression in more detail, we collected samples every 4 h from 40 h to 64 h after CD4+ T cell activation and found that the initial recovery of b-arrestin 1 seemed to precede that of Bcl-2 (Fig. 2a, right). To investigate whether b-arrestin 1 regulates Bcl2 expression in these cells, we obtained CD4+ T cells from Arrb1–/– and wild-type mice, activated them in vitro and assessed Bcl2 mRNA and Bcl-2 protein at various intervals thereafter. As noted above, Bcl2 showed a dynamic pattern of regulation after activation of wild-type CD4+ T cells, with a gradual and significant decrease during the first 2 d and recovery to original expression by day 3 (Fig. 2b). Arrb1–/– CD4+ T cells had significantly less Bcl2 mRNA than wild-type cells did, even at time zero, indicating that b-arrestin 1 is required for normal Bcl2 expression in physiological conditions (Fig. 2b). Moreover, the dynamic pattern of Bcl2 expression after activation of wild-type CD4+ T cells was completely abolished in Arrb1–/– cells (Fig. 2b), indicating that b-arrestin 1 is critically involved in the regulation of Bcl2 after CD4+ T cell activation. Indeed, the amount of Bcl2 mRNA in Arrb1–/– cells was similar to the lowest noted during activation of wild-type CD4+ T cells (Fig. 2b). We further examined CD4+ T cells obtained from Arrb1tg mice and found that b-arrestin 1 protein recovered faster in Arrb1tg CD4+ T cells after activation, such that at 48 h after activation the cells contained significantly more b-arrestin 1 than wild-type cells did (Fig. 2c, right). Notably, at this time point, Bcl2 mRNA and Bcl-2 protein were also higher in Arrb1tg CD4+ T cells than in cells from wild-type mice (Fig. 2c), consistent with the critical function of b-arrestin 1 in the regulation of Bcl2 expression. Additionally, as b-arrestin 2 protein changed in a way similar to b-arrestin 1 during activation of these cells, we also investigated the expression of Bcl2 in CD4+ T cells from Arrb2–/– mice and found that its expression pattern was normal (Fig. 2d). These data indicate b-arrestin 1 but not b-arrestin 2 specifically regulates Bcl2 expression. The critical function of b-arrestin 1 in the positive regulation of Bcl2 expression probably accounts for the lower survival of Arrb1–/– CD4+ T cells, as the apoptosis features in these cells were very similar to those of Bcl2knockout CD4+ T cells13. We used Jurkat T cells to investigate whether b-arrestin 1 promotes Bcl2 expression through direct regulation of its transcription. We found that transient transfection of Arrb1-specific siRNA or the Arrb1 gene significantly decreased or increased, respectively, Bcl2 mRNA and Bcl-2 protein (Supplementary Fig. 4a online), consistent with the results reported above (Supplementary Fig. 3b). Furthermore, the increased Bcl-2 protein caused by overexpression of Arrb1 could be 3 Bcl2 Bax 100 50 0 Time (h) 0 ** ** 24 48 72 150 100 50 0 Time (h) 0 24 48 72 100 50 0 Time (h) 0 ** ** 24 ** 48 150 d ** 100 50 0 Time (h) 0 ** ** 72 0 ** 24 48 Arr1 72 –/– 7 H4Ac 6 H3Ac 5 4 3 2 1 0 24 48 72 WT Epigenetic regulation of Bcl2 expression by p300 Histone acetylation is regulated by the activities of histone acetyltransferase and histone deacetylase. Studies have shown that 0 24 48 Arr1tg 72 u2 . u2 5 × . 1 u1 0 × 0 4 . 1 u1 5 × 0 4 .0 10 u0 × 4 .5 10 4 × 10 4 0. 5 0 × 1. 1 0 05 1. × 1 5 0 1. × 5 75 10 1. × 5 7 1 1. 6 × 0 5 81 10 1. × 1 5 86 0 5 1. × 1 91 0 5 × 10 5 c Histone acetylation ratio WT blocked by either the mRNA transcription inhibitor actinomycin D or the protein synthesis inhibitor cycloheximide (Supplementary Fig. 4b,c). These data are consistent with the idea that b-arrestin 1 promotes Bcl2 expression by increasing its transcription. CREB34 and NF-kB35 are transcription factors known to regulate Bcl2 expression. However, overexpression of Arrb1 did not cause any significant increase in CREB- or NF-kB-mediated transcriptional activity, as assayed by reporter plasmids (Supplementary Fig. 4d,e). Therefore, it seems unlikely that b-arrestin 1 regulates Bcl2 expression through direct activation of these two transcription factors. Another mechanism by which b-arrestin 1 could promote gene expression is by increasing local acetylation of histone H4 at target promoter regions22. Acetylation of histone H4 at the Bcl2 promoter changed dynamically after CD4+ T cell activation (Fig. 3a), which paralleled the changes of Bcl2 expression (Fig. 2). For example, acetylation of histone H4 at the Bcl2 promoter region gradually reached its lowest point at approximately 48 h after CD4+ T cell activation and then recovered to its normal amount at 72 h. As a control, acetylation of histone H3 at the Bcl2 promoter was unchanged. Consistent with our earlier results that Bcl2 expression was much lower in Arrb1–/– CD4+ T cells and remained low after T cell activation, we noted a similar pattern for acetylation of histone H4 at the Bcl2 promoter in Arrb1–/– CD4+ T cells, as it remained similar to the lowest acetylation found in wild-type CD4+ T cells during their activation (Fig. 3b). However, acetylation of histone H4 at the Bcl2 promoter was significantly higher at 48 h after activation in Arrb1tg CD4+ T cells than in wild-type cells (Fig. 3c), a result reminiscent of the faster recovery of expression of Arrb1 and Bcl2 at the same time point in these cells (Fig. 2). Further, b-arrestin 1 promoted acetylation of histone H4 in a region approximately spanning from 15,000 base pairs upstream to 186,000 base pairs downstream of the Bcl2 transcription start site (Fig. 3d). These data indicate that b-arrestin 1 is critically involved in promoting acetylation of histone H4 at the Bcl2 locus, which is most probably the mechanism of upregulation of Bcl2 expression by b-arrestin 1. 4 b 150 Relative H4Ac 150 Relative H3Ac a Relative H4Ac Figure 3 Promotion of acetylation of histone H4 at the Bcl2 locus by b-arrestin 1. Chromatinimmunoprecipitation analysis of splenic CD4+ T cells activated with anti-CD3 and anti-CD28. (a) Analysis of acetylated histone H4 (H4Ac; left) and acetylated histone H3 (H3Ac; right) in wildtype mice. (b,c) Analysis of acetylated histone H4 in Arrb1–/– and wild-type mice (b) and in Arrb1tg and wild-type mice (c). Values are relative to the acetylation at time 0, set as 100%. (d) Chromatin immunoprecipitation and quantitative PCR analysis of the acetylation of histones H4 and H3 at and surrounding the Bcl2 locus in Arrb1–/– and wild-type mice. Data represent the ratio of acetylation at a specific position of the Bcl2 locus (horizontal axis) in wild-type CD4+ T cells to that in Arrb1–/– CD4+ T cells and are normalized to the corresponding input controls. 0, start point of transcription; u, upstream of the start point. **, P o 0.01, versus the corresponding control. All data are pooled from three independent experiments. Relative H4Ac © 2007 Nature Publishing Group http://www.nature.com/natureimmunology ARTICLES Distance (base pairs) b-arrestin 1 can be recruited to promoters of specific target genes and promote local acetylation of histone H4 by specifically binding to p300 (ref. 22), a histone acetyltransferase36. Thus, we investigated the function of p300 in b-arrestin 1–mediated acetylation of histone H4 at the Bcl2 promoter and as a means of regulating Bcl2 transcription. Consistent with the specific function for b-arrestin 1 in the regulation of Bcl2 transcription, we found that b-arrestin 1 but not b-arrestin 2 or the b-arrestin 1 cytoplasmic mutant (Q394L) increased acetylation of histone H4 of the Bcl2 promoter region (Supplementary Fig. 5 online). Furthermore, acetylation of histone H4 at the Bcl2 promoter and Bcl2 transcription were stimulated considerably by overexpression of p300 in Jurkat cells. These effects of p300 were augmented by coexpression of b-arrestin 1 and were blocked by coexpression of Arrb1-specific siRNA (Supplementary Fig. 5c,e). Moreover, a dominant negative mutant of p300 strongly attenuated the stimulatory effect of b-arrestin 1 on both acetylation of histone H4 at the Bcl2 promoter region and Bcl2 transcription (Supplementary Fig. 5c,e). These data indicate that p300 is critical in b-arrestin 1–mediated hyperacetylation of histone H4 at the Bcl2 promoter region and in Bcl2 transcription. Autoimmune demyelinating disease and b-arrestin 1 Regulation of CD4+ T cell apoptosis by cell death–signaling molecules is critical for regulating the frequency and activity of autoreactive CD4+ T cells; disturbance of these cells may contribute to inflammatory pathologies in autoimmune conditions. To assess the pathophysiological relevance of b-arrestin 1 function in CD4+ T cells, we used the EAE mouse model37 to investigate whether b-arrestin 1 regulates the pathogenesis of autoimmune disease. We induced EAE in Arrb1–/–, Arrb1tg and wild-type mice (all on the same C57BL/6 background) by injecting a peptide of rat myelin oligodendroglial glycoprotein (MOG) amino acids 35–55. EAE disease onset was delayed significantly in Arrb1–/– mice (wild-type, 9.7 ± 2.9 d; Arrb1–/–, 16.3 ± 3.0 d) and was accompanied by significantly lower clinical scores (maximum clinical score: wild-type, 3.2 ± 0.2; Arrb1–/–, 1.4 ± 0.4; Fig. 4a). In contrast, although there was no significant difference in the timing of disease onset (wild-type, 9.7 ± 2.9 d; Arrb1tg: 8.7 ± 0.8 d), Arrb1tg mice had greater clinical severity (maximum clinical score: Arrb1tg, 4.2 ± 0.3; wild-type, 3.2 ± 0.2). The differences between the experimental groups ADVANCE ONLINE PUBLICATION NATURE IMMUNOLOGY ARTICLES Arr1tg 3 Arr1 5 –/– 2 1 0 3 WT 2 –/– Arr1 1 0 0 2 4 6 8 10 12 14 16 18 20 Time after immunization (d) Luxol fast blue 0 2 4 6 8 10 12 14 16 18 20 Time after immunization (d) c H&E 20 H incorporation (c.p.m. × 102) b Arr1tg 4 –/– Arr1 5 ** 0 No stim MOG d Live cells (%) Arr1tg Arr1 –/– WT Arr1tg 100 –/– Arr1 75 50 ** 25 * ** 0 24 48 0 24 48 Time (h) 0 MOG CD4 T cells MOG CD8 T cells were highly significant by the linear regression method38 and by twoway analysis of variance (P o 0.001; Fig. 4a). In spinal cord sections, we found a notable paucity of inflammatory lesions and demyelination in Arrb1–/– tissues and more severe inflammatory lesions and demyelination in Arrb1tg tissues than in wild-type tissues (Fig. 4b). Given that b-arrestin 1 expression in CD4+ T cells was critically involved in regulating the apoptosis of these cells, we sought to determine whether dysregulated apoptosis might contribute to the effects of b-arrestin 1 on the disease progression and severity of EAE. We obtained splenocytes from Arrb1–/–, Arrb1tg and wild-type mice in which EAE had been induced, stimulated the cells with MOG peptide and evaluated their proliferation by [3H]thymidine incorporation assay. MOG stimulation greatly increased the proliferation of Arrb1tg splenocytes to more than double the proliferation of wild-type cells, but the MOG-stimulated proliferation was significantly attenuated in Arrb1–/– cells (Fig. 4c). We also tested the viability of MOGspecific T cells isolated from mice with EAE. Consistent with the results reported above, MOG-specific CD4+ T cells from Arrb1tg mice had a significantly higher survival rate, whereas those from Arrb1–/– b 350 300 ** Control MS 250 200 150 100 50 0 c 250 Relative BCL2 mRNA a Relative ARRB1 mRNA © 2007 Nature Publishing Group http://www.nature.com/natureimmunology Arr1tg 10 3 WT WT ** 15 200 * Figure 4 Critical involvement of b-arrestin 1 in EAE. (a,b) Induction of EAE in Arrb1–/–, Arrb1tg and wild-type mice. (a) Clinical scores of mice monitored daily for signs of disease, assigned according to disease severity. Left, each line represents the average scores of 10 Arrb1–/– mice, 10 Arrb1tg mice and 14 wild-type mice. Right, linear regression analysis of the data at left. P o 0.001. Data are pooled from experiments done four times. (b) Sections of spinal cords from mice 20 d after EAE induction; sections were fixed and were stained with hematoxylin and eosin (H&E) to demonstrate the degree of inflammation and with Luxol fast blue to show the degree of demyelination. Original magnification, 200 (Luxol fast blue) or 100 (hematoxylin and eosin). Images are representative of three to four mice per group. (c) Proliferation assay of splenocytes from Arrb1tg, Arrb1–/– and wild-type mice maintained for 8 d after EAE induction, assessed as [3H]thymidine incorporation with or without (No stim) restimulation with MOG peptide. **, P o 0.01, versus the wild-type control. Data are pooled from three independent experiments. (d) Viability of CD4+ and CD8+ T cells purified after MOG restimulation of splenocytes isolated from mice maintained for 8 d after EAE induction; cells were then cultured in medium and assessed by propidium iodide and annexin V exclusion. *, P o 0.05, and **, P o 0.01, versus the wild-type control. Data are pooled from three independent experiments. Control MS Clone 1 Control Arrb1si 150 Actin 100 Arrb1 Arrb2 50 Bcl-2 mice were more susceptible to apoptosis (Fig. 4d). In addition, we analyzed the percentage of CD62L+, CD44+ and CD4+CD25+Foxp3+ regulatory T cells in peripheral CD4+ T cell populations of both naive mice (weeks 7–8 and 15–16) and mice with EAE (weeks 7–8). We found no significant differences among the three groups (wild-type, Arrb1–/– and Arrb1tg), indicating that the proportion of naive, memory and regulatory T cell subsets of CD4+ T cells is not selectively affected by b-arrestin 1 (Supplementary Fig. 6 online). These data suggest that b-arrestin 1 has an important function in the induction and severity of EAE by altering the survival and functional properties of MOG-specific CD4+ T cells. The expression of b-arrestin 1 in splenocytes is upregulated when mice are induced to develop EAE39. We further confirmed that b-arrestin 1 expression in CD4+ T cells was much higher after EAE induction (Supplementary Fig. 7 online). Given the positive function of b-arrestin 1 in promoting CD4+ T cell survival and the finding that its expression was much higher after EAE induction, we sought to determine whether and to what extent b-arrestin 1 expression affects the functional properties of CD4+ T cells and myelin-reactive T cells in patients with multiple sclerosis. We purified CD4+ T cells from samples of peripheral blood mononuclear cells obtained from 14 patients with relapsing-remitting multiple sclerosis (12 female and 2 male; all 21–55 years of age) and Clone 2 Control Arrb1si d Clone 1 Clone 2 100 75 * 50 ** 25 0 Time (h) 0 24 48 Live CD4 T cells (%) WT 4 Live CD4 T cells (%) 5 Clinical score Clinical score a Control Arrb1si 100 75 * 50 ** 25 0 Time (h) 0 24 48 0 Figure 5 Critical involvement of b-arrestin 1 in the pathogenesis of multiple sclerosis. (a,b) Quantitative RT-PCR analysis of ARRB1 mRNA (a) and BCL2 mRNA (b) in CD4+ T cells purified from samples of peripheral blood mononuclear cells obtained from 14 patients with relapsing-remitting multiple sclerosis (MS) and 14 healthy controls. Values are relative to control values, set as 100%. *, P o 0.05, and **, P o 0.01, versus the healthy control. (c) Immunoblot analysis of b-arrestin 1 and Bcl-2 in lysates of MBP-specific CD4+ T cell clones from patients with multiple sclerosis; clones were infected with lentivirus carrying ARRB1-specific siRNA (Arrb1si) or empty vector (Control). Actin, loading control. Data are representative of three independent experiments. (d) Viability of MBP-specific CD4+ T cells infected with lentivirus expressing ARRB1-specific siRNA or empty vector and then cultured (time, horizontal axes). Viable cells with green fluorescent protein fluorescence were identified by propidium iodide exclusion. *, P o 0.05, and **, P o 0.01, versus the empty vector control. Data are pooled from three independent experiments. NATURE IMMUNOLOGY ADVANCE ONLINE PUBLICATION 5 © 2007 Nature Publishing Group http://www.nature.com/natureimmunology ARTICLES 14 gender- and age-matched healthy controls. We used quantitative RT-PCR to evaluate the transcription of ARRB1 and BCL2 in these cells. We detected significantly higher transcription of ARRB1 and BCL2 in CD4+ T cells from patients with multiple sclerosis than in those from healthy people (Fig. 5a,b). In addition, when we analyzed six independent T cell clones reactive to myelin basic protein (MBP), we found that clones derived from patients with multiple sclerosis had higher expression of ARRB1 and BCL2 than did clones generated from healthy people (data not shown). Furthermore, when we ‘knocked down’ b-arrestin 1 expression in MBP-specific CD4+ T cell clones generated from patients with multiple sclerosis40 by a lentivirusmediated RNA-mediated interference approach, we found much less expression of Bcl-2 (Fig. 5c). MBP-specific CD4+ T cells infected with lentivirus carrying ARRB1-specific siRNA were much more susceptible to apoptosis than were cells infected with a control lentivirus (Fig. 5d). These data collectively indicate that CD4+ T cells specifically involved in multiple sclerosis have higher expression b-arrestin 1, which may contribute to the altered survival and aberrant functional properties of these inflammatory T cells in multiple sclerosis. DISCUSSION The immune system is a very dynamic system in mammals, and cell apoptosis in this system is critically regulated by various intracellular and extracellular signaling mechanisms. Our results here have demonstrated that b-arrestin 1 specifically regulates the survival and homeostasis of CD4+ T cell and thus affects adaptive immune responses. Although we found that impaired Akt activation in activated Arrb1–/– CD4+ T cells might contribute to the impaired survival of these activated cells, the survival function of b-arrestin 1 may instead be attributed mainly to its nuclear function through epigenetic regulation of Bcl2 expression in both naive and activated CD4+ T cells. We also found that b-arrestin 1 is critically involved in the induction and severity of autoimmune demyelinating disease and streptozotocininduced type 1 diabetes (data not shown). In the autoimmune demyelinating disease, the encephalitogenic CD4+ T cells had higher expression of b-arrestin 1 that seemed to promote the survival of these cells. These results suggest a critical function for b-arrestin 1 in CD4+ T cell survival and autoimmunity regulation. Our mechanistic analyses showed that b-arrestin 1 epigenetically regulates Bcl2 expression in CD4+ T cells. Bcl-2 accomplishes important functions in cell survival and death by controlling the integrity of mitochondrial membranes and the release of proapoptotic molecules residing in the mitochondria7. In T cells, Bcl2 expression thus should be critically controlled; otherwise, T cell homeostasis and immune responses will probably be disturbed. The regulation mechanism for Bcl2 expression, however, remains mostly unknown. Here we found that b-arrestin 1 promoted acetylation of histone H4 at the Bcl2 locus and Bcl2 expression in both naive and activated CD4+ T cells. We also found that this mechanism is conserved between mice and humans. Thus, in addition to demonstrating a previously unknown mechanism regulating CD4+ T cell survival, our results also provide an epigenetic view of Bcl2 regulation, which has not explored before to our knowledge. We noted involvement of b-arrestin 1 in T cell survival and homeostasis mainly for peripheral CD4+ but not CD8+ T cells and thymocytes. In thymocytes, b-arrestin 1 expression was very low compared with that of splenocytes or lymphocytes, which could explain why b-arrestin 1 had little effect on the thymocytes. Although there was no substantial difference in b-arrestin 1 protein in peripheral CD4+ and CD8+ T cells (data not shown), the effects of b-arrestin 1 on CD4+ and CD8+ T cell survival were very different. CD4+ T cells deficient in b-arrestin 1 had much lower survival than wild-type cells 6 had, whereas Arrb1–/– CD8+ T cells had no notable defect in survival. Mechanistic analysis showed that b-arrestin 1 located in the nucleus upregulated acetylation of histone H4 of the Bcl2 promoter and thus Bcl2 expression; overexpression of a cytoplasmic mutant of b-arrestin 1 (Q394L) did not have any effect on Bcl2 expression. We also examined whether the subcellular distribution of b-arrestin 1 in CD4+ and CD8+ T cells is different. We found that for both naive and activated cells, CD4+ T cells had much more nuclear b-arrestin 1 than CD8+ T cells had (data not shown). Thus, the difference in the subcellular distribution of b-arrestin 1 in CD4+ and CD8+ T cells might account for the differential effect of b-arrestin 1 on the survival of CD4+ and CD8+ T cells. Activation of G protein–coupled receptors recruits both b-arrestin 1 and b-arrestin 2 to the cell membrane, and subsequent interactions between phosphorylated G protein–coupled receptors and the b-arrestins induce receptor endocytosis and signal attenuation. However, accumulating evidence also indicates possible functional differences between the two b-arrestin subtypes as well as their distinct receptor specificity41. Here we have shown that b-arrestin 1 but not b-arrestin 2 specifically promoted acetylation of histone H4 of the Bcl2 locus and Bcl2 expression. This difference between b-arrestin 1 and b-arrestin 2 is consistent with published results22 and is presumably due to their different subcellular distributions. Their functional differences notwithstanding, both b-arrestin 1 and b-arrestin 2 seem to regulate immune responses; our results here have shown that b-arrestin 1 promoted CD4+ T cell survival and autoimmune conditions, whereas another published study has reported that b-arrestin 2–deficient mice have greatly reduced clinical symptoms of asthma mainly because of the effect of b-arrestin 2 on immune cell migration25. It thus seems that, collectively, b-arrestins have positive functions in adaptive immune responses, albeit through different mechanisms. It has been shown that b-arrestin 1 is directly involved in the regulation of gene expression and that this effect can be enhanced by signals from G protein–coupled receptors22. Expression of the delta opioid receptor has been detected after CD4+ T cells are activated in vitro42. Notably, we have found that additional activation of delta opioid receptor induced Bcl2 expression in a b-arrestin 1–dependent way (unpublished data). These data characterize b-arrestin 1 as an important signaling regulator that may directly relay environmental signals to influence CD4+ T cell survival, thus affecting the adaptive immune response. That function, considered in addition to the intrinsic function of b-arrestin 1 in CD4+ T cells, indicates important mechanisms by which the immune system may be modulated by b-arrestin 1 in physiological conditions. Survival of CD4+ T cells is directly linked to the number and function of CD4+ T cells, known to be important in immune system homeostasis and autoimmune conditions. We have demonstrated that CD4+ T cells and MBP-reactive T cells of patients with multiple sclerosis had higher expression of b-arrestin 1 than those of healthy people. Overexpression of ARRB1 in CD4+ T cells may be associated with hyperactivity of CD4+ T cells as one of the immunological features of multiple sclerosis43, although this awaits further investigation. Nevertheless, one therapeutic strategy for the treatment of multiple sclerosis and some other human autoimmune diseases is to deplete or reduce the number and function of CD4+ T cells. Clinical trials have indicated that therapeutic reduction or depletion of CD4+ T cells by monoclonal antibodies may favorably alter the clinical course of multiple sclerosis, although the effect is not statistically significant44,45. It may thus be prudent to further evaluate the function of b-arrestin 1 in therapeutic strategies targeting CD4+ T cells for the treatment of autoimmune diseases such as multiple sclerosis. ADVANCE ONLINE PUBLICATION NATURE IMMUNOLOGY ARTICLES METHODS © 2007 Nature Publishing Group http://www.nature.com/natureimmunology Study subjects. Subjects volunteering for this study were patients from the outpatient clinic of Huashan Hospital of Fudan University and Ruijin Hospital of JiaoTong University who were diagnosed with clinically defined relapsingremitting multiple sclerosis. Blood samples were obtained from subjects after informed-consent procedures were completed in accordance with the guidelines of local institutional review boards. All healthy subjects for this study were volunteers from the Shanghai Blood Center and personnel of the Institute for Biochemistry and Cell Biology; all provided informed consent. Mice, cell lines, tranfection and lentivirus infection. C57BL/6 mice were from the Shanghai Laboratory Animal Center (Chinese Academy of Sciences). Arrb1–/– and Arrb2–/– mice were on a C57BL/6 background. Arrb1tg mice were generated in the laboratory of G.P.; expression of hemagglutinin-tagged human Arrb1 was under control of the human cytomegalovirus promoter (L.Z., R. Yang, J. Chai and G.P., unpublished data). Arrb1tg mice used here were backcrossed onto the C57/BL6 background for more than nine generations and did not manifest autoimmune conditions, as determined by autoantibody titers and assessment of clinically overt disease. The animal protocol was approved by the institutional animal use committee of the Shanghai Institutes for Biological Sciences (Chinese Academy of Sciences). All mice were maintained in pathogen-free conditions and were genotyped before use. Jurkat and HEK293 cells (American Type Culture Collection) were maintained in RPMI 1640 medium and MEM (Gibco-BRL), respectively. Jurkat cells were transfected with Nucleofector (Amaxa). By calcium phosphate precipitation, HEK293 cells were cotransfected with pNF-kB-Luc or pCREB-TA-Luc, pRL-TK (Clontech) and other plasmids. At 36 h after transfection, luciferase activity was measured with the Dual Luciferase Reporter Assay system normalized to control luciferase activity. Cells treated with forskolin (10 ng/ml; Sigma) or recombinant human tumor necrosis factor (10 ng/ml; PeproTech) served as positive controls. Antibodies, reagents, plasmids and siRNA. Mouse anti-Bcl-2 (7; 610539) and anti-Bax (3; 610983), phycoerythrin-conjugated antibodies to mouse CD4 (RM4-5; 553048), CD8 (53-6.7; 553032), CD44 (IM7; 553134), B220 (RA3-6B2; 553089) and Vb6 (RR4-7; 553194), and fluorescein isothiocyanate–conjugated antibodies to mouse CD4 (RM4-5; 01064D), CD8 (53-6.7; 553030), CD3 (1452C11; 553061), CD25 (7D4; 553072) and Vb8.1,2 (MR5-2; 553185) were from BD Biosciences. Allophycocyanin-conjugated anti–mouse CD62L (MEL-14-H2.100; 130-091-805) was from Miltenyi Biotec; the allophycocyanin-conjugated antimouse, rat Foxp3 Staining Set was from eBioscience; antibodies to acetylated histone H4 (06-866) and to acetylated histone H3 (06-599) were from Upstate Biotechnology; IRDye 800CW–conjugated, affinity-purified anti-mouse IgG (610-131-121) and anti-rabbit IgG (600-431-384) were from Rockland; anti-b-actin (1A4; A2547), actinomycin D and dexamethasone were from Sigma; anti-Erk (9122) and antibody to Erk phosphorylated at Ser217 and Ser221 (9121L) were from Cell Signaling; and anti-Akt (KC-5A05) and antibody to Akt phosphorylated at Ser473 (KC-5A04) were from Kangchen. Staphylococcal enterotoxin B was from Biotinge Biomedicine. Plasmids expressing cytomegalovirus b-galactosidase and hemagglutinin-tagged b-arrestin 1 (long form24), b-arrestin 2 and b-arrestin 1 mutant (Q394L) were generated as described33,46. Construction of the pBS-U6-Arrb1, pBS-U6-Arrb2 and pBS-U6-nonspecific siRNA plasmids has been described33,47. Plasmids expressing wild-type p300 and a dominant negative mutant of p300 (deletion of the first cysteine- or histidine-rich region) were from Upstate Biotechnology. Human MBP peptides were synthesized and purified by the Peptide Core Laboratory of M.D. Anderson Cancer Center; peptide purity was over 90%. Cell purification, activation and culture. Splenic cells stained with purified rat anti–mouse CD4 (RM4-5; 553043) or purified rat anti–mouse CD8a (53-6.7; 553027; both from BD Biosciences) were purified with goat anti–rat IgG microbeads, separation columns and an AutoMACS sorter (Miltenyi Biotec), yielding a purity of over 95% by flow cytometry. Purified CD4+ T cells were activated with plate-bound purified hamster monoclonal anti–mouse CD3e (CD3 e-chain; 5 mg/ml; 145-2C11; 553057) and hamster monoclonal anti-mouse CD28 (4 mg/ml; 37.51; 553294; both from BD Biosciences) in RPMI 1640 medium supplemented with 2 mM L-glutamine, 5 mM b-mercaptoethanol, 100 U/ml of penicillin and 10% (vol/vol) FCS. MBP-specific CD4+ T cells were NATURE IMMUNOLOGY ADVANCE ONLINE PUBLICATION maintained in RPMI 1640 medium with MBP peptides48 (20 mg/ml) and recombinant human IL-2 (100 U/ml; PeproTech). For analysis of cell survival, T cells were cultured in RPMI 1640 medium without additional cytokines. At various times, percent viable cells was determined by propidium iodide and annexin V exclusion, or by propidium iodide exclusion only for green fluorescent protein–positive cells, with an Annexin V FLUOS Staining kit (Roche Molecular Biochemicals). T cells from human blood samples were labeled with purified mouse anti–human CD4 (S3.5; MHCD0400; Caltag) and were purified with goat anti–mouse IgG microbeads, separation columns and an AutoMACS sorter (Miltenyi Biotec), yielding a purity of over 90% by flow cytometry. Flow cytometry. Cells were resuspended in PBS containing 1% (wt/vol) BSA (Sigma-Aldrich). For surface staining of CD4, CD8, CD3, B220, T cell receptor Vb8.1,2 and Vb6, cells were incubated for 1 h on ice with fluorochromeconjugated antibodies to various cell surface markers at the recommended dilutions for isotype control antibodies. Stained cells were washed and were analyzed with a FACSAria (BD Biosciences). Reverse transcription and quantitative real-time PCR. Total RNA was extracted from cultured cells with TRIzol (Invitrogen) according to the manufacturer’s instructions. Oligo(dT) priming and superscript III reverse transcriptase (Invitrogen) were used for reverse transcription of purified RNA. All gene transcripts were quantified by quantitative PCR with Brilliant SYBR Green QPCR Master Mix and a Light Cycler apparatus (Stratagene). Primer pairs are listed in Supplementary Methods online. Induction and evaluation of EAE. The encephalitogenic peptide of MOG corresponding to residues 35–55 (BioAsia Biotechnology) used to induce EAE had a purity of 95%. Acute EAE was induced by a subcutaneous immunization with 300 mg of the MOG peptide in complete Fruend’s adjuvant containing heatkilled Mycobacterium tuberculosis (H37Ra strain; 5 mg/ml; BD Diagnostics). Pertussis toxin (200 ng/mouse; List Biological Laboratories) in PBS was administered intravenously on the day of immunization and 48 h later. Mice 6–8 weeks of age were weighed and examined daily for disease symptoms; they were assigned scores for disease severity with the following EAE scoring scale: 0, no clinical signs; 1, limp tail; 2, paraparesis (weakness, incomplete paralysis of one or two hindlimbs); 3, paraplegia (complete paralysis of two hindlimbs); 4, paraplegia with forelimb weakness or paralysis; and 5, moribund state or death. Tissues for histological analysis were removed from mice 20 d after immunization and were immediately fixed in 4% (wt/vol) paraformaldehyde. Paraffinembedded sections of spinal cord 5–10 mm in thickness were stained with Luxol fast blue or with hematoxylin and eosin and were examined by light microscopy. The degree of demyelination and inflammatory infiltration was quantified according to a published procedure49 on an average of three transverse sections of spinal cord per mouse for a total of three to four mice per group. Chromatin immunoprecipitation and immunoblot. Chromatin-immunoprecipitation assays were done according to the manufacturer’s instructions (Upstate Biology). The presence of the target gene promoter sequences in both the input DNA and the recovered DNA immunocomplexes was detected by quantitative PCR. Data were normalized to those obtained with the corresponding DNA input control. The primer pairs for specific promoter regions were from –1,000 base pairs to approximately +500 base pairs of the transcription start site of the gene. Primer pairs are listed in Supplementary Methods. For immunoblot analysis, protein bands were visualized by enhanced chemiluminescence. In some experiments, blots were incubated with IRDye 800CW– conjugated secondary antibody (Rockland) and infrared fluorescence images were obtained with the Odyssey infrared imaging system (Li-Cor Bioscience). Proliferation assays and cytokine measurement. In proliferation assays, mouse splenocytes (5 105 per well) were cultured in triplicate in complete DMEM in 96-well flat-bottomed plates. Cells were cultured for 72 h in the presence or absence of MOG peptide (5 mg/ml). Cells were pulsed with 1 mCi [3H]thymidine during the final 16–18 h of culture before being collected. Incorporation of [3H]thymidine was measured as c.p.m. with a b-plate counter. For cytokine measurement, CD4+ T cells (2 105 cells/well) were stimulated for 24 h in 96-well plates with anti-CD3 and anti-CD28 (both from BD Biosciences) as described above (Cell purification, activation and culture). Supernatants were collected for the measurement of IL-2 production by enzyme-linked 7 ARTICLES immunoassay according to the manufacturer’s instructions (Pierce). A standard curve was generated with known amounts of purified recombinant mouse IL-2. Statistical analysis. Quantitative data are expressed as mean ± s.e.m. Statistical significance was determined by one-way analysis of variance followed by the Bonferroni post-hoc test for multiple comparisons or the two-tailed Student’s t-test. A P value of less than 0.05 was considered statistically significant. The linear regression method38 and two-way analysis of variance were used for EAE experiments. © 2007 Nature Publishing Group http://www.nature.com/natureimmunology Note: Supplementary information is available on the Nature Immunology website. ACKNOWLEDGMENTS We thank R.J. Lefkowitz (Duke University Medical Center) for rabbit polyclonal anti-b-arrestin (A1CT) and for Arrb1–/– and Arrb2–/– mice; Y. Shi, J. Cai and X. Liu for discussions; and S. Xin, Y. Li, G. Ding, P. Wu and S. Chen for technical assistance. Supported by the Ministry of Science and Technology (2003CB515405 and 2005CB522406), the National Natural Science Foundation of China (30021003, 30623003, 30625014, 30400230, 30430650 and 30571731), the Shanghai Municipal Commission for Science and Technology (06ZR14098, 04JC14040, 04DZ14902 and PJ200500330), the Shanghai Municipal Health Bureau (LJ06046), the Shanghai Leading Academic Discipline Project (T206) and the Chinese Academy of Sciences (KSCX2-YW-R-56). AUTHOR CONTRIBUTIONS Y.S. designed and did the experiments and prepared the manuscript; Y.F. and J.K. designed and did the experiments and analyzed the data; C.L. assisted with cell purification, activation and culture; Z.L., Z.G. and C.L. provided the blood samples from patients with multiple sclerosis; D.L. prepared the manuscript; W.C. and J.Q. assisted with the induction of EAE; E.B. and L.Z. assisted with cell purification, activation and culture; and J.Z.Z. and G.P. supervised all studies and the preparation of the manuscript. COMPETING INTERESTS STATEMENT The authors declare no competing financial interests. Published online at http://www.nature.com/natureimmunology/ Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions 1. Marrack, P. & Kappler, J. Control of T cell viability. Annu. Rev. Immunol. 22, 765–787 (2004). 2. Stockinger, B., Kassiotis, G. & Bourgeois, C. Homeostasis and T cell regulation. Curr. Opin. Immunol. 16, 775–779 (2004). 3. Strasser, A. & Pellegrini, M. T-lymphocyte death during shutdown of an immune response. Trends Immunol. 25, 610–615 (2004). 4. Bidere, N., Su, H.C. & Lenardo, M.J. Genetic disorders of programmed cell death in the immune system. Annu. Rev. Immunol. 24, 321–352 (2006). 5. Hughes, P., Bouillet, P. & Strasser, A. Role of Bim and other Bcl-2 family members in autoimmune and degenerative diseases. Curr. Dir. Autoimmun. 9, 74–94 (2006). 6. Marsden, V.S. & Strasser, A. Control of apoptosis in the immune system: Bcl-2, BH3-only proteins and more. Annu. Rev. Immunol. 21, 71–105 (2003). 7. Hengartner, M.O. The biochemistry of apoptosis. Nature 407, 770–776 (2000). 8. Strasser, A., O’Connor, L. & Dixit, V.M. Apoptosis signaling. Annu. Rev. Biochem. 69, 217–245 (2000). 9. Bouillet, P. et al. Proapoptotic Bcl-2 relative Bim required for certain apoptotic responses, leukocyte homeostasis, and to preclude autoimmunity. Science 286, 1735–1738 (1999). 10. Strasser, A., Harris, A.W. & Cory, S. Bcl-2 transgene inhibits T cell death and perturbs thymic self-censorship. Cell 67, 889–899 (1991). 11. Hildeman, D.A. et al. Activated T cell death in vivo mediated by proapoptotic Bcl-2 family member Bim. Immunity 16, 759–767 (2002). 12. Grayson, J.M., Zajac, A.J., Altman, J.D. & Ahmed, R. Cutting edge: increased expression of Bcl-2 in antigen-specific memory CD8+ T cells. J. Immunol. 164, 3950–3954 (2000). 13. Veis, D.J., Sorenson, C.M., Shutter, J.R. & Korsmeyer, S.J. Bcl-2-deficient mice demonstrate fulminant lymphoid apoptosis, polycystic kidneys, and hypopigmented hair. Cell 75, 229–240 (1993). 14. Strasser, A. et al. Enforced BCL2 expression in B-lymphoid cells prolongs antibody responses and elicits autoimmune disease. Proc. Natl. Acad. Sci. USA 88, 8661– 8665 (1991). 15. Dewire, S.M., Ahn, S., Lefkowitz, R.J. & Shenoy, S.K. b-Arrestins and cell signaling. Annu. Rev. Physiol. 69, 483–510 (2007). 16. Wilbanks, A.M. et al. Beta-arrestin 2 regulates zebrafish development through the hedgehog signaling pathway. Science 306, 2264–2267 (2004). 17. Chen, W. et al. Beta-arrestin 2 mediates endocytosis of type III TGF-beta receptor and down-regulation of its signaling. Science 301, 1394–1397 (2003). 8 18. Chen, W. et al. Activity-dependent internalization of smoothened mediated by b-arrestin 2 and GRK2. Science 306, 2257–2260 (2004). 19. Perry, S.J. & Lefkowitz, R.J. Arresting developments in heptahelical receptor signaling and regulation. Trends Cell Biol. 12, 130–138 (2002). 20. Luttrell, L.M. et al. Activation and targeting of extracellular signal-regulated kinases by b-arrestin scaffolds. Proc. Natl. Acad. Sci. USA 98, 2449–2454 (2001). 21. DeFea, K.A. et al. The proliferative and antiapoptotic effects of substance P are facilitated by formation of a b-arrestin-dependent scaffolding complex. Proc. Natl. Acad. Sci. USA 97, 11086–11091 (2000). 22. Kang, J. et al. A nuclear function of b-arrestin1 in GPCR signaling: regulation of histone acetylation and gene transcription. Cell 123, 833–847 (2005). 23. Ferguson, S.S., Barak, L.S., Zhang, J. & Caron, M.G. G-protein-coupled receptor regulation: role of G-protein-coupled receptor kinases and arrestins. Can. J. Physiol. Pharmacol. 74, 1095–1110 (1996). 24. Parruti, G. et al. Molecular analysis of human b-arrestin-1: cloning, tissue distribution, and regulation of expression. Identification of two isoforms generated by alternative splicing. J. Biol. Chem. 268, 9753–9761 (1993). 25. Walker, J.K. et al. Beta-arrestin-2 regulates the development of allergic asthma. J. Clin. Invest. 112, 566–574 (2003). 26. Wang, Y. et al. Association of b-arrestin and TRAF6 negatively regulates Toll-like receptor–interleukin 1 receptor signaling. Nat. Immunol. 7, 139–147 (2006). 27. DiSanto, J.P., Guy-Grand, D., Fisher, A. & Tarakhovsky, A. Critical role for the common cytokine receptor g chain in intrathymic and peripheral T cell selection. J. Exp. Med. 183, 1111–1118 (1996). 28. Nakajima, H., Shores, E.W., Noguchi, M. & Leonard, W.J. The common cytokine receptor g chain plays an essential role in regulating lymphoid homeostasis. J. Exp. Med. 185, 189–195 (1997). 29. Ben-Sasson, S.Z., Makedonski, K., Hu-Li, J. & Paul, W.E. Survival and cytokine polarization of naive CD4+ T cells in vitro is largely dependent on exogenous cytokines. Eur. J. Immunol. 30, 1308–1317 (2000). 30. Kane, L.P. & Weiss, A. The PI-3 kinase/Akt pathway and T cell activation: pleiotropic pathways downstream of PIP3. Immunol. Rev. 192, 7–20 (2003). 31. Gendron, S., Couture, J. & Aoudjit, F. Integrin a2 b1 inhibits Fas-mediated apoptosis in T lymphocytes by protein phosphatase 2A-dependent activation of the MAPK/ERK pathway. J. Biol. Chem. 278, 48633–48643 (2003). 32. Scott, M.G. et al. Differential nucleocytoplasmic shuttling of b-arrestins. Characterization of a leucine-rich nuclear export signal in b-arrestin2. J. Biol. Chem. 277, 37693–37701 (2002). 33. Wang, P., Wu, Y., Ge, X., Ma, L. & Pei, G. Subcellular localization of b-arrestins is determined by their intact N domain and the nuclear export signal at the C terminus. J. Biol. Chem. 278, 11648–11653 (2003). 34. Jambal, P. et al. Cytokine-mediated down-regulation of the transcription factor cAMPresponse element-binding protein in pancreatic b-cells. J. Biol. Chem. 278, 23055–23065 (2003). 35. Tamatani, M. et al. Tumor necrosis factor induces Bcl-2 and Bcl-x expression through NFkB activation in primary hippocampal neurons. J. Biol. Chem. 274, 8531–8538 (1999). 36. Ogryzko, V.V., Schiltz, R.L., Russanova, V., Howard, B.H. & Nakatani, Y. The transcriptional coactivators p300 and CBP are histone acetyltransferases. Cell 87, 953–959 (1996). 37. Sospedra, M. & Martin, R. Immunology of multiple sclerosis. Annu. Rev. Immunol. 23, 683–747 (2005). 38. Bettelli, E. et al. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 441, 235–238 (2006). 39. Vroon, A., Lombardi, M.S., Kavelaars, A. & Heijnen, C.J. Changes in the G-proteincoupled receptor desensitization machinery during relapsing-progressive experimental allergic encephalomyelitis. J. Neuroimmunol. 137, 79–86 (2003). 40. Zang, Y.C., Hong, J., Rivera, V.M., Killian, J. & Zhang, J.Z. Human anti-idiotypic T cells induced by TCR peptides corresponding to a common CDR3 sequence motif in myelin basic protein-reactive T cells. Int. Immunol. 15, 1073–1080 (2003). 41. Oakley, R.H., Laporte, S.A., Holt, J.A., Caron, M.G. & Barak, L.S. Differential affinities of visual arrestin, b arrestin1, and b arrestin2 for G protein-coupled receptors delineate two major classes of receptors. J. Biol. Chem. 275, 17201–17210 (2000). 42. Nguyen, K. & Miller, B.C. CD28 costimulation induces delta opioid receptor expression during anti-CD3 activation of T cells. J. Immunol. 168, 4440–4445 (2002). 43. Zhang, J. & Hutton, G. Role of magnetic resonance imaging and immunotherapy in treating multiple sclerosis. Annu. Rev. Med. 56, 273–302 (2005). 44. van Oosten, B.W. et al. Treatment of multiple sclerosis with the monoclonal anti-CD4 antibody cM-T412: results of a randomized, double-blind, placebo-controlled, MR-monitored phase II trial. Neurology 49, 351–357 (1997). 45. Rep, M.H. et al. Treatment with depleting CD4 monoclonal antibody results in a preferential loss of circulating naive T cells but does not affect IFN-g secreting TH1 cells in humans. J. Clin. Invest. 99, 2225–2231 (1997). 46. Wang, P. et al. Beta-arrestin 2 functions as a G-protein-coupled receptor-activated regulator of oncoprotein Mdm2. J. Biol. Chem. 278, 6363–6370 (2003). 47. Sun, Y., Cheng, Z., Ma, L. & Pei, G. Beta-arrestin2 is critically involved in CXCR4mediated chemotaxis, and this is mediated by its enhancement of p38 MAPK activation. J. Biol. Chem. 277, 49212–49219 (2002). 48. Ota, K. et al. T-cell recognition of an immunodominant myelin basic protein epitope in multiple sclerosis. Nature 346, 183–187 (1990). 49. Zappia, E. et al. Mesenchymal stem cells ameliorate experimental autoimmune encephalomyelitis inducing T-cell anergy. Blood 106, 1755–1761 (2005). ADVANCE ONLINE PUBLICATION NATURE IMMUNOLOGY