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From Basic Discoveries to Medical Innovation

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Editors: Tianna Hicklin, Ph.D. and Sean Sanders, Ph.D.; Proofing: Bob French; Design: Tom Wight, MidAtlantic Publishing Services. Cover image provided courtesy of the Laboratory for Mucosal Immunity, RCAI RIKEN. This image has been digitally altered from original version for publication purposes. © 2012 by The American Association for the Advancement of Science. All rights reserved. September 26, 2012.

scientists are still searching for a deeper understanding of the intricacies of the immune system and the relationships between host and pathogen. and bacteria. draw from one another’s expertise. this defense mechanism can also turn against itself. and colleagues—while also having a global impact. Tianna Hicklin. and Sean Sanders. and drive immunology forward to find new ways of improving human health. We are encouraged to see institutions such as RCAI look toward the future and bring together top-notch researchers from around the world to interpret important new findings in immunology.D. RCAI has published numerous research papers that have significantly advanced our knowledge in different areas of immunology. Many challenges lie ahead for immunologists as the field becomes increasingly multidisciplinary and as advancing life-science technologies enable larger and larger data sets to be collected and analyzed. Despite rapid progression in the field of immunology over the past few decades. One institution that has focused on unraveling some of the remaining questions in immunology is the RIKEN Research Center for Allergy and Immunology (RCAI). However. causing a range of autoimmune diseases. Ph. immune system homeostasis. parasites. In this booklet. It’s an issue that affects each one of us personally—including our friends. the protective role of probiotics. Our most effective defense against these microscopic invaders is our immune system. and the role of immune cells in cancer and autoimmune diseases.D. Science/AAAS Custom Publishing Office 2 Credit: © istockphoto. we invite you to read a sampling of RCAI publications that have shed light on topics such as immune cell development and differentiation. We are constantly bombarded by environmental insults that can affect our health. Editors. New discoveries hold the potential for uncovering novel strategies to strengthen our immunological defense mechanisms and prevent their malfunctioning. from the relatively innocuous irritants that cause allergies to potentially deadly viruses. family. Since . Ph.DEEPENING OUR UNDERSTANDING IN IMMUNOLOGY Human health is undeniably a topic of intense research focus today.

RCAI’s immunology research is highly ranked. and Science Translational Medicine. and clinical trials investigating a natural killer T (NKT) cell therapy for cancer. connect scientists around the world. with a high citation index score. Masaru Taniguchi. Ph.. M. cell signaling. the number of citations per paper has been consistently above 40.D.D. Director RIKEN Research Center for Allergy and Immunology 3 . tumor immunity. and is important for preventing a variety of diseases. MIWI will serve as a worldwide network for the next generation of human immunology research by virtue of its focus on integrative medical immunology. genetic and epigenetic regulation. RCAI scientists have made many discoveries in basic immunological research using in vitro and ex vivo techniques and animal models. This Science reprint collection booklet summarizes some of RCAI’s major research findings over the last decade and introduces a selection of RCAI papers published in Science. The research conducted during RCAI’s first 11 years has opened the door to new directions in human immunology. Credit: Photo courtesy of RIKEN RCAI. Building on the groundwork laid by RCAI. moreover. and compares favorably with other premier immunology institutions around the world. including the development of “humanized” mice. In 2011.THE RIKEN RESEARCH CENTER FOR ALLERGY AND IMMUNOLOGY CELEBRATES ITS FIRST DECADE The RIKEN Research Center for Allergy and Immunology (RCAI) has been one of Japan’s premier strategic natural science research institutes since its establishment in 2001. a new human immunology consortium of laboratories that uses humanized mice. including molecular imaging. RCAI researchers have had a consistently strong track record. RIKEN has decided to establish a new research center for integrative medical science that will launch in April 2013. more than 30 percent of our papers published yearly are found in journals with an impact factor greater than 10. RCAI launched the Medical Immunology World Initiative (MIWI). Science Signaling. Since the center’s inception. immune system development. and the development of new therapies and diagnostic tools. Moreover. and the current endeavors of RCAI investigators continue to spearhead global immunology research. The immune system is critical for maintaining homeostasis in the body. preclinical work on a cedar pollen allergy vaccine. Two key principles have guided the philosophy and direction of RCAI research in its first decade: 1) creating new paradigms in basic immunology and 2) bridging basic immunology findings and medical innovation. RCAI scientists explore many areas of immunology. is a key target for developing therapeutics. mucosal immunity. and improve human well-being. continuously publishing papers in distinguished scientific journals. RCAI has focused on developing ways to relate basic research findings to understanding the human immune system. In its second decade. our researchers have also seen significant progress with translational research studies.

inventions. research on the early immune system is finding that effects that are prenatal or even earlier can influence immune system development. you lose homeostasis. “We have an immune system to fight infections. and the need for individualized and effective immunotherapies. pollutants such as cigarette smoke. Investigating Earlier and Broader Influences In fact. For instance in mice. An explanation for this global pattern. coordinating interactions between the external world of pathogens. and allergens. Early exposure to the normal commensal microorganisms of mice resulted in normal NKT cell levels. the products of immunology research have transformed basic science and clinical practice. the complex interaction between genes and environment. Richard Blumberg. chair of the Department of Immunology. or clinical therapies. receptors and signaling. Immunology continues to be a dynamic field of research that provides tools and inspiration to clinical and basic research. and infectious agents such as bacteria can influence immune development. which are heritable changes in the accessibility of DNA to transcription factors through modifications to nucleotides or histone proteins. leading the researchers to conclude that “age-sensitive contact with commensal microbes” established appropriate microbial tolerance. allergy. Philipps University. This model of interdependency underlies current immunology research. whether the topic is early immune development. This effect was transmitted to a second generation. A hyperhygienic environment in early life inhibits the development of a proper balance between the actions of T effector and T regulatory cells. Marburg. University of Washington. Department of Clinical Chemistry and Molecular Diagnostics.THE INTERCONNECTION. mouse models show that maternal and even grandmaternal exposures to nutrients. In fact. and our internal physiology from organs to genes. high throughput techniques and a variety of in vivo models to address these challenges. and not if it occurred in adulthood. certain concentrations of the methyl donor folate in the maternal diet resulted in epigenetic effects to genes linked to allergic airway disease. Today. Normalization of the immune system occurred only when microbial exposure was early in life. and inflammation-related diseases. A great deal of current immunology research is driven by the global rise in rates of autoimmune disorders. and was associated with epigenetic effects on cytokine genes such as interferongamma and interleukin-4. commensal microorganisms. Immunology research in the 21st century is discovering that the innate and adaptive immune systems are more intricately connected than previously thought. From antibodies to vaccines. 4 . immunologists are addressing major medical and scientific challenges such as the global rise in inflammatory disease. Immunology researchers are using powerful.” says Joan M. Renz’s group found that prenatal exposure to the soil bacterium Acinetobacter lwoffi protected against asthma in a mouse model. gene-environment interactions. For example. The next steps in immunology research are identifying the specific agents. better-functioning adult immune system. In spring 2012. chief of the Division of Gastroenterology. professor of Laboratory Medicine. I mmunology has a rich history of groundbreaking discoveries. and the timing and type of exposure that give protective effects against later immune dysregulation. INTERDEPENDENCE. the entire immune system is now viewed as a complex communication hub. autoimmunity. These transgenerational influences are explained by epigenetic effects. and allergy. from Edward Jenner’s successful smallpox vaccinations in the 18th century to Susumu Tonegawa’s Nobel Prize-winning explanation of antibody gene recombination 200 years later. and if you don’t test the immune system with exposure to pathogens. Germany. is the hygiene hypothesis—that early exposure to a variety of antigens builds a stronger. experimental findings using an animal model provided support for the hygiene hypothesis. says Harald Renz. resulting in immune dysregulation. Researchers are also looking for the targets of early environmental exposure. Goverman. Hepatology and Endoscopy of Brigham and Women’s Hospital in Boston and colleagues found that germ-free mice are more susceptible to inflammatory conditions resembling colitis and asthma and traced this susceptibility to increased invariant natural killer T cells (NKT). AND INFLUENCE OF THE IMMUNE SYSTEM Immunology is a field of discoveries. and breakthroughs. Seattle.

Discovery of the immune functions of TLRs was awarded the 2011 Nobel Prize for Physiology or Medicine. particular compositions of the microbiome. microRNA regulation. Activation of TLRs by ligand binding initiates kinasedependent signal transduction cascades that lead to gene expression responses mediated by transcription factors such as NFKB. and the amount of microbiome diversity.such as stem and progenitor immune system cells. schizophrenia. T cell receptors. and neuroendocrine interactions. and current research is investigating both sides of this communication. gut. so posttranscriptional and posttranslational modifications have a bigger impact in creating different phenotypes. future research should also focus on other receptors connected to inflammation such as tyrosine kinase-associated receptors and other RNA. the main method for this type of research is sampling and characterizing the human microbiome. and other immune system elements determine which cells to attack and which to tolerate. but Alam advises more attention to downstream gene expression effects such as alternative splicing. Self and Nonself Distinguishing between pathogens and commensals and self and nonself is a fundamental function of the immune system. with the help of adaptor proteins. This will determine which microbes are our friends and which are our foes—the basic task of the immune system. particularly the toll-like receptors (TLRs). and secretion of IgA. multiple sclerosis. differentiation of helper T cells. These transmembrane and intracellular proteins. Nucleic acid-sensing receptors are particularly important in understanding autoimmune disease since many of these are an inappropriate reaction to RNA or DNA. These microbes have a strong impact early in life beyond the risk of allergy to risk of conditions such as type I diabetes. including microbes that normally colonize the mucosa of the respiratory system. This is fertile ground for research. In mice. “This is a field to watch. obesity and metabolic syndrome. bind to foreign molecules such as bacterial lipopolysaccharides or viral RNAs and signal pathogen invasion.” Research on commensal gut microbes has found that colonization of the gastrointestinal tract. psychological factors such as stress. Analysis is underway to discover associations between the presence of specific microbes. host disease. In humans.” he says. longitudinal changes in the human microbiome. and protein modification. and depression. “A big area is microbial exposure. he says. and skin. National Institutes of Health Human Microbiome Project. Says Alam. “It’s simple logic. Much research on the response to receptor signaling has focused on changes in gene transcription. When this functionality goes awry. Although we have a lot to learn from TLRs. for example by high throughput pyrosequencing in the U. Professor Alam says that a current area of research is investigating just how the TLR pathways lead to the activation of inflammation. head of allergy and immunology at National Jewish Health and professor of medicine at University of Colorado Denver.and DNA-sensing receptors. major histocompatibility complexes.” says Renz. Substantial research with the goal of modulating the immune system has elucidated many of the mechanisms by which antibodies. begins immediately after birth.” says Rafeul Alam. and conditions ranging from obesity to autoimmune disease. Active research areas include studying how immune development and maintenance are affected by specific species and strains of microbes. This is a symbiotic relationship that regulates growth of commensal bacteria that aid in digestion and produce vitamins and other nutrients for the host. neurodegenerative disease. a topic that is particularly urgent given the global health burden of chronic inflammatory conditions such as diabetes and inflammatory bowel disease. Crosstalk clearly occurs between the microbiota and the immune system.” 5 . Alam says we need additional research on how pathogen signals reach and trigger intracellular TLRs such as TLR7 and TLR9. They are finding that factors affecting immune development include “food and dietary habits. the result can be autoimmunity. development of T regulatory cells. “Higher mammals have fewer genes in their genomes than other organisms. “Work on epigenetics so far has just scratched the surface. intestinal colonization promotes development of immune responses including production of antimicrobial peptides.S. Recent attention has focused on pattern recognition receptors. mainly by species in the phyla Firmicutes and Bacteroidetes. Even a correctly functioning immune system can cause transplant rejection or graft vs.” Immunology researchers are also broadening their definition of the environment.

” For example. An integrated. senior vice president of strategic development. The success of Herceptin and similar therapies has launched dozens of biotechnology companies and clinical trials. Goverman. but the clinical proof-of-concept has motivated additional cell-based therapy development. in 2010. isolating. cell-free vaccines and other treatments based on tumor-associated antigenic peptides identified from a patient’s resected tumor.” Examples are highly specific. Future therapies. Stallergenes. “will simultaneously address the immunosuppressive. soluble factors.Directing the Immune System for Therapy As our understanding of the internal and external communications and responses of the immune system grows.S. The therapy stimulates the patient’s own peripheral blood mononuclear cells to launch a specific attack against cancerous cells. Murphy.” she says. and board member of the Danish Graduate School of Immunology. Murphy says. angiogenic. Poul Sørensen. or marking cellular components. This has the potential to lead to treatment breakthroughs for other diseases. . This cellular flexibility is a challenge in large-scale cell culturing. Loyola University Health System in Maywood. long-lasting responses. Köhler shared the 1984 Nobel Prize in Physiology or Medicine revolutionized basic research by providing tools for precisely identifying. On the cell-based side. oral immunotherapy is a major trend.” Other therapies are also developing in multiple directions. the method which Dendreon must use in their Provenge therapy. says. F. invasive. Paris. and pollen allergy. Niels Kaj Jerne. a monoclonal antibody therapy used to treat malignant B-cell lymphomas has shown some promise with multiple sclerosis and diabetes. Learning to modulate immune system communications in this situation will have clinical implications beyond infectious disease. for example treatment for specific allergies. says these therapies are promising. and Georges J. for which César Milstein. the Seattle company Dendreon received U. Rachna Shah.” 6 Cancer immunotherapy is “coming of age. says Shah. says that current cancer immunotherapy is based on research showing that cancerous cells create an immune microenvironment that suppresses antitumor activity. “Both patients and providers are interested in seeing if these treatments can make a difference in symptoms. who studies autoimmune diseases such as multiple sclerosis. peanut. However. with recent trials showing promise in sublingual therapy for egg. immune cell plasticity also explains immunotherapy successes. but the double-edged sword in any type of cell-based immunotherapy is the inherent plasticity of immune cells. Learning about its mechanism of action has also informed multiple sclerosis research. citing more than 40 different immunotherapies currently in testing in over 60 clinical trials. Illinois notes that studies on complementary and alternative therapies such as traditional Chinese medicine to treat allergies are becoming more common. Development of monoclonal antibodies. and signaling pathways designed to break tolerance and reactivate antitumor immunity to induce potent. Southern Research Institute. “Cancer therapy is driving the development of therapy for autoimmune diseases and other immune disorders. Commercialization of Provenge remains rocky. director of Cancer Therapeutics and Immunology. Monoclonal antibody technology has led to highly specific therapies such as the breast cancer treatment trastuzumab (Herceptin). We are learning how easily immune cells change function. multipronged immune response is required to eliminate cancer. Birmingham. “We’re learning we can reprogram the immune system with lasting effects. Influenza and other viral infections can induce a cytokine storm in which strong production of complement factors and proinflammatory cytokines brings excessive numbers of inflammatory cells to the lungs.” says Joseph Murphy. sipuleucelT (Provenge). Alabama. and hypoxic nature of cancer. so combinatorial approaches are likely to be the most effective. he says. who wrote an extensive 2010 review of cancer immunotherapy. Says Sørensen. He says that new cancer therapies will be “based on a sophisticated knowledge of immune-suppressive cells. In conventional therapy. the list of potential therapeutic targets and technologies expands. for example converting from anti-inflammatory to proinflammatory. Research on the pathogenesis of infectious diseases such as influenza and SARS could contribute to the development of immunomodulating therapies. ultimately impairing lung function. an allergist with Gottlieb Memorial Hospital. Food and Drug Administration approval for their prostate cancer treatment.

Credit: © 7 .

Hiroshi Ohno and his colleagues discovered that a transcription factor. Takashi Saito observed the dynamic movement of signaling molecules during T cell activation and discovered that activation is initiated by small complexes called “T cell receptor (TCR) microclusters. when they found that not only Ca2+. they identified two types of Zn2+ signaling: early. Here. and late. 3) Mucosal Immunity The adaptive immune system has two separate but interrelated components: systemic and mucosal. maintenance. Creating New Paradigms 1) Immune System Development As the immune system develops. 16). Much of classical immunology focuses on the systemic compartment. the progeny of multipotent hematopoietic stem cells (HSCs) become increasingly restricted in their lineage potential and acquire specialized functions. The group could also visualize T cell activation modulated by costimulatory molecules. They found that the Runx complex represses the expression of the transcription factor Th-POK during thymocyte differentiation. however. They determined that Zn2+ signaling plays a role in cell migration. allowing the development of CD8+ cytotoxic T cells rather than CD4+ helper cells (see full paper on page 19) (3. the center has also developed programs with a translational component. but also zinc (Zn2+) ions can act as signaling molecules. Although basic research has always been a focus and strength of RCAI. 8). The “lymph-node equivalent” in the intestine are Peyer’s patches (PPs). 2) Immune Cell Signaling Immune cells use receptor-mediated signaling at multiple stages in their development/differentiation to direct lineage choices and acquisition of effector functions. 18). which are separated from the intestinal contents by a single layer of specialized epithelial cells. Further. These findings have provided key insights into how the spatiotemporal patterns of signaling events affect the initiation. During the 11-year directorship of Masaru Taniguchi. and connective tissue formation (9–12). Visualization of signaling events at the single-molecule level is now possible using highly sensitive fluorescence microscopes developed by RCAI’s Makio Tokunaga (14). Tomohiro Kurosaki’s group has dissected the molecular events in B cell signaling. but the mechanisms by which M cells differentiate and carry out this function have not been fully elucidated. His group later discovered that the transcription factor Bcl11b determines the T cell lineage (see full paper on page 15) (2). a sampling of the pioneering studies and programs that have contributed to RCAI’s mission are briefly described. dendritic cell activation. 6). They identified Stim1/Stim2 as critical for calcium (Ca2+) influx. and the Ser/Thr kinase extracellular signal-regulated kinase (ERK) as essential for early B cell development and plasma cell differentiation (see abstract on page 23) (7.HIGHLIGHTS OF RCAI RESEARCH AND PROGRAMS: 2001–2012 Since its establishment in 2001. Changes in expression levels or posttranslational modification—such as phosphorylation—of signaling molecules propagate signals within immune cells. a key event in many signaling pathways (5. protection at mucosal surfaces is also essential for survival. Scientists have long believed that lymphoid lineage precursors lack the ability to mature into myeloid lineage cells. Spi-B. A surprising discovery was made by Toshio Hirano and his RCAI team. which is induced by the expression of Zn2+ transporters (13). the Research Center for Allergy and Immunology (RCAI) has been at the cutting edge of immunology research and has focused on two goals: creating new paradigms in basic immunology and developing medical innovations. Hiroshi Kawamoto’s research showing that early thymic progenitors can give rise to myeloid cells has called this dogma into question (1). 4). is essential for M-cell differ- 8 .” in which TCRs and various signaling proteins are assembled (15. and regulation of T cell activation (17. RCAI researchers have made a big impact on understanding how the immune system works.” similar to that observed during Ca2+ influx. Ichiro Taniuchi’s group provided further insight into what drives T cell differentiation. however. which involves a “Zn2+ wave. among which are antigen-sampling M cells that deliver antigen to PPs to initiate mucosal immune responses. allergic reactions.

Ishikawa also identified a human leukemia stem cell (LSC) by transplanting human acute myeloid leukemia (AML) cells into NSG mice. but induce distinct cell fates. His team could observe. EGF induces cell division. and quantify the in vivo migration of Bcl6-expressing antigen-specific B cells to the germinal center. this group created an improved recipient mouse (hSCF Tg NSG) that expresses human stem cell factor (SCF). but are typically in a dormant state and therefore resistant to cell cycle-dependent chemotherapy (29–31). coli O157 from entering the bloodstream. which results from IgA with reduced bacteria-binding capacity. bacteria from the genus Bifidobacterium produced acetate that prevented Shiga toxin generated by enterohemorrhagic E. thereby protecting the mice (21). they demonstrated that environmental cues in the gut promote the selective differentiation of IgA-supporting T follicular B helper (TFH cells) from Foxp3+ T cells (see abstract on page 25) (23). Epidermal Growth Factor (EGF) and Heregulin (HRG) both share extracellular signal-regulated kinase (ERK) signaling pathways. In collaboration with Shohei Hori. in collaboration with Chiba University. in collaboration with Leonard Shultz (Jackson Laboratory). Fagarasan’s group also recently found that skewing of the microbiota. The intestinal immune system has intimate and important interactions with the bacterial flora in the gut. who utilized two-photon microscopy to track B cells by moni- toring the expression of Bcl6 with a surrogate fluorescent marker. while HRG induces differentiation. When newborn mice were transplanted with human hematopoietic stem cells (HSCs). and Osamu Ohara. RIKEN RCAI Developing Innovations in Biomedical Sciences 1) Humanized Mice RCAI scientists are working to develop innovative experimental models for investigating the human immune system and disease. which fuses biology and mathematics. a group led by Sidonia Fagarasan investigated the role of immunoglobulin A (IgA) in maintaining the symbiotic balance between gut microbiota and the host immune system (22). New imaging technologies have the potential to become important tools in systems biology since the information obtained can be digitized and quantified. track. RCAI researchers Fumihiko Ishikawa. Haruhiko Koseki. Using integrated multi-“omics” approaches. 2) Natural Killer T Cell Therapy A clinical trial of a natural killer T (NKT) cell targeted therapy for patients with advanced non-small-cell lung cancer is being conducted by RCAI’s Masaru Taniguchi. —Takashi Saito. a process dependent upon interaction with Bcl6-expressing TFH cells (26). One example is systems immunology. The LSCs propagate the leukemia. Ohno’s team studied the “probiotic” effect of these interactions. RCAI’s Mariko Okada. respectively. In this trial.entiation (19) and that glycoprotein-2 (GP-2) serves as a bacterial uptake receptor (20). in collaboration with Boris Kholodenko (University College Dublin). a cytokine that is critical for the maintenance of stem and progenitor cell activities (28). Sixty percent of treated patients (10/17) showed a significantly longer median survival compared to the 9 . autologous dendritic cells pulsed with the NKT cell-activating ligand α-galactosylceramide were administered intravenously. They discovered that in a mouse model. A step in this direction was taken by Takaharu Okada. drives the generalized activation of B and T cells in mice that develop B cell-dependent autoimmune disease (see full paper on page 11) (24). The group found that EGF and HRG induce either transient or sustained cytoplasmic ERK activity. 4) Systems Immunology RCAI is actively creating new immunology fields that are integrated with other scientific fields as well as new technologies. human cytotoxic T cells successfully developed and became functional in these “humanized” mice (27). has used mathematical modeling to tackle the question of how cell fate is determined. More recently. Moreover. His group is currently identifying genes differentially expressed between normal HSCs and LSCs to identify potentially new therapeutic targets for AML (see abstract on page 24). and that discrimination of these signals controls cell fate (25). Deputy Director. have generated immunodeficient mice (NOD/SCID/IL2rgKO (NSG)) that express a human class I antigen.

The number of patient samples deposited in the Japanese clinical archive. 17ra9 (2010). F. expanded trials of the NKT cell therapy are planned. 589 (2008). 971 (2006). Med. 6. 3) Primary Immunodeficiency Clinical Archive To promote awareness and improve treatment options for Primary Immunodeficiency (PID). M.. Yokosuka et al. rcai.. ra25 (2011). 159 (2008). S. Signal. 226 (2009). T. 2. Kanaya et al... Sci. M. and this treatment is now offered as a new therapeutic option under the advanced medical care assessment system approved by the Japanese Ministry of Health. immune regeneration. 3. 13022 (2010). —Shigeo Koyasu. M. 822 (2008). Baba et al. 28. Imamoto. D. Exp. Cell 141. M. Kawamoto et al. 12. N. RCAI is also working toward establishing an Asian PID network and has established an integrated bioinformatics platform for PID called RAPID (rapid. “PIDJ. T. Hase et al. 13. mucosal flora and epigenetic analysis. Sci. 9. 768 (2008).” which was established by RCAI. 23. Nature Biotechnol. the U.S..rcai. 543 (2011).S. this treatment will be covered in part by national health insurance (32). J. 209. K. Fagarasan et al. e3642 (2008). RCAI established an interdisciplinary biomedical research platform and international consortium of research groups that share a common interest in human immunology. Setoguchi et al.. 2768 (2012)... Exp. T. 21. Immunity 34. Yokosuka et al. T. Five researchers have been selected thus far to conduct pioneering work in the following new areas: stem cells and aging reversal.. Hashimoto-Tane et al. 107. 7. Saito et al. S..3 versus 4. Based on these promising results. Nature Methods 5. Immunol. 919 (2011). H. Science 329. T. Matsumoto et al. PLoS ONE. 16. Science Translational Medicine 2. 20. 1113 (2008). Yokosuka et al.. Nature Immunol. 884 (2010). 3. S. 1424 (2002).. 1488 (2009). 149 (2008). 32. 1201 (2012). Science and a PID mutation annotation server. Hirano et al. K. 206. Kitamura et al. 2) Young Chief Investigator Program (YCI) RCAI launched the new Young Chief Investigator (YCI) Program to provide a career path for young investigators (under 40 years old) conducting multidisciplinary research that bridges immunology with other fields. 6. Yasuda et al.. Y. 18. and RCAI. RIKEN RCAI References 1. 29. Ikawa et al. supported in part by the Jeffrey Modell Foundation. S. S. 13. 14. Nature 429. Yamashita et al.. RIKEN RCAI MIWI is to use integrative immunological approaches to obtain fundamental knowledge about the human immune system and the underlying mechanisms of disease development and to discover new principles for diagnosis and treatment. H. J. 26.6 months).. 22.. Nature 462. 30. the Institute of Bioinformatics in India. 93 (2010). 17. 729 (2012). Adv. Mutation@A Glance (rapid. T. Immunity 29. two departments from Zurich University. Wada et al. 5. the Pasteur Institute. 2492 (2009). Nature 469. Fukada et al.HIGHLIGHTS OF RCAI RESEARCH AND PROGRAMS: 2001–2012 untreated group (29.. Imperial College London.riken.. Nature Immunol. 31.. T. the Institute of Medical Science. 25. Proc. Research Coordinator. 1315 (2007). 24. U. A YCI will head an independent laboratory but will have access to mentors who are specialists in related fields.. Fukuda et al. Nature Immunol. S. 275 (2010). the University of Tokyo (IMSUT).A. Yasuda et al. T. For this purpose. Shultz et al. Immunity 34. and multi system-wide analysis. A. Deputy Director. The goal of 10 . 9.. 9. R. 15. Med. Saito et al.. Sakata-Sogawa. T. 10.. Science 298. RCAI has been expanding its collaborative network with the clinical study group from 13 Japanese universities. Immunol. Y.. 8. and the Kazusa DNA Research Institute. T. 1351 (2009). Nature Immunol. Immunity 28. Motohashi et al. Takagi et al.. Nakakuki et al. Acad. 485 (2012). Muroi et al. K. continues to increase each year. 97. L. Nature Immunol. S. 28. development of the interface between integrative biology and mathematics. 961 (2011). 11. Tokunaga. 499 (2008). Nine institutions are currently affiliated with MIWI: the Immunology Frontier Research Center of Osaka University (IFReC). Natl. 4. —Toshitada Takemori. Thus. 25. Labor and Welfare.. National Institutes of Health (NIH).. 1253 (2005). Nishida et al.. Toward the Future 1) Medical Immunology World Initiative (MIWI) To understand the process of disease development and to identify critical events that change human health. Science 336. Immunity 34.. Tsuji et al. Blood 119. 4. Y. Science 319. 7. 182. Nature Biotechnol. 703 (2011). J. 81 (2008).. Kitano et al. 298 (2004).riken. INSERM/Necker Hospital. 27. RCAI launched the zMedical Immunology World Initiative (MIWI) as a new human immunology platform that includes humanized mice. Nature 452. Ishikawa et al.

1D). about 90% of the sequences from WT and Pdcd1–/– mice had mutations and high ratios of replacement (R) to silent (S) mutations in complementarity-determining region 1 (CDR1) and CDR2.8 × 108 and 4. the calculated affinity maturation index (11. 2Department of Biochemistry. However. The altered repertoire of the IgA plasma cells in gut could result from changes in the turnover of IgAs at their inductive or residential places. Pdcd1–/– mice had significantly more peanut agglutinin–positive (PNA+) Fas+ GC B cells and IgA+ B cells in PPs (Fig. Together. but this diminished after antibiotic treatment (Fig. In contrast. quantitative real-time fluorescence polymerase chain reaction analyses of several important B cell markers for dark-zone and light-zone GC B cells did not show significant differences between Pdcd1–/– mice and WT mice (fig. the frequency of BrdU-labeled PCs did not differ significantly between Pdcd1–/– and WT mice (Fig. The total numbers of “healthy” bacteria. S1F). Namely. Japan. We next determined the turnover of IgAproducing cells in LP based on 5-bromo-2′deoxyuridine (BrdU) incorporation. 3 AK project. The pulse-chase BrdU experiment revealed considerable differences in dynamics of IgA GCs in Pdcd1–/– mice. Both WT and Pdcd1–/– mice had a diverse IgA repertoire. Vietnam. the results indicate that alterations of IgA compartment in Pdcd1–/– mice have an impact on symbiotic relations between host and commensal bacteria in the gut. which results in dysregulated selection of IgA precursor cells in the germinal center of Peyer’s patches. PD-1 plays a critical role in regulation of antibody diversification required for the maintenance of intact mucosal barrier. E-mail: sidonia-f@rcai. Together. 15). hereafter. However. Japan. in which B cell interaction with T follicular helper (TFH) cells induces the expression of activationinduced cytidine deaminase (AID) (2.2* Mikako Maruya. PD-1 deficiency leads to roides (9) were not detectable or markedly reduced in Pdcd1–/– mice. D and E). Tsurumi. S2B). 2D). 1F). We investigated whether PD-1 regulates microbial communities and IgA production in the gut. the concentration of free IgA in intestinal secretions was higher in Pdcd1–/– than in WT mice (Fig. These results were confirmed by 16S ribosomal RNA (rRNA) gene pyrosequencing of cecal contents (fig. RIKEN Yokohama 1-7-22. 1F). respectively) (Fig. such as Bifidobacterium and Bacte- T 1 Laboratory for Mucosal Immunity. Most of the IgA+ B cells are generated in the GCs of Peyer ’s patches (PPs) and arrive at the LP as plasmablasts [B220 – IgA+ MHCII + (major histocompatibility class II–positive). 2.1* Keiichiro Suzuki. A comparable proportion of IgA+ GC cells from Pdcd1–/– and WT mice were in cell cycle. we sequenced the immunoglobulin heavy chain (IgH) genes in single sorted IgA-producing cells.1* Thinh H. The diversification of IgA repertoire by somatic hypermutation (SHM). Five days after the removal of BrdU. This difference in the IgA repertoire may be the result of the altered composition of the microflora in Pdcd1–/– mice. 1B). whereas the frequency of BrdU+ PBs in WT mice remained unchanged (Fig. Yoshida Sakyo-ku. were increased about 400-fold in Pdcd1–/– mice.18 × 108 in WT and Pdcd1–/– mice. Compared with WT mice. Flow cytometric analyses of fecal bacteria revealed. PCs) (fig. in both Pdcd1–/– and WT mice. Hanoi.The Inhibitory Receptor PD-1 Regulates IgA Selection and Bacterial Composition in the Gut Shimpei Kawamoto. PBs down-regulate the B cell receptor and MHCII and differentiate into plasma cells (B220– IgA+/lowMHCIIlowCD138+. yet Pdcd1–/– mice had an enrichment of IgA-producing cells with the IgH locus belonging to non-VH1 family genes (Fig.4 Sidonia Fagarasan1† Immunoglobulin A (IgA) is essential to maintain the symbiotic balance between gut bacterial communities and the host immune system. bacteria of the Enterobacteriaceae family.1.1. 1C). Intestinal IgA production occurs via both T helper cell–dependent and independent pathways (1). respectively. as signs of antigen-mediated selection in their IgH genes. In contrast. Pdcd1–/– mice had a 93 to 95% reduction in the number of anaerobic bacteria compared with WT mice (average 2. the IgAs produced in PD-1–deficient mice have reduced bacteria-binding capacity. 1E). S1G). The proliferation of IgA-producing cells in LP was assessed using fluorescent ubiquitination-based cell-cycle indicator (Fucci) mice. Indeed. Kyoto 6068501. Kyoto University. most of the proliferating cells were PBs (13. 4Department of Immunology and Genomic Medicine. Next. respectively).3 Yasuko Doi. these results indicate enhanced commensal-driven turnover of IgA-producing cells in LP of Pdcd1–/– mice. B. we examined the PPs of Pdcd1–/– mice. 1F). Kato. Graduate School of Medicine. Yokohama 230-0045. PD-1 deficiency perturbs the balance of bacterial communities in the gut. however. that the proportion of bacteria coated with IgA (bound IgAs) was reduced in Pdcd1–/– mice (10) (Fig. Research Center for Allergy and Immunology. 12) was lower in IgA-producing cells from LP of Pdcd1–/– mice (Fig. PD-1 deficiency generates an excess number of T follicular helper (TFH) cells with altered phenotypes.riken. S2A). The frequencies and the absolute numbers of IgA-producing cells in the lamina propia (LP) were comparable in Pdcd1–/– and WT mice (fig. he primary function of immunoglobulin A (IgA) is to maintain homeostasis at mucosal surfaces. *These authors contributed equally to this work. To assess the features of LP IgA. there were significantly more BrdU+ PBs in the LP of Pdcd1–/– mice than in those of WT mice (Fig. Cell death was assessed by detection of caspase activation. Although we observed a modest increase in the frequency of light-zone B cells in the PP GCs of Pdcd1–/– mice (16) (fig. 1A). takes place mostly in specialized microenvironments called germinal centers (GCs).1 Lucia M. however. Consequently. which causes alterations of microbial communities in the gut.86 × 108 and 0. which were minor representatives in the WT mice. S1. S1. however. and then BrdU was removed from their drinking water. S2. S1F).1 Yumi Tsutsui. compared with those in framework regions 1 to 3 (FWR1-3) (fig. Although the total number of bacteria cultured from the lumen of the small intestine was comparable between PD-1–deficient mice (Pdcd1–/–) and wild-type (WT) mice (4. Ten days after the initiation of BrdU administration. 5 days after the initiation of BrdU labeling (Fig. The higher turnover of Pdcd1–/– PBs may be the result of increased apoptosis. Mice were continuously fed BrdU for 10 days. 3). In the LP. †To whom correspondence should be addressed. and the frequency of PBs in the cell cycle was comparable for Pdcd1–/– mice and WT mice (fig. and F). Here we provide evidence that the inhibitory co-receptor programmed cell death–1 (PD-1) regulates the gut microbiota through appropriate selection of IgA plasma cell repertoires. antibody-mediated autoimmune diseases (5–7). A and C). In contrast. Similar to humans. Kyoto 606-8501. we observed a significant increase in frequency of caspasehi PBs and PCs in the LP of Pdcd1–/– mice. Graduate School of Medicine. PBs] (13). Thus. Yoshida Sakyo-ku. 1st Ton That Tung. Kyoto University. A to C). 1G and fig.7 × 108 bacteria per g of intestinal content. 5 days after the 11 . Hanoi Medical University. as assessed by Fucci or a 12-hour BrdU pulse test (17) (fig. Note that the incidence of diseases in PD-1–deficient mice varies among mouse colonies and depends on AID (8). Compared with WT mice. S1A). the frequency of GC and IgA+ B cells that incorporated BrdU reached a plateau of 95 and 80%. C. Thus. the frequency of BrdU + PBs was reduced by half in Pdcd1–/– mice. TFH cells express high amounts of the inhibitory coreceptor programmed cell death–1 (PD-1) (4). Tran. in which the cells in S-G2-M phase of the cell cycle are fluorescently marked (14).1. however. hereafter. Japan.

2 0. Mean T SEM (n = 3 to 5 mice per group). (F) Frequency of BrdU and (G) Caspase Student’s t test was used for all statistical analyses.Days 0 0 5 10 15 20 25 45 60 PB PC sorbent assay (ELISA). Data points represent individual mice. ND.05. (A) Small intestine microbiota composition in 3-month** 4 80 old. Pdcd1-/. (E) Representative charts of clonal diversity. calculated as frequency of transcripts of a given in-frame VH-(N)-D-(N)-JH rearrangement of GC IgA+ B cells in PPs of Pdcd1–/– and WT mice.PC 1 not detected.6 0. .5 0. Means T SEM (n = 16 mice per group). Two-tailed unpaired Student’s t test was used for all statistical analyses. replacement in CDR1 and CDR2.IgA+ E WT n=71 PP BrdU + (%) B220+ IgA+ 100 75 50 25 0 * * 80 60 40 20 0 Pdcd1 -/n=77 0 5 10 15 20 25 45 60 Days Fig. Means T SEM (n = 3 to 5 mice per group). specific pathogen–free (SPF) Pdcd1–/–and WT mice. Data are combined from two independent sets of experiments. (D) Frequency of BrdU+ GC B cells (PNA+Fas+) and B220+IgA+ B 12 cells from PPs of Pdcd1–/– and WT mice as determined by flow cytometry. The gut microbial community comVH10 VH5 WT Pdcd1 -/position and the repertoires of LP-resident –/– BrdU F G Chase IgA-producing cells are altered in Pdcd1 WT 100 Pdcd1-/mice. n = 3 mice per group. RCDR.001.01. (D) IgH V family usage and (E) the affinity maturation of IgAcytometry. Two-tailed unpaired + hi three mice per group) is indicated.3 0. Increased numbers and enhanced dynamics of GC and IgA+ B cells in PPs of Pdcd1–/– mice. LZ (light-zone AIDlow) and DZ (dark-zone AIDhi) are shown. are shown. **P < 0.0 50 25 72% 0 55% * C Free IgA (μg/ml) * 107 108 109 250 200 150 100 50 n=141 * VH1 VH2 VH3 VH4 VH6 VH7 VH8 VH9 n=165 VH11 VH12 VH13 VH14 No. 2. The number of sequences analyzed (pooled from experiments.05. Stotal. 1. The number of productive sequences compared is indicated. Con* ** WT PB 3 tents of the entire small intestine were pooled. Data shown are combined from two independent sets of regions 1 to 3 (FWR1-3) (12).01. 100 mm. A WT LZ DZ B WT B220 + 5 10 Pdcd1-/LZ DZ Pdcd1 -/7.IgA coated bacteria (%) A Anaerobic *** * ND B 75 D *** Aerobic Bacteroidaceae Eubacterium Bifidobacterium Lactobacillus Staphylococcus Streptococcus Enterobacteriaceae 103 104 105 106 WT Pdcd1 -/12% 16% 8% E WT Pdcd1-/RCDR/Stotal+RCDR 0.4 0.1 0. Data 2 WT PC 40 from two experiments.2 Fas 14 BCL6 PD-1 C PNA+ Fas+ Cell number x104 120 100 80 60 40 20 0 AID WT Pdcd1 -/- CD3 AID PNA IgA LP caspasehi (%) LP BrdU+ (%) B220 D 100 BrdU Chase WT GC Pdcd1-/. (B) The percentages of fecal bacteria coated with 20 IgA as determined by flow cytometry and (C) free IgA concen0 trations in the feces as determined by enzyme-linked immuno.GC WT IgA+ Pdcd1-/. (C) Absolute numbers of indicated B cell populations isolated from PPs of WT and Pdcd1–/– mice. bacteria/g small intestine content WT Pdcd1-/- 0 Fig.PB ** were identified with standard microbiological methods. IgA PBs and PCs from the LP of Pdcd1–/– and WT mice as determined by flow *P < 0. silent mutations in both CDRs and in framework peritoneal B1 cells. Scale bars. (A) Representative sections of the PPs and (B) flow cytometric profiles of PP cells from WT and Pdcd1–/– mice stained as indicated to reveal the structure and characteristics of GCs. **P < 0. and bacteria 60 Pdcd1-/. *P < 0. ***P < 0. Note that some of the BrdU+ PCs may be also derived from producing cells from the LP of Pdcd1–/–and WT mice.

2 0.4 0.01. (B) Absolute numbers of major T cell populations and (C) the ratio of TFH cells to GC B cells in the PPs of Pdcd1–/– and WT mice. the proportion of TFH cells producing interferon-g (IFN-g) was increased in PPs of Pdcd1–/– mice. D is the diversity region.5 0. was higher in all CD4+ T cell subsets. (A) Representative flow cytometric profiles and plots of PP cells from WT and Pdcd1–/– mice stained for the indicated markers. and deregulation of TFH cells leads to inappropriate GC B cell selection and humoral autoimmunity (16–19).026 85 6. The ratio of TFH to GC B cells in Pdcd1–/– mice was twice as high as that in WT mice (Fig.046 E Expression (relative to Gapdh) 3 IL-21 ** 2 1 65 30 1.5 709 493 305 114 85. Relative amounts of mRNA normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are shown. **P < 0.53 99 0. the frequency of labeled GC and IgA+ B cells was reduced by half in Pdcd1–/– mice. Thus. In order to better characterize how TFH cells may contribute to the altered GC responses observed in Pdcd1–/– mice. and Fig. CXCR5 TFH 39 30. were significantly increased in PPs of Pdcd1–/– mice (Fig. we decided to evaluate the ability of PD-1–deficient TFH cells to support IgA generation in gut in vivo.6 7.8 Ratio TFH/GC C 0.8 BCL6 TFH 0.3 IL-10 0 5 4 3 2 1 0 IL-17 IFN-γ * WT Pdcd1-/- 77 18 99 1 79 14 98 1. TFH cells from Pdcd1–/– mice produced less IL-21 compared with those from WT mice.2 0 13 . The significant drop in BrdU+ cells in PPs of Pdcd1–/– mice could be the result of increased cell death in situ. as previously reported (21) (Fig. The expression of BCL6. whereas most of the BrdU+ cells remained in the PPs in WT mice (Fig. (D) Flow cytometric profiles and (E) mRNA expression of indicated cytokines in PP T cell subsets.012 0.4 1010 715 541 87. In fact. Such an abbreviated transit time likely results in impaired clonal selection and expansion of IgA+ B cells in GCs of PPs.1 0. However. Numbers represent percentage of cells in the quadrants or gates.3 0. Expansion of activated T cells and skewed cytokine profiles of TFH cells in PP GCs of Pdcd1–/– mice. which is required for the production of interleukin-21 (IL-21). the expression of interferon regulatory factor 4 (IRF4).18 1 0 Pdcd1 -/.046 0. Furthermore. Means T SEM of at least two independent experiments. including CXCR5hiICOShi (high levels of ICOS.4 54. The frequency and absolute number of CD4+ T cells. We then asked why PD-1 deficiency causes such changes in the dynamics of GC B cells in PPs. Means T SEM (n = at least 5 mice per group). 2A). In contrast.4 1.3 53. *P < 0.3 42. The data suggest that the increased turnover of IgA+ B cells in PPs of Pdcd1–/– mice may be because they pass through the GCs more rapidly.7 66. A and B.removal of BrdU. 3C). Thus. 3. N is a random nucleotide. Two-tailed unpaired Student’s t test was used for all statistical analyses.8 15.1 0.17 1.4 99 0.4. E and F). a transcription factor required for TFH differentiation. It is well established that the number and activation status of T cells—and TFH cells in particular—play a fundamental role in shaping GC biology. 20).45 IRF4 CXCR5 0.11 D TFH WT 3. as shown by the reduction of the frequency of clonally related IgA sequences [identical VH-(N)-D-(N)-JH where VH is the variable region of the immunoglobulin heavy chain. Indeed. as well as within GCs.001. ***P < 0. the increased number of GC B cells in Pdcd1–/– mice likely results from more inflow of B cells into GCs. the cytokine that promotes growth and differentiation of IgA+ B cells into PCs (19. was reduced in TFH as well as CXCR5hiICOSint cells from Pdcd1–/– mice. Also. S2.5 21. Taken together. 3. 2E). including the TFH cells in PPs of Pdcd1–/– mice (Fig. D and E). we assessed TFH cells in the PPs of Pdcd1–/– mice.6 IL-21 IFN. S2D). PP CXCR5hiICOShi TFH cells were isolated from Pdcd1–/– and WT mice and were adoptively transferred into T cell–deficient (lacking the / 6. Numbers in the graphs indicate the geometric mean fluorescence intensity of BCL6 and IRF4 in the corresponding color-coded T cell subset gates. Indeed.05.2 TCR β CXCR5 CXCR5 6. the frequency and numbers of IgD+ cells with an early GC phenotype (B220+IgD+IgA+/–GL7+Fas+CCR6+) (18) were higher in PP of Pdcd1–/– mice (fig. B cells compete for T cell help before entry into GCs. there were no differences in the frequency of caspasehi GCs and IgA+ cells for WT and Pdcd1–/– mice (fig.5 0. 2D). 3A). and JH is the joining region of the immunoglobulin heavy chain] in Pdcd1–/– mice compared with WT mice (Fig.γ TFH Fig. our results suggest that PD-1 deficiency is associated with an increased import and export of B cells into the GC. 3.8 Cell number x104 A WT B 60 50 40 30 20 10 * *** ** CXCR5+ CXCR5- Pdcd1 -/CD4 ICOS 11. proteininducible costimulator) TFH cells.0 WT Pdcd1-/- 19.

J. we found that serum from Pdcd1–/– mice contained antibodies specific for components of commensal bacteria. Suzuki. 1. 243 (2010). Okazaki. U.CD3e subunit.7. However. I. 13. 17927 . 20 . Hiai. B. Immunol. Slack . by Grants-in-Aid for Scientific Research in Supported Priority Areas (S.Vinuesa.. Natl. J. Rev. 5. J. U. S.. D and E). P. We O. Immunol.C. Immunol.. 132 . Murahashi and T. 141 Immunity 11 . M. . Allen. Supported Grants-in-Aid materials.1217718 Published 27 April 2012 10. L. 487 (2008).1217718 Published 27 April 2012 10.001. 853 (2005). G. Med.8.4 0. B and C)..H. 319 (2001). 319 395 (2001).L. 4. al. and the Pdcd1–/– TFH cells maintained their skewed cytokine profile (Fig.1126/science. M. Science 325.1126/science.T.Tangye. It was interesting that the IgAs induced by Foxp3+ T cells had significantly fewer mutations and low affinity maturation compared with those induced upon transfer of TFH cells (Fig. the results indicate that (i) the quality of gut IgAs critically depends on the number and the nature of TFH cells in PPs. Cell 132 . Kanagawa. Gut 38 .T. Immunity . E. TFH = 175. Materials and methods available as supplementary 12.2 0. 348 (1996). S1 to S4 References (25 –34) References (25–34) 9December December 2011. Science Nishimura alExp. (2010). Victora et al.(2000).Kwon Good-Jacobson et al.M.(2010). Cd3e–/– mice transferred with PD-1– deficient TFH cells generated very few IgAsecreting cells in the LP compared with mice injected with WT TFH cells (Fig. 617 24. In Pdcd1–/– mice. Minato. . H.H. 16. 315. O. (2011).1126/science. 5. der der Waaij. 19. 18. WT Foxp3+. al Proc. . TFH cells from Pdcd1–/– mice are impaired in their ability to support the generation of IgA plasma cells in gut.accepted accepted16 16 March 2012 9 2011. T.C. 20.. we propose that the skewed gut microbial communities that result from the dysregulated selection of IgAs drive the expansion of self-reactive B and T cells. 22. 348 (1996). Annu. M. S. 10. B.D. G.D. Similar to what we observed in Pdcd1–/– mice. et al. On the other hand. Med.1217718 .S. 11. Immunol. 21.. Weiet etal al Nat. 264 (2011). 2.2 CD3 AID DAPI LP IgA 2. Uduman. 183 . Kelsoe. L. Pdcd1–/–. 4F. 19.K. Proc.. the gut microbiota induce hyperactivation of the systemic immune system (fig. 4. D and E.S.1217718 10. T..N. which indicated a breach of normal mucosal-systemic compartmentalization (fig. Muramatsu alJ. G. T.).Rev. Victoraet etal al 143.Cell Immunol.F. Blood 5181 (2010).S. The few IgAs induced by Pdcd1–/– TFH cells had increased turnover (which mirrored the intact Pdcd1–/– mice) and a comparable number of mutations in their VH genes and affinity maturation index with those induced with the help of WT TFH cells (Fig. 2. A. L. 941 (2009). Hershberg. R. 6.R. Hiai..) and by RIKEN President’s Research in Fund Priority Areas (S.. On the basis of the data presented here.Science A. Nat. Kleinstein. and fig. Rev. D.. 20.M. Cyster. 395 (2011). 9.Nishimura. M.F.S.. 14.References Fagarasan.H.Med. and fig. Wei et et al. G. References and Notes Notes O.D. 243 O. Nature 354. (2006). C. S3. 11.) and Special Researchers Discretionary Program (S. materials Science 13.F. Science 8.389 J. M. Tsuji et al. ***P < 0..31 Nat. 1488 (2009). G. J.. Schwickert et al . U. 291 208. van der Waaij. Kleinstein.Int.G. C. E. Okazaki. **P < 0. H. 23. Yuvaraj . T = 177. Thus. K. S. 528 (2007).org/cgi/content/full/336/6080/485/DC1 www. Natl. Slack et al. Sci. Muramatsu M. 16. R. 11.Immunity H. Immunity 31. Sci. Casola for inspiring discussions. H. G. 208 . Kelsoe. inspiring Taniuchi. as previously reported (22). Mesander. T. Uduman. Exp.77 WT TFH 18 WT TFH Pdcd1-/.sciencemag. 22. 5099 (2007). 535 21. 102. Hershberg. Mackay. Taniuchi. 592 (2010)..Annu.K. 1.. et .5. H. Honjo. 5099 5. (F) Total numbers of indicated cells from the PPs of Cd3e–/– mice at 3 months after the reconstitution with the T cells indicated. Numbers represent percentage of cells in the indicated gates. M. Two-tailed unpaired Student’s t test was used for all statistical analyses. Supplementary Materials Supplementary Materials www. (A) Representative flow cytometric profiles of cells isolated from PPs and LP stained for the indicated markers. commensal-driven selection in the LP. Acknowledgments: We thank T. L.Sutherland Murahashi for and T. 11070 10. J. 941 (2009). 12(2009). 683 (2008). . Good-Jacobson et al. I. 4871 (2009). S1 to and S4 Methods Figs.01.3 0.sciencemag. van Waaij. Kanagawa. Acad. Shlomchik.. These observations suggest that a significant fraction of PCs in Pdcd1–/– mice may be generated with the help provided by TFH cells derived from Foxp3+ T cells.van A. H. Blood Online. the absolute number of PP TFH cells was much higher in Cd3e–/– mice that received Pdcd1–/– TFH cells than in mice injected with WT TFH cells. Tsuji alNat. accepted16 16 March 2012 9 2011. M. accepted March 2012 10.62 9. did not expand much but converted into IL-21–producing TFH cells in PPs.R. T.A. and S. 683 are (2008). H. 195 (2006). Honjo.transferred with: Fas 0. S3. Kanagawa. N.materials Materials and methods are available as supplementary on Science Online. 2011.4 WT Foxp3+ Pdcd1-/-Foxp3+ B220 RCDR /Stotal +RCDR IgA DAPI Mutations/VH gene Cell number x104 Fecal IgA (μg/ml) C 120 100 80 60 40 20 0 D ** 10 8 6 4 2 0 E *** TFH Foxp3+ -/- F 0. Acad. Mei et al. 853 (2005). Med. Immunol.T. Limburg. Taken together. D. Immunol. T. 20. J. 487 (2009). U.and Kawamoto.. 4.G.. Immunol. both PD-1–deficient and sufficient Foxp3+ T cells. 100 mm. Immunol.F. Mackay. E.0 80 60 40 20 0 PP CD4+ T 20 15 10 5 PP TFH *** ** ** WT Pdcd1 -/- -/- TFH Foxp3+ - TFH Foxp3+ Cd3e transferred with: Cd3e transferred with: TFH Foxp3+ -/- 0 TFH Foxp3+ Cd3e transferred with: Fig. Limburg.A. Minato. Immunol. Cell 102. A to C). S.T.3. K. 18. C. C. Yuvaraj et al. Nishimura et al. G. 14. and fig..1126/science.4. K.manuscript. M.D. J. . 11.1126/science. Tang.(2005). Immunol. 208..N. Weiss. 3. Kanagawa. Weiss. S4. Cyster. Kaji reading of the manuscript. 12 1488 (2009). Minato. 28. M. Honjo. Honjo. J. (1999). (ii) PD-1 deficiency interferes with the selection of B cells in GCs.Trends J. Honjo. Tang.. 5181 (2010).1 0. M. and (iii) in addition to selection in PP GCs. Scale bars. Nose. Rajewsky. Mei eton al. 179 553 . Haynes et al. Nature 354. Cd3e–/–) mice. and F. Sakaue-Sawano et al. 102. 264 (2011). 141 (1999). Nishimura. suggestions. The number of sequences analyzed: WT.Science C.Okazaki Ley et al . N. 116. 389 (1991). 27. Okazaki. Science 323.J. Jacob. 17. 28. and Y. G. Mesander. Exp. M. E. Nose. Moser. N. J. T = 153. A and B). The results indicate that TFH cells in Pdcd1–/– mice preclude the generation of IgAsecreting cells in gut. Okazaki.T. L. T. Cell 102. IgAs appear to undergo a second.Nat. D and E).1217718 10. Immunol. Fund (S. thank T.). Shlomchik. S4. S. Schwickert et Tangye.(2005). U. 4. Immunol. 116 Cell. B. Injection of TFH cells from both WT and Pdcd1–/– mice into Cd3e–/– mice reconstituted the GC B cells and B220+IgA+ B cells in PPs (Fig. data reported in this paper are the main The paper and the supplementary are tabulated in theby main paper andfor theScientific supplementary materials. . S.) andPostdoctoral by RIKEN President ’s Discretionary (S. 12. K. Moser. TFH = 171.1 18 4. U. Rev. Pdcd1–/– Foxp3+. 528 (2007).5 0.. A. E. 11070 9..7 0. 23. and critical comments.T.transferred with: B Pdcd1-/. 4871 15. 183 . Vinuesa. 7. (2008). T. 4. M. Okada.) and Special Postdoctoral Researchers Program (S.G. 24. 208 1243 (2011). S.M. Immunol. 1243 (2011).TFH 24 Cd3e-/. G. critically reading of the and Y.discussions. H. Indeed. 4. 15. (D) Absolute number of somatic mutations in VH genes and (E) the affinity maturation of the IgA-producing cells from the LP of Cd3e–/– mice at 3 months after the reconstitution with the T cells indicated. 617 (2009). Fagarasan. et al. T. . Casola for suggestions.. Honjo. 553 (2000). Sutherland for critically comments. T. and critical S.Science 325. Int. Cell 143 . 592 (2010).Acknowledgments: Okada. N. March 9 December accepted 2012 Published 2012 9December December 2011. 535 (2010).A.A.. Okada.Trends H. Ley et al. Okazaki et alet . 6. . J. which. van der Waaij. Miyajima and D. the LP may serve not only as reservoir of PC but also as a site where proliferating PB are reselected to fit the geographical distribution of dynamic bacterial communities along the Materials and Methods Materials Figs. along with T and B cell hyperplasia. Immunol. as in SHM-defective AIDG23S mice (23). Miyajima and D. 291.. Rajewsky. Jacob. Kaji for technical assistance. 14 A PP Cd3e-/. Suzuki. Gut P. Exp. Allen..E. Kawamoto. and Okada. A and B). 17. A and C) (24). H.. 11. Immunol. Sakaue-Sawano et al. Nat. S.TFH PNA (B220+ gate) PP 5. Minato. Kwon et al. 4. 315. S3A). Means T SEM (n = 2 to 3 mice per group). Haynes et et al.K. Our studies have implications for how modulation of PD-1 expression promotes tolerance or uncontrolled immune reactions leading to autoimmunity. (1991). The data reported in this paper fortabulated technical in assistance.. 38C. 195(2007). K. Science 323.H. C. (B) Sections of the PPs and small intestine stained as indicated and (C) fecal IgA levels as determined by ELISA from the Cd3e–/– mice 3 months after reconstitution with TFH cells and Foxp3+ T cells from WT and Pdcd1–/– mice.. supported the generation of LP IgAs after the transfer into Cd3e–/– mice (Fig..

with the potential toaddressed. which is thought to serve as the critical checkpoint for preTCR formation in progenitors. Bythe several criteria. together with our other results.maincells vitro expansion (Fig. T. By several criteria.3 Hiroshi Kawamoto1* In early T cell development. whereas the latter stage GFP kinase and interleukin (IL)–7 (fig. and their gene expression first critical checkpoint in(iv) T cell development (13). GFP step. We and others have recently identified the next stage. (FMS-like tyrosine + cells and NKligand). or Pa xMolecular 5 genesGenetics. IgH chain gene. 1C). The molecular mechanisms that drive this determination step remain unclarified. and B cells (1–6). Upon closer unlimited analysis on vitrocells expansion (Fig. We show that. Nihon University School of Medicine. cell (iii) development.of Such peared tocapacity. these arrested B cell progenitors – – to DN4 (double-negative CD4 CD8 )lineage that canunbe undergo self-renewal and remain B tracked by surface The DN2 along stage committed.may NK be a critical checkpoint for T D). whereas Tin cell associated progenitors not occur thislineage culture–system. S3E). The DP cells genCD25 ture nearly could identical be maintained by using c-kit erated reducing concentration IL-7selfapcells atby the time of the passage (fig. and natural killer (NK) cells (7. because long-term culthe FFDN2 cells appeared identical to DN2mt + + ture :could be maintained using c-kit cells (i) they gave rise toby authentic ab CD25 T cells cells at the time to of a passage (fig. RIKEN Research Center for Allergy and Immunology.1 Ryo Kominami. 3Division of Cell Regeneration and Transplantation. potential to develop can be subdivided into two stages based on transother lineages. committed. whereas in the control FFDN2 cells could not generate Bwith cellsTSt-4 (fig. Flt3L (FMS-like ure of TCR gene rearrangement enforced kinase ligand). 2C figs. ditions in theisolated feeder-free culture system. cells. and Bc l11b were note. These cells not express FFDN2 cells could not generate Bdid cells (fig. does not require additional environmental factors in this feeder-free culture system.riken. while essentially in these cultures developed up to the1G) abTCRtaining c-kit and CD25 expression (Fig. the final yield of DP cells maintain long-term culture. These results demonstrated that abTCR+ cells can be generated from prethymic progenitors in a “feeder-free” culture system and that the TCRb-selection. Japan. progenitors developing toward T cells were arrested and the arrested cells entered a self-renewal cycle. we found 1E). Notably. and 1B). the stage date no such checkpoint has been identified for before that step under particular conditions. S1). Yokohama 230-0045. Japan. DCs. Furthermore. – + profiles similar to those of DN2mt cells (Fig. IL-7 S5). Unlike the B cell to demonstrate developmental arrest at lineage. Of genes such as lck. DCs. Our study thus identifies the earliest checkpoint during T cell development and shows that it is Bcl11b-dependent. essentially mainDN generated thewhile feeder-free condition. Cells in the panel) thus DN2mt named them cells (feederfraction possessed potential to c-kit+CD25+ DN2-like free-cultured cells). where cells were cultured stroindicating that dedifferentiation to more primitive mal cells expressing DLL4 (TSt-4/DLL4). 2 Department of Schooldevelopof Medical Ebf . GFP– Unlike to the cell lineage. where cells were cultured with TSt-4 stromal cells expressing DLL4 (TSt-4/DLL4).primitive 1 (Sfpi1) indicating that dedifferentiation to more and C/EBPdid a. with thephenotype. the FFDN2 cells appeared identical to DN2mt cells: (i) they gave rise to authentic ab T cells when transferred to a TSt-4/DLL4 stromal cocul- ture system (fig. Niigata University. whereas in the control group. Among genes up-regulated by our induction system. 1C). The DN2 stage step. S7). dendritic cells (DCs). including myeloid and T cell (9–11). S3. Therefore. cells through the DN2-determination After 1D and fig. generation of CD4+CD8+ double-positive (DP) cells was observed (fig. Furthermore. We gene rearrangement. S7). including myeloid. C and D). a T cell lineage–specific transcription factor origi15 .2 Yoshimoto Katsura. is unlikely that this arrest is due to tyrosine the failSCF It (stem cell factor). DN We After 7 days of to culture. of Niigata 951-8510. 1A. gene rearrangement. 1F). S5). S2). pT a. S1). Although thecells kinetics of DPCells cell growth freshly isolated DN2mt (fig. Niigata 951-8510. Tokyo 173-8610. Of CD8 double-positive (DP) cells eration of CD4 note. Nihon University School of undergo self-renewal and remain B lineage unMedicine. 1A. when murine hematopoietic progenitors were cultured on immobilized Notch ligand DLL4 protein in the presence of a cocktail of cytokines including interleukin-7. these arrested B cell progenitors Advanced Medical Research Center. S3.1 Kiyokazu Kakugawa. ture system (fig. 12).An Essential Developmental Checkpoint for Production of the T Cell Lineage Tomokatsu Ikawa. cells are generated from multipotent hematopoietic stem cells through a series of differentiation steps. Reduced concentrations of interleukin-7 promoted T cell lineage determination. S3. and a expressing CD4+ CD8+ DPcomparable stage (Fig. date no such checkpoint hasmyeloid been identified for cells and T NK 1F). are determined the Tcells cell lineage (7at . be authentic DP cells. This case illustrates that such a critical developmental checkpoint exists at the step when uncommitted B cell progenitors become determined T Laboratory for Lymphocyte Development. GFP (DLL4) protein in –the presence the cytokines potential. Upon closer analysis on DN cells generated in the feeder-free condition. expression was notvarious observed in FFDN2 cells generated testing cytokines and Notch ligand from conprogenitors from plck-GFP mice (Fig. deletion of E2a . stage (Fig. develop E-mail: along *To whom correspondence should be kawamoto@rcai. 1A.1 Satoshi Hirose. we observed that these cells resembled DN2mt cells (maintained c-kithighCD25+) (Fig. and DCs (fig. because long-term cul+ + was (fig. This step is thought to be the can subdivided into in two stages based on transfirstbe critical checkpoint T cell development (13). 2B). 12 ). Often transcription factors regulate cell lineage determination steps. term N the cells. (iii) intracellular step between these the DN2-determination T cell receptor (TCR) bstep chain protein to was step. The first step in this pathway is the generation of progenitors that have lost erythroid/megakaryocyte potential but retain the capacity to generate other hemopoietic cells. we searched for an expressed (Fig. Tokyo 173-8610. GFP+ DN3 appear on arrest day 3 after reduction (Fig. A similar arrest and selfrenewal of progenitors were observed in thymocytes of mice deficient in the transcription factor Bcl11b. Niigata University.1 Asako Shibano-Satoh. mental arrest before formation a functional Japan. S10). GFP expression Sca-1 (LKS) cells from 13 days post-coitum (dpc) was not fetal observed FFDN2 cells generated from murine liver in with immobilized Delta-like 4 progenitors isolated from plck-GFP (Fig. to potential. 8). E2a. Japan. – genic green fluorescent protein (GFP) expression ) c-kit+ We cultured lineage-negative (Lin controlled the proximal lck post-coitum (plck) promoter Sca-1+ (LKby S) cells from 13 days (dpc) (lck is afetal src family kinase selectively expressed murine liver with immobilized Delta-like 4 cells retainof non-T lineage by T cells).showed S2). maintaining non-T lineage potentials. (ii) they retained the indicated that the DN2-determination potential to produce macrophages (Fig. (ii) they retained the designate these two DN2mt (myeloid-T) potential to (T-lineage produce macrophages K and DN2t determined) (Fig. they give renewal together with because our other results. 1A. 3Division of Cell Regeneration and Transplantation. taining c-kit and CD25 expression (Fig. cells. whereas the latter stage GFP the cell lineage before the initiation of+ T CR are determined to the T cell lineage (7. myeloid-lineage transcription factors PU. We were cultured lineage-negative (Lin ) c-kit + 1D and fig.1 Rumi to the B cell lineage. Advanced Medical Research Center. S11). remained the stage (Fig. including thatFlt3L for myeloid cells. rise to CD4 and CD8 single-positive (SP) cells when transferred to a fetal thymus organ culture system (fig. Yokohama 230-0045. S4). the most critical step for development of the T cell lineage is now thought to be at the point where myeloid potential is terminated. expression ofof a culture. E-mail: kawamoto@rcai. right cells (maintained c-kithighCD25 freshlyand isolated cells FFDN2 (fig. tem. stages other lineages. we focused on Bcl11b. step 1B). This determination is thought be not the expressed (Fig. 2A).2 Kyoko Masuda. 2 1 *To whom correspondence should be addressed. progenitors retaining the potential to generate myeloid and natural killer lineages are eventually determined to a specific T cell lineage.1. SCF (stem cell factor). maintain long-term culture. right panel) and thus named them FFDN2 cells (feederfree-cultured DN2-like cells). left panel). in the + was that in the the TSt-4/DLL4 c-kitdelayed CD25+compared fraction with possessed potential to feeder cell culture system. group. In the the Tof cell before the initiation TCR case B lineage cell differentiation. S6). Astages and B). and DCs (fig. genic green fluorescent protein expression This case illustrates that such a(GFP) critical developcontrolled by the proximal promoter mental checkpoint exists at lck the(plck) step when un(lck is a src kinase selectively expressed committed Bfamily cell progenitors become determined cells retain lineage by TB cells). RIKEN Research case for ofAllergy B cell deletion of Japan. Such selfwhen transferred TSt-4/DLL4 stromal coculrenewal capacity. C and intracellular To investigate the molecular mechanisms of T cell receptor (TCR) b chain protein was not T cell lineage determination. the B non-T cell lineage. Ebf . Immunology. including myeloid and T cell (9 –11). In the Laboratory for Lymphocyte Development. in which the T cell progenitors have lost B cell potential but are still able to generate myeloid cells.TCR In this induction not prevent the cells developmental (fig. and interleukin because (IL)–7 (fig. S6). Department of Molecular Genetics. proceed mental arrest before formation of they a functional through developmental stages referred to as DN1 IgH chain gene. S3E). developmental potential to and that of S8 and S9). S4).showed Tcf1. gen+ + progenitors did not occur in this culture system. As T cell progenitors develop. including that for cells. A and B). and (iv) their gene expression environmental cue that could the arrested profiles were similar to those of drive DN2mt cells (Fig. proceed designate these two stages DN2mtthey (myeloid-T) through developmental stages referred toterm as DN1 and DN2t (T-lineage determined) and the – to DN4 (double-negative CD8–) that can be step between these stagesCD4 the DN2-determination tracked bydetermination surface phenotype. Japan.riken. FFDN2 cells an almost unlimited in markedly up-regulated (Fig. functional TCR b chainat gene did After 7 days cells remained the DN not prevent the developmental arrest (fig. A reliable way to substantiate that a given step is critical for the development of a lineage is to demonstrate developmental arrest at the stage 1 before that step under particular conditions. left panel). 1G) and a we observed that these cells resembled DN2mt + developmental potential comparable to that of ) (Fig. FFDN2 It is unlikely thisdifferentiation arrest is due to the failthat cells that initiate when the ure of TCR gene rearrangement concentration of IL-7 is reducedbecause on day enforced 7 of culexpression of ato functional b chain genesysdid ture (10 ng/ml 1 ng/ml).observed FFDN2 cells an almost in was (fig. Graduate School of Medical and Dental Sciences. Center anddifferentiation. We sought to identify the step at which progenitors become fully committed to the T cell lineage and what regulates this transition. leads Graduate to an early and Dental Sciences. or Pax5 genes leads to an early developAs T cell progenitors develop. (DLL4) protein in the presence of themice cytokines 1E).


7 51. support mental factors in this feeder-free culture T cell differentiation up to the DP stage.52 1. whereas in there was little effect frequently observed cell S14A).genotypes. DP and cells TCRb gene rearrangement enhanced −/− thymocytes of Bcl11b mice and cultured (Fig. the absence of −/− that the Bc l11b DN2 cells that developed in the Bcl11b had a severe impact on the generation of thymus ab did dedifferentiate intowas more primitive thymic Tnot cells. indicating that self-renewing fetal liver expansion cells into by transferring Bcl11b is not so prominent vivo. we observed nearly complete detransduced cells could give rise to DN3 cells even velopmental arrest at the DN2 stage.Flow In right panels. T cell (14the ). 4B).4 73. a difference could irradiated B6Ly5.44 0. (D) Generation of macrophages NKdays. (A) Flow cytometric analysis of fetal CD45.4 0. Similar to was ex vivo fetal 4A). We carefully reexamined phenotype of Bcl11b deficiency is lethal the around the neofetal thymus cells Bcl11b-deficient mice natal period ( 15). although the possibility 17 -/. (B) each Absolute numbers of total c-kit versus CD25 of cells gated in and upper panels.2) were into8 lethally – after transfer are shown. CD4atand CD8there single-positive (SP) cells when transferred to a fetal thymus organ culture arrest at(fig. There no phenotype (Fig. conditions.3 Mac1 Mac1 82.6 16. Although kinetics of DP cell growth hibit impaired thymocyte development around was delayed compared with that in the TSt-4/DLL4 the DN3 immature SP stage because an infeeder cellto culture system.1). Atthat 8 weeks be due to the limited niche space in the thymus defor after transfer. fromfor the indicated For each more than and five mice. cells mice cultured on TSt-4/DLL4 stromal cellsand for 30 versus CD25 of fetal liver cells from Bcl11b + fetal liver cells. The Bcl11bThe developmental steps just formatransduced cells could give rise to after DN3the cells even tion preTCR stage) and ab (DP in theof presence of (DN3 a high concentration ofTCR IL-7 (Fig. the final yield of of DP cells ability to rearrange the V segments was nearly identical (fig. More than five mice mice were individually analyzed. the Vb to Db gene segments (15).5 5. For each whereas myeloid-lineage –associated apoptotic death. cultures.08 7.6 16.3 11. (C) Flow cytometric thymocytes and DN2 cells in 18 dpc fetuses of mean the indicated More than five mice −/− versus CD25analyzed of fetal liver LKS cells from Bcl11b mice cultured on TSt-4/DLL4 stromal cells for 30 individually for each group. The DP cells genb to D b gene (15).4 S14B). where T cells are conup-regulate Bcl11b.4 5. S10). and loss of B ability to (fig.2 − by our induction we focused Bcl11b.73 1. BcDN2 l11b−/− Bc l11b−/− cells. In contrast.6 73. For group. because they found 18 dpc.2) were transferred into lethally macrophage ( and NK liver cell markers byBcl11b flow cytometry.52 4.1 16.1 F4/80 F4/80 C C CD25 D D M M NK cells NK cells +/+ +/+ Bcl11b Bcl11b E E CD25 CD25 12. increase in thymic B cells in the recipients the − DN2 features DN2mt Bcl11b−//− fetal cells liverexhibited cells (fig. 3.7 Bcl11b+/+/0.1in congenic mice.7 CD25 55.0 16. To whether the that decultures and in the Bclinvestigate 11b−/− mice suggested −/− progenitors is velopmental arrest of Bc l11b the arrest in the cultures may be due to a failure to seen in the adult thymus. 3.04 CD25 2. Similar to FFDN2 cells.4 1. (D) shown Generation macrophages and cells NK cells −/− cells in the presence of M-CSF + panel) or IL-15 (right panel) and analyzed for well) 7 daysBcl11b with TSt-4 (left fetal liver cells. althoughthis the possibility. are shown CD4 versus mice. demonstrating were equivalent to DN2mt cells. and representative after transfer are shown. The appearance that occurred in the DLL4/IL-7 cultures.-/- 10 0 88 88 84.-/Bcl11b Bcl11b TCR TCR CD4 CD4 c-kit c-kit . Bcl11bafter transfer.4 2. fraction shown.52 1.04 12. only a under TSt-4/DLL4 which can support few DP cells (Fig. more than five thymocytes and DN2 cells in 18 dpc fetuses of the indicated Bcl11b genotypes. cells continued toon proliferate a T cell lineage –specific transcription factor origieven after 4 weeks. 3B). and representative data are shown.13 0. the of cytes may explain the previous findings that Bcl11b had a severe impact on the generation of loss-of-function mutations the Bc l11b gene are thymic abT cells. The The similar of arrest in same is true for stage cells generated in the DLL4/IL-7 the BcThese l11b−/− mice suggested that cultures and (fig. results in the cultures due to a failure to the arrest segregation to the may gdT be cell lineage occurs up-regulate Bcl11b.enhanced 17). possibility stillretrovirally remains that the gd T cells thatcDNA had been genwe transduced Bcl11b into feerated from leakyand ” DN3 cells these underwent comtal liver LKS“cells cultured transduced pensatory proliferation.0 6. that the S11). we observed nearly complete early progenitors. 3E).1 NK1. 4C). profiles of cells gated on thymocytes CD3–CD4–CD8 [triple negative (TN)] irradiated mice (Ly5.4 84. vivo. the arrested genes werecell suppressed (Fig.73 3. (ACD8 ) Flow cytometric of fetal /− c-kit versus CD25 of cells−gated in Profiles upper panels.2 CD45. lymphomas on the generation of in gdmurine T cellsT (fig. and the + SD isBcl11b shown. indicating Bcl11b As − has beenliver reported (16).6 2.0 6. we produced mice −/− (Fig. Similar to ex vivo fetal /− T cell differentiation up − to the DP stage.63 0. are shown for CD4 versus of 18 dpc fetal analysis thymocytes. Bcl11b deficiency isarrest lethalin around the neoThe similar stage of the DLL4/IL-7 natal period (15 ).39 7. Bcl11b is essential for Profiles T cell lineage determination. a difference that that the b-selection. (E)(Ly5. arrested cells even 4 weeks. recipients of the that occurred in the DLL4/IL-7 /− fetal cells (fig. indicating that self-renewing expansion progenitors in a “feeder-free ” culture system and is not soTCR prominent in vivo.4 11.5 49.59 4. including potential to develop into around macrothe DN3 to N immature SP stage because ancell inphages and K cells (Fig. For each group. rearrange potential S12).35 −/− mice. Bcl11b−/system. on the generation of gd T cells (fig. and thymocytes from Bcl11b Fig. These results suggested that identified in human T cell lymphoblastic the segregation to the gdT acute cell lineage occurs before the DN2mt stage. 12. was a developmental progenitors is velopmental ofthere Bcl11b arrestin at the the adult DN2 stage (Fig. demonstrating progenitors the DN2-determination step enter a that Bcl11b at expression eliminated the DN2 arrest self-renewal cycle. fetal thymocytes. whereas myeloid-lineage –associated −/− Bc l 11b DN2 cells. representative are shown. of indicating /− cells. 3D). 3A). The c-kit+ CD25 cells shown in (C) were cultured (200 cells per from for cultured macrophage and NK cellcells markers flow cytometry.5 49.0 31. at arrest 18 dpc.+ nally identified a tumor suppressor in murine expressing CD4+as CD8 DP stage (Fig.4 4. few (Fig.44 -/.35 CD8 TCR CD8 CD25 Fig. the There was no that Bcl11b expression eliminated DN2 arrest increase in thymic B cells in thecultures.52 2. E) Fetal cells from or Bcl11b (Ly5. moreprofiles than five were individually analyzed. cells under DLL4/IL-7 conditions. which is thought tocould serve be duecritical to the limited niche space in the thymus for as the checkpoint for preTCR formation in early progenitors. The induced by for g irradiation (14) and that chromosame is true cells generated in the DLL4/IL-7 somal aberration disrupting Bcl11b gene was cultures (fig. 2C and figs.1 16. c-kit −/− Data are representative ofLKS three independent experiments.7 55. from To investigate whether the and de−/− found that.7 66.8 3C).+/- CD8 CD8 2.+/- 25. from profiles the indicated mice. S13). Bcl11b-deficient mice exBc l11b DN2 cells features of DN2mt hibit impaired the thymocyte development cells. more than five mice were individually analyzed. Fetal liver cells from Bcl11b or Bcl11b−/− mice (Ly5. In such thymocytes of Bcl11b mice and cultured cellsthe continued toDN2 proliferate cultures. To examine this possibility.63 0. Data are or representative of three well) for 7 days with TSt-4 in theby presence of M-CSF (left panel) IL-15 (right panel) andindependent analyzed for +/+ −/− experiments.8 TCR 4. and loss primitive of B cell thymusand did N not into more potential (fig. −/− T cell lymphoma (14exhibited ). whereas there little effect progenitors in vivo. including the potential to develop into macrothat the Bcl11b− DN2 cells that developed in the phages K dedifferentiate cells (Fig. and TCR b critical gene rearrangement cells that to pass these points succumb to (Fig. and representative profiles are shown. tinuously generated. of self−/− renewing among l11babsence thymoAs hasprogenitors been reported (16Bc ).7 4.0 73.13 5. 3E). the arrested DN2 cells genes were suppressed (Fig. cultured thesewith DN2 cells velopmental arrest We at the DN2 stage. The c-kit CD25+ cells in (C)of were cultured (200 per from Bcl11b−/− of Data cultured are representative three independent experiments.3 CD3 CD3 TN Gated TN Gated 0. such Often transcription factors regulate cellIn lineage / determination steps.59 4. Despite this.3 2. Despite this.8 2.1 congenic mice.7 Bcl11b 66. progenitors in S12).6 0 c-kit c-kit NK1. 3C). To examine before the DN2mt stage. Similar to FFDN2 of cells.6 2. cytometric reconstituted of recipient mice 8 weeks – fraction are shown.6 2.26 3. stage) serve as checkpoints was (16. their DN2 was surface were after equivalent to maintaining DN2mt cells. Among genes up-regulated A A CD4 CD4 Bcl11b+/+ Bcl11b+/+ 2.6 0 2 -/. (C) Flow cytometric analysis ofdays.stage These results demonstrated system absolute of be DN2 cells was not prethymic increased abTCR+ number cells can generated from (Fig.4 Bcl11b-/Bcl11b-/0. the DN2 (Fig. CD8 of 18 dpc group. 3B).6 73. thymocytes from Bcl11b mice were individually analyzed. 3D). and the mean + SD is shown.8 B B x105 305 x10 30 20 20 10 total total +/++/+ +/. (B) Absolute numbers of were total analysis of were c-kit individually analyzed for each group.0 16. of with only a in the presence of a high concentration IL-7 (Fig. of mice cells gated on CD3–CD4 CD8– [triple negative (TN)] data are are shown. and 4A).08 0. Data mice are representative of three independent +/+ of reconstituted irradiated mice Flow cytometric profiles thymocytes of transferred recipient mice weeks experiments. 4C).3 90.3 51.4 0 x105 85 x10 6 8 4 6 2 4 DN2 DN2 c-kit c-kit +/++/+ +/. maintaining their DN2 surface nally identified a tumor suppressor in murine phenotype (Fig. was a developmental rise to that.3 0. which can system. We the phenotype of erated by carefully reducing reexamined the concentration of IL-7 apfetal thymus cells from Bcl11b-deficient micegive and peared to be authentic DP cells. S14A). S14B).7 82.26 0. The At 8 weeks cells under DLL4/IL-7 conditions. S13). Bcl11b-deficient mice exS8 andlymphoma S9). does not additional environunder TSt-4/DLL4 conditions. In right panels.1). 4B). Bcl11b is essential for T cell lineage determination. 3A).4 5.9 4.39 0. the seen thymus.6 31. where T cells are conabsolute number of DN2 cells waschimeric not increased tinuously generated. Werequire cultured these DN2 cells progenitors.3 12.9 4.-/- 25. we produced chimeric mice we retrovirally transduced Bcl11b cDNA into fe−/− fetalthese livertransduced cells into by liver transferring Bcand l11bcultured tal LKS cells irradiated B6Ly5.5 3.

Nature179 452 764 (2008)..Wakabayashi K. Agata. Biochem. Katsura. Heavey. al.1126/science. 200 797 (2004). 169. 30. 1 (2001). Int. S. L. 12. Nature 452. It is quite likely that the A A recent recent study study demonstrated demonstrated that that Bcl11b Bcl11b is is reduction in IL-7 signaling is a physiological expressed in the T cell – like lymphoid expressed in the T cell–like lymphoid cells cells of of mediator of this step. C. 17 ). Wakabayashi et al. Boehmer. T. 401 . Y. 11. 2. Busslinger.. A. M. 201us (2005). 127 (2006). Rev. 452 Nat.. Nat. Erman. 764 (2008). was partially supported by J. Japan. Yamasaki for providing us with reagents Supporting Online Material and protocols. Kawamoto. The appearance of selfdriven progenitors driven checkpoint checkpoint at at which which T T cell cell − progenitors /− thymorenewing progenitors among Bcl11b terminate non-T-lineage non-T-lineage potential in order order to bebeterminate potential in to cytes may explain previous findings that come determined to the T cell lineage (fig.1 1 or or suggests that that progression throughis the DN2C/ /EBP EBPa a. Y. L. was was overexpressed overexpressed in in thymothymoa cells that caused fail to pass these enter points succumb to cytes cytes and and caused the the cells cells to to enter a a self-renewal self-renewal apoptotic cell 19 death. G. thymus. 20. . type killer erated from “leaky ” DN3 cells underwent comleukemia cases leukemia proliferation. Nat. Ikawa. Agata. 2. Q. 19. B. et al .3699 (2007). M. Immunol. Nat. Exp. diminished IL-7. 200 . Y. Education. T. Kawamoto. G. J. 203 (2008). Exp.Int. 8. This J. It It is quite quite likely likely that that the the cycle in vivo ). Wada etNat. H. Lu. Bell. al. References 2 March 2010. 4. Rev.M.. 2. 533 (2003). Wright. J. T. Y. von J. Y.. NatureSports. Rev. L. Rev. Y. A. Katsura. Japan. Wakabayashi et al. Adolfsson J. von Boehmer. Immunol. Immunol. 127 (2002). accepted 12 May 2010 Published 2 July July 2010 Published www. Immunol. 169. induced by g irradiation (14) and that chromosuggests suggests that that progression progression through through the the DN2DN2somal aberration disrupting Bc l11b gene was determination step is instructed by environmental determination step is instructed by environmental identified in human T cell acute lymphoblastic leukemia cases (18) because the acquisition of cytes and caused the cells is to enter a self-renewal signals signals in in the the thymus. we propose propose that that Bcl11b Bcl11b arose arose in in phyphyBcll1b is thought to be a transcriptional repressor we speculate that Bcl11b directly suppresses J. 6. 9 (2008). and and such suppression suppression critical for C that such is critical for determination step is instructed by environmental differentiation differentiation toward toward the the T T cell cell fate. Y. such as PU. and (2009). Yu. B. Immunol. H. T. 598 (2003). 556 (1999).. Y. Katsura. 4. 193 10. Kawamoto.. 30 . M. Leukemia 19. ). 10. 10. Considering Considering that that come determined to the T cell lineage (fig. Rev. Rothenberg. and a known known oncogene. and Culture. Y. Rev. Immunol. for providing with reagents 19. 179 . myeloid-lineage associated genes. Immunol. 9 . Singer. Nature 24 .1126/science. 349 (2004). 263 P. Opin. Erman. Murre. E. signals in the thymus. Immunol. diminished triggered by an extrinsic cue. and S. J. Immunol. Pongubala et al. Busslinger.Trends Y. such as P myeloid-lineage–associated genes. and to P. 20. 20 . Nature . Y. V.Katsura. H. 22. 9. Nutt. A.. Immunol. Q. 797 (2004).. 9.Grant-in-Aid S1 to s16 for Young Scientists (A) from the Ministry of Education. Cell 121 . Y. H..1188995 Materials and Methods Figs S1 to s16 18 . J. Considering that (fig. 22. Y. Opin. 127 (2002). Murre. J. Kawamoto. Moore. L. Med. Nat. 4.preexisting M. Yui. Laiosa. Science 327. Res. Y. ilar was recently observed when L stage) serve as critical checkpoints ( 16 . because because the the acquisition acquisition of of pensatory self-renewal capacity is regarded as the first step self-renewal capacity issteps regarded as the first step The developmental just after the formain development. 18.Rolink. . 295452 (2005). . C.. speculate that Bcl11b directly triggered by an–extrinsic cue. cases ( (18 18). Lu. step. A. Sports. Fehling. K.sciencemag. and S. U. C. 295 (2005). propose that Bcl11b arose20 in.protocols. Kawamoto.. V. Katsube. Kawamoto. S15). a simin leukemia leukemia development. et al. Park. Nature 459. In context. Notes Cell 121. Guo et alScience.Katsura. 301 M.. 12. Bcll1b is thought to be a transcriptional represBcll1b is thought to be a transcriptional represOur finding that that Bcl11b up-regulation can be sor. 9. 9 (2008). H. E. 14. 24 . Because Because a a Bcl11b Bcl11b homolog homolog has not not matically down-regulated at the transition from been been found found in in animals animals other other than than vertebrates vertebrates the DN2 towe the DN3 stage (20). ab TCR ilar outcome outcome was recently observed when Lmo2 mo2.Wakabayashi Yu. Kadesch. S15). H. Immunol. Adolfsson et al. In contrast. 11. J. McCormack 15. 16. 705 (2006). Rev. 16. Rolink. H. (2005). Rev. et al. P. 6. Immunol. Annu. H. M. Immunol. S. The authors are grateful to C. 193 J. H. S16).A recent study demonstrated that Bcl11b is logeny to construct a new lineage distinct from expressed in the T cell –like lymphoid the preexisting innate type killer cells. E..of and andet toal P. Curr. accepted 12 May 2010 2 accepted 12 May 2010 Supporting Online Material 2 March March 2010. 6. T. L. Curr. Stadtfeld. Takahama.Young Scientists (A) from the Ministry of . 13. Bell. weH. H. et Bhandoola. Przybylski et et al al . ). Immunol. Rev. Ikawa. (fig. Nat.1188995 10. the manuscript. C. Nature 13. Rothenberg. 1 (2001). IL-7.1188995 the manuscript. Wright. 7. S16). Kadesch. logeny to construct a new lineage distinct from 2. 15. V. 203 (2008). Bhandoola. Nature . Kawamoto. Commun. 768 (2008).sciencemag. Trends T. 533 A.. 796 (2009). Nat. cells. Science. Katsura. M. Med. K. Guo 17. 18. Laiosa. Y. 301 . M. 19. . Kawamoto. Culture. 401 . 263 Immunol. the arrested cycle cycle in in vivo vivo ( (19). 201 Res. progenitors at the DN2-determination step enter a The The present present study study thus thus defines defines a a Bcl11bBcl11bself-renewal cycle. diminished IL-7. 17. Y. G. we we speculate Bcl11b directly suppresses suppresses sor.Masuda J. (2009). 556 (1999). 3519 (2002). J. 1. J. come determined to the T cell lineage (fig. Yui. because because the the IL-7R IL-7R is is dradradriven checkpoint at which T cell progenitors matically matically down-regulated down-regulated at at the the transition transition from from terminate non-T-lineage potential in order to bethe the DN2 DN2 to to the the DN3 DN3 stage stage ( (20 20). signaling is a physiological reduction in (19 IL-7 reduction in IL-7 signaling is a physiological The present study thus defines a Bcl11bmediator mediator of of this this step. A. Ikawa. B. Y. 14. Singer. T. McCormack .J.1126/science. Biochem. The authors are grateful to C. P. J.. . L. 598 (2003). oncogene. H. critical 20. 9. A. Immunol. M. 3699 (2007). Grant-in-Aid for . 879 (2010). (2003). Feigenbaum. because the IL-7R has is dralamprey lamprey ( (21 21). cells of lamprey (21). Burrows Science for 327 . Nat. 796 21. Leukemia Biophys. Katsura. (1997). 5. V. Y. Graf. T. This work was partially supported by Materials and Methods Figs. 705 (2006). Feigenbaum. Katsura. 349 (2004). Immunity (2009). Immunol. Murre. the innate Y. K. J. Stadtfeld. Biophys. phy1. Immunol. H.Ikawa.. Przybylski etYamasaki al. Immunol. 7. fate. Immunity Commun. 5. 8. Murre. 768 (2008). 21. S16). Nat. alH. . Fehling. Rev. 3. In this this a (DP simtion of preTCR (DN3 stage) andcontext. Y. 2010. Moore. Immunol. (1997). Nutt. . Pongubala et al . 20 . References and 4. Katsube. ). Immunol. 879reading (2010). 459. 6. H. Takahama. H. Burrows for critical reading of www. M. 127 (2006). B. Graf. et J. loss-of-function mutations the Bcl11b gene Our finding finding that that Bcl11b in up-regulation can are be Our Bcl11b up-regulation can be frequently observed in murine T cell lymphomas triggered by an extrinsic cue..Masuda al. ). J. A. S15).P. M. Wada et al. E. 3. 3519 (2002). Park. Because a Bcl11b homolog has not beenReferences found in and animals Notesother than vertebrates (fig. 8. 8 . Annu. 9.

CD4+CD8+ double-positive (DP) thy- CD4+CD8– helper and CD4–CD8+ cytotoxic T cells. he peripheral T cell repertoire is formed after developing thymocytes have undergone a series of developmental selection processes. A Runx-binding sequence within the Th-POK locus acts as a transcriptional silencer that is essential for Th-POK repression and for development of CD8+ T cells. CD8+. This gives rise to two functionally distinct subsets: R1f/f:R3f/f:Cd4 74 5 *These authors contributed equally to this work. Tsurumi-ku. ‡To whom correspondence should be addressed. Japan Science and Technology Agency (JST).1* Sawako Muroi. Japan. (A) CD4 and CD8 expression in lymph node abT cells from mice with indicated genotypes. T A mocytes undergo positive selection through T cell receptor (TCR) interaction with major histocompatibility complex (MHC) proteins. 4-1-8 Honcho. class II° control mice (lane 2). WA 98195– 7650. Kamigyo-ku. Yokohama 230-0045. Identification of the transcription factors network in CD4 and CD8 lineage choice provides insight into how distinct T cell subsets are developed for regulating the adaptive immune system. 2Precursory Research for Embryonic Science and Techonology (PRESTO). 1 Laboratory for Transcriptional Regulation. 19 .riken. whereas cells expressing class I–restricted TCRs differentiate into the cytotoxic lineage linage – and silence CD4 expression (1–3). Intracellular staining of IL-4 and IFN-g analyzed at 6 hours after re-stimulation of cells that were cultured for 5 days after initial TCR stimulation.2 Kaori Akiyama. Kawaguchi. (C) Cell numbers of mature thymocytes and splenocytes showing CD4+CD8– abT cells in class II+ control mice (lane 1). 5).2 Chieko Tezuka. (B) CD4 and CD8 expression in mature thymocytes and LN TCRb+ T cells either in the presence (II+) or absence (II°) of I-A MHC class II molecules. Numbers in the plots in (A). USA. revealed that its expression is essential and sufficient for development of helper-lineage cells (4. E-mail: taniuchi@rcai. 1-7-22 Suehiro-cho.TCRβ+ splenocytes R1+/+:R3+/+:Cd4 65 1 R1f/f:R3+/+:Cd4 13 2 R1+/f:R3f/f:Cd4 74 24 C 100 mature CD4+CD8thymocytes 10 cell number (x106) 34 85 2 17 cell number (x104) 10 R1+/+:R3f/f:Cd4 82 6 R1 446/ 446: R3+/+:Cd4 34 3 Δ Δ R1 +/ 446: R3f/f:Cd4 78 20 Δ R1 446/ 446: R3f/f:Cd4 96 3 Δ Δ 1 1 CD4 11 62 1 1 0.Repression of the Transcription Factor Th-POK by Runx Complexes in Cytotoxic T Cell Development Ruka Setoguchi. Seattle. 1959 NE Pacific Street.1* Yoshinori Naoe. Japan. Cells expressing MHC class II–restricted TCRs differentiate into the helper lineage and cease CD8 expression.1 Tsukasa Okuda. and class II° Runx1D446/D446:Runx3 f/f:Cd4 mice (lane 4). (D) Expression of CD154 at 42 hours after in vitro TCR stimulation of control CD4+. class II+ Runx1D446/D446:Runx3 f/f:Cd4 mice (lane 3).1*† Masashi Tachibana. Th-POK. 3Kyoto Prefectural University of Medicine. Saitama 332-0012. We report the redirected differentiation of class I–restricted thymocytes into CD4+CD8– helper-like T cells upon loss of Runx transcription factor complexes. University of Washington. †Present address: Department of Immunology. Th-POK expression and genetic programming for T helper cell development are actively inhibited by Runx-dependent silencer activity.2‡ Mouse CD4+CD8+ double-positive (DP) thymocytes differentiate into CD4+ helper-lineage cells upon expression of the transcription factor Th-POK but commit to the CD8+ cytotoxic lineage in its absence. mature thymocytes (HSA. Japan.TCRβhigh) LN TCRβ+ cells D 42 21 5 1 II+ 65 1 IIo 9 2 60 II+ 1 4 IIo CD4+ 34 88 38 93 2 3 31 CD8+ R1Δ446/Δ446: R3f/f:Cd4 95 3 67 31 95 4 88 6 1 0 0 3 CD4 class Irestricted CD4+CD8- 20 32 4 CD154 IL-4 CD8 IFN-γ CD4+CD8. (B). allowing for cytotoxic T cell differentiation. and (D) indicate the percentage of cells in each quadrant or region. RIKEN Research Center for Allergy and Immunology.1. Differentiation of class I–restricted cells into CD4+CD8– helper-like cells by loss of Runx complex function. Kawaramachi-Hirokoji.3 Ichiro Taniuchi1. 1. gain or loss of function of the BTB/POZ domain–containing zinc finger transcription factor. Thus.1 1 2 3 4 0. and class I–restricted CD4+CD8– cells. Kyoto 602-8566.1.1 1 2 3 4 CD8 B cont. Recently. Error bars indicate standard deviation.

The numbers of transgenic founders expressing GFP among the total transgenic founders are indicated at right. control IgG 3. Runx proteins possess a conserved Val-Trp-Arg-Pro-Tyr (VWRPY) motif at the C-terminal end. 0 0 0 0 4+ 8+ 4+ 8+ 4+ 8+ 4+ 4+8int GFP Cbfβ GFP Cbfβ Runx1D446/D446 (lane 1 2 3 4 5 6 7 8 9 f/f Wt R1f/f:Cd4 R3f/f:Cd4 Cbfβf/f:Cd4 3). A marked reduction of splenic CD8+ T cells in Runx1D446/D446:Runx3f/f:Cd4 mice (Fig. (C) Relative Th-POK expression abundances after reconstitution (lane 8) mice and in CD4+ and CD8+ peripheral T cells in mice of the of Runx complex function. De-repression A B C 100 700 160 of Th-POK by loss of 120 Runx complex func600 80 100 120 tion. Black boxes represent exons. and Cbf b f/f:Cd4 thymocytes. allowing the recruitment of the Groucho/TLE co-repressor proteins to their target genes (10. with those in red indicating evolutionarily conserved Runx motifs. The regions at 1 kb upstream of exon Ia (UP1) and the TCRb enhancer (TCRb) were used as negative and positive controls. Relative Th-POK expression (arbitrary units) A 10 kb Lenep Relative Th-POK expression (arbitrary units) C Th-POK Thymocytes ATG TAA Relative Th-POK expression (arbitrary units) Splenocytes CD8+SP 8 CD69-DP 0 CD4+CD8int 53 CD4+SP 52 CD4+ 50 CD8+ 4 Ia Ib II III a RBS 1 2 GFP Kp Δ674 bp 2 51 70 50 85 72 3/3 a b c d 15. 2. (B) ChIP experiment showing binding of Runx complexes to RBS-1 and RBS-2 in the indicated cell subsets. HindIII (H). respectively. EcoRV (RV). Cbf b protein (6). and numbers in parenthesis indicate mean fluorescent intensity of GFP in GFP+ cells. The restriction sites shown are Eco47III (E47).6 kb GFP 593 bp c 3/6 e f g H 562 bp RV 5. Lane 9 in (A) indicates Th-POK expression in control CD4+CD8– SP vector encoding Cbfb (Cbfb). Identification and characterization of RBSs at the Th-POK locus.5 kb b E47 RV 1/1 GFP GFP 0 21 26 (610) 0 12 (203) 0 H 6. KpnI (Kp). Input 2. . and XhoI (X). 1A and fig. The dashed line indicates nontransgenic littermate control. we used the Cre/loxPmediated conditional gene inactivation (7) to clarify Runx complex function in silencing of the Cd4 gene (8) and recently reported that the combined inactivation of Runx1 and Runx3 in DP thymocytes resulted in a dramatic loss of CD8+ T cells (9). One representative result out of three experiments is Cbfb f/f:Cd4 mice were transduced with control retroviral vector (GFP) or shown. we introduced the Runx1D446 allele (12) that generates a mutant Runx1 protein lacking the VWRPY motif on a Runx3deficient background (Runx3f/f:Cd4 mice) (13). Runx1 :Cd4 (lane 2).Runx transcription factor complexes are composed of heterodimers for one of three Runx proteins and their obligatory non–DNA-binding partner. Cbf b f/f:lck (lane 7). Runx1D446/D446:Runx3 f/f:Cd4 (lane 6). Because of the embryonic or neonatal lethality of mice deficient for any of Runx family genes.4 kb GFP GFP GFP 0 9 22 (624) 0 23 (380) 0 d 2/2 XX B DP 1 2 3 Runx sites mutated 1 16 26 12 47 25 Thymocytes CD4+SP CD8+SP 1 2 3 1 2 3 Splenocytes 4+T 1 2 3 1 e 2/2 8+T 2 3 0 18 48 1 87 3 RBS1 RBS2 UP1 TCRβ 1. The maps for each reporter transgene construct (Tg-a to Tg-g) are indicated. and each green bar represents the signal intensity of an individual oligonucleotide probe in a ChIP-on-chip experiment. Runx3 :Cd4 (lane 4+84+8int 4). Runx1 f/f:Runx3 f/f:Cd4 (lane 5). anti-Cbfβ2 Ab f 3/3 1 60 83 47 80 36 g 1/1 20 20 Fig. (C) Histograms showing the GFP expression in the indicated T cell subsets from representative transgenic founder for each construct. S1) indicated that VWRPY-dependent repres- Fig. Blue boxes represent RBSs. (A and B) Rela500 80 tive Th-POK expression 60 60 80 abundances (normal60 40 40 ized to hprt) in sorted 40 CD69– DP thymocytes 40 20 20 (A) from wild-type (lane 20 f/f 1). Numbers in the histogram indicate the percentage of GFP+ cells. (A) The structure of the murine Th-POK locus is shown at the top.1 kb 3. 11). Purified CD4+CD8– and CD4+CD8int cells from indicted genotype (B). 3. To test whether VWRPY- dependent repression might be involved in the loss of CD8+ T cells. Circles represent putative Runx motifs.

A 15. We next examined the functional properties of these CD4+CD8– cells. 3A) and contain two or one conserved Runx motifs. and ThGFP:SD GFP .8 0. Purified CD4+CD8– and CD4+CD8int cells were transduced with a retroviral vector encoding Cbfb or with an empty vector control. 2A). 1D). We conclude from these results that class I–restricted CD4+CD8– cells that develop in Runx mutant animals are functionally helperlike T cells. remaining B Fig. To better understand the functional activities of RBS-1 and RBS-2 in light of these results.5-kb genomic fragment encompassing the RBSs and exons Ia and Ib was linked to a green fluorescent protein (GFP) reporter transgene cassette (Tg-a in Fig. 1A). 2C). expression of Th-POK was markedly reduced upon re- expression of Cbf b in CD4+CD8int cells. 1. Th-POK expression was not detected in control CD69– DP thymocytes.28) expression abundances – Δ + /GFP:S 10 in sorted CD69 DP thymocytes showing derepression of Th-POK 0 upon deletion of the Th+/+ SΔ/+ Cbf β f/f: GFP POK silencer. In these experiments. Th-POKGFP 34.4 1. Because ectopic expression of Th-POK has been shown to redirect class I–restricted cells to become CD4+CD8– cells (4. 5).2 1. we used these mice for further analyses. and Th-POK hemizygous transgenic (Th-POK Tg) 20 mice. Because the leaky CD4–CD8+ subset that escaped Cre-mediated recombination (9) was less apparent in Runx1D446/D446:Runx3f/f:Cd4 mice (Fig. To understand mechanisms underlying Runxmediated repression of Th-POK. 2B) that still developed in Cbf b f/f:Cd4 mice because of the slow turnover of Cbf b protein after inactivation of the Cbf b gene (13).sion by Runx1 was involved in the generation of CD8+ T cells. Potentially. respectively. Essential require. including a strain in which the Cbf b gene is conditionally inactivated by either a LckCre or a Cd4-Cre transgene (13). GFP expression was first detected in postselection CD4+CD8int thymocytes and was upregulated in CD4+ SP thymocytes. respectively (Fig.5 Th-POK GFP .3 0.A ment of the Th-POK siTCRβ+ LN cells RBS-1 RBS-2 ATG TAA Ia Ib lencer for development +/+ SΔ/+ Th-POK Tg Th-POK 64.8 matic structure of the Th-POKSΔ Th-POK locus and tarGFP geted alleles Th-POKSD.1 kb upstream and ~7. with no detectable effect in CD4+CD8– cells (Fig. we performed transgenic reporter assays. These results indicated that the absence of Runx complexes forced the majority of class I–restricted cells to differentiate into CD4+CD8– T cells. 3A). a 40-fold increase in Th-POK transcript abundances was detected in CD69– DP thymocytes in which Runx complexes were disrupted either by combined Runx1 mutations with a Runx3 deficiency or by loss of Cbf b protein (Cbfbf/f:Lck mice) (Fig. (D) GFP Lck expression from the ThPOKGFP and Th-POKGFP:SD alleles in indicated thymocyte subsets. These were observed in control CD4+ T cells as well as in class I–restricted CD4+CD8– cells. 3B). In contrast. 3A and figs. In contrast. Relative Th-POK expression (arbitrary units) CD4 21 . By using ChIP analysis in T cell subsets. we measured the expression of Th-POK in several Runx mutant mice.4 kb downstream of exon Ia (Fig. we examined whether Runx complexes directly associate with the Th-POK locus.9 93. These results suggest that Runx-mediated Th-POK repression operates in peripheral CD8+ T cells. respectively) are located ~3. A modest Th-POK de-repression by inactivation of Runx1 alone indicated a redundant function of Runx3 in the repression of Th-POK. 1D).7 1. Although there was a marked decrease in CD4+CD8– T cell numbers in control class II–deficient mice. Although Th-POK mRNA was undetectable in control CD8+ T cells and in CD8+ T cells deficient for Runx1 or Runx3.4 0. the ligand for CD40. Dashed and bold lines indicate GFP expression in control mice and Th-POK+/GFP (+/GFP) or ThPOK+/GFP:SD (+/GFP:SD) mice. (B) CD4 and CD8 ex. B and C). the predominance of CD4+CD8– T cells persisted in class II–deficient Runx1D446/D446:Runx3f/f:Cd4 mice in both the thymus and the periphery (Fig. we confirmed an association between Runx complexes and these two regions (Fig. Th-POKSD +/GFP 30 heterozygous (SD/+). respectively.9 3. it was absent in both wild-type CD4+ T cells and in class I–restricted CD4+CD8– cells (Fig. the loss of CD8+ T cells could occur either by a developmental block of class I–restricted cells or by a redirection of class I– restricted cells toward the CD4+CD8– lineage. revealing that the binding of Runx complexes to these regions did not correspond with Th-POK repression.7 of CD8+ T cells. but not in control CD8+ T cells (Fig. although high interferon-g (IFN-g) production was detected in control CD8+ T cells. it was present in Cbf b-deficient CD4+CD8int T cells (Fig.3 0. We therefore next examined whether Th-POK repression could be restored in these CD4+CD8int cells upon reexpression of Cbf b protein. The numbers in parenthesis indicate mean fluorescent intensity of GFP in total CD69– DP thymocytes. Using a ChIP-on-chip (ChIP indicates chromatin immunoprecipitation) approach with an antibody against Cbf b2.C CD69-CD4+CD8+ CD4+CD8CD4-CD8+ D CD69 DP thymocytes 40 pression in lymph node (6. However. Consistent with a previous report (4). Exons and loxP Th-POKGFP:SΔ POK sequences are indicated as black boxes and black CD8 triangles. One of the characteristic features of CD4+ helper-lineage T cells is the early induction of CD154. In all transgenic mouse founders obtained with Tg-a. S2 and S3). (A) Sche96. binding of Runx complexes to RBS-1 and RBS-2 was detected in both Th-POK–expressing and nonexpressing cells. we detected two regions occupied by Runx complexes within the Th-POK locus. To determine whether CD4+CD8– cells that emerge in Runx mutant mice are class II–restricted or redirected class I–restricted cells. 4. we crossed Runx1D446/D446:Runx3f/f:Cd4 mice onto a MHC class II–deficient background (14). Distal and proximal Runx-binding sequences (RBS-1 and RBS-2. (C) Relative Th-POK (17.79) abT cells from wildtype (+/+). after TCR stimulation (15) and the production of interleukin-4 (IL-4).

and tinguish differences TCR signaltinguish qualitative differences in TCR signaling. We therefore refer to RBS-1 as References andiNotes 1. Identification of 13. 91 (2004). Cells Zou. 83... S5). I. Littman. 11. J. et al. Tillman. Natl. Germain.20. F. Blood . E.. Although GFP expression from the ThPOKGFP locus was not detected in CD69– DP thymocytes. J. 91 (2004). Sarafova et al . . K. 151 . indicating that RBS-1 is a transcriptional silencer required to repress the reporter gene in CD8 lineage cells. 2497 (1993). Rajewsky. an inhibitor of Runx-dependent Cd4 silencer acP. Cell. 280 (2004). Runx complexes with the Th-POK silencer in 523 (1999).. H.Noelle. S. C. He et al21 . The antagonistic interplay between primary 9. Jameson. Levanon et al. we deleted either 5′ or 3′ sequences as well as RBS-1 from the 15. R. Rothenberg. Caudy. Rev. Deletion of RBS-1 from one Th-POK allele led to the loss of peripheral CD8+ T cells (Fig. 5581 (1997). M.S.(2000). Cell 111 . 17 W. ing. 685 (1999). . E. L.. Papaioannou. L. 685 (1999). JST. Rothenberg. 1.D. 1. Ellmeier. 5581 (1997). 1. 17K.. Further studies on the regulatory pathways cytotoxic-lineage mature thymocytes (Fig. Singer.high in splenic CD4+ T cells.1945 Proc.sciencemag. 83. 1417 (1991). T. S3) was found to be required for positive transcriptional regulatory activity (as in the Tg-c and Tg-d constructs). Johnson. Ledbetter. Y.. (1993). Orkin. R. Further studies on will the regulatory Our results reveal that helper lineage–specific of Th -POK repression shed lightpathways on how of Th-POK repression will shed light are on conhow signals initiated by external stimuli expression of Th-POK is regulated by the RBS-1 signalsinto initiated by externalinstimuli consilencer. It is therefore possible that TCR signals 22. Immunol. Taniuchi et al . 4405 (2007). .. Waldschmidt. B. K. Proc. Immunol.19.1151844 22 . 3C). We 22. 4.. E. A.5-kb fragment thus contains the major cis-regulatory regions that direct expression of Th-POK in the helper lineage. Science 253 1417 (1991). Med. Immunol. G. S1 and to S5 S1 to S5 References Th-POK repression may require class II–specific Figs. Immunol. Hogquist. Taniuchi. 75 grateful R.562 (2004). Blood 562 (2004). 4A) resulted in uniform de-repression of GFP in CD69– DP thymocytes. Natl.A. M. Y.. D. repression operates in all pre-selection DP thymo. 506 (2000). 1155 (1993). Med.. To further narrow down the critical Th-POK regulatory regions. R. 95. Miele. Nature 404 . thymocytes. L. Rajewsky. etJ. Immunol. 6. C.A. deletion of a 674-bp fragment of RBS-1 (Tg-b) resulted in GFP expression both in CD4+ helperlineage and in CD8+ cytotoxic-lineage cells. Aronson. J. Johnson. J. 1749 (2007). 8. Annu.Cell. References TCR signals during a specified time window. Cell 73. K. 1155 (1993). ity of silencer activity is not regulated at the level 21 .Rev. (2002). 179 4405 (2007). The accession number for antagonism of Th-POK silencer activity and by grants from PRESTO. cells (16. D. 12. Adv. Ledbetter. Orkin. Rev. Immunol. Singer. D. K. deletion of RBS-1 (Th-POKGFP:SD locus in Fig.179 57 . V. Y.10. 5. Med. H. L. 621 (2002). Papaioannou. 57 (2000). 13. play between these two factors. . S. Immunol. A.Aruffo. Egawa. R. Y.1126/science. Caudy. Zou. Genes Cells 4. S. 8. Adv. J. Cell 73. GFP expression in mice heterozygous for Th-POKGFP allows us to examine expression of Th-POK at the single-cell level. 3. Nat. Rev. I. 826 (2005). X. M. The 15. 139 (2003). Th-POK was recently described as 11. Th-POK and Runx complex target genes will 14. A. T. Nat. Gergen. The accession number for mouse Th-POK silencer is EU371956 in GenBank. H. R. K. J. U. Science 253V. 204 . D. Nishimura Naoe et al. Annu. Tillman. 139 (2003). C. Egawa. Wildt et al J. Gu. D. Nat. (1998).Bosselut. 18. Yasutomo. 17 October 2007. consistent with an antagonistic inter. K. Littman. Noelle. T. K. Littman. Doyle. V. Acad. (2007). T. 441 (2007). Germain. D. followed by Th-POK qualitative silencer activity acts asin a sensor to dishigh GFP expression in both helper.. Nat.R. J. Grusby. S. Efficient repression of GFP in CD8+ T cells by a 562-bp fragment of RBS-1 (fig. Glimcher. Jameson. A. Ellmeier. 2. C. We further investigated Th-POK de-repression by using mice in which the coding sequence for Th-POK was replaced with the gfp gene (Th-POKGFP locus). Littman and W. .H. Sci.are Sarafova etto alD. Y.103 17. 15. X. Nat. Liu. Sun et al. Nat. Liu. U. Biol.75 (2005). Nature 433. Immunol. Uniform de-repression of Th-POK in CD69– 18. R. Immunity 235 . Littman23 and W. S4). Naoe. To examine the physiological function of the RBS-1 silencer. V. We are grateful to the D. H. S.S. alExp. 5. S. Bosselut. Hogquist. J. . E. J. R. 6. 4B) and to the Th-POK de-repression in CD69– DP thymocytes (Fig. network regulating lineage specification of DP 16. of Runx complex binding. This work was supported by grants fromof PRESTO. Taniuchi. Material and Methods Methods lineage (19–21). Rev. Blechman. POK silencer may therefore have a central role 7. Biol. . Ellmeier for cytes. H.Ito. Immunol. Mol.(2007).Nishimura Gergen. S2) in the context of Tg-e construct required Runx motifs (Tg-f and Tg-g) (Fig. Ito. Gu. mouse Th-POK silencer is EU371956 in GenBank. Immunol. Sawada. lencer indicates that silencer-mediated Th-POK 20. Immunity . Littman. 373 (2005). Yasutomo. 11590 two opposing fates are induced in progenitor to be necessary for specification of the helper www.. 151. 17. 21. whose activity depends on binding of verted genetic programs the cell are nucleus. R. we deleted the 674-bp KpnIEco47III sequences from the Th-POK locus by homologous recombination in embryonic stem (ES) cells (Fig. 280 (2004). 621 9. K. 4D). Immunol.A. C. 4A and fig. M. R.. Roy.5-kb fragment. Fisher.. A. E. I. Annu. N. Immunol. . Exp. Ellmeier for critical reading of manuscript.(2005). J. A. F. Exp. R. di i h ll l Runx complexes.103 204 1749 (2007). P. Exp. S. whereas it was almost undetectable in splenic CD8+ T cells (Fig. help to further unravel the transcription factors 15.. Genet. D. 19. B. The association of References and Notes 2. T. Cell 111. Glimcher.Naoe J. J. Grusby. consistent with Runx-dependent activity of RBS-1 silencer. A. JST. M. cells expressing Th-POK indicates that specific3. the Th-POK silencer (fig. Aruffo. 441 (2007). 4C).Mol. Immunol. in regulating Th-POK silencer activity. Acad. 2497 E. 523 (1999). R. Miele. Taniuchi al. S.12. M. Nature 404. D. R. 21. N. Aronson. Fuchs. (1998). A. R. Naoe. Genet. 7. Starr.17 Blechman. R. Fuchs.sciencemag. 1945Y. Fisher. Wildt et al. A. K. S. Y. Immunol. 14. reversal of silencer-mediated Material Figs. et al . 95. 204 . Waldschmidt. 11590 Levanon et al. W. 506 (2000). Starr. T. Whereas RBS-2 (fig. I. D. tivity (18). . Bosselut. M. Given that susSupporting Online Material tained class II–specific TCR signals are thought Supporting Online Material www. Sci. X. R. Med. R. Immunol. 8 .Doyle. Sawada. J. C. This work was supported after engagement of MHC class II result in critical reading the manuscript. K.. R. L. S. that interact with Runx factors bound to the Th6. L. Annu. Additional molecules 4.. accepted 20 December 2007 Our results suggest that a mechanism regulating Published 8 February 2008 Th-POK silencer activity acts as a sensor to dis. 16. M. 17). 8. 17.Genes Y. 3C). thus induce Th-POK expression. J. Nat. Roy.. 204 lineage-determining factors is often observed when 10. R. Bosselut. DP thymocytes upon deletion of the Th-POK si.

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Toronto. where it induces downstream signals.utoronto. † Present address: Faculty of Life Sciences. E-mail: saito@rcai. in vivo T cell responses were significantly impaired. Research Center. Elford. Japan. † To whom correspondence should be addressed. Toronto.1* Douglas G. University Health Network and Department of Medical Biophysics. Chiba (T. 230-0045. such as TFH cells.2† Midori Unno.1* Noriko Komatsu.3 Tasuku Honjo. CA 94080.H. Chiba University Graduate School of Medicine.1 Hiromitsu Hara.4* Keiichiro Suzuki.) SCIENCE | VOL 311 | 31 MARCH 2006 | PAGES 1927-1932 25 . Chiba City. Thus. University of Toronto. ǁ To whom correspondence should be addressed. Yokohama. in the absence of IRAK-4. Sakyo-ku. The nature of these TFH cells in Peyer’s patches has been elusive. § Present address: Amgen San Francisco.4‡ Arata Takeuchi. 620 University Avenue.2* Shimpei Kawamoto. Minatojima-minami-machi. (S.3§ǁ Takashi Saito1ǁ IRAK-4 is a protein kinase that is pivotal in mediating signals for innate immune responses.Y. Japan. Kanagawa 230-0045. Tsurumi-ku.1 Nien-Jung Chen.3 Denis Bouchard. RIKEN Research Center forAllergy and Immunology. 1-5-5. Kyoto 606-8501. Tsurumi. University of Manchester. Here. South San Francisco. Kyoto University. we report that IRAK-4 signaling is also essential for eliciting adaptive immune responses. 230-0045. Japan. ‡ Present address: Faculty of Pharmacy.). Yokohama City. RIKEN. Yokohama 1-7-22. including protein kinase CΘ activation through the association with Zap70. Japan. Suite 706. Thus. 1 2 Laboratory for Cell Signaling. 2 Research Unit for Immune Homeostasis. Japan. Yokohama.1 Osami Kanagawa.1. 4 Department of Immunology and Genomic Medicine. Research Center for Allergy and Immunology.1 Shinobu Suzuki.). Our findings suggest that T cells use this critical regulator of innate immunity for the development of acquired immunity. Yokohama 1-7-22. 3 Advanced Medical Discovery Institute. Canada. environmental cues present in gut Peyer’s patches promote the selective differentiation of distinct helper T cell subsets. Tokyo 202-8585.) SCIENCE | VOL 323 | 13 MARCH 2009 | PAGES 1488-1492 A Critical Role for the Innate Immune Signaling Molecule IRAK-4 in T Cell Activation Nobutaka Suzuki.2 Sho Yamasaki. Institute for Breast Cancer Research.Preferential Generation of Follicular B Helper T Cells from Foxp3+ T Cells in Gut Peyer’s Patches Masayuki Tsuji.2 Jun-ichiro Suzuki. BMA 3F. Tsurumi.3 Alisha R. 1-7-22 Suehiro-cho.1 Tadashi Yokosuka. Ohashi. 230-0045. Yokohama 1-7-22. Kobe 650-0047. IRAK-4 is recruited to T cell lipid rafts. USA. (S. Tsurumi.-C. 1 Laboratory for Mucosal Immunity.riken. Millar. University Health Network. Nishitokyo-shi.1 Christine Mirtsos. Manchester M13 9PT. (W. Ontario M5G 2C1. Oxford Road. C-2259 Michael Smith Building. Musashino University. Upon T cell receptor stimulation. K. Japan. sidonia-f@rcai. 620 University Avenue. 3 Laboratory for Autoimmune Regulation. Japan. RIKEN.4 Shohei Hori. E-mail: shohei@rcai. 1-1-20 Shin-machi. *Present address: Nihon Schering K. Suite 706. 1120 Veterans Boulevard. suggesting that IRAK-4 may be involved in direct signal cross talk between the two systems. 1-8-1 Inohana.2 Wen-Chen Yeh. Graduate School of Medicine. Here. *These authors contributed equally to this work.riken. we demonstrate that suppressive Foxp3+CD4+ T cells can differentiate into TFH cells in mouse Peyer’s patches.F. Chuo-ku.3 Pamela S.riken. Canada. Ontario M5G 2C1. Yokohama. This signaling pathway was found to be required for optimal activation of nuclear factor κB.2† Sidonia Fagarasan1† Most of the immunoglobulin A (IgA) in the gut is generated by B cells in the germinal centers of Peyer’s patches through a process that requires the presence of CD4+ follicular B helper T (TFH) cells. 4 Department of Molecular Genetics.S. wyeh@uhnres.1 Thomas Calzascia. Chuo-ku. The conversion of Foxp3+ T cells into TFH cells requires the loss of Foxp3 expression and subsequent interaction with B cells.

These results identify ssRNA as a ligand for TLR7 and suggest that cells of the innate immune system sense endosomal ssRNA to detect infection by RNA viruses. E-mail: nagata@genetic. Tsurumi-ku. Milk fat globule epidermal growth factor (EGF) factor 8 (MFG-E8 ) is a protein that binds to apoptotic cells by recognizing phosphatidylserine and that enhances the engulfment of apoptotic cells by macrophages. and suffered from glomerulonephritis as a result of autoantibody production. Single-stranded RNA (ssRNA) molecules of nonviral origin also induce TLR7-dependent production of inflammatory cytokines. Many apoptotic lymphocytes were found on the MFG-E8—/— tingible body macrophages. but the pathways linking virus recognition to IFN induction remain poorly understood. These data demonstrate that MFG-E8 has a critical role in removing apoptotic B cells in the germinal centers and that its failure can lead to autoimmune diseases. Diebold. 2-2 Yamada-oka. 5 Laboratory of Genetics. Japan. Osaka 565-0871. Yokohama.3 Hiroaki Hemmi. Japan. We report that tingible body macrophages in the germinal centers of the spleen and lymph nodes strongly express MFG-E8.4 Kay Miyasaka.5 Katsuyuki Aozasa. Japan. Graduate School of Frontier Biosciences. with the formation of numerous germinal centers.1 Masato London Research Institute.6* Apoptotic cells expose phosphatidylserine and are swiftly engulfed by macrophages. Integrated Biology Laboratories. 2 Department of Pathology.3 Yasuo Uchiyama. Japan Science and Technology Corporation. 1 Immunobiology Laboratory. Kanagawa 2300045. 2 Department of Host Defense. Here. Research Institute for Microbial Diseases. Plasmacytoid dendritic cells produce vast amounts of IFN-α in response to the wild-type influenza virus.3 Shigekazu Nagata1. Suita City.2. Japan.5. *To whom correspondence should be addressed.1. Caetano Reis e Sousa1* Interferons (IFNs) are critical for protection from viral infection. The MFG-E8—/— mice developed splenomegaly. 1 Department of Genetics. E-mail: caetano@cancer.2 Shizuo Akira.2 Masato Koike.2. Suita.1 Tsuneyasu Kaisho. and 3 Department of Cell Biology and Neuroscience.Autoimmune Disease and Impaired Uptake of Apoptotic Cells in MFG-E8–Deficient Mice Rikinari Hanayama. SCIENCE | VOL 304 | 21 MAY 2004 | PAGES 1147-1150 Innate Antiviral Responses by Means of TLR7Mediated Recognition of Single-Stranded RNA Sandra S. Cancer Research UK. Osaka 565-0871. 4 Akira Innate Immunity Project. Japan. 6 Core Research for Evolutional Science and Technology. Osaka 565-0871. Osaka University. 4 Laboratory for Innate Cellular Immunity. *To whom correspondence should be addressed. but they were not efficiently engulfed. London WC2A 3PX. RIKEN Research Center for Allergy and Immunology. Osaka SCIENCE | VOL 303 | 5 MARCH 2004 | PAGES 1529-1531 26 . Yokohama City. 3 RIKEN Research Center for Allergy and Immunology. Osaka 1-7-22 Suehiro-cho. Kanagawa 230-0045. Japan Science and Technology UK. Osaka 565-0871. Yamadaoka 3-1. we show that this requires endosomal recognition of influenza genomic RNA and signaling by means of Toll-like receptor 7 (TLR7) and MyD88. Osaka University Medical School. ERATO.

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†To whom correspondence should be addressed.1 Hitoshi Nagaoka. Japan. Graduate School of Medicine. 1 Department of Medical Chemistry. Yoshida. 3 Department of Pathology and Biology of Disease. Reduction of bacterial flora by antibiotic treatment of AID—/— mice abolished ILF hyperplasia as well as the germinal center enlargement seen in secondary lymphoid tissues. E-mail: honjo@mfour. *These authors contributed equally to this work. Kyoto University. 2 RIKEN Research Center for Allergy and Immunology.Critical Roles of Activation-Induced Cytidine Deaminase in the Homeostasis of Gut Flora Sidonia Fagarasan. Tsurumi-ku. Kanagawa 230-0045. these results suggest that SHM of ILF B cells plays a critical role in regulating intestinal microflora. Yokohama. Kyoto Hiroshi Hiai. Because an inability to switch to immunoglobulin A on its own does not lead to a similar phenotype.1* Keiichiro SCIENCE | VOL 298 | 15 NOVEMBER 2002 | PAGES 1424-1427 28 .3 Tasuku Honjo1† Activation-induced cytidine deaminase (AID) plays an essential role in class switch recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin We report here that deficiency in AID results in the development of hyperplasia of isolated lymphoid follicles (ILFs) associated with a 100-fold expansion of anaerobic flora in the small intestine. Japan.2*Masamichi Muramatsu.

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