Introduction

　The immune system protects our life from invasions of various pathogens, e.g. bacteria and virus, and also from various diseases caused by mutations, e.g. tumorigenesis and neurodegeneration. The most basic feature of the immune system is self-tolerance and immune response against non-self antigens. While immune responses are important for protection of our life, excess activation of the immune reactions against self-antigens and pathogens causes various immune diseases such as autoimmunity and allergic reaction. Thus understanding of the regulation of immune system is very important. However, it is a very complex system and many problems still remain to be studied.

　Dendritic cells (DCs) are professional antigen presenting cells (APCs) and act as sentinels in peripheral tissues, continuously sampling antigens, and initiate immune responses by presenting antigens to T cells upon encounters with microbial products or tissue damages. In contrast, DCs in the thymus play a major role in central tolerance of T cells, which is achieved by inducing apoptosis of self-reactive T cells. A variety of DC subsets have been described based on their cell surface phenotype and morphology and are widely distributed in various tissues including lymphoid such as the thymus, spleen and lymph node and non-lymphoid tissues such as the skin, liver and gut. However, the developmental origin and functions of DC subtypes have been a controversial issue. Differences in the cytokine and transcriptional factor requirements suggest different developmental pathways for DCs. In this study, I investigate roles of Oncostatin M (OSM), a member of the IL-6 family cytokines, for the development of DCs in the fetal thymus and for the immune response of peripheral DCs.

Results and discussions

1. Development of dendritic cells in the fetal thymus

1-1. A unique feature of thymic DCs at fetal stage

　T cell development occurs most actively in the fetal thymus and thymic DCs are involved in T cell selection. A unique feature of thymic DCs is that they can be derived from intrathymic T/DC precursors and stay in the thymus. However, the exact origin of thymic DCs and factors involved in the DC development still remain unclear. Most DCs are CD8α+CD11b- in the adult thymus, but I found that DCs in the fetal thymus are negative for CD8α and partially positive for CD11b. These CD8α-CD11b+ cells express co-stimulatory molecules and MHC class II molecule at high levels and exhibit strong APC activity. Thus, these results suggest that CD11b+ DCs are functionally maturated APCs and are involved in T cell selection in the fetal thymus.

1-2. Thymic epithelial cells (TECs) induce the development of fetal-type DCs from thymocytes

　In our laboratory, E. Esashi established a fetal thymic epithelial cell line, ORTEC, which depends on OSM for proliferation, and reported that OSM receptor (OSMR)-positive thymic epithelial cells were located at the cortico-medullary junction where T cell selection occurs actively. Thus I examined whether thymic DCs were induced from thymocytes by ORTEC and fount that ORTEC conditioned medium (ORTEC-CM) induced generation of CD11b+ DCs from intrathymic T cell progenitors and these DCs exhibited strong APC activity. This indicates that TECs produce a soluble factor that induces thymic DC development.

1-3. Identification of a factor required for DC development and function

　To identify a factor produced by ORTEC, I analyzed the expression of cytokines known to stimulate DC development or to maintain survival of thymocytes by using RT-PCR and microarray analysis. Among various cytokines I examined, only IL-18, which is known to activate the TRAF6 signaling pathway, was clearly expressed in ORTEC and the thymus. I found that the IL-18 receptor subunits were expressed on T cell progenitors and that IL-18 induced the development of CD11b+ DCs from T cell progenitors. Furthermore, I found a significant reduction of CD11b+ DCs in TRAF6-deficient fetal thymus, indicating that TRAF6 is required for the thymic DC development. However, as no such reduction was found in IL-18 receptor-deficient fetal thymus, an additional cytokine that activates TRAF6 is involved in the DC development in vivo. I also showed that the IL-18-induced DCs exhibited strong APC activity and preferentially bound CD4+CD8+T (DPT) cells. Moreover, apoptotic T cells were frequently found in the conjugates formed by these DCs. Taken together, these results strongly suggest that CD11b+ DCs induced by TEC-derived IL-18 are actively involved in T cell selection by inducing apoptosis of DPT cells in the fetal thymus.

2. OSM negatively regulates peripheral immune response via DCs

2-1. OSM-deficient mice exhibit enhanced Th1 response

　OSM-deficient mice are born according to the Mendelian rule of inheritance, appear normal at birth and develop normally. Most of immune cells including T cells are developed and distributed normally in OSM-deficient mice. However, the development of thymic CD4+ macrophages, which efficiently phagocytosed apoptotic thymocytes in the thymus, was impaired. In accordance with the reduction of professional scavenger of apoptotic cells in the thymus, apoptotic cells were accumulated in OSM-deficient thymus. It is well known that impaired clearance of apoptotic cell leads autoimmune disease. In fact, OSM-deficient mice exhibited a symptom of autoimmunity by aging. However, it was not clear whether autoimmunity of OSM-deficient mice was caused solely by the impaired clearance of apoptotic thymocytes in the thymus. Therefore I considered another reason for autoimmunity of OSM-deficient mice.

　T cells are composed of CD8+ T cells (cytotoxic T cells; Tc) and CD4+ T cells (helper T cells; Th). CD4+ T cells are further divided into Th1 cells and Th2 cells and those subsets regulate distinct types of immune response. Th1 cells, which predominantly secrete IFN-γ, regulate cell-mediated immunity, including the activation of innate immunity. Conversely, Th2 cells, which secrete IL-4, IL-5, IL-6, IL-10 and IL-13, regulate antibody-mediated immunity (humoral immunity). I found that OSM-deficient mice exhibited prolonged IFN-γ production in serum after lipopolysaccaride (LPS) administration into intraperitoneum (i.p.). On the other hand, IL-10 production was reduced in OSM-deficient mice. These results indicated that Th1 responses were augmented in OSM-deficient mice by microbial infection. I assessed the Th1/Th2 balance of OSM-deficient mice by in vitro splenocytes culture and found that OSM-deficient splenocytes produced more IFN-γ and less IL-10 than those of WT. Therefore I concluded that OSM-deficient mice exhibit enhanced Th1 response.

2-2. OSM is a negative regulator of IL-12 production from DCs

　Differentiation of Th1 cells requires IL-12 that induces IFN-γ production in T cells. DCs capture foreign antigens and migrate into the secondary lymphoid organs such as the spleen and lymph nodes. DCs then present the antigen by MHC class II to CD4+ T cells and produce IL-12 efficiently for inducing Th1 response. As Th1 response was enhanced in OSM-deficient mice, I assessed whether IL-12 production by LPS stimulation was increased in OSM-deficient mice. As expected, I found that IL-12 production was enhanced in OSM-deficient splenocytes and DCs. In co-culture of CD4+ T cells and DCs, which were isolated from WT or OSM-deficient spleen, in the presence of LPS, OSM-deficient DCs induced production of more IFN-γ and less IL-10 from CD4+ T cells than WT DCs. Activation of DCs was augmented and prolonged in OSM-deficient spleen by LPS injection. Taken together, it is indicated that OSM-deficient DCs are defective in the negative regulation after activation. Using bone marrow-derived DCs (BMDCs), I found that DCs expressed OSM. More interestingly, OSMR expression was dramatically up-regulated in BMDCs and splenic DCs by LPS stimulation. I also found that OSM suppressed IFN-γ production in OSM-deficient splenocytes. It is known that Th1 cells produce OSM as well as IFN-γ. Therefore, it is suggested that OSM negatively regulates Th1 response via directly acting on DCs by both autocrine and paracrine mechanisms.

　OSM-deficient mice develop autoimmune diseases. Here, I show that OSM-deficient mice are biased to Th1 response upon microbial infection. In addition, clearance of apoptotic thymocytes is impaired in OSM-deficient thymus. These results suggest that autoimmunity in OSM-deficient mice is induced by alteration in the central (thymus) as well as peripheral (spleen) immune response.

　本論文は5つのChapterからなる。Chapter 1でのGeneral introduction、Chapter 2でのMaterials & methodsに続き、Chapter 3とChapter 4では研究成果が、Chapter 5ではGeneral discussionが記されている。

　申請者の所属する研究室では、造血組織に発現する多機能性サイトカインであるオンコスタチンM(OSM)に注目して、血液・免疫系の発生・分化の分子機構の研究を展開している。申請者は、OSMの発現が主要な免疫器官である胸腺や脾臓に強く認められることに着目し、本論文では、免疫系の調節において中心的な役割を果たす樹状細胞(Dendritic cell; DC)の発生、分化、機能に対するOSMの作用について詳細に解析している。

　Chapter 3では、胸腺内T細胞分化機構の解明を目的として、OSM反応性の胸腺上皮細胞(Thymic epithelial cell; TEC)による胸腺樹状細胞(Thymic DC)の分化機構及び機能について解析している。T細胞は胸腺で分化成熟し、その分化機構には胸腺内微小環境が重要な役割を果たしている。胸腺におけるT細胞以外の細胞群として、TEC、Thymic DC、胸腺マクロファージ(Thymic macrophage; Thymic Mφ)が存在し、TECはT細胞の正の選択に、Thymic DCはT細胞の負の選択に、Thymic Mφは選択機構により殺されたT細胞の処理に寄与していると考えらえている。しかし、これらの細胞群は存在頻度が極めて低いために、その性状および作用に関しては未だ不明な部分が多い。本研究では、T細胞分化が劇的に起こる胎児胸腺のDCが、成体胸腺DCとは異なる表現型(CD11b陽性)であることを明らかにした。さらに、このCD11b陽性のDCが、OSM受容体陽性のTECから分泌される液性因子によって胸腺内の未分化T細胞から分化誘導されることを明らかにし、そのDC分化誘導因子としてIL-18を同定した。IL-18で誘導されたDCは、抗原提示能が高く、未選択のT細胞と強く結合してアポトーシスを誘導することから、T細胞の負の選択に積極的に関与することが示唆された。本研究によって、機能的なThymic DCをin vitroで分化誘導する系が確立されたことにより、T細胞の負の選択機構の研究が進展することが大いに期待される。

　Chapter 4では、OSM遺伝子欠損(OSM KO)マウスが自己免疫症状を自然に発症することから、末梢の免疫応答におけるDCとOSMの役割について解析している。自己免疫疾患の発症機構は非常に複雑であり、その発症機構の解明は免疫学にとって最も重要な課題の一つである。OSM KOマウスは、TECの低形成及び貪食能の高いThymic Mφの減少により胸腺内に多くの死細胞が蓄積することで、自己免疫疾患を発症すると考えられるが、胸腺での異常が自己免疫疾患に直接寄与することの証明はこれまでにない。そこで、申請者は、末梢の免疫応答性を検討した。免疫応答は細胞性免疫を誘導するTh1と抗体産生を誘導するTh2という機能の異なるヘルパーT細胞のバランスで制御されている。OSM KOマウスを免疫刺激すると、ヘルパーT細胞の反応性が野生型と大きく異なり、Th1優位な応答性を示した。DCはIL-12を産生してTh1分化を誘導することが知られているが、OSM KOマウスのDCでは、その活性化が亢進かつ持続して起こりIL-12を過剰に産生することでTh1偏向性を示すことが明らかとなった。活性化したDCにおいてOSM受容体の発現が増強していたこと、活性化Th1細胞やDCがOSMを分泌したこと、さらにOSMによりTh1応答がin vitroで抑制されたことから、OSMが活性化したDCに作用しTh1応答を負に制御していることが示唆された。以上の結果から、OSM KOマウスの自己免疫疾患の発症には、末梢におけるDCを介した免疫応答のアンバランスが関与していることが強く示唆された。本研究により、OSMはDCを介して過剰な免疫応答を抑制するサイトカインであることが明らかとなり、免疫制御の新たな仕組みが明らかにされた。これは、自己免疫疾患の治療法の開発にも貢献する重要な研究成果である。

　なお、本論文Chapter 3は江指永二、秋山泰身、井上純一郎、宮島篤との共同研究、Chapter 4は江指永二、森川吉博、宮島篤との共同研究であるが、申請者が主体となって実験及び考察を行ったもので、申請者の寄与が十分であると判断する。したがって、博士(理学)の学位を授与できると認める。