The intestine is continually exposed to antigens from food proteins and from commensal or pathogenic bacteria. Strict regulation is thus required in the intestine, and oral tolerance represents a unique aspect of the intestinal immune system. Oral tolerance has been classically defined as the specific suppression of cellular or humoral immune responses to an antigen (Ag) by means of prior administration of Ag through the oral route. The response likely evolved as an analog of self tolerance to prevent hypersensitivity reactions to food proteins. Oral tolerance results in antigen-specific T cell deletion, anergy, or induction of regulatory T cells (Tregs). Food hypersensitivity presumably results from either a failure in establishing oral tolerance or a breakdown in existing tolerance. Furthermore, since oral tolerance is a potent way of inducing regulatory cells towards specific Ag, the idea of using the oral route to induce tolerance to Ag involved in autoimmune diseases becomes an important clinical application of the phenomenon.

Chapter 1. Three different CD103+ dendritic cells (DC) subsets in MLN have distinct function in intestinal immune regulation

DC have been revealed as important regulators in oral tolerance induction. It has been proposed that oral tolerance requires mesenteric lymph nodes (MLN), and CD103+ DC and PD-L1+ DC in the MLN are suggested to be critical for the induction of oral tolerance. However, the relationship of these DC subsets remains unclear. Therefore we aimed to clarify the phenotypes and functions of MLN DC subsets in relation to oral tolerance induction. Flow cytometric analysis demonstrated that CD103+ DC in MLN are divided into distinct three populations by CD11b and PD-L1 expression. CD103+CD11b+PD-L1+ DC and CD103+CD11b-PD-L1+ DC prominently expressed CCR7, which is the chemokine receptor required to migrate to MLN from the lamina propria. CD103+CD11b+PD-L1+ DC presented orally administrated Ag to CD4+ T cells and strongly induced T cell proliferation. On the other hand, CD103+CD11b-PD-L1+ DC prominently expressed retinaldehyde dehydrogenase 2 (Raldh2) compared to other CD103+ DC subsets, and strongly induced Foxp3 expression in CD4+ T cells by producing retinoic acid. CD103+CD11b-PD-L1- DC could not present orally administrated Ag, but promptly induced IFN-γ production in CD4+ T cells via IL-12 independent mechanism in vitro. These results suggested that the three CD103+ DC subsets have distinctive functions, and may play different roles in inducing oral tolerance.

Chapter 2. IL-10 and IL-27-producing DC capable of enhancing IL-10 production of T cells are induced in oral tolerance 1)

In addition to MLN, Peyer's patch (PP) is also an important site for establishing oral tolerance. PP DC from tolerized mice induced IL-10 production but not Foxp3 expression in co-cultured T cells. The number of CD11b+DC increased after ingestion of Ag, and CD11b+ DC prominently expressed IL-10 and IL-27 compared with CD11b- DC. These results suggested that IL-10 and IL-27 producing CD11b+ DC are increased by interaction with antigen specific T cells in PP, and these PP CD11b+ DC act as inducers of IL-10 producing T cells in oral tolerance.

Chapter 3. Th2 suppressive arginase 1 expressing CD11b+ DC are induced in PP after oral Ag administration

Food allergies presumably result from either a failure to establish oral tolerance, or a breakdown in existing tolerance. Allergy results in an excessive Th2-type immune response, characterized by IL-4, IL-13, and IL-5. Therefore, during oral Ag administration, suppression of excessive IL-4 production may be necessary to establish oral tolerance and prevent the onset of food allergy. In addition to the role of PP DC for inducing IL-10 producing T cells, we also found that PP DC from tolerized mice could suppress excessive IL-4 production in T cells. PP DC from tolerized mice prominently expressed arginase 1, and suppressed IL-4 secretion by CD4+ T cells via arginase 1. Arginase 1 expression in PP DC was increased after oral Ag administration, and the expression was restricted to CD11b+ DC. PP CD4+ T cells prominently expressed IL-4 compared to SPL or MLN CD4+ T cells, and arginase 1 expression in DC was induced by IL-4 in vitro. These observations suggested that after oral Ag administration, PP T cells abundantly produce IL-4, and IL-4 induced arginase 1 expression in PP CD11b+ DC. Then PP CD11b+ DC suppress excessive IL-4 production by arginase 1, establishing IL-4-arginase 1 negative feedback loop.

Chapter 4. Th2 suppressive arginase 1 expressing neutrophils are accumulated in PP after oral Ag administration

During examination of CD11b+ DC, we also found that neutrophils were increased in PP after oral Ag administration, and accumulated around T cells in the intrafollicular region (IFR). Numbers of neutrophils in blood was also increased after oral Ag administration, and it was suggested that fibroblastic reticular cells (FRC) in OVA-fed PP could chemoattract neutrophils. These results suggested the possibility of stepwise attraction of neutrophils; Ag-specific CD4+ T cell response promotes certain chemokine secretion from FRC, which promotes neutrophil migration from blood to PP IFR. Similar to CD11b+ DC, accumulated PP neutrophils prominently expressed arginase 1, and suppressed production of IL-4 via arginase 1. Arginase 1 expression in neutrophils was induced by IL-4 in vitro. These results suggested that after oral Ag administration, FRC in PP recruited neutrophils, and these recruited neutrophils also were involved in establishing the IL-4-arginase 1 negative feedback loop.

Chapter 5. Stromal cells in gut-associated lymphoid tissue have distinct immunoregulatory function

Non-hematopoietic stromal cells provide structural support to the lymphoid organs. Recent studies have shown that stromal cells also have a crucial role in tolerance induction in the periphery. T cell zone of lymphoid tissue is delineated by FRC and forms a scaffold to provide essential guidance cues to cells of the immune system. It is suggested that stromal cells play important roles in shaping tissue-specific immune responses; however, intestinal tissue-specific phenotypes of stromal cells remain unclear and immunoregulatory function of PP-stromal cells have not been reported. Therefore, the characteristics of stromal cells in MLN and PP, in particular FRC (gp38+CD31-CD45- cells) were examined, focusing on T cell response. For comparison, double-negative cells (DNC; gp38-CD31-CD45- cells) were isolated from mouse MLN and PP. MLN-FRC prominently expressed cyclooxygenase-2 (COX2) compared with PP-FRC or DNC. MLN-FRC strongly suppressed CD4+ T cell proliferation but PP-FRC showed only weak suppression. MLN-FRC suppressed CD4+ T cell proliferation depending partly on COX2 activation. It was reported that cultured lymph node-FRC suppressed T cell proliferation dependent on nitric oxide synthase 2 (NOS2). However, we found that NOS2 expression in freshly isolated MLN- and PP-FRC was extremely lower than that in DNC, and that NOS2 was not essential for suppressive function of T cell response by MLN-FRC. PP-FRC prominently expressed Raldh2 compared with MLN-FRC, and PP-FRC induced Foxp3 expression in CD4+ T cells via producing retinoic acid. These results suggested that phenotypes and functions of FRC are distinct between MLN and PP, and they are involved in intestinal immune response in different manners.

Conclusion

DC have been thought to critical for inducing intestinal tolerance, and this study showed distinct DC subsets have distinct immunoregulatory function in different tissues. In addition to DC, it was shown that neutrophils and stromal cells are also involved in immune regulation. This study suggested that more types of cells than previously assumed communicate with each other, and are involved in intestinal immune tolerance.

Figure 1. Suggested model of cellular network in intestinal immune regulation

腸管における免疫抑制機構として経口摂取された抗原に対する免疫応答を抑制する経口免疫寛容が知られているが、その誘導メカニズムについては不明な点が多い。本研究は経口免疫寛容誘導モデルを中心に、腸管免疫反応の場である腸間膜リンパ節(MLN)およびパイエル板(PP)におけるT細胞応答の抑制に関与する細胞種やその抑制メカニズムについて解明を試みたもので、序論および5章と総合討論からなる。

研究の背景と目的が述べられている序論に続き、第1章では、MLNにおける樹状細胞(DC)の機能について述べられている。MLNにおけるCD103を発現するDC(CD103+ DC)はさらに表面分子CD11b, PD-L1発現により、3つのCD103+ DCサブセットに区分されることが示された。抗原を経口投与したマウス由来のCD103+CD11b+PD-L1+ DCはT細胞の増殖を強く誘導した。一方でCD103+CD11b-PD-L1+ DC はレチノイン酸を介してT細胞におけるFoxp3発現を強く誘導することが示された。 またCD103+CD11b-PD-L1- DC はT細胞のIFN-γ産生を強く誘導することが確認された。これらの結果より、3つのCD103+ DCサブセットはそれぞれ異なる性質を持っており、経口免疫寛容誘導においても異なるはたらきで寄与する可能性が示された。

第2章では、経口免疫寛容誘導時のPP DCによる、T細胞のIL-10産生誘導について述べられている。抗原の経口摂取によりPPにおいてCD11b+ DCの数が増加することが示されたことから、これらCD11b+ DCの機能について解析した。PP CD11b+ DCはIL-10, IL-27を高産生しており、抑制性サイトカインであるIL-10を高産生するT細胞を誘導することが示された。

第3章では、経口免疫寛容誘導時のPP CD11b+ DCによるT細胞のIL-4抑制について述べられている。抗原の経口摂取により誘導されるPP CD11b+ DCはarginase 1活性を介してT細胞のIL-4産生を抑制することが示された。CD11b+ DCのarginase 1発現は抗原の経口摂取後に上昇し、IL-4により誘導されることが示された。これらの結果からPPにおいては抗原の経口摂取後、T細胞がIL-4を産生し、IL-4がCD11b+ DCのarginase 1発現を誘導、続いてarginase 1高発現 CD11b+ DCがarginase 1依存的にT細胞のIL-4産生を抑制、といったIL-4-arginase 1の負のフィードバックループを形成することで過剰なTh2応答を回避している可能性が示された。

第4章では、経口免疫寛容誘導時の好中球によるT細胞のIL-4抑制について述べられている。抗原の経口摂取によりPPにおいてCD11b+ DCに加えて好中球も増加することが示され、好中球のPPへの遊走には線維芽細網細胞(FRC)が関与していることが示唆された。CD11b+ DCと同様に、PPにて増加した好中球はarginase 1を高発現しており、arginase 1活性を介してT細胞のIL-4産生を抑制することが示された。これらの結果から、抗原の経口摂取後PPにおけるFRCは血中から好中球を誘引し、PPに遊走した好中球はCD11b+ DCと同様にIL-4-arginase 1の負のフィードバックループを形成することで過剰なTh2応答を回避している可能性が考えられた。

第5章では、リンパ組織の支持構造を構成する細胞であるストローマ細胞、特にFRCについて述べられている。MLN-FRC は一部COX2依存的にT細胞増殖を強く抑制する一方でPP-FRCはレチノイン酸産生を介してT細胞のFoxp3発現を誘導することが示された。これらの結果より、MLNとPPにおけるFRCはその性質や機能が異なり、これらの異なるメカニズムで腸管の免疫反応調節に寄与している可能性が示された。

総合討論では、腸管における免疫抑制反応について、細胞のネットワークやストローマ細胞を中心とした免疫環境に焦点をあて、考察がなされている。

本研究では、腸管において、異なる部位で異なるDCサブセットが異なる免疫制御機能を有することを示したほか、好中球やストローマ細胞も免疫制御に寄与していることを示し、腸管免疫抑制メカニズムを解明する上で新たな知見を提示したもので、学術的・応用的に貢献するところが少なくない。よって審査委員一同は、本論文が博士(農学)の学位論文として価値あるものと認めた。