The mucosal surfaces are the biggest part of immune system represented by an ample area of tissues immunologically active that is covered by fine epithelial barriers. As the majority of infections are initiated at mucosal sites, the mucosal immune system is considered to be crucial line of defense against infectious disease. Despite the fact that the mucosal immune system is constituted for anatomical and functionally distinct compartments located far way from each other, the oral ingestion of antigen induces humoral and cellular responses not strictly to the site of antigen introduction but in the other mucosal compartments as well. In the gastrointestinal tract (GI), the episodes responsible for the induction of most of antigen-specific responses take place at specific sites in the mucosa that are characterized by the presence of organized lymphoid tissues. These tissues are consisted of lymphoid follicles such as Peyer's patch (PP) and mesenteric lymph node (MLN), which are constituted basically by antigen-presenting cells (APCs), immature B cells and adjacent T cells. Antigens in general are effectively taken up from the lumen in the specialized region called follicle-associated epithelium (FAE), located in the PP, structure considered to be crucial for the induction of Ag-specific mucosal immune response. The FAE localizes such uptake to sites where antigens and pathogens can be efficiently processed and presented for induction of appropriate immune response. This process named as transcytosis is performed by specialized cells called microfold cells (M cells). However, the molecular mechanism involved in the uptake and trancytosis of luminal antigen and microbes by M cells is not clear. In this study, I evaluated the efficiency of mucosal vaccine targeting to M cells via specific transcytotic receptors and the roles of gut-associated lymphoid tissue (GALT) in the induction of mucosal immune response through M cell targeted antigen delivery. Firstly, the results demonstrated that the glycoprotein 2 (GP2) specifically expressed on the apical plasma membrane of M cells serves as a transcytotic receptor for mucosal antigens. GP2 protein selectively bound to enteropathogenic bacteria Salmonella enterica serovar typhimurium, by recognizing FimH-expressing type 1 pili on the bacterial outer membrane and induced efficient bacteria translocation from lumen to adjacent lymphoid tissues. However, M cells in Gp2-/- mice displayed defect in transcytosis of type1-piliated bacteria by M cells, resulting in attenuation of immune response in PPs. Likewise, both mucosal and systemic immune responses against tetanus toxoid (TT) expressed by recombinant S. typhimurium were efficiently induced in Gp2+/+ mice, but not Gp2-/- mice, after oral administration. Thus, it is possible assume that GP2 is a novel transcytotic receptor on M cells for type I piliated bacteria that is prerequisite for mucosal immunosurveillance. These findings can be taken as a cornerstone to define the molecular basis of antigen transport by M cells. As M cell demonstrated to be extremely efficient in transporting living particulate antigen, next, I assess the efficacy of soluble antigen delivery through PP and Villous M cells and the functions of GALT in the induction of mucosal immune response. By taking advantage of our previously established monoclonal antibody (NKM 16-2-4) that recognizes specifically murine M cells located in the epithelium of both Peyer's patches (PP M cells) and intestinal villi (villous M cells), via the recognition of M cell specific carbohydrate moiety containing α (1,2) fucose, PP-, MLN-, and PP and MLN double-deficient, and WT mice were orally immunized with TT conjugated with NKM 16-2-4 (NKM 16-2-4-TT) or control antibody (Rat IgG-TT). High levels of TT-specific serum IgG antibody responses were induced in all animals by oral administration with NKM 16-2-4-TT but not Rat IgG-TT. However, TT-specific intestinal IgA antibody responses were not induced in the PP- and double- deficient mice. Low levels of antigen-specific response were induced in the MLN-lacking mice compared to WT mice, but it was not statistically significant. These results suggest that PP is essential in the induction of Ag-specific mucosal IgA response after oral immunization with NKM 16-2-4 -TT. As the fucosylation in PP M cells and villous M cells is distinctly regulated by α (1,2) fucosyltransferases FUT1 and FUT2, respectively, FUT2-deficient mice were immunized with the complex NKM 16-2-4-TT in order to clarify the role of the uptake via villous M cells in the induction of mucosal immune response. All FUT2-deficient mice induced nice levels of TT-specific IgA antibody responses, but failed or induced low levels of TT- specific IgG response. These results highlight the importance of vaccine-delivery to PP and villous M cells in promoting the induction of antigen-specific mucosal and systemic immune responses. Taken all together, the results provided for this study demonstrated that: (1) GP2-dependent transcytotic pathway would provide a new strategy for development of M-cell-targeted vaccines; (2) the PP is crucial for induction and regulation of Ag-specific IgA mucosal response against soluble antigen orally delivered via α (1,2) fucose moiety; and, finally, (3) the antigen-delivery to both PP and villous M cells might be important strategy for generation of antigen-specific mucosal immunity.

本論文に記す研究ではまず、小腸パイエル板M細胞上に特異的に発現するGP2が、トランスサイトーシスにより細菌をパイエル板のリンパ組織へ提示することで、結果として膜面での抗原特異的免疫反応を惹起するという新たな証拠を示す。また本研究では、可溶性抗原がα(1,2)フコース含有炭化水素複合体によりM細胞を透過して届けられる際、パイエル板が抗原特異的IgA応答の誘導に欠くことのできない役割を果たすことを示す。さらに、絨毛M細胞上のα(1,2)フコース含有炭化水素複合体はIgA応答の誘導には寄与しておらず、一方でパイエル板M細胞上のα(1,2)フコース含有炭化水素複合体は、TT-NKM16-2-4複合体の経口投与による免疫感作後の全身性免疫の活性化には関与していないことを示す。最後に、本研究の結果より、可溶性抗原の粘膜ワクチンはパイエル板と絨毛のM細胞を標的とした場合に、全身系と粘膜系のいずれの応答の誘導に対してもより効果的であることが明確に示唆された。

すなわち、本研究で得られた知見は、我々のM細胞に対する免疫生物学的理解を深め、粘膜免疫応答の惹起におけるGALTの役割を明らかにし、消化管において上皮層の防御を超えて粘膜抗原を輸送する制御装置としてのM細胞の重要性を確固たるものにし、M細胞標的型粘膜ワクチンの新規開発に繋がる方策を与えるものである、学位の授与に値するものと考えられる。