Antigen-specific regulatory T cells are detected in Peyer's patches after the interaction between T cells and dendritic cells loaded with orally administered antigen
Introduction
Various substances enter the digestive tract which begins at the mouth and ends at the anus, and thus various antigens are found in this area. Of these antigens, those that are pathogenic and cause harm to the organism are prevented from entry or expelled from the body. However, the body does not mount an immune response to substances such as food antigens and indigenous intestinal bacteria that are not only harmless but also beneficial to the organism, rather, immune tolerance is induced. This phenomenon is known as oral tolerance. An understanding of this phenomenon will open the door to treatments for inflammatory bowel disease, said to occur as a result of failure to become tolerant to indigenous intestinal bacteria, as well as for other autoimmune disorders. Moreover, as the capacity to artificially prevent oral tolerance would pave the way to the development of oral vaccines for various infectious diseases, clarification of the mechanisms of this phenomenon are eagerly awaited.
The principal mechanism of oral tolerance is thought to be the formation of antigen-specific CD25+CD4+ regulatory T cells (Treg cells) in the vital organ known as Peyer's patches (PPs) in gut associated lymphoid tissue (GALT) (Mowat, 2003, Strobel and Mowat, 2006, Weiner, 1997) in the small intestine. However, the details of how these Treg cells are formed in GALT are not yet clear. In addition, the literature contains a report of Treg cell number increasing following oral administration of an antigen (Zhang et al. 2001); however, it is not clear from this report how or where the orally administered antigen is presented to T cells or whether Treg cells are induced. Furthermore, it has been discovered in recent years that dendritic cells (DCs) play an important role in the maintenance of immune tolerance in peripheral T cells (Coombes and Powrie 2008). Three main populations of DCs exist in PP, namely, CD11b+, CD8α+, and CD11b−CD8α− (double negative; DN) (Iwasaki and Kelsall, 2000, Iwasaki and Kelsall, 2001). In vitro, CD11b+ DC induces IL-10-producing CD4+T cells, and alongside CD8α+ DCs and DN DCs induces IL-12-producing CD4+T cells, also known as Th1 cells. These DCs are known to be spread unevenly within PP. The fact that the DCs in PP and those in the spleen possess different function with respect to differentiation of naïve T cells, plus the fact that oral administration of Flt3 ligand, a growth factor known to specifically simulate and cause propagation of DCs, brings about more efficient induction of oral tolerance, suggests that the DCs present in PP play an important role in inducing oral tolerance (Viney et al. 1998). There are several reports in the literature that examine whether DCs residing in PP and other GALT can induce CD25+CD4+ Treg cells (Benson et al., 2007, Coombes et al., 2007, Mucida et al., 2007, Sun et al., 2007). However, none contain in vivo data regarding the site at which homologous antigen is captured, thereby causing DCs to become regulatory, nor is there data on the site at which the antigen is presented to naïve T cells, thereby causing induction of Treg cells.
Hence, the authors orally administered the antigen fluorescence-labelled ovalbumin (OVA) to Balb/c mice, and examined by fluorescence microscopy the kinetics by which OVA reaches the intestine, and whether OVA is trapped by DCs in PP. Also investigated was the point of contact in PP between T cells and the DCs that ingested OVA. OVA-specific naïve T cells prepared from OVA TCR transgenic mice were transferred to Balb/c mice, then isolated from PP. By doing so, it was possible to investigate, by examining genetic expression measured by real-time PCR, whether the above T cells were being induced to differentiate into Treg cells. Results showed the possibility that DCs induce oral antigen-specific Treg cells in PP in vivo, and that these T cells strongly express CCR9, an intestinal homing marker.
Section snippets
Mice
Female Balb/c mice were purchased from SLC (Shizuoka, Japan) and OVA323–339 TCR transgenic mice from Jackson Laboratory (Bar Harbor, USA). All mice were raised in an SPF environment in the animal rooms in the Department of Allergy and Rheumatology of the Faculty of Medicine, the University of Tokyo, and were used in the experiments from eight to ten weeks postpartum. All experiments were conducted with the approval of the Medical Ethics Council of the University of Tokyo.
Fluorescence labelling and oral administration of antigen
Mice were
Kinetics of antigen-presenting cells in PP in mouse administered FITC-labelled OVA
The kinetics by which orally administered antigen is ingested by antigen-presenting cells is unknown. Hence, we administered 30 mg of FITC-labelled OVA to Balb/c mice and visualized the kinetics of FITC-positive cells in PP using a fluorescence microscope. We orally administered FITC-labelled OVA to 4 groups of Balb/c mice (3 mice/group), and PPs were isolated from mice of each group 3, 6, 12, and 24 h after the administration. As shown in Fig. 1, FITC-positive cells appear in the subepithelial
Discussion
Regulatory T cells produced in GALT are thought to play an important role in the mechanism of oral tolerance. Whether a systemic immune response or oral tolerance is induced to an oral antigen is decided through a process whereby antigen presenting cells in GALT process an antigen and present it to T cells. Many reports exist in the literature confirming DCs to be the principal antigen-presenting cells in this process (Coombes and Powrie 2008). Accordingly, visualization of the means by which
Acknowledgements
We thank Dr. K. Sagawa, Dr. Y. Tanno, and other members of Division of Allergy and Rheumatology, University of Tokyo for helpful discussion. We are deeply grateful to Prof. A. Yamada (Kyorin University School of Medicine) for kind encouragement.
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