Elsevier

Immunobiology

Volume 216, Issue 3, March 2011, Pages 416-422
Immunobiology

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

https://doi.org/10.1016/j.imbio.2010.06.008Get rights and content

Abstract

Systemic immune tolerance is induced for orally administered antigen, and this phenomenon is called oral tolerance. However, the mechanism of oral tolerance has not been completely elucidated. It has been suggested that antigen presentation and generation of regulatory T cells in Peyer's patches (PPs) are important for induction of oral tolerance. Hence, we orally administered fluorescence-labelled antigen to mice and examined kinetics of the antigen and interaction between antigen-loaded dendritic cells and T cells. It was visualized that dendritic cells in PP rapidly take up antigen. We next transferred antigen-specific naïve T cells from T cell receptor transgenic mice and administered the antigen orally. Antigen-specific T cells accumulated in IFR in PP and DCs that have ingested antigen come in contact with antigen-specific T cells in IFR. The accumulated T cells were then collected and analyzed for the pattern of gene expression by real-time PCR, which revealed a gene expression pattern similar to that of FoxP3-positive regulatory T (Treg) cells. CCR9, an intestinal homing marker, was also strongly expressed. These results suggest that DCs that have captured oral antigens in PPs locally induce antigen-specific naïve T cells to differentiate into Treg cells with the intestinal homing phenotype.

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 CD11bCD8α (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.

References (26)

  • A.M. Faria et al.

    Oral tolerance: mechanisms and therapeutic applications

    Adv. Immunol.

    (1999)
  • J. Huehn et al.

    Homing to suppress: address codes for Treg migration

    Trends Immunol.

    (2005)
  • S. Sakaguchi et al.

    Regulatory T cells and immune tolerance

    Cell

    (2008)
  • H.L. Weiner

    Oral tolerance: immune mechanisms and treatment of autoimmune diseases

    Immunol. Today

    (1997)
  • M.J. Benson et al.

    All-trans retinoic acid mediates enhanced T reg cell growth, differentiation, and gut homing in the face of high levels of co-stimulation

    J. Exp. Med.

    (2007)
  • C.A. Byersdorfer et al.

    Visualization of early APC/T cell interactions in the mouse lung following intranasal challenge

    J. Immunol.

    (2001)
  • J.L. Coombes et al.

    A functionally specialized population of mucosal CD103+ DCs induces Foxp3+ regulatory T cells via a TGF-beta and retinoic acid-dependent mechanism

    J. Exp. Med.

    (2007)
  • J.L. Coombes et al.

    Dendritic cells in intestinal immune regulation

    Nat. Rev. Immunol.

    (2008)
  • C.M. Freeman et al.

    CCR8 is expressed by antigen-elicited, IL-10-producing CD4+CD25+ T cells, which regulate Th2-mediated granuloma formation in mice

    J. Immunol.

    (2005)
  • S. Hori et al.

    Control of regulatory T cell development by the transcription factor Foxp3

    Science

    (2003)
  • A. Iellem et al.

    Unique chemotactic response profile and specific expression of chemokine receptors CCR4 and CCR8 by CD4(+)CD25(+) regulatory T cells

    J. Exp. Med.

    (2001)
  • A. Iwasaki et al.

    Localization of distinct Peyer's patch dendritic cell subsets and their recruitment by chemokines macrophage inflammatory protein (MIP)-3alpha, MIP-3beta, and secondary lymphoid organ chemokine

    J. Exp. Med.

    (2000)
  • A. Iwasaki et al.

    Unique functions of CD11b+, CD8 alpha+, and double-negative Peyer's patch dendritic cells

    J. Immunol.

    (2001)
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