Elsevier

Experimental Eye Research

Volume 85, Issue 5, November 2007, Pages 626-636
Experimental Eye Research

Transforming growth factor β-producing Foxp3+CD8+CD25+ T cells induced by iris pigment epithelial cells display regulatory phenotype and acquire regulatory functions

https://doi.org/10.1016/j.exer.2007.07.015Get rights and content

Abstract

The ocular pigment epithelial (PE) cells convert T cells into T regulators (Tregs) in vitro. The PE-induced Tregs fully suppress activation of bystander responder T cells. Iris PE (IPE) cells from anterior segment in the eye produce costimulatory molecules and transforming growth factor β (TGFβ) that is delivered to CD8+ Tregs. We have now examined whether T cells exposed to cultured IPE express CD25 and Foxp3, and to determine if the CD25+ IPE-exposed T cells display regulatory functions in vitro. We have found that cultured B7-2+ IPE converted CTLA-4+ T cells into CD25+ Tregs that suppress the activation of bystander T cells. The CD8+ IPE-induced Tregs constitutively expressed CD25. Through TGFβ–TGFβ receptor interactions, the IPE converted these T cells into CD25+ Tregs that express Foxp3 transcripts. The CD8+ IPE-induced Tregs produced immunoregulatory cytokines, e.g., interleukin-10 and TGFβ. In addition, IPE-exposed T cells that downregulated Foxp3 mRNA failed to acquire the regulatory function. In conclusion, ocular pigment epithelial cells convert CD8+ T cells into CD25+ Tregs by inducing the transcription factor Foxp3. Thus, T cells that encounter ocular parenchymal cells participate in the T-cell suppression.

Introduction

Ocular immune privilege protects the delicate internal structures of the eye from the blinding consequences of innate and adaptive immune inflammation (Niederkorn, 2002, Streilein, 2003). One of the factors responsible for creating ocular immune privilege is the pigment-containing epithelium of the iris, the ciliary body, and the neural retina. The ocular pigment epithelial (PE) cells contribute to the integrity of the blood-ocular barrier, and thereby secure immune privilege within the eye. T cells that encounter PE cells in vitro are inhibited from undergoing TCR-triggered activation (Sugita and Streilein, 2003, Sugita et al., 2004) and are converted into regulators (Yoshida et al., 2000). We recently showed that iris PE (IPE) cells produce B7 costimulatory molecules and membrane-bound transforming growth factor β (TGFβ) that is delivered to CD8+ T regulators (CD8+ IPE-induced Tregs) (Sugita et al., 2006a). The CD8+ IPE-induced Tregs engage CTLA-4+ bystander T cells by B7 interactions for targeted delivery of membrane-bound TGFβ. Importantly, IPE-induced Tregs express their own B7 and membrane-bound TGFβ in order to suppress bystander T cells. In addition, we showed that thrombospondin-1 (TSP-1) produced by IPE and the IPE-induced Tregs binds and activates TGFβ (Futagami et al., 2007). The TSP-1 is essential to the induction of eye-specific Tregs and the subsequent suppression of bystander T cells in vitro. The CD8+ T cells that encounter ocular PE express immunoregulatory molecules to achieve suppression of T-cell activation.

CD4+CD25+ T regulatory cells have emerged as a unique population of suppressor T cells that maintain peripheral immune tolerance (Sakaguchi, 2000, Shevach, 2000). These regulatory T cells develop spontaneously in the thymus and suppress T- and B-cell activation in vitro by a cell contact-dependent mechanism (Mason and Powrie, 1998, Nakamura et al., 2001, Suri-Payer et al., 1998). More importantly, mice depleted of CD4+CD25+ T cells are vulnerable to a variety of autoimmune diseases (Maloy and Powrie, 2001, McHugh and Shevach, 2002, Salomon et al., 2000). Mice that are thymectomized on their 3rd postnatal day (a procedure that depletes their CD4+CD25+ T-cell population) develop immune-mediated inflammation in a variety of organs, including the uveal tract of the eye (Asano et al., 1996, Takeuchi et al., 1998). This finding raises some critical questions: (I) can the ocular PE cells induce regulatory T cells that express CD25 molecules, (II) can the PE-induced Tregs be induced or converted from peripheral naturally arising CD25+ Tregs, and (III) if this latter situation occurs, which molecules and/or cytokines are responsible for the conversion?

Because the T cells exposed to ocular PE (PE-induced Tregs) suppress bystander effecter T cells, we questioned whether the PE-induced Tregs are related to, or perhaps even derived from, the natural CD25+ T-cell population. We designed a series of experiments to examine this possibility. We found that T cells exposed to IPE proliferate due to TCR-trigged activation via costimulatory interactions. The T cells exposed to IPE are converted into CD25+ T regulatory cells. The Tregs that emerge in the presence of IPE are not necessarily derived from naturally arising CD25+ Tregs. Instead, IPE-induced Tregs can arise independently from T cells that are CD25 precursors. These eye-derived CD25+ Tregs greatly express Foxp3 transcripts through TGFβ–TGFβ receptor interactions, and produce immunoregulatory cytokines.

Section snippets

Mice

Adult C57BL/6 mice, purchased from CLEA Japan Inc. (Tokyo, Japan), were used as donors of lymphoid cells and ocular PE. Mice of the C57BL/6 background with disrupted CD28 genes were purchased from Jackson Laboratories. Drs. Philip J. Lucas and Ronald E. Gress (National Cancer Institute, Bethesda, MD) kindly provided dominant negative TGFβ type II receptor (TGFβ RII) transgenic mice (Lucas et al., 2000). Dr. James P. Allison (Univ. California at Berkeley) kindly provided C57BL/6 background CTLA-4

IPE cells promote T-cell proliferation in vitro after 24-h culture

We first examined whether cultured IPE cells suppress or promote T-cell activation. To assess the influence of IPE on T-cell proliferation, different types of T-cell assays were performed. The initial results showed that primary cultured IPE cells promote T-cell proliferation after 24-h culture with a low concentration (0.01–0.1 μg/ml) of anti-CD3 antibodies (Fig. 1A). IPE also profoundly promoted the proliferation of the CD8+ population induced by anti-CD3 after 24-h culture, whereas IPE did

Discussion

In this study, we showed that iris pigment epithelium converts CD8+ T cells into CD25+ Tregs by inducing the transcription factor Foxp3. The CD8+CD25+ IPE-induced Tregs fully suppress bystander T cells in vitro. We suspect that the peripherally arising CD25+ Tregs that develop in the presence of ocular resident cells/tissues protect the delicate internal structures of the eye from intraocular inflammation. Mice possess a population of native T regulators that express surface markers for CD4 and

Acknowledgments

The authors thank Professor J. Wayne Streilein of the Schepens Eye Research Institute for many useful ideas that contributed to this project. Dr. Streilein passed away on March 15, 2004. We greatly appreciate the expert technical assistance of Ms. Tomoko Yoshida and Mrs. Ikuyo Yamamoto.

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