Functional expression of B7H1 on retinal pigment epithelial cells

Abstract

The interaction of programmed death-1 (PD-1) expressed on T cells with its ligands B7H1 (PDL1) and B7DC (PDL2) is known to be a mechanism of T cell inhibition. In the present study, we examined whether human or murine retinal pigment epithelial (RPE) cells express B7H1 and B7DC, and if so, whether these molecules expressed on RPE cells play an inhibitory role via interaction with T cells. The transcriptional levels and surface expression of B7H1 and B7DC on human RPE cell line (ARPE-19), RPE cells freshly isolated from healthy human subjects, and murine RPE cells were studied by reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry. In addition, T cells from healthy subjects were cultured with ARPE-19 for 72 h in the presence or absence of monoclonal antibody (mAb) to B7H1 or B7DC, and cytokine production (IFN-γ, IL-8, and MCP-1) was measured. Messenger RNA and cell surface protein expression of B7H1 and B7DC were demonstrated on non-stimulated ARPE-19 and freshly isolated human RPE cells, and the expression of these molecules was predominantly upregulated by treatment with IFN-γ. In murine RPE cells, B7H1 expression was detected only when stimulated with IFN-γ. IFN-γ, IL-8, and MCP-1 production by T cells co-cultured with IFN-γ-untreated or -treated ARPE-19 was significantly enhanced in the presence of anti-B7H1 mAb. These data suggest that B7H1 expressed on RPE cells plays an immunosuppressive role in ocular inflammation, which may contribute to immune privilege in the posterior segment of the eye.

Introduction

Retinal pigment epithelium (RPE) located between the choroidal blood supply and the photoreceptor cell layer of the neural retina plays a critical role in maintaining immune privilege in the posterior segment of the eye. In addition to its role as a blood–retinal barrier, it secretes immunosuppressive factors and expresses FasL on the cell surface (Gabrielian et al., 1999, Holtkamp et al., 2001, Liversidge et al., 1988). Moreover, RPE expresses no major histocompatibility complex (MHC) class II molecules under normal circumstances, although the expression of these molecules can be induced by exposure to IFN-γ (Liversidge et al., 1988). Since the major T cells that recognize retinal self-antigens are CD4⁺ T cells, expression of MHC class II molecules on the RPE would promote activation of self-reactive T cells that enter the eye to elicit autoimmune attack. Indeed, it has been shown that IFN-γ-stimulated RPE cells can present antigen to T cells and induce the proliferation of retinal antigen-specific T cells (Liversidge et al., 1988, Osusky et al., 1997, Percopo et al., 1990). However, RPE cells are known to express different levels of MHC class II molecules depending on the concentration of IFN-γ, and T cell activation is regulated positively or negatively by the MHC class II expression level of RPE (Sun et al., 2003). Additionally, activated human RPE cells are able to produce several cytokines and chemokines, and are capable of regulating normal T cells (Elner et al., 1997, Willermain et al., 2000).

Two signals regulating T cell activation are widely accepted. Signal 1 consists of interaction of T cell receptor (TCR) with antigen presented by the MHC on the surface of antigen-presenting cells (APCs). Signal 2 consists of engagement of costimulatory receptors on T cell via ligands present on the surface of APCs (Alegre et al., 2001, Bretscher, 1999). A primary costimulatory signal is delivered through the CD28 receptor after engagement of its ligand B7-1 (CD80) or B7-2 (CD86) on APC. Engagement of CTLA-4 (CD152) by the same B7-1 or B7-2 ligand results in attenuation of T cell responses. Recently, molecular homologs of B7-like ligands have been identified, B7H1 (also termed PDL1) and B7DC (also termed PDL2). PD-1, their receptor, is a 55 kDa type 1 transmembrane protein belonging to the CD28/CTLA-4 family (Ishida et al., 1992). These B7 family members have been shown to down-regulate T cell activation through PD-1 (Carter et al., 2002, Freeman et al., 2000, Latchman et al., 2001). Cross-linking of PD-1 with B7H1 or B7DC results in decreased IFN-γ, IL-10, IL-4 and IL-2 secretion (Freeman et al., 2000, Latchman et al., 2001). Thus, B7H1 or B7DC leads to diminished immune responses by regulating T cell activation, and the two molecules may have overlapping functions. Whether they are expressed on human RPE cells and involve regulating T cell activation as well as cytokine and chemokine production has not been investigated. In the present study, we demonstrate that human RPE cells express B7H1 and B7DC, and that B7H1 expressed on RPE cells plays an inhibitory role via interaction with T cells.