Efficient presentation of myelin oligodendrocyte glycoprotein peptides but not protein by astrocytes from HLA-DR2 and HLA-DR4 transgenic mice

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Abstract

The role of astrocytes in the pathogenesis of multiple sclerosis (MS) is not well understood. Astrocytes may modulate the activity of pathogenic T cells by presenting myelin antigens in combination with pro- or anti-inflammatory signals. Astrocytes have been shown to present myelin basic protein (MBP) and proteolipid protein (PLP) to T cells, but it has remained unresolved whether astrocytes present myelin oligodendrocyte glycoprotein (MOG), which has been implicated as an important autoantigen in MS.

Here, we asked whether astrocytes presented MOG to T cells. To closer model presentation of human MOG by astrocytes in MS patients, we generated astrocytes from transgenic mice expressing the MS-associated MHC class II alleles HLA-DR2 (DRB1*1501) and HLA-DR4 (DRB1*0401).

The results show that IFN-γ-activated HLA-DR2 and HLA-DR4 expressing astrocytes efficiently presented immunodominant and subdominant MOG peptides to T cells. The hierarchy of the presented MOG epitopes was comparable to that of professional APCs, including dendritic cells and microglia. Importantly, astrocytes were poor at processing and presenting native MOG protein. Furthermore, astrocytes induced a mixed Th1/Th2 cytokine response in MOG-specific T cells, whereas dendritic cells induced a predominantly Th1 cell response.

Collectively, the results suggest that astrocytes may modulate anti-MOG T cell responses in the CNS.

Introduction

The etiology of multiple sclerosis (MS), the most common demyelinating disease of the central nervous system (CNS) in humans, is not well understood. However, it is widely believed that an erroneous attack of T cells on myelin antigens in the CNS contributes to the pathogenesis of this disease (Zamvil and Steinman, 1990, Steinman, 1996, Martin et al., 1992). Amongst the autoantigens most widely studied in MS are MBP, PLP, and, more recently, MOG (Rivers and Schwentker, 1935, Zamvil et al., 1985, Sobel et al., 1990, Amor et al., 1994). The role of MOG in MS is less well understood, in particular in the context of autoreactive T cells restricted by HLA-DR2.

Activation of autoreactive T cells in the CNS is dependent on the interaction with local antigen presenting cells (APC)(Benveniste, 1997, Shrikant and Benveniste, 1996, Greter et al., 2005, Heppner et al., 2005). Monocyte-macrophage-like microglia cells and dendritic cells function as professional APCs, as they constitutively express MHC class II molecules and, to a varying degree, costimulatory molecules (Gehrmann et al., 1993, Jander and Stoll, 1996, Sedgwick et al., 1993, Juedes and Ruddle, 2001, Olson et al., 2001). On the other hand, astrocytes are the most abundant cells in the CNS and represent the non-professional class of CNS-resident APCs. Astrocytes do not constitutively express MHC class II molecules, however, MHC class II expression can be induced with IFN-γ, and further modulated by TNF-α Wong et al., 1984, Vass and Lassmann, 1990, Panek et al., 1992, Dong et al., 1999). Surface expression of the B7-family of costimulatory molecules and of CD40 on astrocytes is controversial (Satoh et al., 1995, Nikcevich et al., 1997, Soos et al., 1999, Tan et al., 1998). IFN-γ-stimulated murine astrocytes activate myelin-specific memory CD4+ T cells and can prime naive antigen-specific T cells (Nikcevich et al., 1997, Soos et al., 1999, Tan et al., 1998). Therefore, in vitro activated astrocytes have the capacity to stimulate autoreactive T cells, and it has been suggested that astrocytes may promote CNS inflammation. However, the paucity of evidence supporting astrocyte APC function in situ in the CNS, and the ability of astrocytes to express anti-inflammatory molecules such as TGF-β has lead some groups to challenge this view (Horwitz et al., 1999, Gimsa et al., 2004, Bechmann et al., 2002). Nonetheless, if astrocytes were to play a prominent role in specifically modulating the activity of neuroantigen-reactive T cells in the CNS they should be able to present immunodominant and subdominant myelin antigen epitopes to T cells. Along these lines, work from several groups showed that mouse astrocytes expressing murine MHC class II molecules can process and present immunodominant PLP and MBP epitopes to T cells (Nikcevich et al., 1997, Tan et al., 1998, Soos et al., 1998). However, these results are contrasted by data showing inefficient processing and presentation of other proteins, such as ovalbumin (OVA), by astrocytes (Sedgwick et al., 1991, Matsumoto et al., 1992, Weber et al., 1994). The processing and presentation of MOG, in particular by astrocytes expressing MS-associated HLA-DR2 molecules, has remained unresolved. Therefore, in this study we investigated whether astrocytes, derived from transgenic mice expressing the human MHC class II molecules HLA-DR2 and HLA-DR4 in the absence of endogenous mouse MHC class II, processed and presented MOG epitopes to encephalitogenic T cells, and how the presentation of MOG epitopes by astrocytes compared to that of professional APCs.

We determined the CD4+ T cell epitopes of human MOG by utilizing astrocytes derived from mice transgenic for human HLA-DR2 (DRB1*1501) and HLA-DR4 (DRB1 *0401) molecules in the absence of endogenous murine MHC class II.

The data show that IFN-γ-activated astrocytes efficiently presented MOG peptides to T cells, comparable with professional APCs, but that they were poor at processing and presenting native MOG protein. Furthermore, astrocytes induced a mixed Th1/Th2 cytokine response in MOG-specific T cells, whereas dendritic cells induced a predominantly Th1 cell response.

Section snippets

Mice, antigens, peptides

HLA-DR2 (DRB1*1501) and HLA-DR4 (DRB1*0401) transgenic mice were generated as described previously (Ito et al., 1996, Rich et al., 2004) and bred at CWRU under specific pathogen-free conditions. All animal procedures were conducted according to guidelines of the Institutional Care and Use Committee (IACUC) of CWRU. Female mice were injected s.c. at 6–10 weeks of age with 100 μg human recombinant MOG1-125 (rhMOG) in complete Freund's Adjuvant (CFA). CFA was prepared by mixing incomplete Freund's

HLA-DR2- and HLA-DR4 transgenic astrocytes present immunodominant MOG epitopes to CD4+ T cells

To determine the presentation of MOG epitopes by astrocytes expressing the human MHC class II molecules HLA-DR2 and HLA-DR4, we generated astrocytes from neonatal HLA-DRB1*1501 and HLA-DRB1*0401 transgenic mice, respectively, and established primary cell lines as described in Material and methods. Astrocyte cultures were essentially pure and free of contamination with microglial cells as verified by immunofluorescence staining for GFAP (> 98% positive cells), absence of CD68 and Mac-1 surface

Discussion

We show that astrocytes expressing the MS-associated MHC class II molecules HLA-DR2 and HLA-DR4 are highly efficient at presenting immunodominant MOG epitopes. However, while astrocytes were highly efficient at presenting MOG peptides, they were defective at presenting MOG epitopes derived from whole MOG protein (Fig. 5). Moreover, astrocytes induced a mixed Th1/Th2 cytokine profile as compared to DCs, which preferentially induced pro-inflammatory Th1 cytokines. Collectively, the data suggest

Acknowledgments

We thank Dr. Neal Guentzel for reviewing the manuscript and Rocio Guardia-Wolf for excellent technical assistance.

This work was supported by grants NS42809 and AR45918 from the National Institutes of Health, RG 3322 from the National Multiple Sclerosis Society, and a grant from the Wadsworth Foundation to T.G.F.

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