Role of astrocytes in antigen presentation and naive T-cell activation
Introduction
The central nervous system (CNS) has long been considered an immunologically privileged site since it does not have its own lymphatic circulation, it is isolated from the immune system by the blood–brain barrier (BBB) and because CNS cells lack or express negligible levels of MHC class I and class II molecules. However, this view has been challenged by the observation that activated T-cells readily cross the BBB and circulate throughout the CNS where they assume immune surveillance functions (Wekerle et al., 1986, Cross et al., 1990, Hickey et al., 1991). The ability of CNS resident cells to serve as APCs for the incoming T-cells has been suggested by bone-marrow chimera experiments in which adoptive transfer of myelin-specific CD4+ T-cells, that were MHC compatible with resident CNS cells but mismatched with bone-marrow derived cells, could induce experimental autoimmune encephalomyelitis (EAE) (Myers et al., 1993). However, the strongest evidence suggesting a central role for CNS cells as APCs is the fact that myelin-specific T-cells transferred after an in vitro activation step, home to and remain within the CNS to initiate an inflammatory process, whereas similarly activated T-cells with irrelevant specificity can initially be detected in the CNS but quickly disappear (Wekerle et al., 1986, Hickey et al., 1991).
Which resident CNS cell populations perform an APC function is a much debated issue since the CNS is devoid of dendritic cells, the paradigm of professional APC (Hart and Fabry, 1995, Shrikant and Benveniste, 1996). CNS macrophages and microglial cells are strong candidates as they express class II MHC molecules or are induced to do so in the presence of IFN-γ and can express co-stimulatory B7.1 and B7.2 molecules both in vitro and in vivo (Williams et al., 1994, Hart and Fabry, 1995, Windhagen et al., 1995, Shrikant and Benveniste, 1996). Indeed, these cells have been shown to promote CD4+ T-cell proliferation (Matsumoto et al., 1992, Cash and Rott, 1994).
Astrocytes are the other candidates for an APC function in the CNS. They constitute the most abundant glial cell type and are in close proximity to the BBB. Astrocytes do not constitutively express MHC molecules in vivo (Matsumoto et al., 1986, Matsumoto et al., 1992) but MHC class I expression occurs during in vitro culture conditions. MHC class II expression on astrocytes is inducible upon exposure to IFN-γ or viruses both in vitro (Fontana et al., 1986, Massa et al., 1986, Massa et al., 1987) and in vivo (Vass and Lassmann, 1990). Activated astrocytes also express ICAM1 molecules (Frohman et al., 1989, Kraus et al., 1992, Satoh et al., 1995) and CD44 (Haegel et al., 1993), thereby promoting adhesion to T-cells. Recent work also indicates that mouse astrocytes can also be induced to express B7 molecules by IFN-γ in vitro (Nikcevitch et al., 1997, Soos et al., 1999) and therefore express all the molecular attributes of genuine APCs. Indeed, astrocytes can present antigen to CD4+ T-cell clones in an MHC-restricted fashion (Fontana et al., 1984, Fierz et al., 1985, Nikcevitch et al., 1997, Soos et al., 1999).
During demyelinating diseases such as multiple sclerosis (MS) and EAE, activated T lymphocytes once in the CNS enhance MHC class I and class II expression on astrocytes through secretion of IFN-γ. Their location on the brain side of the BBB allows early encounter with lymphocytes entering the CNS. Moreover, astrocytes from EAE susceptible strains of mice or rats show a higher expression of MHC class II molecules than those from resistant strains, reinforcing the notion that astrocytes play a role in the disease process (Massa et al., 1987).
Autoreactive Th1 CD4+ T-cells are central to the induction of EAE. Although less studied, CD8+ T lymphocytes are present in the lesions in great numbers. Their involvement in the disease process is undisputed but their role appears to be complex. On the one hand they contribute to the severity of the lesions, and on the other hand they limit the number of relapses (Koh et al., 1992) and protect against reinduction of disease (Jiang et al., 1992). However their depletion has no effect on the timing of induction of EAE and spontaneous recovery from the first episode of the disease (Sedgwick et al., 1987).
In this context, we have addressed the question of whether naive CD8+ or CD4+ T-cells from TCR transgenic (TCR-Tg) mice can be activated by primary astrocyte cultures. We show in vitro that astrocytes can stimulate CD4+ T-cells only in the presence of IFN-γ. Moreover, astrocytes have the ability to present Ag to CD8+ T-cells, leading to T-cell proliferation. We show that the addition of IFN-γ is an important, but not always essential, condition for antigen presentation to naive CD8+ T cells.
Section snippets
Mice
Four αβ TCR-Tg mouse lines were used as a source of naive antigen-specific T-cells. CD4+ T-cells were isolated from 6.5 TCR-Tg mice, which express a TCR specific for the influenza virus hemagglutinin (HA) 110–119 peptide presented by I–Ed (Kirberg et al., 1994), or from HNT-TCR mice expressing a TCR specific for the HA126–138:I–Ad complex (Scott et al., 1994, Liblau et al., 1996). CD8+ T-cells originated from F5 mice expressing a TCR specific for the influenza virus nucleoprotein (NP) 366–374
Characterization of naive T-cells
Highly purified CD4+ or CD8+ T-cells from TCR-Tg mice were prepared and used as responding cells. In order to assess the functional status of the T-cells, CD4+ or CD8+ T-cells from the TCR-Tg mice were incubated with an anti-CD62L mAb or a control mAb. As shown in Fig. 1, both CD4+ and CD8+ T-cells express high levels of CD62L indicative of a naive phenotype. Labeling experiments using anti-CD25, -CD69, -CD44 and -CD45RB mAbs confirmed this non activated state (Pihlgren et al., 1996, Pardigon
Discussion
In the present study, we have analyzed the capacity of IFN-γ-treated or untreated murine astrocytes to stimulate antigen-specific proliferation of purified naive CD4+ and CD8+ T-cells. We show that only IFN-γ-treated astrocytes induce naive CD4+ T-cell proliferation. This effect is dependent on the induction by IFN-γ of both MHC class II and B7 molecules expression on astrocytes. Furthermore, astrocytes can sustain antigen-specific proliferation of naive CD8+ T-cells originating from two
Acknowledgements
This work was supported by INSERM, the French Research Ministry (ACC-SV), ARSEP and ‘la Ligue contre la Sclèrose en plaques’. We thank Dr. P. Linsley for providing the CTLA-4-Ig molecule, Drs. A. Barcon and M. Mallat for help with astrocyte cultures, Dr. A. Trautmann for critical reading of the manuscript, N. Bercovici and N. Pardigon for their help and support, and Mrs C. Chretien-Dumont for expert secretarial assistance.
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Anne Cornet and Estelle Betelli contributed equally to this work.