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Brain Advance Access published online on July 10, 2008
Brain, doi:10.1093/brain/awn148
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© The Author (2008). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Naïve CD8 T-cells initiate spontaneous autoimmunity to a sequestered model antigen of the central nervous system

Shin-Young Na1, Yi Cao1,*, Catherine Toben1, Lars Nitschke1,{dagger}, Christine Stadelmann2, Ralf Gold3,{ddagger}, Anneliese Schimpl1,{ddagger} and Thomas Hünig1,{ddagger}

1Institute for Virology and Immunobiology, University of Würzburg, D-97078 Würzburg, 2Institute for Neuropathology, University of Göttingen, D-37073 Göttingen and 3Department of Neurology at St Josef Hospital, Ruhr-University Bochum, D-44791 Bochum Bochum, Germany

Correspondence to: Thomas Hünig, Institute for Virology and Immunobiology, University of Würzburg, Verbacher Straße 7, D-97078 Würzburg, Germany E-mail: huenig{at}vim.uni-wuerzburg.de


   Summary
Top
 Summary
Introduction
 Material and methods
 Results
 Discussion
 Supplementary material
 References
 
In multiple sclerosis, CD8 T-cells are thought play a key pathogenetic role, but mechanistic evidence from rodent models is limited. Here, we have tested the encephalitogenic potential of CD8 T-cells specific for the model antigen ovalbumin (OVA) sequestered in oligodendrocytes as a cytosolic molecule. We show that in these ‘ODC-OVA’ mice, the neo-self antigen remains invisible to CD4 cells expressing the OVA-specific OT-II receptor. In contrast, OVA is accessible to naïve CD8 T-cells expressing the OT-I T-cell receptor, during the first 10 days of life, resulting in antigen release into the periphery. Introduction of OT-I as a second transgene leads to fulminant demyelinating experimental autoimmune encephalomyelitis with multiple sclerosis-like lesions, affecting cerebellum, brainstem, optic nerve and spinal cord. OVA-transgenic oligodendrocytes activate naïve OT-I cells in vitro, and both major histocompatibility complex class I expression and the OT-I response are further up-regulated by interferon-{gamma} (IFN-{gamma}). Release of IFN-{gamma} into the circulation of ODC-OVA/OT-I double transgenic mice precedes disease manifestation, and pathogenicity of OT-I cells transferred into ODC-OVA mice is largely IFN-{gamma} dependent. In conclusion, naïve CD8 T-cells gaining access to an ‘immune-privileged’ organ can initiate autoimmunity via an IFN-{gamma}-assisted amplification loop even if the self-antigen in question is not spontaneously released for presentation by professional antigen presenting cells.

Key Words: autoimmune encephalitis; multiple sclerosis; cytotoxic T-cell response; demyelinating disease; blood–brain barrier

Abbreviations: BBB, blood–brain barrier; EAE, experimental autoimmune encephalomyelitis; MBP, myelin basic protein; ODC-OVA mice, mice expressing ovalbumin in oligodendrocytes

.

Received April 10, 2008. Revised June 4, 2008. Accepted June 12, 2008.


   Introduction
Top
 Summary
 Introduction
Material and methods
 Results
 Discussion
 Supplementary material
 References
 
Multiple sclerosis, the most common autoimmune disorder of the central nervous system (CNS), afflicts 1/800 of the Caucasian population (Compston et al., 2003Go). Although distinct sub-types of the disease exist with regard to progression, severity of various neurological signs and histopathological presentation, this has not yet been transferred to individualized treatment strategies. Untreated multiple sclerosis invariably leads to irreversible and severe disability, sometimes only after decades. The anti-inflammatory and immunosuppressive therapies which are presently available can delay the course of some forms of multiple sclerosis, but none of them are curative and some even have major side effects.

Classical animal models of multiple sclerosis rely on immunization with CNS-derived antigens in adjuvants, thereby favouring the activation of CD4 T-cells and the induction of auto-antibodies. Besides activated CD4 T-cells of the pro-inflammatory T-helper 1 type (Hafler et al., 1996Go; Lassmann and Ransohoff, 2004Go), IL-17-producing CD4 T-cells play a key pathogenetic role in these models (Hofstetter et al., 2005Go; Park et al., 2005Go; Bettelli et al., 2007Go). In the human disease itself, however, CD8 T-cells are increasingly receiving attention as major mediators of autoimmune attack (Friese and Fugger, 2005Go). For example, CD8 T-cells are over-represented in chronically inflamed multiple sclerosis plaques (Booss et al., 1983Go; Hauser et al., 1986Go; Babbe et al., 2000Go), and often found in close association with oligodendrocytes and demyelinated axons (Neumann et al., 2002Go). Furthermore, pronounced oligoclonal expansions of CD8 T-cells in blood, brain and cerebrospinal fluid of multiple sclerosis patients (Babbe et al., 2000Go; Crawford et al., 2004Go; Skulina et al., 2004Go), and a skewed repertoire of CD8 T-cells producing pro-inflammatory cytokines (Laplaud et al., 2004Go) suggest an important role of this subset in the aetiology of multiple sclerosis.

Several transgenic mouse models have been developed to study the pathogenetic role of CD8 T-cells in autoimmune destruction of the CNS. Some of these systems have employed T-cell receptor (TCR)-transgenic T-cells recognizing natural or model brain auto-antigens in a major histocompatibility complex (MHC) class I-restricted manner (Cornet et al., 2001Go; Huseby et al., 2001Go). However, in transgenic mice expressing such class I-restricted TCR together with the relevant antigen in the CNS, either self-tolerance (Perchellet et al., 2004Go) or early death due to an attack on enteric glial cells (Cornet et al., 2001Go) precluded the analysis of experimental autoimmune encephalomyelitis (EAE) development. On the other hand, Fournier and colleagues recently demonstrated a pathogenetic role for endogenous CD8 T-cells in EAE observed in mice over-expressing the co-stimulatory ligand CD86 on microglia cells (Brisebois et al., 2006Go).

Here, we employ a novel model for CNS-specific autoimmunity to evaluate the capacity of CD8 T-cells to initiate and execute myelin destruction. This model employs ovalbumin (OVA) sequestered as a neo-self antigen in the cytosol of oligodendrocytes. In contrast to other transgenic models for tissue-specific autoimmune diseases employing OVA as a self-antigen (Kurts et al., 1997bGo; Gallegos and Bevan, 2004Go; Lee et al., 2007Go), these ‘ODC-OVA mice’ express the target antigen neither in the thymus nor in stromal cells of peripheral lymphoid or non-lymphoid tissues. We show that endogenously generated CD8 T-cells expressing the MHC class I-restricted, OVA-specific OT-I receptor are highly encephalitogenic in ODC-OVA mice. OVA-specific CD4 T-cells, in contrast, remain fully ignorant of the self-antigen unless OVA-specific CD8 T-cells first release OVA from oligodendrocytes for peripheral presentation. The positive feedback loop initiated by OT-I cells upon antigen recognition on oligodendrocytes involves and largely depends on the production of the pro-inflammatory cytokine interferon-{gamma} (IFN-{gamma}) by the auto-reactive CD8 T-cells. Importantly, initiation of this auto-reactive vicious circle requires access of cytotoxic T-cells to OVA-expressing oligodendrocytes within the first 2 weeks of life, operationally defining the time point at which the blood–brain barrier (BBB) becomes impermeable for naïve CD8 T-cells.


   Material and methods
Top
 Summary
 Introduction
 Material and methods
Results
 Discussion
 Supplementary material
 References
 
Mice
ODC-OVA transgenic mice were generated as described (Cao et al., 2006Go), using an OVA cDNA under the control of an oligodendrocyte-specific myelin basic protein (MBP) promoter construct, containing the 6.5 kb proximal part of the MBP promoter (Forghani et al., 2001Go; Farhadi et al., 2003Go). OT-I (Hogquist et al., 1994Go), OT-II (Barnden et al., 1998Go) and RAG-1–/– mice were crossed with ODC-OVA tg mice. C57BL/6 mice were purchased from Harlan-Winkelmann, Borchen, Germany. All mice were housed in a specified pathogen-free animal facility at the Institute for Virology and Immunobiology, Würzburg.

Adoptive transfer of OT-I or OT-II cells
MACS (Miltenyi Biotech, Bergisch-Gladbach, Germany)-purified OT-I CD8, OT-I/IFN-{gamma}-deficient CD8 or OT-II CD4 cells (7 x 106) were transferred i.p. into 2-week-old ODC-OVA/RAG-1–/– or RAG-1–/– mice. The mice were weighed and scored for clinical signs of EAE on a daily basis. Disease severity was assessed using a scale ranging from 0 to 5 (Gold et al., 1995Go): 0, normal; 1, limp tail; 2, partial hind leg paralysis; 3, total hind leg paralysis; 4, hind and front limb paralysis; 5, moribund or dead.

For OVA-specific proliferation, purified CD8 or CD4 cells were labelled with 10 µM carboxyfluoroscein succinimidyl ester (CFSE) for 5 min at RT and 5 x 106 cells were transferred into recipient mice of various ages. After 3 days of cell transfer, lymphocytes were isolated and measured by flow cytometry.

Histology and immunohistochemistry
Brain and spinal cord were fixed in 4% paraformaldehyde in phosphate buffer solution (PBS) and embedded in paraffin. Paraffin sections were stained with hematoxilin/eosin and Luxol Fast Blue.

Immunohistochemistry was performed with 5 µm paraffin sections as described (Linker et al., 2002Go). T-cells were labelled with anti-CD3 (Serotec; Wiesbaden, Germany), macrophages with rat anti-mouse MAC-3 (BD Pharmingen) followed by anti-rat IgG-biotinylated secondary antibody (Vector via Linavis, Wertheim, Germany).

Preparation of oligodendrocytes
Brains were taken from 2- to 5-day-old mice and washed twice in PBS. Brains were digested with 0.25% trypsin (Invitrogen) in the presence of 40 µg/ml DNase (Sigma) and the reaction was stopped by adding Dulbecco's modified eagle's medium (DMEM) with 10% FCS. Cells were re-suspended in DMEM with 10% FCS and plated onto tissue culture flasks coated with poly L-lysine (Sigma). After 10 days microglia cells were detached from the primary culture by gentle shaking and oligodendrocyte precursors were harvested with strong shaking of the flasks. Cells were cultured in Neurobasal medium (Gibco) with 10 ng/ml platelet-derived growth factor (PDGF, Sigma) for 4 days and PDGF was withdrawn for an additional 3 days. Oligodendrocytes were stained with mouse anti-2',3' cyclic nucleotide 3'phosphodiesterase (CNPase) antibody (Sternberger Monoclonals via Szabo Scandic, Vienna, Austria) and rabbit anti-OVA antiserum (Sigma, Deisenhofen, Germany) was used for OVA staining. As secondary antibodies anti-mouse Alexa 594 and anti-rabbit Alexa 488 (Molecular Probes) were used. For co-culture experiments, PDGF withdrawal oligodendrocytes were incubated with OT-I CD8, OT-I/IFN-{gamma}-deficient CD8 or OT-II CD4 T cells in the presence or absence of 10 U/ml IFN-{gamma} (R&D).

Leucocyte isolation from spinal cord
Spinal cords were taken from WT or ODC-OVA/OT-I double transgenic mice, mashed through a cell strainer, re-suspended in 30% percoll and loaded with 70%, 45% percoll gradient. A single layer of leucocytes was collected between 45% and 30% percoll gradient and analysed by flow cytometry.

Fluorescence activated cell sorting analysis
Cells were stained for fluorescence activated cell sorting analysis using the following Abs (all from BD Pharmingen): anti-CD4-PE, anti-CD4-FITC, anti-CD4-APC, anti-CD25-PE, anti-CD8{alpha}-FITC, anti-CD8{alpha}-APC, anti-CD69-FITC, anti-CD69-PE, anti-CD45-FITC, anti-Mac1-PE, anti-I-Ab-biotin, anti-H-2Kb-biotin, anti-V{alpha}2-PE, anti-V{alpha}2-biotin, anti-Vβ5-biotin, anti-B7.1-biotin, anti-B7.2-biotin. All biotinylated Abs were followed by streptavidin-cychrome. Data were collected on a FACScalibur (Becton Dickinson, Heidelberg, Germany) and analysed using CellQuestTM software (Becton Dickinson). All histograms and dot plots use logarithmic scales.

Statistical analysis
Statistical analysis was performed by the logrank-test GraphPad Prism 3.0 (GraphPad Software, San Diego, California).


   Results
Top
 Summary
 Introduction
 Material and methods
 Results
Discussion
 Supplementary material
 References
 
Spontaneous CD8-mediated EAE in ODC-OVA/OT-I double transgenic mice
Initial characterization of ODC-OVA mice using RT–PCR, western blotting and immune histology had revealed an exclusive expression of OVA in the oligodendrocytes of brain and spinal cord, and no evidence for ectopic expression in the thymus or in peripheral lymphoid and non-lymphoid organs (Cao et al., 2006Go; and data not shown). Furthermore, CD4 cells developing in ODC-OVA mice expressing the H-2 IAb-restricted, OVA323–339-specific OT-II TCR remained completely ignorant of OVA, as assessed by the frequencies and phenotypes of OT-II thymocytes and peripheral T-cells, and by the unimpaired in vitro reactivity of the transgenic T-cells to OVA (Cao et al., 2006Go). Not surprisingly, no infiltration or inflammation of the CNS was observed in ODC-OVA/OT-II double transgenic mice (Cao et al., 2006Go).

Since oligodendrocytes from ODC-OVA mice express ovalbumin as a cytosolic molecule (Fig. 1A), we considered the possibility that release of the antigen by membrane shedding was insufficient to allow presentation by MHC II-expressing APC. However, the low but significant amount of MHC I molecules expressed by oligodendrocytes (Agresti et al., 1998Go; and Fig. 1B) might suffice for recognition by OVA-specific CD8 T-cells, and an autoimmune attack on the CNS.


Figure 1
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  		Fig. 1 Expression of OVA, MHC and co-stimulatory ligands by oligodendrocytes derived from ODC-OVA mice. (A) Cytoplasmic localization of OVA revealed by two-colour staining of cultured oligodendrocytes with rabbit anti-OVA (green) and CNPase (red). (B) Expression of MHC class I, but not MHC class II antigens or CD28 ligands on cultured oligodendrocytes assessed by flow cytometry. Thin lines indicate isotype control staining.

 
When ODC-OVA mice were crossed with OT-I mice expressing a transgenic TCR-specific for the OVA-derived SIINFEKL peptide presented by H-2 Kb on most of their CD8 T-cells (Hogquist et al., 1994Go), double transgenic pups initially appeared healthy. Between days 12 and 19, however, severe locomotor defects set in (Fig. 2A). Signs included not only ascending paralysis of the extremities, as is observed in classical EAE models mediated by pro-inflammatory Th1 and Th17 CD4 T-cells, but also uncoordinated limb movements and tremor as clinical signs of cerebellar affliction (see videos S1 and S2). These features resemble signs first considered as multiple sclerosis-specific neurological defects by Charcot more than a century ago (Charcot, 1872Go). The disability of ODC-OVA/OT-I double transgenic mice progressed rapidly to a stage where they were unable to take up milk and had to be euthanized.


Figure 2
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  		Fig. 2 Development of lethal EAE in ODC-OVA/OT-I double transgenic mice. (A) Disease onset and mean disease score. n = 35 for ODC-OVA, n = 40 for OT1, n = 48 for RAG-1 WT and 15 for RAG-1–/–. (B) Histology of cerebellum and spinal cord. Numbers indicate disease scores on a 1–5 scale (see Materials and Methods). LFB: Luxol fast blue (myelin).

 
OT-I mediated EAE is independent of CD4 or B-lymphocytes
While the majority of ODC-OVA/OT-I double transgenic mice came down with fulminant neurologic disease, about 10% of the animals remained free of obvious signs (Fig. 2A), and even fewer recovered after only mild manifestations of disease (not shown). In order to examine possible modulatory or helper effects of other lymphocyte populations generated in ODC-OVA/OT-I double transgenic mice, we additionally introduced RAG-1 deficiency into these animals. Under these circumstances, all mice experienced fulminant EAE between days 12 and 16 of life and either died or had to be euthanized. It is likely that the increased incidence of disease in RAG-1-deficient double transgenic mice is due to the removal of regulatory T-cells expressing endogenous TCR, as previously shown in a CD4-mediated TCR transgenic EAE system (Olivares-Villagomez et al., 1998Go). Furthermore, this result indicates that CD4 T-cells, B-cells or CD8 T-cells with specificities other than OT-I are not required for the development of lethal CNS-specific autoimmunity directed by the neo-self antigen OVA expressed in the cytosol of oligodendrocytes.

Infiltration, inflammation and demyelination of the CNS in spontaneously unfolding CD8 T-cell-mediated EAE
To characterize disease progression at the level of tissue damage, double transgenic mice were sacrificed at various stages of disease, ranging from symptom-free (grade 0) to locomotory disability affecting all extremities (grade 4). With regard to the brain, the cerebellum (Fig. 2B), the brain stem, and the optic nerve (not shown) were particularly affected. As shown by HE staining and immune histology in Fig. 2B, affection of the cerebellum is characterized by progressive severe spongiform degeneration of tissue architecture, influx of T-cells (the OT-I cells), up-regulation of the MAC-3 antigen indicative of microglia/macrophage activation, and loss of myelin. T-cell infiltration, microglia/macrophage activation and loss of myelin are also observed in the spinal cord where, especially with regard to infiltration with OT-I cells, pathology unfolds with accelerated kinetics as compared to the brain. Importantly, and in contrast to classical CD4 T-cell mediated EAE induced by MOG-peptide immunization (Linker et al., 2005Go), many axons remained structurally intact as visualized by axon-specific silver staining and immune histology (Supplementary Fig. S1). Furthermore, the scarcity of amyloid precursor protein aggregates within the demyelinated areas indicates that the preserved axons still contain functional retrograde transport machinery (Supplementary Fig. S1). Using three-colour immunofluorescent staining of cryosections from affected tissues, we were also able to directly demonstrate the close association of OT-I cells and oligodendrocytes in the cerebellum and spinal cord of sick double transgenic mice, and the accumulation of OT-1 cells in the white matter (Supplementary Fig. S2). Together, these findings are reminiscent of human multiple sclerosis plaques (Neumann et al., 2002Go), and are in line with a direct cytotoxic attack on oligodendrocytes rather than an indirect, inflammation-driven destruction of both, the myelin sheaths and the neurons.

Infiltrating CD8 T-cells and activated microglia cells were further characterized by flow cytometry (Fig. 3). Single-cell suspensions were prepared from the spinal cords of healthy ODC-OVA mice, and of score 4 EAE ODC-OVA/OT-I double transgenic mice. Lymphocytes and macrophages/microglia cells were identified using the pan-leucocyte marker CD45 and the macrophage/microglia marker CD11b (Fig. 3A). Both populations were markedly increased in double transgenic mice and exhibited signs of activation. Thus, the macrophage/microglia population had strongly up-regulated both MHC classes I and II molecules as well as the co-stimulatory ligands CD80 and CD86, as compared to CD11b positive cells found in single transgenic mice (Fig. 3A). With regard to the CD8 T-cells, which were only present in significant numbers in the spinal cords of mice undergoing EAE, both the early activation marker CD69 and CD25, the alpha chain of the IL-2 receptor, were strongly expressed (Fig. 3B).


Figure 3
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  		Fig. 3 Up-regulation of activation markers on microglia and infiltrating CD8 T-cells in ODC-OVA/OT-I double transgenic mice. Leukocytes were isolated from spinal cord of a double transgenic mouse with grade 4 EAE, and from a single transgenic littermate control. (A) Expression of MHC classes I and II molecules and of CD28 ligands on CD45 and CD11b expressing cells within a live scatter gate. Thin lines indicate isotype control staining. (B) Expression of CD25 and CD69 on CD8 cells identified within a live scatter gate.

 
Access of OT-I cells to SIINFEKL/Kb expressing oligodendrocytes is restricted to the first 10 days of life
The fulminant demyelination observed in ODC-OVA/OT-I double transgenic mice indicated that in contrast to MHC class II-restricted OT-II cells (Cao et al., 2006Go), MHC class I-restricted OT-I cells do gain access to OVA-expressing oligodendrocytes. However, the dramatic destruction mediated by the effector arm of the response does not provide information on the site of initial priming with OVA. In other transgenic models of autoimmunity such as the RIP-mOVA model for type I diabetes, this occurs in the lymph node draining the organ which expresses the model self-antigen (Kurts et al., 1996Go).

To test for the presence of OVA-presenting APC, OT-I cells labelled with the covalent dye CFSE were transferred to ODC-OVA mice or to non-transgenic littermates. When analysed 3 days later, extensive cell division (Fig. 4A) and CD69 up-regulation (not shown) were observed when the recipient ODC-OVA mice were 7 or 10 days old at the time of transfer. This result is consistent with spontaneous leakage of OVA from the CNS in a free or cell-bound form, as has been reported for OVA drained to the pancreatic lymph node of RIP-mOVA mice (Kurts et al., 1996Go). Alternatively, OT-I T-cells first release OVA from oligodendrocytes, which then leads to activation of further transferred cells in the draining lymphatic tissue.


Figure 4
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  		Fig. 4 Visibility of OVA for transferred OT-I and OT-II cells in vivo. 5 x 106 CFSE-labelled OT-I or OT-II cells were i.p. injected into ODC-OVA single transgenic mice at the ages indicated. (A) Access of OT-I cells is limited to the first 10 days of life. CFSE profiles of CD8-gated cervical and mesenteric lymph node cells recovered 3 days after transfer are shown. (B) OT-II cells fail to respond to transgenic OVA. CFSE profiles and CD69 expression are shown for CFSE-gated cervical lymph node cells from ODC-OVA mice 5 days after transfer (left). As a positive control, wild type mice received 100 µg OVA in saline 1 day before cell transfer (right). (C) OT-II cells respond to transgenic OVA when co-transferred with OT-I cells. CFSE profiles and CD69 expression of cervical lymph node cells recovered from wild type (right panels) or ODC-OVA mice 5 days after transfer are shown. Typical results from individual mice are shown. Between three and seven mice were analysed per group.

 
Surprisingly, transfer of CFSE-labelled OT-I cells into ODC-OVA mice at 12 days of age or later failed to reveal any antigen presentation in draining or peripheral lymph nodes (Fig. 4A), in spite of continued OVA-expression in the oligodendrocytes of the CNS (Cao et al., 2006Go). Since DC presenting antigens via MHC class I persist for over a week unless removed by a strong CTL response (Hermans et al., 2000Go), this result is unlikely to be explained by cessation of spontaneous OVA-release from the CNS at that point of time. More likely, the BBB may become sufficiently developed by 2 weeks of age to terminate access to the CNS for naïve OT-I cells, which is required to release the sequestered self-antigen.

Importantly, the undivided phenotype of OT-I cells recovered from mesenteric (Fig. 4A) and other peripheral lymph nodes (not shown) after transfer into ODC-OVA mice at 2 weeks of age or later also reveals that OT-I cells do not encounter the SIINFEKL peptide on stromal cells of peripheral lymph nodes, as has been reported for transgenic mice expressing cytosolic OVA under the intestinal fatty acid binding protein promoter (Lee et al., 2007Go).

CD8-mediated antigen release from oligodendrocytes is required for OVA-presentation in draining lymph nodes
Next, we tested the hypothesis that the presence of stimulatory OVA-derived peptides in peripheral lymph nodes depended on initial antigen release mediated by OT-I cells entering the brain. For this purpose, we employed OT-II cells as a source of indicator cells detecting OVA presented by professional APC but unable to recognize OVA-expressing oligodendrocytes. CFSE-labelled OT-II cells (depleted of CD25+ cells to avoid potential suppressive effects of nTreg cells) did not respond with proliferation or up-regulation of CD69 in cervical lymph nodes of 10 days old ODC-OVA recipient mice, which provide a highly stimulatory environment for OT-I cells (Fig. 4B versus A). The functionality of the readout system was confirmed by the ability of soluble OVA injected 1 day earlier to drive OT-II proliferation (Fig. 4B). In keeping with our earlier findings with OT-II/ODC-OVA double transgenic mice (Cao et al., 2006Go), these results show that OVA expressed by oligodendrocytes in ODC-OVA mice is not constitutively presented to CD4 T-cells in peripheral lymphoid organs.

Next, we tested whether the transfer of naïve OT-I cells would lead to the release of OVA into the periphery, as analysed by the response of co-transferred OT-II cells. Indeed, as shown in Fig. 4C, proliferation and the up-regulation of CD69 in OT-II cells were readily observed in ODC-OVA mice injected with both OT-I and OT-II cells, supporting the hypothesis that OT-I cells release OVA from oligodendrocytes, leading to its presentation by MHC-II positive cells, most likely DC, in the periphery.

Finally, we confirmed the notion that OT-I cells are required to release OVA from the brain by performing OVA-specific ELISA on sera from 9- to 12-day-old ODC-OVA single transgenic and 12- to 16-day-old ODC-OVA/OT-I double transgenic mice. Circulating OVA was exclusively detected in the latter, reaching 25 ng/ml, whereas single transgenic mice showed values below the level of detection (0.6 ng/ml; data not shown).

Oligodendrocytes can directly activate OT-I but not OT-II cells in vitro
The ‘split ignorance’ observed for OT-I and OT-II T-cells with regard to initial recognition of OVA supported our hypothesis that antigen sequestration in the oligodendrocytes themselves provided access to the MHC class I but not to the MHC class II antigen processing pathway. When naïve OT-I cells were co-cultured with oligodendrocytes derived from ODC-OVA mice, a significant fraction up-regulated the early activation marker CD69, indicative of antigen recognition (Fig. 5A), in spite of the lack of detectable CD28 ligands (Fig. 1B). Moreover, pre-treatment of the oligodendrocytes with IFN-{gamma} not only resulted in up-regulation of Kb molecules at the cell surface (Agresti et al., 1998Go; and own unpublished data), but also in enhanced activation of OT-I cells (Fig. 5A). No T-cell activation was observed using oligodendrocytes from non-transgenic control mice. As expected from the MHC class II negative status of oligodendrocytes (Fig. 1B), OT-II cells also did not respond to either wild type or OVA-transgenic oligodendrocytes (Fig. 5C). To test whether endogenous, OT-I derived IFN-{gamma} was also able to enhance the antigen presenting capacity of ODC-OVA oligodendrocytes, we also employed OT-I cells from IFN-{gamma}-deficient mice (Fig. 5A). This markedly reduced the response of OT-I cells, indicating an important, but not essential role of IFN-{gamma} in increasing the visibility of the sequestered OVA to CD8 T-cells. To fully rule out the possibility that in this in vitro system, oligodendrocyte-derived OVA was recognized after cross-presentation by contaminating dendritic or microglia cells, the ODC-OVA transgene was introduced into B10.D2 mice which express the H-2d instead of the H-2b alleles of class I and class II antigen-presenting molecules. Oligodendrocyte cultures derived from transgenic and non-transgenic mice of both MHC types were mixed 4 days before adding OT-I cells to detect the presence of stimulatory Kb/SIINFEKL complexes (Fig. 5B). The results showed a strict requirement of OVA expression in H-2b cells, ruling out a major contribution of cross-presentation of acquired antigen to the observed in vitro activation of OT-I cells.


Figure 5
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  		Fig. 5 IFN-{gamma} amplifies activation of OT-I cells by OVA-expressing oligodendrocytes. (A) Induction of CD69 on OT-I cells co-cultured with oligodendrocytes from transgenic but not from wild type mice is amplified by exogenous and endogenous IFN-{gamma}. (B) Activation of OT-I cells by co-cultured OVA-expressing oligodendrocytes does not depend on cross-presentation. Oligodendrocyte cultures were derived from 2 day old wild-type and transgenic H-2b (C57BL/6) or H-2d (B10.D2) mice. At the time of separation from microglia (day 10 of culture), cultures were mixed as indicated, and freshly prepared OT-I cells were added 4 days later. OT-I cells were stained after another 3 days in culture. (C) OT-II cells are not activated by transgenic or wild type oligodendrocytes. OT-II cells were co-cultured with oligodendrocytes for 4 days before analysis.

 
Role of CD8 T-cell derived IFN-{gamma}
The ability of IFN-{gamma} to up-regulate presentation of endogenous OVA to OT-I cells in vitro prompted the question whether this cytokine would play a role during EAE development in vivo. Indeed, IFN-{gamma} was detectable in the plasma of double transgenic mice even before the onset of discernable disease signs, as was TNF which synergizes with IFN-{gamma} in promoting up-regulation of MHC I proteins on oligodendrocytes (Agresti et al., 1998Go) (Fig. 6A).


Figure 6
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  		Fig. 6 IFN-{gamma} contributes to OT-I mediated EAE in vivo. (A) Detection of IFN-{gamma} and TNF in the blood of ODC-OVA/OT-I double transgenic mice. Serum samples were taken from ODC-OVA single transgenic, and from ODC-OVA/OT-I double transgenic mice before disease onset (day 912), or with grades 3–4 EAE, and analysed by ELISA. (B) A 7 x 106 OT-I cells were transferred i.p. to 2-week-old ODC-OVA transgenic (left, circles) or non-transgenic (left, triangles) RAG-1-deficient mice. In the right panel, OT-I cells from IFN-{gamma}-deficient donors were transferred to RAG-1-deficient ODC-OVA transgenic mice. Weight (shown as deviation from the day of transfer) and disease scores (not shown) were monitored on a daily basis. (C) Histopathology of a RAG-1-deficient ODC-OVA mouse with grade 4 EAE 11 days after transfer of WT OT-I cells (left), or 12 days after transfer of IFN-{gamma}-deficient OT-I cells (right).

 
In order to directly address whether OT-I transgenic CD8 T-cell-derived IFN-{gamma} plays a role in EAE development, we utilized an adoptive transfer system for OT-I mediated OVA-specific EAE. Since ODC-OVA mice with an intact immune system do not undergo EAE upon transfer of naïve OT-I cells (see Discussion), we tested RAG-1-deficient mice as recipients. Indeed, we found that i.p. transfer of 7 x 106 purified naive OT-I cells into 2-week-old RAG-1° ODC-OVA mice induced weight loss (Fig. 6B) and severe EAE signs (not shown) in all recipients.

Histological analysis performed on sections of brain and spinal cord during the acute phase of OT-I mediated adoptive-transfer EAE showed massive infiltration of the cerebellum, the medulla and the spinal cord by CD3 positive T-cells (representing the transferred OT-I cells which were derived from RAG-1-deficient TCR transgenic donors), and by macrophages, along with extensive demyelination (Fig. 6C). It is worth noting that in contrast to OT-I cells, OT-II CD4 cells were unable to infiltrate the CNS or induce disease in RAG-1-deficient ODC-OVA mice even when derived from RAG-1-deficient OT-II donor mice which are free of regulatory T-cells (Supplementary Fig. S3). This result lends further support to our conclusion that without an initial attack of CD8 cells on the oligodendrocytes sequestering the antigen, OVA remains invisible to CD4 T-cells.

This adoptive transfer system was then used to test the encephalitogenic capacity of OT-I cells lacking an intact IFN-{gamma} gene. Only 2 of 12 ODC-OVA/RAG-1-deficient mice that had received IFN-{gamma}-deficient OT-I cells developed EAE as evidenced by weight loss (Fig. 6B) and clinical signs (not shown), and had to be euthanized. One mouse was transiently affected but recovered, while the remaining mice remained healthy. Histological analysis of recipients of IFN-{gamma}-deficient OT-I cells, having undergone only mild and transient EAE, revealed much less tissue damage and demyelination in the brain and spinal cord as compared to mice having received OT-I cells with an intact IFN-{gamma} gene. Nevertheless, some T-cell infiltration and activated microglia cells or macrophages, indicated by up-regulation of MAC-3, were observed in recipients of IFN-{gamma}-deficient OT-I cells as well. Inflammation and tissue damage were more prominent in the spinal cord than in the brain, suggestive of a slower progression of the ascending disease in the absence of IFN-{gamma}. Together, these results support the conclusion that IFN-{gamma} plays a central but not exclusive role in OT-I mediated inflammation of the CNS of ODC-OVA mice.


   Discussion
Top
 Summary
 Introduction
 Material and methods
 Results
 Discussion
Supplementary material
 References
 
We show here that without help by other T- or B-lymphocyte subsets or support by ‘danger signals’ addressing the innate immune system, CD8 T-cells specific for a protein sequestered in oligodendrocytes are able to mount a spontaneous, lethal demyelinating attack on the CNS. The resulting phenotype of EAE is novel, since both the histopathology and clinical features are dominated by cerebellar and brainstem lesions in which extensive demyelination proceeds in the presence of a marked preservation of ‘naked’ axons, a histological feature known from human multiple sclerosis plaques (Neumann et al., 2002Go) but absent from lesions caused by pro-inflammatory CD4 T-cells via recruitment and activation of phagocytic cells (Linker et al., 2005Go). CD8 T-cell-dominated effector mechanisms are also suggested by the more destructive, spongiform histopathology observed in ODC-OVA/OT-I double transgenic mice.

The in vivo activation of MHC class I-restricted OT-I, but not of class II-restricted OT-II cells, in ODC-OVA mice indicates that the antigen is initially provided by class II negative cells, most likely the oligodendrocytes themselves, rather than by cross-presentation on professional APC in which both the MHC class I and class II pathways of antigen presentation are operative. This is in contrast to the RIPmOVA model, where OVA derived from pancreatic β cells is constitutively cross-presented to both OT-I and OT-II cells by DC in the draining lymph nodes (Kurts et al., 1997aGo). In terms of human multiple sclerosis, this could mean that in the absence of effective peripheral suppressor mechanisms, CD8 T-cells specific for CNS-specific antigens (Crawford et al., 2004Go; Dressel et al., 1997Go) may mount cytotoxic responses without the need for cross-presentation or triggering of innate receptors in the target tissue. Such a scenario is compatible with the recent findings of increased importance of CD8 T-cells in human disease (Friese and Fugger, 2005Go; Metz et al., 2007Go).

The notion that priming of OT-I cells occurs directly on oligodendrocytes seems at odds with the known requirement for co-stimulatory ligands for the activation of naive CD8 T-cells and their absence on oligodendrocytes (Odeberg et al., 2005Go). However, this rule does not strictly apply to high-affinity TCR such as OT-I, as we show here directly in our in vitro experiments using CD80/86-negative oligodendrocytes as stimulator cells. Furthermore, in another autoimmune model also using OT-I cells, Lee et al. (2007Go) recently showed that a cytosolic OVA transgene expressed under the control of the intestinal fatty acid binding protein promoter allows ‘promiscuous’ expression of SIINFEKL/Kb on lymph node stroma cells, driving proliferation (and eventual deletion) of OT-I cells without providing co-stimulatory ligands. While in our system, this mechanism of ectopic expression in lymph node stromal cells does not seem to play a role as evidenced by the absence of an OT-I response in recipients above 12 days of age (Fig. 4), these data provide another example for the activation of OT-I cells without the need for classical co-stimulation.

In addition to a lack of promiscuous expression in lymph node stroma, our ODC-OVA mice also do not express the antigen in medullary epithelial cells of the thymus, as is the case for many (but not all) natural and some transgenic tissue-specific antigens. Besides our inability to detect OVA mRNA or protein by sensitive assays in thymic tissue (Cao et al., 2006Go; and data not shown), this is illustrated by the thymic phenotype of rare ODC-OVA/OT-I double transgenic mice with intact RAG genes which escape the disease. Thymocytes of those mice are indistinguishable with regard to cellular composition and cell surface marker expression from thymocytes of single-transgenic OT-I mice (data not shown).

The lack of OVA expression in the thymus of ODC-OVA mice may be surprising given the use of an (albeit truncated) MBP promoter to drive OVA expression, and the well-known presence of both foetal and adult forms of MBP in medullary thymic epithelial cells of rats (Wekerle et al., 1996Go) and mice (Fritz and Zhao, 1996Go). However, ectopic expression of tissue-specific antigens relies, inter alia, on the accessibility of the relevant genes to the transcriptional machinery dedicated to that task (Derbinski et al., 2005Go), a prerequisite possibly lacking in ODC-OVA mice due to the integration site of the transgene.

In contrast to many, but not all tissue-specific antigens, OVA expressed as a neo-self antigen in ODC-OVA mice is, therefore, not ectopically expressed in the thymus or periphery, providing the opportunity to study the unfolding of autoimmunity without interference by this tolerizing mechanism. Abolition of thymic expression of natural tissue-specific self-antigens does indeed result in spontaneous autoimmunity (DeVoss et al., 2006Go; Gavanescu et al., 2007Go), and conversely, thymic expression of a single organ-specific self-antigen can prevent the development of autoimmunity to this organ (DeVoss et al., 2006Go). Interestingly, mice lacking thymic expression of tissue-specific antigens due to a defect in the AIRE protein develop autoimmunity even without a detectable contribution of microbial ‘danger signals’ to the break down of self-tolerance, illustrating the potential to develop truly spontaneous autoimmunity to self-antigens when central tolerance fails (Gray et al., 2007Go).

An important and only poorly resolved issue is the entry of CD8 T-cells into the brain via the BBB, i.e. the endothelial lining of cerebral blood vessels and perivascular macrophages (Man et al., 2007Go). While for CD4 T-cells, a series of phenotypic and functional changes have been defined from peripheral activation to infiltration of and retention in the CNS (Flugel et al., 2001Go), little is known about CD8 T-cells. There is, however, agreement not only on increased T-cell traffic but also on an enhanced proportion of CD8 T-cells in parenchymal perivascular spaces of the brain under inflammatory conditions (Man et al., 2007Go). While our present experiments do not address CD8 T-cell migration through a fully developed BBB, they illustrate the devastating effects of unimpeded access of even naïve CD8 T-cells specific for a cytosolic protein sequestered within oligodendrocytes early in life. Furthermore, they operationally define the closing of the BBB for naïve CD8 T-cells at around 2 weeks of life. Future experiments will address the conditions under which OT-I cells can traverse the BBB and attack oligodendrocytes in adult mice.

One intriguing possibility regarding the cues that guide CD8 T-cells to the CNS comes from a recent report showing that in adult mice expressing an MHC class I-restricted transgenic TCR specific for an HA peptide on most of their T-cells, intra-cerebral injection of the cognate peptide in soluble form will initiate CNS infiltration by the circulating HA-specific CD8 T-cells. In this model, peptide-loaded MHC class I molecules expressed on the luminal side of endothelial cells appear to trigger transmigration (Galea et al., 2007Go). This result illustrates the ability of endothelial cells loaded from without with a pre-formed class I-restricted peptide to trigger transmigration through the BBB, but does not address the issue whether such peptides derived from sequestered or released self-antigens or from infectious agents actually reach that site. In our ODC-OVA mice, such spontaneous release does not occur as evidenced by the exclusion of OT-I cells from the CNS after the first 2 weeks of age in spite of the continued presence of OVA in the cytosol of all oligodendrocytes throughout adult life (data not shown). It will be of interest to see, however, whether an independent inflammatory stimulus will lead to OVA cross-presentation by the vascular endothelium in ODC-OVA mice.

Another open question is the role of other lymphocyte populations in controlling OT-I mediated EAE. Thus, the increased disease penetrance in ODC-OVA/OT-I double transgenic mice that additionally lack the ability to generate endogenous T- and B-cells, and the ability of adoptively transferred naïve OT-I cells to cause EAE in RAG-1-deficient, but not in ODC-OVA mice with an intact lymphocyte compartment, is in line with dominant suppressor mechanisms. Such protective regulatory T-cells could be immune-deviated OT-I cells themselves, as was suggested for keratinocyte-specific CD8 cells in the postnatal period (Alferink et al., 1999Go). However, because initial OVA release and transfer to MHC-II positive APC are still functional in young single transgenic ODC-OVA mice after transfer of OT-I cells without development of EAE, it seems more likely that ‘natural’ MHC-II-restricted Treg cells with specificity for other oligodendrocyte antigens co-released with OVA could interfere with the progression of myelin destruction in a bystander fashion. Alternatively (or perhaps additionally), pre-activation of the OT-I cells in the lymphopenic recipient mice during homeostatic expansion may contribute to their pathogenicity. Indeed, the transient activation of OT-I cells expanding in a lymphopenic setting leads to their transient acquisition of an effector–memory phenotype allowing rapid IFN-{gamma} secretion and cytotoxicity in response to antigenic stimulation (Goldrath et al., 2000Go).

Finally, our results point to the importance of IFN-{gamma} for CD8-mediated EAE. For CD4-driven disease, both exacerbating and ameliorating effects of this cytokine have been described (Renno et al., 1998Go; Balabanov et al., 2007Go). Here, we show that OT-I mediated EAE in ODC-OVA mice critically depends on IFN-{gamma} production by the CD8 cells themselves, and demonstrate that in vitro, OT-I-derived IFN-{gamma} enhances recognition of the cytosolic OVA-protein on oligodendrocytes. It has been shown by others that the MHC-I up-regulating effect of IFN-{gamma} is further boosted by TNF (Agresti et al., 1998Go). Although not directly addressed by our experiments, we expect that the concomitant systemic release of both, IFN-{gamma} and TNF in mice undergoing OT-I mediated EAE will dramatically increase Kb-restricted OVA-presentation by oligodendrocytes in vivo, driving a forward amplifying loop of OT-I activation. Whether the subsequent release of OVA—shown here by its MHC class II-restricted presentation in the periphery—is due to a direct cytotoxic attack by activated OT-I cells, or due to cytotoxicity mediated by cytokine-activated innate immune cells, is presently under investigation. In any case, in a setting with a polyclonal T-cell repertoire, such antigen release will lead to the recruitment of T-cells specific for additional endogenous antigens presented by resident APC. With regard to antigen released by a CD4 T-cell mediated attack, myeloid DC seem to be particularly efficient in promoting MHC class II-restricted epitope spreading in the brain (Bailey et al., 2007Go), but analogous experiments using CD8 cells as primary or secondary autoimmune cells have not been carried out. The use of the model antigen OVA and the availability of MHC class I- and class II-restricted transgenic TCRs specific for OVA itself, but also for endogenous myelin antigens, open new possibilities for the dissection of the initiating and propagating events in EAE involving both classes of T-cells.


   Supplementary material
Top
 Summary
 Introduction
 Material and methods
 Results
 Discussion
 Supplementary material
References
 
Supplementary material is available at Brain online.


   Footnotes
 
*Present address: Laboratory of Molecular and Experimental Pathology, Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, P.R. China Back

{dagger}Present address: Department of Genetics, University of Erlangen, D-91058 Erlangen, Germany Back

{ddagger}Shared senior authorship Back


   Acknowledgements
 
This work was supported by Deutsche Forschungsgemeinschaft through SFB 581, and by Hertie Foundation through the Institute for MS Research and a joint grant to R.G. and T.H. C.S. is supported by the Medical Faculty of Goettingen (junior research group). The technical assistance of Annett Horn, Kirsten Stark and Alexander Zant is gratefully acknowledged.


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