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Constitutive expression of a costimulatory ligand on antigen-presenting cells in the nervous system drives demyelinating disease¹

SIMONE P. ZEHNTNER^*,2, MARCEL BRISEBOIS^,2, ELISE TRAN^*, TREVOR OWENS^*, and SYLVIE FOURNIER^,3

^* Neuroimmunology Unit, Montreal Neurological Institute, and
Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada

³Correspondence: Department of Microbiology and Immunology, McGill University, 3775 University St., Montreal, Quebec, H3A 2B4 Canada. E-mail: sylvie.fournier{at}mcgill.ca

SPECIFIC AIMS

To determine the role of organ-restricted antigen presentation in driving autoimmune diseases, we studied transgenic mice that constitutively express the costimulatory ligand B7.2 on resident antigen-presenting cells of the nervous tissue.

PRINCIPAL FINDINGS

1. B7.2 is constitutively expressed in the nervous tissue of B7.2 transgenic line 31 mice
To study the functional consequences of expression of a costimulatory ligand in a target organ of autoimmune disease, we used B7.2 transgenic mouse lines 27 and 31. Immunohistochemical studies and flow cytometric analyses of line 31 mice lacking mature /ß T cells (TCRß -/- mice) or deficient in CD28 expression (CD28-/- mice) revealed elevated expression of B7.2 on microglia and spinal root macrophages (Fig. 1 B). Levels of B7.2 expression on microglial cells in line 31 mice that contained T cells were equivalent (Fig. 1A ). B7.2 expression on microglia in line 27 mice was similar to that in wild-type animals (Fig. 1B ). B7.2 is therefore constitutively expressed on nervous system resident antigen-presenting cells of line 31 mice.

View larger version (42K): [in this window] [in a new window]   Figure 1. Constitutive B7.2 expression in the nervous tissue of line 31 mice. B7.2 expression on Mac-1high-gated CD45low microglia and CD45high macrophages from the spinal cord of 3-month-old wild-type littermates (WT), line 27 and preclinical and symptomatic line 31 mice (A), and WT littermates, line 27 and line 31 TCRß-/- mice (B). Right-hand panels show B7.2 expression on isolated cells with microglial morphology in the spinal cord and macrophage-like morphology in spinal roots of symptomatic (A) and TCRß-/- (B) line 31 mice.

2. B7.2 transgenic line 31 mice develop demyelinating disease
At 3 months of age or later, line 31 mice exhibited dysfunction of their hind limbs, suggesting a neurological disorder (Fig. 2 A). Histological and immunohistochemical analysis of the nervous tissue of disease-affected mice revealed mononuclear cell infiltrates composed of CD8+, CD4+, and MHC class II+ cells in spinal cord and roots, and severe demyelination. Cellular infiltrates were detectable before onset of clinical symptoms. In contrast, neurological symptoms were never observed in control line 27 mice (Fig. 2A ). These results indicate a correlation between elevated constitutive expression of B7.2 on microglial cells and susceptibility to spontaneous disease development.

View larger version (44K): [in this window] [in a new window]   Figure 2. B7.2 transgenic line 31 mice develop T cell-mediated neurological disease. Line 31 (n=38), line 27 mice (n=29), and TCRß-/- line 31 mice (n=38) were examined weekly for neurological symptoms. At 3 months of age or later (median=136 days), line 31 mice showed clinical symptoms. No disease was observed in line 27 or TCRß-/- line 31 mice (A). Insert: line 31 mice displayed deterioration of hind limb control and coordination. In adoptively transferred TCRß-/- animals, flow cytometric analysis of CD8 and TCRß expression showed T cell infiltrates only in symptomatic line 31 TCRß -/- mice reconstituted with line 31 T cells (B). Immunohistochemical staining of B7.2 in corresponding animals illustrated B7.2 expression only on microglial cells (arrowhead and insert) in control TCRß -/- line 31 mice (C). In adoptively transferred animals, infiltrating T cells (arrows and insert) were also observed in symptomatic TCRß -/- line 31 animals. No B7.2 staining or cellular infiltrates were observed in adoptively transferred C57Bl/6 TCRß -/- mice.

3. T cells and expression of B7.2 in the nervous tissue are both required for disease development
TCRß -/- line 31 animals never developed disease or showed mononuclear cell infiltrates in the spinal cord parenchyma or roots, indicating that T cells are required for the development of the demyelinating disease (Fig. 2A ). Moreover, T cells isolated from line 31 mice transferred disease into line 31 TCRß -/- mice but not into C57Bl/6 TCRß -/- mice (Fig. 2B, C ), implying that B7.2 expression in the nervous tissue is also a requirement for disease induction.

4. CD8+ T cells dominate nervous tissue infiltrates
There was a dramatic skewing toward CD8+ T cells in the nervous tissue of line 31 mice, becoming more marked with clinical manifestations of disease. CD8+ T cells exhibited a phenotype consistent with recent activation (CD44^high, CD62L^low, CD11b/Mac-1^int), and a high proportion produced IFN-. mRNA for the proinflammatory cytokine TNF- and for the cytotoxic effector molecules perforin and granzyme B were elevated in the spinal cord of line 31 mice with clinical disease. Taken together, these results indicate that accumulation of memory-effector CD8+ T cells in the nervous tissue correlates with the symptomatic state in line 31 mice.

CONCLUSIONS AND SIGNIFICANCE

In this study we have shown that mice that constitutively express the costimulatory ligand B7.2 on microglia and peripheral root macrophages spontaneously develop demyelinating disease. These findings represent the first demonstration that dysregulated expression of a costimulatory ligand in the nervous system is required for the spontaneous development of an autoimmune demyelinating disease.

Our study demonstrates that two events are required for the development of organ-specific autoimmunity: antigen presentation in the context of costimulation in the target organ and access of T cells to it. We were able to separate these experimentally. In line 27 and 31 mice, transgene-derived B7.2 expression on T cells leads to a similar increased frequency of memory T cells in peripheral lymphoid organs. The frequency of memory T cells likely contributes to the development of the neurological disease in line 31 mice, because memory T cells have an increased ability to cross the blood–brain barrier. The lack of disease development in line 27 mice, which showed a slightly higher frequency of spinal cord T cells compared with wild-type mice, indicates that enhanced T cell trafficking through the nervous tissue is not sufficient to initiate an autoimmune response in the absence of effective antigen presentation in the target tissue. The necessary role played by B7.2 expression on microglia in the development of this autoimmune neurological disorder is highlighted by the induction of pathological changes upon transfer of T cells from line 31 mice into TCRß -/- line 31 mice but not into C57Bl/6 TCRß -/- mice.

Our data support the following model for the development of autoimmune disease in the nervous system (Fig. 3 ). T cells access healthy nervous tissue to exercise immune surveillance, as in other tissues. Increased frequency of memory T cells, such as it occurs during infection, could increase access of peripheral T cells to the nervous tissue. T cells that have specificities for self-antigen(s) presented by microglial cells, instead of being anergized as previously shown by others, engage in productive antigen recognition and are reactivated because of high constitutive expression of the costimulatory ligand B7.2 by these resident antigen-presenting cells. They proliferate, express cytotoxic effector functions, and promote demyelination. This progresses to overt disease. It is likely that T cells encounter antigens that they recognize in tissues other than the nervous system in line 31 mice. However, lack of B7.2 expression on resident antigen-presenting cells in those tissues precludes their responses.

View larger version (42K): [in this window] [in a new window]   Figure 3. Schematic diagram. Costimulatory ligand expression by CNS resident microglia is required for spontaneous development of autoimmune demyelinating disease. 1) T cells enter the healthy CNS to perform immune surveillance. 2) In the healthy CNS, T cells that interact with microglia are anergized due to a lack of costimulation. 3) Activation of microglia as it occurs during infection, up-regulates costimulatory ligands such as B7.2. T cells specific for myelin peptides presented in the context of B7.2 by activated microglial cells are reactivated, leading to destructive autoimmunity.

The fact that CD8+ T cells dominate the pathology of disease in line 31 mice is of interest. Myelin-specific CD8+ T cells, in addition to CD4+ T cells, are implicated as mediators of CNS autoimmunity in multiple sclerosis, an inflammatory disease of the CNS, and Guillain-Barré syndrome, an inflammatory disease of the peripheral nervous system.

Our findings lend weight to models for induction of autoimmunity through infection, which is the most potent stimulus for B7 expression. Individuals whose MHC genetics permit presentation of cross-reactive epitopes to a self-reactive T cell repertoire may be pushed over a disease threshold by costimulation in the target tissue. Our demonstration that expression of B7 on nervous system myeloid cells induces demyelinating disease validates a key component of this hypothesis.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0199fje doi: 10.1096/fj.03-0199fje

² S.P.Z. and M.B. contributed equally to this work.

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