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The role of cellular immune response in Theiler's virus-induced central nervous system demyelination
Journal of Neuroimmunology 147 (2004) 73 – 77 www.elsevier.com/locate/jneuroim
The role of cellular immune response in Theiler’s virus-induced central nervous system demyelination M. Kariuki Njenga a,*, Cristina Marques a, Moses Rodriguez b a
Department of Veterinary Pathobiology, University of Minnesota, 1971 Commonwealth Avenue, St. Paul, MN, 55108, USA b Departments of Neurology and Immunology, Mayo Clinic, Rochester, MN, USA
Abstract Theiler’s murine encephalomyelitis virus (TMEV) persists in spinal cord white matter of susceptible mice (e.g., SJL/J), resulting in chronic inflammation and demyelination. Reconstitution of severe combined immunodeficient (SCID) mice with CD4+ T- or CD8+ Tlymphocytes results in extensive TMEV-induced demyelination, and depletion of CD8+ T-lymphocytes in the early or late phase of the disease decreases the extent of demyelination, indicating that the cellular immune response against the virus plays a key role in myelin destruction. In susceptible mice, the demyelinated lesions are characterized by infiltration of a large numbers of B- and Tlymphocytes; whereas in mice resistant to TMEV-induced demyelination (e.g., C57BL/6), virus clearance requires infiltration of between 2.9 105 and 5.7 105 CD8+ T-lymphocytes and between 3.4 105 and 6.1 105 CD4+ T-lymphocytes per mouse in the brain 5 – 9 days post infection. Transgenic expression of capsid proteins of TMEV abrogates resistance in C56BL/6 mice, rendering the mice susceptible to TMEV persistence and demyelination. Comparison of the kinetics of virus replication and B- and Tlymphocyte infiltration in mice lacking key adhesion molecules (L-selectin (L-sel / ), P-selectin (P-sel / ), intracellular adhesion molecule-1 (ICAM-1 / ), or leukocyte function-associated antigen-1 (LFA-1 / )) demonstrates a role for individual adhesion molecules in recruitment of immune cells into central nervous system (CNS), but the role is not significant to prevent eventual virus clearance. D 2003 Elsevier B.V. All rights reserved. Keywords: Theiler’s syndrome; Demyelination; Cellular immune response; Adhesion molecules
Theiler’s murine encephalomyelitis virus (TMEV), a positive stranded RNA virus belonging to the Picornaviridae family induces a central nervous system (CNS) demyelinating disease in mice that serves as a model for multiple sclerosis (MS) in humans. Intracerebral inoculation of susceptible strains of mice with TMEV induces a biphasic CNS disease characterized by acute encephalitis in the first 2 weeks, followed by a chronic phase in which the virus persists in glial cells and macrophages, resulting in progressive inflammatory myelin destruction in the white matter of the spinal cord (Lipton, 1975; Rodriguez et al., 1987). As in MS, there is strong involvement of cellular immune response in the TMEV-associated myelin destruction and neurologic deficits observed in mice infected with the virus. For example, depletion of CD8+ T-lymphocytes in
* Corresponding author. Tel.: +1-612-625-2719; fax: +1-612-6255203. E-mail address: [email protected] (M.K. Njenga). 0165-5728/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2003.10.042
early or late phase of the disease decreases the extent of demyelination, whereas reconstitution of severe combined immunodeficient (SCID) mice with CD4+ T- or CD8+ Tlymphocytes results in extensive TMEV-induced demyelination (Rodriguez et al., 1996; Rodriguez and Sriram, 1988). The prevailing opinion is that B- and T-lymphocytes play paradoxical functions in the TMEV-induced CNS disease; participating in the virus clearance in CNS cells during the acute phase of the disease, but aggravating the demyelinating process in the chronic phase of the disease. This is supported by the fact that the effective immune response observed in mice resistant to TMEV-induced demyelination (e.g., C57BL/6) is characterized by infiltration of a large number of CD4+ T- and CD8+ T-lymphocytes in the CNS soon after intracerebral infection (3 –7 days post infection) (Monteyne et al., 1997). This rapid recruitment of T lymphocytes results in the decline of virus titers and complete viral clearance within 21 days (Borrow et al., 1992; Fiette et al., 1993; Lindsley and Rodriguez, 1989; Nicholson et al., 1996; Pullen et al., 1993; Rodriguez et al.,
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1993). On the other hand, the demyelinated lesions in the spinal cord white matter are characterized by infiltration of a large numbers of B- and T-lymphocytes, suggesting that recruitment of these cells into the CNS is an important step in the process of myelin destruction (Clatch et al., 1986; Clatch et al., 1985; Dal Canto and Lipton, 1977; Kurtz et al., 1995; Rodriguez et al., 1996; Yauch and Kim, 1994). We have been interested in the factors involved in Band T-lymphocyte trafficking into the CNS following TMEV inoculation, in particular, the activation and antigen specificity of these cells, and the role of adhesion molecules in the process. The evidence so far indicates that the immune responses to capsid antigens of TMEV (particularly VP1, VP2, and VP3) contribute to protection against virus persistence in resistant strains of mice; however, the immune response also contributes to myelin destruction once the virus has persisted in susceptible strains of mice. For example, studies have shown that a small peptide within the VP2 capsid protein of TMEV, VP2121 – 130 is the immunodominant epitope recognized by 55– 75% of CD8+ T-lymphocytes infiltrating the CNS of resistant strains of mice, and an epitope located in VP1 (VP1233244) was identified as the main class IIrestricted T-lymphocyte antigen in demyelinated lesions of SJL/J mice (Yauch et al., 1994). In addition, important antibody epitopes have been shown to be located in the VP1 protein (VP112 – 25, VP1146 – 160, and VP1262 – 276), VP2 protein (VP22 – 16 and VP2165 – 179), and VP3 protein (VP324 – 37) (Inoue et al., 1994; Kim et al., 1992). To more specifically address this question, we generated two lines of transgenic mice expressing the viral coding sequences encompassing the VP1 capsid protein or the combined VP2, VP3, and VP4 capsid proteins and one line of transgenic mice expressing the non-capsid TMEV proteins (combined 2A + 2B + 2C + 3A +3B + 3C + 3D), each under the control of class I major histocompatibility complex promoter in order to allowed viral peptides to be assessed as self-antigens during thymic lymphocyte selection in the mice (Lin et al., 2002). The two lines of transgenic mice expressing TMEV capsid proteins, generated in a resistant H-2b background, were tolerized to the viral transgenes and became susceptible to virusinduced demyelination following intracerebral inoculation with TMEV, whereas the mice expressing the non-capsid viral proteins remained resistant to TMEV-induced demyelination. These findings indicated that coding regions in the capsid proteins of TMEV were important for protection. Tolerance in the TMEV transgenic mice occurred in the context of CD8+ class I-restricted responses, because antibody and class II-restricted responses appeared unaffected in the TMEV capsid proteins transgenic mice (Lin et al., 2002). The antigen specificity of the expressed transgene was demonstrated by the finding that CNSinfiltrating CD8+ T-lymphocytes obtained from TMEVinfected VP1 transgenic mice could not lyse VP1-trans-
fected C57SV (H-2b) target cells, whereas the CD8+ Tlymphocytes lysed VP2-transfected target cells. Because the TMEV capsid protein transgenic mice developed focal demyelination, this implied that CD8+ T-lymphocyte responses directed against these epitopes were not required for myelin injury, a result consistent with other data indicating that CD8+ T-lymphocytes are not required for demyelination because mice lacking class I MHC and CD8+ T-lymphocytes can develop TMEV-induced demyelination (Fiette et al., 1993; Rodriguez et al., 1993). Researchers showed previously that mice lacking class I MHC or CD8+ T-lymphocyte function showed TMEVinduced demyelination but they develop less severe neurologic deficits, because of compensatory spontaneous remyelination and up-regulation of sodium channels (Rivera-Quinones et al., 1998). This suggests that CD8+ T-lymphocytes may be one of the factors responsible for damaging axonal surfaces to prevent myelin repair. Following intracerebral TMEV inoculation, naive B- and T-lymphocytes are selected from precursor cells in the primary lymphoid organs, becoming activated and losing their lymphoid organ homing receptors in order to migrate to CNS (Dailey et al., 1982; Mackay, 1993; Rigby and Dailey, 2000). We used mice with targeted deletion of Lselectin (L-sel / ), leukocyte-function associated antigen-1 (LFA-1 / ), or both intracellular adhesion molecule-1 and P-selectin (ICAM-1/P-sel / ) to investigate the role of adhesion molecule in immune cells recruitment following virus infection in the CNS. The knockout mice from a resistant H-2b genetic background were infected with TMEV intracerebrally and the number of virus-specific CD8+ T-lymphocytes, CD4+ T-lymphocytes, and B-lymphocytes in the brain, blood, cervical lymph nodes and spleen evaluated 9 days after infection. Both the total number of lymphocytes infiltrating each mouse, determined using the 6-Am bead microsphere method (Dittel and LeBien, 1995; Wolf et al., 1993), and the proportion of each lymphocyte subtype (as percent of total mononuclear cells) were calculated. The average number of lymphocytes infiltrating the brains of TMEV-infected mice were between 7.0 105 and 1.5 106 B-lymphocytes, between 3.4 105 and 6.0 105 CD4+ T-lymphocytes, and between 2.9 105 and 5.7 105 CD8+ T-lymphocytes per mouse. However, there was a 1.5- to 2-fold decrease in the number of CD4+ T- and CD8+ T-lymphocytes in the brains of ICAM-1/ Psel / and L-sel / mice when compared to non-mutant C57BL/6 mice. In the cervical lymph nodes, there was a dramatic 5- to 11-fold decrease in B-lymphocytes, CD4+ T-lymphocytes, and CD8+ T- lymphocytes in L-sel / mice, whereas there was no change in the ICAM-1/P-sel / mice when compared to non-mutant mice. There were no changes in the number of B-lymphocytes, CD4+ T-lymphocytes or CD8+ T-lymphocytes in blood of the knockout mice, but there was a threefold increase in the number of CD4+ T-
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Fig. 1. Representative FACScan dotplots showing the absence of TMEV-specific CD8+ T-lymphocytes in cervical lymph nodes of wild-type C57BL/6, ICAM1/P-sel / , and L-sel / mice. Mononuclear cells were isolated from tissue homogenate of brains, cervical lymph nodes, blood, or spleens from four to five mice of each strain, and stained with R-phycoerythrin Db:VP2121 – 130 tetramer for one h, followed by FITC-conjugated anti-CD8 as described previously (Johnson et al., 1999). The VP2121 – 130 epitope is the immunodominant CD8+ T-lymphocyte epitope in the CNS of TMEV-infected C57BL/6 mice. Ten thousand cells were analyzed per sample after gating for the lymphocyte population. Despite the presence of a large number of CD8+ T-lymphocytes in the cervical lymph nodes (20 – 30% of the mononuclear cells), there were few or no detectable virus-specific CD8+ T-lymphocytes. The data is representative of four independents experiments.
lymphocytes in both L-sel / and ICAM-1/P-sel / mice in the spleen. As expected, there was higher recruitment or retention of virus-specific CD8+ T-lymphocytes in the brain (63 –68% of all CD8+ T-lymphocytes), compared to the blood (13 – 16%) and spleen (1.3 – 6%) in both knockout and non-mutant mice (Fig. 1). A surprising finding was that there were few or no virus-specific CD8+
T-lymphocytes in the cervical lymph nodes (Fig. 1), suggesting that CD8+ T-lymphocyte priming following infection in the brain may occur either within the brain, or in other distant lymphoid organs. In contrast, the proportion of CD4+ T-lymphocytes in the brain, cervical lymph nodes, blood, and spleen were comparable in all strains of mice (Fig. 2). The ICAM-1/P-sel / mice
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Fig. 2. Representative FACScan dotplots showing proportions of CD4+ T-lymphocytes in the brain, cervical lymph nodes, blood, and spleen of wild-type C57BL/6, ICAM-1/P-sel / , and L-sel / mice. Mononuclear cells were isolated from tissue homogenate of brains, cervical lymph nodes, blood, or spleens from four to five mice of each strain, and stained with anti-CD4 antibody. Ten thousand cells were analyzed per sample after gating for the lymphocytes. The data is representative of four independent experiments.
allowed virus persistence in the spinal cord for up to 21 days after infection, whereas the wild-type mice cleared the virus. Levels of virus-specific IgG’s were consistently lower in L-sel / mice when compared to wild-type mice up to 28 days after infection, whereas ICAM-1/P-sel / mice showed a drop in virus-specific IgG levels only at 21 days post infection. The levels of infectious TMEV, as measured by plaque assay at 3, 7, 14, and 28 days after infection, were between 4 and 6 log10 PFU of virus per gram of CNS tissues at day 3 and 7 post infection, and then decreased to undetectable levels by day 14 after infection in the knockout and non-mutant C57Bl/6 mice.
Taken together, results suggest a minor individual role for ICAM-1/P-sel and LFA-1 adhesion molecules, which is not significant enough to prevent virus clearance from the CNS.
L -sel,
Acknowledgements This research was supported by the P01-NS 38468 and NS 32129 grants from the National Institutes of Health and a grant from the Academic Health Center of the University of Minnesota. We thank Rebecca LaRue,
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Jeremy Alley, and Laurie Brewer for their technical support. References Borrow, P., Tonks, P., Welsh, C.J.R., Nash, A.A., 1992. The role of CD8+ T cells in the acute and chronic phases of Theiler’s murine encephalomyelitis virus-induced disease in mice. J. Gen. Virol. 73, 1861 – 1865. Clatch, R.J., Melvold, R.W., Miller, S.D., Lipton, H.L., 1985. Theiler’s murine encephalomyelitis virus (TMEV)-induced demyelinating disease in mice is influenced by the H-2D region: correlation with TMEV-specific delayed-type hypersensitivity. J. Immunol. 135, 1408 – 1414. Clatch, R.J., Lipton, H.L., Miller, S.D., 1986. Characterization of Theiler’s murine encephalomyelitis virus (TMEV)-specific delayed-type hypersensitivity responses in TMEV-induced demyelinating disease: correlation with clinical signs. J. Immunol. 136, 920 – 927. Dailey, M.O., Fathman, C.G., Butcher, E.C., Pillemer, E., Weisman, I., 1982. Abnormal migration of T lymphocyte clones. J. Immunol. 128, 2134 – 2136. Dal Canto, M.C., Lipton, H.L., 1977. Multiple sclerosis. Animal model: Theiler’s virus infection in mice. Am. J. Pathol. 88, 497 – 500. Dittel, B.N., LeBien, T.W., 1995. The growth response to IL-7 during normal human B cell ontogeny is restricted to B-lineage cells expressing CD34. J. Immunol. 154, 58 – 67. Fiette, L., Aubert, C., Brahic, M., Rossi, C.P., 1993. Theiler’s virus infection of beta 2-microglobulin-deficient mice. J. Virol. 67, 589 – 592. Inoue, A., Choe, Y.K., Kim, K.S., 1994. Analysis of antibody responses to predominant linear epitopes on Theiler’s murine encephalomyelitis virus. J. Virol. 68, 3324 – 3333. Johnson, A.J., Njenga, M.K., Hansen, M.J., Kuhns, S.T., Chen, L., Rodriguez, M., Pease, L.R., 1999. Prevalent class I-restricted T cell-response to the Theiler’s virus epitope Db:VP2121-130 in the absence of endogenous CD4 help, tumor necrosis factor alpha, gamma interferon, perforin, or costimulation through CD28. J. Virol. 73, 3702 – 3708. Kim, B.S., Choe, Y.K, Crane, M.A., Jue, C.R., 1992. Identification and localization of a limited number of predominant conformation-independent antibody epitopes of Theiler’s murine encephalomyelitis virus. Immunol. Lett. 31, 199 – 205. Kurtz, C.I., Sun, X.M., Fujinami, R.S., 1995. B-lymphocyte requirement for vaccine-mediated protection from Theiler’s murine encephalomyelitis virus-induced central nervous system disease. J. Virol. 69, 5152 – 5155. Lin, X., Njenga, M.K., Johnson, A.J., Pavelko, K.D., David, C.S., Pease, L.R., Rodriguez, M., 2002. Transgenic expression of Theiler’s murine encephalomyelitis virus genes in H-2b mice inhibits resistance to virusinduced demyelination. J. Virol. 76, 7799 – 7811.
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Lindsley, M.D., Rodriguez, M., 1989. Characterization of the inflammatory response in the central nervous system of mice susceptible or resistant to demyelination by Theiler’s virus. J. Immunol. 142, 2677 – 2682. Lipton, H.L., 1975. Theiler’s virus infection in mice: an unusual biphasic disease process leading to demyelination. Infect. Immun. 11, 1147 – 1155. Mackay, C.R., 1993. Homing of naive, memory and effector lymphocytes. Curr. Opin. Immunol. 5, 423 – 427. Monteyne, P., Bureau, J.F., Brahic, M., 1997. The infection of mouse by Theiler’s virus: from genetics and immunology. Immunol. Rev. 159, 163 – 176. Nicholson, S.M., Dal Canto, M.C., Miller, S.D., Melvold, R.W., 1996. Adoptively transferred CD8+ T lymphocytes provide protection against TMEV-induced demyelinating disease in BALB/c mice. J. Immunol. 156, 1276 – 1283. Pullen, L.C., Miller, S.D., Dal Canto, M.C., Kim, B.S., 1993. Class Ideficient resistant mice intracerebrally inoculated with Theiler’s virus show an increased T cell response to viral antigens and susceptibility to demyelination. Eur. J. Immunol. 23, 2287 – 2293. Rigby, S., Dailey, M.O., 2000. Traffic of L-selectin-negative T cells to sites of inflammation. Eur. J. Immunol. 30, 98 – 107. Rivera-Quinones, C., McGavern, D., Schmelzer, J.D., Hunter, S.F., Low, P.A., Rodriguez, M., 1998. Absence of neurologic deficits following extensive demyelination in class I-deficient murine model of multiple sclerosis. Nat. Med. 4, 187 – 193. Rodriguez, M., Sriram, S., 1988. Successful therapy of Theiler’s virusinduced demyelination (DA strain) with monoclonal anti-Lyt-2 antibody. J. Immunol. 140, 2950 – 2955. Rodriguez, M., Oleszak, E., Leibowitz, J., 1987. Theiler’s murine encephalomyelitis: a model of demyelination and persistence of virus. Crit. Rev. Immunol. 7, 325 – 366. Rodriguez, M., Dunkel, A.J., Thiemann, R.L., Leibowitz, J., Zjlstra, M., Jaenisch, R., 1993. Abrogation of resistance to Theiler’s virus-induced demyelination in H-2b mice deficient in h2-mcroglubulin. J. Immunol. 151, 266 – 276. Rodriguez, M., Pavelko, K.D., Njenga, M.K., Logan, W.C., Wettstein, P.J., 1996. The balance between persistent infection and immune cells determines demyelination. J. Immunol. 157, 5699 – 5709. Wolf, M.L., Weng, W.K., Stieglbauer, K.T., Shah, N., LeBien, T.W., 1993. Functional effect of IL-7-enhanced CD19 expression on human B cell precursors. J. Immunol. 151, 138 – 148. Yauch, R.L., Kim, B.S., 1994. A predominant viral epitope recognized by T cells from the periphery and demyelinating lesions of SJL/J mice infected with Theiler’s virus is located within VP1233 – 244. J. Immunol. 153, 4508 – 4519. Yauch, R.L., Palma, J.P., Yahikozawa, H., Koh, C.S., Kim, B.S., 1998. Role of individual T-cell epitopes of Theiler’s virus in pathogenesis of demyelination correlates with the ability to induce a Th1 response. J. Virol. 72, 6169 – 6174.
The role of cellular immune response in Theiler’s virus-induced central nervous system demyelination M. Kariuki Njenga a,*, Cristina Marques a, Moses Rodriguez b a
Department of Veterinary Pathobiology, University of Minnesota, 1971 Commonwealth Avenue, St. Paul, MN, 55108, USA b Departments of Neurology and Immunology, Mayo Clinic, Rochester, MN, USA
Abstract Theiler’s murine encephalomyelitis virus (TMEV) persists in spinal cord white matter of susceptible mice (e.g., SJL/J), resulting in chronic inflammation and demyelination. Reconstitution of severe combined immunodeficient (SCID) mice with CD4+ T- or CD8+ Tlymphocytes results in extensive TMEV-induced demyelination, and depletion of CD8+ T-lymphocytes in the early or late phase of the disease decreases the extent of demyelination, indicating that the cellular immune response against the virus plays a key role in myelin destruction. In susceptible mice, the demyelinated lesions are characterized by infiltration of a large numbers of B- and Tlymphocytes; whereas in mice resistant to TMEV-induced demyelination (e.g., C57BL/6), virus clearance requires infiltration of between 2.9 105 and 5.7 105 CD8+ T-lymphocytes and between 3.4 105 and 6.1 105 CD4+ T-lymphocytes per mouse in the brain 5 – 9 days post infection. Transgenic expression of capsid proteins of TMEV abrogates resistance in C56BL/6 mice, rendering the mice susceptible to TMEV persistence and demyelination. Comparison of the kinetics of virus replication and B- and Tlymphocyte infiltration in mice lacking key adhesion molecules (L-selectin (L-sel / ), P-selectin (P-sel / ), intracellular adhesion molecule-1 (ICAM-1 / ), or leukocyte function-associated antigen-1 (LFA-1 / )) demonstrates a role for individual adhesion molecules in recruitment of immune cells into central nervous system (CNS), but the role is not significant to prevent eventual virus clearance. D 2003 Elsevier B.V. All rights reserved. Keywords: Theiler’s syndrome; Demyelination; Cellular immune response; Adhesion molecules
Theiler’s murine encephalomyelitis virus (TMEV), a positive stranded RNA virus belonging to the Picornaviridae family induces a central nervous system (CNS) demyelinating disease in mice that serves as a model for multiple sclerosis (MS) in humans. Intracerebral inoculation of susceptible strains of mice with TMEV induces a biphasic CNS disease characterized by acute encephalitis in the first 2 weeks, followed by a chronic phase in which the virus persists in glial cells and macrophages, resulting in progressive inflammatory myelin destruction in the white matter of the spinal cord (Lipton, 1975; Rodriguez et al., 1987). As in MS, there is strong involvement of cellular immune response in the TMEV-associated myelin destruction and neurologic deficits observed in mice infected with the virus. For example, depletion of CD8+ T-lymphocytes in
* Corresponding author. Tel.: +1-612-625-2719; fax: +1-612-6255203. E-mail address: [email protected] (M.K. Njenga). 0165-5728/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2003.10.042
early or late phase of the disease decreases the extent of demyelination, whereas reconstitution of severe combined immunodeficient (SCID) mice with CD4+ T- or CD8+ Tlymphocytes results in extensive TMEV-induced demyelination (Rodriguez et al., 1996; Rodriguez and Sriram, 1988). The prevailing opinion is that B- and T-lymphocytes play paradoxical functions in the TMEV-induced CNS disease; participating in the virus clearance in CNS cells during the acute phase of the disease, but aggravating the demyelinating process in the chronic phase of the disease. This is supported by the fact that the effective immune response observed in mice resistant to TMEV-induced demyelination (e.g., C57BL/6) is characterized by infiltration of a large number of CD4+ T- and CD8+ T-lymphocytes in the CNS soon after intracerebral infection (3 –7 days post infection) (Monteyne et al., 1997). This rapid recruitment of T lymphocytes results in the decline of virus titers and complete viral clearance within 21 days (Borrow et al., 1992; Fiette et al., 1993; Lindsley and Rodriguez, 1989; Nicholson et al., 1996; Pullen et al., 1993; Rodriguez et al.,
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1993). On the other hand, the demyelinated lesions in the spinal cord white matter are characterized by infiltration of a large numbers of B- and T-lymphocytes, suggesting that recruitment of these cells into the CNS is an important step in the process of myelin destruction (Clatch et al., 1986; Clatch et al., 1985; Dal Canto and Lipton, 1977; Kurtz et al., 1995; Rodriguez et al., 1996; Yauch and Kim, 1994). We have been interested in the factors involved in Band T-lymphocyte trafficking into the CNS following TMEV inoculation, in particular, the activation and antigen specificity of these cells, and the role of adhesion molecules in the process. The evidence so far indicates that the immune responses to capsid antigens of TMEV (particularly VP1, VP2, and VP3) contribute to protection against virus persistence in resistant strains of mice; however, the immune response also contributes to myelin destruction once the virus has persisted in susceptible strains of mice. For example, studies have shown that a small peptide within the VP2 capsid protein of TMEV, VP2121 – 130 is the immunodominant epitope recognized by 55– 75% of CD8+ T-lymphocytes infiltrating the CNS of resistant strains of mice, and an epitope located in VP1 (VP1233244) was identified as the main class IIrestricted T-lymphocyte antigen in demyelinated lesions of SJL/J mice (Yauch et al., 1994). In addition, important antibody epitopes have been shown to be located in the VP1 protein (VP112 – 25, VP1146 – 160, and VP1262 – 276), VP2 protein (VP22 – 16 and VP2165 – 179), and VP3 protein (VP324 – 37) (Inoue et al., 1994; Kim et al., 1992). To more specifically address this question, we generated two lines of transgenic mice expressing the viral coding sequences encompassing the VP1 capsid protein or the combined VP2, VP3, and VP4 capsid proteins and one line of transgenic mice expressing the non-capsid TMEV proteins (combined 2A + 2B + 2C + 3A +3B + 3C + 3D), each under the control of class I major histocompatibility complex promoter in order to allowed viral peptides to be assessed as self-antigens during thymic lymphocyte selection in the mice (Lin et al., 2002). The two lines of transgenic mice expressing TMEV capsid proteins, generated in a resistant H-2b background, were tolerized to the viral transgenes and became susceptible to virusinduced demyelination following intracerebral inoculation with TMEV, whereas the mice expressing the non-capsid viral proteins remained resistant to TMEV-induced demyelination. These findings indicated that coding regions in the capsid proteins of TMEV were important for protection. Tolerance in the TMEV transgenic mice occurred in the context of CD8+ class I-restricted responses, because antibody and class II-restricted responses appeared unaffected in the TMEV capsid proteins transgenic mice (Lin et al., 2002). The antigen specificity of the expressed transgene was demonstrated by the finding that CNSinfiltrating CD8+ T-lymphocytes obtained from TMEVinfected VP1 transgenic mice could not lyse VP1-trans-
fected C57SV (H-2b) target cells, whereas the CD8+ Tlymphocytes lysed VP2-transfected target cells. Because the TMEV capsid protein transgenic mice developed focal demyelination, this implied that CD8+ T-lymphocyte responses directed against these epitopes were not required for myelin injury, a result consistent with other data indicating that CD8+ T-lymphocytes are not required for demyelination because mice lacking class I MHC and CD8+ T-lymphocytes can develop TMEV-induced demyelination (Fiette et al., 1993; Rodriguez et al., 1993). Researchers showed previously that mice lacking class I MHC or CD8+ T-lymphocyte function showed TMEVinduced demyelination but they develop less severe neurologic deficits, because of compensatory spontaneous remyelination and up-regulation of sodium channels (Rivera-Quinones et al., 1998). This suggests that CD8+ T-lymphocytes may be one of the factors responsible for damaging axonal surfaces to prevent myelin repair. Following intracerebral TMEV inoculation, naive B- and T-lymphocytes are selected from precursor cells in the primary lymphoid organs, becoming activated and losing their lymphoid organ homing receptors in order to migrate to CNS (Dailey et al., 1982; Mackay, 1993; Rigby and Dailey, 2000). We used mice with targeted deletion of Lselectin (L-sel / ), leukocyte-function associated antigen-1 (LFA-1 / ), or both intracellular adhesion molecule-1 and P-selectin (ICAM-1/P-sel / ) to investigate the role of adhesion molecule in immune cells recruitment following virus infection in the CNS. The knockout mice from a resistant H-2b genetic background were infected with TMEV intracerebrally and the number of virus-specific CD8+ T-lymphocytes, CD4+ T-lymphocytes, and B-lymphocytes in the brain, blood, cervical lymph nodes and spleen evaluated 9 days after infection. Both the total number of lymphocytes infiltrating each mouse, determined using the 6-Am bead microsphere method (Dittel and LeBien, 1995; Wolf et al., 1993), and the proportion of each lymphocyte subtype (as percent of total mononuclear cells) were calculated. The average number of lymphocytes infiltrating the brains of TMEV-infected mice were between 7.0 105 and 1.5 106 B-lymphocytes, between 3.4 105 and 6.0 105 CD4+ T-lymphocytes, and between 2.9 105 and 5.7 105 CD8+ T-lymphocytes per mouse. However, there was a 1.5- to 2-fold decrease in the number of CD4+ T- and CD8+ T-lymphocytes in the brains of ICAM-1/ Psel / and L-sel / mice when compared to non-mutant C57BL/6 mice. In the cervical lymph nodes, there was a dramatic 5- to 11-fold decrease in B-lymphocytes, CD4+ T-lymphocytes, and CD8+ T- lymphocytes in L-sel / mice, whereas there was no change in the ICAM-1/P-sel / mice when compared to non-mutant mice. There were no changes in the number of B-lymphocytes, CD4+ T-lymphocytes or CD8+ T-lymphocytes in blood of the knockout mice, but there was a threefold increase in the number of CD4+ T-
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Fig. 1. Representative FACScan dotplots showing the absence of TMEV-specific CD8+ T-lymphocytes in cervical lymph nodes of wild-type C57BL/6, ICAM1/P-sel / , and L-sel / mice. Mononuclear cells were isolated from tissue homogenate of brains, cervical lymph nodes, blood, or spleens from four to five mice of each strain, and stained with R-phycoerythrin Db:VP2121 – 130 tetramer for one h, followed by FITC-conjugated anti-CD8 as described previously (Johnson et al., 1999). The VP2121 – 130 epitope is the immunodominant CD8+ T-lymphocyte epitope in the CNS of TMEV-infected C57BL/6 mice. Ten thousand cells were analyzed per sample after gating for the lymphocyte population. Despite the presence of a large number of CD8+ T-lymphocytes in the cervical lymph nodes (20 – 30% of the mononuclear cells), there were few or no detectable virus-specific CD8+ T-lymphocytes. The data is representative of four independents experiments.
lymphocytes in both L-sel / and ICAM-1/P-sel / mice in the spleen. As expected, there was higher recruitment or retention of virus-specific CD8+ T-lymphocytes in the brain (63 –68% of all CD8+ T-lymphocytes), compared to the blood (13 – 16%) and spleen (1.3 – 6%) in both knockout and non-mutant mice (Fig. 1). A surprising finding was that there were few or no virus-specific CD8+
T-lymphocytes in the cervical lymph nodes (Fig. 1), suggesting that CD8+ T-lymphocyte priming following infection in the brain may occur either within the brain, or in other distant lymphoid organs. In contrast, the proportion of CD4+ T-lymphocytes in the brain, cervical lymph nodes, blood, and spleen were comparable in all strains of mice (Fig. 2). The ICAM-1/P-sel / mice
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Fig. 2. Representative FACScan dotplots showing proportions of CD4+ T-lymphocytes in the brain, cervical lymph nodes, blood, and spleen of wild-type C57BL/6, ICAM-1/P-sel / , and L-sel / mice. Mononuclear cells were isolated from tissue homogenate of brains, cervical lymph nodes, blood, or spleens from four to five mice of each strain, and stained with anti-CD4 antibody. Ten thousand cells were analyzed per sample after gating for the lymphocytes. The data is representative of four independent experiments.
allowed virus persistence in the spinal cord for up to 21 days after infection, whereas the wild-type mice cleared the virus. Levels of virus-specific IgG’s were consistently lower in L-sel / mice when compared to wild-type mice up to 28 days after infection, whereas ICAM-1/P-sel / mice showed a drop in virus-specific IgG levels only at 21 days post infection. The levels of infectious TMEV, as measured by plaque assay at 3, 7, 14, and 28 days after infection, were between 4 and 6 log10 PFU of virus per gram of CNS tissues at day 3 and 7 post infection, and then decreased to undetectable levels by day 14 after infection in the knockout and non-mutant C57Bl/6 mice.
Taken together, results suggest a minor individual role for ICAM-1/P-sel and LFA-1 adhesion molecules, which is not significant enough to prevent virus clearance from the CNS.
L -sel,
Acknowledgements This research was supported by the P01-NS 38468 and NS 32129 grants from the National Institutes of Health and a grant from the Academic Health Center of the University of Minnesota. We thank Rebecca LaRue,
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