Human HLA-DR transgenes protect mice from fatal virus-induced encephalomyelitis and chronic demyelination

Abstract

We evaluated the participatory role of human HLA-DR molecules in control of virus from the central nervous system and in the development of subsequent spinal cord demyelination. The experiments utilized intracranial infection with Theiler's murine encephalomyelitis virus (TMEV), a picornavirus that, in some strains of mice, results in primary demyelination. We studied DR2 and DR3 transgenic mice that were bred onto a combined class I-deficient mouse (beta-2 microglobulin deficient; beta2m(0)) and class II-deficient mouse (Abeta(0)) of the H-2(b) background. Abeta(0).beta2m(0) mice infected with TMEV died within 18 days of infection. These mice showed severe encephalomyelitis due to rapid replication of virus genome. In contrast, transgenic mice with insertion of a single human class II major histocompatibility complex (MHC) gene (DR2 or DR3) survived the acute infection. DR2 and DR3 mice controlled virus infection by 45 days and did not develop spinal cord demyelination. Levels of virus RNA were reduced in HLA-DR transgenic mice compared to Abeta(0).beta2m(0) mice. Virus-neutralizing antibody responses did not explain why DR mice survived the infection and controlled virus replication. However, DR mice showed an increase in gamma interferon and interleukin-2 transcripts in the brain, which were associated with protection. The findings support the hypothesis that the expression of a single human class II MHC molecule can, by itself, influence the control of an intracerebral pathogen in a host without a competent class I MHC immune response. The mechanism of protection appears to be the result of cytokines released by CD4(+) T cells.

FIG. 1.

Spinal cord pathology following TMEV infection (18 days). Glycol methacrylate plastic-embedded sections were stained with modified erichrome/cresyl violet stain. (A) Absence of gray matter pathology in B6 mouse. (B) Presence of inflammation and neuronal injury in the gray matter of an Aβ⁰.β2m⁰ mouse. (C) Absence of gray matter pathology in an Aβ⁰.β2m⁰.DR2 mouse. (D) Absence of gray matter pathology in an Aβ⁰.β2m⁰.DR3 mouse. (E) Presence of inflammation in the gray matter pathology in a β2m⁰ mouse. (F) Presence of inflammation in the gray matter pathology in an Aβ⁰ mouse.

FIG. 2.

Spinal cord pathology following TMEV infection (45 days). Glycol methacrylate plastic-embedded sections were stained with modified erichrome/cresyl violet stain. (A) Absence of white matter pathology in a B6 mouse. (B) Presence of inflammation and demyelination in a β2m⁰ mouse. (C) Absence of white matter pathology in an Aβ⁰.β2m⁰.DR2 mouse. (D) Absence of white matter pathology in an Aβ⁰.β2m⁰.DR3 mouse. (E) Positive control showing demyelination in an SJL/J mouse. (F) Positive control showing demyelination in a B10.Q mouse.

FIG. 3.

Pathology of the brain. Pathological analysis of brain areas (cerebellum, brain stem, cortex, hippocampus, striatum, corpus callosum, and meninges) at 18 days and 45 days following TMEV infection. The mouse strains shown are B6, β2m⁰, Aβ⁰, Aβ⁰.β2m⁰, Aβ⁰.β2m⁰.DR2, and Aβ⁰.β2m⁰.DR3. Pathological qualitative scores from 0 to 4 are described in Materials and Methods. Each point represents one mouse. There were no differences between the strains in the distribution of brain pathologies at 18 days after infection. At the 45-day time point, data were not available (NA) for Aβ⁰.β2m⁰ mice because all of these mice were dead (or moribund and thus sacrificed for humane reasons) by 18 days after infection. These data are representative of two of the three experiments performed for each strain at each time point.

FIG. 4.

Virus antigen-positive cells. Virus antigen-positive cells were determined by immunoperoxidase staining and expressed as the percentage of spinal quadrants showing virus antigen-positive cells in either the gray matter or the white matter. Twenty to thirty spinal cord quadrants were analyzed for each mouse. Analysis was done 18 days and 45 days after infection. Each bar represents one animal. (A) B6 mice (18 days). (B) β2m⁰ mice (18 days). (C) Aβ⁰ mice (18 days). (D) Aβ⁰.β2m⁰ mice (18 days). (E) Aβ⁰.β2m⁰.DR2 mice (18 days). (F) Aβ⁰.β2m⁰.DR3 mice (18 days). (G) B6 mice (45 days). (H) β2m⁰ mice (45 days). (I) Aβ⁰ mice (45 days). (J) Aβ⁰.β2m⁰.DR2 mice (45 days). (K) Aβ⁰.β2m⁰.DR3 mice (45 days). (L) N/A, no animals are available because all Aβ⁰.β2m⁰ mice died from TMEV infection by day 18.

FIG. 5.

Virus RNA expression. Levels of virus RNA expression in mice at 7, 18, and 45 days following TMEV infection analyzed independently in the brain and spinal cord. Levels of viral capsid RNA message were quantified by Light Cycler PCR using primers for capsid VP2. Each symbol represents the level from a single mouse. The levels of expression of GAPDH RNA were consistent among the groups (brains, log₁₀ 7.40 ± 0.03 virus copies; spinal cords, log₁₀ 7.01 ± 0.03 virus copies). The dotted line represents the sensitivity of the assay. Symbols along or below the line indicate animals with minimal or no detection of virus RNA.

FIG. 6.

Virus-specific humoral immune responses. Shown are the results of an ELISA for serum IgG antibodies at 18 and 45 days after infection directed against purified TMEV antigens in various strains of mice. Negative controls are from mice not infected with TMEV.

FIG. 7.

FACS analyses of BILs. Mice were infected for 7 days with TMEV, and then BILs were isolated using a Percoll gradient. Cells were double labeled for CD4 or CD8 and analyzed by cell sorting. The number shown in the lower left quadrant of each panel represents the percentage of BILs positive for the CD4 marker. Note that no CD8⁺ T cells were present in the BILs from either Aβ⁰.β2m⁰.DR3 (A, B, and C) or β2m⁰ (D, E, and F) mice.

FIG. 8.

BILs from Aβ⁰.β2m⁰.DR3 mice contain activated CD4⁺ T cells and express similar inflammatory cytokines to B6 mice. (A) RNA transcripts of cytokines in brains of mice at 7 and 18 days postinfection were measured by quantitative real-time RT-PCR. Data are shown as a percentage of transcripts relative to the B6 mice for each gene, and all data are normalized to GAPDH. Each bar represents data from five animals. *, P < 0.05; **, P < 0.001. (B) Flow cytometry of BILs from an Aβ⁰.β2M⁰.DR3 mouse. CD4⁺ T cells from the left panel are gated, and CD69 expression levels are shown on the right panel. The gray histogram represents the isotype control antibody.