The role of CD8(+) T cells and major histocompatibility complex class I expression in the central nervous system of mice infected with neurovirulent Sindbis virus

Abstract

Little is known about the role of CD8(+) T cells infiltrating the neural parenchyma during encephalitis induced by neurovirulent Sindbis virus (NSV). NSV preferentially infects neurons in the mouse brain and spinal cord; however, it is generally accepted that neurons can express few if any major histocompatibility complex (MHC) class I molecules. We evaluated the possible roles and interactions of CD8(+) T cells during NSV encephalitis and demonstrated that MHC class I antigen (H2K/D) was expressed on endothelial cells, inflammatory cells, and ependymal cells after intracerebral inoculation of NSV. No immunoreactivity was observed in neurons. On the other hand, in situ hybridization with probes for MHC class I heavy chain, beta2 microglobulin, and TAP1 and TAP2 mRNAs revealed increased expression in a majority of neurons, as well as in inflammatory cells, endothelial cells, and ependymal cells in the central nervous system of infected mice. NSV-infected neurons may fail to express MHC class I molecules due to a posttranscriptional block or may express only nonclassical MHC class I genes. To better understand the role CD8(+) T cells play during fatal encephalitis induced by NSV, mice lacking functional CD8(+) T cells were studied. The presence or absence of CD8 did not alter outcome, but absence of beta2 microglobulin improved survival. Interestingly, the intracellular levels of viral RNA decreased more rapidly in immunocompetent mice than in mice without functional CD8(+) T cells. These observations suggest that CD8(+) T cells may act indirectly, possibly via cytokines, to contribute to the clearance of viral RNA in neurons.

FIG. 1

Double immunofluorescence microscopy in the CNS of C57BL/6 mice (A, C, D, E, and F) and B2m-KO mice (B) after infection with NSV. Panels A, B, C, and D show double staining for MHC class I (H-2Kb/H-2Db) antigen (green) and SV antigen (red). Panel E shows double staining for F4/80 antigen (green) and SV antigen (red). Panel F shows double staining for H-2Kb/H-2Db antigen (green) and F4/80 antigen (red). At 3 days after infection, H-2Kb/H-2Db antigen was detected in endothelial cells of C57BL/6 mice (A) but not in B2m-KO mice (B). SV-antigen-positive neurons shown in panel A (red) did not demonstrate MHC class I immunoreactivity. At 5 days after infection (C and D), the numbers of MHC class I immunoreactive cells increased, and cells that were positive for MHC class I and SV (arrowheads) were detected. Note that SV-antigen-positive cells with neuronal morphology do not show immunoreactivity for MHC class I. At 5 days after infection (E), F4/80-antigen-positive cells (green) with engulfed SV antigen (red) accumulated. Most F4/80-positive cells (red) detected in NSV-infected foci show MHC class I (green) immunoreactivity (F). (A, B, C, E, and F) Ventral horn of lumbar spinal cords; (D) thalamus. (A, B, C, D, and F) Magnification, ×322; (E) magnification, ×403.

FIG. 2

Northern blot analysis of mRNAs for the MHC class I molecules and peptide transporters in the brains of C57BL/6 and B2m-KO mice at 1, 3, 5, 7, and 10 days after intracerebral inoculation of 1,000 PFU of NSV. C, control uninfected mice; HC, MHC class I heavy chain.

FIG. 3

In situ hybridization for MHC class I and peptide transporter mRNA in the cerebral cortex of C57BL/6 mice infected with NSV. Heavy chain mRNA was expressed in neurons in uninfected mice (A) at very low levels. β2 microglobulin (B), TAP1 (C), and TAP2 (D) mRNAs were barely detectable. Expression of heavy chain (E and F), β2 microglobulin (G and H), TAP1 (I and J), and TAP2 (K) mRNA was increased at 5 days after NSV infection. Signals were detected in the cytoplasms of neurons, glial cells, and inflammatory cells. (A, B, C, D, E, G, and I) Magnification, ×77; (F, H, J, and K) magnification, ×155.

FIG. 4

In situ hybridization with strand-specific probes. The lumbar spinal cords of uninfected C57BL/6 mice (A, D, G, and J) and C57BL/6 mice at 5 days after infection with NSV (B, C, E, F, H, I, K, and L) was stained with heavy chain probes (A, B, and C), β2 microglobulin probes (D, E, and F), TAP1 probes (G, H, and I), and TAP2 probes (J, K, and L). (A, B, D, E, G, H, J, and K) Antisense probe; (C, F, I, and L) sense probe. Magnification in all panels, ×97.

FIG. 5

Survival of B2m-KO (solid triangle), CD8-KO (solid circle), and immunocompetent (open box) C57BL/6 mice after infection with NSV. Eleven-week-old mice were inoculated intracerebrally with 1,000 PFU of NSV in 0.03 ml of Hanks balanced salt solution. Groups of 20 mice (A) or 19 mice (B) were examined.

FIG. 6

Replication of NSV in the CNS of B2m-KO (solid triangle), CD8-KO (solid circle), and immunocompetent (open box) C57BL/6 mice after infection with NSV. (A) Amount of infectious virus in brain; (B) amount of infectious virus in spinal cord. Each time point represents the geometric mean and standard deviation for three mice.

FIG. 7

Graph of viral RNA in the CNS of B2m-KO, CD8-KO, and immunocompetent C57BL/6 mice after infection with NSV. (A) Relative amount of viral RNA in brain; (B) relative amount of viral RNA in spinal cord. Each time point represents the geometric mean and standard deviation for three mice.

FIG. 8

Graph of neutralizing antibody response of B2m-KO, CD8-KO, and immunocompetent C57BL/6 mice after infection with NSV.