Role of viral persistence in retaining CD8(+) T cells within the central nervous system

Abstract

The continued presence of virus-specific CD8(+) T cells within the central nervous system (CNS) following resolution of acute viral encephalomyelitis implicates organ-specific retention. The role of viral persistence in locally maintaining T cells was investigated by infecting mice with either a demyelinating, paralytic (V-1) or nonpathogenic (V-2) variant of a neurotropic mouse hepatitis virus, which differ in the ability to persist within the CNS. Class I tetramer technology revealed more infiltrating virus-specific CD8(+) T cells during acute V-1 compared to V-2 infection. However, both total and virus-specific CD8(+) T cells accumulated at similar peak levels in spinal cords by day 10 postinfection (p.i.). Decreasing viral RNA levels in both brains and spinal cords following initial virus clearance coincided with an overall progressive loss of both total and virus-specific CD8(+) T cells. By 9 weeks p.i., T cells had largely disappeared from brains of both infected groups, consistent with the decline of viral RNA. T cells also completely disappeared from V-2-infected spinal cords coincident with the absence of viral RNA. By contrast, a significant number of CD8(+) T cells which contained detectable viral RNA were recovered from spinal cords of V-1-infected mice. The data indicate that residual virus from a primary CNS infection is a vital component in mediating local retention of both CD8(+) and CD4(+) T cells and that once minimal thresholds of stimuli are lost, T cells within the CNS cannot survive in an autonomous fashion.

FIG. 1

Presence of vRNA in brains of V-1- and V-2-infected mice. Total RNA was prepared from brains of four individual mice infected with either V-1 (A) or V-2 (B) at days 7, 11, 33, and 63 p.i. as indicated. Sequences from the viral N RNA and host HPRT RNA (A and B) were amplified and analyzed by gel electrophoresis. PCR products from days 33 and 63 p.i. were further amplified using a nested set of primers for the viral N gene (C). M, marker; N, naive control mouse; −, no RNA used during the cDNA synthesis.

FIG. 2

Presence of vRNA in spinal cords of V-1- and V-2-infected mice. Total RNA was prepared from spinal cords of four individual mice infected with either V-1 (A) or V-2 (B) at each of the indicated time points. Sequences from the viral N RNA and host HPRT RNA (A and B) were amplified and analyzed by gel electrophoresis. PCR products from days 33 and 63 p.i. were further amplified using a nested set of primers for the viral N gene (C). M, marker; N, naive control mouse; −, no RNA used during the cDNA synthesis.

FIG. 3

Presence of virus-specific CD8⁺ T cells in the brain. Brain mononuclear cells were isolated from six to eight mice sacrificed at days 7, 10, 33, and 70 p.i. as indicated and pooled prior to analysis. Live mononuclear cells were gated based on forward and side scatter (FSC and SSC) analysis, and representative gates are shown for each time point (A). Brain mononuclear cells were stained directly ex vivo with MAb to CD8 and the L^d-pN tetramer reagent (B). Numbers in the right quadrants represent percentages of tetramer⁺ CD8⁺ T cells (upper number) and tetramer⁻ CD8⁺ T cells (lower number). The data presented in panel B were used to calculate the total number of infiltrating CD8⁺ T cells (C) and total number of tetramer⁺ CD8⁺ T cells (D) per brain.

FIG. 4

Presence of virus-specific CD8⁺ T cells in the spinal cord. Mononuclear cells were isolated from the spinal cords of V-1- and V-2-infected mice (n = 6 to 8) at the indicated time points and pooled prior to ex vivo staining with anti-CD8 MAb and the L^d-pN tetramer reagent (A). Numbers shown in the right quadrants represent percentages of tetramer⁺ CD8⁺ T cells (upper number) and tetramer⁻ CD8⁺ T cells (lower number). The data presented in panel A were used to calculate the total number of infiltrating CD8⁺ T cells (B) and total number of tetramer⁺ CD8⁺ T cells (C) per spinal cord.

FIG. 5

Presence of CD4⁺ T cells in the brain and spinal cord. Mononuclear cells were isolated from brains and spinal cords of V-1- and V-2-infected mice at the indicated time points (n = 6 to 8). For each time point, mononuclear cells from brains were pooled as one set and mononuclear cells from spinal cords were pooled as a separate set prior to ex vivo staining with anti-CD4 MAb (A). The data presented in panel A were used to calculate the average number of infiltrating CD4⁺ T cells on a per-brain basis (B) as well as the average number of CD4⁺ T cells per spinal cord (C).