Pathogenic role for virus-specific CD4 T cells in mice with coronavirus-induced acute encephalitis

Abstract

Acute viral encephalitis is believed to result from direct virus destruction of infected cells and from virus-induced host immune response, but the relative contribution of each remains largely unknown. For example, C57BL/6 (B6) mice infected with mouse hepatitis virus (JHM strain, JHMV) develop severe encephalitis, with death occurring within 7 days. Here, we show that the host response to a single JHMV-specific immunodominant CD4 T-cell epitope is critical for severe disease. We engineered a recombinant JHMV with mutations in the immunodominant CD4 T-cell epitope (rJ.M(Y135Q)). Infection of naïve B6 mice with this virus resulted in mild disease with no mortality. However, introduction of a CD4 T-cell epitope from Listeria monocytogenes into rJ.M(Y135Q) generated a highly virulent virus. The decrease in disease severity was not due to a switch from Th1 to Th2 predominance in rJ.M(Y135Q)-infected mice, an effect on CD8 T-cell function, or differential expression of tumor necrosis factor-alpha by JHMV-specific CD4 T cells. These results show that the response to a single virus-specific CD4 T-cell epitope may contribute to a pathogenic host response in the setting of acute viral disease and that abrogation of this response ameliorates clinical disease without diminishing virus clearance.

Figure 1

Schematic diagram of recombinant JHMV constructs. Recombinant rJ, rJ.M_Y135Q, and rJ.M_Y135Q.LLO₁₉₀ were engineered as described in Materials and Methods. A mutation to abrogate recognition by epitope M133-specific CD4 T cells (Y135Q) was introduced into the M gene in rJ.M_Y135Q. The LLO190 epitope from LM was introduced into gene 4 of rJ.M_Y135Q to generate rJ.M_Y135Q.LLO₁₉₀.

Figure 2

Detection of epitope M133- and LLO190-specific CD4 T cells in the CNS of infected mice. Cells were harvested from the brains of 5- to 6-week-old B6 mice 7 days after i.n. infection with rJ (A), rJ.M_Y135Q (B), or rJ.M_Y135Q.LLO₁₉₀ (C). JHMV-specific CD4 T cells were identified by intracellular staining for IFN-γ after stimulation with cognate peptides and FACS analysis as described in Materials and Methods. Individual mice were analyzed in these assays. The percentage of IFN-γ⁺ and IFN-γ⁻ CD4 T cells is shown. Note that the Y135Q mutation in the M protein abrogated recognition by epitope M133-specific CD4 T cells and variant peptide M133-Y135Q did not elicit a de novo CD4 T-cell response (B, right panel). The CD4 T-cell response was analyzed in 6 to 12 experiments.

Figure 3

Kinetics of virus production in 17Cl-1 cells and thermal stability of recombinant viruses. A: 17Cl-1 cells were infected with recombinant viruses at 1 plaque forming unit/cell. Cells and supernatant were harvested at the indicated times, and titers were measured by plaque assay on HeLa-MHVR cells. Each virus was assayed in two to three independent experiments. B: Cell-free virus was incubated in solutions at a pH of 6, 7, or 8 at 37°C. Aliquots were removed at the indicated time points and titered on HeLa-MHVR cells. The fraction of virus surviving at each time point is shown. Data are representative of three independent experiments.

Figure 4

Mortality, weight loss, and virus titers in B6 mice infected with recombinant viruses. A: B6 mice were infected with rJ (n = 23), rJ.M_Y135Q (n = 23), or rJ.M_Y135Q.LLO₁₉₀ (n = 23) and monitored for survival. B: Eight rJ-, eight rJ.M_Y135Q.LLO₁₉₀-, and nine rJ.M_Y135Q-infected mice were weighed daily. Weights relative to weight at onset of experiment are shown for mice surviving at each time point. C: A total of 87 B6 mice were used for determining CNS virus titers with six to nine mice assayed at the indicated time points for each virus. Significant decreases in virus titers were detected in the CNS of rJ.M_Y135Q.LLO₁₉₀-infected mice at day 7, when compared with mice infected with rJ or rJ.M_Y135Q (**P < 0.005). At day 12, virus was cleared from two of six mice infected with rJ.M_Y135Q or rJ.M_Y135Q.LLO₁₉₀; average titers are shown for those mice with detectable virus.

Figure 5

Inflammation and virus antigen in the CNS of mice infected with rJ or rJ.M_Y135Q. Brains were harvested from rJ- or rJ.M_Y135Q-infected mice and fixed in zinc formalin. Sagittal sections were prepared and stained with hematoxylin and eosin (A and B) or for virus antigen (C and D) as described in Materials and Methods. A–D: Photomicrographs of the dorsal midbrain. Similar numbers of infiltrating parenchymal and perivascular cells and of infected cells were detected in the CNS of mice infected with rJ (A and C) or rJ.M_Y135Q (B and D). Scale bar = 50 μm. E: Numbers of neutrophils (CD45^hiF4/80⁻Ly6G⁺) and macrophages/microglia (CD45^int/hiF4/80⁺Ly6G⁻, “macrophages”) were determined as described in Materials and Methods. Seven rJ- and eight rJ.M_Y135Q-infected mice were analyzed in these assays. F: Total numbers of CD8 and CD4 T cells in the infected CNS were assayed as described in Materials and Methods. Seven and 15 rJ-, 7 and 19 rJ.M_Y135Q-, and 4 and 11 rJ.M_YB5Q.LL0₁₉₀-infected mice were analyzed for CD8 T cells and CD4 T cells, respectively.

Figure 6

Mortality and weight loss in BALB/c, LM-immune, and RAG1^−/− mice infected with recombinant viruses. Mice were infected with rJ, rJ.M_Y135Q, or rJ.M_Y135Q.LLO₁₉₀ and monitored for mortality (A, C, and E) and weight loss (B and D). In B and D, weight relative to weight at onset of experiment is shown for mice surviving at each time point. A and B: Eight BALB/c mice each were infected with rJ, rJ.M_Y135Q, or rJ.M_Y135Q.LLO₁₉₀ and monitored for survival and weight loss. C and D: Eight 5-week-old B6 mice were inoculated intraperitoneally with LM as described in Materials and Methods. Twenty-eight days later, four mice each were inoculated intranasally with either rJ or rJ.M_Y135Q.LLO₁₉₀ and monitored for survival and weight loss. Data are representative of three independent experiments. E: rJ-, rJ.MY135Q-, and rJ.M_Y135Q.LLO₁₉₀-infected RAG1^−/− mice (n = 4 for each virus) were monitored for mortality. Data are representative of two independent experiments.

Figure 7

Response to subdominant JHMV-specific CD4 T-cell epitopes in the CNS of infected mice. Lymphocytes were harvested from the brains of 5- to 6-week-old B6 mice 7 days after infection with rJ, rJ.M_Y135Q, or rJ.M_Y135Q.LLO₁₉₀. JHMV-specific CD4 T cells were identified by intracellular staining for IFN-γ. Individual mice were analyzed in these assays. Eight to 12 mice infected with each virus were analyzed in two to three independent experiments. A: Numbers of cells responding to individual epitopes are shown. B: The number of total virus-specific CD4 T cells in each mouse was calculated by summing the number of cells responding to the individual epitopes indicated in A. The total number of virus-specific CD4 T cells in the CNS was significantly less in mice infected with rJ.M_Y135Q than in the CNS of mice infected with rJ or rJ.M_Y135Q.LLO₁₉₀ (**P < 0.0001, *P = 0.011).

Figure 8

Virus-specific CD8 T-cell numbers and activity in the JHMV-infected CNS. Mononuclear cells were harvested from the brains of 5- to 6-week-old B6 mice 7 days p.i. as described in Materials and Methods. A: Cells were stained for surface CD8 and with D^b/S510 tetramer S510. The percentage of tetramer⁺ and tetramer⁻ CD8 T cells is shown. B: JHMV-specific CD8 T cells were identified by intracellular staining for IFN-γ after stimulation with peptide S510 and FACS analysis. Individual mice were analyzed in these assays. The percentage of IFN-γ⁺ and IFN-γ⁻ CD8 T cells is shown. C: Four B6 mice were infected with rJ or rJ.M_Y135Q. Cells were prepared from the CNS at day 7 p.i. and analyzed for TNF-α and IFN-γ expression after stimulation with peptide S510 as described in Materials and Methods. The results are expressed as the percentage of TNF-α⁺/IFN-γ⁺ CD4 T cells. None of the differences between samples were statistically significant. D and E: CNS-derived mononuclear cells were prepared from mice infected with rJ (n = 3) or rJ.M_Y135Q (n = 3) at 7 days p.i. Ex vivo cytolytic activity was assayed at the indicated E:T ratios using EL-4 target cells coated with 1 μmol/L peptide S510 or irrelevant peptide. Background cytolysis was < 8% in this experiment and was subtracted from specific release of peptide-coated targets. E:T ratios are displayed as either total populations or percentage of epitope S510-specific cells as determined by D^b/S510 tetramer staining. Average cytolysis/2000 tetramers is shown in E. None of the differences between samples were statistically significant. Data are representative of three independent experiments.

Figure 9

TNF-α expression in the infected CNS. A: RNA was harvested from the brains of mice infected with rJ (n = 3) or rJ.M_Y135Q (n = 3) at each time point. The amount of TNF-α mRNA was quantified by real-time RT-PCR as described in Materials and Methods. B: Five B6 mice were infected with rJ, rJ.M_Y135Q, or rJ.M_Y135Q.LLO₁₉₀ and sacrificed at day 7 p.i. Cells were prepared from the CNS and analyzed for TNF-α and IFN-γ expression after stimulation with the indicated peptides by intracellular cytokine staining as described in Materials and Methods. The results are expressed as the percentage of TNF-α⁺/IFN-γ⁺ CD4 T cells. None of the differences between samples were statistically significant.