Yellow fever virus encephalitis: properties of the brain-associated T-cell response during virus clearance in normal and gamma interferon-deficient mice and requirement for CD4+ lymphocytes

Abstract

Viral encephalitis caused by neuroadapted yellow fever 17D virus (PYF) was studied in parental and gamma interferon (IFN-gamma)-deficient (IFN-gamma knockout [GKO]) C57BL/6 mice. The T-cell responses which enter the brain during acute fatal encephalitis of nonimmunized mice, as well as nonfatal encephalitis of immunized mice, were characterized for relative proportions of CD4+ and CD8+ cells, their proliferative responses, and antigen-specific expression of cytokines during stimulation in vitro. Unimmunized mice accumulated only low levels of T cells within the brain during fatal disease, whereas the brains of immunized mice contained higher levels of both T-cell subsets in response to challenge, with CD8+ cells increased relative to the CD4+ subset. The presence of T cells correlated with the time at which virus was cleared from the central nervous system in both parental and GKO mice. Lymphocytes isolated from the brains of challenged immunized mice failed to proliferate in vitro in response to T-cell mitogens or viral antigens; however, IFN-gamma, interleukin 4 (IL-4), and, to a lesser extent, IL-2 were detectable after stimulation. The levels of IFN-gamma, but not IL-2 or IL-4, were augmented in response to viral antigen, and this specificity was detectable in the CD4+ compartment. When tested for the ability to survive both immunization and challenge with PYF virus, GKO and CD8 knockout mice did not differ from parental mice (80 to 85% survival), although GKO mice exhibited a defect in virus clearance. In contrast, CD4 knockout and Igh-6 mice were unable to resist challenge. The data implicate antibody in conjunction with CD4+ lymphocytes bearing a Th1 phenotype as the critical factors involved in virus clearance in this model.

Figures

FIG. 1

FACS analysis of CD3+ CD4+ T cells isolated from brains of immunized and virus-challenged mice. (A and B) CD3+ CD4+ cells from brains of parental mice on days 4 and 5 postchallenge. (E and F) CD3+ CD8+ cells on the same days. (C and D) CD3+ CD4+ cells on days 4 and 5 and (G and H) CD3+ CD8+ cells from GKO mice.

FIG. 2

Production of IL-4 as measured by CTLL cell assay for parental and GKO mice. Media were collected from cultures of brain-associated lymphocytes (harvested on days 4 and 5) that were stimulated for 2 to 3 days in the presence of control (no mitogen), ConA, or anti-CD3 antibody. The media were assayed for stimulation of CTLL cells as described in Materials and Methods. The results are expressed as the percentage of the maximal stimulation observed with recombinant IL-2. The values represent means + standard errors of the means. The asterisks indicate significant differences from control cultures: ∗, P < 0.005; ∗∗, P < 0.005 (Mann-Whitney test). The difference between anti-CD3-stimulated samples of GKO and parental mice was not significant (P > 0.05).

FIG. 3

Analysis of IFN-γ expression in CD4+ and CD8+ compartments of brain-associated lymphocytes by intracellular staining. Cells were harvested from the brains of immunized, virus-challenged parental mice on day 4 or 5 following challenge, incubated for 2 days with various stimuli, and then processed as described in Materials and Methods. (A) Dot plots for typical experiments in which unstimulated and phorbol myristate acetate (PMA)-stimulated cells were used to establish the range of stimulation in the assay. (B) Percentages of CD4+ or CD8+ cells expressing IFN-γ in response to different stimuli (U, unstimulated; mock, SW-13 cell antigen; PYF, viral antigen; PMA, phorbol ester). The results represent composite means (± the standard errors of the means) for three separate experiments involving 16 samples for each stimulus. The differences in the levels of IFN-γ among CD4+ cells were significant for PYF and PMA versus unstimulated and mock-stimulated samples (P < 0.005 [Wilcoxon rank order test]).

FIG. 4

Inflammatory cytokine expression in parental and GKO mice during acute encephalitis with PYF. Brains were harvested on day 5 postinoculation, and RT-PCR assay for GAPDH and cytokine mRNAs (IL-1β, TNF-α, and IFN-γ) was performed as described in Materials and Methods. pBS, target PCR product generated from a plasmid containing cDNA of the respective mRNAs; (−) RT, reactions in which RT was not done prior to PCR amplification; WT, wild-type parental mice; GKO, GKO mice. The results for two male and two female mice of each strain with encephalitis (PYF) and for uninfected controls are shown.

FIG. 5

In vitro proliferation assay of brain-associated lymphocytes from parental (A and B) and GKO (C and D) mice which had been immunized and virus challenged. (A) Lymphocytes were isolated on day 5 postchallenge. The cells were stimulated with viral antigen (Viral Ag), SW-13 cell antigen (Control Ag), or medium alone for 2 and 3 days. (B) Cells harvested from three mice on day 5 postchallenge were pooled and tested for stimulation by ConA or anti-CD3 antibody after 2 to 4 days. (C) Cells were harvested on day 5 postchallenge and stimulated with viral antigen, control antigen, or medium. The results after 3 to 5 days of stimulation are shown. (D) Cells from three mice on day 5 postchallenge were pooled and stimulated with ConA or anti-CD3 antibody, and proliferation was measured on day 2 or 3 following stimulation. The error bars indicate standard deviations.

FIG. 6

Viral replication in brains of parental and GKO mice. Brain-associated virus was measured as described in Materials and Methods. (A) Virus burdens (mean ± standard error of the mean [SEM]) in unimmunized mice after i.c. inoculation with 104 PFU of PYF. Three to five mice were tested for each time point. (B) Results for immunized parental and GKO mice that had been challenged with 104 PFU of PYF. Brain-associated virus (mean ± SEM) was measured for between 4 and 12 mice for each time point. The differences in the average values were significant for day 4 (P < 0.005) and day 7 (P < 0.05) (Wilcoxon rank order test). C571, unimmunized mice used as the control for peak virus burden.