CD8 T cells protect adult naive mice from JEV-induced morbidity via lytic function

Abstract

Following Japanese encephalitis virus (JEV) infection neutralizing antibodies are shown to provide protection in a significant proportion of cases, but not all, suggesting additional components of immune system might also contribute to elicit protective immune response. Here we have characterized the role of T cells in offering protection in adult mice infected with JEV. Mice lacking α/β-T cells (TCRβ-null) are highly susceptible and die over 10-18 day period as compared to the wild-type (WT) mice which are resistant. This is associated with high viral load, higher mRNA levels of proinflammatory cytokines and breach in the blood-brain-barrier (BBB). Infected WT mice do not show a breach in BBB; however, in contrast to TCRβ-null, they show the presence of T cells in the brain. Using adoptive transfer of cells with specific genetic deficiencies we see that neither the presence of CD4 T cells nor cytokines such as IL-4, IL-10 or interferon-gamma have any significant role in offering protection from primary infection. In contrast, we show that CD8 T cell deficiency is more critical as absence of CD8 T cells alone increases mortality in mice infected with JEV. Further, transfer of T cells from beige mice with defects in granular lytic function into TCRβ-null mice shows poor protection implicating granule-mediated target cell lysis as an essential component for survival. In addition, for the first time we report that γ/δ-T cells also make significant contribution to confer protection from JEV infection. Our data show that effector CD8 T cells play a protective role during primary infection possibly by preventing the breach in BBB and neuronal damage.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1. T cells play a significant role in protection from JEV infection.

[A] Survival kinetics of JEV infected WT B6 and Rag1-null mice over time. n = 10–13 mice. [B] Survival kinetics of JEV infected WT B6 and TCRδ-null mice over time. n = 15–20 mice. [C] Survival kinetics of JEV infected WT B6 and TCRβ-null mice over time. n = 12–18 mice. [D] Relative weight loss of JEV infected TCRβ-null mice as compared to uninfected TCRβ-null (mock) and infected WT B6 mice (mean ± SE, n > 10). [E] Clinical score for JEV infected WT B6 and TCRβ-null mice along with uninfected TCRβ-null (mock) mice (mean ± SE, n > 10). [F] Distribution of leukocyte subsets per brain in uninfected WT B6, infected WT B6 and infected TCRβ-null mice (mean ± SE, n as shown). [G] Frequencies of CD44highCD69+ activated cells in brains of infected WT B6 mice (mean ± SE, n as shown). [H] Frequencies of CD44highCD69+ JEV-specific cells in spleens of infected WT B6 mice (mean ± SE, n as shown). $ = p

Fig 2. JEV-specific IgM, IgG and neutralizing antibody levels.

[A] JEV-specific IgM levels in WT B6 and TCRβ-null mice prior to (day 0) and post-infection on indicated days. n = 6 mice. [B] JEV-specific IgG levels in WT B6 and TCRβ-null mice prior to (day 0) and post-infection on indicated days. n = 6 mice. [C] Virus neutralization titers (PRNT50) in infected WT B6, TCRβ-null and TCRδ-null shown as reciprocal log 2 values. n = 5 mice per group.

Fig 3. JEV burden in the blood and brains of B6 and TCRβ-/- mice.

[A] JEV levels in blood at day 1 post infection (mean ± SE, n > 8). JEV levels in brain tissue at day 7 [B] and day 12 [C] post infection respectively (mean ± SE, n ≥ 8). [D-G] Representative images of brains after injection of Evans blue in WT B6 and TCRβ-null mice. [H] Pooled data to show relative levels of Evans blue in B6 (black bars) and TCRβ-null (gray bars) in mock and JEV-infected mice (mean ± SE, n > 5 mice). ψ = p

Fig 4. Cytokine mRNA levels in the brains of infected B6 and TCRβ-/- mice.

Relative mRNA levels of IL-1β [A], IL-6 [B], TNFα [C] and IFNγ [D] normalized to GAPDH in the brain homogenates of B6 and TCRβ-null mice infected with JEV on day 7 and 12 post-infection (mean ± SE, n > 7). $ = p

Fig 5. Presence of T cells is essential for protection from primary JEV infection, but CD4 T cells appear less critical.

[A] Survival kinetics of mock and JEV infected TCRβ-null with or without transfer of naïve T cells from WT B6 mice as indicated (n > 8). [B] Survival kinetics of mock and JEV infected TCRβ-null mice with purified naïve T cell or total spleen cell transfers from WT B6 mice (n = 7 for sorted naïve T cell transfer, for other groups n > 8). [C] Survival kinetics following JEV infection in WT B6 and MHCII-null mice over time (n = 14). [D] Relative weight loss of JEV infected MHCII-null mice as compared to infected WT B6 mice (mean ± SE, n = 14). [E] Clinical score for JEV infected WT B6 and MHCII-null mice (mean ± SE, n = 14). [F] Survival kinetics of mock and JEV infected TCRβ-null mice with or without transfer of naïve T cells from MHCII-null or WT B6 mice (n > 8). $ = p

Fig 6. Absence of CD8 cells, but not IFNγ, influences the outcome of JEV infection.

[A] Survival kinetics following JEV infection in WT B6 and IFNγ-null mice over time (n > 8). [B] Survival kinetics of mock or JEV infected TCRβ-null mice with or without transfer of naïve T cells from IFNγ-null or WT B6 mice (n > 8). [C] Survival kinetics following JEV infection in WT B6 and TAP1-null mice over time (n = 14). [D] Relative weight loss of JEV infected TAP1-null mice as compared to infected WT B6 mice (mean ± SE, n = 14). [E] Clinical score for JEV infected WT B6 and TAP1-null mice (mean ± SE, n = 14). [F] Titers of JEV in brains of infected WT B6, beige, TAP1-null and TCRδ-null mice day 12 post-infection. Each symbol represents data from individual mice. [G] Virus neutralization titers from WT B6, TAP1-null and beige mice day 12 post-infection (mean ± SE, n = 5). [H] Survival kinetics of mock or JEV infected TCRβ-null mice with or without transfer of naïve T cells from TAP1-null or WT B6 mice (n > 8). $ = p

Fig 7. Beige defect makes mice susceptible to JEV infection.

[A] Survival kinetics following JEV infection in WT B6 and beige mice over time (n > 8). [B] Relative weight loss of JEV infected beige mice as compared to infected WT B6 mice (mean ± SE, n = 14). [C] Clinical score for JEV infected WT B6 and beige mice (mean ± SE, n = 14). [D] Survival kinetics of mock or JEV infected TCRβ-null mice with or without transfer of naïve T cells from beige or WT B6 mice (n > 8). $ = p