Qualitative and quantitative characteristics of rotavirus-specific CD8 T cells vary depending on the route of infection

Abstract

CD8 T-cell response provides an important defense against rotavirus, which infects a variety of systemic locations in addition to the gut. Here we investigated the distribution, phenotype, and function of rotavirus-specific CD8 T cells in multiple organs after rotavirus infection initiated via the intranasal, oral, or intramuscular route. The highest level of virus-specific CD8 T cells was observed in the Peyer's patches of orally infected mice and in the lungs of intranasally infected animals. Very low levels of virus-specific CD8 T cells were detected in peripheral blood or spleen irrespective of the route of infection. Rotavirus-specific CD8 T cells from Peyer's patches of orally infected mice expressed high levels of CCR9, while CXCR6 and LFA-1 expression was associated with virus-specific CD8 T cells in lungs of intranasally infected mice. Oral infection induced the highest proportion of gamma interferon(-) CD107a/b(+) CD8 T cells in Peyer's patches. When equal numbers of rotavirus-specific CD8 T cells were transferred into Rag-1 knockout mice chronically infected with rotavirus, the donor cells derived from Peyer's patches of orally infected mice were more efficient than those derived from lungs of intranasally infected animals in clearing intestinal infection. These results suggest that different routes of infection induce virus-specific CD8 T cells with distinct homing phenotypes and effector functions as well as variable abilities to clear infection.

FIG. 1.

Distribution of virus-specific CD8 T cells after infection via various routes. On day 7 after p.o., i.n., or i.m. infection of mice with RRV, lymphocytes were isolated from the indicated organs (see Materials and Methods) and stained with anti-CD3, anti-CD8, Vp6 tetramer, and Vp7 tetramer to determine the total percentage of tetramer⁺ CD8 T cells in each organ. Displayed are means and SEM for five mice in each group. The experiment was repeated four times with similar results. *, P < 0.05 (ANOVA).

FIG. 2.

Expression of lymphocyte homing markers on rotavirus-specific CD8 T cells following RRV infection by different routes. Lymphocytes were isolated on day 7 after infection from the indicated organs and stained with anti-CD8, anti-CD3, VP6 tetramer, and VP7 tetramer, along with antibodies against CCR9, LFA-1, and CXCR6 to determine the frequency of tetramer⁺ CD8 T cells expressing each of these markers. The percentage of total tetramer⁺ cells expressing each marker was calculated based on results with VP6 and VP7 tetramers. (A) CCR9 expression on tetramer⁺ CD8 T cells. (B) LFA-1 expression on tetramer⁺ CD8 T cells. (C) CXCR6 expression on tetramer⁺ CD8 T cells. Standard errors and the ANOVA test were based on five mice in each group. Displayed are means and SEM for five mice in each group. The experiment was repeated four times with similar results. *, P < 0.05 (ANOVA).

FIG. 3.

Expression of IFN-γ and CD107a/b in virus-specific CD8 T cells from PP of mice infected via different routes. (A) Simultaneous staining of intercellular IFN-γ and CD107a/b after ex vivo peptide restimulation. PP lymphocytes from p.o., i.n. and i.m. infected mice were incubated with or without a mixture of VP6 and VP7 peptides for 6 h and then stained for intracellular IFN-γ and CD107a/b. Displayed are cells gated on the CD3⁺ CD8⁺ lymphocyte population. (B) Total percentage of combined Vp6 and Vp7 peptide-specific IFN-γ⁺ CD107a/b⁺, IFN-γ⁻ CD107a/b⁺, and IFN-γ⁺ CD107a/b⁻ cell subsets in CD8 T cells from PP. *, P < 0.05; **, P < 0.01 (Student's t test) (C) Proportion of each cell subset (IFN-γ⁺ CD107a/b⁺, IFN-γ⁻ CD107a/b⁺, and IFN-γ⁺ CD107a/b⁻) out of total Vp6 and Vp7 peptide-specific CD8 T-cell populations. The graphs show results from an experiment with five mice in each group, which was repeated three times with similar results.

FIG. 4.

Fecal shedding of rotavirus Ag from chronically infected Rag-1 KO mice following adoptive transfer of RRV immune or naive CD8 T cells. CD8 T cells were isolated from PP or lungs of p.o. or i.n. infected mice, respectively, and transferred into Rag-1 mice chronically infected with EC murine rotavirus. Each recipient mouse received 5 × 10⁵ CD8 T cells from PP or 6.5 × 10⁵ CD8 T cells from lungs; both sets of T cells contained 0.15 × 10⁵ Vp6 and Vp7 tetramer⁺ CD8 T cells. Control mice received 5 × 10⁵ CD8 T cells from PP or 6.5 × 10⁵ CD8 T cells from lungs of uninfected mice. Stool samples were collected on days 1, 3, 5, 8, 10, 12, 14, 16, 20, and 28 post-adoptive transfer and tested for rotavirus Ag by ELISA. The graph is based on results of an experiment with four mice in each group, which was repeated twice with similar results.