Epitope-specific regulatory CD4 T cells reduce virus-induced illness while preserving CD8 T-cell effector function at the site of infection

Abstract

The role of epitope-specific regulatory CD4 T cells in modulating CD8 T-cell-mediated immunopathology during acute viral infection has not been well defined. In the murine model of respiratory syncytial virus (RSV) infection, CD8 T cells play an important role in both viral clearance and immunopathology. We have previously characterized two RSV epitope-specific CD4 T-cell responses with distinct phenotypic properties. One of them, the IA(b)M(209)-specific subset, constitutively expresses FoxP3 and modulates CD8 T-cell function in vitro. We show here that the IA(b)M(209)-specific CD4 T-cell response regulates CD8 T-cell function in vivo and is associated with diminished RSV-induced illness without affecting viral clearance at the site of infection. Achieving the optimal balance of regulatory and effector T-cell function is an important consideration for designing future vaccines.

FIG. 1.

CD4 T-cell responses to RSV M and M2. Lung lymphocytes were isolated at day 7 postinfection and stained with tetramers and phenotyping antibodies to identify specific CD4 T cells (A) or stimulated with MHC class II-restricted CD4 T-cell epitope-containing peptides (M₂₀₉ or M2₂₅) in the presence of costimulators and brefeldin A for 5 h and then stained for lineage phenotype and cytokines intracellularly (B). Representative flow plots are gated on live CD4⁺ CD8⁻ T cells. Frequencies of specific or cytokine-producing T cells refer to the total CD4 T-cell population. The cytokine-producing cell frequency is background subtracted, which is based on irrelevant OVA peptide stimulation. The data (mean ± the standard deviation [SD], n = 5 to 6/group) represent three independent experiments and are compared by using the Student t test.

FIG. 2.

Frequency and CD127 expression of RSV-specific CD8 cells in peripheral blood after administration of rAd5-M/M2. Mice were immunized with KLH or KLH-conjugated peptides M₂₀₉ or M2₂₅ and then immunized with rAd5-M/M2 intramuscularly. (A) Peripheral blood samples were collected 10 days after rAd5-M/M2 immunization and stained with tetramers and phenotyping antibodies to assess the frequency of RSV tetramer-specific CD8 T cells. M₂₀₉ peptide-immunized mice had a lower frequency of CD8 T-cell responses after rAd5-M/M2 than M₂₅ peptide- or KLH-immunized mice. A representative plot is gated on CD4⁻ CD8⁺ T cells. The percentages of specific CD8 T cells refer to total CD8 T-cell population. (B) We also assessed the frequency of CD127-expressing specific CD8 T cells in mice immunized with M₂₀₉ or M₂₅ peptides. The percentages refer to the specific CD8 T cells. The data (mean ± the SD, n = 5/group) are representative of three independent experiments and were compared by using the Student t test. Filled bars indicate the KLH-only-immunized control groups, hatched bars indicate M₂₀₉-KLH-immunized mice, and dotted bars indicate M2₂₅-KLH-immunized mice.

FIG. 3.

Frequency and cytokine production of RSV-specific CD8 T cells from the spleen after RSV challenge. Mice were immunized with KLH or KLH-conjugated peptides, followed by rAd5-M/M2 administration intramuscularly, and then challenged with live RSV intranasally. Spleen lymphocytes were isolated at 7 day postchallenge and stained with tetramers and phenotyping antibodies to identify specific CD8 T cells by flow cytometry (A) or stimulated with CD8 epitope-containing peptides in the presence of costimulators and brefeldin A for 5 h and then stained for lineage phenotype and cytokine expressions intracellularly (B). The frequencies of specific CD8 T cells or cytokine-producing cells refer to CD8 T-cell population. The cytokine-producing cell frequency is the background (ranging from 0.01 to 0.08%) subtracted based on irrelevant OVA peptide stimulation. The data (mean ± the SD, n = 5/group) are representative of three independent experiments and compared by using the Student t test. Filled bars indicate the KLH-only-immunized control groups, hatched bars indicate M₂₀₉-KLH-immunized mice, and dotted bars indicate M2₂₅-KLH-immunized mice. We also assessed the CD8 T-cell responses in the spleen in unimmunized mice depleted of CD4 T cells just prior to RSV challenge to remove the impact of CD4 T-cell modulation on the CD8 T-cell responses. Naive mice were treated with anti-CD4 antibody (□) or isotype control (▪) and then challenged with RSV. Spleen lymphocytes were isolated at day 7 postchallenge. Frequencies of specific CD8 T cells (C) and cytokine-producing CD8 T cells (D) from CD4-depleted or control mice stimulated with epitope-containing peptides were assessed as described in panels A and B.

FIG. 4.

Frequency and cytokine production of specific CD8 cells from the lung after RSV challenge. Mice were immunized with KLH or KLH-conjugated peptides, followed by rAd5-M/M2 administration intramuscularly, and then challenged with RSV intranasally. Lung lymphocytes were isolated at day 7 postinfection and stained with tetramers and phenotyping antibodies to identify specific CD8 T cells by flow cytometry (A) or stimulated with CD8 epitope-containing peptides in the presence of costimulators and brefeldin A for 5 h and then stained for lineage and cytokine expression intracellularly (B). Frequencies of specific CD8 T cells or cytokine-producing cells refer to the total CD8 T-cell population. The cytokine-producing cell frequency is the background (ranging from 0.03 to 0.2%) subtracted based on irrelevant OVA peptide stimulation. The data (mean ± the SD, n = 5/group) are representative of three independent experiments. Filled bars indicate the KLH-only-immunized control groups, hatched bars indicate M₂₀₉-KLH-immunized mice, and dotted bars indicate M2₂₅-KLH-immunized mice. We then assessed CD8 T-cell responses in the lungs in unimmunized mice depleted of CD4 T cells just prior to RSV challenge to remove the impact of CD4 T-cell modulation on the CD8 T-cell responses. Naive mice were treated with anti-CD4 antibody (□) or isotype control (▪) and then infected with RSV. Lung lymphocytes were isolated at day 7 postinfection. Frequencies of specific (C) or cytokine-producing (D) CD8 T cells from CD4-depleted or control mice stimulated with epitope-containing peptides were assessed as described in panels A and B. (E) Isolated lung lymphocytes were cultured with CD8 epitope peptide M2₈₂- and M₁₈₇-loaded P815 and EL-4 cells, respectively, for 3 h. Dead P815 and EL-4 cells after culturing were assessed with flow cytometry by annexin V staining. The percentages of specific killings refer to total P815 and EL-4 cell populations, with nonspecific cell death subtracted based on irrelevant OVA peptide-loaded P815 and EL-4 cell cultures. The data (mean ± the SD, n = 5/group) are representative of three independent experiments. (F) Supernatant of ground lung tissue isolated at days 4 and 7 postinfection were plated on HEp-2 cell layer and cultured for 4 days. The virus titers are expressed as PFU per gram of lung tissue. The limit of detection is 1.8 log₁₀ PFU/gram of tissue. The data (mean ± the SD, n = 5/group) are representative of two independent experiments. All data were statistically compared by using the Student t test.

FIG. 5.

Weight loss after RSV challenge following immunization and boost. Mice were weighed daily after virus challenge (challenge day = day 0). Shown are the percentages of baseline weight. The data (mean ± the SD, n = 5/group) are representative of three independent experiments.

FIG. 6.

Phenotype and cytokine expressions of CD4 and CD8 T cells in the spleen and lung. Mice were primed with KLH or KLH-conjugated peptides, followed by rAd5-M/M2 administration intramuscularly, and then challenged with RSV intranasally. (A) Lung and spleen lymphocytes were isolated at day 7 postinfection and stained with tetramers and phenotyping antibodies to identify specific CD8 T cells and their phenotypes by flow cytometry. Representative plots are gated on IA^bM₂₀₉-specific CD4 T cells. (B) Frequencies of PD-1 high or PD-1 low with CD62L expression cells refer to the gated population or stimulated with CD4 epitope peptide M₂₀₉ in the presence of costimulators and brefeldin A for 5 h and then stained for lineage phenotype and intracellular cytokines. The ratio of CD4 T cells producing IFN-γ and IL-2 are the background subtracted based on irrelevant OVA peptide stimulation. The data (mean ± the SD, n = 5/group) are representative of three independent experiments and compared by using the Student t test.