Interleukin-4 diminishes CD8(+) respiratory syncytial virus-specific cytotoxic T-lymphocyte activity in vivo

Abstract

Although interleukin-4 (IL-4) has been implicated in respiratory syncytial virus (RSV)-enhanced disease, the mechanism by which it modulates immune responses to primary RSV infection remains unclear. We have developed a system to investigate the effect of IL-4 on RSV epitope-specific cytotoxic T-lymphocyte (CTL) effector function in vivo, using an H-2K(d)-restricted RSV M2 epitope. BALB/c mice were infected with recombinant vaccinia virus (rVV) constructed to express RSV M2 protein (vvM2) alone or coexpress M2 and IL-4 (vvM2/IL-4). Splenocytes were assessed for M2-specific CTL activity in a direct (51)Cr release assay and intracellular gamma interferon (IFN-gamma) production by fluorescence-activated cell sorting analysis. Mice infected with vvM2/IL-4 had less M2-specific primary CTL activity than those infected with vvM2. M2-specific CTL frequency, as measured by M2 peptide-induced intracellular IFN-gamma production, was diminished in the vvM2/IL-4 group, partially accounting for the reduction of CTL activity. Mice immunized with either construct were challenged intravenously with RSV 4 weeks postimmunization, and direct CTL were measured. These results demonstrate that local expression of IL-4, at the time of antigen presentation, diminishes the cytolytic activity of primary and memory CD8(+) RSV-specific CTL responses in vivo.

FIG. 1

Kinetics of viral replication and cytokine production in the lungs and spleens. Mice were sacrificed on days 2, 4, 6, and 10 postinfection. vvM2 (■) and vvM2/IL-4 (○) viral replication was measured in the lung (A) and spleen (B) by standard plaque assay. IL-4 protein in the lung (C) and spleen (D) was measured by ELISA. Solid bars, vvM2; hatched bars, vvM2/IL-4. The limit of detection for virus replication is 1.8 log₁₀ PFU/g of tissue. Data are representative of three independent experiments with four mice per group (n = 4).

FIG. 2

IFN-γ levels in mice infected with vvM2 (solid bars) or vvM2/IL-4 (hatched bars) on days 2, 4, 6, and 10 postinfection. IFN-γ was measured from spleen supernatant by ELISA. Data represent two independent experiments.

FIG. 3

M2-specific CTL activity. (A) CTL activity in splenocytes was measured by determination of specific lysis on day 6 postinfection in mice injected with 5 × 10⁶ PFU of vvIL-4 (▴), vvM2 (■), or vvM2/IL-4 (○) (P <0.05 between vvM2 and vvM2/IL-4 for E:T ratios of 100:1 and 50:1). (B) LU measured on a per-spleen basis. Solid bars, vvM2; hatched bars, vvM2/IL-4 (P <0.05). Results are representative of six independent experiments.

FIG. 4

Anti-IL-4 treatment restores CTL activity. Direct CTL activity was measured in splenocytes on day 6 postinfection. Mice were injected with vvM2 (■), vvM2/IL-4 (○), or vvM2/IL-4 plus anti-IL-4 (▴). Results are representative of three independent experiments.

FIG. 5

M2-specific CTL activity of mice coinjected with vvM2 and vvIL-4. CTL activity was measured in splenocytes on day 6 postinfection. Mice were coinjected with vvM2 plus vvIL-4 or vvM2 plus vvLac. Scatter plot data are values for individual mice at E:T = 100:1, and the horizontal bar indicates the arithmetic mean of the group. The data are derived from three independent experiments, each designated by a unique symbol. P = 0.001 by Mann-Whitney test.

FIG. 6

Western blot analysis of M2 protein expression in infected HEp-2 cells at 24 and 48 h postinfection. RSV polyclonal antibody was used to detect M2 expression in vvIL-4-, vvM2-, and vvM2/IL-4-infected cells; 20 μg of protein was loaded into each lane. Levels of M2 expression were similar for the two vectors.

FIG. 7

Kinetics of splenic CTL activity. Mice were injected with vvM2 (■) or vvM2/IL-4 (○) and sacrificed on days 4, 6, and 10 postinfection. Data represent two independent experiments (P <0.05 between groups for days 4 and 6).

FIG. 8

Intracellular cytokine staining of M2 peptide-stimulated spleen cells. Mice were primarily infected (1° CTL) or immunized (2° CTL) with vvM2 or vvM2/IL-4; 2 × 10⁶ spleen cells were stimulated with FLU 147-155 (TYQRTRALV) or RSV M2 82-90 (SYIGSINNI) peptide for 8 h in the presence of monensin and analyzed for IFN-γ production by flow cytometry. Data for primary CTL are representative of averages compiled from five independent experiments with n = 4 or 5 per group; data for secondary CTL are representative of two independent experiments, n = 4 (P > 0.05 between groups for both primary and secondary CTL responses).

FIG. 9

Memory CD8⁺ T-cell activity. Mice were injected with medium (▴) or with 5 × 10⁶ PFU of vvM2 (■) or vvM2/IL-4 (○). Four weeks later, all mice were challenged with 10⁷ PFU of live RSV intravenously and CTL activity in splenocytes was measured on day 5 after challenge (P < 0.05 between vvM2 and vvM2/IL-4 for E:T = 100:1 and 50:1).