Effects of type I interferons on the adjuvant properties of plasmid granulocyte-macrophage colony-stimulating factor in vivo

Abstract

While administration of granulocyte-macrophage colony-stimulating factor (GM-CSF) can induce the local recruitment of activated antigen-presenting cells at the site of vaccine inoculation, this cellular recruitment is associated with a paradoxical decrease in local vaccine antigen expression and vaccine-elicited CD8+ T-cell responses. To clarify why this cytokine administration does not potentiate immunization, we examined the recruited cells and expressed inflammatory mediators in muscles following intramuscular administration of plasmid GM-CSF in mice. While large numbers of dendritic cells and macrophages were attracted to the site of plasmid GM-CSF inoculation, high concentrations of type I interferons were also detected in the muscles. As type I interferons have been reported to damp foreign gene expression in vivo, we examined the possibility that these local innate mediators might decrease plasmid DNA expression and therefore the immunogenicity of plasmid DNA vaccines. In fact, we found that coadministration of an anti-beta interferon monoclonal antibody with the plasmid DNA immunogen and plasmid GM-CSF restored both the local antigen expression and the CD8+ T-cell immunogenicity of the vaccine. These data demonstrate that local innate immune responses can change the ability of vaccines to generate robust adaptive immunity.

FIG. 1.

Plasmid GM-CSF intramuscular inoculation in mice. (A) GM-CSF protein expression in muscle of plasmid GM-CSF-inoculated mice. Mice (n = 4 mice; 8 muscles/group) were inoculated with 50 μg plasmid GM-CSF, sham plasmid, or PBS. At 4 h and 1, 2, 3, and 7 days postinoculation, muscles were isolated and homogenized and GM-CSF expression was measured by ELISA. A significant amount of GM-CSF protein was measured in plasmid GM-CSF-injected muscles (P < 0.01; t test) and reached a peak on day 3 following inoculation. Data are expressed as the mean ± standard error. (B) The CD8⁺ T-cell response elicited by a plasmid DNA immunogen was suppressed by plasmid GM-CSF pretreatment. Three groups of mice (n = 6 mice/group) were inoculated intramuscularly with 50 μg plasmid GM-CSF, 50 μg sham plasmid, or PBS and 3 days later with a plasmid gp120 vaccine construct. p18-specific CD8⁺ T-lymphocyte responses were then monitored by staining peripheral blood lymphocytes with a D^d/p18 tetramer and an anti-CD8 MAb. The plasmid GM-CSF-pretreated group had a threefold-lower peak vaccine-elicited CD8⁺ T-cell response on day 12 following plasmid gp120 inoculation than the other two groups (P < 0.01; t test). Data are expressed as the mean percentage of tetramer-positive CD8⁺ T cells ± standard error. The results shown are representative of three experiments performed.

FIG. 2.

Pretreatment with plasmid GM-CSF suppressed expression of a plasmid DNA immunogen. Mice (n = 4 mice/group) were inoculated with 50 μg plasmid GM-CSF, sham plasmid, or PBS on day −3 and with 50 μg plasmid Luc on day 0. Luc expression was monitored by IVIS over the ensuing 21 days. The amount of Luc expression on day 7 was significantly lower in the plasmid GM-CSF-pretreated group than in the PBS group and also lower on day 14 following plasmid Luc inoculation than in both the PBS- and sham-pretreated control groups (P < 0.05; t test). Luc expression is shown as mean ± standard error. The results shown are representative of six experiments performed.

FIG. 3.

Plasmid DNA and plasmid GM-CSF recruited monocytes and APCs at the site of inoculation. Mice (n = 3 mice; 6 muscles/group) were inoculated intramuscularly with 50 μg sham plasmid, 50 μg plasmid GM-CSF, or PBS, and quadriceps muscles were harvested at 4 h and on days 1, 2, 3, and 7 postinoculation. Cells were isolated from muscle by collagenase digestion, stained with MAbs, and analyzed by flow cytometry. The frequencies of cells per muscle are shown for F4/80⁺ monocytes; CD3⁻, CD19⁻, CD4⁻, CD8⁻, B220⁻, Gr1⁻, and CD11c⁺ mDCs; and CD3⁻, CD19⁻, Gr1⁺, CD11c^int, and B220⁺ pDCs. mDCs were present in larger numbers in muscle of plasmid GM-CSF-inoculated mice on day 7 than in the other groups (P < 0.01; t test). The results shown are representative of two experiments performed.

FIG. 4.

Administration of plasmid GM-CSF transiently increased levels of inflammatory chemokines and cytokines in the muscle at the site of inoculation. Mice (n = 4 mice; 8 muscles/group) were inoculated with 50 μg plasmid GM-CSF, sham plasmid, or PBS. At 4 h and on days 1, 2, 3, and 7 following inoculations, muscles were isolated, homogenized, and assessed for IL-6, MCP-1, TNF-α, and IL-12p70 using a CBA assay. Significant amounts of cytokines were measured for IL-6, MCP-1, and TNF-α but not IL-12p70 at 4 h following the inoculation in the GM-CSF group (P < 0.01; t test). Increased amounts of MCP-1 and TNF-α were measured for 3 days following inoculation. Data are expressed as the mean ± standard error. The results shown are representative of four experiments performed.

FIG. 5.

Administration of plasmid GM-CSF increased levels of IFN-β in the muscle at the site of inoculation. Mice (n = 4 mice; 8 muscles/group) were inoculated with 50 μg plasmid GM-CSF, sham plasmid, or PBS. At 4 h and 1, 2, 3, and 7 days postinoculation, muscles were isolated and homogenized and IFN-α, IFN-β, and IFN-γ were measured by ELISA or CBA assay. Significant amounts of IFN-α and IFN-β secretion were measured in plasmid GM-CSF-injected muscles (P < 0.01; t test). Data are expressed as the mean ± standard error. The results shown are representative of four experiments performed.

FIG. 6.

Administration of anti-IFN-α and anti-IFN-β MAbs enhanced expression of plasmid Luc in mice pretreated with plasmid GM-CSF. (A) Three groups of mice (n = 4 mice/group) were administered 50 μg plasmid Luc by intramuscular inoculation and were also given 250 μg anti-IFN-β or isotype control (ISO) MAb intraperitoneally at the same time. Luc expression was monitored by IVIS over the ensuing 21 days, and expression is expressed as the mean ± standard error. No significant differences in Luc expression were observed between the three groups of mice (P = 0.49; t test). (B) Mice (n = 4 mice/group) were inoculated intramuscularly with 50 μg plasmid GM-CSF on day −3 and 50 μg plasmid Luc on day 0. Coincident with the plasmid Luc inoculation, 250 μg anti-mouse IFN-α, anti-mouse IFN-β, or isotype control MAb was injected intraperitoneally, Luc was monitored by IVIS over the ensuing 28 days and is expressed as the mean ± standard error. Compared to the isotype control group, Luc expression was significantly increased for 14 days in the anti-IFN-β- but not anti-IFN-α-administer group (P < 0.05; t test). The results shown are representative of four experiments performed.

FIG. 7.

Anti-gp120 antibody responses in plasmid GM-CSF-pretreated, plasmid gp120 DNA-vaccinated mice. Four groups of mice (n = 6 mice/group) were inoculated on day −3 with either 50 μg plasmid GM-CSF or sham plasmid alone. On day 0, all mice were inoculated with 50 μg plasmid gp120 DNA. Groups of plasmid GM-CSF-pretreated mice also received 250 μg anti-IFN-β MAb or the same quantity of isotype control (Cont) antibody intraperitoneally. On day 28, sera were obtained from the mice and anti-gp120 antibody titers were evaluated by ELISA. The geometric mean titer (GMT) ± standard error is shown for each group. The results shown are representative of three experiments performed.

FIG. 8.

Anti-IFN-β antibody treatment augmented CD8⁺ T-cell responses. Three groups of mice (n = 6 mice/group) received either no pretreatment (A) or pretreatment with 50 μg plasmid GM-CSF on day −3 (B). All mice were then immunized with 50 μg plasmid gp120 on day 0. Mice then either were untreated or were inoculated with 250 μg anti-mouse IFN-β antibody or an isotype control (Iso Cont) antibody. gp120-specific cellular immune responses were monitored by D^d/p18 tetramer staining of peripheral blood CD8⁺ T cells. Peak immune responses were significantly higher in the anti-IFN-β antibody-treated group (P < 0.01; t test). Data are expressed as the mean ± standard error. The results shown are representative of three experiments performed.