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. 2008 Dec 23;105(51):20428-33.
doi: 10.1073/pnas.0811139106. Epub 2008 Dec 15.

IL-10 and PD-L1 operate through distinct pathways to suppress T-cell activity during persistent viral infection

David G Brooks  1 , Sang-Jun Ha, Heidi Elsaesser, Arlene H Sharpe, Gordon J Freeman, Michael B A Oldstone
Affiliations

IL-10 and PD-L1 operate through distinct pathways to suppress T-cell activity during persistent viral infection

David G Brooks et al. Proc Natl Acad Sci U S A. .

Abstract

Suppression of T-cell responses by host-derived regulatory factors is a key event leading to viral persistence. Antibody blockade of either IL-10 or programmed death-ligand 1 (PD-L1) during viral persistence enhances T-cell function and reduces viral titers. Because blockade of these immunoregulatory networks represents a powerful approach to establish immune control during persistent infection, it is important to determine whether these immunoinhibitory factors act independently or jointly and if combined blockade of these factors further enhances T-cell immunity and viral clearance. Herein, we demonstrate that the IL-10 and PD-L1 immunosuppressive pathways are mechanistically distinct. As a result, simultaneous blockade of IL-10 and PD-L1 was significantly more effective in restoring antiviral T-cell responses than blockade of either alone, and led to substantially enhanced control of an established persistent viral infection. Thus, combinatorial blockade of multiple immune-regulatory molecules may ultimately restore the T-cell responses required to tip the balance from viral persistence to immune-mediated control or elimination of persistent infection.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
IL-10 and PD-L1 act through distinct pathways to suppress antiviral immunity. (A) WT and IL-10 KO mice were infected with LCMV-Cl 13 and the level of PD-1 expression on virus-specific CD8 T cells was quantified on day 5 after infection (at a time when viral titers were similarly high in the two mouse strains). Flow plots are gated on CD8 and LCMV-GP33–41 tetramer+ cells to specifically identify virus-specific T cells. Data are representative of four mice per group and of two experiments. (B) PD-L1 (Upper) and PD-L2 (Lower) expression on dendritic cells on day 5 after LCMV-Cl 13 infection of WT (gray) and IL-10 KO (black line) mice. (C) Bar graphs demonstrate the geometric mean fluorescence intensity (GMFI) of PD-L1 (Upper) and PD-L2 (Lower) expression on dendritic cells, B cells, and macrophages in WT and IL-10 KO mice. Bars indicate the average ± SD of four to five mice per group. *, P < 0.05. (D) IL-10 RNA expression was quantified in the spleens of uninfected WT mice (naïve) and in WT and PD-L1 KO mice on day 5 after LCMV-Cl 13 infection (at a time when viral titers were equivalent in the WT and knockout mice). Bars indicate the average ± SD of five to six mice per group. *, P < 0.05 compared to LCMV-Cl 13 infected mice.
Fig. 2.
Fig. 2.
Blockade of multiple immune regulatory molecules further enhances T-cell immunity in the absence of CD4 T-cell help when compared to either treatment alone. (A) Mice were depleted of CD4 T cells immediately before LCMV-Cl 13 infection and the frequency of LCMV-GP276–286 and LCMV-GP33–41 tetramer-positive cells was measured in the indicated tissues 3 days following the final antibody treatment. Dot plots are gated on CD8 T cells and the number in each plot indicates the frequency of tetramer+ cells in the spleen, liver, or lung. Data are representative of three to four mice per group and of two experiments. (B) Total number of LCMV-GP276–286 and LCMV-GP33–41 tetramer-positive cells in the indicated tissues was calculated based on (A). *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C) The mice treated with antibodies were sacrificed at 1 week after initial treatment. Ki-67 expression was observed on DbGP276–286 tetramer-positive cells. The numbers in each plot indicate the frequency of Ki-67-positive cells. Data are representative of two independent experiments. (D) The ability of LCMV-specific CD8+ T cells to produce IFNγ and degranulate was measured 3 days following the completion of therapy. Splenocytes were stimulated with the indicated peptides in the presence of αCD107a/b antibodies and then costained for IFNγ. Dot plots are gated on CD8 T cells and the numbers represent the frequency of positive cells in each quadrant. Data are representative of two independent experiments. (E) Flow plots illustrate the frequency of TNFα and IFNγ producing GP33–41 and GP276–286 specific CD8+ T cells in the spleen 3 days after the cessation of therapy. Dot plots are gated on CD8 T cells and the numbers represent the frequency of positive cells in each quadrant. The numbers in parentheses indicate the frequency of cells that express both TNFα and IFNγ. Data are representative of two independent experiments.
Fig. 3.
Fig. 3.
Dual IL-10R/PD-L1 blockade restores function to previously exhausted T cells. (A) LCMV-Cl 13-infected mice were treated with (i) isotype control antibody, (ii) anti-IL-10R-blocking antibody alone, (iii) anti-PD-L1-blocking antibody alone, or (iv) cotreated with anti-IL-10R and anti-PD-L1-blocking antibodies. Antibody therapy was initiated on day 25 after infection and administered every 3 days for a total of five treatments. The flow plots illustrate the frequency of Thy1.1+ tg virus-specific CD8 T cells (P14 cells) in the spleen on day 40 after LCMV-Cl 13 infection (i.e., day 3 after treatment). The graph on the right illustrates the number of P14 cells following each treatment regimen. Circles represent individual mice within each group and the line indicates the average for each group. *, P < 0.05 compared to all other treatment groups. Data are representative of four to five mice per group and of two experiments. (B) Flow plots illustrate the percentage of IFNγ- and TNFα-producing P14 cells in the spleen on day 40 after LCMV-Cl 13 infection (i.e., day 3 after treatment). The bar graph represents the average fold increase ± SD in the number of TNFα-producing P14 cells in each treatment group compared to isotype treatment (which is set to 1). *, P < 0.05 compared to all other treatment groups. Data are from the same experiment as shown in (A) and are representative of four to five mice per group and of two experiments.
Fig. 4.
Fig. 4.
Enhanced control of persistent viral infection following dual IL-10R/PD-L1 blockade. (A) LCMV-Cl 13-infected mice were treated with the indicated antibodies and serum viral titers were quantified on day 25 (gray circles, before treatment) and day 40 (white circles, 3 days following cessation of therapy). Each circle represents a single mouse within each group and the graph contains data from three experiments. The black circles indicate mice in which liver viral titers were quantified in parallel, as shown in (B). The dashed line indicates the level of detection of the assay (200 PFU/ml serum). The number above each group represents the average fold decrease in virus titers between day 25 and day 40. *, significant (P < 0.05) decrease in viral titers compared to isotype treatment. **, significant (P < 0.05) decrease in virus titers compared to all other treatment groups. (B) Liver virus titers were quantified on day 40 after LCMV-Cl 13 infection (3 days following the completion of antibody therapies).
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