Interplay between regulatory T cells and PD-1 in modulating T cell exhaustion and viral control during chronic LCMV infection

Abstract

Regulatory T (T reg) cells are critical for preventing autoimmunity mediated by self-reactive T cells, but their role in modulating immune responses during chronic viral infection is not well defined. To address this question and to investigate a role for T reg cells in exhaustion of virus-specific CD8 T cells, we depleted T reg cells in mice chronically infected with lymphocytic choriomeningitis virus (LCMV). T reg cell ablation resulted in 10-100-fold expansion of functional LCMV-specific CD8 T cells. Rescue of exhausted CD8 T cells was dependent on cognate antigen, B7 costimulation, and conventional CD4 T cells. Despite the striking recovery of LCMV-specific CD8 T cell responses, T reg cell depletion failed to diminish viral load. Interestingly, T reg cell ablation triggered up-regulation of the molecule programmed cell death ligand-1 (PD-L1), which upon binding PD-1 on T cells delivers inhibitory signals. Increased PD-L1 expression was observed especially on LCMV-infected cells, and combining T reg cell depletion with PD-L1 blockade resulted in a significant reduction in viral titers, which was more pronounced than that upon PD-L1 blockade alone. These results suggest that T reg cells effectively maintain CD8 T cell exhaustion, but blockade of the PD-1 inhibitory pathway is critical for elimination of infected cells.

Figure 1.

T reg cells have an activated phenotype during chronic viral infection. Analysis was done on splenocytes from naive uninfected mice, mice infected with LCMV Arm, and analyzed at least 100 d after acute infection and LCMV cl-13 chronically infected mice (mice transiently depleted of CD4 T cells and infected with LCMV cl-13, with analysis done at least 40 d after infection). (A) Percentage of T reg cells (Foxp3⁺) among CD4 T cells. (B) Absolute number of T reg cells in spleen. (C) Frequency of Vβ5⁺ cells among T reg cells. Mice infected with LCMV cl-13 and analyzed 21 d later were included as positive controls (22–40% Vβ5⁺ cells). (D) Graph shows MFI of CD25 expression on T reg cells. (E) Histograms show representative expression of different markers by T reg cells and numbers represent MFI. Data are a compilation of 4 experiments (A and B) or show representative results from 3 experiments (C–E; n = 3–4 mice per group). Error bars indicate SEM. Non-parametric Mann Whitney test, where *, P < 0.05; **, P < 0.01; NS = not significant.

Figure 2.

Exhausted CD8 T cells expand and undergo phenotypic changes upon T reg cell ablation. (A) Experimental outline. (B) Total numbers of activated T cells in the spleen of PBS-treated or T reg cell–depleted (DT) mice chronically infected with LCMV. (C) Number of LCMV-specific (Db GP276) CD8 T cells among PBMCs before and after DT treatment. (D and E) Absolute numbers (D) and frequency (E) of LCMV-specific (Db GP276) CD8 T cells within total CD8 T cell population. (F) Absolute numbers of LCMV DbGP276-specific CD8 T cells in the spleen at different days after T reg cell ablation. (G) Proliferative activity of LCMV-specific (Db-GP276) CD8 T cells. Numbers show MFI of Ki67 expression. (H) Phenotype of LCMV-specific (Db GP276) CD8 T cells in spleen. Numbers show MFI of different CD8 T cell markers. Data are a compilation of 5 independent experiments with 3–5 mice per group. Error bars indicate SEM. Non-parametric Mann Whitney test, where *, P < 0.05; ***, P < 0.001; NS = not significant.

Figure 3.

LCMV-specific CD8 T cells regain effector function upon T reg cell ablation in chronically infected mice. LCMV chronically infected Foxp3^DTR knock-in mice were depleted of T reg cells for 10 d by DT administration as in Fig. 2. (A) Absolute numbers of CD8 T cells in spleen producing IFN-γ. (B) MFI of IFN-γ production by CD8 T cells after in vitro restimulation with various LCMV peptides. (C) Percentage among DbGP276-specific cells that express IFN-γ in spleen. (D) Frequency of CD8 T cells producing both IFN-γ and TNF after in vitro restimulation with LCMV peptides. (E) Degranulation and surface expression of CD107 a/b in CD8 T cells after in vitro restimulation with GP276 LCMV peptide. (F) Granzyme B expression on LCMV DbG276-specific CD8 T cells in spleen. (G) Ex vivo cytotoxic activity of splenic CD8 T cells measured by ⁵¹Cr release from MC57 target cells unpulsed (control) or pulsed with a mix of LCMV peptides (GP33, GP276, and NP396). Data are a compilation of 5 independent experiments with 3–5 mice per group. Error bars indicate SEM. Non-parametric Mann Whitney test, where *, P < 0.05; ***, P < 0.001.

Figure 4.

Rescue of LCMV-specific CD8 T cell responses by T reg cell ablation is greater than by blockade of the PD-1 pathway. LCMV chronically infected Foxp3^DTR knock-in mice were depleted of T reg cells for 10 d by DT administration as in Fig. 2 or mice received 3 doses of PD-L1 blocking antibody, every 3 d. (A and B) Dot plots (A) show frequency in the same mouse and graphs (B) show number of LCMV-specific (Db GP276) CD8 T cells among PBMCs before and after treatment. (C and D) Dot plots show frequency (C) and graphs show number (D) of LCMV-specific (Db GP33) CD8 T cells in different organs 11 d after treatment. Data are a compilation of 3 independent experiments with 3–5 mice per group. Error bars indicate SEM. Non-parametric Mann Whitney test, where *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS = not significant.

Figure 5.

Cognate antigen is necessary for activation of antiviral T cells after T reg cell depletion. (A) Experimental outline. (B) Total numbers of activated T cells in the spleen of PBS-treated or T reg cell–depleted (DT) mice 100 d after infection with LCMV Arm. (C) Phenotype of total CD8 T cells in spleen. (D) Absolute numbers of LCMV-specific (Db GP276) CD8 T cells. (E) Absolute numbers of cells in the spleen that produce IFN-γ after in vitro restimulation with various LCMV peptides. (F) Phenotype of splenic LCMV-specific (Db GP276) CD8 T cells. A representative of 3 independent experiments is shown, with 5–6 mice per group. Error bars indicate SEM. Non-parametric Mann Whitney test, where ***, P < 0.001; NS = not significant.

Figure 6.

Co-stimulation and CD4 T cells are required for the rescue of exhausted CD8 T cells upon T reg cell depletion. Foxp3^DTR knock-in mice chronically infected with LCMV cl-13 received DT for 10 d (as in Fig. 2). CTLA-4 Ig was administered every other day during T reg cell depletion starting at day −2, and CD4 T cells were depleted upon administration of CD4 antibody on days −2 and −1 of T reg cell ablation. (A) Representative dot plots show expression of B7.1 and B7.2 on splenic DCs after 8 d of T reg cell ablation. (B and C) Frequency of splenic CD8 T cells that co-express IFN-γ and TNF (B) and absolute numbers of CD8 T cells that produce IFN-γ (C) after in vitro restimulation with LCMV peptides. (D) Frequency of LCMV-specific (Db-GP276) and CD127-expressing splenic CD8 T cells. (E) Graphs show B7.1 and B7.2 MFI on CD11b⁺ splenic DCs after 8 d of T reg cell ablation preceded or not by conventional CD4 T cell depletion. The data are representative of 3 independent experiments with 3–5 mice per group. Error bars indicate SEM. Non-parametric Mann Whitney test, where *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 7.

Inability to clear virus coincides with PD-L1 up-regulation. Foxp3^DTR knock-in mice chronically infected with LCMV received DT for 10 d (as in Fig. 2). (A) Viral load after 10 d of T reg cell depletion. (B) Dot plots show PD-1 expression on DbGP276-specific CD8 T cells. (C) PD-L1 expression on splenic DCs after 7 d of T reg cell ablation. (D) Graph shows IFN-γ in serum before and at day 5 after initiation of DT treatment. Dashed line indicates the limit of detection of this assay. (E) Representative dot plots show intracellular staining of LCMV nucleoprotein (NP) and PD-L1 expression on splenic DCs. Staining of DCs in uninfected mice is shown as a control for specificity of NP staining and for basal PD-L1 expression. PD-L1 expression is shown as MFI on infected (NP positive) or noninfected (NP negative) DCs. Data are a compilation of 3 experiments (A) or show representative results of 2–3 experiments (B–E) with 3–6 mice per group. Error bars indicate SEM. (A) Non-parametric Mann Whitney test, where NS = not significant. (D) Wilcoxon matched pairs test, where *, P < 0.05. (E) Student’s t test, where **, P < 0.01; ***, P < 0.001.

Figure 8.

Essential role for PD-L1 blockade in viral control after T reg cell depletion. Foxp3^DTR knock-in mice chronically infected with LCMV received DT for 10 d (as in Fig. 2) in combination with PD-L1 blocking antibody. PD-L1 antibody was administered on days 1, 4, and 7 of DT treatment. (A) Viral titers in serum before and after treatment. (B) Fold reduction in serum viral titer after treatment. (C) Absolute numbers of splenic CD8 T cells that produce IFN-γ after in vitro restimulation with LCMV peptides. (D) Absolute numbers of CD8 T cells co-expressing IFN-γ and TNF after in vitro restimulation with GP276 LCMV peptide. (E) Representative dot plots showing frequency of CD8 T cells producing cytokines as in D. Data are a compilation (A and B) or show representative results of 3 independent experiments, with 4–6 mice per group. Error bars indicate SEM. Non-parametric Mann Whitney test, where *, P < 0.05; ***, P < 0.001; NS = not significant.

Figure 9.

Transient T reg cell depletion improves CD8 T cell rescue and viral control when combined with PD-L1 blockade without causing overt disease in LCMV chronically infected mice. (A) Foxp3^DTR knock-in mice chronically infected with LCMV received continuous DT (on days 0, 1, 4, and 7) and/or PD-L1 antibody (on days 1, 4, and 7), as in Fig. 8. Mice weight after 11 d of treatment, relative to initial weight before treatment. (B) As in A, but mice received transient DT treatment (on days 0, 1, and 4). Mice weight after 12 d of treatment. (C) Representative dot plots show frequency of T reg cells in PBMC before and after continuous or transient DT treatment. (D) Absolute numbers of LCMV-specific (Db GP276) CD8 T cells within the total splenic CD8 T cell population. (E and F) Frequency of CD8 T cells producing both IFN-γ and TNF (E) and absolute numbers of CD8 T cells in the spleen that produce IFN-γ after in vitro restimulation with LCMV peptide GP276 (F). (G) Viral titers in serum before and after treatment. (H) Fold reduction in serum viral titer after treatment. A representative of 3 independent experiments is shown, with 6 mice per group. Error bars indicate SEM. Non-parametric Mann Whitney test, where *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS = not significant.