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Hantavirus-Driven PD-L1/PD-L2 Upregulation: An Imperfect Viral Immune Evasion Mechanism

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Hantavirus-Driven PD-L1/PD-L2 Upregulation: An Imperfect Viral Immune Evasion Mechanism

Martin J Raftery et al. Front Immunol.

Abstract

Viruses often subvert antiviral immune responses by taking advantage of inhibitory immune signaling. We investigated if hantaviruses use this strategy. Hantaviruses cause viral hemorrhagic fever (VHF) which is associated with strong immune activation resulting in vigorous CD8+ T cell responses. Surprisingly, we observed that hantaviruses strongly upregulate PD-L1 and PD-L2, the ligands of checkpoint inhibitor programmed death-1 (PD-1). We detected high amounts of soluble PD-L1 (sPD-L1) and soluble PD-L2 (sPD-L2) in sera from hantavirus-infected patients. In addition, we observed hantavirus-induced PD-L1 upregulation in mice with a humanized immune system. The two major target cells of hantaviruses, endothelial cells and monocyte-derived dendritic cells, strongly increased PD-L1 and PD-L2 surface expression upon hantavirus infection in vitro. As an underlying mechanism, we found increased transcript levels whereas membrane trafficking of PD-L1 was not affected. Further analysis revealed that hantavirus-associated inflammatory signals and hantaviral nucleocapsid (N) protein enhance PD-L1 and PD-L2 expression. Cell numbers were strongly reduced when hantavirus-infected endothelial cells were mixed with T cells in the presence of an exogenous proliferation signal compared to uninfected cells. This is compatible with the concept that virus-induced PD-L1 and PD-L2 upregulation contributes to viral immune escape. Intriguingly, however, we observed hantavirus-induced CD8+ T cell bystander activation despite strongly upregulated PD-L1 and PD-L2. This result indicates that hantavirus-induced CD8+ T cell bystander activation bypasses checkpoint inhibition allowing an early antiviral immune response upon virus infection.

Keywords: CD86; PD-1; PD-L1; PD-L2; bystander activation; hantaviruses; viral immune evasion.

Figures

Figure 1
Figure 1
Levels of sPD-L1, sPD-L2, hantavirus-specific IgG and NETs in sera from hantavirus-infected patients. Sera from normal healthy individuals or convalescent hantavirus-infected patients (after the viremic phase) was tested by ELISA for levels of (A) sPD-L1 and (B) sPD-L2. Error bars represent the mean ± SD (****p < 0.0001, ***p < 0.001, paired Student's t-test). (C) Sequential sera samples from patients with PUUV (black) or DOBV (blue) were tested for sPD-L1. Red samples also tested additionally positive for hantavirus RNA and are therefore acute infections. (D) Levels of sPD-L1 in patients with kidney failure or in convalescence were further analyzed. Convalescent sera were separated into early convalescent (IgM dominant) or late convalescent (IgG dominant). Error bars represent the mean ± SD (*p < 0.05, paired Student's t-test). (E) The level of NETs in sera from normal healthy individuals or convalescent hantavirus-infected patients was determined as previously described (27). Error bars represent the mean ± SD (***p < 0.001, paired Student's t-test). (F) Spleen sections from uninfected or HTNV-infected humanized mice were stained for human PD-L1 (red) and nuclei (blue). HTNV-infected spleen sections show large areas of human cells with enhanced PD-L1 expression in comparison to uninfected spleen sections (upper left and right panel; inserts show higher magnification of cells; bars represent 100 μm). Slides from uninfected and HTNV-infected humanized and unreconstituted mice animals (N = 3 each group; 12 total) were analyzed using ImageJ to determine the intensity of human PD-L1 staining (Lower panel). Error bars represent the mean ± SEM (****p < 0.0001, paired Student's t-test). The samples from unreconstituted mice were used to determine the background staining. No significant difference was found in background staining in HTNV-infected or uninfected unreconstituted mice.
Figure 2
Figure 2
Mature DC phenotype after hantavirus infection. Immature DCs were infected with HTNV at MOI of 1.5 and incubated for 4 days before staining for (A) maturation markers and (B) costimulatory markers. The results shown are representative of three independent experiments using three different donors.
Figure 3
Figure 3
Hantavirus-induced upregulation of PD-L1 and PD-L2 on immature DCs. (A) Immature DCs were infected with HTNV at a MOI of 1.5 and incubated for 4 days before staining for PD-1, PD-L1 or PD-L2. (B) Immature DCs infected as for (A) were stained for members of the B7 family other than PD-L1/PD-L2. The results shown are representative of three independent experiments using three different donors. Positive controls are given in the lower panel (B7-H2 and B7-H3 from HUVEC, B7-H4 from HEK293 cells transfected with a B7-H4 plasmid).
Figure 4
Figure 4
Increase in PD-L1 and PD-L2 transcripts but not cellular uptake in hantavirus-infected immature DCs. (A) Immature DCs were infected with HTNV at MOI of 1.5 and incubated for 4 days or exposed to IFN-α for 6 h at 2,000 U/ml before being harvested. Subsequently, RNA was isolated and the number of indicated transcripts quantified by qPCR according to the delta-delta-Ct (ddCt) method. (B) Immature DCs infected as for (A) were incubated with PE-coupled anti-PD-L1 antibody at 4°C 1 h or at 37°C for 4 h before being washed and analyzed by flow cytometry. Uptake was calculated by subtracting MFI at 37°C from MFI at 4°C. Uptake of HTNV infected cells was then compared to uninfected cells. Results are derived from three independent experiments, error bars represent the mean ± SD.
Figure 5
Figure 5
Control of PD-L1 expression by inflammatory stimuli. (A) Immature DCs were exposed to the following inflammatory stimuli before staining for PD-L1: type I IFN (IFN-α at 1,000 U/ml), type II IFN (IFN-γ at 1,000 U/ml), poly(dA:dT), UV-inactivated VSV or poly(I:C) for 24 h. The results shown are representative of three independent experiments using three different donors. (B) Huh7.5 cells (control), Huh7.5 cells permanently expressing a constitutively active form of RIG-I (RIG-CA) or Huh7.5 cells stimulated with IFN-γ at 1,000 U/ml for 24 h were stained for PD-L1 and analyzed by flow cytometry. Results are derived from three independent experiments, error bars represent the mean ± SEM (*p < 0.05, paired Student's t-test). (C) HEL cells were transfected with HTNV N-expressing plasmids or empty plasmids (Control). After 2 days cells were stained for PD-L1. Results are given as a percentage of control and are derived from three independent experiments, error bars represent the mean ± SD.
Figure 6
Figure 6
Upregulation of functional PD-L1 and PD-L2 on HTNV-infected endothelial cell lines. (A) HUVECs were infected with HTNV at a MOI of 1.5 and incubated for 4 days before staining for PD-L1 or PD-L2. The results shown are representative of 4 independent experiments using 4 different donors. (B) Human primary fibroblasts (Fi301) cells were infected at a MOI of 1.5 and incubated for 12, 24 or 48 h before staining for PD-L1 or MHC class I molecules. Results are derived from three independent experiments (*p < 0.05, **p < 0.01, paired Student's t-test). (C) HUVECs infected as for (A) were mixed with allogeneic CD4+ cells at a ratio of 1:4 and treated with PHA at 5 μg/ml. After 2 days the number cells was measured by MTT dye test (EZ4U-test). Results are derived from three independent experiments using three different donors, error bars represent the mean ± SD.
Figure 7
Figure 7
Monocyte-dependent bystander activation of CD8+ T lymphocytes by hantavirus. PBMCs isolated from blood of healthy human donors were mock-infected or infected with HTNV. After 3–4 days cells were analyzed by flow cytometry. (A) HTNV-specific increase in CD69+ cells in the CD4+ and CD8+ subset of CD3+ cells after 4 days of incubation. Results are from three independent experiments. Error bars represent the mean ± SEM (*p < 0.05, paired Student's t-test). (B) PBMCs from HLA-A2+ HCMV-seropositive healthy human donors were exposed to HTNV or HSV-1 for 4 days before being stained for HCMV-specific CD3+ cells using a pp65 loaded tetramer reagent (CMVpp65TET). Degranulation was determined by CD107a staining. Results are derived from three independent experiments. Error bars represent the mean ± SEM (*p < 0.05, paired Student's t-test).
Figure 8
Figure 8
Dependency of hantavirus-induced bystander activation on costimulatory CD86 molecules. (A) Immature DCs were infected with HTNV at a MOI of 1.5 and incubated for 4 days or exposed to IFN-α for 6 h at 2,000 U/ml before being harvested. Subsequently, cellular RNA was isolated and the number of indicated cytokine-encoding transcripts quantified by qPCR according to the delta-delta-Ct (ddCt) method. (B) PBMCs treated with anti-IL15 (20 μg/ml) or anti-IFN-α (20 μg/ml) and PBMCs depleted of CD14+ cells were exposed to HTNV at a MOI of 1.5 for 4 days before CD69 expression on CD8+ cells was measured by cytofluorimetric analysis. Results are derived from three independent experiments, error bars represent the mean ± SEM (*p < 0.05, ***p < 0.001, 1 way ANOVA test with Bonferroni correction). (C) PBMCs treated with anti-CD86 or anti-MHC (both 10 μg/ml) were exposed to HTNV at a MOI of 1.5 for 4 days before CD69 expression on CD8+ CD45RO+ cells was determined by cytofluorimetric analysis. Error bars represent the mean ± SEM (*P < 0.05, Student's t-test). (D) PBMC infected with MOI 1.5 of TULV or HTNV were analyzed 3 days post infection for the expression of CD86 on the surface of CD14+ cells. (E) Sera from normal healthy individuals or convalescent hantavirus-infected patients were tested by ELISA for levels of sCD86. Error bars represent the mean ± SD (****p < 0.0001, paired Student's t-test).

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