PD-L1 expression induced by the 2009 pandemic influenza A(H1N1) virus impairs the human T cell response

Abstract

PD-L1 expression plays a critical role in the impairment of T cell responses during chronic infections; however, the expression of PD-L1 on T cells during acute viral infections, particularly during the pandemic influenza virus (A(H1N1)pdm09), and its effects on the T cell response have not been widely explored. We found that A(H1N1)pdm09 virus induced PD-L1 expression on human dendritic cells (DCs) and T cells, as well as PD-1 expression on T cells. PD-L1 expression impaired the T cell response against A(H1N1)pdm09 by promoting CD8⁺ T cell death and reducing cytokine production. Furthermore, we found increased PD-L1 expression on DCs and T cells from influenza-infected patients from the first and second 2009 pandemic waves in Mexico City. PD-L1 expression on CD8⁺ T cells correlated inversely with T cell proportions in patients infected with A(H1N1)pdm09. Therefore, PD-L1 expression on DCs and T cells could be associated with an impaired T cell response during acute infection with A(H1N1)pdm09 virus.

Figure 1

PD-L1 is expressed on human dendritic cells and T cells, whereas PD-1 is expressed only on T cells after exposure to A(H1N1)pdm09 virus. PBMCs were stimulated with A(H1N1)pdm09 virus (pH1N1), seasonal influenza virus (H3N2), staphylococcal enterotoxin B (SEB), or synthetic TLR7 agonist (CL264); PD-L1 and PD-1 expression on DCs and T cells was analyzed by flow cytometry. (a) Fold increase in PD-L1 expression on conventional (cDCs) and plasmacytoid dendritic cells (pDCs), CD4⁺, and CD8⁺ T cells after 18 h of stimulus. M: medium. (b) PBMCs were stimulated with live or UV-inactivated pH1N1 for 18 h; virus and PD-L1 expression was measured on cDCs and pDCs. (c) Kinetics of PD-L1 and PD-1 expression on CD4⁺ and CD8⁺ T cells induced by pH1N1 or SEB. PBMCs were stimulated with pH1N1 for 2 h, then cycloheximide (CHX) was added for another 16 h, and PD-L1 expression on CD4⁺(d), CD8⁺ T cells (e), pDCs (f), and cDCs (g) was measured by flow cytometry. (n = 5 donors, error bars indicate standard error of the mean (SEM)). *P < 0.05, **P < 0.01, and ***P < 0.001 by one way ANOVA test with Bonferroni posttest.

Figure 2

PD-L1 signaling blockade decreased CD8⁺ T cell death in vitro but did not have an effect on T cell proliferation in response to A(H1N1)pdm09 virus. PBMCs from healthy individuals were stimulated with A(H1N1)pdm09 for 18 h, washed, labeled with CFSE, and treated on days 0, 3, and 5 with a blocking anti-PD-L1 antibody or an isotype control. Cells were incubated for 7 days, and T cell proliferation and cell death were determined. SEB was used as a control. (a) Representative histograms of the CD4⁺ T cell CFSE dilution from one individual. (b, c) T cell proliferation expressed as the percentage of CFSE⁺ dividing cells. (d) Representative plot of Annexin V and 7-AAD staining to evaluate CD8⁺ T cell apoptosis, which was gated from CD2⁺ and CD8⁺ cells. (e, f) Percentage of early apoptotic (Annexin V⁺ 7-AAD⁻) T cells. (n = 7, error bars indicate SEM). *P < 0.05 by Student's t-test.

Figure 3

PD-L1 blocking increased in vitro IFN-γ, IL-10, and TNF production, predominantly by CD4⁺ T cells in response to A(H1N1)pdm09 virus. Cytokine levels in the supernatants (SN) of PBMCs cultured for 7 days as described in Figure 2 and PBMCs stimulated with hemagglutinin (HA) for 7 days were measured with a Th1/Th2/Th17 human cytometric bead array kit (CBA). The production of IFN-γ (a), IL-10 (b), and TNF (c) by PBMCs is shown. Isolated memory CD4⁺ T cells (T_m) and sorted cDCs were co-cultured with or without PD-L1 blocking for 7 days, and cytokine production in the SNs was measured; IFN-γ (d), IL-10 (e), and TNF (f) levels are shown. Results are duplicates from 3 independent experiments and error bars indicate SEM. pH1N1: A(H1N1)pdm09 virus; SEB: staphylococcal enterotoxin B. *P < 0.05 by Student's t-test.

Figure 4

PD-L1 expression is increased on dendritic cells and T cells from PBMCs of patients with acute influenza infection. Cell proportions and surface PD-L1 expression on cDCs and pDCs (a–d) and CD4⁺ and CD8⁺ T cells (e–h) from cryopreserved PBMCs from patients with confirmed infection with A(H1N1)pdm09 virus (pH1N1+), patients with influenza-like illness but with a negative RT-PCR result for pandemic H1N1 influenza (pH1N1−), and healthy controls (HC, n = 10; error bars indicate SEM) were analyzed by flow cytometry. MFI: mean fluorescence intensity; pH1N1: A(H1N1)pdm09 virus. *P < 0.05, **P < 0.01, and ***P < 0.001 (Mann-Whitney test).

Figure 5

PD-L1 expression on CD8⁺ T cells is associated with a decreased T cell proportion in patients with acute A(H1N1)pdm09 viral infection. Correlations between PD-L1 expression on the proportions of CD8⁺ and CD4⁺ T cells in PBMCs from pH1N1+ patients are shown. Correlations between PD-L1 expression on CD8⁺ T cells and the proportion of total CD4⁺ (a) and CD8⁺ (b) T cells in PBMCs in pH1N1+ patients are indicated. Spearman correlation (ρ) and P values are shown in each graph. MFI: mean fluorescence intensity; pH1N1: A(H1N1)pdm09 virus.