Programmed death-1-induced interleukin-10 production by monocytes impairs CD4+ T cell activation during HIV infection

Abstract

Viral replication and microbial translocation from the gut to the blood during HIV infection lead to hyperimmune activation, which contributes to the decline in CD4+ T cell numbers during HIV infection. Programmed death-1 (PD-1) and interleukin-10 (IL-10) are both upregulated during HIV infection. Blocking interactions between PD-1 and programmed death ligand-1 (PD-L1) and between IL-10 and IL-10 receptor (IL-10R) results in viral clearance and improves T cell function in animal models of chronic viral infections. Here we show that high amounts of microbial products and inflammatory cytokines in the plasma of HIV-infected subjects lead to upregulation of PD-1 expression on monocytes that correlates with high plasma concentrations of IL-10. Triggering of PD-1 expressed on monocytes by PD-L1 expressed on various cell types induced IL-10 production and led to reversible CD4+ T cell dysfunction. We describe a new function for PD-1 whereby microbial products inhibit T cell expansion and function by upregulating PD-1 levels and IL-10 production by monocytes after binding of PD-1 by PD-L1.

Figure 1

PD-1 expression is upregulated in CD16⁻ and CD16⁺ monocyte subsets during HIV infection. (a) MFI of PD-1 expression on total monocytes (CD14⁺ cells), CD14⁺CD16⁻ monocytes and CD14⁺CD16⁺ monocytes in PBMCs from viremic (n = 23), aviremic (n = 13) and healthy (n = 14) subjects analyzed ex vivo by flow cytometry. Representative flow cytometry histograms of each category of donors are shown in this panel. APC, allophycocyanin. (b) PD-1 MFI (as measured in a) in relation to viral load in viremic subjects. *P < 0.05, **P < 0.001 and ***P < 0.0001.

Figure 2

PD-1 expression in monocytes is upregulated by TLR ligands of bacterial origin and inflammatory cytokines and correlate with IL-10 concentrations in the blood of viremic subjects. (a) Flow cytometry analysis of the ratio of PD-1 MFI on CD14⁺CD16⁻ and CD14⁺CD16⁺ monocytes (n = 3), incubated for 48 h with LPS, LTA, CpG DNA, single-stranded RNA (ssRNA), TNF, IL-1β or IL-6 and HIV-1 R5 strain (ADA), to PD-1 MFI on unstimulated monocytes in PBMCs from healthy subjects. (b) Flow cytometry analysis of the ratio of PD-1 MFI on monocytes in PBMCs from healthy subjects (n = 3), incubated for 48 h with sera from healthy or viremic subjects, to PD-1 MFI on monocytes incubated with RPMI medium supplemented with commercial human serum. Representative results of three experiments are shown in this figure. *P < 0.05. (c) ELISA analysis of IL-10 in the serum of HIV-infected donors (n = 39) were and flow cytometry analysis of PD-1 expression on monocytes from the same donors assessed ex vivo. Error bars represent s.d.

Figure 3

Specific PD-1 triggering induces IL-10 production by monocytes. (a–c) IL-10 concentrations, as measured by ELISA 24 h after incubation of monocytes with 10 μg ml⁻¹ antibody to PD-1, polyclonal goat IgG (n = 7; (a)), antibody to HLA-I or antibody to HLA-II (b) in the presence or absence of 5 or 10 μg ml⁻¹ polymyxin-B ((c) n = 3). (d) Flow cytometry plots showing IL-10 expression in monocytes incubated with 10 μg ml⁻¹ antibody to PD-1 or 1 μg ml⁻¹ LPS; 5 μg ml⁻¹ monensin was added 1 or 18 h later (n = 3). (e,f) IL-10 secreted by monocytes incubated for 24 h with 10 μg ml⁻¹ antibody to PD-1 for 0, 3, 6, 24 and 48 h (e) or with a range of concentrations of antibody to PD-1 (f); n = 3 for each). (g) IL-10 expression as measured by flow cytometry and ELISA at 24 h after incubation of CD16⁻ and CD16⁺ monocytes with 10 μg ml⁻¹ antibody to PD-1. For flow cytometry analysis, 5 μg ml⁻¹ monensin was added or not 18 h after stimulation. Cell concentrations were 1 ×10⁶ cells per ml. (h) IL-10 expression, as measured by flow cytometry and ELISA at 48 h after monocyte incubation with PD-L1–expressing (in the presence or absence of antibody to PD-L1), PD-L2–expressing or Mock (empty vector)-expressing Cos-7 cells at a ratio of 1:4. Monensin was added or not 24 h after the incubation with Cos cells (n = 4). Owing to subject variability, ELISA results are expressed as the fold increase in IL-10 production by monocytes cultured with Cos-Mock cells. (i) IL-10 secreted by monocytes (n = 3) incubated in the presence of 75 pg ml⁻¹ LPS, 0.5 μg ml⁻¹ antibody to PD-1 or both LPS and antibody to PD-1. Error bars represent s.d. *P < 0.05.

Figure 4

IL-10 production by monocytes from viremic subjects is specifically induced by PD-1–PD-L1 interaction. (a) IL-10 expression, as measured by flow cytometry in monocytes from viremic or healthy subjects when PBMCs (n = 11) were incubated for 40 h in the presence or absence of 10 μg ml⁻¹ blocking antibody to PD-L1. Sixteen hours before the end of the incubation period, 5 μg ml⁻¹ monensin was added. (b) Representative flow cytometry plots of PD-1 and IL-10 expressed by CD16⁻ and CD16⁺ monocytes subsets (n = 11). Gates are defined on the basis of the background fluorescence obtained with an isotype control antibody of the phycoerythrin-conjugated antibody to IL-10 and showed in c. (c) IL-10 production by monocytes, as measured by flow cytometry, after the incubation in the presence of a blocking antibody to PD-L1. Owing to the variable levels of IL-10 production between subjects, results are reported as the fold decrease of the percentage of cells expressing IL-10 as compared to untreated cells. IL-10 production by the latter are normalized to 1. *P ≤ 0.01 and **P ≤ 0.001.

Figure 5

IL-10 production induced by PD-1 triggering on monocytes inhibits CD4⁺ T cell proliferation and cytokine production. (a) Flow cytometry plots showing CD4⁺ T cells proliferation when CFSE-labeled PBMCs (n = 3) were incubated with 5 ng ml⁻¹ SEB, CMV or HIV gag peptides (50 ng ml⁻¹ per peptide), in the presence or absence of 10 μg ml⁻¹ antibody to PD-1, blocking antibody to IL-10R or both. (b) Antigen-specific proliferation measured in PBMCs, as described in a in the presence of isolated monocytes treated with antibody to PD-1. (c) Antigen-specific proliferation measured in PBMCs as described in b, except monocytes were treated with antibody to PD-1 for 12 h (n = 6). (d) Proliferation by CD4⁺ T cells from healthy subjects (n = 3) incubated for 24 h with beads coated with antibody to CD3, antibody to CD28 and either PD-L1–specific or isotype control antibody the presence or absence of 5 ng ml⁻¹ IL-10 or the supernatants of PD-1–stimulated monocytes. 1, unstimulated; 2, incubated with IL-10; 3, CD3-specific antibody (anti-CD3), CD28-specific antibody (anti-CD28) and isotype control antibody stimulation; 4, anti-CD3, anti-CD28 and PD-L1 stimulation; 5, anti-CD3, anti-CD28, isotype control and IL-10 stimulation; 6, anti-CD3, anti-CD28, PD-L1 and IL-10 stimulation; 7, stimulation with anti-CD3, anti-CD28, isotype control and unstimulated monocyte supernatant (NSMS); 8, stimulation with anti-CD3, anti-CD28, PD-L1 and NSMS; 9, stimulation with anti-CD3, anti-CD28, isotype control and PD-1–stimulated monocyte supernatant (PSMS); 10, stimulation with anti-CD3, anti-CD28, isotype control and PSMS in the presence of antibody to IL-10R; 11, stimulation with anti-CD3, anti-CD28, PD-L1 and PSMS; 12, stimulation with anti-CD3, anti-CD28, PD-L1 and PSMS in the presence of antibody to IL-10R. *P < 0.05, **P < 0.005 and ***P < 0.0005. Error bars represent s.d.

Figure 6

Phosphorylation of STAT-3 by IL-10 produced by PD-1–triggered monocytes and correlation of PD-1, PD-L1 and IL-10R expression in CD4⁺ T cells. (a) P-STAT3 expression measured by flow cytometry in CD4⁺ T cells from healthy subjects (n = 3) treated or not with 10 μg ml⁻¹ of blocking antibody to IL-10R for 1 h and stimulated, over 15 min, with 50 ng ml⁻¹ of IL-10 or with monocytes prestimulated with antibody to PD1 (ratio of five monoytes to one CD4⁺ T cell) in the presence or the absence of antibody to IL-10R. (b–d) Correlation between PD-1 and IL-10R (b), PD-L1 and IL-10R (c) and PD-1 and PD-L1 (d) expression, as measured by flow cytometry in PBMCs labeled with antibody to CD3, antibody to CD4, antibody to PD-L1, antibody to IL-10R and/or antibody to PD-1. (e) PD-1 and PD-L1 expression on IFN-γ⁺CD4⁺ and CD154⁺CD4⁺ T cells, as detected by flow cytometry in PBMCs from viremic subjects (n = 9) incubated in the presence or absence of CMV and HIV peptides. Brefeldin-A was added 1 h later (5 h before the intracellular staining (ICS). *P < 0.05 and **P < 0.005. Error bars represent s.d.