Immune suppression by neutrophils in HIV-1 infection: role of PD-L1/PD-1 pathway

Abstract

HIV-1 infection is associated with a progressive loss of T cell functional capacity and reduced responsiveness to antigenic stimuli. The mechanisms underlying T cell dysfunction in HIV-1/AIDS are not completely understood. Multiple studies have shown that binding of program death ligand 1 (PD-L1) on the surface of monocytes and dendritic cells to PD-1 on T cells negatively regulates T cell function. Here we show that neutrophils in the blood of HIV-1-infected individuals express high levels of PD-L1. PD-L1 is induced by HIV-1 virions, TLR-7/8 ligand, bacterial lipopolysaccharide (LPS), and IFNα. Neutrophil PD-L1 levels correlate with the expression of PD-1 and CD57 on CD4+ and CD8+ T cells, elevated levels of neutrophil degranulation markers in plasma, and increased frequency of low density neutrophils (LDNs) expressing the phenotype of granulocytic myeloid-derived suppressor cells (G-MDSCs). Neutrophils purified from the blood of HIV-1-infected patients suppress T cell function via several mechanisms including PD-L1/PD-1 interaction and production of reactive oxygen species (ROS). Collectively, the accumulated data suggest that chronic HIV-1 infection results in an induction of immunosuppressive activity of neutrophils characterized by high expression of PD-L1 and an inhibitory effect on T cell function.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1. Neutrophils from HIV-1 infected individuals express elevated levels of surface PD-L1.

A, B) Representative histogram of PD-L1 expression on CD15+ neutrophils (A) and CD14+ monocytes (B) in fresh blood obtained from a healthy donor (HD) and HIV-1-infected individual. C, D) PD-L1 expression on CD15+ neutrophils (C) and CD14+ monocytes (D) of HDs (N = 12), HIV-1-infected patients on ART (N = 17), and subjects not treated with ART (off ART; N = 20). MFI, mean fluorescent intensity. E, F) PD-L1 expression on CD15+ neutrophils (E) and CD14+ monocytes (F) of HD (N = 12) and HIV-1-infected individuals with viral loads <2,000 vRNA copies/ml of plasma (N = 21), >2,000 vRNA copies/ml of plasma(N = 19), or elite controllers (EC: <50 copies/ml; off ART; N = 4). Statistical analysis on C-F was performed using the Mann Whitney rank sum test. G, H) PD-L1 expression on CD15+ neutrophils (G) (N = 5) and CD14+ monocytes (H) (N = 4) of HIV-1-infected individuals prior to and following the administration of ART.

Figure 2. Neutrophil suppression of T cell function is mediated in part via the PD-L1/PD-1 pathway.

A) PBMCs of HIV-1-infected individuals display higher frequency of CD15+ LDNs compared to healthy donors (N = 13 and 29). Statistical analysis was performed using the Mann Whitney rank sum test. B) Depletion of CD15+ neutrophils co-segregating with the PBMC fraction results in an enhanced frequency of antigen-specific IFNγ-producing cells. Representative intracellular cytokine staining of CD8+ T cells from HIV-1-positive subject. PBMCs or CD15+ cell-depleted PBMCs were incubated with HIV-1-Gag peptide pool for 24 hrs. C) Summary of normalized data of four experiments using independent HIV-1-infected donors. Mann Whitney rank sum test. D) Depletion of CD15+ cells from PBMCs results in an increase of IFNγ production in culture supernatant in response to stimulation with HIV-1 Gag (N = 3). E) CD3+ T cells were isolated from HIV-1-infected subjects with high PD-L1 expression and stimulated with α-CD3 and α-CD28 antibodies. Neutrophils were incubated with activated T cells at a 5∶1 ratio in the presence of anti-PD-L1 blocking antibody or isotype control for 24 hours. The experiment was performed in the presence of ROS scavengers SOD and catalase. IFNγ release into media was determined by ELISA. A representative experiment of three independent experiments using separate HIV-1-infected donors is presented; statistical significance was analyzed using Student's t-test.

Figure 3. PD-L1 expression on neutrophils is induced by HIV-1 virions, IFNα, TLR-7 ligand R848, and LPS.

A, B) PBMCs or PMNs were stimulated with IFNα (1,000 U/ml), R848 (5 µg/ml), or vehicle (Veh) for 24 hours and PD-L1 expression on CD15+ LDNs and PMNs was determined by flow cytometry. Representative of three separate experiments using separate donors; statistical significance was analyzed using Student's t-test. C) PBMCs or PMNs were incubated with AT-2-inactivated HIV virions (AT-2 HIV) at indicated doses or control microvesicles from untransfected cell cultures for 24 hours. D) Representative histogram of PD-L1 expression on PMNs incubated for 24 hrs with LPS (100 ng/mL). E) Summary of PD-L1 expression on PMNs in the presence or absence of LPS (N = 3); statistical significance was analyzed using Student's t-test.

Figure 4. PD-L1 expression on neutrophils correlates directly with PD-1 and CD57 expression on T cells and inversely with the expression of CD3ζ chain on T cells.

CD3+CD8+ and CD3+CD4+ T cells from PBMCs were stained for PD-1 (A), CD57 (B), and CD3ζ (C). A) Correlation between PD-1 expression on CD8+ and CD4+ T cells and PD-L1 expression on neutrophils in blood. B) Correlation between the percentage of CD57+ cells of total CD3+CD8+ and CD3+CD4+ T cells and PD-L1 expression on neutrophils in blood. C) Flow cytometry analysis of the expression of CD3ζ chain on T cells and correlation between the percentage of CD3+ CD3ζ high population and PD-L1 expression on neutrophils. Spearman's rank correlation coefficients (R) and p values are indicated for each correlation; lines represent linear regression analysis.

Figure 5. Neutrophil PD-L1 expression correlates with elevated levels of markers of neutrophil degranulation in vivo.

A–C) PD-L1 expression on neutrophils correlates with plasma levels of arginase-1 (A), neutrophil gelatinase-associated lipocalin (NGAL) (B), and myeloperoxidase (MPO) (C) (N = 16). Analyzed by Pearson product-moment correlation test; D'Agostino & Pearson omnibus normality test of normal distribution of data passed for all populations (α = 0.05). D–F) PD-L1 expression on neutrophils correlates with plasma levels of G-CSF (D), GM-CSF (E), and MCP-1/CCL2 (F) in HIV-1-infected donors (N = 13).