Immune Suppression by Neutrophils in HIV-1 Infection: Role of PD-L1/PD-1 Pathway

<div><p>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.</p></div

Neutrophil PD-L1 expression correlates with elevated levels of markers of neutrophil degranulation <i>in vivo</i>.

<p>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).</p

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

<p>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.</p

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

<p>A) PBMCs of HIV-1-infected individuals display higher frequency of CD15<i>⁺</i> 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 <i>t</i>-test.</p

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.

<p>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 <i>p</i> values are indicated for each correlation; lines represent linear regression analysis.</p

Correlations between NK cell subset activation and CD4+ T cell counts in HIV+ subjects.

<p>Correlations between NK cell subset activation and CD4+ T cell counts in HIV+ subjects.</p

Correlations of NK cell activation in healthy controls and ART-treated HIV disease.

<p>Correlations between the percentages of CD107a-expressing NK cells and co-expression of CD38 and HLA-DR on NK cells in healthy controls (A) and HIV+ subjects (B), between the percentages of CD107a-expressing NK cells and NKG2D-expressing NK cells in healthy controls (C) and HIV+ subjects (D), and between the percentages of NKG2D-expressing NK cells and co-expression of CD38 and HLA-DR on NK cells in healthy controls (E) and HIV+ subjects (F).</p

Clinical characteristics.

<p>Clinical characteristics.</p

NK cells and subsets distribution.

<p><b>Blood samples were stained with fluorescent-labeled antibodies and tested by flow cytometry</b>. (A) Representative dot plots, showing the gating strategies used to assess NK cells, and CD56dimCD16+ or CD56brightCD16− NK cell subsets. The median frequency (B) and absolute count (C) of NK cells were shown in immunologic non-responders, immunologic responders, and healthy controls. The median frequencies of CD56dimCD16+ (D) or CD56brightCD16− subsets (E) in NK cells were shown in the three study groups.</p

NK cell activation in treated HIV disease.

<p>(A) Dot plots represent the gating strategies from one representative donor of each study group. The median frequencies of CD38+HLA-DR+ NK cells (B) and NK cell subsets (C) among healthy controls, immunologic responders, and immunologic non-responders. The median frequencies of CD107a+ on NK cells (D) and NK cell subsets (E) in healthy controls, immunologic responders, and immunologic non-responders. The median frequencies of NKG2D+ on NK cells (F) and NK cell subsets (G) in healthy controls, immunologic responders, and immunologic non-responders.</p