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. 2014 Oct 3;9(10):e109103.
doi: 10.1371/journal.pone.0109103. eCollection 2014.

Characterization of programmed death-1 homologue-1 (PD-1H) expression and function in normal and HIV infected individuals

Preeti Bharaj  1 , Harendra Singh Chahar  1 , Ogechika K Alozie  2 , Lizette Rodarte  1 , Anju Bansal  3 , Paul A Goepfert  3 , Alok Dwivedi  4 , N Manjunath  1 , Premlata Shankar  1
Affiliations

Characterization of programmed death-1 homologue-1 (PD-1H) expression and function in normal and HIV infected individuals

Preeti Bharaj et al. PLoS One. .

Abstract

Chronic immune activation that persists despite anti-retroviral therapy (ART) is the strongest predictor of disease progression in HIV infection. Monocyte/macrophages in HIV-infected individuals are known to spontaneously secrete cytokines, although neither the mechanism nor the molecules involved are known. Here we show that overexpression of the newly described co-stimulatory molecule, PD1 homologue (PD-1H) in human monocyte/macrophages is sufficient to induce spontaneous secretion of multiple cytokines. The process requires signaling via PD-1H as cytokine secretion could be abrogated by deletion of the cytoplasmic domain. Such overexpression of PD-1H, associated with spontaneous cytokine expression is seen in monocytes from chronically HIV-infected individuals and this correlates with immune activation and CD4 depletion, but not viral load. Moreover, antigen presentation by PD-1H-overexpressing monocytes results in enhanced cytokine secretion by HIV-specific T cells. These results suggest that PD-1H might play a crucial role in modulating immune activation and immune response in HIV infection.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. PD-1H expression in subsets of human PBMC.
a) PBMCs co-stained for PD-1H along with Monocytes (CD14), T cell (CD3), and B cell (CD19) markers were analyzed by flow cytometry (n = 7). The overlay histogram shows PD-1H expression on CD14, CD3 and CD19 gated cells. The grey filled histogram represents isotype control. b) Cumulative data from seven donors showing PD-1H expression levels expressed as mean fluorescence intensity (MFI). c) Isolated monocytes were cultured for the indicated times and examined for PD-1H expression by flow cytometry (n = 5).
Figure 2
Figure 2. Effect of TLR ligands and cytokines on monocyte PD-1H expression.
a) Isolated monocytes were treated with the indicated TLR ligands [TLR1/2: PAM3CSK4 (1 µg/ml), TLR2: HKLM (108 cells/ml), TLR3: Poly I:C (2 µg/ml), TLR4: LPS (200 ng/ml), TLR5: flagellin (1 µg/ml), TLR6/2: FSL1 (0.5 µg/ml), TLR7: Imiquimod (2 µg/ml), TLR8: ssRNA40 (2 µg/ml), TLR9: ODN2006 (5 µM)] or cytokines (c) and examined for PD-1H expression by flow cytometry. The bar graphs and dose response curves represent data (mean±SD) from 3 replicates. * indicates p<0.05 (One way ANOVA followed by Tukey test). b,d) Dose dependent effect of TLRs and cytokines was determined on five different donors. Mean +/- SD for 5 donors is shown.
Figure 3
Figure 3. Spontaneous cytokine secretion by monocytes upon overexpression of PD-1H.
Monocytes were nucleofected with control or PD-1H expression plasmid without or along with PD-1H siRNA or nucleofected with cytoplasmic domain deficient PD-1H as indicated and the culture supernatants tested for cytokine secretion by cytometric bead array (CBA) or ELISA. a) Representative result with monocytes from one donor (out of 10 donors tested) is shown. b) Supernatants from a) were also tested for individual cytokines by ELISA. Cumulative data from 10 donors are shown c) Cytokine secretion by monocytes in presence of PD-1H, PD-1H lacking cytoplasmic domain, PD-1H with its specific siRNA, vector alone and mock treatment as assessed by ELISA. ND, not detected.
Figure 4
Figure 4. Overexpression of PD-1H on monocytes in HIV infection.
A representative histogram (a) and cumulative data (b) of PD-1H expression on CD14 gated PBMC from normal and HIV-infected donors. MFI, mean fluorescence intensity. EC, elite controllers. C, controllers. * indicates p<0.05, Kruskal Wallis test followed by Dunn multiple comparison tests. c) PD-1H expression on CD14+ (classic) and CD16+ (activated) monocytes from HIV+ donors. Each horizontal line represents expression on CD14+ and CD16+ monocytes from the same individual. d) The mRNA expression level for indicated cytokines in ex vivo-isolated monocytes from normal and HIV-infected individuals was assessed by real time PCR. Mean + SD from PBMCs from five donors. (PD-1HHI MFI: 1600-4500; PD-1HLO MFI 700-1500). *  =  p<0.05 (One way ANOVA followed by Tukey test).
Figure 5
Figure 5. The level of PD-1H expression on monocytes in HIV-infected individuals correlates with immune activation and CD4 T cell depletion, but not viral load.
Correlation of PD-1H expression on monocytes with (a) immune activation/exhaustion markers CD38, PD-1 and HLA-DR on CD4 and CD8 T cell subsets (b) viral loads and (c) CD4 counts. (n = 35). r and p values determined by Spearmans non parametric test values. p<0.05 were considered significant.
Figure 6
Figure 6. Stimulation with PD-1H- overexpressing monocytes enhances HIV gag-specific IFN-γ response by T cells.
Monocytes from HIV-infected individuals were untreated or nucleofected with control plasmid or PD-1H expression plasmid in presence of scrambled siRNA or PD-1H siRNA. After 24 h of transfection, the monocytes were pulsed with HIV clade B gag peptide pool, and used to stimulate autologous T cells. After overnight stimulation, IFN-γ secretion was measured by ELISA. Each symbol represents an individual HIV donor. Mo  =  monocytes. No pep  =  stimulation with monocytes without peptide. ND =  not detected. siSc  =  transfection with control scrambled siRNA; siPD-1H  =  transfection with PD-1H siRNA. Control pl  =  transfection with control plasmid; PD-1H pl  =  transfection with PD-1H expression plasmid.
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