Upregulation of PD-L1 on monocytes and dendritic cells by HIV-1 derived TLR ligands
Meier, Angelaa; Bagchi, Aranyab; Sidhu, Harlyn Ka; Alter, Galita; Suscovich, Todd Ja; Kavanagh, Daniel Ga; Streeck, Hendrika; Brockman, Mark Aa; LeGall, Sylviea; Hellman, Judithb; Altfeld, Marcusa
aPartners AIDS Research Center, Massachusetts General Hospital, Division of AIDS, Harvard Medical School, Boston, Massachusetts, USA
bDepartment of Anesthesia and Critical Care, Massachusetts General Hospital, Boston, Massachusetts, USA.
Received 22 August, 2007
Revised 21 November, 2007
Accepted 26 November, 2007
Increased PD-L1 expression has been reported in HIV-1-infected individuals, but the mechanisms leading to PD-L1 upregulation remain to be elucidated. Here we demonstrate that HIV-1-derived Toll-like receptor (TLR)7/8 ligands can induce MyD88-dependent upregulation of PD-L1 on plasmacytoid dendritic cells, myeloidic dendritic cells and monocytes. These data suggest a mechanism through which HIV-1-derived TLR ligands might contribute to the functional impairment of virus-specific PD-1-positive T cells by inducing the upregulation of PD-L1 on antigen-presenting cells.
Chronic viral infections are characterized by the persistence of antigen, leading to general immune activation and functional impairment of virus-specific T cells [1–6]. More recently, it has been demonstrated that these functionally impaired T cells upregulate PD-1 [1–4]. Interestingly, blocking the interaction of PD-1 with its ligand, PD-L1 (B7-H1) that is expressed on a variety of cell subsets including dendritic cells and monocytes, allowed PD-1-positive T cells to regain their proliferative capacity [1–4,7]. PD-L1 expression levels have been demonstrated to correlate directly with viral load in HIV-1-infected individuals . The mechanism by which persistent HIV-1 replication induces the upregulation of PD-L1 on antigen-presenting cells (APC) remains to be elucidated. Here we demonstrate that the upregulation of PD-L1 on dendritic cells and monocytes can be induced by HIV-1-derived TLR7/8 ligands [9,10] and is dependent on the TLR adaptor molecule MyD88.
Freshly isolated peripheral blood mononuclear cells (PBMC) from 15 HIV-1-negative individuals were stimulated with either a TLR9 ligand (ODN02216 5 μM; InvivoGen, San Diego, California, USA), synthetic TLR7/8 ligands CL097 (1 μg/ml; InvivoGen) and Imiquimod (1 μg/ml; InvivoGen), or HIV-1-derived TLR7/8 ligands (uridine-rich HIV-1-derived single-strand RNA sequences, 15 μg/ml), as described previously . The study was approved by the MGH Institutional Review Board, and each study subject gave informed written consent. After 20 h of stimulation with the respective TLR ligands, upregulation of PD-L1, CD80 and CD86 was assessed using seven-color flow-cytometry. PBMC were labelled with a CD86-Biotin antibody, washed and stained with an antibody-cocktail containing CD3/CD19/CD56 -Alexa 700, CD14-APC-Cy7, CD11c-APC, CD123-PeCy5, PD-L1-PeCy7, CD80-FITC (all BD Biosciences, San Jose, California, USA), and streptavidin-Cascade Yellow (Molecular Probes, Carlsbad, California, USA). After fixation in Fix&PermA (Caltag, Carlsbad, California, USA), samples were acquired on an LSRII flow cytometer. Unpaired two-tailed Student's t-tests were employed to assess the statistical significance of differences.
Incubation of human PBMC with the previously described HIV-1-derived uridine-rich ssRNA resulted in the upregulation of PD-L1 on APC, including monocytes (expressing TLR7 and TLR8), myeloidic dendritic cells (expressing TLR8) and, to a lesser extent, plasmacytoid dendritic cells (expressing TLR7; Fig. 1a). In contrast, no upregulation of PD-L1 was observed on either of these APC when cells were incubated with control oligonucleotides in which all uridines where replaced by adenosines (U-to-A variants; Fig. 1a), indicating that the HIV-1-derived ssRNA oligonucleotides were detected by TLR, as previously shown . This was further supported by the lack of PD-L1 upregulation on bone marrow-derived macrophages derived from mice deficient for MyD88 (MyD88KO), a crucial signalling protein for TLR7 and TLR8, whereas bone marrow-derived macrophages derived from wild-type mice upregulated PD-L1 in response to the HIV-1-derived TLR ligands (data not shown). In line with the results using HIV-1-encoded TLR7/8 ligands, the synthetic TLR7 and TLR7/8 agonists Imiquimod and CL097, and the TLR9 agonist ODN02216 also induced upregulation of PD-L1 on human APCs (Fig. 1a).
In addition to the upregulation of PD-L1, CD80 and CD86, two ligands for CTLA-4, were also significantly upregulated on plasmacytoid dendritic cells in response to the HIV-1-derived TLR ligands (as shown for CD80 in Fig. 1b). In contrast to this predominantly U variant-specific upregulation of CD80 and CD86 on plasmacytoid dendritic cells, myeloidic dendritic cells and monocytes also upregulated these two molecules, but not PD-L1, in response to control U-to-A oligonucleotides (Fig. 1b), indicating the presence of additional receptors other than TLR7/8 on those cells capable of sensing HIV-1-derived oligonucleotides.
In chronic HIV-1 infection, expression levels of PD-L1 (B7-H1) correlate directly with viral load levels , indicating a potential direct effect of the virus on PD-L1 expression. The mechanisms by which PD-L1 is upregulated in these settings are, however, not well understood. Here, we demonstrate for the first time that stimulation of dendritic cells and monocytes with viral TLR7/8 ligands, including HIV-1-derived ssRNA, can lead to the upregulation of PD-L1, providing a potential mechanism involved in the described upregulation of PD-L1 in chronic HIV-1 infection. This upregulation of PD-L1 by HIV-1-derived oligonucleotides was specific for the uridine-rich TLR7/8 ligands and depended on MyD88, whereas the U-to-A variants of these oligonucleotides also induced the upregulation of other co-stimulatory molecules including CD80 and CD86 on monocytes and myeloidic dendritic cells, but not plasmacytoid dendritic cells, indicating the presence of other RNA sensing receptors in these cells.
Viral infections, including lymphocytic choriomeningitis virus, hepatitis C virus and HIV-1, can lead to the upregulation of PD-1 expression on virus-specific T cells [1–4], sometimes starting during primary infection . The interaction of PD-1 with its ligand, PD-L1, results in the functional impairment of PD-1-positive virus-specific T cells . Most importantly, the blockade of this interaction between PD-1 and PD-L1 has been shown to reconstitute T-cell function [1,3,4], and to enhance the immune control of lymphocytic choriomeningitis virus viremia in vivo . Activation of dendritic cells or monocytes by TLR ligands can result in secondary activation of T cells in vitro , and pathogen-encoded TLR ligands have been proposed to contribute to the unspecific immune activation observed during chronic persistent infections, such as HIV-1 . The plasmacytoid dendritic cell response to viral infections might therefore have different effects on T-cell immunity at different stages of infection. While virus-encoded TLR ligands enhance the ability of plasmacytoid dendritic cells to prime virus-specific T cells in primary infection, persistent TLR-mediated stimulation of plasmacytoid dendritic cells in chronic infection might contribute to unspecific immune activation, whereas PD-L1 expression on plasmacytoid dendritic cells downmodulates the function of virus-specific effector T cells that preferentially express PD-1.
Further studies are needed to explore the regulatory network between pathogen-induced activation of APCs, resulting in unspecific T-cell activation, and the expression of inhibitory receptors/ligands such as PD-1 on virus-specific T-cell subsets. A better understanding of these pathways will be crucial for the design of immunotherapeutic interventions aimed at decreasing the unspecific hyperactivation of the immune system that has been strongly associated with HIV-1 immunopathology, while increasing virus-specific T-cell immunity that might contribute to the control of viral replication.
The authors would like to thank all patients participating in this study.
Sponsorship: This study was supported by the National Institutes of Health (1R21AI071806-01A2).
Conflicts of interest: None.
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