Tim-3 is a Marker of Plasmacytoid Dendritic Cell Dysfunction during HIV Infection and Is Associated with the Recruitment of IRF7 and p85 into Lysosomes and with the Submembrane Displacement of TLR9

Jordan Ari Schwartz, Kiera L. Clayton, Shariq Mujib, Hongliang Zhang, A. K. M. Nur-ur Rahman, Jun Liu, Feng Yun Yue, Erika Benko, Colin Kovacs and Mario A. Ostrowski
J Immunol April 15, 2017, 198 (8) 3181-3194; DOI: https://doi.org/10.4049/jimmunol.1601298

Abstract

In chronic diseases, such as HIV infection, plasmacytoid dendritic cells (pDCs) are rendered dysfunctional, as measured by their decreased capacity to produce IFN-α. In this study, we identified elevated levels of T cell Ig and mucin-domain containing molecule-3 (Tim-3)–expressing pDCs in the blood of HIV-infected donors. The frequency of Tim-3–expressing pDCs correlated inversely with CD4 T cell counts and positively with HIV viral loads. A lower frequency of pDCs expressing Tim-3 produced IFN-α or TNF-α in response to the TLR7 agonists imiquimod and Sendai virus and to the TLR9 agonist CpG. Thus, Tim-3 may serve as a biomarker of pDC dysfunction in HIV infection. The source and function of Tim-3 was investigated on enriched pDC populations from donors not infected with HIV. Tim-3 induction was achieved in response to viral and artificial stimuli, as well as exogenous IFN-α, and was PI3K dependent. Potent pDC-activating stimuli, such as CpG, imiquimod, and Sendai virus, induced the most Tim-3 expression and subsequent dysfunction. Small interfering RNA knockdown of Tim-3 increased IFN-α secretion in response to activation. Intracellular Tim-3, as measured by confocal microscopy, was dispersed throughout the cytoplasm prior to activation. Postactivation, Tim-3 accumulated at the plasma membrane and associated with disrupted TLR9 at the submembrane. Tim-3–expressing pDCs had reduced IRF7 levels. Furthermore, intracellular Tim-3 colocalized with p85 and IRF7 within LAMP1+ lysosomes, suggestive of a role in degradation. We conclude that Tim-3 is a biomarker of dysfunctional pDCs and may negatively regulate IFN-α, possibly through interference with TLR signaling and recruitment of IRF7 and p85 into lysosomes, enhancing their degradation.

Introduction

IFN-α is a key factor in the immune response to invading pathogens; it promotes immune readiness and restricts viral replication (1–3). Plasmacytoid dendritic cells (pDCs) account for 0.1–0.5% of all PBMCs and are the major IFN-α–producing cell (4, 5). Pathogen-associated ssRNA or unmethylated DNA recognized by endosomal TLR (TLR7 and TLR9, respectively) rapidly activate pDCs (6, 7). Agonistic activation of TLR7 or TLR9 triggers a bifurcating signaling cascade comprising the noncanonical NF-κB pathway and PI3K-dependent phosphorylation and nuclear translocation of IRF7, resulting in rapid production of TNF-α and IFN-α (4, 8–10).

Many cancers, autoimmune disorders, and chronic infections are associated with decreased responsiveness of pDCs to TLR agonists (5, 11–14). This dysfunction of the pDC response is well documented in the context of HIV type 1 infection (15–21). The role of pDCs during HIV infection is complex and not fully understood (5, 7). pDCs are the primary IFN-α–producing cell during HIV infection and contribute to the underlying immune activation driving disease progression (17, 22–25). pDC dysfunction is first observed in acute infection when the frequency of circulating pDCs in the periphery decreases, and those remaining in circulation exhibit a decreased capacity to produce IFN-α (15–19, 21, 26). pDC dysfunction persists into chronic HIV infection and correlates with disease-progression markers, such as decreasing CD4+ T cell counts and elevated viral loads (19, 20, 22, 27, 28). Combined antiretroviral therapy (cART) partially restores pDC numbers and function but not to preinfection levels (17, 26, 29). The causes of pDC dysfunction during HIV infection remain controversial. O’Brien et al. (30) suggested that HIV infection continually activates pDCs, leading to a persistently immature phenotype. Other investigators showed that HIV virions at physiologically relevant titers are poor pDC agonists (6, 31, 32), from which we infer that pDC dysfunction may have a different source. In an effort to better understand pDC dysfunction, we sought to identify and characterize a marker of dysfunction on pDCs during HIV infection.

Chiba et al. (33) reported that dysfunctional pDCs at tumor sites in a murine model expressed T cell Ig and mucin-domain containing molecule-3 (Tim-3). Tim-3 expression is found on a wide array of immune cells, including cells of the classical lymphoid and myeloid lineages, and it is involved in regulating numerous cellular functions (34). On CD4+ and CD8+ T cells, Tim-3 serves as a marker of activation and exhaustion and has been of recent interest as a target for immunotherapy (35, 36). Conversely, Tim-3 is constitutively expressed on resting macrophages and NK cells and is downregulated upon activation (37–39). Multiple mechanisms were proposed for Tim-3 to exert its function. In mouse DCs, Tim-3 selectively binds high mobility group box 1 (HMGB1) with high affinity, interfering with its ability to augment TLR activation (33). In human kidney cells, Tim-3 shuttles the survival factor NUR77 to autophagosomes (40), thus enhancing its degradation, whereas in mouse mast cells, Tim-3 perpetuates activation signals, enhancing their degranulation (41). Thus, Tim-3 function appears to be context and cell type specific. Recently, Tim-3 was identified on human pDCs at the Human Leukocyte Differentiation Antigen 10 workshop (42) but, to our knowledge, no study has investigated the regulation of function of Tim-3 in these specialized cells. Given the differences in expression profiles and functions of Tim-3 in different cell types, we undertook studies to interrogate the role of Tim-3 expression on human pDCs in the context of pDC activation and HIV infection.

Materials and Methods

Ethics statement

Written informed consent was obtained in accordance with the institutional ethics board guidelines for conducting clinical research at the University of Toronto, St. Michael’s Hospital, and Maple Leaf Clinic. Study approval was provided by the research ethics boards of the University of Toronto and St. Michael’s Hospital. Human samples were obtained by peripheral blood draw or leukapheresis at St. Michael’s Hospital, Maple Leaf Medical Clinic, or the University of Toronto by a nurse or certified phlebotomist. All participants were older than 18 y of age at the time of inclusion in this study.

Clinical study design

Human blood or leukapheresis samples were processed by Ficoll-Paque (GE Healthcare, Little Chalfont, U.K.) overlay and centrifugation at 500 × g for 30 min. Buffy coat layers were collected and washed twice in PBS by centrifugation for 5 min at 400 × g. PBMCs were processed for analysis directly (as described below) or were resuspended in complete R10 medium (RPMI 1640 + 10% FBS + penicillin + streptomycin, + l-glutamine; Thermo Fisher Scientific, Waltham, MA) and stimulated in vitro (as described below).

Viruses, agonists, cytokines, and IFN-α blocking Abs

HIV BaL was a generous gift from Dr. A. Cochrane (University of Toronto). pNL4-3 was obtained from the National Institutes of Health (NIH) AIDS Reagent Program and was transfected into the HEK 293T cell line (American Type Culture Collection, Manassas, VA) using Fugene HD (Promega, Madison, WI) transfection reagent. All strains of HIV were amplified in U87 CD4+CCR5+ or U87 CD4+CXCR4 cells (NIH AIDS Reagent Program), purified using a Fast-Trap Lentivirus Purification system (Millipore, Billerica, MA), as per the manufacturer’s instructions, and stored in R10 at −80°C until use. The concentration of each HIV strain was measured by p24 ELISA (Leidos, Frederick, MD). HIV was used at a concentration of 10 ng/ml of p24. Influenza A PR/8/34 (H1N1) (Charles River, North Franklin, CT) was used at a multiplicity of infection = 0.5. Sendai virus Cantell strain (Charles River) was used at 18 hemagglutinin units/ml. Imiquimod (R837) was used at 2.5 μg/ml, and CpG (ODN2216) was used at 12.5 μg/ml (InvivoGen, San Diego, CA). Other reagents used in culture included recombinant IFN-α2a (PBL Assay Science, Piscataway, NJ), TNF-α, IL-6 (R&D Systems, Minneapolis, MN), and IL-1β (BioLegend, San Diego, CA). Blockade of IFNAR was performed using IFNAR2 blocking Ab (10 μg/ml; PBL Assay Science).

Isolation of pDCs

PBMCs, obtained as described above, were used for isolation of pDCs by negative selection (Miltenyi Biotec, Bergisch Gladbach, Germany), as per the manufacturer’s instructions. A second application to the column was required to reach purity > 90%, as determined by flow cytometry identifying pDCs as BDCA2+CD123+. pDCs were suspended in complete R10 medium supplemented with 20 ng/ml IL-3 (R&D Systems).

In vitro stimulation

Enriched pDCs from HIV− donors were cultured in complete R10 medium supplemented with 20 ng/ml IL-3 (R&D Systems), at a concentration of 2 × 104 cells per well, in a 96-well flat-bottom plate in a final volume of 200 μl in a humidified incubator at 37°C with 5% CO2. PBMCs were cultured in complete R10 medium supplemented with 20 ng/ml IL-3 at 2 × 106 cells per well in 500 μl in a 24-well plate in a humidified incubator at 37°C with 5% CO2. Brefeldin A (GolgiPlug; BD Biosciences, Franklin Lakes, NJ) was added to the culture 6 h prior to the end of the incubation period at 1 μg/ml to retain cytokines. Then samples were prepared for flow cytometry.

Inhibitor experiments

To test the effect of molecular pathways on Tim-3 expression, enriched pDCs from donors not infected with HIV were incubated in vitro in the presence of IFN-α or imiquimod plus R406 (Selleck Chemicals, Houston, TX) at 1 μM, LY 294002 (Sigma-Aldrich, St. Louis, MO) at 1 μM, SB 203508 (Sigma-Aldrich) at 1 μM, Torin 1 (Tocris Bioscience) at 1 μM, ruxolitinib (Cayman Chemical, Ann Arbor, MI) at 500 nM or DMSO (Sigma-Aldrich) to the maximal concentration for 24 h. Then cells were prepared for flow cytometry.

Tim-3 siRNA

Freshly isolated pDCs were treated with small interfering RNA (siRNA) as previously described by Smith et al. (43). Briefly, freshly isolated pDCs were seeded at 1 × 105 pDCs per 100 μl in a 96-well flat-bottom plate. Transfection mix was prepared using three 20-bp Tim-3–targeting siRNA sequences starting at mRNA nt 201, 348, and 640 (accession number NM_032782 [http://www.ncbi.nlm.nih.gov/genbank]) (prepared by GenePharma, Shanghai, China). A nonspecific scramble siRNA sequence was prepared as a control at equivalent concentrations. Starting from 20 μM siRNA stock, 3.2 μM siRNA mixture diluted in PBS was mixed 50:50 with DOTAP Liposomal Transfection Reagent (Roche, Mannheim, Germany) and incubated at room temperature for 15 min. siRNA mixture was applied to pDCs at a final concentration of 160 nM siRNA. siRNA-treated pDCs were incubated at 37°C for 24 h. pDCs were then seeded at 2 × 104 pDCs per 200 μl in the presence of imiquimod or medium alone for an additional 24 h, followed by the collection of supernatant for quantification of IFN-α by ELISA (Mabtech, Cincinnati, OH) and preparation of FACS for analysis of Tim-3 knockdown.

Anti–Tim-3 Ab experiments

To test the effects of anti–Tim-3 Abs on pDC function, enriched pDCs from donors not infected with HIV were cultured in a 96-well plate and incubated in vitro for 20 h with imiquimod at 37°C. Samples were washed three times in R10 warmed to 37°C and seeded in a new 96-well flat-bottom plate. In experiments in which cross-linking was performed, the new 96-well flat-bottom plate was coated overnight with PBS supplemented with 10 μg/ml anti–Tim-3 Ab clone 344801 (R&D Systems) or F38-2E2 (BioLegend) or isotype Ab Rat IgG1 (R&D Systems) or Mouse IgG1 (BioLegend) for 18 h at 4°C, followed by one round of washing with PBS. The Ab-coated plate was warmed to 37°C, and previously activated pDCs were added to the plate for 30 min, followed by 8 h of stimulation with Sendai virus. In experiments in which soluble anti–Tim-3 Ab treatment was used, previously activated pDCs were seeded in a new 96-well flat-bottom plate, and 10 μg/ml anti–Tim-3 Ab clone 344823 (R&D Systems) or F38-2E2 (BioLegend) or Ab Rat IgG2A Isotype Control (R&D Systems) or Mouse IgG1 (BioLegend) was added to the culture for 30 min, followed by 8 h of stimulation in the presence of Sendai virus. GolgiPlug was added for the final 6 h of stimulation. pDCs were prepared for intracellular staining (ICS), as described below.

Preparation of PBMCs and pDCs for flow cytometry

PBMCs or pDCs were washed once in chilled PBS and incubated in the dark at room temperature with PBS containing LIVE/DEAD Violet for 10 min (Thermo Fisher Scientific), followed by a wash, and blocking in PBS containing 2% FBS for 10 min. Surface staining was performed using BDCA2 FITC (Miltenyi Biotec), CD123 PE-Cy7, CD86 PerCP-Cy5.5 (BioLegend), or Tim-3 PE (R&D Systems) in PBS containing 2% FBS buffer at 4°C for 20 min. Cells were washed twice; if no ICS was required, the cells were resuspended in 1% formalin. For ICS, cells were incubated in BD Cytofix/Cytoperm solution (BD Biosciences) for 10 min at 4°C. Cells were washed twice with BD Perm/Wash Buffer and stained for IFN-α allophycocyanin (Miltenyi Biotec), TNF-α BV650, or PE (BioLegend) in BD Perm/Wash for 20 min at 4°C. Cells were washed two more times with BD Perm/Wash, followed by resuspension in PBS. Analysis for the clinical prospective study was performed using a BD FACSCanto II. All other samples were acquired on a BD Fortessa, and data were analyzed using FlowJo v10.0.8r1 software (TreeStar).

Confocal microscopy

The confocal technique was adapted from Clayton et al. (44). Briefly, following stimulation, pDCs were affixed onto coverslips using poly-l-lysine. pDCs were fixed in 4% methanol-free paraformaldehyde (Thermo Fisher Scientific) and washed twice with PBS before being permeabilized with ice-cold 100% methanol. Slides were blocked using 10% BSA and washed three times prior to the addition of goat anti-human Tim-3 polyclonal Ab (R&D Systems), rabbit anti-human IRF7 Ab (Santa Cruz Biotechnology, Dallas, TX), rabbit anti-human PI3K p85 Ab (Thermo Fisher Scientific), mouse anti-human TLR9 Ab (Thermo Fisher Scientific), mouse anti-human LAMP1 Ab (Santa Cruz Biotechnology), or mouse anti-human EEA1 Ab (BD Biosystems) in a 3% BSA solution. After three washes, Alexa Fluor 488 anti-goat IgG Ab, Alexa Fluor 594 anti-rabbit IgG Ab, and Alexa Fluor 647 anti-mouse IgG Ab were applied in 3% BSA solution. Slides were washed with DAPI (Sigma-Aldrich) to stain the nuclei. Cells were mounted in Pro-Long Gold Antifade Mountant (Thermo Fisher Scientific). Full three-dimensional z-stacks were collected for each slide on a Zeiss LSM 700 microscope using a 60× 1.4 NA oil immersion lens. Images were made using Fiji (ImageJ version 2.0.0), and colocalization was assessed using Imaris version 8.3 (Bitplane).

Statistical analysis

All graphs and statistics were generated using GraphPad Prism 6 (GraphPad, La Jolla, CA).

Results

Elevated Tim-3 expression on pDCs from untreated HIV-infected donors correlates with disease progression

We asked whether Tim-3, which was identified on dysfunctional murine pDCs in tumor models (33), is expressed on dysfunctional human pDCs during HIV infection. Ex vivo PBMCs were examined in 8 uninfected volunteers (HIV−) and 26 HIV-infected donors: 14 were untreated (HIV+Rx−), and 12 were receiving cART (HIV+Rx+) (Table I). pDCs were defined by their high expression of BDCA2 and CD123 (Fig. 1A). Similar to what was reported previously (15, 18, 20, 21, 26, 27, 45, 46), untreated HIV infection was associated with a reduced frequency of circulating pDCs, whereas cART partially restored levels similar to those observed in uninfected controls (median pDC frequency [interquartile range (IQR)] for HIV− = 0.2850% of total PBMCs [0.2350–0.4550], HIV+Rx− = 0.1950% of total PBMCs [0.1168–0.2875], HIV+Rx+ = 0.2550% of total PBMCs [0.1425–0.2950]) (Fig. 1B). Untreated HIV infection was associated with an elevated frequency of Tim-3–expressing pDCs, which was decreased in donors undergoing cART (median percentage of pDC Tim-3+ [IQR] for HIV− = 17.45% [15.05–22.58], HIV+Rx− = 40.15% [34.28–44.58], HIV+Rx+ = 32.45% [15.43–39.25]) (Fig. 1C, 1D). The frequency of Tim-3 expression on pDCs from HIV+Rx− volunteers correlated negatively with CD4 count (r = −0.5736, p = 0.0359) and positively with viral loads (r = +0.7802, p = 0.0025) (Fig. 1E, 1F). We performed a secondary analysis based on the frequency of Tim-3 expression and identified two statistical outliers. These statistical outliers had the highest frequency of Tim-3 expression and were among the donors with the lowest CD4 count and highest viral load. Exclusion of the outliers from our analysis abrogated the correlation between CD4 count and frequency of Tim-3 expression and lowered the correlation between viral load and Tim-3 (r = +0.6545, p = 0.0336).

View this table:
Table I. Donor characteristics for clinical study
FIGURE 1.
FIGURE 1.

HIV infection is associated with elevated expression of Tim-3 on pDCs. Blood samples were collected from 26 HIV-infected donors and 8 uninfected controls. PBMCs were stained for ex vivo analysis by flow cytometry. pDCs were defined as BDCA2+CD123+LIVE/DEAD Violet−. (A) Representative dot plots showing how pDCs were identified. Donors were stratified into one of three groups based on infection and treatment status: HIV−, HIV+Rx−, and HIV+Rx+. (B) The frequency of ex vivo PBMCs identified as pDCs are shown for each donor. (C) Dot plots for the quantification of Tim-3+ pDCs are shown for one representative donor for each group. (D) Summary of Tim-3 expression for all donors. Expression of Tim-3 on gated pDCs of HIV+Rx− donors plotted against CD4 count (E) and viral load (F) for donors with detectable virus. (G) Ex vivo PBMCs were stimulated in vitro for 8 h in the presence of CpG, imiquimod, or Sendai virus, and IFN-α and TNF-α production was measured by ICS. Statistical analysis for comparisons between groups in (B) and (D) was performed using the Mann–Whitney U test and two-way ANOVA (G). Error bars represent median and IQR in (B), (D), and (G). Correlation in (E) and (F) was measured by the Spearman correlation test, showing the linear regression line of best fit. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Tim-3+ pDCs from HIV-infected donors exhibit deficiencies in IFN-α production

Ex vivo PBMCs were stimulated in vitro, and pDC cytokine production was examined by ICS. Similar to previous reports (15–20), pDCs from HIV-infected donors displayed significant dysfunction. A lower percentage of pDCs from HIV-infected individuals produced IFN-α and TNF-α in response to TLR7 agonists, imiquimod, and Sendai virus compared with uninfected controls but not in response to the TLR9 ligand ODN2216 (CpG) (Fig. 1G).

Next, we sought to understand how Tim-3 expression relates to production of IFN-α or TNF-α from pDCs. Ex vivo pDCs from untreated HIV-infected individuals were stimulated in vitro with CpG, imiquimod, or Sendai virus and assessed for function and activation (Fig. 2A). We observed that Tim-3− pDCs produced IFN-α, in response to all stimuli, at a higher frequency than did Tim-3+ pDCs (Fig. 2B). Because Tim-3 is considered an activation marker, we assessed it alongside another pDC activation marker, CD86. In this case, pDCs demonstrated higher IFN-α production from CD86− pDCs if stimulated with TLR7, but not TLR9, agonists, whereas Tim-3+ pDCs made less IFN-α with all stimuli. In contrast to IFN-α, Tim-3+ cells produced TNF-α at the same levels as Tim-3− cells.

FIGURE 2.
FIGURE 2.

Tim-3 expression is associated with a decreased frequency of IFN-α– and TNF-α–producing pDCs from HIV-infected donors. PBMCs from HIV-infected donors not receiving cART were incubated in vitro for 8 h in the presence of CpG, imiquimod, or Sendai virus. (A) Dot plots show a representative experiment. BDCA2+CD123+LIVE/DEAD Violet− gated pDCs were evaluated for production of IFN-α and TNF-α versus Tim-3 or CD86. (B) Graphs summarizing data from five donors where BDCA2+CD123+LIVE/DEAD Violet− pDCs were compared based on surface markers. In the left panels, pDCs were split into two groups (Tim-3+ and Tim-3−). In the right panels, pDCs were split into two groups (CD86+ and CD86−). The frequency of IFN-α– or TNF-α–producing pDCs was measured independently within each group for their response to each stimulus. *p < 0.05, **p < 0.01, paired t test.

Tim-3 is upregulated on pDCs following TLR stimulation

To determine the effect of activation on Tim-3 regulation, pDCs from individuals not infected with HIV were cultured for 24 h in the presence of HIV BaL, HIV NL4-3, influenza PR8, CpG, imiquimod, or Sendai virus. Influenza PR8, CpG, imiquimod, and Sendai virus significantly increased the percentage of pDCs expressing Tim-3 and their median fluorescent intensity (MFI) of Tim-3 compared with the medium control (Fig. 3A). HIV BaL and HIV NL4-3 did not enhance Tim-3 expression compared with the medium-only control. To define the kinetics of the pDC Tim-3/cytokine response, pDCs were analyzed 6, 12, 18, 24, and 48 h after stimulation. CpG is shown as a representative stimulus in Fig. 3B and had similar activation dynamics to imiquimod and Sendai virus stimulation; however, all stimuli had varying temporal kinetics (Fig. 3C). Temporally, TNF-α was produced before IFN-α. The bulk of IFN-α produced in response to CpG, imiquimod, and Sendai virus seen after 6 h of exposure was from pDCs coproducing TNF-α, a trend similar to what was reported previously (47). At the 12- and 18-h time points for those three stimuli, there is a shift toward IFN-α production independent of TNF-α. IFN-α production declines between 18 and 48 h postactivation with CpG, imiquimod, and Sendai virus, preceding the enhancement of Tim-3 expression. Notably, there was a strong correlation between the maximum percentage of IFN-α– or TNF-α–producing pDCs and Tim-3 expression observed at 48 h after activation (Fig. 3D).

FIGURE 3.
FIGURE 3.

pDC-activation kinetics reveal Tim-3 is an activation marker expressed after IFN-α and TNF-α production. pDCs obtained through negative selection from fresh PBMCs isolated from volunteers not infected with HIV were stimulated in vitro for 24 h to investigate the relationship between Tim-3 and activation in response to HIV BaL, HIV NL4-3, influenza PR8, CpG, imiquimod, or Sendai virus. (A) Summary of pooled flow cytometry data from six independent experiments, with each treatment represented at least three times. Percentage of pDCs positive for Tim-3 (upper panel). The MFI induced by each treatment (lower panel). (B and C) The dynamics of Tim-3 expression was assessed alongside IFN-α and TNF-α production using flow cytometry in a time-course experiment with samples collected at 6, 12, 18, 24, and 48 h poststimulation. (B) Representative flow plots for CpG stimulation. (C) Summary of results from a representative time-course experiment for each treatment in which IFN-α, TNF-α, and Tim-3 were evaluated. Data points represent the mean value for each treatment, and error bars display the SD of the triplicate samples. (D) Maximum values for IFN-α and TNF-α production observed from two time-course experiments were graphed against maximum Tim-3 expression obtained for each treatment. Statistical analyses were performed using the paired t test to compare each group with the medium-alone treatment (A) or Pearson correlation analysis (D). *p < 0.05, **p < 0.01.

IFN-α contributes to the upregulation of Tim-3 but not CD86

Because Tim-3 expression was typically preceded by a cycle of cytokine production, we hypothesized that pDC-generated cytokines may induce Tim-3 expression directly. We screened a panel of typical cytokines produced by pDCs, including IFN-α, TNF-α, IL-1β, and IL-6. When IFN-α was added to the culture, the percentage of Tim-3–expressing pDCs increased to 49.3 ± 4.24% compared with 24.85 ± 2.33% with culture medium alone (Fig. 4A). No other cytokines tested had any effect on Tim-3 expression. As little as 0.1 ng/ml IFN-α was sufficient to induce a significant change in Tim-3 expression (Fig. 4B). Notably, none of the cytokines tested significantly affected the expression of CD86. In a subsequent series of experiments, pDCs were activated by CpG, imiquimod, or Sendai virus in the presence of an IFN-α receptor (IFNAR)-blocking Ab to confirm a role for IFN-α feedback in Tim-3 induction in pDCs. Anti-IFNAR Abs significantly reduced the percentage and MFI of Tim-3 expression on pDCs in response to CpG, imiquimod, and Sendai virus stimulation (Fig. 4C). Anti-IFNAR Ab had the opposite effect on CD86, with increased intensity of CD86 expression on pDCs in response to CpG; however, it had no other effect on CD86 percentage or intensity in response to any other stimulus tested.

FIGURE 4.
FIGURE 4.

IFN-α contributes to elevated Tim-3 expression. (A) pDCs obtained through negative selection of fresh PBMCs isolated from a donor not infected with HIV were cultured and stimulated in the presence of IFN-α, TNF-α, IL-1β, or IL-6 at 10 ng/ml or imiquimod positive control for 24 h and screened for Tim-3 and CD86 expression by flow cytometry. (B) Exogenous IFN-α was titrated to determine the effects of IFN-α on Tim-3 expression. (C) IFNAR-blocking Ab (10 μg/ml) was used to determine the effects of IFNAR signaling on Tim-3 or CD86 expression in response to CpG, imiquimod, and Sendai virus stimulation. Tim-3 and CD86 expression is shown based on their frequency on pDCs (upper panels) and MFI (lower panels). Error bars represent SD. Treatments were performed in triplicates. Each graph shows a single representative experiment from at least two independent experiments. Statistical analyses in (A) and (B) were performed using one-way ANOVA with the Bonferroni multiple-comparison test to compare each group with the medium-alone treatment. (C) Two-way ANOVA with the Bonferroni multiple-comparison test was used to compare the effects of treatment on each stimulus. *p < 0.05, **p < 0.01, ****p < 0.0001.

To further interrogate the regulation of Tim-3 expression on pDCs, we used a panel of small molecule inhibitors that target signaling pathways known to regulate pDC function: PI3K/mammalian target of rapamycin, MAPK, spleen tyrosine kinase, and JAK1/JAK2. pDCs were incubated with imiquimod or IFN-α for 24 h in the presence or absence of specific pathway inhibitors and examined for Tim-3 and CD86 expression. A reduction in the percentage of Tim-3–expressing pDCs in response to imiquimod and IFN-α was detected by inhibiting JAK1/JAK2 using ruxolitinib, and PI3K using LY294002 (Fig. 5). None of the inhibitors tested affected the expression of CD86 in response to imiquimod stimulation.

FIGURE 5.
FIGURE 5.

JAK and PI3K pathways regulate the expression of Tim-3. pDCs obtained through negative selection of PBMCs isolated from volunteers not infected with HIV were stimulated with imiquimod (A) or IFN-α (10 ng/ml) (B) in the presence of DMSO, R406, LY 294002, Torin 1, SB 203580, or ruxolitinib for 24 h. The value obtained from each inhibitor is reported relative to the DMSO control. Each inhibitor was used in three independent experiments, and the results of each experiment were pooled together. *p < 0.05, **p < 0.01, one-way ANOVA with the Bonferroni multiple-comparison, each group versus DMSO control.

Tim-3–expressing pDCs are refractory to restimulation

Given the preceding data, we hypothesized that Tim-3 expression may mark a population of pDCs that are refractory to further stimulation. pDCs from uninfected participants were stimulated with CpG, imiquimod, or Sendai virus for 20 h, washed, and subjected to a second (8-h) round of stimulation with CpG, imiquimod, or Sendai virus. Representative data are shown for pDCs following the second restimulation (Fig. 6); data are organized by rows, which represent the stimuli used for the first stimulation, and columns, which represent the second stimulation. We observed that the more Tim-3 expressed after the first stimulation, the less IFN-α is induced from the second stimulus. This was true for all treatment combinations examined. For example, if pDCs are stimulated first with imiquimod, Tim-3 expression increased to 78.19% (third row, first dot plot in Fig. 6), and the maximum percentage of IFN-α–producing pDCs observed after a second stimulation with Sendai virus was 4.39%. In contrast, 10.92% of the unstimulated pDCs express Tim-3, and subsequent stimulation with Sendai virus resulted in 23.07% of the pDCs producing IFN-α.

FIGURE 6.
FIGURE 6.

Tim-3–expressing pDCs produce less IFN-α upon secondary stimulation. pDCs obtained through negative selection of fresh PBMCs isolated from a donor not infected with HIV were stimulated in the presence of CpG, imiquimod, Sendai virus, or medium alone for 20 h. Then pDCs were washed twice with warmed R10, stimulated a second time (8 h) with CpG, imiquimod, Sendai virus, or medium alone, and analyzed for Tim-3, IFN-α, and TNF-α by flow cytometry. A representative experiment of three independent experiments is shown. Dot plots are shown following the second round of stimulation. The flow panels are sorted with the rows representing the first round of stimulation and the columns representing the second round of stimulation. Each treatment was performed in triplicate and in three independent experiments.

Knockdown of Tim-3 is associated with increased IFN-α secretion

We posited that if Tim-3 plays a role in pDC dysfunction, then knocking it down should reverse dysfunction. We used the liposome-based transfection reagent DOTAP to deliver Tim-3 siRNA or scramble siRNA control. Tim-3 siRNA partially knocked down Tim-3 in the presence of imiquimod stimulation (Fig. 7A). IFN-α secretion in response to imiquimod was increased in Tim-3 siRNA–treated pDCs compared with those treated with scramble siRNA (60,209 ± 12,602 and 38,886 ± 6976 pg/ml IFN-α, respectively, *p > 0.05) (Fig. 7B).

FIGURE 7.
FIGURE 7.

Tim-3 siRNA knockdown increases the secretion of IFN-α from pDCs. pDCs obtained through negative selection of PBMCs from volunteers not infected with HIV were cultured in the presence of DOTAP-encapsulated siRNA for 20 h at a density of 1 × 106 pDC per milliliter. pDCs were then seeded at 2 × 104 pDCs per 200 μl and treated with imiquimod or medium alone for 24 h. (A) Tim-3 expression was measured by flow cytometry after siRNA treatment and subsequent stimulation with imiquimod and is shown in a representative graph in which scramble siRNA (dashed gray line), Tim-3 siRNA (black line), and fluorescence minus one control (light gray line) are shown for a representative experiment. Graphical analyses of the frequency of pDCs expressing Tim-3 (lower left panel) and MFI of Tim-3 expression (lower right panel) are shown for a representative donor. (B) IFN-α secretion was detected by ELISA. Each treatment was performed in triplicate in three independent experiments. Error bars represent the SD. *p < 0.05, ***p < 0.001, two-way ANOVA with the Bonferroni multiple-comparison test.

Anti–Tim-3 Ab has no effect on pDC activation when delivered in the presence of stimuli

Next we investigated a potential mechanism of action for the function of Tim-3. We theorized that Abs targeting Tim-3 should have measureable functional consequences on activation. Previous studies showed that Tim-3 cross-linking via certain Abs modulates the activation of T cells, NK cells, monocytes, and DCs (33, 35, 37, 39). For example, cross-linking Tim-3 on NK cells with the 344801 (801) or 344823 (823) anti–Tim-3 Ab inhibits NK cell function, presumably through Tim-3 signaling (39). We examined whether Tim-3 Abs would restore IFN-α or TNF-α production. pDCs were cultured in a 96-well flat-bottom plate and stimulated with imiquimod for 20 h to upregulate Tim-3. pDCs were then treated with anti–Tim-3 Abs, either by seeding them in a new 96-well flat-bottom plate coated with 801, which is known to induce cross-linking stimulation, or by transferring them to a 96-well flat-bottom plate with medium supplemented with 823. Activated anti–Tim-3 Ab–treated pDCs were further stimulated with Sendai virus for 8 h. We observed no consistent effect from anti–Tim-3 Abs 801 or 823 (Fig. 8) or clone F38-2E2 (data not shown) on IFN-α or TNF-α.

FIGURE 8.
FIGURE 8.

Anti–Tim-3 Ab provided in the soluble or cross-linking form has no effect on IFN-α and TNF-α production in the presence of activation. pDCs obtained through negative selection from fresh PBMCs isolated from donors not infected with HIV were used to assess the effect of Tim-3 Abs on IFN-α and TNF-α production. pDCs were first stimulated with imiquimod for 20 h. (A) For cross-linking anti–Tim-3 Ab treatments, activated pDCs were reseeded in a 96-well flat-bottom plate coated overnight with 10 μg/ml of 801 at 4°C. (B) For soluble anti–Tim-3 Ab treatments, activated pDCs were reseeded in a new 96-well flat-bottom plate and treated with 10 μg/ml 823. (A and B) pDCs were rested in the presence of Ab for 30 min and then Sendai virus was added to the culture for an additional 8-h stimulation. IFN-α and TNF-α production were measured by flow cytometry at 8 h following the second stimulation. Each treatment was performed in duplicate or triplicate and in at least two independent experiments; a representative experiment is shown. Error bars represent SD. Statistical analysis was performed by two-way ANOVA and the Bonferroni multiple-comparison test to compare differences between groups.

Tim-3 associates with p85 and IRF7 within LAMP1+ lysosomes and areas of displaced TLR9

We demonstrated no effect on pDC activation or function from cross-linking or soluble Abs to Tim-3. Thus, we investigated the potential for Tim-3 to function intracellularly. Chiba et al. (33) found that Tim-3 colocalized with HMGB1, which they presumed would interfere with TLR signaling. We used confocal microscopy to visualize Tim-3 and TLR9 intracellularly. Prior to activation, Tim-3 expression was low and dispersed throughout the cytoplasm, whereas TLR9 staining was uniform, forming a ring encircling the cell around the submembrane region. Following activation with imiquimod, Tim-3 staining accumulates on the plasma membrane, and submembrane TLR9 staining becomes discontinuous proximal to regions of high Tim-3 expression, giving the appearance of displacement (Figs. 9A, 10A). When colocalization of Tim-3 and TLR9 was measured, we found a significant decrease in overlap, consistent with displacement (Fig. 10B).

FIGURE 9.
FIGURE 9.

Tim-3 displaces TLR9 and colocalizes with p85 and IRF7. pDCs isolated through negative selection from the PBMCs of donors not infected with HIV were stimulated with imiquimod for 24 h. (A) Tim-3 (green), TLR9 (red), and nuclear DAPI stain (blue) were visualized by confocal microscopy (original magnification ×640). Merged images contain Tim-3, TLR9, and DAPI. Enlarged images in boxed areas show Tim-3 (upper right panel) and TLR9 (lower right panel) at 24 h postactivation at approximately 2.5 times the original magnification. Tim-3 also was assessed with p85 (B) and IRF7 (C) at 12 and 24 h postimiquimod stimulation using confocal microscopy (original magnification ×640). Scale bar, 10 μm. Arrowheads indicate points of interest shared between two images. Representative images were selected from a minimum of seven three-dimensional z-stacks per condition and are representative of two independent experiments.

FIGURE 10.
FIGURE 10.

Intensity and colocalization analysis of Tim-3, TLR9, p85, and IRF7. Confocal microscopy data presented in Fig. 9 were analyzed quantitatively to evaluate the staining intensity of Tim-3 (A), p85 (C), and IRF7 (D) for untreated samples, as well as at 12 and 24 h postimiquimod stimulation. Colocalization between Tim-3 and TLR9 (B), Tim-3 and p85 (C), and Tim-3 and IRF7 (D) was measured by Pearson’s colocalization test for untreated samples, as well as at 12 and 24 h postimiquimod stimulation. Graphs are shown for a representative experiment with a minimum of 15 cells per condition. The data represent analysis of full three-dimensional z-stacks. Box error bars represent minimum and maximum. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA and the Bonferroni multiple-comparison test were used to compare differences between groups (A, C, and D); Student t test (B).

Tim-3 was shown to interact with the p85 subunit of PI3K (48), which regulates IRF7 and IFN-α production (10). In resting pDCs, p85 was found along the cell membrane, in the cytoplasm, and within the nucleus. (Fig. 9B). At 24 h after imiquimod stimulation, p85 was mainly cytoplasmic and showed greater colocalization with Tim-3 (Fig. 10C). Given the observed changes in the p85 staining profile following activation, we similarly investigated the effects of activation on IRF7. Prior to activation, IRF7 staining was found throughout the cytoplasm and was excluded from the nucleus. At 12 h after activation with imiquimod, IRF7 was within the cytoplasm and the nucleus, and the intensity of staining decreased (Figs. 9C, 10D). Tim-3 colocalized with IRF7 before and after activation.

pDCs are endocytotic cells, so we questioned whether Tim-3 had any relationship with endocytosis pathways. Tim-3 demonstrated minimal localization to the early endosomal marker EEA1 (Fig. 11A, Supplemental Fig. 1). However, Tim-3 exhibited a relationship with the lysosomal marker LAMP1. We saw triple colocalization of Tim-3, LAMP1, and p85 or IRF7, indicating the presence of p85 and IRF7 with Tim-3 in the lysosomes (Fig. 11B, 11C, bottom panels).

FIGURE 11.
FIGURE 11.

Tim-3, p85, and IRF7 colocalize with LAMP1. pDCs isolated through negative selection from the PBMCs of donors not infected with HIV were visualized by confocal microscopy (original magnification ×640). (A) pDCs stimulated for 24 h with imiquimod were stained for Tim-3 (green), EEA1 (red, left panels) or LAMP1 (red, right panels). (B) pDCs stimulated for 24 h with imiquimod are shown (upper panels) with p85 (green), EEA1 (red, left panels), or LAMP1 (red, right panels) in separate panels or merged together. Lower panels: Tim-3 (green), p85 (red), and LAMP1 (blue) are shown in separate panels and merged together. (C) pDCs stimulated for 12 h with imiquimod are shown (upper panels) with IRF7 (green), EEA1 (red, left panels), or LAMP1 (red, right panels) in separate panels and merged together. Lower panels: Tim-3 (green), IRF7 (red), and LAMP1 (blue) are shown in separate panels and merged together. Scale bars, 10 μm. Representative images were selected from a minimum of seven three-dimensional z-stacks per condition and are representative of two independent experiments.

To determine the relationship between surface and intracellular Tim-3, pDCs were stimulated for 12 h with imiquimod and pulsed with a Tim-3 Ab for 30 or 60 min prior to preparation for confocal microscopy. Tim-3 rapidly colocalized with LAMP1+ lysosomes (colocalization score 30-min pulse: 0.5694 ± 0.0997, 60-min pulse: 0.6217 ± 0.0512) but not with EEA1+ endosomes (colocalization score 30-min pulse: 0.3676 ± 0.0665, 60-min pulse: 0.3529 ± 0.04749) (Fig. 12). Internalized Tim-3 demonstrated colocalization with IRF7 (colocalization score 30-min pulse: 0.6031 ± 0.04214, 60-min pulse: 0.6115 ± 0.0628). Thus, surface Tim-3 may become internalized and facilitate the degradation of IRF7.

FIGURE 12.
FIGURE 12.

Surface Tim-3 internalizes and colocalizes with IRF7 and LAMP1. pDCs isolated through negative selection from the PBMCs of donors not infected with HIV were stimulated for 12 h with imiquimod and pulsed for 30 and 60 min prior to the end of stimulation with anti-Tim-3 Ab. Confocal microscopy was used to visualize the expression of Tim-3 (green), IRF7 (red), and EEA1 (blue-top) or LAMP1 (blue-bottom) from pDCs (original magnification ×640) (upper panels). Scale bars, 10 μm. Representative images were selected from a minimum of six three-dimensional z-stacks per condition and are representative of two independent experiments. Summary graphs are shown for a single experiment performed in duplicate (lower panels). Colocalization scores were determined using the Pearson colocalization test. Statistical analysis was performed by Student t test.

Discussion

In this study, we investigated Tim-3, a marker typically associated with T cell exhaustion (35, 36), as a possible biomarker of dysfunction. A higher frequency of pDCs from HIV-infected donors, which were dysfunctional in response to TLR and virus stimulation, expressed Tim-3. Thus, Tim-3 served as reliable biomarker of pDC dysfunction. Tim-3 was inducible by in vitro activation via TLR9 or TLR7 agonists. In vitro–activated pDCs expressing Tim-3 were defective in IFN-α and TNF-α production. Of note, the strength of the pDC stimulus directly correlated with greater Tim-3 expression and subsequent dysfunction. Surprisingly, exogenous HIV virions did not induce much Tim-3 pDC expression, which correlated with the poor ability of exogenous HIV virions to stimulate pDCs. This indicates that, in HIV-infected individuals, HIV may not be directly inducing Tim-3 on pDCs, although previous work showed that cell-associated HIV is a more potent inducer of IFN-α (31, 49). Thus, cell-associated HIV might be an activation stimulus for Tim-3 induction in vivo. There was a strong association between HIV viral load and Tim-3 expression. It is possible that with chronic HIV infection and declining CD4 counts, the associated enhanced susceptibility to other virus infections could also be a pDC-activating stimulus.

pDC dysfunction was defined in our study cohorts using in vitro activation of PBMCs and direct detection of cytokine production by ICS (17). This technique allowed for the detection of Tim-3 and cytokine production on a single-cell level. There was evidence of pDC dysfunction in HIV-infected donors in response to TLR7 agonists, but dysfunction was not observed in response to the TLR9 agonist, CpG. Previous reports of pDC dysfunction in HIV-infected individuals in response to TLR9 agonists were interpreted with experiments that used HSV (15, 26), whereas studies using CpG stimulation as the TLR9 agonist, similar to ours, did not demonstrate significant dysfunction (16, 21). Future experiments to clarify this discrepancy are warranted.

To our knowledge, this is the first study to comprehensively investigate Tim-3 on pDCs. Chiba et al. (33), using a murine model, showed that some tumor-associated pDCs express Tim-3; however, they did not investigate the regulation of Tim-3 induction or its effects on cytokine production at the protein level. In humans, we found that Tim-3 was inducible on pDCs by agonistic TLR stimulation or through addition of exogenous IFN-α. Induction of Tim-3 by agonistic TLR stimulation was preceded by a cycle of activation characterized by a dynamic relationship between TNF-α and IFN-α production in which TNF-α was produced by pDCs before IFN-α. Tim-3 was upregulated after TNF-α and IFN-α production had peaked, indicating that it is upregulated later in activation. Tim-3 was inducible by IFN-α but not TNF-α. Both TLR and IFN-α induction of Tim-3 were PI3K dependent. The importance of IFN-α was further confirmed by the addition of exogenous cytokines, blockade of IFNAR, and an inhibitor of JAK1/JAK2.

Under all of the conditions tested in this study, Tim-3+ pDCs had a diminished capacity to produce IFN-α. This was observed in our examination of ex vivo–activated pDCs from HIV-infected donors, as well as of pDCs isolated from uninfected donors that were used to assess the dynamics of pDC activation and restimulation. We found a strong correlation between the level of Tim-3 expression on pDCs and defective IFN-α production. Of note, pDCs stimulated by Sendai virus demonstrated some capacity to produce IFN-α from Tim-3+ pDCs, indicating that Tim-3–expressing pDCs could have some functional capacity with certain virus stimuli. This apparent inability of Tim-3–expressing pDCs to produce IFN-α could represent a different stage of differentiation of pDCs after activation, such as one toward Ag presentation. However, we found that Tim-3–expressing pDCs, in contrast to Tim-3–expressing macrophages (50), are unable to phagocytose apoptotic T cells, suggesting that they behave differently in terms of Ag-presenting capability (data not shown). Further studies in this regard are warranted.

The mechanism by which Tim-3 exerts its function on pDCs is unknown. Previous studies investigating a role for Tim-3 using anti–Tim-3 Abs differed widely in their mode of delivery and the final outcomes reported. For example, in murine pDCs, soluble anti–Tim-3 Ab increased TLR signaling (33). The investigators interpreted that the Ab blocked Tim-3 from degrading HMGB1. Conversely, cross-linking Abs inhibited NK-mediated killing (39), and this was interpreted as inducing signals downstream from Tim-3–inhibited NK function. In our experiments, multiple clones of anti–Tim-3 Abs were tested with no observed effect on pDC function, regardless of whether they were delivered as soluble or plate bound (cross-linking) forms. These findings suggest that Tim-3 may not be operating through a direct signaling pathway in pDCs. However, we observed that the submembrane region, which is usually populated by a rim of TLR9, was displaced by Tim-3. Interestingly, Chiba et al. (33) proposed a mechanism of action for Tim-3 in DCs and pDCs, whereby HMGB1 is sequestered away from TLRs, thus preventing their activation. It is possible that exogenous Tim-3 Abs may not have access to this intracellular process. Furthermore, our observations of rapid internalization of Tim-3 directly into lysosomes suggest that a function for Tim-3 may be the transfer of agonists into acidic lysosomes, bypassing TLR activation. Interestingly, the transfer of CpG to lysosomes is associated with decreased IFN-α production (51, 52). Further studies of Tim-3 trafficking in the pDC response are warranted.

Balasubramanian et al. (40) demonstrated an ability for Tim family proteins to direct intracellular proteins for lysosomal degradation. Although we did not conclusively link Tim-3 to the degradation of p85 or IRF7, we found a pattern of colocalization and redistribution among cellular compartments consistent with a role in degradation. After activation, Tim-3 colocalized with p85, which, in turn, associated with LAMP1. It is unclear what role Tim-3 has in the redistribution of p85 or the extent to which the redistribution of p85 effects IRF7, given its importance in the regulation of IRF7 (10). Further, the intensity of IRF7 decreased after activation, which is potentially explained by p85 redistribution and/or by enhanced degradation of IRF7, both of which may be mediated by Tim-3. Despite decreased IRF7 levels, Tim-3 remained colocalized with IRF7, which is suggestive of a close relationship. The accumulation of Tim-3 within lysosomes, where it forms a focal point with p85 and IRF7, further implicates it in the degradation of these critical factors in pDC function. Lastly, the high level of colocalization with recently internalized Tim-3 and IRF7 within lysosomes provides further evidence that Tim-3 may act to regulate these factors. Future studies should be conducted to validate the role of Tim-3 in the regulation of p85 and IRF7.

We identified Tim-3 as a reliable biomarker for pDC dysfunction that is upregulated during HIV infection even after cART, indicating ongoing pDC abnormalities with therapy. Our findings implicate Tim-3 in the negative regulation of IFN-α production from pDCs. Furthermore, we found evidence that Tim-3 may exert its function through bypassing TLR activation and facilitating the degradation of proteins important for IFN-α production. The lack of any effect of Tim-3 Abs on pDC function would make Tim-3 Ab therapies problematic. Alternatively, because immune activation enhances HIV disease progression, Tim-3–expressing pDCs could limit ongoing activation. The relevance of Tim-3 as a biomarker of pDC dysfunction should be examined in other inflammatory and autoimmune conditions.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Dr. Vineet Joag, Dr. Rupurt Kaul, and Dr. Eleanor Fish for helpful discussions and critical review of the manuscript. We acknowledge all of the donors who volunteered for this study, as well as the nurses and phlebotomists who collected the samples. HIV-1 BaL was a generous gift from Dr. Alan Cochrane. The following reagents were obtained through the NIH AIDS Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, NIH: pNL4-3 from Dr. Malcolm Martin and U87+CD4+CCR5 and U87+CD4+CXCR4 from Dr. HongKui Deng and Dr. Dan R. Littman.

Footnotes

  • This work was supported by Canadian Institutes of Health Research Grant HIG-133050.

  • The sequences in this article have been submitted to GenBank (http://www.ncbi.nlm.nih.gov/genbank) under accession number NM_032782.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    cART
    combined antiretroviral therapy
    DC
    dendritic cell
    HMGB1
    high-mobility group box 1
    ICS
    intracellular staining
    IFNAR
    IFN-α receptor
    IQR
    interquartile range
    MFI
    median fluorescent intensity
    NIH
    National Institutes of Health
    pDC
    plasmacytoid dendritic cell
    siRNA
    small interfering RNA
    Tim-3
    T cell Ig and mucin-domain containing molecule-3.

  • Received July 27, 2016.
  • Accepted February 8, 2017.

References

  1. ↵
    1. Wang B. X.,
    2. E. N. Fish
    . 2012. The yin and yang of viruses and interferons. Trends Immunol. 33: 190–197.
    OpenUrlCrossRefPubMed
    1. González-Navajas J. M.,
    2. J. Lee,
    3. M. David,
    4. E. Raz
    . 2012. Immunomodulatory functions of type I interferons. Nat. Rev. Immunol. 12: 125–135.
    OpenUrlCrossRefPubMed
  3. ↵
    1. Stetson D. B.,
    2. R. Medzhitov
    . 2006. Type I interferons in host defense. Immunity 25: 373–381.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Gilliet M.,
    2. W. Cao,
    3. Y. J. Liu
    . 2008. Plasmacytoid dendritic cells: sensing nucleic acids in viral infection and autoimmune diseases. Nat. Rev. Immunol. 8: 594–606.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Swiecki M.,
    2. M. Colonna
    . 2015. The multifaceted biology of plasmacytoid dendritic cells. Nat. Rev. Immunol. 15: 471–485.
    OpenUrlCrossRefPubMed
  6. ↵
    1. Beignon A. S.,
    2. K. McKenna,
    3. M. Skoberne,
    4. O. Manches,
    5. I. DaSilva,
    6. D. G. Kavanagh,
    7. M. Larsson,
    8. R. J. Gorelick,
    9. J. D. Lifson,
    10. N. Bhardwaj
    . 2005. Endocytosis of HIV-1 activates plasmacytoid dendritic cells via Toll-like receptor-viral RNA interactions. J. Clin. Invest. 115: 3265–3275.
    OpenUrlCrossRefPubMed
  7. ↵
    1. Fitzgerald-Bocarsly P.,
    2. E. S. Jacobs
    . 2010. Plasmacytoid dendritic cells in HIV infection: striking a delicate balance. J. Leukoc. Biol. 87: 609–620.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    1. Guiducci C.,
    2. R. L. Coffman,
    3. F. J. Barrat
    . 2009. Signalling pathways leading to IFN-alpha production in human plasmacytoid dendritic cell and the possible use of agonists or antagonists of TLR7 and TLR9 in clinical indications. J. Intern. Med. 265: 43–57.
    OpenUrlCrossRefPubMed
    1. Wang Y.,
    2. M. Swiecki,
    3. S. A. McCartney,
    4. M. Colonna
    . 2011. dsRNA sensors and plasmacytoid dendritic cells in host defense and autoimmunity. Immunol. Rev. 243: 74–90.
    OpenUrlCrossRefPubMed
  10. ↵
    1. Guiducci C.,
    2. C. Ghirelli,
    3. M. A. Marloie-Provost,
    4. T. Matray,
    5. R. L. Coffman,
    6. Y. J. Liu,
    7. F. J. Barrat,
    8. V. Soumelis
    . 2008. PI3K is critical for the nuclear translocation of IRF-7 and type I IFN production by human plasmacytoid predendritic cells in response to TLR activation. J. Exp. Med. 205: 315–322.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    1. Demoulin S.,
    2. M. Herfs,
    3. P. Delvenne,
    4. P. Hubert
    . 2013. Tumor microenvironment converts plasmacytoid dendritic cells into immunosuppressive/tolerogenic cells: insight into the molecular mechanisms. J. Leukoc. Biol. 93: 343–352.
    OpenUrlAbstract/FREE Full Text
    1. Kwok S. K.,
    2. J. Y. Lee,
    3. S. H. Park,
    4. M. L. Cho,
    5. S. Y. Min,
    6. S. H. Park,
    7. H. Y. Kim,
    8. Y. G. Cho
    . 2008. Dysfunctional interferon-alpha production by peripheral plasmacytoid dendritic cells upon Toll-like receptor-9 stimulation in patients with systemic lupus erythematosus. Arthritis Res. Ther. 10: R29.
    OpenUrlCrossRefPubMed
    1. Summers K. L.,
    2. A. M. Marleau,
    3. J. L. Mahon,
    4. R. McManus,
    5. I. Hramiak,
    6. B. Singh
    . 2006. Reduced IFN-alpha secretion by blood dendritic cells in human diabetes. Clin. Immunol. 121: 81–89.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Van Brussel I.,
    2. E. A. Van Vré,
    3. G. R. De Meyer,
    4. C. J. Vrints,
    5. J. M. Bosmans,
    6. H. Bult
    . 2011. Decreased numbers of peripheral blood dendritic cells in patients with coronary artery disease are associated with diminished plasma Flt3 ligand levels and impaired plasmacytoid dendritic cell function. Clin. Sci. 120: 415–426.
    OpenUrl
  15. ↵
    1. Kamga I.,
    2. S. Kahi,
    3. L. Develioglu,
    4. M. Lichtner,
    5. C. Marañón,
    6. C. Deveau,
    7. L. Meyer,
    8. C. Goujard,
    9. P. Lebon,
    10. M. Sinet,
    11. A. Hosmalin
    . 2005. Type I interferon production is profoundly and transiently impaired in primary HIV-1 infection. J. Infect. Dis. 192: 303–310.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    1. Martinson J. A.,
    2. A. Roman-Gonzalez,
    3. A. R. Tenorio,
    4. C. J. Montoya,
    5. C. N. Gichinga,
    6. M. T. Rugeles,
    7. M. Tomai,
    8. A. M. Krieg,
    9. S. Ghanekar,
    10. L. L. Baum,
    11. A. L. Landay
    . 2007. Dendritic cells from HIV-1 infected individuals are less responsive to toll-like receptor (TLR) ligands. Cell. Immunol. 250: 75–84.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Tilton J. C.,
    2. M. M. Manion,
    3. M. R. Luskin,
    4. A. J. Johnson,
    5. A. A. Patamawenu,
    6. C. W. Hallahan,
    7. N. A. Cogliano-Shutta,
    8. J. M. Mican,
    9. R. T. Davey Jr..,
    10. S. Kottilil,
    11. et al
    . 2008. Human immunodeficiency virus viremia induces plasmacytoid dendritic cell activation in vivo and diminished alpha interferon production in vitro. J. Virol. 82: 3997–4006.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    1. Huang J.,
    2. Y. Yang,
    3. M. Al-Mozaini,
    4. P. S. Burke,
    5. J. Beamon,
    6. M. F. Carrington,
    7. K. Seiss,
    8. J. Rychert,
    9. E. S. Rosenberg,
    10. M. Lichterfeld,
    11. X. G. Yu
    . 2011. Dendritic cell dysfunction during primary HIV-1 infection. J. Infect. Dis. 204: 1557–1562.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    1. Barblu L.,
    2. K. Machmach,
    3. C. Gras,
    4. J. F. Delfraissy,
    5. F. Boufassa,
    6. M. Leal,
    7. E. Ruiz-Mateos,
    8. O. Lambotte,
    9. J. P. Herbeuval,
    10. ANRS EP36 HIV Controllers Study Group
    . 2012. Plasmacytoid dendritic cells (pDCs) from HIV controllers produce interferon-α and differentiate into functional killer pDCs under HIV activation. J. Infect. Dis. 206: 790–801.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    1. Donhauser N.,
    2. K. Pritschet,
    3. M. Helm,
    4. T. Harrer,
    5. P. Schuster,
    6. M. Ries,
    7. G. Bischof,
    8. J. Vollmer,
    9. S. Smola,
    10. B. Schmidt,
    11. German Competence Network HIV/AIDS
    . 2012. Chronic immune activation in HIV-1 infection contributes to reduced interferon alpha production via enhanced CD40:CD40 ligand interaction. PLoS One 7: e33925.
    OpenUrlCrossRefPubMed
  21. ↵
    1. Kaushik S.,
    2. F. Teque,
    3. M. Patel,
    4. S. H. Fujimura,
    5. B. Schmidt,
    6. J. A. Levy
    . 2013. Plasmacytoid dendritic cell number and responses to Toll-like receptor 7 and 9 agonists vary in HIV type 1-infected individuals in relation to clinical state. AIDS Res. Hum. Retroviruses 29: 501–510.
    OpenUrlCrossRefPubMed
  22. ↵
    1. Lehmann C.,
    2. M. Lafferty,
    3. A. Garzino-Demo,
    4. N. Jung,
    5. P. Hartmann,
    6. G. Fätkenheuer,
    7. J. S. Wolf,
    8. J. van Lunzen,
    9. F. Romerio
    . 2010. Plasmacytoid dendritic cells accumulate and secrete interferon alpha in lymph nodes of HIV-1 patients. PLoS One 5: e11110.
    OpenUrlCrossRefPubMed
    1. Li Q.,
    2. J. D. Estes,
    3. P. M. Schlievert,
    4. L. Duan,
    5. A. J. Brosnahan,
    6. P. J. Southern,
    7. C. S. Reilly,
    8. M. L. Peterson,
    9. N. Schultz-Darken,
    10. K. G. Brunner,
    11. et al
    . 2009. Glycerol monolaurate prevents mucosal SIV transmission. Nature 458: 1034–1038.
    OpenUrlCrossRefPubMed
    1. Kwa S.,
    2. S. Kannanganat,
    3. P. Nigam,
    4. M. Siddiqui,
    5. R. D. Shetty,
    6. W. Armstrong,
    7. A. Ansari,
    8. S. E. Bosinger,
    9. G. Silvestri,
    10. R. R. Amara
    . 2011. Plasmacytoid dendritic cells are recruited to the colorectum and contribute to immune activation during pathogenic SIV infection in rhesus macaques. Blood 118: 2763–2773.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    1. Shang L.,
    2. L. Duan,
    3. K. E. Perkey,
    4. S. Wietgrefe,
    5. M. Zupancic,
    6. A. J. Smith,
    7. P. J. Southern,
    8. R. P. Johnson,
    9. A. T. Haase
    . 2016. Epithelium-innate immune cell axis in mucosal responses to SIV. Mucosal Immunol.
  26. ↵
    1. Finke J. S.,
    2. M. Shodell,
    3. K. Shah,
    4. F. P. Siegal,
    5. R. M. Steinman
    . 2004. Dendritic cell numbers in the blood of HIV-1 infected patients before and after changes in antiretroviral therapy. J. Clin. Immunol. 24: 647–652.
    OpenUrlCrossRefPubMed
  27. ↵
    1. Donaghy H.,
    2. A. Pozniak,
    3. B. Gazzard,
    4. N. Qazi,
    5. J. Gilmour,
    6. F. Gotch,
    7. S. Patterson
    . 2001. Loss of blood CD11c(+) myeloid and CD11c(−) plasmacytoid dendritic cells in patients with HIV-1 infection correlates with HIV-1 RNA virus load. Blood 98: 2574–2576.
    OpenUrlAbstract/FREE Full Text
  28. ↵
    1. Lehmann C.,
    2. J. M. Harper,
    3. D. Taubert,
    4. P. Hartmann,
    5. G. Fätkenheuer,
    6. N. Jung,
    7. J. van Lunzen,
    8. H. J. Stellbrink,
    9. R. C. Gallo,
    10. F. Romerio
    . 2008. Increased interferon alpha expression in circulating plasmacytoid dendritic cells of HIV-1–infected patients. J. Acquir. Immune Defic. Syndr. 48: 522–530.
    OpenUrlCrossRefPubMed
  29. ↵
    1. Benlahrech A.,
    2. A. Yasmin,
    3. S. J. Westrop,
    4. A. Coleman,
    5. A. Herasimtschuk,
    6. E. Page,
    7. P. Kelleher,
    8. F. Gotch,
    9. N. Imami,
    10. S. Patterson
    . 2012. Dysregulated immunophenotypic attributes of plasmacytoid but not myeloid dendritic cells in HIV-1 infected individuals in the absence of highly active anti-retroviral therapy. Clin. Exp. Immunol. 170: 212–221.
    OpenUrlCrossRefPubMed
  30. ↵
    1. O’Brien M.,
    2. O. Manches,
    3. R. L. Sabado,
    4. S. J. Baranda,
    5. Y. Wang,
    6. I. Marie,
    7. L. Rolnitzky,
    8. M. Markowitz,
    9. D. M. Margolis,
    10. D. Levy,
    11. N. Bhardwaj
    . 2011. Spatiotemporal trafficking of HIV in human plasmacytoid dendritic cells defines a persistently IFN-α-producing and partially matured phenotype. J. Clin. Invest. 121: 1088–1101.
    OpenUrlCrossRefPubMed
  31. ↵
    1. Lepelley A.,
    2. S. Louis,
    3. M. Sourisseau,
    4. H. K. Law,
    5. J. Pothlichet,
    6. C. Schilte,
    7. L. Chaperot,
    8. J. Plumas,
    9. R. E. Randall,
    10. M. Si-Tahar,
    11. et al
    . 2011. Innate sensing of HIV-infected cells. PLoS Pathog. 7: e1001284.
    OpenUrlCrossRefPubMed
  32. ↵
    1. Lo C. C.,
    2. J. A. Schwartz,
    3. D. J. Johnson,
    4. M. Yu,
    5. N. Aidarus,
    6. S. Mujib,
    7. E. Benko,
    8. M. Hyrcza,
    9. C. Kovacs,
    10. M. A. Ostrowski
    . 2012. HIV delays IFN-α production from human plasmacytoid dendritic cells and is associated with SYK phosphorylation. PLoS One 7: e37052.
    OpenUrlCrossRefPubMed
  33. ↵
    1. Chiba S.,
    2. M. Baghdadi,
    3. H. Akiba,
    4. H. Yoshiyama,
    5. I. Kinoshita,
    6. H. Dosaka-Akita,
    7. Y. Fujioka,
    8. Y. Ohba,
    9. J. V. Gorman,
    10. J. D. Colgan,
    11. et al
    . 2012. Tumor-infiltrating DCs suppress nucleic acid-mediated innate immune responses through interactions between the receptor TIM-3 and the alarmin HMGB1. Nat. Immunol. 13: 832–842.
    OpenUrlCrossRefPubMed
  34. ↵
    1. Han G.,
    2. G. Chen,
    3. B. Shen,
    4. Y. Li
    . 2013. Tim-3: an activation marker and activation limiter of innate immune cells. Front. Immunol. 4: 449.
    OpenUrlCrossRefPubMed
  35. ↵
    1. Jones R. B.,
    2. L. C. Ndhlovu,
    3. J. D. Barbour,
    4. P. M. Sheth,
    5. A. R. Jha,
    6. B. R. Long,
    7. J. C. Wong,
    8. M. Satkunarajah,
    9. M. Schweneker,
    10. J. M. Chapman,
    11. et al
    . 2008. Tim-3 expression defines a novel population of dysfunctional T cells with highly elevated frequencies in progressive HIV-1 infection. J. Exp. Med. 205: 2763–2779.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    1. Anderson A. C.,
    2. N. Joller,
    3. V. K. Kuchroo
    . 2016. Lag-3, Tim-3, and TIGIT: co-inhibitory receptors with specialized functions in immune regulation. Immunity 44: 989–1004.
    OpenUrlCrossRefPubMed
  37. ↵
    1. Zhang Y.,
    2. C. J. Ma,
    3. J. M. Wang,
    4. X. J. Ji,
    5. X. Y. Wu,
    6. J. P. Moorman,
    7. Z. Q. Yao
    . 2012. Tim-3 regulates pro- and anti-inflammatory cytokine expression in human CD14+ monocytes. J. Leukoc. Biol. 91: 189–196.
    OpenUrlAbstract/FREE Full Text
    1. Möller-Hackbarth K.,
    2. C. Dewitz,
    3. O. Schweigert,
    4. A. Trad,
    5. C. Garbers,
    6. S. Rose-John,
    7. J. Scheller
    . 2013. A disintegrin and metalloprotease (ADAM) 10 and ADAM17 are major sheddases of T cell immunoglobulin and mucin domain 3 (Tim-3). J. Biol. Chem. 288: 34529–34544.
    OpenUrlAbstract/FREE Full Text
  39. ↵
    1. Ndhlovu L. C.,
    2. S. Lopez-Vergès,
    3. J. D. Barbour,
    4. R. B. Jones,
    5. A. R. Jha,
    6. B. R. Long,
    7. E. C. Schoeffler,
    8. T. Fujita,
    9. D. F. Nixon,
    10. L. L. Lanier
    . 2012. Tim-3 marks human natural killer cell maturation and suppresses cell-mediated cytotoxicity. Blood 119: 3734–3743.
    OpenUrlAbstract/FREE Full Text
  40. ↵
    1. Balasubramanian S.,
    2. S. K. Kota,
    3. V. K. Kuchroo,
    4. B. D. Humphreys,
    5. T. B. Strom
    . 2012. TIM family proteins promote the lysosomal degradation of the nuclear receptor NUR77. Sci. Signal. 5: ra90.
    OpenUrlAbstract/FREE Full Text
  41. ↵
    1. Phong B. L.,
    2. L. Avery,
    3. T. L. Sumpter,
    4. J. V. Gorman,
    5. S. C. Watkins,
    6. J. D. Colgan,
    7. L. P. Kane
    . 2015. Tim-3 enhances FcεRI-proximal signaling to modulate mast cell activation. J. Exp. Med. 212: 2289–2304.
    OpenUrlAbstract/FREE Full Text
  42. ↵
    1. Gurka S.,
    2. S. Dirks,
    3. J. Photiadis,
    4. R. A. Kroczek
    . 2015. Expression analysis of surface molecules on human thymic dendritic cells with the 10th HLDA Workshop antibody panel. Clin. Transl. Immunology 4: e47.
    OpenUrl
  43. ↵
    1. Smith N.,
    2. P. O. Vidalain,
    3. S. Nisole,
    4. J. P. Herbeuval
    . 2016. An efficient method for gene silencing in human primary plasmacytoid dendritic cells: silencing of the TLR7/IRF-7 pathway as a proof of concept. Sci. Rep. 6: 29891.
    OpenUrl
  44. ↵
    1. Clayton K. L.,
    2. M. S. Haaland,
    3. M. B. Douglas-Vail,
    4. S. Mujib,
    5. G. M. Chew,
    6. L. C. Ndhlovu,
    7. M. A. Ostrowski
    . 2014. T cell Ig and mucin domain-containing protein 3 is recruited to the immune synapse, disrupts stable synapse formation, and associates with receptor phosphatases. J. Immunol. 192: 782–791.
    OpenUrlAbstract/FREE Full Text
  45. ↵
    1. Feldman S.,
    2. D. Stein,
    3. S. Amrute,
    4. T. Denny,
    5. Z. Garcia,
    6. P. Kloser,
    7. Y. Sun,
    8. N. Megjugorac,
    9. P. Fitzgerald-Bocarsly
    . 2001. Decreased interferon-alpha production in HIV-infected patients correlates with numerical and functional deficiencies in circulating type 2 dendritic cell precursors. Clin. Immunol. 101: 201–210.
    OpenUrlCrossRefPubMed
  46. ↵
    1. Soumelis V.,
    2. I. Scott,
    3. F. Gheyas,
    4. D. Bouhour,
    5. G. Cozon,
    6. L. Cotte,
    7. L. Huang,
    8. J. A. Levy,
    9. Y. J. Liu
    . 2001. Depletion of circulating natural type 1 interferon-producing cells in HIV-infected AIDS patients. Blood 98: 906–912.
    OpenUrlAbstract/FREE Full Text
  47. ↵
    1. O’Brien M.,
    2. O. Manches,
    3. C. Wilen,
    4. R. Gopal,
    5. R. Huq,
    6. V. Wu,
    7. N. Sunseri,
    8. N. Bhardwaj
    . 2016. CD4 receptor is a key determinant of divergent HIV-1 sensing by plasmacytoid dendritic cells. PLoS Pathog. 12: e1005553.
    OpenUrlCrossRefPubMed
  48. ↵
    1. Lee J.,
    2. E. W. Su,
    3. C. Zhu,
    4. S. Hainline,
    5. J. Phuah,
    6. J. A. Moroco,
    7. T. E. Smithgall,
    8. V. K. Kuchroo,
    9. L. P. Kane
    . 2011. Phosphotyrosine-dependent coupling of Tim-3 to T-cell receptor signaling pathways. Mol. Cell. Biol. 31: 3963–3974.
    OpenUrlAbstract/FREE Full Text
  49. ↵
    1. Schmidt B.,
    2. B. M. Ashlock,
    3. H. Foster,
    4. S. H. Fujimura,
    5. J. A. Levy
    . 2005. HIV-infected cells are major inducers of plasmacytoid dendritic cell interferon production, maturation, and migration. Virology 343: 256–266.
    OpenUrlCrossRefPubMed
  50. ↵
    1. Nakayama M.,
    2. H. Akiba,
    3. K. Takeda,
    4. Y. Kojima,
    5. M. Hashiguchi,
    6. M. Azuma,
    7. H. Yagita,
    8. K. Okumura
    . 2009. Tim-3 mediates phagocytosis of apoptotic cells and cross-presentation. Blood 113: 3821–3830.
    OpenUrlAbstract/FREE Full Text
  51. ↵
    1. Guiducci C.,
    2. G. Ott,
    3. J. H. Chan,
    4. E. Damon,
    5. C. Calacsan,
    6. T. Matray,
    7. K. D. Lee,
    8. R. L. Coffman,
    9. F. J. Barrat
    . 2006. Properties regulating the nature of the plasmacytoid dendritic cell response to Toll-like receptor 9 activation. J. Exp. Med. 203: 1999–2008.
    OpenUrlAbstract/FREE Full Text
  52. ↵
    1. Honda K.,
    2. Y. Ohba,
    3. H. Yanai,
    4. H. Negishi,
    5. T. Mizutani,
    6. A. Takaoka,
    7. C. Taya,
    8. T. Taniguchi
    . 2005. Spatiotemporal regulation of MyD88-IRF-7 signalling for robust type-I interferon induction. Nature 434: 1035–1040.
    OpenUrlCrossRefPubMed
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