Dendritic Cell Dysfunction During Primary HIV-1 Infection

Abstract

Dendritic cells have critical roles for generating and fine-tuning adaptive immune responses and for regulating immune activity through cytokine secretion. In this study, we analyzed functional properties of dendritic cells in primary human immunodeficiency virus type 1 (HIV-1) infection. We found substantial disarray of the functional properties of myeloid and plasmacytoid dendritic cells in acute HIV-1 infection, which included defective antigen-presenting and cytokine secretion properties and was associated with a distinct surface expression profile of immunomodulatory dendritic cell receptors from the leukocyte immunoglobulin-like receptor family. These data indicate that key functional properties of dendritic cells are compromised during primary HIV-1 infection.

Primary human immunodeficiency virus type 1 (HIV-1) infection is a transient acute illness that typically occurs within 2–3 weeks of viral transmission and is characterized by extremely high levels of viral replication. Subsequently, viral loads spontaneously decline to a set point, which is predictive of the ensuing clinical disease progression. Immune responses occurring during these earliest stages of HIV-1 infection are attractive targets for immunologic interventions aiming at reducing host susceptibility to HIV-1 infection, and are a subject of active investigations [1]. Previous studies have shown that systemic release of acute-phase proteins [2], a proinflammatory cytokine storm [3], and activation of natural killer cells contribute to the initial innate immune defense against HIV-1. HIV-1–specific cytotoxic T cells typically emerge as the first sign of systemic adaptive immune activity against HIV-1 [4] and play an important role in the partial immunologic control of HIV-1 replication that is achieved at the viral set point.

Dendritic cells are a specialized group of professional antigen-presenting cells that have critical functions for regulating immune activation and for priming effector cell responses; these cells are therefore of outstanding interest for the development of effective HIV-1 vaccination strategies [5]. Two classes of dendritic cells are distinguished: myeloid dendritic cells (mDCs) have strong antigen-presenting properties and influence immune activation through secretion of regulatory cytokines, whereas plasmacytoid dendritic cells (pDCs) can exert antiviral activities mainly by producing abundant amounts of interferon (IFN) α. During primary HIV-1 infection, both of these cell types have important roles for sensing HIV-1 antigen, presenting viral antigen to T and B cells and fine tuning functional properties of adaptive effector cell responses [1]. Prior work has shown that in advanced stages of HIV-1 infection, dendritic cell numbers are diminished and have reduced functional antigen-presenting properties [6, 7] with altered cytokine secretion characteristics [8]. It is currently unclear whether these changes develop late during the disease owing to prolonged, long-term exposure to high viral loads or reflect an early functional defect that occurs during primary HIV-1 infection. Because all HIV-1–specific vaccine candidates under development rely on dendritic cells for antigen presentation and effector cell priming, the understanding of dendritic cell characteristics during the early phase of host-pathogen interactions is particularly important. In the present study, we show that during acute HIV-1 infection, mDCs have weaker abilities for antigen presentation and secretion of interleukin (IL) 12p70 and tumor necrosis factor (TNF) α; this occurs in conjunction with substantially reduced secretion of IFN-α in pDCs. Overall, these data provide strong evidence for functional defects in dendritic cells during primary HIV-1 infection.

Materials and Methods

Patients.

Study individuals were recruited based on protocols approved by the Institutional Review Board of the Massachusetts General Hospital, after providing written consent.

Phenotypic Analysis of Dendritic Cells.

Peripheral blood mononuclear cells (PBMCs) freshly isolated from whole blood samples were stained with blue viability dye and antibodies against CD11c, HLA-DR, lin, CD123, CD86, CD83, and CD40 and a panel of antibodies recognizing LILRA2, LILRB1, LILRB2, LILRB3, and LILRA4. Afterward, cells were washed, fixed, and analyzed by flow cytometry (Supplementary Figure 1).

Dendritic Cell Isolation.

mDCs were isolated from whole PBMC samples by positive selection with Blood Dendritic Cell Antigen (BDCA)-1– and BDCA-3–specific antibodies, using isolation kits from Miltenyi Biotech (Miltenyi). The purity of mDCs was >90%.

Cytokine Secretion Assays.

Fresh PBMCs were stimulated with Toll-like receptor 7/8 ligands CL097 (5 μg/mL; InvivoGen) for 16 hours in the presence of brefeldin A. After addition of blue viability dye, fixation, and permeabilization, intracellular cytokine staining was performed using antibodies against IFN-α (Cell Sciences), TNF-α (BioLegend), IL-6 (eBioscience), and IL-12p70 (Miltenyi Biotec), as described elsewhere [9]. Cells were analyzed on an LSRII cytometer using FACSDiva software after gating on the viable cells.

Mixed Lymphocyte Reactions.

T-cell proliferation assays were preformed in 96-well microtiter plates. Purified mDCs were mixed with allogeneic carboxyfluorescein diacetate succinimidyl ester (CFSE)–labeled T cells (isolated with a T-cell enrichment kit; StemCell Technologies) at ratios of 1:50 or 1:100, as appropriate. After 6 days of culture, cells were analyzed by flow cytometry.

Statistics.

Data are presented as box-and-whisker plots, reflecting the minimum, maximum, and the 25th, 50th and 75th percentiles. Differences were tested for statistical significance with Mann–Whitney U tests.

Results

To analyze functional characteristic of dendritic cells during primary HIV-1 infection, we focused on a cross-sectional analysis involving 17 HIV-1–infected patients (mean age, 37 years; range, 25–51 years; male-female ratio, 14:3). Seven of these patients met criteria for “acute HIV-1 infection” (stage III–IV of the classification system proposed by Fiebig et al [10]) and had mean CD4 cell counts of 403/μL (range, 317–831) with average viral loads of 221 000 copies/mL (range, 100 000–822 000). Ten of our study subjects were identified during “early-stage” HIV-1 infection (Fiebig stage V–VI) and had mean CD4 cell counts of 444/μL (range, 392–1483) with average viral loads of 6290 copies/mL (range, 182–124 000). A cohort of 11 HIV-1–uninfected individuals (mean age, 24 years (24–48); male-female ratio, 6:5) was recruited for control purposes.

We initially focused on analyzing mDCs, which have key roles for priming and regulating antigen-specific effector cell immune responses. The relative proportion of mDCs, defined as Lin^-HLA-DR⁺CD11c⁺ events, was significantly reduced in patients with primary infection compared with the control population (Figure 1A). To investigate whether these quantitative decreases are associated with qualitative and functional changes, we used mixed lymphocyte reactions to analyze the antigen-presenting properties of mDCs. For this purpose, isolated mDCs from the study subjects were mixed with CFSE-labeled allogeneic T cells from identical, HIV-1 negative donors, and proliferative T cells were assessed on day 6 by flow cytometry. As summarized in Figure 1B, we noticed significantly reduced abilities of mDCs to prime allogeneic CD4 and CD8 T-cell responses in patients detected during acute HIV-1 infection, but stimulatory properties of mDCs from patients with early-stage HIV-1 infection were not different from those in the HIV-1 negative control cohort. Notably, the antigen-presenting properties of mDCs from all patients with primary HIV-1 infection were positively correlated with the corresponding CD4 T-cell counts and inversely correlated with the respective viral loads (Figure 1C).

Figure 1.

Altered functional properties of myeloid dendritic cells (mDCs) in primary human immunodeficiency virus type 1 (HIV-1) infection. A, Relative frequencies of mDCs in individuals with acute (Fiebig stage III/IV) or early HIV-1 infection (Fiebig stage V/VI) and in HIV-1–negative persons (HIV-). B, Proportions of proliferating allogeneic CD4 and CD8 T cells after exposure to mDCs from persons with acute or early HIV-1 infection and HIV-1–negative control subjects. C, Correlation between stimulatory activities of mDCs from persons with primary HIV-1 infection and corresponding CD4 cell counts or HIV-1 viral loads; Pearson’s correlation coefficient is indicated. D, Intracellular secretion of cytokines in mDCs from indicated study groups after stimulation with Toll-like receptor 7/8 ligands. IL, interleukin; TNF, tumor necrosis factor. E, Surface expression of indicated immunomodulatory leukocyte immunoglobulin-like receptor (LILR) on dendritic cells in acute or early HIV-1 infection and HIV-1–negative control subjects.

We subsequently focused on analyzing cytokine secretion properties of mDCs in our study cohorts. Using a flow cytometry–based approach allowing single-cell analysis, we observed that the relative proportions of mDCs secreting IL-12p70 or TNF-α were significantly reduced during acute- and early-stage HIV-1 infection, whereas frequencies of IL-6 secreting mDCs were similar between the patient groups (Figure 1D). In contrast, intensities of cytokine secretion on a per-cell level were not diminished during primary infection, and, in fact, tended to be higher for IL-6 and IL-12p70 in patients identified during stage III/IV of primary infection (Figure 1D). To analyze whether these functional alterations were associated with phenotypic correlates, we assessed the surface expression of costimulatory molecules and immunomodulatory receptors from the leukocyte immunoglobulin-like receptor (LILR) family, which can act as activating or inhibitory regulators of dendritic cell function. Although no changes were observed for the costimulatory molecules CD86, CD83, and CD40 between the different study cohorts (data not shown), we observed strong down-regulation of LILRA2, a stimulatory dendritic cell receptor [9], in conjunction with up-regulation of LILRB2, an inhibitory receptor [9], on mDCs from patients with acute HIV-1 infection (Figure 1E).

We subsequently analyzed functional characteristics of pDCs, which can modulate host defense through secretion of abundant amounts of proinflammatory cytokines. The relative proportion of pDCs was substantially reduced during all stages of primary HIV-1 infection (Figure 2A). Moreover, the proportion of IFN-α–secreting cells, as well as the per-cell intensity of IFN-α production, was substantially decreased in persons with acute and early-stage HIV-1 infection. Proportions of IL-6– and TNF-α–secreting pDCs were similar between patients from primary HIV-1 infection and HIV-1–negative controls, but per-cell intensities of cytokine production tended to be higher in patients with primary HIV-1 infection, specifically with regard to IL-6 (Figure 2B). Again, functional changes in pDCs were not associated with alterations in the surface expression of costimulatory molecules (data not shown); however, we found that LILRA4, a receptor that modulates cytokine secretion in pDCs [11] was down-regulated during acute stages of primary HIV-1 infection (Figure 2C).

Figure 2.

Reduced cytokine secretion by plasmacytoid dendritic cells (pDCs) in primary human immunodeficiency virus type 1 (HIV-1) infection. A, Frequencies of pDCs in persons with acute HIV-1 infection (Fiebig stage III/IV) or early HIV-1 infection (Fiebig stage V/VI) and HIV-1–negative control subjects (HIV-). B, Intracellular secretion of cytokines in pDCs from indicated study subjects after stimulation with Toll-like receptor 7/8 ligands. IFN, interferon; IL, interleukin. C, Surface expression of leukocyte immunoglobulin-like receptor (LILR) A4 on pDCs during acute or early HIV-1 infection in comparison with HIV-1–negative control subjects.

Discussion

As the most effective professional antigen-presenting cells, dendritic cells are likely to play key roles in immune defense against HIV-1 infection. In persons with spontaneous control of HIV-1 replication, circulating mDCs have superior antigen-presenting properties [9], whereas stimulatory activities of mDCs in patients with progressive HIV-1 infection are suppressed [6], suggesting that specific functional characteristics of dendritic cells are required for effective immune control of HIV-1. In this study, we demonstrate a complex functional and phenotypic disarray of mDCs and pDCs during primary HIV-1 infection. Most importantly, we found that 2 major functional hallmarks of dendritic cells, the antigen-presenting properties of mDC and IFN-α secretion in pDCs, were inhibited during acute HIV-1 infection. Interestingly, the functional decline of the stimulatory activities of mDCs occurred only during acute HIV-1 infection (stage III/IV) and was not visible in persons with early-stage infection (stage V/VI), suggesting that acute HIV-1 infection is associated with a transient and reversible defect in antigen-presenting properties of mDCs. In contrast, inhibition of IFN-α secretion in pDCs was noted during all analyzed stages of primary infection, although it was most pronounced in persons with acute HIV-1 infection; this may indicate that the functional impairment of IFN-α secretion in pDCs starts during acute infection but continuously persists during the subsequent disease process.

It is noteworthy that the most significant functional alterations of dendritic cells occurred during acute HIV-1 infection at the time of maximal HIV-1 viral loads and high-level immune activation. This suggests that the functional suppression of dendritic cells at this time is related to high levels of circulating viral particles and the associated massive release of proinflammatory cytokines. Indeed, previous work has shown that exposure to repeated antigenic stimulation [12] or high dosages of cytokines [13] can induce a state of functional unresponsiveness in pDCs that makes such cells refractory to antigenic stimulation ex vivo. In addition, it is important to recognize that functional inhibition of dendritic cells in primary HIV-1 infection was associated with a distinct expression profile of immunomodulatory receptors from the LILR family, which can significantly influence antigen-presenting and cytokine secretion properties of dendritic cells [9, 14]. The observed down-regulation of the activating receptor LILRA2 and up-regulation of inhibitory receptor LILRB2 on mDCs in acute HIV-1 infection is likely to cause a net reduction in the functional antigen-presenting properties of mDCs and may therefore contribute to the diminished allostimulatory functions of dendritic cells in primary HIV-1 infection [9]. Moreover, altered expression of LILRA4, a dominant modulator of cytokine secretion in pDCs [11] is likely to play a prominent role for the functional impairment in IFN-α secretion of pDCs during primary HIV-1 infection.

Our results differ from those of another study in which no evidence for defective functional properties of mDCs and pDCs during acute HIV-1 infection was found [15]. In part, these differences may be due to alternative experimental protocols for assessing dendritic cell function. For instance, other investigators analyzed allogeneic T-cell proliferation after dendritic cell stimulation using radioactive incorporation of tritium on day 5 of culture, whereas we used a CFSE-based assay that allows for measuring proliferative responses over the entire period of culture. Moreover, in comparison to the study by Sabado et al [15], mixed lymphocyte reactions in our study were performed in more patients with acute HIV-1 infection (stage III/IV) and may therefore have been more sensitive for detecting functional antigen-presenting defects of dendritic cells. Finally, the cytokine secretion of dendritic cells in our study was analyzed intracellularly on the single-cell level using flow cytometry instead of quantifying bulk amounts of extracellularly secreted cytokines in the culture supernatants, which may be influenced by the effects of the secreted cytokines on cellular activation and cytokine release.

In summary, our data provide strong evidence for a functional inhibition of mDCs and pDCs during acute HIV-1 infection. These observations extend our understanding of immunologic alterations occurring during the earliest stages of HIV-1 infection and suggest that functional defects of dendritic cells during primary HIV-1 infection may contribute to the ultimate failure to immunologically control HIV-1 infection.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online (http://www.oxfordjournals.org/our_journals/jid/).  Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.