CD2 Distinguishes Two Subsets of Human Plasmacytoid Dendritic Cells with Distinct Phenotype and Functions

Toshimichi Matsui, John E. Connolly, Mark Michnevitz, Damien Chaussabel, Chun-I Yu, Casey Glaser, Sasha Tindle, Marc Pypaert, Heidi Freitas, Bernard Piqueras, Jacques Banchereau and A. Karolina Palucka
J Immunol June 1, 2009, 182 (11) 6815-6823; DOI: https://doi.org/10.4049/jimmunol.0802008

Abstract

Plasmacytoid dendritic cells (pDCs) are key regulators of antiviral immunity. They rapidly secrete IFN-α and cross-present viral Ags, thereby launching adaptive immunity. In this study, we show that activated human pDCs inhibit replication of cancer cells and kill them in a contact-dependent fashion. Expression of CD2 distinguishes two pDC subsets with distinct phenotype and function. Both subsets secrete IFN-α and express granzyme B and TRAIL. CD2high pDCs uniquely express lysozyme and can be found in tonsils and in tumors. Both subsets launch recall T cell responses. However, CD2high pDCs secrete higher levels of IL12p40, express higher levels of costimulatory molecule CD80, and are more efficient in triggering proliferation of naive allogeneic T cells. Thus, human blood pDCs are composed of subsets with specific phenotype and functions.

Dendritic cells (DCs)5 represent a complex system of cells with different anatomical localization, distinct subsets, and distinct functions (1, 2). Two main DC pathways bearing common and unique functions have been recognized, i.e., myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) (2, 3). The pDCs have been described independently in the late 1970s as plasmacytoid monocytes and plasmacytoid T cells. In the 1990s, pDCs were identified as DCs (4, 5) and as the natural IFN-producing cells (6, 7, 8). Indeed, upon viral encounter, pDCs rapidly secrete considerable amounts of type I IFNs (6, 7) as well as numerous chemokines (9). pDCs can be activated by 1) viruses (6, 7); 2) IL-3 and CD40L (IL-3/CD40L) (5, 10), possibly originating from mast cells triggered for example by parasites; and 3) by bacterial components in the form of CpG DNA (11, 12). Once activated, pDCs induce the expansion and differentiation of Ag-specific memory B and T lymphocytes, yielding plasma cells (13) and CTLs (14, 15). There is evidence that pDCs are involved in tolerance induction (16, 17, 18). pDCs stimulated via TLR or CD40 induce IL-10-secreting regulatory CD4+ T cells owing to the expression of ICOS ligand (19) as well as suppressor CD8+ T cells (20). Finally, pDCs activated via IL-3/CD40L can also drive type 2 polarization of CD4+ T cells (10).

pDCs play a key role in disease pathogenesis. Their alteration appears central to immune dysfunction underlying systemic lupus erythematosus (SLE), a prototype autoimmune disease characterized by a break of tolerance to self-Ags (21, 22, 23). pDC accumulate in skin lesions of SLE (24) where they become activated and secrete type I IFN, thereby contributing to tissue damage. Early studies demonstrated that immune complexes are potent stimuli for IFN-α secretion by pDCs in a FcR (CD32)-dependent manner (25, 26). Indeed, chromatin and/or ribonucleoprotein-containing immune complexes can be internalized by pDCs via FcγRIIa, reach the endosomal compartment, and activate IFN-α secretion through TLR9- and/or TLR7-dependent pathways (27, 28). Sera from SLE patients can also induce IFN-α secretion in a TLR7/8-dependent manner (29). The aberrantly produced IFNs are major effectors in the pathogenesis of autoimmunity, mainly by inducing an unabated maturation of peripheral mDCs (22) that can stimulate autoreactive T cells. DNA-containing immune complexes can also activate pDCs to produce IFN-α via the high mobility group box 1 protein (HMGB1) activation of TLR9 through a mechanism involving the Ig superfamily member RAGE, receptor for HMGB1 (30). Similar observations were made in psoriasis (31). There, antimicrobial peptide LL37 (also known as CAMP) has been shown as the key factor that mediates pDC activation (32). LL37 converts inert self-DNA into a potent trigger of IFN production by binding the DNA to form aggregated and condensed structures that are delivered to and retained within early endocytic compartments in pDCs to trigger TLR9 (32). Accumulation of pDCs has also been found in cancer, most particularly in breast cancer, where it has been suggested to represent an independent prognostic factor associated with poor outcomes as measured by overall survival and time to disease progression (33). Furthermore, in vivo signaling via TLR ligands such as CpG (TLR9) (34) or imiquimod (TLR7) (35) leads to tumor regression both in mice and humans, possibly via mobilization of pDCs. These observations raise a question as to the role of pDCs in tumor microenvironment. The analysis of pDC transcripts revealed several unusual features including the presence of granzyme B (36), an effector molecule of CTLs and NK cells. We show here that pDCs display cytotoxic properties and are composed of subsets with distinct phenotype and functions.

Materials and Methods

Isolation of DCs

PBMCs were isolated from buffy coats by Ficoll centrifugation. Monocytes, T cells, B cells, and RBC were depleted using respective mAbs coupled to microbeads (CD3, CD14, CD19, and glycophorin A; Miltenyi Biotec). Enriched PBMCs were labeled with CD11c-allophycocyanin, HLA-DR-QR, CD123-PE, and a mixture of FITC- or PE-coupled mAbs against lineage markers: CD3, CD14, CD16, CD19, CD20, CD56 (BD Biosciences). In some experiments, cells were also stained additionally with CD2-PE-Cy7 (39C1.5; Beckman Coulter). Alternatively, pDCs were sorted using a lineage mixture, HLA-DR, BDCA-2 (AC144; Miltenyi Biotec), and CD2 (S5.2; BD Biosciences). Cells were sorted using either FACSVantage or FACSAria (BD Biosciences). NK cells were sorted from nondepleted PBMCs using CD16-FITC and CD56-PE (BD Biosciences).

Cell culture

Cultures were established in endotoxin-free medium consisting of RPMI 1640 supplemented with 10% (v/v) heat-inactivated FCS, 10 mM HEPES, 2 mM l-glutamine, penicillin, and streptomycin. Sorted total pDCs, mDCs, and NK cells were seeded at 5 × 105 cells/ml in medium containing 20 ng/ml IL-3 (R&D Systems) and 200 ng/ml CD40L (R&D Systems), 6 μg/ml CpG (InvivoGen), or 1 hemagglutination unit/culture well (corresponding to ∼104 viral particles) Influenza A/PR8/34 virus (Charles River Laboratories). Sorted CD2high and CD2low pDCs were seeded at 1.25 × 105 cells/ml unless otherwise indicated. After 18–24 h of culture, cells and supernatants were harvested for further analysis.

ELISA

Secretion of granzyme B was determined using either 1) a sandwich ELISA kit (Research Diagnostic) following the manufacturer’s protocol or 2) Luminex assay. Secretion of TRAIL was determined using an ELISA kit (Research Diagnostic).

Cytokine multiplex analysis

Cell culture supernatants from activated pDCs were diluted 1/2.5 with culture medium and analyzed using the Beadlyte cytokine assay kit (Millipore) as per the manufacturer’s protocol. Assay plates were run on the Bio-Plex Luminex 100 XYP instrument (Bio-Rad) for analysis. Cytokine concentrations were calculated using Bio-Plex Manager 3.0 software with a five-parameter curve-fitting algorithm applied for standard curve calculations.

Microarrays

Total RNA was extracted from sorted pDC subsets of two different normal donors. After two rounds of linear amplification, cRNA was hybridized on Affymetrix HG-U133A GeneChip arrays. Specific hybridization intensity, probe set signal values, and present/absent calls were computed using GeneChip Operating Software version 1.0.0.046. Normalization of signal values per chip was achieved using the GeneChip Operating Software version 1.0.0.046 global method of scaling to the target intensity value of 500/GeneChip. Gene expression was analyzed with GeneSpring 7.1 software. Transcripts were ranked based on fold change and absolute difference in expression between the two study conditions. Each probe set was previously normalized with a per chip normalization to the 50th percentile and a per gene normalization to the median of each gene. (This per chip normalization was applied to these data.) GEO accession no. GSE15215; http://www.ncbi.nlm.nih.gov/projects/geo/query/acc.cgi?acc = GSE15215.

51Cr release assay

Harvested total pDCs, mDCs, NK cells, and CD2highCD2low pDCs were used as effectors. Effectors were cocultured with 51Cr-labeled K562 at 100:1 E:T ratio or 30:1 E:T ratio. After 18 h of culture, supernatants were analyzed. Percentage of specific lysis was calculated as (cpmexperiment − cpmspontaneous release)/(cpmmaximum release − cpmspontaneous release).

Confocal imaging

Five × 105 cells were washed twice with ice-cold PBS plus 1% BSA, fixed for 20 min in 4% paraformaldehyde, and permeabilized with PBS/0.05% saponin/1% BSA. Endogenous biotin was blocked using endogenous biotin- blocking reagent (Invitrogen). Cells were then stained with FITC-conjugated anti-lysozyme (Research Diagnostic) and either biotin-conjugated anti-BDCA2 (Miltenyi Biotec) or anti-granzyme B (BD Biosciences) followed by treatment with either Alexa Fluor 568-labeled streptavidin or goat anti-mouse IgG2a. respectively (Invitrogen). Washed cells were then mounted onto slides in Vectashield (Vector Laboratories) containing TOPRO-2 (Invitrogen). Cells were then imaged using a Leica TCS-NT/SP1 confocal microscope equipped with three lasers (argon, krypton, and helium neon) capable of three fluorescence channels and transmitted light detection. Images were acquired on a Leica DMIRBE microscope with the ×63 Plan APO objective using Leica TCS version 2.1 software.

Live cell microscopy

Activated CD2high or CD2low pDCs were mixed on ice with K562 cells in RPMI 1640 medium with 10% human AB serum at a ratio of 2:1 in flat-bottom 48-well plates and allowed to settle on ice for 10 min. Plates were then transferred to a 37°C/5% CO2 live cell imaging chamber. Cells were imaged either in bright field or 488-nm florescence every 10 s for 8 h using an Olympus IX-70 microscope at ×10 magnification (Olympus ×10 PlanFl aperture 0.30). Time course images were assembled into stacks using MetaMorph version 6.2r4 (Universal Imaging). For presentation purposes, Quicktime (Apple Computer) movies were generated from assembled stacks. For the purpose of size compression, every tenth frame was inserted into the movie and displayed at a frame rate of 1 frame every 1/10 s, for a total acceleration of ∼1600 times. The percentage of pDCs interacting with K562 cells at any given time was determined through an analysis of time-course image stacks. Total cells in the frame at a given time point as well as cells interacting with K562 cells were quantitated using the Manual Object Count Tool in the MetaMorph software package. Frames before and after any given time point were used as a reference to identify cells as they entered clusters. Percent interaction is expressed as the ratio of bond pDCs over the total number of pDCs in the frame for that time point. To monitor tumor cell killing, K562 cells were labeled with calcein-AM (Invitrogen), washed, and cocultured with CD2high pDCs. Killing was assessed by loss of calcein-AM florescence over time by live cell florescence microscopy.

Electron microscopy

Cells were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate (pH 7.4) for 1 h at room temperature, washed in cacodylate buffer, and postfixed in 1% osmium tetroxide in the same buffer for 1 h at room temperature. Cells were stained en bloc in 2% uranyl acetate in 50 mM sodium maleate (pH 5.2) for 1 h at room temperature, then dehydrated in ethanol and embedded in EMbed 812 epoxy resin (all EM reagents from Electron Microscopy Sciences). Ultrathin sections were cut on a Reichert Ultracut E microtome and collected on formvar- and carbon-coated nickel grids. Sections were stained with 1% lead citrate followed by 2% uranyl acetate and examined in a Tecnai 12 Biotwin electron microscope (FEI). Digital images were recorded using a Morada charge-coupled device camera (Olympus Soft Imaging Solutions).

Thymidine incorporation assay

Harvested total pDCs and mDCs were used as effectors. Effectors were cocultured with K562 cells for 18 h at a ratio of 100:1 E:T ratio. After 18 h, cell were pulsed with 1 μCi/well of [3H]thymidine for 3 days. After 3 days, cocultures were harvested for the incorporation of [3H]thymidine with liquid scintillation counter (PerkinElmer).

Statistical analysis

The nonparametric Mann-Whitney U test and paired Wilcoxon test were used as indicated.

Results

Activated blood pDCs display cytotoxic properties

pDCs and mDCs were sorted from PBMCs as LINnegHLA-DR+CD11c−CD123+ cells (pDCs) or LINnegHLA-DR+CD11c+CD123− cells (mDCs) as described earlier (9). pDCs were analyzed either fresh or after overnight activation with three well-characterized activators: IL-3/CD40L, influenza virus (PR8, H1N1), or CpG A. Freshly isolated pDCs, but not mDCs, show transcription of granzyme B consistent with earlier studies (36) at levels similar to those observed in cultured Ag-specific CD8+ T cells (data not shown). However, although CD8+ T cells show transcription of at least four genes coding different types of granzymes (37), pDCs express uniquely granzyme B (data not shown). Resting pDCs express the granzyme B protein (data not shown) and its expression and secretion to culture supernatant is considerably increased after activation (data not shown).

Freshly isolated blood pDCs do not kill efficiently chronic myeloid leukemia-derived K562 cells (data not shown). However, pDCs activated with IL-3/CD40L kill K562 cells as measured by the release of 51Cr (mean ± SEM = 29 ± 5% specific lysis, range 10–46%, n = 8; Fig. 1⇓A). Specific lysis in cocultures of K562 cells with pDCs was significantly higher than that observed with mDCs (mean ± SEM = 7 ± 2.4% specific lysis; range, 0–17%, p < 0.005; Fig. 1⇓A). The killing of K562 cells by pDCs required high E:T ratio (E:T ratio = 100:1) and long E:T interaction (18 h). pDCs also demonstrated cytotoxic function against cancer cells when activated with influenza virus (mean ± SEM = 22.5 ± 2.5% specific lysis, n = 4) or CpG (Fig. 1⇓B). CpG-activated pDCs were capable of killing multiple tumor-derived cell lines including K562 cells, breast cancer cells (1806 cells), and melanoma cells (Colo829 cells; Fig. 1⇓, B and C). Furthermore, tumor cells cocultured with activated pDCs showed a significant (p < 0.0001) decrease in thymidine incorporation (mean ± SEM = 25.4 × 103 ± 1.8 × 103 cpm vs 5.1 × 103 ± 0.89 × 103 cpm for K562 cells cultured without or with activated pDCs, respectively; Fig. 1⇓D). These results indicate that activated pDCs can halt the growth of tumor cells in vitro. Although the killing of target cells by pDCs as measured in a chromium release assay appears not very efficient (high E:T ratio and long incubation time), pDCs are able to abrogate tumor cell growth almost completely (Fig. 1⇓D). These results suggest that pDC might be important in the control of tumor growth.

FIGURE 1.
FIGURE 1.

pDCs kill malignant cells. A and B, Killing of K562 cells was analyzed in an 18-h 51Cr release assay with pDCs activated with IL-3 and CD40L (A), influenza virus, or CpG (B). Specific lysis (ordinate). Paired Wilcoxon test. Color code indicates experiments with cells from the same donor. Inert plot shows killing by NK cells isolated from the same donors. C, Killing of breast cancer (1806) and melanoma (Colo8290) cells by CpG-activated pDCs and mDCs, 18-h 51Cr release assay, and specific lysis (ordinate). Duplicate wells from one experiment are representative of two experiments. D, Inhibition of malignant cell replication on 3-day coculture. Thymidine incorporation (ordinate). Duplicate (or triplicate) wells from four different experiments. E, Fluorescence microscopy analysis of conjugates between CD45PE-labeled K562 cells (red) and CD123FITC/HLA-DRFITC-labeled pDCs (green; 1:2 ratio; 45 min). F, Flow cytometry analysis of conjugate formation between CD45PE-labeled K562 cells (red) and CD123FITC/HLA-DRFITC-labeled pDCs (green; 1:2 ratio; 45 min) (upper right quadrant).

CD2 distinguishes two fractions of blood pDCs

The killing of K562 by activated pDCs could be blocked by EGTA, indicating Ca2+ dependence (data not shown). Supernatants from activated pDCs or from pDC/K562 cocultures did not kill K562 cells, indicating the cell contact dependence (data not shown). Accordingly, live microscopy demonstrated that activated pDCs form tight clusters with K562 within 45 min of coculture (Fig. 1⇑E). However, not all pDCs appeared bound with K562 cells, suggesting the presence of two subsets.

Accordingly, flow cytometric conjugate formation assays, where CD45PE-labeled K562 cells are coincubated with CD123FITC/HLA-DRFITC-labeled activated pDCs, showed that only a fraction of pDCs (27% in the experiment displayed in Fig. 1⇑F) were engaged in conjugates with target cells.

An extensive analysis of T cell and NK cell markers on pDCs defined as lineageneg HLA-DR+CD11cnegCD123+ (data not shown) or as HLA-DR+CD11cnegBDCA-2+ (Fig. 2⇓, A and B) revealed differential expression of CD2 (38, 39, 40, 41). In PBMC staining, CD2high pDCs show CD2 staining intensity within the range of that in T cells (Fig. 2⇓A). Analysis of 14 healthy donors showed that 12–43% of blood pDCs express CD2 at high levels (mean ± SD: 22.5 ± 8.7%; Fig. 2⇓C). Given that pDCs represent ∼0.25 ± 0.1% of adult PBMCs, CD2high pDCs represent ∼0.05% of mononuclear cells. CD2high pDCs were also detected in cell suspensions prepared from tonsils as BDCA-2+CD2high double-positive cells (mean, 0.77% of cell suspension; range, 0.36–1.54%; mean, 22.8% of BDCA-2+ pDCs; range, 16.7–32%; Fig. 2⇓D). Giemsa staining of sorted CD2high pDCs revealed morphology characteristic of pDCs (data not shown). Similarly, both subsets display pDC ultrastructure in electron microscopy with rich endoplasmic reticulum (Fig. 3⇓A). Furthermore, after sort and overnight culture with IL-3/CD40L or with CpG A, CpG B, C, or live influenza virus, CD2high pDCs retain the expression of CD2 while CD2low pDC do not acquire expression of CD2 (Fig. 3⇓B), suggesting that CD2 on pDCs is not a mere marker of cell activation.

FIGURE 2.
FIGURE 2.

CD2 expression discriminates two subsets of pDCs. A, PBMCs staining shows the lack of CD2 expression by blood CD19+ B cells (upper plots), expected peak of expression on CD3+ T cells (middle plots), and a subset of BDCA2+ pDCs that show CD2 staining within the range of staining on T cells (lower plots). Representative of the analysis of PBMCs from three donors. B, pDC subsets distinguished by CD2 expression can also be defined among HLA-DR+BDCA2+ (left panel) and CD11cneg (middle panel) cells in pre-enriched PBMCs. C, Frequency of two subsets within the total pDC population. D, BDCA-2+CD2+ pDCs in single-cell suspension prepared from tonsils; representative of three independent experiments.

FIGURE 3.
FIGURE 3.

CD2high pDCs and CD2low pDCs show similar ultrastructure. A, CD2low (left panels) and CD2high (right panels) pDCs were fixed and processed for transmission electron microscopy. Note the abundance of endoplasmic reticulum in both subsets of cells (arrows). Bar, 2-μm. B, CD2 staining in flow cytometry on pDC subsets presort, after sort, and after an 18-h activation with CpG. C, Sorted CD2low (upper panels) and CD2high (lower panels) were cultured overnight at 30,000 cells/well with either 1 live hemagglutinin unit/well PR8 virus or 6 μg/ml CpG. Harvested cells were analyzed by flow cytometry for their surface expression of CD2. Upper panels, CD2low pDC isotype control, CD2 staining after sort, CD2 staining after PR8 exposure, and CD2 staining after CpG exposure. Lower panels, CD2high pDCs after PR8 exposure. Representative experiment of four performed testing different conditions of pDC activation is shown.

Thus, pDCs can be separated according to CD2 expression, and CD2high and CD2low pDCs display comparable morphology and ultrastructure.

CD2high pDCs express lysozyme

A microarray analysis of sorted CD2high and CD2low subsets of pDCs revealed that the most significantly overexpressed transcript in CD2high pDCs was lysozyme (139-fold overexpression). This is unexpected as lysozyme is known to be a product of myeloid cells such as neutrophils, monocytes as well as epithelial cells and plays a role in the breakdown of bacterial cell walls (42) (Fig. 4⇓A). Accordingly, CD2high pDCs exclusively expressed lysozyme in their intracellular compartments (Fig. 4⇓B), thus confirming the existence of two distinct pDC subsets. However, adding lysozyme to cocultures of CD2low pDCs and K562 cells was not sufficient to reproduce K562 cell killing (data not shown). Interestingly, cells costaining with anti-BDCA-2 and anti-lysozyme Abs could be observed in biopsies of melanoma tumors from patients (Fig. 4⇓C). Transcription of several other molecules could distinguish the two subsets (Fig. 4⇓, A and D). Beside lysozyme, the top five transcripts overexpressed in CD2high pDCs included Gas7, a member of growth arrest-specific genes expressed in neurons (43) and possibly involved in dendrite formation (44); AXL, a member of the Tyro 3 family of receptor tyrosine kinases with anti-inflammatory activities (45) that is involved in type I IFN-mediated suppression of TNF production (46); CHST6, a gene encoding an enzyme carbohydrate sulfotransferase (47); and RGC32, a member of response genes to complement (48), whose expression was found up-regulated in blood cells of patients with hyper-IgE syndrome (49) (Fig. 4⇓A). The transcription pattern of CD2low pDCs was strikingly different and the most overexpressed transcript NSEP1 encodes YB-1 (50), a transcription factor involved in the regulation of the cell cycle (50) as well as in biological effects of IFN-γ (51, 52). Interestingly, YB-1 protein appears to interact with different viruses including HIV (53, 54). Other molecules implicated in cell cycle regulation were also transcribed by CD2low pDCs including CDKN2D, an inhibitor of cyclin-dependent kinases (55) and CCNL2, cyclin L2 (56). Finally, the top transcripts also encompassed molecules relevant for endoplasmic reticulum such as COPE (57) (Fig. 4⇓D) and RRBP1 ribosome-binding protein (58) (data not shown).

FIGURE 4.
FIGURE 4.

CD2high pDCs and CD2low pDCs show distinct transcriptional profiles. A, Top 10 genes overexpressed in CD2high pDCs compared with CD2low pDCs (determined by fold change). Microarray scaled expression data; two experiments with pDC subsets sorted from two different donors. B, Sorted pDCs subsets were stained with FITC-conjugated anti-lysozyme mAb and either biotin- conjugated anti-BDCA2 or anti-granzyme-B mAb followed by treatment with either Alexa Fluor 568-labeled streptavidin or goat anti-mouse IgG2a, respectively. Images were acquired on a Leica DMIRBE microscope with the ×63 Plan APO objective using Leica TCS version 2.1 software. C, Staining of melanoma tumor. D, Top 10 genes overexpressed in CD2low pDCs compared with CD2high pDCs (determined by fold change). Microarray scaled expression data; two experiments with pDC subsets sorted from two different donors. Inset plot shows genes with expression values <1000.

Thus, pDC subsets distinguished by CD2 display distinct and unique transcription and protein expression profiles.

Activated CD2high pDCs form tight clusters with other cells

Both pDC subsets responded to activation via CD40 with increased transcription and expression of granzyme B and TRAIL (data not shown). Cell tracking in live microscopy indicated that, after 1 h, ∼30% of CD2high pDCs, but <10% of CD2low pDCs, were capable of interacting with K562 cells (Fig. 5⇓A, movie Fig. S16). After 8 h of observation, up to 80% of CD2high pDCs were interacting with K562 cells when <20% of CD2low pDCs interacted with K562 (Fig. 5⇓A). The interactions between CD2high pDCs and K562 cells involved several pDCs bound to a K562 target cell (Fig. 5⇓B) and lead in many instances to K562 cell death (movie Fig. S2). The formation of very tight clusters with other cells might permit pDCs to “screen” their surface and may serve as means to eliminate cells and/or capture Ags for example through nibbling (59) as it has been described for mDCs. Thus, CD2high pDCs could represent a link between the innate and the adaptive immunity in the context of cancer.

FIGURE 5.
FIGURE 5.

CD2high pDCs strongly bind target cells. A, Activated CD2high or CD2low pDCs were mixed with K562 cells at 2:1 ratio and monitored in a 37°C/5% CO2 live cell imaging chamber. The percentage of pDCs interacting with K562 cells was determined through an analysis of time-course image stacks. Percent interaction is expressed as the ratio of bound pDCs over the total number of pDCs in the frame for that time point. B, K562 cell (green) surrounded by tightly bound CD2+ pDCs.

Activated CD2high pDCs induce proliferation of naive allogeneic T cells

We next analyzed the capacity of CD2high and CD2low pDCs to present Ag to T cells. CD2high and CD2low pDCs were pulsed with influenza virus and cocultured for 6 days with CFSE-labeled autologous CD4+ and CD8+T cells. As shown in Fig. 6⇓A, both subsets were able to trigger proliferation of autologous T cells to influenza virus. To determine their capacity to launch primary immune responses, pDC subsets were activated with CpG and used in standard MLR. As shown in Fig. 6⇓B, CD2high pDCs were more efficient than CD2low pDCs in the induction of naive allogeneic T cell proliferation as measured by thymidine incorporation. These data were further confirmed by the analysis of T cell division by CFSE dilution (Fig. 6⇓C). The superiority of CD2high pDCs in stimulating MLR was observed with purified CD4+ T cells (mean ± SEM = 18 ± 3.5% vs 1.6 ± 0.6% for CD2high and CD2low pDCs, respectively, p = 0.02; Fig. 6⇓D) and with CD8+T cells (mean ± SEM = 4 ± 0.6% vs 0.3 ± 0.1% for CD2high and CD2low pDCs, respectively, p = 0.009; Fig. 6⇓D). However, despite their lower proliferation rate, on a per cell basis, T cells exposed to CD2low pDCs produced equivalent levels of IL-2, IL-10, and IFN-γ as those exposed to CD2high pDCs (Fig. 6⇓E).

FIGURE 6.
FIGURE 6.

CD2 expression discriminates two subsets of pDCs, one of which is immunogenic. A, Sorted pDC subsets were pulsed with heat-inactivated influenza virus and used in cocultures with CFSE-labeled autologous T cells. T cell proliferation (CFSE dilution) was measured at day 6. B–D, Sorted pDC subsets were activated with CpG and plated with naive allogeneic T cells. T cell proliferation was measured by thymidine incorporation (B; representative experiment of three performed) or by CFSE dilution (C; representative experiment) (D; four independent experiments). E, Supernatants of pDC/T cell cocultures were analyzed at day 5 for cytokine secretion.

Upon challenge with live Influenza virus or CpG, CD2high pDCs and CD2low pDCs secreted comparable amounts of IFN-α, IP-10, and MIP1-α (Fig. 7⇓A and data not shown). Interestingly, CD2high pDCs activated with live influenza virus secreted higher levels of IL12p40 than CD2low pDCs isolated from the same donor (Fig. 7⇓B). A similar difference in IL12 p40 secretion could be observed after activation with CpG (data not shown). Flow cytometry analysis demonstrated the same levels of MHC class I or MHC class II molecule expression by the two pDC subsets (Fig. 7⇓C). Both subsets also express CD83 typically found on mature DCs (data not shown). Analysis of costimulatory molecules expression showed that CD2high pDCs can up-regulate CD80 after activation with CpG while CD2low pDCs do so poorly (Fig. 7⇓D). Expression of CD86 is more variable and both subsets can up-regulate CD86 although CD2high pDCs show higher intensity of staining (Fig. 7⇓E).

FIGURE 7.
FIGURE 7.

Activated CD2high pDCs and CD2low pDCs show unique and distinct features. A and B, Cytokine secretion (ng/ml, ordinate) by pDC subsets after activation with influenza virus (Luminex analysis). A, Representative of eight experiments; B, five different donors. Color indicates pDC subsets isolated from the same donor; 25,000–30,000 cells/well. C–E, Flow cytometry analysis of protein expression by activated pDC subsets: MHC class I and class II molecules (B; two different donors); CD80 (C; two different donors), and CD86 (D; representative of four different donors). Values indicate mean fluorescence intensity.

Thus, upon activation, pDC subsets defined by high expression of CD2 secrete higher levels of IL12 p40 and show higher expression of CD80 and a distinct capacity to trigger naive T cell expansion.

Discussion

The present study demonstrates that human pDCs are composed of two subsets, which can be distinguished by the expression of CD2. These two subsets are found not only in blood but also in tonsils. In addition to their circulation in the blood and secondary lymphoid organs such as tonsils, CD2high pDCs can be detected in some tumor biopsies, suggesting their potential involvement in tumor immunosurveillance. Both subsets secrete IFN-α and express the cytotoxic molecules granzyme B and TRAIL. The CD2high pDCs are potent in initiating T cell immune responses. In contrast, the CD2low pDCs appear to display a limited capacity to induce allogeneic T cell proliferation. These different functional properties of CD2high pDCs and CD2low pDCs are associated with distinct transcription profiles, differential secretion of IL12p40, and differential expression of costimulatory molecule CD80 on activation.

In concordance with other studies, granzyme B inhibitor does not seem to affect cytotoxic function of pDCs. Granzyme B-mediated killing of target cells seems to be completely dependent on the presence of perforin (60, 61, 62), although an alternative mechanism has been suggested (63). Because pDCs do not express perforin, they must either use another protein that can substitute or they use granzyme for biological function other than killing. Certain acute viral (HIV, EBV) infections seem associated with elevated plasma levels of granzymes A and B (64). Although it was considered to be associated with an ongoing CTL activation (64), the source of extracellular granzyme B in these patients may need to be revisited in the view of our results. Furthermore, elevated granzyme B plasma levels were also observed in patients with parasite infection with Plasmodium falciparum (65) and in severe bacterial infection (66). These in vivo observations are consistent with high secretion triggered in vitro by pDC activation via CD40 and TLR ligands, respectively. Recent studies demonstrate the involvement of granzyme B in extracellular matrix remodeling via cleavage of vitronectin, laminin, and fibronectin (67). That may lead in vitro to anoikis, cell death due to lack of extracellular contact (67). This, along with their capacity to secrete granzyme B, could point to a novel role of pDCs in inflammation as well as tumor development and progression.

Thus, the killing mechanism remains to be established. Three recent studies in the human and in the mouse suggested a role of TRAIL-mediated cytotoxicity exerted by cells with pDC properties (68, 69, 70). Our preliminary results corroborate the potential role of TRAIL (data not shown); however, the inhibition is not complete, suggesting contribution of other mechanisms. The killing is unlikely to be due to contaminating NK cells since it requires a high E:T target ratio and is slow (NK cell-mediated lysis usually occurs at 4 h). We did not detect IFN-γ transcription or secretion as well as we did not detect perforin (data not shown).

Resting CD2high pDCs highly transcribe genes involved in the immune functions such as lysozyme, AXL, or fractalkine receptor. Another feature is the expression of two genes linked to Th2 polarization (ALOX5) and Th2 signature such as IgE (RGC32). Thus, CD2high pDCs might be responsible for the capacity of pDCs to induce type 2 immunity (10). Conversely, CD2low pDCs highly transcribe genes involved in cell cycle regulation and in secretory functions. Nonactivated CD2high pDCs also show 2-fold higher transcription of CD2 as compared with nonactivated CD2low pDC (supplemental Fig. 3). Whereas upon activation both subsets express MHC class molecules and CD83, CD2high pDCs up-regulate CD80. Although both subsets secrete similar levels of cytokines and chemokines such as IL-6, IL-8, TNF α, IFN α, IP-10, MIP1-α, and MIP1-β on activation, CD2high pDCs secrete IL12p40. Preliminary results show 2-fold enhanced transcription of p19 in microarrays of activated CD2high pDCs (data not shown). Thus, the identification of common and unique transcripts further supports the notion of two pDC subsets in humans.

The differential expression of lymphoid-related genes (RAG1 and Ig rearrangement products) (71) or proteins (CD4) (72) and Ly49Q (73) has been used to demonstrate the existence of murine pDC subsets. Similar to our results in humans, murine pDC subsets differ in their capacity to trigger allogeneic T cell proliferation and to secrete IL12p70 and IFN-γ (71).

pDCs have been shown to arise from both myeloid and lymphoid-committed hematopoietic progenitor cells (74, 75). The CD2 subset clearly demonstrates the prevalent expression of myeloid-related genes, thus suggesting that pDCs originating from committed myeloid progenitors might have distinct functions than those originating from committed lymphoid progenitors. Thus, both in humans and mice, pDCs constitute a heterogeneous population of cells which display a remarkable array of immune functions. The existence of two pDC subsets might provide a framework for explanation of a diverse array of immune responses elicited by pDCs ranging from CD8+ T cell tolerance/anergy, generation of regulatory T cells, to Th2 polarization.

Acknowledgments

We thank Elizabeth Kraus and Sebastien Coquery for help in flow cytometry and cell sorting; Windy Allman for help with microarrays; Sophie Paczesny for help with analysis of Ag-specific CD8+T cells; Cindy Samuelsen, Carson Harrod, and Nicolas Taquet for administrative and facility support; Dr. Gerard Zurawski for critical reading of this manuscript; and Dr. Michael Ramsay for continuous support.

Disclosures

The authors have no financial conflict of interest.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 This work was supported by Baylor Health Care Systems Foundation and the National Institutes of Health (U19 AIO57234 and CA78846 to J.B.). J.B. is the recipient of the Caruth Chair for Transplantation Immunology Research. A.K.P. is the recipient of the Michael A. Ramsay Chair for Cancer Immunology Research.

  • ↵2 T.M., J.E.C., and M.M. contributed equally.

  • ↵3 M.P. passed away in 2008.

  • ↵4 Address correspondence and reprint requests to Dr. A. Karolina Palucka and Dr. Jacques Banchereau, Baylor Institute for Immunology Research, 3434 Live Oak, Dallas, TX 75204. E-mail addresses: karolinp{at}baylorhealth.edu and jacquesb{at}baylorhealth.edu

  • ↵5 Abbreviations used in this paper: DC, dendritic cell; pDC, plasmacytoid DC; mDC, myeloid DC; SLE, systemic lupus erythematosus.

  • ↵6 The online version of this article contains supplemental material.

  • Received June 24, 2008.
  • Accepted March 22, 2009.

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