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The Journal of Immunology, 2006, 177: 1028-1039.
Copyright © 2006 by The American Association of Immunologists

Activation of Plasmacytoid Dendritic Cells with TLR9 Agonists Initiates Invariant NKT Cell-Mediated Cross-Talk with Myeloid Dendritic Cells1

Carlos J. Montoya*,{dagger}, Hyun-Bae Jie§, Lena Al-Harthi*, Candice Mulder*, Pablo J. Patiño{dagger}, María T. Rugeles{dagger}, Arthur M. Krieg{ddagger}, Alan L. Landay2,* and S. Brian Wilson2,§

* Department of Immunology and Microbiology, Rush University Medical Center, Chicago, IL 60612; {dagger} Group of Immunovirology, Biogenesis Corporation, University of Antioquia, Medellin, Colombia, South America; {ddagger} Coley Pharmaceutical Group, Wellesley, MA 02481; and § Diabetes Unit, Massachusetts General Hospital, Boston, MA 02139


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
CD1d-restricted invariant NK T (iNKT) cells and dendritic cells (DCs) have been shown to play crucial roles in various types of immune responses, including TLR9-dependent antiviral responses initiated by plasmacytoid DCs (pDCs). However, the mechanism by which this occurs is enigmatic because TLRs are absent in iNKT cells and human pDCs do not express CD1d. To explore this process, pDCs were activated with CpG oligodeoxyribonucleotides, which stimulated the secretion of several cytokines such as type I and TNF-{alpha}. These cytokines and other soluble factors potently induced the expression of activation markers on iNKT cells, selectively enhanced double-negative iNKT cell survival, but did not induce their expansion or production of cytokines. Notably, pDC-derived factors licensed iNKT cells to respond to myeloid DCs: an important downstream cellular target of iNKT cell effector function and a critical contributor to the initiation of adaptive immune responses. This interaction supports the notion that iNKT cells can mediate cross-talk between DC subsets known to express mutually exclusive TLR and cytokine profiles.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Activation of the innate immune response is crucial to control the early invasion of pathogens and the subsequent establishment of adaptive responses. Myeloid dendritic cells (mDCs)3 capture Ags in peripheral tissues and migrate to secondary lymphoid nodes to instruct naive T cells, while plasmacytoid DCs (pDCs) enter to the lymphoid nodes directly via the blood (1, 2, 3, 4). Resting DCs that capture self-Ags in the steady state are able to induce tolerance, (5, 6) whereas in the presence of inflammation, these same cells are able to initiate adaptive immune responses (7). A significant component of this discrimination is controlled by pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs) (8, 9). The TLR family is the best-characterized class of PRRs. TLR recognition of PAMPs initiates inflammatory responses by orchestrating the recruitment of leukocytes and the activation of stromal and resident innate cells.

Resident DCs are among the first cells to be activated after pathogen invasion. These APCs are central to the activation of NK cells and to T lymphocyte activation (10, 11, 12). A major activation pathway by which DCs mature occurs via TLR recognition of PAMPs (4, 13). Importantly, there are several functional classes of DCs, each of which expresses a different complement of TLRs (14, 15). This is what has given rise to the notion that DC subsets occupy special distinct niches responding to specific types of pathogens through expression of distinct sets of TLRs (9, 16, 17).

Once invading pathogens activate innate cells expressing the necessary TLR, such as viruses interacting with TLR9 on pDCs, these cells rapidly produce IFNs, chemokines, and cytokines (8). After activation, these DCs undergo a complex modulation of chemokine responsiveness, and maturation during migration into T cell areas of secondary lymphoid organs where they activate T cells (18, 19, 20).

pDCs selectively express TLR7 and TLR9 and are specialized cells that rapidly produce massive amounts of type I IFN (IFN-I) following viral infection (21, 22). This burst of IFN-I secretion is important for both pDC and mDC differentiation and migration, and the ability of mDC to cross prime antiviral CTL (1, 7, 23, 24, 25). In addition to specific viruses, bacterial DNA and the archetypal TLR9 agonist, CpG oligodeoxyribonucleotides (ODNs), induce the activation and maturation of pDCs and mDCs, stimulate monocytes and B cells, and enhance Th1 cytokine production by activated T and NK cells (4, 26, 27). There are three distinct classes of CpG ODNs which are TLR9 agonists with different patterns of immune activation. They are the A-, B- and C-class CpG ODNs, which differ in that the A-class ODNs activate pDC to mature and to secrete high levels of IFN-{alpha}, but do not strongly activate B cells; the B-class ODNs activate B cells and mature pDCs, but only weakly induce pDC secretion of IFN-{alpha}, and the C-class ODNs exhibit the combined but attenuated effects of both the A and B classes.

Interestingly, CD1d-restricted invariant NK T (iNKT) cells, a subset of lymphocytes considered innate-like, make important functional contributions to these very same immune processes, including the CpG ODN-dependent responses, despite not expressing detectable TLRs. Almost all iNKT cells recognize glycolipid Ags presented by CD1d. The regulation of mDC maturation and cytokine secretion by iNKT cells is an important component of their effector function (28, 29, 30, 31, 32, 33) Recently, Steinman and coworkers (34) have argued that this interaction is a major control mechanism for this process that is independent of TLR signaling. It is not known how TLR9-induced signals affect iNKT cells; however, the activation of iNKT cells in vivo clearly can lead to the subsequent activation of mDCs, B cells, monocytes, NK cells, and T cells (30, 35, 36, 37) Thus, it seems reasonable to speculate that the immune responses regulated either by activated iNKT cells or TLR agonist-activated pDCs would overlap.

Despite these overlapping regulatory activities between DCs and iNKT cells, their possible interaction during the human immune response has not been explored extensively. Moreover, most of the regulatory studies in vitro have been with human DCs derived from monocytes incubated with recombinant cytokines (GM-CSF, IL-4, and/or TNF-{alpha}), and it is unclear how immature DCs and iNKT cells, normally present in peripheral blood, may be activated by TLR agonists and interact with one another to modulate immune responses. Thus, using the model of innate immune activation with the TLR9 agonists CpG ODNs, we evaluated the interaction between CpG ODN-activated pDCs, iNKT cells, and mDCs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Abs and reagents

Fluorochrome-labeled mAbs against human molecules 6B11, CD3, CD4, CD8, CD11c, CD19, CD25, CD38, CD40, CD69, CD86, CD45RO, CD123, CD154, HLA-DR, perforin, IFN-{gamma}, IL-4, TNF-{alpha}, lineage markers (anti-CD3, CD14, CD16, CD19, CD20, and CD56) and isotype control Abs were obtained from BD Pharmingen.

FITC-labeled anti-Vbeta11 and PE-labeled anti-V{alpha}24 were obtained from Beckman Coulter/Immunotech. Fc{gamma}R blocking reagent, anti-PE magnetic beads, and isolation kits for mDCs and pDCs were obtained from Miltenyi Biotec.

Recombinant human (h) IFN-{alpha} and neutralizing mAbs against IFN-{alpha}, IFN-beta, and CD118 were obtained from PBL Biomedical Laboratories. Neutralizing Abs against IL-6 and TNF-{alpha} were from BD Pharmingen, and anti-IP-10 was obtained from R&D Systems. PMA and ionomycin were obtained from Sigma-Aldrich; {alpha}-galactosylceramide ({alpha}-GalCer) was from Kirin Brewery.

CpG ODNs

Synthetic endotoxin-free ODNs were provided by Coley Pharmaceutical Group (lowercase letters, phosphorothioate linkage; capital letters, phosphodiester linkage 3' of the base; underlined letters, CpG dinucleotides):

A-class CpG ODN 2216 5'-ggGGGACGATCGTCgggggG-3'; A-class control ODN 2243 5'-ggGGGAGCATGCTGgggggG-3'; B-class CpG ODN 2006 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3'; C-class CpG ODN 2395 5'-TCGTCGTTTTCGGCGCGCGCCG-3'; B- and C-class control ODN 2137 5'-TGCTGCTTTTGTGCTTTTTGCTT-3'.

Isolation and culture of mononuclear cells

Heparinized whole-blood samples were obtained from healthy adult donors after obtaining informed consent for an approved protocol Institutional Review Board ORA#0306110. PBMCs were isolated by Ficoll gradient (BioWhittaker). The viability of PBMCs was determined by trypan blue exclusion.

PBMCs (1 x 106/ml) were suspended in complete culture medium (RPMI 1640 supplemented with 10% of heat-inactivated FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine). ODNs were used at concentrations of 0.04–4 µg/ml; PMA at 50 ng/ml and ionomycin at 500 ng/ml. Cultures were incubated at 37°C in 5% CO2. Culture supernatants for ELISA were collected and stored at –80°C until the measurement of cytokine levels.

Conditioned supernatants from PBMCs or cells incubated for 24 h either with or without 4 µg/ml A-class control ODN or A-class CpG ODN were collected, filtered with a 0.22-µm filter, and stored at –20°C until their use with fresh cells.

Cytokine blockade

Freshly isolated cells resuspended in complete medium (1 x 106/ml) were cultured, stimulated with/without A-class CpG ODN (4 µg/ml), rh-IFN-{alpha} (5000 U/ml), or PBMC-conditioned supernatant (dilutions: 1/20, 1/10, 1/5, and 1/2). For blockade, neutralizing Abs were added 1 h before the addition of the stimuli: anti-IFN-{alpha} (10,000 U/ml), anti-IFN-beta (3,000 U/ml), anti-IFN-IR (CD118, 10 µg/ml), anti-TNF-{alpha} (10 µg/ml), anti-IL-6 (0.1 µg/ml), and anti-IP-10 (10 µg/ml).

Isolation of DC and iNKT cells

BDCA-1 positive (mDC) or BDCA-2 positive (pDC) cells were magnetically isolated from 7 x 107 PBMCs per the manufacturer’s specifications (BDCA-1 and BDCA-2 DC isolation kits; Miltenyi Biotec), using an AutoMACS (Miltenyi Biotec) set to run the POSSELD software program. Negative selection of pDCs and B cells was done with BDCA-2 and CD19 microbeads per the manufacturer’s recommendations. The number and viability of these purified DC subgroups were determined by trypan blue exclusion, and purity assessed by flow cytometry.

To isolate iNKT cells, 8 to 10 x 107 PBMCs were incubated 20 min on ice with 10 µg of PE labeled-anti invariant NKT cell mAb (clone 6B11; BD Pharmingen) and 100 µl of FcR{gamma}-blocking reagent (Miltenyi Biotec). The 6B11+ cells were then isolated using anti-PE microbeads (Miltenyi Biotec) as per manufacturers specifications and were magnetically isolated using the program Posseld on an AutoMACS instrument. Viability and purity were determined as described for DCs.

Coculture of purified iNKT cells, pDCs, and mDCs

Purified iNKT cells (30K cells) were cultured alone, with either pDCs (20K cells) or mDCs (20K cells) in 0.5 ml of complete culture medium and incubated either with/without A-class or control ODN; after 24 h of culture, cells were analyzed by flow cytometry.

Culture of purified pDCs with A-class CpG ODN

Freshly isolated BDCA-2-positive cells were cultured in 24-well plates (2.5 x 105 pDC/ml) and incubated with 4 µg/ml A-class CpG ODN during 24 h at 37°C/5% CO2. Then, the pDC-conditioned supernatants were collected centrifuged 10 min/500 x g/RT, filtered with a 0.22-µm filter, and stored at –20°C until their use for incubation with purified iNKT cells and for analysis of cytokine production by ELISA.

Incubation of purified iNKT cells with pDC-conditioned supernatant

Purified iNKT cells (2.5 x 105 cells/ml) were incubated for 24 h/37°C/5% CO2 with or without pDC-conditioned supernatant (dilution 1/2 with complete culture medium). Cells were washed with RPMI 1640 medium and resuspended in complete culture medium before the coculture with {alpha}-GalCer-loaded mDCs.

Coculture of preactivated iNKT cells and {alpha}-GalCer-loaded mDC

Purified mDCs were resuspended in complete medium and incubated with or without {alpha}-GalCer (10, 20, 50, or 100 ng/ml) for 24 h/37°C/5% CO2. In parallel, iNKT cells were preincubated with or without pDC-conditioned supernatants and then cocultured with the {alpha}-GalCer-loaded mDCs at a ratio of 25K:10K, respectively. After 24 h of incubation, 100 µl of culture supernatant was gently collected from each well and stored at –20°C until analysis by ELISA. Then, 100 µl of fresh complete culture medium was added to each well, and the plate was incubated for another 24 h at 37°C/5% CO2. Afterward, 1 µCi per well of [3H]thymidine was added,and the plate was again incubated overnight; finally, the culture was harvested after 24 h of culture, and incorporated cpms were counted using a 1205 Beta Plate (Pharmacia).

Flow cytometry

Phenotypic analysis of iNKT cells, mDCs, and pDCs was performed by three- or four-color flow cytometry. For iNKT cell characterization, the following combinations of mAbs were used: Vbeta11 FITC/V{alpha}24PE, Vbeta11 FITC/6B11 PE, or 6B11 PE/CD3 PcP. CD4-APC and 6B11-PE was used for iNKT cell subset analysis. Evaluation of mDC and pDC was done with the combinations Lin FITC/CD11c PE/HLA-DR PcP and Lin FITC/CD123 PE/HLA-DR PcP, respectively. PBMCs were labeled with CFSE per the manufacturer’s specifications (Invitrogen Life Technologies/Molecular Probes).

Intracellular stainings were performed following the manufacturer’s recommendations (BD Pharmingen). Brefeldin A solution (1 x 40 µl per well; BD Biosciences) was added during the last 6–12 h of culture. After the staining for extracellular Ags, cells were incubated with 500 µl of 1x permeabilizing solution 2 (BD Biosciences) during 10 min/room temperature/dark. Isotype-matched control Abs were included for all experiments as controls for nonspecific binding. Because of the low frequency of iNKT cells and DCs in PBMCs, from 2.5 to 5 x 105 total gated cells were analyzed for each data point. Dead cells were gated out by forward and side scatter. Flow cytometry was performed using the FACSCalibur (BD Biosciences) and analyzed with CellQuest software (BD Biosciences).

Detection of cytokines by ELISA

Commercially available kits were used to determine cytokine concentrations in culture supernatants, and assays were performed per the manufacturer’s specifications; IFN-{alpha}, TNF-{alpha}, IL-6, and IL-12 were obtained from Pierce Endogen; and IFN-{gamma}, IP-10, IL-4, IL-7, IL-15, and IL-18 were obtained from R&D Systems.

Statistical analysis

Data are shown as a mean ± SD of three or more independent experiments. Statistical analysis for the comparison of different stimuli was performed using Student’s two-tailed t test. A value of p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CpG ODN-stimulated PBMCs activate iNKT cells in culture

To evaluate the interaction of pDCs, mDCs, and iNKT cells during the development of TLR9-dependent innate responses, PBMCs were incubated with different classes of CpG ODNs or their respective ODN controls. CpG ODNs induce the expression of activation markers on particular cell types (4, 27, 38) Thus, the expression of CD40 on pDCs and mDCs, CD69 on iNKT cells, and CD86 on B lymphocytes was determined. Incubation of PBMCs with A-class CpG ODN induced the highest expression of CD40 on both pDCs and mDCs, while B and C classes of C CpG ODNs led to the highest expression of CD86 on B cells (Table I). Unexpectedly, CpG ODN markedly stimulated CD69 expression on iNKT cells. When compared with A- and B-class ODNs, C-class CpG ODNs induced an intermediate level of expression of CD40 and CD69 on DCs and iNKT cells, respectively.


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  		Table I. Effect of different classes of ODNs on the expression of activation markers by immune cells (n = 4)a

 
Because A-class CpG ODN was the most potent ODN for inducing activation markers on DCs and iNKT cells and is thought to be pDC selective, this ODN was selected for further study. Dose-response experiments indicated that the incubation of PBMCs with 4 µg/ml A-class CpG ODNs induced the highest secretion of IFN-{alpha} and expression of CD40 on the positive control pDC population and CD69 on iNKT cells (Fig. 1). Consequently, this concentration elicited equivalent maximal responses in both the iNKT and pDC populations; the A-class CpG ODN was selected to evaluate the interaction between pDC and iNKT cells.


Figure 1
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  		FIGURE 1. Dose-response of innate immune cells to A-class CpG ODN. PBMCs were incubated for 24 h with different concentrations of A-class ODNs (control and CpG ODN), and the expression of activation markers and the secretion of IFN-{alpha} were evaluated by flow cytometry and ELISA, respectively. A, During the analysis, mononuclear cells were included in a region (R1) created in the forward vs side scatter, and used to define the lineage-negative/HLA-DR-positive cells (R2); in the later region, the pDCs were determined as the cells bright for CD123 (R3) and mDCs as cells bright for CD11c. In the later subpopulation the expression of CD40 was analyzed (upper panel, representative dot plots for pDCs). For iNKT cells, the lymphocytes (R1) positive for both CD3 and 6B11 were defined (R2), and in this later region, the expression of CD69 was evaluated (lower panel). B, The incubation with 4 µg/ml A-class CpG ODN induced the highest secretion of IFN-{alpha} and expression of CD40 on pDC and CD69 on iNKT cells. Data are reported as means ± SD (n = 4). *, p < 0.05

 
To determine whether treatment of PBMCs with A-class CpG could expand the iNKT cell population, the frequency of these T cells in PBMCs was measured after 18, 24, 36, 48, and 72 h of cell culture. There were no significant differences in the percentage of iNKT cells observed over the time course of activation (data not shown). Because human iNKT cell subsets are CD4+, CD8{alpha}{alpha}+, CD4–/CD8{alpha}{alpha}– (double negative, DN), it was important to determine whether the relative proportions and activation status of these subsets in response to A-class CpG ODNs stimulation were subset specific. Treatment of PBMCs with A-class CpG ODNs did not significantly modify the proportion of CD4+, CD8+, DN and double-positive (DP) iNKT cells or selectively activate any particular subset (Fig. 2 and data not shown).


Figure 2
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  		FIGURE 2. Frequency of iNKT cell subgroups in culture of PBMCs with A-class ODNs. PBMCs were incubated for 24 h with 4 µg/ml A-class ODNs (control and CpG ODN) and the expression of CD4 and CD8 on iNKT cells was evaluated by flow cytometry. A, iNKT cells were detected in the lymphocyte region (R1) as cells DP for Vbeta11 and V{alpha}24 (R2); in this later subpopulation, the frequency of iNKT cell subgroups was defined according to the expression of the molecules CD4 and CD8 and the conditions of PBMC stimulation. B, Summary of the data showing that, in comparison to PBMCs not stimulated or incubated with A-class control ODN, the incubation with A-class CpG ODN did not significantly modify the proportion of CD4+, CD8+, and DN and DP iNKT cells. Data are reported as means ± SD (n = 5).

 
The combination anti-V{alpha}24/anti-Vbeta11 identifies a population of T cells whose TCR coexpression for both chains is highly enriched for iNKT cells but not specific for the invariant V{alpha}24J{alpha}18 TCR {alpha}-chain (39). To validate that the population of T cells identified for the donors used in this work were iNKT cells, the frequency of iNKT cells was determined in parallel using the combination anti-Vbeta11 with the invariant chain CDR3 loop-specific mAb 6B11. For all donors used there were no significant differences between the frequencies of V{alpha}24/Vbeta11+ T cells and 6B11/Vbeta11+ T cells (Fig. 3A). Hence, the frequency of iNKT cell subsets was unaffected by activation of PBMCs with CpG ODNs.


Figure 3
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  		FIGURE 3. Flow cytometry detection of iNKT cells and impact of A-class ODNs on their expression of activation markers. A, For the analysis of iNKT cells in PBMCs, different combinations of mAbs were used; despite that the combination of anti-Vbeta11 and the clone 6B11 (anti-CDR3 loop of invariant {alpha}chain) more specifically detects the iNKT cells, there were no significant differences regarding the use of the combination anti-V{alpha}24/anti-Vbeta11. B, PBMCs were incubated for 24 h with 4 µg/ml A-class ODNs (control and CpG ODN) and the expression of CD25, CDCD38, CD45RO, CD69, CD154 and HLA-DR was evaluated by flow cytometry. In these representative histograms from one of five healthy subjects, it is shown that the incubation with A-class CpG ODNs up-regulated the expression of CD38 and CD69, while the expression of CD45RO and HLA-DR was not up-regulated. More than 80% of iNKT cells express CD45RO, a phenotypical marker of memory cells.

 
To further explore the effect of CpG ODNs on iNKT cells, the expression of other T cell activation markers was evaluated (Fig. 3B). When compared with basal conditions or control ODNs, A-class CpG ODNs significantly up-regulated the expression on iNKT cells of the early activation markers CD38 and CD69; the expression of CD25, CD154 and HLA-DR was unaffected (Fig. 3B and Table II). As expected, the T cell memory marker CD45RO was expressed on >80% of iNKT cells, independent of the condition of cell culture. The pattern of expression of these proteins after 18, 48, and 72 h of incubation with A-class control or CpG ODN, was identical with those at 24 h. Hence, CpG ODN treatment selectively induced iNKT cell activation markers, with A-class CpG ODNs being the most potent.


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  		Table II. Expression of activation markers on iNKT cells incubated with A-class CpG ODN (n = 5)a

 
A-class CpG ODN-mediated activation did not stimulate iNKT cell proliferation, cytokine secretion, or perforin expression

TCR- and/or cytokine-mediated activation of iNKT cells induces the rapid release of cytokines (40, 41) Because incubation with A-class CpG ODNs induced the expression of activation markers on iNKT cells, their ability to induce cytokine and perforin expression was evaluated by intracellular staining. Treatment of PBMCs with CpG ODNs did not increase IFN-{gamma}, IL-4, TNF-{alpha}, or perforin expression by iNKT cells (Fig. 4). PBMCs were incubated for 6 h with PMA/ionomycin as a positive control of activation, which significantly increased the expression of all three cytokines and perforin by iNKT cells. Notably, A-class ODN treatment induced CD69 expression to the same extent as did treatment with PMA and ionomycin. To determine whether the activation with CpG ODNs leads to either an early or late production of these molecules, we evaluated their expression after 12, 18, 48, and 72 h of incubation, and there was no significant differences when compared with the expression observed after 24 h.


Figure 4
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  		FIGURE 4. Impact of A-class ODNs on cytokine and perforin production by iNKT cells. PBMCs were incubated for 24 h with 4 µg/ml A-class ODNs (control and CpG ODN) and the expression by iNKT cells of IFN-g, IL-4, TNF-{alpha}, perforin and CD69 was evaluated by intracellular staining and flow cytometry. As a control for positive activation, the PBMCs were incubated by 6 h with PMA (50 ng/ml) and ionomycin (500 ng/ml). It is shown that, despite the stimulation with A-class CpG ODN up-regulating the expression of CD69, the incubation with this CpG ODNs did not increase the expression IFN-{gamma}, IL-4, TNF-{alpha} or perforin by iNKT cells. Data are reported as means ± SD (n = 3).

 
CpG ODN-dependent activation of iNKT cells is mediated by soluble factors

A-class CpG ODNs characteristically activate pDCs to produce massive amounts of IFN-I ({alpha}, beta, and {omega}) and stimulate their own maturation (4). This class of cytokines also is thought to be integral to the activation of other innate and adaptive immune cells such as NK cells, monocytes, and T lymphocytes (42, 43, 44, 45). To test whether the activation of iNKT cells was dependent on soluble factors, PBMCs were incubated with CpG ODNs for 24 h, and the concentration of cytokines that are characteristic of CpG ODN activation was determined (46). When compared with unstimulated PBMCs, CpG ODN treatment markedly increased the secretion of IFN-{alpha} and IP-10, and the production of TNF-{alpha} and IL-6 was modestly elevated. There was no significant increase in the secretion of other possible activators of iNKT cells such as IL-7, IL-12, IL-15, IL-18, and IFN-{gamma} (Table III). These results are consistent with other published findings (46).


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  		Table III. Concentration of cytokines in supernatants from PBMCs cultured with A-class CpG ODNa

 
To evaluate the potential role for IFN-I on iNKT cell activation, PBMCs were incubated with A-class CpG ODN with/without a mixture of mAbs to block the effect of IFN-I (neutralizing anti-IFN-{alpha}, neutralizing anti-IFN-beta, and neutralizing anti-IFN-I receptor (CD118)). Although incubation with CpG ODNs alone significantly increased the expression of CD69 on iNKT cells, the coincubation with CpG ODN and IFN-I blocking mixture reduced the expression of CD69 x 45% (p = 0.024) (Table IV). The effect of this blocking mixture was very heterogeneous in the healthy controls evaluated (range of CD69 reduction, 15–67%). As a positive control for these experiments, PBMCs were incubated with rh-IFN-{alpha} with/without the same blocking mixture. In all cases, rh-IFN-{alpha} up-regulated the expression of CD69 (56% ± 15%), while the blocking mixture was 95–100% effective at inhibiting iNKT cell activation by rh-IFN-{alpha}. This suggested that other cytokines/chemokines/soluble factors were potentially important for inducing activation markers on iNKT cells.


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  		Table IV. Role of IFN-I on A-Class CpG ODN-mediated activation of iNKT cells

 
The contributions of IFN-I and other soluble mediators of CpG ODN-induced activation of iNKT cells were confirmed using PBMC-conditioned supernatants. Fresh PBMCs were incubated 24 h with/without A-class CpG ODN, rh-IFN-{alpha}, PBMC-conditioned supernatant, and the anti-IFN-I mixture. The conditioned medium and rh-IFN-{alpha} consistently induced higher expression of CD69 than did optimal doses of CpG ODNs (Fig. 5A). The anti-IFN-I blocking mixture completely inhibited the expression of CD69 induced by rh-IFN-{alpha}, and only partially blocked the expression induced by CpG ODN or PBMC-conditioned supernatant.


Figure 5
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  		FIGURE 5. Role of cytokines on the A-class CpG ODN-mediated activation of iNKT cells. A, PBMCs were incubated 24 h with A-class CpG ODN (4 µg/ml), rh-IFN-{alpha} (5,000 U/ml) or supernatant from CpG ODN-stimulated PBMCs (dilution 1/2 with complete culture medium); the activation mediated by IFN-I was blocked with a combination of anti-IFN-{alpha} (10,000 U/ml), anti-IFN-beta (3,000 U/ml) and anti-CD118 (10 µg/ml). Then, the expression of CD69 on iNKT cells was evaluated by flow cytometry, gating on the iNKT cells with the mAbs 6B11 and anti-CD3. Blocking the IFN-I partially inhibited the expression of CD69 induced by CpG ODNs or PBMC-conditioned supernatant. Data are reported as means ± SD (n = 3). B, PBMCs cultured with A-class CpG ODNs were coincubated with neutralizing Abs against IFN-a (10,000 U/ml), TNF-{alpha} (10 µg/ml), IL-6 (0. 1 µg/ml), and IP-10 (10 µg/ml). After 24 h, the expression of CD69 on iNKT cells was determined by flow cytometry. The neutralization of IFN-{alpha} and TNF-{alpha} significantly decreased the CpG ODN-induced CD69 expression, while there was an additive effect observed when all the neutralizing Abs were used in combination. Data are reported as means ± SD (n = 3).

 
Because A-class CpG ODN stimulation of PBMCs also induced the secretion of IP-10, IL-6, and TNF-{alpha} in addition to IFN-{alpha}, neutralizing Abs against IP-10, IL-6, and TNF-{alpha} were used to evaluate the contributions these cytokines/chemokines in the activation of iNKT cells. Only neutralization of IFN-I and TNF-{alpha} had moderate yet equivalent inhibitory effects (Fig. 5B). Neutralization of IP-10 and IL-6 had no effect on expression of CD69 by iNKT cells. Simultaneous neutralization of all four cytokines had an additive effect but was only 40–50% effective at reducing CpG ODN-induced expression of CD69. This suggests that IFN-I and TNF-{alpha} were important for iNKT activation but other soluble factor(s) contribute to this process.

A-class CpG ODN-mediated activation of purified iNKT cells

Treatment of PBMCs with CpG ODNs activates several different subpopulations of leukocytes. To clarify the interaction mediated by soluble factors between CpG ODN-activated pDCs and iNKT cells, highly purified populations of cells were isolated using Miltenyi magnetic beads and an AutoMacs instrument. The purified pDCs were incubated for 24 h with A-class CpG ODN, and the pDC-conditioned supernatants were used to treat purified iNKT cells (Fig. 6A). The incubation of purified iNKT cells with pDC-conditioned supernatants resulted in the same up-regulated expression of CD69 (as well as CD38, data not shown), while the coincubation with combinations of neutralizing Abs was again only partially effective at inhibiting this response (Fig. 6B). It is important to note that the concentration of cytokines in supernatants from CpG ODN-stimulated purified pDC is lower than in supernatants from PBMCs. This is likely a consequence of using the BDCA2 Ab for positive selection of pDC. Although highly selective for this cell subset, the Ab inhibits subsetquent in vitro responses of pDC to TLR-dependent activation. However, the stimulation of purified pDC with CpG still resulted in secretion of the same cytokines/chemokines as was observed with PBMC-derived supernatants (IFN-{alpha}, PBMC = 1,247 + 477 pg/ml, pDC = 449 + 64 pg/ml; IP10, PBMC = 1,681 + 310 pg/ml, pDC = 917 + 466 pg/ml; TNF-{alpha}, PBMC = 489 + 208 pg/ml pDC = 263 + 79 pg/ml; IL-6, PBMC = 216 + 20 pg/ml, pDC = 167 + 50 pg/ml). Thus, the same concentration of neutralizing Abs used for the PBMC experiments was used for supernatants from purified pDC; because the concentration of secreted IFN-{alpha} and other cytokines was lower than that seen in PBMC, it is less likely that insufficient Abs were used. Hence, alleviating the concern that insufficient neutralization occurred. Using this reconstituted system neutralization by anti-IFN-I was modestly more effective when compared with the ability to inhibit the effect of conditioned medium prepared from CpG ODN-treated PBMCs. As was seen using conditioned medium from CpG ODN-treated PBMCs, maximal blockade of pDC-derived conditioned medium was only 50% effective and there was no contribution by neutralization of IP-10 and IL-6. Thus, the induction of activation markers on iNKT cells by CpG ODNs can be reproduced using highly purified pDC and iNKT cell systems suggesting that the active factors are released directly by pDCs. However, it should be noted that there might still be some contribution to this effect by rare contaminating cells (4).


Figure 6
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  		FIGURE 6. Neutralization of IFN-{alpha} and TNF-{alpha} partially inhibits the expression of CD69 on purified iNKT cells incubated with supernatants from CpG ODN-stimulated purified pDCs. A, The iNKT cells and pDCs were purified from PBMCs using anti-6B11 PE plus anti-PE microbeads or anti-BDCA-2 microbeads, respectively, and the POSSELD program of AutoMACS. In these representative dot plots, the percentage of pDCs and iNKT cells is shown in the positive fraction after the magnetic isolation. B, To obtain pDC-conditioned supernatants, pDCs were purified with anti-BDCA-2 microbeads and incubated by 24 h with A-class CpG ODN; these supernatants were collected, filtered (0. 22 µm), and used for the coincubation with purified iNKT cells and neutralizing Abs against IFN-{alpha} (10000 U/ml), TNF-{alpha} (10 µg/ml), IL-6 (0.1 µg/ml), and/or IP-10 (10 µg/m). The incubation of purified iNKT cells with pDC-conditioned supernatants up-regulated the expression of CD69, which was partially blocked by the neutralization of IFN-{alpha} and TNF-{alpha}. This graphic shows representative data from one of three independent experiments.

 
iNKT cells activated in a pDC-dependent manner are licensed to interact with mDC

Recent results indicate that mDC and iNKT cell cross-talk is important and that mDCs need to be activated in a TLR-dependent fashion to present self or foreign Ag to quiescent iNKT cells (33, 47, 48) Interestingly, human mDCs, but not pDCs express CD1d (4, 49) To evaluate the contribution of CD1d to CpG ODN-mediated iNKT cell activation, highly purified subpopulations of pDCs, mDCs, and iNKT cells were isolated and cocultured with/without ODNs (control and CpG). The purity of iNKT cells was always >85%, while the purity of mDCs and pDCs was >90% (Fig. 7A). Consistent with the observation that iNKT cells do not express TLR9, treatment of purified iNKT cells with A-class CpG ODNs did not result in the up-regulation of CD69 expression (Fig. 7B). Similar results were observed when purified iNKT cells and mDCs were cocultured and incubated without stimulation or with control and A-class CpG ODNs (p = 0.437 and p = 0.382). When iNKT cells were cocultured with pDCs and incubated with A-class CpG ODNs, the expression of CD69 on iNKT cells significantly increased in comparison with unstimulated cells or incubated with control ODNs (p = 0.020 and 0.007, respectively) (Fig. 7B). Thus, CpG ODN-mediated activation of iNKT cells is pDC dependent, and quiescent iNKT cells fail to respond to unstimulated mDCs when purified and cultured together in vitro.


Figure 7
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  		FIGURE 7. Role of ODNs on the activation of purified iNKT cells cocultured with purified DCs. A, The pDCs, mDCs and iNKT cells were purified from PBMCs using anti-BDCA-2 microbeads, anti-BDCA-1 microbeads, and anti-6B11 PE and anti-PE microbeads, respectively, and POSSELD program of AutoMACS. In these representative dot plots, it is shown the percentage of these leukocyte subpopulations in whole PBMCs, the negative fraction,and in the positive fraction, before and after the magnetic isolation, respectively. B, The purified iNKT cells were cocultured 24 h with/without purified mDCs, purified pDCs and A-class ODNs (control and CpG), and the expression of CD69 on iNKT cells was determined by flow cytometry. Just the coculture of purified iNKT cells with purified pDCs and incubated with A-class CpG ODN increased significantly the expression of CD69 on iNKT cells. These dot plots are representative of one of four independent experiments.

 
iNKT cells are strongly activated by TCR-mediated CD1d-restricted signals, particularly when mDCs are used as APCs. It also is well known that {alpha}-GalCer is presented by CD1d and a potent activator of iNKT cells (49, 50). To determine whether pDC-derived conditioned medium could license iNKT cells to respond to mDCs, purified iNKT cells were treated with conditioned or control medium and then cocultured with mDCs plus increasing doses of {alpha}-GalCer. First, purified iNKT cells were incubated with/without pDC-conditioned supernatant (dilution 1/2 in complete culture medium), and after 24 h of incubation, iNKT cells were washed, and cocultured with purified mDC with/without {alpha}-GalCer (0, 10, 20, and 50 ng/ml). Strikingly, only those iNKT cells that had been primed with conditioned medium from CpG ODN-treated pDCs were able to respond to {alpha}-GalCer-loaded mDCs. These preactivated iNKT cells acquired the ability to respond to {alpha}-GalCer in a dose-dependent fashion by secreting IFN-{gamma}, and to much lesser extent, IL-4, and vigorously proliferating (Fig. 8). In comparison, iNKT cells incubated with conditioned medium from control ODN-incubated pDCs did not secrete cytokines or proliferate. Thus, soluble factors produced by CpG ODN-stimulated pDCs enhance the CD1d-restricted activation of iNKT cells and licensed these T cells to interact with mDCs.


Figure 8
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  		FIGURE 8. Role of CpG ODN-activated pDCs on the CD1d-restricted activation of iNKT cells. A, Purified iNKT cells were incubated by 24 h with/without supernatants from CpG ODN-stimulated pDCs (dilution 1/2); after, these preincubated iNKT cells were washed and cocultured by triplicate with purified mDCs previously incubated by 24 h with/without {alpha}-GalCer (0, 10, 20, and 50 ng/ml). After 24 h of coculture, supernatants (100 µl per well) were collected and replaced with fresh complete culture medium. The concentration of IFN-{gamma} and IL-4 was measured in theses supernatants by ELISA; there was an increase in the secretion of IFN-g and IL-4 when the iNKT cells were preincubated with pDC-conditioned supernatants, and in this condition of stimulation it was observed a dose-response effect for the production of IFN-{gamma} regarding the concentration of {alpha}-GalCer used for the incubation with purified mDCs. This graphic shows representative data from one of four independent experiments. B, Twenty-four hours after the collection of supernatants, 1 µCi of [3H]thymidine was added to each well and, after an incubation overnight, the cell culture plate was harvested and the proliferation of the iNKT cells was determined measuring the cpm in a beta counter. In this representative figure of four independent experiments, it is shown that, when purified iNKT cells were preincubated with pDC-conditioned supernatant, there was an increase in the proliferation of iNKT cocultured with {alpha}-GalCer-loaded mDCs.

 
Because CpG ODN enhanced iNKT cell responses to {alpha}-GalCer-loaded mDC and TLR4 ligands have been shown to directly enhance CD1d-dependent Ag presentation to iNKT cells (33), the ability of ODNs to support expansion and survival of iNKT cells was evaluated. PBMCs were stimulated with {alpha}-GalCer in the presence or absence of CpG ODN or LPS, with or without depletion of pDC and B cells (Fig. 9). Interestingly, activation of iNKT cells in the presence of CpG resulted in the preferential expansion and long-term survival of DN iNKT cells. After deletion of pDC and B cells, only CD4+ iNKT cells expanded in response to {alpha}-GalCer. And, as expected, the deletion of TLR9-responding cells had a significant impact on the ability of iNKT cells to respond to {alpha}-GalCer. It also should be noted that, consistent with the results of Brigl et al. (33), LPS induced the most robust expansion of CD4+ and DN iNKT cells (data not shown), but these cells failed to survive in long-term cultures (Fig. 9B).


Figure 9
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  		FIGURE 9. Frequency of expanded iNKT cell subgroups in culture of PBMCs activated with {alpha}-GalCer ODNs. A, Effect of ODNs on iNKT cells subsets after {alpha}-GalCer activation in the presence or absence of pDC and B cells. Whole PBMC, or PBMC where pDC and B cells were removed using anti-BDCA-2 and anti-CD19 microbeads, were labeled with CFSE and cultured for 8 days with {alpha}-GalCer (100 ng/ml) with or without ODNs. Cells were stained with 6B11-PE and CD4-APC to enumerate iNKT cell subsets, and gated on 6B11+ cells to determine the extent of cell division by CFSE dilution. The data are representative of three separate experiments. B, Activation of iNKT cells with {alpha}-GalCer and ODNs results in selective long-term survival of DN iNKT cells. PBMC were activated with control IL-2 (20 U/ml), IL-2, and {alpha}-GalCer + ODNs, or IL-2 and {alpha}-GalCer + LPS (1 µg/ml) and cultured for 21 days. Cells were supplemented with IL-2 and fresh medium every seventh day. The data are representative of four separate experiments.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Freshly isolated resting pDCs express low levels of MHC class I and II and CD86 and do not express detectable levels of CD80 or CD1d. In humans, these cells express CD4 and CD123 but lack the expression of myeloid markers, such as CD11b, CD11c, CD13, and CD33, and are found in blood and secondary lymph organs (4). These cells participate in innate responses to several different types of viruses, including influenza, HSV-family viruses, and HIV (24). (51, 52) pDCs rapidly produce vast amounts of IFN-I within the first 24 h of viral infection (21, 22) This burst of cytokine secretion is thought to be critical for activation of other cells of the innate and adaptive immune systems, including the induction of mDC maturation (4). (23, 24). Over the next 48–72 h, in a process that is dependent on autocrine stimulation by IFN-I, pDCs differentiate into mature DCs capable of stimulating T cells (16). The pDC-dependent T cell effects are thought to be dependent on IFN-I but independent of IL-12. In human DCs, the main source of IL-12 important for activating NK cells and inducing Th1-like T cell responses is thought to be mDCs (53, 54) Consequently, as is the case with TLR profiles, there appears to be clear species differences between human and murine pDC and mDC effector function (4, 9). However, the pDC lineage is unique in the capacity to secrete massive amounts of IFN-I.

The cross-talk between pDCs and mDCs and the ability of pDCs to induce adaptive T cell responses by activating mDCs have recently been demonstrated (23, 24, 51, 55). Treatment of immature mDCs with IFN-I leads to their activation and enhanced production of IL-12, IL-15, and IL-18 (56, 57). A similar IFN-I-dependent or CpG-dependent effect has been noted for NK cell activation (55, 58).

Interestingly, iNKT cell activation is important for the same mDC maturation, NK activation, and antiviral or antitumor responses (28, 29, 31, 32, 36, 59, 60, 61, 62, 63) Yet, iNKT cells do not express detectable levels of TLRs, do not respond directly to CpG ODNs even in the presence of highly purified mDCs (Fig. 7), and human pDCs and/or lymphoid DCs do not express CD1d (14, 49, 64, 65). Activation of iNKT cells by CpG ODNs has been reported to be IL-12 and IL-15 dependent (60, 66, 67). However, despite the significant congruent involvement of iNKT cells and pDCs in these responses, the consequences of pDC activation on iNKT cell function has not been investigated and is poorly understood.

Thus, to determine whether pDC activation can control iNKT cell function, a study of TLR9-dependent activation with CpG ODNs was undertaken. Treatment of PBMCs with CpG ODNs strongly induced activation markers on iNKT cells. This effect was most pronounced for the pDC-selective A-class CpG ODNs (Tables I and II) (38, 68). Even though CpG ODN treatment resulted in the induction of activation markers on iNKT cells to the same extent as PMA/ionomycin, no cell division or cytokine secretion was noted. The activation of iNKT cells was partially dependent on IFN-I and TNF-{alpha}. However, it was noted that other soluble factors are likely to be involved. Although the conditioned medium and Ab blockade experiments support the notion that soluble mediator of the CpG response can preactivate iNKT cells, this does not preclude an important role for cell-to-cell contact. It is likely that direct cell contact with pDC, in part mediated by OX40/OX40L, contributes to the activation of iNKT following CpG exposure (69). Marschner et al. (69) examined the consequences of pDC and iNKT interactions, and their results are in agreement with respect to licensing of iNKT cells, role of IFN-I, and subsequent activation by mDCs. However, these authors found that cell-cell contact, including pDCs that do not express detectable CD1d, was necessary for the effect. In the results presented in this study, licensing of iNKT cells to recognize CD1d on mDCs did not require cell-cell contact of iNKT cells with pDC. These differences may be the result of different experimental protocols. Marschner et al. (69) admixed in semi pure iNKT cells, selected on the basis of V{alpha}24 expression, into PBMC to levels of 10% whereas either PBMC or reconstitution experiments were used in this study.

It is unlikely that IL-6, IL-7, IL-12, IL-15, IL-18, or IFN-{gamma} made any significant contribution to the induction of activation markers on iNKT cells, because these cytokines were absent or not induced when comparing A-class CpG with control ODN treatment (Table III and Figs. 5 and 6). Importantly, exposure of iNKT cells to IFN-I, TNF-{alpha}, and soluble factors licensed these T cells to respond to Ag-loaded mDCs (Figs. 7 and 8), and resulted in the preferential expansion and survival of DN iNKT cells (Fig. 9). Despite the expression of CD1d, the in vivo activation of iNKT cells by mDCs presenting endogenous self-Ags does not occur unless the DCs are activated by either direct bacterial contact, by activation of TLRs (33, 47, 48, 70, 71). Consistent with these observations, freshly isolated human iNKT cells were refractory to TCR-dependent stimulation. This state could be overcome by exposure to pDC-derived conditioned medium. Therefore, it seems reasonable to suggest that in addition to direct effects of pDC-derived cytokines on mDCs, a cross-talk with mDCs mediated by iNKT cells would make significant contributions to the outcome of immune responses following pDC activation. Reciprocal activation of NK cells, mDCs, and pDCs has been reported before. We have previously argued that the ratio of CD4+ to DN iNKT cell function has important consequences for controlling the outcome of an immune response and that a significant component of this regulation occurs by controlling the effector function of mDC (30). It is now appreciated that CD4+ iNKT cells preferentially subserve immunoregulary functions and are significant contributors to asthma and atopic disorders (72, 73, 74, 75, 76). In contrast, DN iNKT cells are thought to be the effectors of iNKT cell-dependent tumor and anti-viral responses (58, 77, 78, 79). Notably, iNKT cells are important for the control of the HSV family of viruses (37, 60, 61, 80). Moreover, two separate immunodeficiencies characterized by a specific loss of iNKT cells with a concomitant susceptibility to HSV family infections have been reported (81, 82, 83, 84). Given that CpG are considered to be useful therapeutic approaches for these diseases, it seems reasonable to speculate that CpG regulation of iNKT cell subset function contributes to their mechanism of action.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
A. M. Krieg is Senior Vice President, R&D CSO, and cofounder of Coley Pharmaceutical Group, which provided the CpGs used in this study. Dr. Krieg is an active scientific collaborator on this work and National Institutes of Health Program Project Grant PO1 AI055793, which funded this work.


   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 in part by National Institutes of Health Grants 5P01AI055793 (to C.J.M., L.A-H., C.M., A.M.K., A.L.L., and S.B.W.) and 2R01AI45051 (to S.B.W.). Back

2 Please address correspondence and reprint requests to Dr. S. Brian Wilson, Diabetes Research Unit, Massachusetts General Hospital, 65 Landsdowne Street, Room 528, Cambridge, MA 02139. E-mail address: bwilson{at}rics.bwh.harvard or Dr. Alan L. Landay, Department of Immunology and Microbiology, Rush University Medical Center, Chicago, IL 60612. E-mail address: alanday{at}rush.edu Back

3 Abbreviations used in this paper: mDC, myeloid dendritic cell; pDC, plasmacytoid DC; PRR, pattern recognition receptor; PAMP, pathogen-associated molecular pattern; IFN-I, type I IFN; ODN, oligodeoxyribonucleotide; iNKT, invariant NK T cell; {alpha}-GalCer, {alpha}-galactosylceramide; DN, double negative; DP, double positive; IP-10, IFN-{gamma}-inducible protein 10; rh, recombinant human. Back

Received for publication October 5, 2005. Accepted for publication May 1, 2006.


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