DEC-205 is a type I C-type lectin receptor (CLR) that is expressed on various APC subsets and has been suggested to bind necrotic and apoptotic cells. Here we study DEC-205 characteristics in plasmacytoid DCs (pDCs) obtained from healthy individuals and assess its ability to mediate antigen presentation by isolating sufficient numbers of pDCs from apheresis material obtained from stage III/IV melanoma patients. The results demonstrate that DEC-205 is expressed on human pDCs. Internalization of DEC-205 after antibody ligation is clathrin- and dynamin-dependent as it is blocked by hypertonic shock or by inhibition of dynamin activity. Antibody targeting to DEC-205 does not affect TLR-induced expression levels of co-stimulatory and MHC molecules, but clearly impairs TLR-induced IFN-α secretion by 40%. We observed that TLR-mediated signaling increases DEC-205 expression levels without affecting receptor internalization. Moreover, human pDCs retained the capacity to present antigens via DEC-205 following TLR activation.
Plasmacytoid DCs (pDCs) are one of the two major subsets of human DCs that circulate in the peripheral blood, and are characterized as CD4+CD45RA+IL-3Rα+ (CD123) ILT3+ILT1− CD11c− lineage− cells 1. PDCs can be further characterized by BDCA-2 and BDCA-4 that are exclusively expressed by human pDCs in peripheral blood and BM 2. Upon stimulation by viruses or bacteria, pDCs become activated and produce large amounts of type I interferon (IFN-α/β) 3.
Because of their high IFN production, pDCs are considered to have an important role in controlling both innate and adaptive immune responses. In a resting stage, pDCs might induce regulatory responses whereas their activated equivalents have stimulatory capacities 4. In the tumor micro-environment of patients with ovarian carcinoma, pDCs with this resting phenotype have been demonstrated to maintain the tumor immunosuppressive environment 5. Activated pDCs on the other hand induce the expansion of antigen-specific CD8+ T cells against the tumor-associated epitope MART-1/melan A26–356 and Th1 CD4+ T-cell populations specific for endogenous antigens 7 and influenza virus 8, 9. During influenza virus infection, pDCs are able to prime virus-specific primary and secondary CD4+ and CD8+ T-cell immune responses in vitro and in vivo 10. Fonteneau et al. described that only pDCs exposed to replicative virus and not to nonreplicating, boiled or ultraviolet irradiated virus can induce these responses 8, suggesting that intracellular virus protein expression is crucial for antigen presentation. In contrast, Di Pucchio et al. have shown that pDCs can induce the expansion of virus-specific CD8+ T cells after uptake of inactivated virus 11.
C-type lectin receptors (CLRs) recognize defined carbohydrate moieties in a Ca2+-dependent manner. Ligand binding to CLRs can be of exogenous or endogenous nature 12. In APCs, CLRs and TLRs together play an important role as pattern-recognition receptors that recognize pathogen-associated molecular patterns 13. Human pDCs express several potential antigen uptake receptors including DCIR 14, 15, BDCA-2 2, 16, 17 and BDCA-4 2, 16, but many of the CLRs that are abundantly expressed in other subpopulations of DCs 18, such as mannose receptor (CD206), Dectin-1 and DC-SIGN 14 are not expressed. As most CLRs are rapidly internalized following ligand binding, CLRs are increasingly employed in vaccination strategies involving targeted delivery of antigens to various DC subsets 19. The selection of the appropriate receptor for targeted delivery is crucial to improve antigen processing and (cross-) presentation for the induction of antigen-specific T cells. The CLR DEC-205 belongs to the macrophage mannose receptor family of C-type lectin endocytic receptors and was first identified in mice 20. DEC-205 has been extensively employed for targeted delivery of antigens to APCs in murine studies, resulting in the presentation of antigenic peptides in MHC classes I and II 21–24. Recent findings have revealed that DEC-205 recognizes dying murine thymocytes, displaying a role for DEC-205 as a potential sensor for apoptotic and necrotic cells 25. The ligand recognized by DEC-205 on dead cells is not known and the role of DEC-205 in antigen cross-presentation associated with dead cells remains to be elucidated.
Blood DCs, including pDCs, Langerhans cells and in vitro cultured immature monocyte-derived DCs express low levels of DEC-205, but expression levels strongly increase upon DC activation, indicating that DEC-205 might be an activation-associated molecule 12, 26–29. In mice DEC-205 expression is restricted to DCs, whereas in humans DEC-205 is much more broadly expressed on multiple cell types 29. The cytoplasmatic domain of the DEC-205 receptor contains protein motifs for endocytosis and late endosome/MHC class II compartment-targeting. These motifs facilitate efficient antigen-loading to MHC class II molecules for antigen presentation to T cells 24. The DEC-205 endocytic pathway does not lead to activation of myeloid DCs. It has been exploited in vitro in man and in vivo, in mice, to deliver specific antigens to DCs, and induces specific CD8+ and CD4+ T-cell responses 22, 23.
It is still unclear whether mature DCs take up and present antigens. There are reports showing that Fc receptors on pDCs are incapable of internalization following pDC maturation, and DEC-205 was reported not to internalize on mature moDCs 30, 31. Recent findings in mice have demonstrated that mature DCs can still endocytose and present antigens 32, 33. In contrast, Young et al. have shown that cDC matured in vivo can take up OVA in vitro, but do not present it to T cells because MHC class II synthesis has stopped. However, pDCs continue to produce MHC class II after maturation and this allows them to present antigens encountered in an activated state 34.
Here, we describe that DEC-205 is expressed by human pDCs and becomes upregulated upon maturation. In contrast to activated monocyte-derived DCs 31, activated pDCs internalize DEC-205 upon receptor ligation. We show that targeting DEC-205 on fresh as well as on activated human pDCs induces antigen presentation and proliferative recall responses. In addition, ligation of DEC-205 by antibodies impaired TLR-induced IFN-α production by human pDCs.
DEC-205 is expressed and internalized by purified human pDCs
To investigate the expression of antigen uptake receptors, pDCs were isolated from the blood of healthy donors using anti-BDCA-4-coated beads and magnetic separation. In accordance with known literature, freshly isolated human pDCs expressed DEC-205 (Table 1), CD32 and the pDC-specific CLR BDCA-2 (Table 1), but did not express other uptake receptors including DC-SIGN, Dectin-1 or mannose receptor (data not shown). Since the natural ligands for DEC-205 are yet unknown, we mimicked ligand binding to DEC-205 by mAb ligation. Upon triggering surface receptor molecules, pDCs internalized CD32, BDCA-2 and DEC-205 as determined by analyzing surface expression levels (Fig. 1). Thus, human pDCs express a repertoire of antigen uptake receptors that are internalized upon receptor triggering, suggesting a role for pDCs in receptor-mediated antigen capture and presentation.
Table 1. Surface receptor expression on human pDCs
To establish whether DEC-205 triggering modulates human pDC activation and function, we studied the effect of DEC-205 triggering on the viability, expression of maturation markers and secretion of cytokines by pDCs. DEC-205 triggering did not affect the viability of human pDCs (Supporting Information Fig. 1A) nor CD40, CD80 and CD86 expression (Fig. 2A and B), nor the expression levels of MHC class I and class II molecules after overnight activation (Fig. 2B). PDCs are known to secrete IFN-α within the first 12 h subsequent to TLR-induced activation. Therefore, we tested the ability of pDCs to secrete IFN-α, TNF-α and IL-6 following DEC-205 and BDCA-2 triggering and subsequent activation by the TLR ligands R848 or CpG C, that bind TLR7 and TLR9 respectively. In accordance with previous studies 2, 35, triggering BDCA-2 impaired TLR-induced IFN-α and TNF-α secretion (IFN-α: mIgG2b-CpG C, 15 847±5319pg/mL; mIgG2b-R848, 3975±1372 pg/mL; BDCA-2-CpG C, 5409±2103 pg/mL; BDCA-2-R848, 999±402 pg/mL) (Fig. 2C) (TNF-α: mIgG2b-CpG C, 10 976±3552 pg/mL; mIgG2b-R848, 9424±5619 pg/mL; BDCA-2-CpG C, 8674±5721 pg/mL; BDCA-2-R848, 3978±2205 pg/mL) (Fig. 2D). Interestingly, pDCs triggered with αDEC-205 antibody showed a ∼40% decreased IFN-α secretion compared to pDCs that were treated with isotype control antibodies upon TLR-induced activation (αDEC-205-CpG C, 9573±4547 pg/mL; αDEC-205-R848, 1412±393.6 pg/mL) (Fig. 2C). This inhibitory effect of the αDEC-205 antibody on IFN-α secretion was concentration-dependent (Supporting Information Fig. 1B and C). In contrast to IFN-α, secretion of IL-6 and TNF-α was not affected by DEC-205 triggering (Fig. 2D).
These data demonstrate that following DEC-205 triggering, pDCs remain sensitive for TLR-mediated signals and develop a mature phenotype, but are impaired to secrete high amounts of IFN-α.
Maturation of human pDCs induces upregulation of DEC-205
Freshly isolated pDCs activated with CpG C or R848 displayed a fully activated phenotype as determined by upregulation of CD80, CD83, CD86 and MHC class I and II molecules (data not shown) and downregulation of the CLRs DCIR 14 and BDCA-2 17 (Fig. 3C). Interestingly, pDCs that were cultured with IL-3 or activated for 24 or 48 h through TLR-9 or TLR-7 showed a significant increase in the expression levels of DEC-205 (Fig. 3A and B). Enhanced expression of the CLR DEC-205 on monocyte-derived DCs after activation has also been noted and hints toward the possibility that DEC-205 expressing DCs sample their environment and take up antigens even in an activated state.
Activated pDCs internalize DEC-205 through a clathrin-dependent mechanism
Freshly isolated pDCs rapidly internalized DEC-205 upon receptor triggering (Fig. 4A). Surprisingly, upon TLR triggering with CpG C or R848 pDCs maintained the capacity to internalize DEC-205 and this even appeared to be enhanced when compared to freshly isolated pDCs (Fig. 4B). Internalization was confirmed by confocal microscopy demonstrating that DEC-205 receptor really accumulates inside the cell after receptor triggering (Fig. 4C).
Subsequently, we sought to determine the mechanism underlying DEC-205 internalization. A classical pathway for receptor-mediated endocytosis is clathrin-dependent 36. Clathrin-dependent endocytosis can be inhibited by incubation of cells in hypertonic media 14. Disruption of clathrin-coated pits in hyperosmolar medium abolished DEC-205 receptor internalization in CpG C-activated pDCs completely (Fig. 4D). Vesicle fission in clathrin-mediated endocytosis is dynamin-dependent. Dynasore is a reversible noncompetitive dynamin inhibitor and is used to study dynamin involvement 37. As expected, DEC-205 internalization could also be inhibited by incubating CpG C-activated pDCs with Dynasore (Fig. 4D). These data indicate that the mechanism of DEC-205 internalization in activated human pDCs is clathrin- and dynamin-dependent. Furthermore, we here show that activated pDCs remain capable of internalizing DEC-205 upon receptor triggering.
Targeting antigen toward DEC-205 on activated pDCs induces T-cell proliferation
Since pDCs rapidly internalize DEC-205 upon triggering, we next assessed whether DEC-205 functions as an antigen uptake receptor on human pDCs by targeted delivery of the model antigen keyhole limpet hemocyanin (KLH). To this end, pDCs and PBLs were isolated from peripheral blood of melanoma patients participating in ongoing vaccination trials that include the use of KLH as a helper antigen. To facilitate KLH targeting we made use of anti-biotin-KLH construct that provides us with a flexible tool to rapidly study various receptors expressed by pDCs. Ultimately, fusion constructs for the generation of vaccines and clinical application should be made for the receptor of choice.
Patient-derived purified pDCs were targeted via DEC-205, DCIR 14 or control IgG using biotinylated antibodies. Subsequently, pDCs were incubated with anti-biotin-antibodies conjugated to KLH and cocultured with fresh patient-derived CD4+ T cells. Proliferation was measured after four days of co-culture. T-cell stimulation with PHA, as a positive control, induced strong T-cell proliferation (35 372 ±1466 cpm). In concordance with previous results 38, pDCs did not present soluble exogenous antigen, as anti-biotin-KLH and mIgG-biotin-anti-biotin-KLH were not able to induce significant proliferative T-cell responses (Fig. 5A). Interestingly, targeting DEC-205 on freshly isolated human pDCs led to proliferative recall T-cell responses, illustrating the capacity of this receptor to specifically deliver antigens in MHC class II loading compartments of pDCs (Fig. 5A). Since DEC-205 is upregulated and internalized by human pDCs following TLR-induced activation, we investigated whether DEC-205 still mediates antigen presentation in fully activated pDCs. Antigen presentation via DEC-205 was compared to DCIR, which is downregulated upon TLR-induced activation. In accordance with the elevated expression levels and the capacity to internalize the receptor upon triggering, we observed that CpG C-activated pDCs induced antigen-specific T-cell responses via DEC-205 (Fig. 5B). In contrast, DCIR could not mediate antigen presentation in activated pDCs (Fig. 5B). Interestingly, we observed a significant increase in KLH-specific T-cell proliferation when DEC-205 was targeted on activated pDCs compared to freshly isolated pDCs (Fig. 5B). These data clearly demonstrate that activated pDCs not only display increased DEC-205 expression levels, but retain the capacity of receptor-mediated antigen uptake, processing and presentation to T cells. Next, we sought to determine the difference in targeting DEC-205 on different DC subsets. Therefore, we isolated pDCs, CD1c+ mDCs and generated immature monocyte-derived DCs and loaded them with KLH via targeting DEC-205. In Fig. 5C, it can be appreciated that the three DC subsets were equally efficient in inducing KLH-specific recall T-cell responses. Moreover, targeting DEC-205 on the different DC subsets with a construct lacking KLH or anti-biotin-KLH alone did not induce specific T-cell activation (Fig. 5C). Taken together, DEC-205 on human pDCs can be targeted to induce antigen-specific proliferative T-cell responses to a similar level as CD1c+ mDCs and monocyte-derived DCs.
In this study, we demonstrated that DEC-205 is expressed by freshly isolated human pDCs and mediates antigen uptake and presentation in a clathrin-dependent manner. Endocytic receptors are generally downregulated in DCs upon activation with maturation stimuli 39. This could partially explain the fact that mature DCs have a decreased capacity to take up and present antigen. DEC-205 seems to be an exception to this rule, as it is upregulated in both human pDCs and monocyte-derived DCs upon maturation 31. However, in contrast to activated moDCs 31, activated human pDCs still internalized DEC-205 in a clathrin- and dynamin-dependent fashion. In mice, Platt et al. have recently shown that BM DCs also capture and present antigens in and activated state 32. This suggests that DEC-205 may perform distinct functions in the various DC subsets.
Many CLRs, such as mannose receptor 40, DCIR 14 and DC-SIGN 41 have the ability to rapidly internalize from the plasma membrane via clathrin-coated vesicles. Our experiments showed that DEC-205 is connected to the clathrin-mediated receptor uptake machinery in human pDCs. Previously it was shown that DEC-205 delivers antigens to late endosomes or lysosomes, where they are degraded. The cytoplasmic domain of DEC-205 contains two functional regions 24 a membrane-proximal region with a tyrosine-based coated-pit sequence for uptake 42 and a distal region with an EDE amino acid triad for targeting to proteolytic vacuoles. Our experiments showed that endocytosis of the exogenous antigen KLH via DEC-205 led to antigen presentation and induced proliferative recall CD4+ T-cell responses. Since pDCs cannot take up soluble antigens in a receptor-independent fashion 1, 43, receptor-mediated uptake is essential for antigen presentation by pDCs. Previously, we demonstrated that both DCIR and CD32-mediated antigen uptake leads to antigen presentation by freshly isolated human pDCs resulting in proliferative recall T-cell responses 14, 38. However, upon pDC activation, DCIR expression levels dramatically drop and CD32-mediated antigen uptake and presentation is hampered 30. These findings are in accordance with the notion that immature APCs take up antigens, undergo a process of activation and then present the encountered antigen to T cells 44. Indeed, in mouse experiments it was demonstrated that simultaneously delivering antigen and maturation stimuli by administration of an anti-CD40 antibody and an anti-DEC-205 antibody/Ag fusion protein induced potent antigen-specific CTL responses in mice 22. Much less is known regarding DEC-205 in man. Surprisingly, we find that human pDCs presented antigen targeted to DEC-205 irrespective of the pDC activation status. Furthermore, in line with increased expression and enhanced internalization, we found that targeting DEC-205 to activated human pDCs induced stronger antigen-specific T-cell responses when compared to targeting DEC-205 to freshly isolated pDCs. Whereas activated pDCs cannot take up and present immune complexes, they do present antigens encountered via DEC-205. This demonstrates that exogenous class II-restricted antigens are processed fundamentally different when taken up via DEC-205 than via CD32. This shows that, for class II-restricted antigens, vaccination strategies involving delivery of antigens to specific surface receptors may result in presentation by steady state and activated pDCs.
Activation of pDCs leads to a variety of phenotypical and functional alterations, including activation-induced antigen processing and increased surface expression of MHC class I and II. Furthermore, co-stimulatory molecules are upregulated, and pDCs acquire the ability to secrete large amounts of cytokines such as type I IFNs. Expression of co-stimulatory molecules CD40, CD80 and CD86 and the secretion of IL-6 remained unaffected after triggering DEC-205 and TLR-induced activation. Interestingly, we demonstrate that DEC-205 triggering on pDCs resulted in a 40% reduction in TLR ligand-induced IFN-α secretion. Previously, we showed that triggering DCIR on human pDCs reduced TLR-9-induced IFN-α secretion 14. Moreover, it has been described that triggering BDCA-2 also exhibits inhibitory effects on TLR-9-induced IFN-α secretion 17. Apart from DCIR and BDCA-2, TLR-9-induced IFN-α secretion remains unaffected upon BDCA-4 and CD32 receptor triggering 2, 30, 35. DEC-205 now represents a third CLR on pDCs that modulates TLR-induced responses. Although this suggests a similar mode of action for all CLRs, there appear to be distinct ways by which CLRs induce signaling. CLRs such as BDCA-2 are associated with immunoreceptor tyrosine-based activation motif (ITAM)-containing adaptor molecules, like the Fc receptor γ chain or DAP12 45. Other CLRs, such as DCIR, induce signaling pathways through activation of protein kinases or phosphatases that interact with their cytoplasmic domains 46. Whether DEC-205 belongs to one of these two categories and whether the various signaling motifs of these receptors induce distinct responses remains to be determined.
Taken together, we show that the endocytic receptor DEC-205 is expressed by human pDCs in a steady state and is upregulated upon maturation. Moreover, targeting DEC-205 on fresh and activated pDCs with specific antibodies coupled to KLH leads to antigen delivery, resulting in presentation and antigen-specific proliferation of T cells. Furthermore, we illustrate that triggering DEC-205 on human pDCs leads to impaired IFN-α secretion after TLR-induced activation. These findings have implications for using DEC-205 as a target receptor for the delivery of antigens and the induction of antigen-specific T-cell responses. Future research will be focused on the identification of the natural endogenous ligands that bind to DEC-205 and the role of DEC-205 in modulating immune responses after antigen uptake and TLR signaling. Identification of the intracellular interaction partners of DEC-205 will reveal how it modulates TLR signaling and whether it resembles other CLRs or follows a distinct pathway. Detailed information about receptor regulation, internalization and signaling is instrumental for the rational design of CLR-targeted therapeutics.
Materials and methods
Buffy coats and patient material were obtained from volunteers after informed consent and according to institutional guidelines. CD1c+ mDCs and pDCs were purified by positive isolation using anti-CD1c- and anti-BDCA-4-conjugated magnetic microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany) and adjusted to 106 cells/mL in X-VIVO-15 (Lonza, Verviers, Belgium) supplemented with 2% of human serum. mDC and pDC purity was routinely up to 95%, as assessed by double staining BDCA-2+/CD123+ for pDCs (Supporting Information Fig. 2A) and CD11c+/CD1c+ for mDCs (Miltenyi Biotec). PDCs were cultured overnight with 10 ng/mL rhIL-3 or activated overnight in the presence of 4 μg/mL R848 (Axxora, Breda, The Netherlands) or 5 μg/mL ODN-CpG-C (M362, Axxora). Monocyte-dervied DCs were generated from adherent PBMCs, and subsequently by culturing in the presence of IL-4 (500 U/mL) and GM-CSF (800 U/mL) (both Cellgenix, Freiburg, Germany). Cells were cultured in X-VIVO 15 medium supplemented with 2% of human serum and harvested on day 6 as immature DC.
The phenotype of pDCs was determined by flow cytometry. The following primary monoclonal antibodies (mAbs) and the appropriate isotype controls were used: anti-HLA DR/DP (clone Q5/13; Becton Dickinson, Mountain View, CA, USA); anti-DEC-205 (clone MG38, eBioscience, Halle Zoersel, Belgium); anti-BDCA-2 (AC144, Miltenyi Biotec) and anti-CD32 (clone AT-10; ABD Serotec, Dusseldorf, Germany) followed by goat-anti-mouse PE (BD, Breda, The Netherlands); mIgG1-PE, mIgG1-APC, anti-HLA-ABC-PE (W6/32), anti-HLA-DR/DP-FITC (Q5/13), anti-CD80-PE, anti-CD86-APC (all BD Bioscience Pharmingen, San Diego, CA, USA); anti-CD40 (Beckman Coulter, Mijdrecht, The Netherlands). MFI and percentage of positive cells are based upon inclusion of cells in a live cell gate (Supporting Information Fig. 2A–D) and determined on a FACSCalibur (BD Biosciences, San Jose, CA, USA). FACS gating strategies for the different experiments in this manuscript are shown in the Supporting Information Fig. 2.
DEC-205 internalization assays
Purified or overnight-activated pDCs were incubated with primary monoclonal anti-DEC-205 (10 μg/mL, clone MG38, IgG2b ebioscience) and anti-BDCA-2 (10 μg/mL, clone AC144, IgG1, Miltenyi Biotec) in PBS, for 30 min on ice. Unbound antibodies were washed away and cells were incubated either for 30 min at 37°C to induce endocytosis or kept at 4°C. Subsequently, isotype-specific secondary antibodies were added (goat anti-mouse Alexa 647, Invitrogen Life Technologies, Breda, The Netherlands). To inhibit clathrin-mediated endocytosis, cells were incubated in hypertonic medium. Incubation in medium containing 450 mM sucrose (Boom, Meppel, The Netherlands) results in disruption of clathrin-coated pits and induces formation of nonfunctional so-called clathrin microcages 14. This process is fully reversible once cells are returned into isotonic media. Incubation in medium containing 80 μM Dynasore (Sigma Aldrich, Zwjindrecht, The Netherlands), a reversible noncompetitive dynamin inhibitor, results in a loss in vesicle fissure.
For confocal imaging, pDCs were allowed to adhere to poly-L-lysine-coated cover slips, fixed in 1% paraformaldehyde in PBS and permeabilized with 0.1% triton-X100. PDCs were incubated for 30 min at 4°C with primary anti-DEC-205 and anti-BDCA-2 mAbs. Unbound antibodies were washed away and cells were incubated at 37°C for 30 min. Then, cells were stained with isotype-specific goat-anti-mouse Alexa-488 and Alexa-647 secondary antibodies (Invitrogen). Cells were mounted with Mowiol (Brunschwig Chemie, Amsterdam, The Netherlands) and were imaged with a Bio-Rad MRC 1024 confocal system operating on a Nikon Optiphot microscope and a Nikon 60× planApo 1.4 oil immersion lens. Pictures were analyzed with Bio-Rad Lasersharp 2000 and Adobe Photoshop 7.0 (Adobe Systems, Mountain View, CA, USA) software.
PDCs were cultured overnight at a concentration of 1×105 pDCs/100 μL/well in a 96-well round bottom plate. Supernatants were collected from pDC cultures after 16 h of activation, and IFN-α and IL-6 production was analyzed by murine monoclonal capture and HRP-conjugated anti-IFN-α antibodies (Bender MedSystems, Vienna, Austria) or anti-IL-6 Abs (Sanquin, Amsterdam, The Netherlands) using standard ELISA procedures. TNF-α production was measured using a human Multiplex kit (Bender MedSystems) according to manufacturer's instructions.
Specific KLH responses
Cellular responses against the protein KLH were measured in a proliferation assay. PBMCs, pDCs, CD1c+ mDCs and immature monocyte-derived DCs were isolated from stage III and IV melanoma patients participating in ongoing vaccination trials with mature monocyte-derived DCs, which were pulsed with KLH to provide T-cell help 47. These patients developed KLH-specific T-cell responses and therefore CD4+ T cells were isolated with a CD4+ T-cell isolation kit (Miltenyi Biotec) according to the manufacturer's instructions. Freshly isolated pDCs, CD1c+ mDCs, immature monocyte-derived DCs and CpG C-activated pDCs were incubated for 45 min at 4°C with biotinylated anti-DEC-205 (clone MG38, eBioscience), biotinylated anti-DCIR (Clone 216110, R&D Systems) or with biotinylated mIgG (1 μg/mL). Finally, KLH coupled to an anti-biotin antibody (10 μg/mL) was added for 30 min at 4°C. As a control, purified T cells were stimulated with PHA (0.5 μg/mL). The purified T cells were plated in a 96-well tissue culture round-bottom microplate with autologous KLH-loaded DCs in a ratio 20:1 (1×105 T cells: 5×103 pDCs). After 4 days of culture, 1 μCi/well of tritiated thymidine was added for 16 h and incorporation was measured in a β-counter. In experiments where both fresh and activated pDCs from the same donor were used, T-cell proliferation data were normalized to account for intra-experimental differences induced by the fact that experiments with fresh and activated cells were performed on consecutive days. Therefore, stimulation indices were calculated by dividing tritium counts of experimental samples by those of the control pDCs-mIgG-KLH sample.
All experiments were performed at least three times and results are shown as the mean±SEM. Data sets were either tested by a one-way ANOVA followed by Tukey's multiple comparison test or by a two-way ANOVA followed by Bonferroni.
This work was supported by grants from the Dutch Cancer Society (KWF 2003-2917, KWF 2004-3126, KWF 2004-3127), The Netherlands Organization for Scientific Research (NWO ZonMW) (Vidi grant 91776363 to J. dV. and Veni grant 916.66.028 to AC), the TIL-foundation, the NOTK-foundation, and from the EU (Cancerimmunotherapy and DC-Thera).
Conflict of interest: The authors declare no financial or commercial conflict of interest.