Functional dichotomy of plasmacytoid dendritic cells: Antigen-specific activation of T cells versus production of type I interferon

Authors


Abstract

Human plasmacytoid dendritic cells (PDC) are believed to link innate and adaptive immunity by producing type I interferon (IFN-I) and triggering adaptive T cell-mediated immunity. However, it remains elusive to which degree both PDC functions are linked. Here we show that CMV antigen targeted to PDC using a CD303 (blood dendritic cell antigen 2, BDCA-2) mAb is rapidly endocytosed and traffics via early sorting endosomes to emerging MHC-enriched compartments. Both processes occur independently of TLR ligand stimulation. Restimulation of CMV-specific CD4+ effector-memory T helper cells by autologous PDC and induction of IFN-I production in PDC are dependent on appropriate stimulation. Type B CpG oligonucleotide (CpG-B)-stimulated PDC efficiently process and present CMV antigen and are thus capable of stimulating CMV-specific effector-memory T helper cells. CpG-A-stimulated PDC produce large amounts of IFN-I and express programmed death receptor-1 ligand 1. CpG-A plus CpG-B-co-stimulated PDC behave like CpG-B-stimulated PDC, suggesting that antigen processing and presentation in PDC is dependent on stimulation that concurrently inhibits IFN-I production. In vivo targeting of antigens to PDC via CD303 combined with appropriate PDC stimulation may allow induction of specific T cell activation.

Supporting Information for this article is available at http://www.wiley-vch.de/contents/jc_2040/2008/37552_s.pdf

Abbreviations:
BMDC:

bone marrow-derived DC

CLSM:

confocal laser scanning microscopy

EEA1:

early endosome antigen 1

IFN-I:

type I interferon

MoDC:

monocyte-derived dendritic cell

PD-L1:

programmed cell death receptor-1 ligand 1

PDC:

plasmacytoid dendritic cell

Introduction

In the last decade numerous efforts have been undertaken to exploit dendritic cells (DC) to improve vaccine efficacy. In most cases autologous monocyte-derived DC (MoDC) have been loaded ex vivo with antigens and subsequently re-administered to the patient. This approach requires cell separation and cell cultivation steps making the whole procedure expensive, time-consuming and potentially less efficient. Many of these limitations could potentially be overcome by a combination of in vivo antigen targeting to DC and in vivo maturation of DC.

Bonifaz et al.1 elegantly demonstrated a novel strategy for in vivo targeting of antigens to DC. The authors used a CD205-specific mAb for targeting of ovalbumin to mouse DC and a CD40-specific mAb for DC maturation. The targeted DC efficiently induced specific CD4+ and CD8+ T cell immunity. Moreover, it could be demonstrated that specific peripheral T cell tolerance can be induced using the same strategy but omitting the DC maturation-inducing CD40-specific mAb 24. In vitro studies using mAb against the human mannose receptor (CD206) 5 and human DC-SIGN (CD209) 6, 7 have indicated that these C-type lectins are suitable endocytic receptors for targeting of human DC. However, none of the mentioned receptors is specifically expressed on immature DC in humans. Furthermore, the maturation status of DC significantly influences the outcome of immune responses. As a consequence, antigen targeting via receptors expressed on both mature and immature DC could lead to situations where both immunization and tolerization occur. However, generally, the induction of either immunity or tolerance is required.

Plasmacytoid DC (PDC) have been shown to be recruited to tumor tissue and subvert tumor immunity by inducing CD8+ regulatory T cells 8, 9. On the other hand, PDC have potent T cell stimulatory capacity in allogeneic mixed lymphocyte reactions upon in vitro maturation and can prime antigen-specific CD8+ and CD4+ T cell responses both in vitro and in vivo10, 11. This indicates that PDC can be used for the induction and maintenance of T cell tolerance as well as for induction of T cell immunity. CD303 is a type II C-type lectin. Using a mouse IgG1-specific CD4+ T cell clone we could previously demonstrate that CD303 is an endocytic receptor mediating antigen uptake and presentation 12. Due to its PDC-restricted expression, CD303 represents an attractive receptor for an in vivo antigen targeting approach. In the present work we have analyzed whether autologous CMV-specific CD4+ effector-memory T helper (Th) cells can be specifically restimulated by PDC if CMV antigen is targeted in vitro to CD303 and to which degree this is dependent on PDC maturation.

Our results are not in complete agreement with the current view that PDC after producing large amounts of type I interferon (IFN-I) in response to microbial stimulation differentiate into mature DC capable of stimulating naive T cells and modulating the adaptive immune response 13. Rather, dependent on stimulation, PDC differentiate with similar kinetics into cells that either produce large amounts of IFN-I and express the co-inhibitory molecule programmed death receptor-1 ligand 1 (PD-L1) or differentiate into cells that produce little or no IFN-I but efficiently process and present antigen.

Results

PDC redistribute MHC class II molecules upon culturing

In conventional immature DC most MHC class II molecules are intracellularly localized in early lysosomal structures termed MIIC 14. Upon maturation intracellular class II molecules are transported via non-lysosomal compartments (CIIV) to the cell surface 14, 15. Here, freshly isolated PDC were cultured for 24 h in the presence of IL-3 alone or in combination with type A CpG oligonucleotide (CpG-A) or CpG-B for maturation. Localization of MHC class II molecules was analyzed during maturation by staining of HLA-DR and lysosome-associated membrane protein 3 (CD63) 16 and confocal laser scanning microscopy (CLSM).

In freshly isolated PDC most MHC class II molecules are present in abundant small intracellular compartments, which do not stain for CD63, indicating that these are non-lysosomal compartments (Fig. 1). After culturing for 12 h, MHC class II molecules are localized to few much larger compartments in the perinuclear cytoplasm, which are CD63+. Therefore, these are presumably lysosomal compartments, which in fact are quite reminiscent of MIIC in immature conventional DC 14. This immediate redistribution of MHC class II and lysosomal compartments seems to be stimulus-independent as it appears in all three cultures. After culturing for 24 and 48 h in the presence of CpG-B, similar to mature conventional DC, PDC extensively up-regulate MHC class II molecules on the plasma membrane. To a much lower extent this is also observed when PDC are cultured with CpG-A or in absence of CpG oligonucleotide. These findings indicate that particularly CpG-B triggers extensive redistribution of MHC class II molecules from lysosomal compartments to the plasma membrane.

Figure 1.

Redistribution of MHC class II and CD63 molecules in PDC. Purified PDC were cultured in the presence of 100 ng/mL IL-3 alone (denoted as medium) or in combination with 5 µg/mL CpG-A or CpG-B, respectively. Cells were stained intracellularly at various time points with biotin-conjugated anti-HLA-DR mAb and streptavidin-Alexa Fluor 633 dye (red) and FITC-conjugated CD63 mAb (green) and subsequently analyzed by CLSM. Confocal images were merged to demonstrate co-localization (yellow). Representative results of three donors are shown.

Differential expression of cytokines, co-stimulatory and co-inhibitory molecules upon CpG-induced maturation of PDC

To elucidate differences between CpG-stimulated and unstimulated PDC with regard to secretion of cytokines and expression of co-stimulatory molecules, freshly isolated PDC were cultured for 24 h in the presence of IL-3 and CpG-A or CpG-B, respectively. The culture supernatants were analyzed for IFN-α, TNF-α, TNF-β, IL-1β, IL-4, IFN-γ, IL-6, IL-8 and IL-10 levels (Fig. 2A). Among these cytokines IL-6, IL-8, TNF-α, IFN-α and to some extent TNF-β are detectable after stimulation with CpG oligonucleotides. Unlike CpG-A, CpG-B induces high levels of IL-6, IL-8 and TNF-α (Fig. 2A). As described previously 17, only CpG-A induces extensive secretion of IFN-I. When PDC are cultured in the absence of CpG oligonucleotide, only IL-8 is secreted in detectable amounts. CpG-B induces high and CpG-A relatively low levels of CD80 and CD86 expression (Fig. 2B). Only minor differences are detectable regarding the expression of CD40.

Figure 2.

Differential cytokine secretion and expression of co-stimulatory molecules in CpG-A- and CpG-B-stimulated PDC. (A) Purified PDC were cultured for 24 h in the presence of 100 ng/mL IL-3 alone or in combination with 5 µg/mL CpG-A or CpG-B, respectively. Culture supernatants were analyzed for secreted IFN-α using a specific ELISA and for secreted IL-1β, IL-6, IL-8, TNF-α and TNF-β using a cytokine bead array assay. (B) Stimulated PDC were analyzed for CD40, CD80, CD86 and PD-L1 expression by flow cytometry. Flow cytometric results are depicted as mean fluorescence intensities (MFI) or percentage of positive cells, respectively, as indicated. Values are presented as means ± standard deviations of three independent experiments with different donors.

Most intriguingly, when analyzing PDC for the expression of PD-L1, a molecule which is known to antagonize T cell activation via programmed cell death 1 receptor ligation 1820, we found that about 30% of CpG-A-activated PDC are positive for this co-inhibitory molecule (Fig. 2B). CpG-B-activated or non-stimulated PDC fail to express PD-L1 after 24 h of culture. Further experiments showed that PD-L1 expression on PDC is IFN-I-dependent and restricted to fresh isolated PDC (Supporting Information Fig. 1). PD-L1 could not be induced when PDC were activated with CpG-B prior to IFN-I challenge.

Antigens internalized via CD303 traffic via early sorting endosomes to lysosomes

It has been previously shown that mAb ligation of CD303 induces rapid internalization of the antibody-receptor complex 12. Here we analyzed intracellular trafficking of internalized CD303 mAb by CLSM. PDC were labeled with biotinylated CD303 mAb and streptavidin Alexa Fluor 633 and then cultured in the presence of IL-3 and for activation in the presence of CpG-B. To track internalization and intracellular trafficking of CD303, PDC were intracellularly stained at various time points for early endosome antigen 1 (EEA1), a marker for early sorting endosomes 21, for HLA-DR, and for CD63 and Lamp-1 (CD107a) 22, both markers for lysosomal compartments. As shown in Fig. 3, after 5–10 min, CD303 mAb is internalized and co-localizes with EEA1 in early endosomes. After 20 min, internalized CD303 mAb is present in numerous EEA1 vesicles, most likely endosomal carrier vesicles. After 15 h, internalized CD303 mAb co-localizes with HLA-DR, CD107a and CD63 in lysosomal compartments. Due to cessation of the Alexa Flour 633 signal beyond 15 h of culturing, further analysis of intracellular CD303 mAb trafficking was not feasible (data not shown).

Figure 3.

CD303 mAb is internalized by PDC and traffics through early sorting endosomes to lysosomes. Purified PDC were stained with biotin-conjugated CD303 mAb and streptavidin-Alexa Fluor 633 dye (red). PDC were cultured in medium supplemented with 100 ng/mL IL-3 and 5 µg/mL CpG-B and harvested at various time points. PDC were stained intracellularly with FITC-conjugated mAb directed against EEA1, HLA-DR, CD107a or CD63, respectively (green), and analyzed by CLSM. Confocal images were merged to demonstrate co-localization (yellow). Representative results of three donors are shown.

Antigen targeted to PDC via CD303 is efficiently presented to CD4+ T cells

We demonstrated before that PDC internalize CD303 mAb and induce a proliferative response in a T cell clone which is specific for a mouse IgG1-derived peptide 12. Instead of a Th cell clone with possibly unusual activation requirements, here we used autologous CD4+ CD45RO+ effector-memory T cells from blood of a CMV-seropositive donor. We analyzed to which degree CMV-specific CD4+ effector-memory T cells can be induced to produce IFN-γ by mature PDC of the same donor, when CMV antigen is endocytosed via CD303 before maturation. For receptor-mediated endocytosis of CMV antigen by PDC via CD303, biotinylated CD303 mAb and anti-biotin mAb, which was conjugated to FITC as well as to a cell lysate of CMV-infected cells, were used as primary and secondary labeling reagent, respectively. Receptor-specific targeting of antigen to PDC via CD303 was controlled by flow cytometry based on the FITC signal.

Freshly isolated PDC were labeled for 10 min with each of the targeting reagents, washed once (Fig. 4A), and then matured by culturing for 24 h in the presence of IL-3 and CpG-B. After PDC maturation, autologous CD4+ CD45RO+ effector-memory T cells were added and co-cultured for 20 h. Stimulation of CMV-specific CD4+ T cells was then analyzed by intracellular staining of IFN-γ. PDC-induced restimulation of CMV-specific CD4+ effector-memory T cells is much more efficient when CMV antigen is antibody-targeted to PDC using CD303 mAb as compared to CD45 mAb or isotype-matched control mAb with irrelevant specificity (Fig. 4B).

Figure 4.

PDC targeted with CMV antigen via CD303 efficiently stimulate autologous CMV-specific CD4+ effector-memory T cells. (A) Purified PDC from CMV-seropositive donors were labeled on ice with biotinylated CD303 mAb (bold line), biotinylated CD45 mAb (faint line) or biotinylated isotype control mAb (dotted line) and FITC-labeled anti-biotin mAb conjugated to CMV antigen. (B) PDC targeted with CMV antigen as described in (A) or PDC labeled with CD303-biotin and anti-biotin mAb conjugated to control antigen were cultured in medium supplemented with 100 ng/mL IL-3 and 5 µg/mL CpG-B for 24 h. Thereafter, PDC (1×105) were co-cultured with purified autologous CD4+ CD45RO+ effector-memory T cells (1×106) for additional 20 h. Brefeldin A was added to the cultures after 16 h to a final concentration of 5 µg/mL. T cell stimulation was analyzed by intracellular staining of IFN-γ and flow cytometric analysis. Representative results of at least three donors are shown. (C) Frequencies of IFN-γ-producing T cells obtained in three (control antigen) and eigth (CMV antigen) independent experiments with PDC from different donors.

To prove that mAb targeting of CD303 by itself does not lead to T cell stimulation, a control antigen (a cell lysate of non-CMV-infected cells) was targeted to PDC using CD303 mAb. No significant T cell stimulation could be observed (Fig. 4B). Also when CMV antigen is continuously present in a tenfold higher concentration as used for antigen targeting during the whole maturation period, frequencies of IFN-γ+ cells are significantly lower as when CMV antigen is targeted to CD303 (data not shown). These results were highly reproducible independent of the cell donor (Fig. 4C). We also examined, whether PDC could cross-prime memory CD8+ T cells after antigen targeting. Using this experimental setting we could not show any specific cross-priming. Apparently, PDC induced extensive non-specific IFN-γ secretion in co-cultured CD8+ T cells (data not shown).

PDC targeted with antigen via CD303 induce a robust proliferative response of antigen-specific CD4+ T cells

CD4+ CD45RO+ effector-memory T cells from blood of a CMV-seropositive donor were cultured with CMV antigen- or control antigen-targeted PDC as described above. T cells were expanded in co-culture with PDC for 10 days in the presence of IL-2 and IL-7. Expansion of CMV antigen-specific CD4+ CD45RO+ effector-memory T cells was analyzed after overnight restimulation using T cell-depleted autologous PBMC as APC and cell lysate of CMV-infected cells by intracellular staining of IFN-γ (Fig. 5). After 10 days of expansion the frequency of IFN–γ-producing CMV antigen-specific CD4+ CD45RO+ effector-memory T cells is more than sixfold higher when CMV antigen is antibody-targeted to PDC using CD303 mAb as compared to isotype-matched control mAb. Antigen-specific T cell proliferation with PDC was also demonstrated by expansion of CFSE-labeled CMV antigen-specific effector-memory T cells (Supporting Information Fig. 2).

Figure 5.

PDC targeted with CMV antigen via CD303 induce a proliferative response in autologous CD4+ CD45RO+ effector-memory T cells. Purified PDC from CMV-seropositive donors were targeted with CMV antigen or control antigen via CD303 as described in Fig. 4. CMV antigen-loaded PDC (1×105) were then co-cultured with autologous CD4+ CD45RO+ effector-memory T cells (1×106) for 10 days in the presence of 10 ng/mL IL-7 and 10 ng/mL IL-2. Thereafter, T cells were restimulated with autologous CD3+ T cell-depleted PBMC (1×105) and 5 µg/mL CMV antigen. As a control, CD4+ CD45RO+ effector-memory T cells were cultured in the presence of IL-2 and IL-7 but in the absence of PDC and stimulated after 10 days with autologous CD3+ T cell-depleted PBMC. This is denoted here as non-stimulated T cells. Restimulation of T cells was analyzed by intracellular staining of IFN-γ and flow cytometric analysis. Data are representative of three independent experiments with different donors.

Antigen targeted to PDC via CD303 is presented to CD4+ T cells for several days

In immature DC, class II molecules are rapidly internalized and recycled, turning over with a half-life of about 10 h. Maturation stimuli induce a rapid and transient boost of class II synthesis, while the half-life of class II molecules increases to over 100 h. These coordinated changes result in the rapid accumulation of a large number of long-lived peptide-loaded MHC class II molecules capable of stimulating T cells even after several days 23. In order to analyze the presentation of CMV antigen peptide-loaded MHC class II molecule on the plasma membrane of maturing PDC, CMV antigen was targeted to PDC via CD303 and PDC were matured for 5–101 h with CpG-B. CMV antigen presentation by matured PDC was then analyzed by co-culturing with autologous CD4+ CD45RO+ effector-memory T cells from blood of a CMV-seropositive donor and intracellular staining of IFN-γ.

As shown in Fig. 6, PDC stimulate CMV antigen-specific CD4+ CD45RO+ effector-memory T cells already 5 h after CMV antigen targeting via CD303 and onset of CpG-B-induced maturation. However, the highest stimulatory capacity is obtained when PDC are matured for 29 h. Thereafter the stimulatory capacity of PDC decreases again. However, even after 101 h, CpG-B-matured PDC were still able to specifically stimulate CMV antigen-specific CD4+ CD45RO+ effector-memory T cells. The decreasing stimulatory capacity after 29 h is most likely not due to cell death. According to propidium iodide staining the viability of PDC ranged 90–95% after 77 h and 80–85% after 101 h (data not shown). Thus, our results indicate that PDC which are targeted with CMV antigen via CD303 and stimulated with CpG-B display long-lived CMV peptide-loaded MHC class II molecules on the cell surface.

Figure 6.

PDC targeted with CMV antigen via CD303 can efficiently present antigen for up to 5 days. Purified PDC (1×105) from CMV-seropositive donors were targeted with CMV antigen via CD303 as described in Fig. 4 and cultured in the presence of 100 ng/mL IL-3 and 5 µg/mL CpG-B. After 5–101 h autologous CD4+ CD45RO+ effector-memory T cells (1×106) were added and co-cultured for 20 h. T cell stimulation was analyzed by intracellular staining of IFN-γ as in Fig. 4. Data are representative of two independent experiments with different donors.

Induction of antigen presentation in PDC is dependent on appropriate stimulation

To test to which degree antigen presentation by PDC is dependent on stimulation of PDC, CMV antigen was targeted to PDC via CD303 and PDC were stimulated for 24 h with CpG-A, CpG-B or CpG-C, respectively. CpG-C has been shown to be less potent than CpG-A and more potent than CpG-B to induce production of IFN-I 24. As shown in Fig. 7A, unlike CpG-B-stimulated PDC, CpG-A-stimulated PDC almost completely failed to induce CMV-specific CD4+ effector-memory T cells to produce IFN-γ. CpG-C-stimulated PDC were more potent than CpG-A-stimulated PDC but less potent than CpG-B-stimulated PDC in this regard.

Figure 7.

Specific restimulation of CD4+ CD45RO+ effector-memory T cells by CMV antigen-targeted PDC is dependent on appropriate stimulation of PDC. (A) Purified PDC from CMV-seropositive donors were targeted with CMV antigen via CD303 as described in Fig. 4 and cultured for 24 h with 100 ng/mL IL-3 alone or as indicated in combination with 5 µg/mL CpG-A, CpG-B or CpG-C, respectively. T cell stimulation was analyzed by intracellular staining of IFN-γ and flow cytometric analysis as in Fig. 4. Data are representative of two independent experiments with different donors. (B) Purified PDC were targeted as in (A) and cultured for 24 h with 100 ng/mL IL-3 and 5 µg/mL CpG-A, CpG-B or CpG-A plus CpG-B, respectively. Culture supernatants were analyzed for secreted IFN-α by ELISA and PDC were co-cultured with autologous effector-memory T cells as above. Representative results of three independent experiments with different donors are shown. (C) Purified PDC were targeted with CMV antigen via CD36 using a biotinylated CD36 mAb as primary reagent. PDC were stimulated for 24 h as indicated and IFN-α production and T cell stimulation was analyzed as in (B). Data are representative of three independent experiments with different donors.

In view of the fact that CpG-C combines submaximal IFN-I with submaximal antigen presentation induction, we were wondering whether co-stimulation of PDC with CpG-A and CpG-B would mimic CpG-C-induced stimulation of PDC. Surprisingly, CpG-A and CpG-B co-stimulated PDC do not produce IFN-I but induce CMV-specific CD4+ effector-memory T cells to produce IFN-γ to a similar degree as CpG-B-stimulated PDC (Fig. 7B). In order to exclude that the results are dependent on CD303 mAb signaling, CVM antigen was targeted to PDC via the scavenger receptor CD36 (Fig. 7C). Again, only CpG-B-matured PDC were able to restimulate T cells, demonstrating that induction of CMV antigen presentation critically depends on appropriate stimulation and is independent of the receptor which is used for antigen targeting to PDC.

To analyze how far the expression of PD-L1 in CpG-A-activated PDC influences T cell restimulation, anti-PD-L1 mAb was added to PDC-T cell co-cultures. No increase of IFN-γ-producing T cells was observed when PD-L1 was blocked (Supporting Information Fig. 3). Thus the low capacity of CpG-A-activated PDC to restimulate effector-memory T cells cannot be deduced to PD-L1 expression.

To investigate to which degree PDC are committed to the CpG-A- or CpG-B-induced functional phenotype, freshly isolated PDC were stimulated for 24 h with CpG-A/CpG-B or without CpG. Thereafter, PDC were washed extensively and restimulated for additional 24 h with the same or the other CpG oligonucleotide, respectively. Supernatants were analyzed for IFN-I production and PDC were analyzed for CD86 expression (Fig. 8). PDC, which encountered a CpG oligonucleotide upon primary stimulation, cannot be induced to produce IFN-I upon secondary stimulation independent of which CpG oligonucleotide is used for primary and secondary stimulation. However, PDC that are cultured in the presence of IL-3 alone in the first 24-h culture period can be induced to produce IFN-I, in fact surprisingly more efficiently by CpG-B as compared to CpG-A. Compared to CpG-A, the expression level of CD86 is higher when CpG-B is used for primary stimulation, independent of which CpG oligonucleotide is used for secondary stimulation. In contrast, up-regulation of CD86 expression in PDC which are cultured in the presence of IL-3 alone in the first 24-h culture period is more apparent after CpG-B as compared to CpG-A stimulation.

Figure 8.

PDC are committed to the CpG-A- or CpG-B-induced functional phenotype. Purified PDC were cultured with 100 ng/mL IL-3 alone or in combination with 5 µg/mL CpG-A or CpG-B for 24 h. Cells were washed intensively in medium and further cultured for additional 24 h as indicated with IL-3 alone, or in combination with 5 µg/mL CpG-A or CpG-B, respectively. Thereafter, culture supernatants were analyzed for secreted IFN-α by ELISA and PDC were analyzed for expression of CD86 by flow cytometry. Values are presented as means ± standard deviations of six independent experiments with different donors.

Discussion

Here we show that efficient antigen processing and presentation for specific stimulation of T cells and IFN-I production are not co-induced but rather differentially induced functions of PDC. PDC in blood are considered as immature DC or pre-DC 25. Analyzing the intracellular localization of MHC class II molecules and of lysosomal markers such as CD63, we observed remarkable differences when compared with immature bone marrow-derived DC (BMDC). In immature BMDC, lysosomal markers and MHC class II molecules are intracellularly co-localized in MIIC 15, 26. In freshly isolated PDC, lysosomal markers and MHC class II molecules are not co-localized but present in distinct intracellular compartments. When cultured in the presence of IL-3, MHC class II molecules and CD63 molecules undergo a redistribution process. Within less than 12 h, both types of molecules accumulate in few much larger perinuclear vesicles reminiscent of MIIC. According to the intracellular localization of MHC class II molecules and lysosomal markers, PDC are distinct from immature BMDC, when freshly isolated, but similar to BMDC after culture-induced differentiation. Thus, PDC represent pre-DC rather than immature DC.

Independent of Toll-like receptor (TLR) ligand stimulation, CD303 mAb is rapidly internalized by PDC and traffics via EEA1+ early endosomes within 15 h to the concomitantly emerging MIIC compartments. Inaba et al.26 showed that immature BMDC internalize HEL antigen, transport it to MIIC within 3 h, but fail to form MHC class II-peptide complexes, even after an incubation period of 24 h. When BMDC are exposed to HEL in the presence of a maturation stimulus, abundant MHC class II-peptide complexes are formed within 3 h in MIIC and MHC class II-peptide complexes are detected on the cell surface after 24 h.

Whether PDC present MHC class II-CMV peptide complexes after receptor targeting of CVM antigen has been analyzed here indirectly by restimulation of CMV-specific CD4+ effector-memory T cells from CMV-seropositive donors. CMV-specific CD4+ effector-memory T cells could be efficiently restimulated by PDC when CMV antigen was targeted to CD303 or CD36 and PDC were stimulated for 24 h with CpG-B, but not with CpG-A. CpG-B-stimulated PDC express CD86 and CD80 at higher levels compared to CpG-A-stimulated PDC. This is unlikely to be the reason for the observed differences in restimulation of CMV-specific CD4+ effector-memory T cells. CMV-specific CD4+ effector-memory T cells are known to lack expression of CD28 27 and their restimulation is therefore rather independent of co-stimulation by CD80 and CD86 molecules. This could be confirmed by lack of any differences in restimulation of CMV-specific CD4+ effector-memory T cells by CpG-B-stimulated versus non-stimulated PDC, when PDC are pulsed with CMV peptides (data not shown). Thus, we conclude that in contrast to CpG-A, CpG-B induces efficient CMV antigen presentation of MHC class II-CMV peptide complexes to CMV-specific CD4+ effector-memory T cells by PDC.

Cella et al.23 showed that newly synthesized MHC class II molecules disappear rapidly from the cell surface of immature MoDC with a half-life of about 10 h. In mature MoDC the half-life of MHC class II molecules is increased more than ten times compared to immature MoDC. We show here that CpG-B-stimulated PDC retain their CMV-specific CD4+ effector-memory T cell stimulation capacity for up to 5 days after targeting of CMV antigen to CD303. This strongly suggests that CpG-B induces differentiation of PDC to functionally mature DC with strong and sustained antigen presentation capabilities.

Unlike CpG-B-stimulated PDC, about 30% of the CpG-A-stimulated PDC express PD-L1 (CD274). PD-L1 18, 28 is known to bind to programmed cell death receptor-1 20, 29 on activated T cells and thereby inhibit T cell proliferation and T cell production of IFN-γ and IL-2 18, 19, 30. Expression of PD-L1 on monocytes and semi-matured DC, but not B cells, is enhanced by IFN-β 30. Direct or indirect induction of PD-L1 expression in PDC by CpG-A but not CpG-B points to a novel role for IFN-I-producing PDC regulating T cell immunity.

Whether CpG oligonucleotides induce production of IFN-I or rather up-regulation of CD80 and CD86 has been shown to be determined by the higher-order structure and intracellular location of the CpG oligonucleotide 31, 32. Activation of TLR9 by the multimeric CpG-A occurs in endosomes and leads exclusively to IFN-I production, whereas monomeric CpG-B oligonucleotides localize to lysosomes and promote CD80 and CD86 expression of PDC. However, CpG-B, when complexed into microparticles, localizes in endosomes and induces IFN-I from PDC, whereas monomeric forms of CpG-A localize to lysosomes and induce up-regulation of CD80 and CD86 expression 31.

CpG-C is distributed in both types of endosomes. Hence, it may be anticipated that CpG-C-stimulated PDC induce restimulation of CMV-specific CD4+ effector-memory T cells and secretion of IFN-I. However, compared to CpG-A- and CpG-B-stimulated PDC, respectively, CpG-C- 24 and 7-allyl-8-oxoguanosine- (loxoribine; data not shown) stimulated PDC neither induce maximum restimulation of CMV-specific CD4+ effector-memory T cells nor maximum secretion of IFN-I. We are currently analyzing whether CpG-C and loxoribine induce a mixed functional phenotype of PDC at the single-cell level or only at the population level. It might well be that CpG-C induces some PDC to produce IFN-I and some PDC to process CMV antigen and present MHC class II-CMV peptide complexes, but rarely or never both functions in the very same PDC.

Interestingly, co-stimulation of PDC with CpG-A and CpG-B induces maximum restimulation of CMV-specific CD4+ effector-memory T cells but no secretion of IFN-I. It appears unlikely that this is due to changes in the intracellular location of CpG-A and CpG-B. Most likely, CpG-A is still present in endosomes and CpG-B in lysosomes. We suspect that robust lysosomal TLR9 signaling by CpG-B inhibits induction of IFN-I secretion by endosomal TLR9 signaling and thereby prevents co-induction of both functions in the very same PDC.

Once PDC have been induced to produce IFN-I by CpG-A stimulation, they can hardly be induced to up-regulate CD86 expression by subsequent CpG-B stimulation. Also, once PDC have been induced to strongly up-regulate CD86 expression, they cannot be induced to produce IFN-I by subsequent CpG-A stimulation. This strongly suggests that both events are induced mutually exclusively rather than consecutively.

PDC produce IFN-I when cultured for 24 h in the presence of IL-3 prior to CpG-B stimulation. This clearly demonstrates that the PDC response is not exclusively determined by the nature of the stimulus, e.g. the higher-order structure of the CpG oligonucleotide, but at least to the same degree by the differentiation stage of the PDC. Redistribution of intracellular compartments upon culturing in the presence of IL-3 (see above) may result in endosomal instead of lysosomal TLR9 signaling by CpG-B. CpG-B may fail to reach lysosomes at this stage.

CpG-A- and CpG-B-stimulated PDC differ only marginally regarding their capability to induce in vitro proliferation of naive allogeneic CD4+ T cells in mixed lymphocyte reactions 31, 33. We suspect that this is due to direct recognition of intact allo-MHC molecules on PDC that is independent on antigen processing and presentation by PDC.

PDC can act in a tolerogenic as well as immunogenic way 34, making this DC subpopulation a promising target for immunotherapy of cancer and autoimmune diseases as well as for educating the immune system to accept transplanted allografts. In vivo targeting of antigen to CD303 should enable specific in vivo delivery of tumor antigens or autoantigens to PDC. Even though mAb triggering of CD303 inhibits IFN-I production in PDC 12, most likely in vivo targeting of antigen to CD303 as such will not determine whether the targeted PDC is immunogenic or tolerogenic. Specific induction of immunogenicity and tolerogenicity in PDC could rather be achieved by e.g. TLR ligands, agonistic CD40 mAb or CD40L-blocking mAb. Recently it has been demonstrated, that efficient presentation of phagocytosed antigen by mouse BMDC is dependent on the presence of stimulating TLR ligands within the very same phagosome that contains the antigen 35. Thus, the TLR ligands and the antigens should be physically associated to ensure that internalized antigens and TLR ligands end up in the very same phagosome, and antigen is efficiently presented.

In conclusion, our results demonstrate that innate and adaptive immunity are not linked at the level of individual PDC by production of large amounts of IFN-I followed by triggering of adaptive T cell-mediated immunity. Rather, both PDC functions are mutually exclusively induced by distinct signals. We suspect that this functional dichotomy is critical for controlling adaptive immunity, maintaining self tolerance and preventing induction of autoimmunity. In vivo targeting of antigens to PDC via CD303 combined with appropriate PDC stimulation may allow stimulation of specific anti-tumor T cell immunity or induction of tolerance 36.

Materials and methods

Media, oligonucleotides, and antigen conjugates

Cells were cultured in X-vivo-15 medium (Biowhittaker/Cambrex, Verviers, Belgium) supplemented with 10% human AB serum (PAA Laboratories, Linz, Austria) at 37°C in humidified atmosphere containing 9% CO2. Depending on the assay, medium was further supplemented as indicated in the particular protocol.

Completely and partially phosphorothioate-modified CpG ODN were obtained from Metabion (Germany). Small letters indicate phosphorothioate linkage and capital letters indicate phosphodiester linkage at the 3′ end of the respective nucleotide: CpG-A (ODN 2216), 5′-ggGGGACGATCGTCgggggG-3′, CpG-B (ODN 2006), 5′-tcgtcgttttgtcgttttgtcgtt-3′; and CpG-C (M352), 5′-TCGTCGAACGTTCGAGATGAT-3′. FITC-conjugated anti-biotin mAb (Miltenyi Biotec, Bergisch Gladbach, Germany) was coupled to CMV antigen or CMV control antigen (CMV complement Fixation Test; Dade Behring, Germany) following standard conjugation chemistry as previously described 37, 38.

Cell preparation

Buffy coats from normal healthy volunteers were obtained from the Institute for Transfusion Medicine (Dortmund Hospital, Germany). PBMC were prepared from buffy coats by standard Ficoll-Paque (GE Healthcare, Germany) density gradient centrifugation.

PDC were isolated from PBMC under sterile conditions by direct magnetic labeling with anti-blood DC cell antigen 4 mAb (AD5-17F6)-conjugated microbeads (BDCA-4 Cell Isolation Kit; Miltenyi Biotec) using PBS/EDTA buffer (Miltenyi Biotec) supplemented with 0.5% human AB serum (PAA Laboratories). Labeled cells were enriched using a high-gradient magnetic cell sorting device (Mini- and Midi-MACS®; Miltenyi Biotec) according to the manufacturer's instructions. PDC purities of 95–98% were routinely obtained, as determined by staining with FITC-conjugated CD303 mAb (AC144; Miltenyi Biotec). PDC-depleted PBMC (1×107–2×107/mL) were cultured in flasks (BD Falcon, Germany) overnight and autologous effector-memory CD4+ T cells were isolated using the memory CD4+ T cell Isolation Kit (Miltenyi Biotec) according to the manufacturer's instructions. As determined by staining with PE-conjugated CD45RO mAb (UCHL-1; Miltenyi Biotec), FITC-conjugated CD45RA mAb (OX-33; Miltenyi Biotec), and allophycocyanin-conjugated CD4 mAb (SK3; Miltenyi Biotec), 95–98% pure CD4+ CD45RO+ CD45RA T cells were routinely obtained. Isolated T cells were used directly after separation, or were cryo-preserved before co-culture with PDC as indicated.

For restimulation of T cells after the proliferation phase, autologous PBMC were depleted of T cells, using CD3 microbeads (Miltenyi Biotec) according to the manufacturer's instructions. T cell-depleted PBMC were cryo-preserved and used for restimulation at time points indicated.

For cryo-preservation, aliquots of T cells or T cell-depleted PBMC were frozen in human AB serum (PAA Laboratories) supplemented with 10% DMSO (Sigma-Aldrich, Germany) at –70°C or in liquid nitrogen at –196°C, respectively. For stimulation analysis, purified PDC (1×105–2×105 in 100 µL/well) were cultured for the indicated time periods in medium supplemented with 100 ng/mL IL-3 (Peprotech, UK) alone, or in combination with 5 µg/mL CpG-A, CpG-B (Metabion), 100 ng/mL IFN-α (PBL Biomedical Laboratories) or anti-IFN-α (a gift from Ilkka Julkunen, Finnland), respectively, as indicated.

Antigen targeting and in vitro restimulation of CD4+ CD45RO+ CD45RA T cells

PDC were isolated from CMV-seropositive donors and labeled for 10 min on ice in PBS/EDTA buffer supplemented with 0.5% AB serum (PAA Laboratories) with biotin-conjugated CD303 mAb (AC144; Miltenyi Biotec), CD45 mAb (5B1; Miltenyi Biotec) and anti-cytokeratin mAb (CK3-21F11; Miltenyi Biotec) as a primary reagent. Cells were washed and labeled for 10 min on ice with CMV- or control antigen-conjugated anti-biotin-FITC mAb (10 µg/mL) as a secondary reagent. Antigen-targeted PDC (1×105 in 100 µL/well) were cultured for 24 h in 96-well flat-bottom plates (TPR, Switzerland) in medium with 100 ng/mL IL-3 (Peprotech) alone, or in combination with 5 µg/mL CpG-A, CpG-B or CpG-C (Metabion), respectively. Subsequently, PDC were co-cultured with isolated autologous effector-memory T cells at a 1:10 ratio (PDC:T cells, 1×106 total cells in 200 µL/well) in 96-well round-bottom plates (TPR) for 20 h with or without 1 µg/mL anti-PD-L1 mAb (eBioscience, USA). After 16 h, brefeldin A was added at a final concentration of 5 µg/mL (Sigma-Aldrich, Germany). IFN-γ production by T cells was analyzed by intracellular staining.

For antigen-dependent T cell proliferation experiments, antigen-targeted PDC and memory-effector T cells were co-cultured at a 1:10 ratio (PDC:T cells) for 10 days or 3 days in 48-well flat-bottom plates (Corning Incorporated) in the presence of 100 ng/mL IL-3, 10 ng/mL IL-2 and 10 ng/mL IL-7 (Peprotech) at a total cell concentration of 1×106 cells per mL. After the expansion phase, T cells were restimulated for 20 h with autologous T cell-depleted PBMC in the presence of 5 µg/mL CMV antigen (Dade Behring). After 16 h, brefeldin A was added at a final concentration of 5 µg/mL (Sigma-Aldrich). IFN-γ production by T cells was analyzed by intracellular staining. In other proliferation experiments, memory-effector T cells were labeled with 10 µmol CFSE (Sigma-Aldrich, Germany) in medium at 37°C for 10 min, washed two times in PBS/EDTA and once in medium before co-culture at the same conditions as described above. CFSE-labeled T cells were co-cultured with PDC for 4 days before analysis by FACS.

Confocal scanning microscopy

For antigen transport analysis after internalization, purified PDC were indirectly labeled for 10 min on ice in PBS/EDTA buffer (Miltenyi Biotec) supplemented with 0.5% human AB serum (PAA Laboratories) with 10 µg/mL biotin-conjugated CD303 mAb (AC144; Miltenyi Biotec) as primary labeling reagent. Cells were washed, labeled for 10 min on ice with Alexa Fluor 633-conjugated streptavidin (Invitrogen, Germany) as secondary reagent and cultured in medium with 100 ng/mL IL-3 alone or in combination with 5 µg/mL CpG-B for the time periods indicated. After cultivation, cells were centrifuged onto coverslips, fixed in PBS containing 3.7% paraformaldehyde for 5 min, washed in PBS containing 0.1% sodium borohydride and permeabilized in saponin buffer (PBS containing 0.5% BSA, 0.5% FCS, 1% fish skin gelatin and 0.1% saponin) for 30 min. Cells were then stained in saponin buffer for 1 h with FITC-conjugated mAb against CD63 (H5C6), CD107a (1D4B), HLA-DR (G46-6) and EEA1 (14) (Becton Dickinson, Germany), respectively. Before analysis, coverslips were washed twice in saponin buffer and PBS, and mounted in Vectrashield (Vector, Burlingame, CA). Cells were immediately analyzed by CLSM (Leica, Germany).

For PDC maturation analysis, purified PDC were cultured for the indicated time periods in medium supplemented with 100 ng/mL IL-3 alone, or with IL-3 and 5 µg/mL CpG-A or CpG-B, respectively. PDC were stained intracellularly as described above using FITC-conjugated CD63 mAb and biotin-conjugated anti-HLA-DR mAb (910/D7; Miltenyi Biotec) as primary reagent, washed three times in saponin buffer and stained with Alexa Fluor 633-conjugated streptavidin (Invitrogen) as secondary reagent.

Flow cytometry

At indicated time points, PDC were washed in PBS/EDTA buffer supplemented with 2% BSA (Kraeber, Germany) and stained for 10 min at 4°C using fluorescence-labeled mAb against human CD40 (3/23), CD80 (3H5), CD86 (2331) (Becton Dickinson) and PD-L1 (MIH1) (eBioscience), respectively. To block FcR-mediated antibody binding, FcR-blocking reagent (Miltenyi Biotec) was added. Dead cells were excluded from analysis according to propidium iodide staining.

IFN-γ production was determined by intracellular IFN-γ staining. Using Inside Stain Kit (Miltenyi Biotec), cells were stained with allophycocyanin-conjugated CD4 mAb (M-T466; Miltenyi Biotec) and PE-conjugated anti-IFN-γ mAb (45-15; Miltenyi Biotec) according to the manufacturer's instructions. For analysis of CFSE-labeled T cells, cells were stained with allophycocyanin-conjugated CD4 mAb (M-T466; Miltenyi Biotec) before analysis. Cells were analyzed using the FACSCaliburTM and CellQuestPro software (Becton Dickinson).

Cytokine detection

For the detection of cytokines, culture supernatants were collected at the time points indicated and analyzed for the presence of IL-1β, IL-4, IL-6, IL-8, IL-10, TNF-α, TNF-β and IFN-γ using Cytokine bead array (human Th1/Th2 cytokine Flow Cytomix; Bender MedSystems, CA, USA). The production of IFN-α was analyzed by ELISA (human IFN-α Module Set; Bender MedSystems, CA, USA) according to the manufacturer's instructions.

Acknowledgements

We thank I. Johnston and Ralph Schaloske for critical reading of the manuscript.

Appendix

Some of the authors are employed by Miltenyi Biotec.

Footnotes

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