Published online before print August 4, 2005
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and Th1 chemokine production
,1
,3
,4
* Laboratory for Immunological Research, Schering-Plough Research Institute, Dardilly, France;
INSERM U590, Centre Léon Bérard, Lyon, France;
DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, California;
Department of Research and Development, Research Group on Lymphoma, EFS Rhône-Alpes Grenoble, La Tronche, France; and
|| University of Minnesota Cancer Center, Minneapolis, Minnesota
1Correspondence: INSERM U590, Centre Léon Bérard, Lyon, France. E-mail: bendris{at}lyon.fnclcc.fr
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(IFN-)-inducible protein (IP)-10/CXC chemokine ligand 10 (CXCL10) and macrophage inflammatory protein-1ß/CC chemokine ligand 4 (CCL4)] or Th2 cells (such as thymus and activation-regulated chemokine/CCL17 and monocyte-derived chemokine/CCL22), respectively. Activated natural killer cells were preferentially recruited by supernatants of virus-activated PDC, and supernatants of CD40L-activated PDC attracted memory CD4+ T cells, particularly the CD4+CD45RO+CD25+ T cells described for their regulatory activities. It is striking that CD40L and virus synergized to trigger the production of IFN- by PDC, which induces another Th1-attracting chemokine monokine-induced by IFN-/CXCL9 and cooperates with endogenous type I IFN for IP-10/CXCL10 production. In conclusion, our studies reveal that PDC participate in the selective recruitment of effector cells of innate and adaptive immune responses and that virus converts the CD40L-induced Th2 chemokine patterns of PDC into a potent Th1 mediator profile through an autocrine loop of IFN-.
Key Words: PDC • migration • memory T cells • NK cells • chemokines
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PDC, similarly to in vitro-derived myeloid human DC [16
], have the capacity to induce T helper cell type 1 (Th1) or Th2 responses, depending on the type of activation, its duration, and the antigen dose [17
18
19
20
21
]. Virus-activated human PDC prime naive CD4+ T cells to differentiate into Th1 cells in an IFN-
-dependent manner [18
, 19
]. Whereas upon CD40 ligand (CD40L) activation, human PDC induce naive CD4+ T cells to secrete type 2 cytokines [17
] by an OX40L-dependent mechanism [22
]. Of note, interleukin (IL)-10 is produced by naive CD4+ T cells primed by virus- or CD40L-activated PDC [17
, 19
], and PDC can induce the development of regulatory CD8+ T cells [23
] and CD4+ CD25+ T cells [24
] in vitro. Studies in mice suggest that PDC may have a role in the generation of CD4+ regulatory T cells [25
26
27
]. It is most important that human and mouse PDC express a different repertoire of TLRs than myeloid DC, suggesting that they may have evolved to respond to different types of pathogens [4
]. Thus, different DC subsets are required to elicit a specific response to a given pathogen [2
].
Th1, Th2, and regulatory T cells express distinct sets of chemokine receptors and display distinct chemotactic responsiveness. More specifically, CXC chemokine receptor 3 (CXCR3) ligands [i.e., monokine induced by IFN- (MIG)/CXC chemokine ligand 9 (CXCL9), IFN--inducible protein (IP)-10/CXCL10, and IFN-inducible T cell- chemoattractant (I-TAC)/CXCL11] and CC chemokine receptor 5 (CCR5) ligands [i.e., macrophage inflammatory protein (MIP)-1/CC chemokine ligand 3 (CCL3), MIP-1ß/CCL4, and regulated on activation, normal T expressed and secreted (RANTES)/CCL5] have been clearly identified as Th1-attracting chemokines, whereas CCR4 ligands [i.e., monocyte-derived chemokine (MDC)/CCL22 and thymus and activation-regulated chemokine (TARC)/CCL17] act as Th2 as well as regulatory T cell-attracting chemokines [28
29
30
31
32
]. Few data have been reported regarding chemokine production by ex vivo-purified DC. Recently, it has been shown that CD40L (or CpG-)- but not virus-activated PDC produce MDC/CCL22 at low levels [33
, 34
], and activated PDC failed to produce TARC/CCL17 [34
]. Conversely, RANTES/CCL5, IL-8/CXCL8, and IP-10/CXCL10 are secreted by virus-activated PDC [34
35
36
], and MIP-1/CCL3 and MIP-1ß/CCL4 are produced by virus-, CpG-, or resiquimod-stimulated PDC [34
35
36
37
]. In addition, it was proposed that IP-10/CXCL10 and MIP-1ß/CCL4 are involved in PDC-induced chemotaxis of activated T cells and natural killer (NK) cells [36
].
In this report, we show that human tonsillar PDC activated with signals that endow them with the ability to promote a type 1 response (virus), secreted type 1-attracting chemokines, and recruited activated NK cells, and PDC, activated by a type 2 signal (CD40L), produced type 2-attracting chemokines and attracted CD4+ CD25+ T cells described for their regulatory activity. Furthermore, we also observed that CD40 and virus synergized to induce a dominant Th1 profile through an IFN- autocrine loop. Collectively, our results suggest that PDC participate in the selective recruitment of effector cells of innate and adaptive immune responses and that they are critically involved in Th1 responses.
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Cytokines and antibodies
Human recombinant IL-3, IFN-2b, and IFN- were purchased, respectively, from R&D Systems (Minneapolis, MN), Schering-Plough Research Institute (Kenilworth, NJ), and Sigma Chemical Co. (St Louis, MO). Blocking antibodies used in this study were mouse monoclonal anti-human IFN-/ß receptor chain 2/CD118 [MMHAR-2, mouse immunoglobulin G2a (mIgG2a); PBL Biomedical Laboratories, Piscataway, NJ] and anti-IFN- (MAB285, mIgG2a, R&D Systems, Minneapolis, MN) antibodies, and the isotype control antibody (mIgG2a) was purchased from R&D Systems (Minneapolis, MN). Monoclonal antibodies (mAb) used for flow cytometry included phycoerythrin (PE)-labeled anti-CD123, -CD80, -CD86, -CCR7, -MIG/CXCL9 (B8-11, mIgG1), -IP-10/CXCL10 (6D4/D6/G2, mIgG2a), and -IFN- (B27, mIgG1, BD PharMingen, San Diego, CA) and fluorescein isothiocyanate (FITC)-labeled anti-CD123 (Miltenyi Biotec, Germany) and anti-IFN-2 (225.2C, mIgG2b, Chromaprobe, Aptos, CA).
Isolation of fresh PDC
Human blood was obtained anonymously from the Etablissement Français du Sang (Lyon, France) after the donor gave informed consent according to the Declaration of Helsinki, specifically indicating the possible research use of the sample if it was not suitable for transfusion use. Isolation of PDC from tonsils (discarded human surgical material obtained anonymously according to institutional regulations in compliance with French law) was performed as described previously [38
]. Briefly, tonsils were cut into small pieces and digested for 30 min at 37°C with collagenase IV (1 mg/ml, Sigma Chemical Co.) and DNase I (50 KU/ml, Sigma Chemical Co.) in RPMI 1640. Lineage marker-negative (Lin–) mononuclear cells were obtained by the Ficoll-Rosetting method (eliminating CD2+ cells), followed by immunomagnetic depletion of contaminating cells reactive to a mixture of anti-CD3 (OKT3 ascites), -CD14 (MOP9.25 ascites), -CD19 (4G7 ascites), -CD20 (1F54 ascites), -CD56 (NKH1, Beckman Coulter, Marseille, France), and -glycophorin A (clone 11E4B7.6, Beckman Coulter) antibodies using goat anti-mouse IgG coupled to magnetic beads (Dynabeads M-450, Dynal, Oslo, Norway). The enriched Lin– cells were further stained with anti-CD11c PE (clone S-HCL-3, Becton Dickinson, San Jose, CA), anti-CD4 PE-Cy5 (clone 13B8.2, Beckman Coulter), and a cocktail of FITC-Lin [anti-CD1a (clone HI149, BD PharMingen), -CD3 (clone UCHT1, Dako, Denmark), -CD14 (clone MOP9, Becton Dickinson), -CD15 (clone C3D-1, Dako), -CD16 (clone NKP15, Becton Dickinson), -CD19 (clone 4G7, Becton Dickinson), -CD20 (clone B-Ly1, Dako), -CD34 (clone 58, Beckman Coulter), -CD35 (clone E 11, BD PharMingen), -CD56 (clone NCAM16.2, Becton Dickinson), and -CD57 (clone HNK-1, Becton Dickinson)] antibodies. Tonsil PDC were fluorescein-actived cell sorter (FACS)®-sorted as Lin– CD4+ CD11c– cells using a FACStarplus® flow cytometer (BD Biosciences, Sunnyvale, CA). All the procedures of staining and sorting were performed in the presence of 0.5 mM EDTA to avoid cell aggregation. Reanalysis of the sorted populations showed a purity >98% (Fig. 1
).
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Figure 1. Purification of human tonsil PDC. Flow cytometry analysis of (A) DC-enriched population from tonsil following Ficoll-Rosetting and immunomagnetic bead depletion of non-DC subsets before cell sorting and (B) FACS-sorted Lin– CD4+ CD11c– PDC according to the combined gate settings shown. Enriched cells were triple-stained with anti-Lin (CD3, CD14, CD15, CD16, CD19, CD20, CD34, CD35, CD56, and CD57) FITC, anti-CD11c PE, and anti-CD4 PE-Cy5 antibodies. The percentage of Lin– cells after sorting varies between 0.05 and 2 % (mean=0.93%; median=0.78%; n=10). Data shown are representative of 10 experiments. FSC/SSC, Forward-scatter/side-scatter, respectively.
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NK cells were enriched from human PBMC of healthy donors following a similar procedure using anti-CD3 (clone OKT3) and anti-CD4 (clone Q4120, Sigma Chemical Co.) mAb instead of anti-CD16 and anti-CD56 mAb. After two rounds of Dynabeads depletion, the purity of CD56+ NK cells was routinely higher than 80%. The enriched cell preparations were assessed for phenotype and chemotaxis.
In vitro stimulation of PDC
For chemokine production, isolated PDC were cultured at 5 x 105 cells/ml in the presence of IL-3 (20 ng/ml), with or without irradiated murine fibroblastic L cells transfected with human CD40L (hCD40L; at a ratio of 10 PDC for one L cell) [39
]; 1 HAU/ml formaldehyde-inactivated human influenza (FLU) virus, strain New Caledonia (kindly provided by Nicholas Kuehn, Aventis Pasteur, Val de Reuil, France); simultaneous IL-3, hCD40L-transfected L cells, and virus; or IFN-2b (1000 U/ml). In some experiments, IFN- was added at a final concentration of 25 ng/ml. For blocking experiments, isolated PDC (5x105 cells/ml) were incubated with IL-3 and virus, with or without hCD40L-transfected L cells, and mouse monoclonal anti-human IFN-/ß receptor chain 2 and anti-IFN- antibodies were used at 20 µg/ml. As IFN- is an endogenous survival factor produced in response to virus, we added IL-3 to the virus group to circumvent the effect of neutralization of type I IFN on PDC death.
To evaluate the chemotactic activities of stimulated PDC-derived supernatants, PDC were cultured at 106 cells/ml in the presence of 1 HAU/ml FLU virus and IFN- (25 ng/ml) or IL-3 (20 ng/ml) and irradiated hCD40L-transfected L cells for 60 h. This activation time was predetermined to have an optimal chemokine production while avoiding chemokine degradation.
Enzyme-linked immunosorbent assay (ELISA) and intracellular staining
MDC/CCL22, TARC/CCL17, MIP-1ß/CCL4, IP-10/CXCL10, MIG/CXCL9, RANTES/CCL5, IL-8/CXCL8, stromal-derived factor 1 (SDF-1)/CXCL12, and IFN- productions were determined by specific quantitative ELISA (R&D Systems, Abington, UK) using cell-culture supernatants at 60 h stimulation. Culture supernatants of CD40L-transfected L cells were negative in all chemokine ELISAs measured. For intracellular flow cytometry analysis, PDC were cultured as described in the presence of IL-3 and hCD40L-transfected L cells with or without FLU virus for 14 h, followed by addition of the GolgiPlug (BD PharMingen) for 8 h. The cells were harvested, stained with FITC- or PE-labeled anti-CD123, and then fixed with 2% paraformaldehyde (Sigma Chemical Co.), permeabilized with 0.5% saponin, and stained with the PE anti-IP-10/CXCL10, -MIG/CXCL9, -IFN-, or FITC anti-IFN-2 antibodies.
Migration assays
Production of cell culture supernatants
Culture supernatants of virus and IFN-- versus IL-3 and CD40L-activated PDC, thereafter referred to as PDC SN1 and PDC SN2, respectively, were harvested following 60 h incubation. Control supernatants were complete medium containing 1 HAU/ml FLU virus and IFN- (25 ng/ml; control SN1) or medium from CD40L-transfected L cells cultured in IL-3 (20 ng/ml; control SN2). After 60 h culture, culture supernatants were recovered, 0.22 µm-filtrated, and used for chemotaxis assays at a 50% final concentration.
In vitro chemotaxis assay
To evaluate the chemotactic activities of 60 h activated PDC-derived supernatants, we used, as target cells, PBMC, T or NK cell-enriched populations, freshly isolated or cultured overnight with IFN-2b (1000 U/ml), or IL-2 (200 ng/ml) and IL-15 (200 ng/ml; R&D Systems, Minneapolis, MN). Cell migration was measured in 24-well Transwell plates (6.5 mm diameter, 3 µm pores, Corning Costar Europe, The Netherlands). Enriched T or NK cells were suspended at 5 x 106 cells/ml in RPMI 1640 containing 5% FCS, kept for 1 h at 37°C (5% CO2), and then 100 µl cell suspension was added to the top chambers (5x105 total cells/well). In the bottom chambers, 600 µl different solutions were prepared: recombinant human chemokines (MIP-1ß/CCL4, TARC/CCL17, MDC/CCL22, MIG/CXCL9, IP-10/CXCL10, and SDF-1/CXCL12, from R&D Systems, Minneapolis, MN, used at 500 ng/ml) in RPMI 1640 containing 5% FCS, control medium (RPMI 1640 containing 5% FCS), the control supernatants (control SN1 and SN2), and the supernatants from activated PDC (PDC SN1 and SN2). Culture supernatants of CD40L-transfected L cells were negative in the chemotaxis assays. The different PDC supernatants were used, diluted 1/2, and distributed into the bottom well. After 1.5 h (for T cell migration) or 2.5 h incubation (for NK cell migration) at 37°C (5% CO2), the transwell inserts were lifted, and cells from the lower chambers were collected in a separate tube; each well was rinsed once with 500 µl of a buffer containing 0.5 mM EDTA and 2% FCS in phosphate-buffered saline. Migrated cells were identified by FACS staining for the expression of CD3, CD4, or CD8 for total T cells; CD3, CD4, and CD45RA or CD45RO for naive versus memory T cells; and CD4, CD45RO, and CD25 for regulatory T cells. NK cells were identified as CD56+ CD3– cells. Each sample was acquired on a FACSCaliburTM for 90 s. In some experiments, purified, neutralizing mAb to MDC/CCL22 (clone 57226.11, R&D Systems, Minneapolis, MN), TARC/CCL17 (clone 540026.11, R&D Systems, Minneapolis, MN), IP-10/CXCL10 (clone 33036.211, R&D Systems, Minneapolis, MN), and MIG/CXCL9 (clone 49106.11, R&D Systems, Minneapolis, MN) or isotype controls (normal mouse IgG1 and IgG2a) were added to the cell supernatants at a final concentration of 50 or 100 µg/ml, 10 min before the start of the assay. Migration inhibition data were determined by subtracting the amount of background migration from the experimental groups. Results were expressed as migration index (ratio chemokine/medium) or as number of migrating cells (mean of duplicate experimental points).
Statistical analysis
Statistical analysis of results was done with two-tailed Student’s t-test. Data are means ± SD, unless otherwise indicated.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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/CCL3, and MIP-1ß/CCL4 transcripts, and CD40L-activated PDC expressed high levels of MDC/CCL22 and low but significant levels of TARC/CCL17 and I-309/CCL1 transcripts (not shown). Similar results were observed with leukemic PDC (not shown).
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Figure 2. Production of IP-10/CXCL10, MIP-1ß/CCL4, MDC/CCL22, and TARC/CCL17 by stimulated PDC. (A) Supernatant of 5 x 105 cells/ml FACS-sorted human tonsillar PDC cultures stimulated with IL-3 and hCD40L-transfected L cells (shaded circles) or virus (open circles) were analyzed for chemokine contents after 60 h activation. Each circle represents an independent experiment (n=10 for IP-10/CXCL10, n=4 for MIP-1ß/CCL4, and n=5 for MDC/CCL22 and TARC/CCL17), and horizontal bars represent the mean. Statistical significance between IL-3/CD40L versus virus activation (P values) is indicated and was performed using the Student’s t-test. (B) Chemokine protein production by human tonsillar PDC was analyzed at different time-points of parallel cultures (12–72 h and 4 days) with virus for IP-10/CXCL10 and MIP-1ß/CCL4 or with IL-3 and hCD40L L cells for MDC/CCL22 and TARC/CCL17.
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In the presence of exogenous IFN-, tonsillar PDC produce MIG/CXCL9
MIG/CXCL9 protein was not detectable in the culture supernatants of (virus- or CD40L-) activated tonsillar PDC. When exogenous IFN- was added to the cultures, CD40L- and virus-activated tonsillar PDC secreted MIG/CXCL9 (13.7±2.2 ng/ml, n=4, and 2.9±2.7 ng/ml, n=4, respectively; Fig. 3
). Exogenous IFN- also increased the production of IP-10/CXCL10 by virus (two- to threefold)- or CD40L-activated tonsillar PDC (3.7- to 18-fold) without significantly affecting MIP-1ß/CCL4, TARC/CCL17, and MDC/CCL22 production (Fig. 3)
. IFN- induction of MIG/CXCL9 and enhancement of IP-10/CXCL10 by CD40L-activated tonsillar PDC were dose-dependent (not shown). Of note, in the absence of activation signals such as CD40L or virus, exogenous IFN- enhanced the production of IP-10/CXCL10 (5.8 ng/ml vs. 0.8 ng/ml) and induced that of MIG/CXCL9 (3.3 ng/ml) without significantly inducing MIP-1ß/CCL4 production (<500 pg/ml) by IL-3-cultured tonsillar PDC.
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Figure 3. Production of MIG/CXCL9, IP-10/CXCL10, MIP-1ß/CCL4, MDC/CCL22, and TARC/CCL17 by stimulated PDC in the presence of exogenous IFN-. Supernatants of 5 x 105 cells/ml FACS-sorted human tonsillar PDC cultures stimulated with IL-3 and hCD40L L cells (black bars) or virus (gray bars) were analyzed for chemokine contents in the presence (hatched bars) or absence (filled bars) of IFN- (25 ng/ml) after 60 h activation. Results are indicated as the quantity of chemokines (ng/ml) ± SD and are representative of four independent experiments.
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(PDC SN1)- or IL-3 and CD40L-activated (PDC SN2) tonsillar PDC were tested in migration assay. Enriched NK cells cultured overnight with IFN-, but not resting NK cells, migrate significantly in response to PDC SN1 (1.9- to 2.1-fold increase, n=3, P=0.03) and to a lower extent, to PDC SN2 (less than 1.3-fold, n=3, P=0.004; Fig. 4
) in a dose-dependent manner (not shown). Priming with IFN- was more efficient than IL-2 and IL-15 to induce NK cells to migrate in response to virus-activated PDC supernatants (not shown). IFN- treatment increased NK cell mobility (Fig. 4 , medium alone) and NK cell chemotactic response to SDF-1/CXCL12 and to a lesser extent, to combined MIG/CXCL9 and IP-10/CXCL10 (Fig. 4)
. Neither untreated nor IFN--treated NK cells migrated in response to TARC/CCL17, MDC/CCL22, MIP-1ß/CCL4, or combined TARC/CCL17 and MDC/CCL22 (Fig. 4
and not shown). Of note, CXCR3 expression on NK cells, but not CCR4, CCR5, or CXCR4, was up-regulated by IFN- (not shown). The migration of IFN--treated NK cells in response to PDC SN1 was brisker than that observed in response to optimal concentrations of IP-10/CXCL10 and MIG/CXCL9, alone or in combination (Fig. 4)
. Indeed, neutralizing mAb to MIG/CXCL9 and IP-10/CXCL10 and their combination only inhibited the migration of IFN--treated NK cells marginally in response to PDC SN1 (not shown). Thus, our data suggest that NK cells require priming by IFN-, which is produced at high levels by PDC exposed to viruses, to migrate in response to virus-activated PDC supernatants and that in addition to MIG/CXCL9 and IP-10/CXCL10, other factors contribute to migration of IFN--primed NK cells.
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Figure 4. Migration of NK cells in response to culture supernatants of 60 h-activated PDC. Resting (open bars) or IFN--treated (solid bars) NK cells were tested in the chemotaxis assay using 3 µm transwells (as described in Materials and Methods) in response to medium, tonsillar PDC SN1 (50%), PDC SN2 (50%), and their respective control SN used at the same percentages (control SN1 and control SN2, i.e., activators alone in the absence of PDC), MIG/CXCL9 (500 ng/ml), IP-10/CXCL10 (500 ng/ml), TARC/CCL17 (500 ng/ml), MDC/CCL22 (500 ng/ml), and SDF-1/CXCL12 (500 ng/ml). After 2.5 h, migrated cells were recovered from the lower chambers, stained with anti-CD56 PE and anti-CD3 PC5 antibodies. Cells were gated on CD3 exclusion, and numbers of migrated CD56+ NK cells in duplicates for the different conditions are represented on the y-axis; one representative experiment shown out of four.
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45% and 55% the migration of memory CD4+CD45RO+ T cells in response to PDC SN2 (P<0.05, n=6 experiments), whereas neutralizing anti-TARC/CCL17 mAb alone had no significant effect (Fig. 5C)
. Phenotype characterization of the memory CD4 T cell population indicated that PDC SN2 and to a lower extent, PDC SN1 induced the migration of CD4+CD45RO+CD25+ T cells [nine (P=0.033)- and sixfold (P=0.013) for PDC SN2 and SN1, respectively] more efficiently than that of CD4+CD45RO+ CD25– T cells [four (P<0.001)- and threefold (P=0.005) for PDC SN2 and SN1, respectively, n=3 (Fig. 5D)
]. Thus, human activated tonsillar PDC produced chemokines attracting preferentially CD4 memory T cells, comprising a significant proportion of CD4+CD45RO+CD25+ T cells.
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Figure 5. Migration of T cell populations in response to culture supernatants of 60 h-activated PDC. Tonsillar PDC SN1 and SN2 (twice diluted) were tested for their chemotactic activities on enriched T cells using 3 µm transwells as described in Materials and Methods. (A) Numbers of migrated CD3+ CD4+ (open bars) and CD3+ CD8+ (solid bars) T cells are indicated. Triple-color stainings for CD3, CD4, and CD8 and appropriate gates allowed the enumeration of migrated CD4+ and CD8+ T cells. Results are from one representative experiment of three performed. (B) Numbers of migrated CD3+ CD4+ CD45RO+ (open bars) and CD3+ CD4+ CD45RA+ (solid bars) T cells are indicated. Triple-color stainings for CD3, CD4, and CD45RO or CD45RA and appropriate gates allowed the enumeration of migrated naive and memory CD4 T cells. Results are from one representative experiment of three performed. (C) Inhibition of PDC SN2-induced chemotaxis of CD3+ CD4+ CD45RO+ T cells after preincubation with a neutralizing anti-TARC/CCL17, anti-MDC/CCL22, or combination of anti-TARC/CCL17 and anti-MDC/CCL22 mAb compared with isotype control (mouse IgG2b). The mean ± SD of six independent experiments is shown (P<0.05). (D) Migration of CD4+ CD45RO+ CD25+ (solid bars) and CD4+ CD45RO+ CD25– (open bars) T cells. Triple-color stainings for CD4, CD45RO, and CD25 and appropriate gates allowed the enumeration of migrated CD4+ CD45RO+ CD25+ versus CD25– T cells. Results are expressed as migration index (ratio SN/medium) and are from one representative experiment of three performed.
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Figure 6. CD40L and virus synergize to promote Th1 chemokine production by PDC. (A) MDC/CCL22, TARC/CCL17, RANTES/CCL5, IL-8/CXCL8, IP-10/CXCL10, and MIP-1ß/CCL4 were measured by ELISA in the supernatants of purified FACS-sorted human tonsillar PDC (5x105 cells/ml) stimulated with IL-3 and hCD40L-transfected L cells, virus, or a combination of IL-3, hCD40L-transfected L cells, and virus for 60 h. Each symbol represents an independent experiment. (B) IP-10/CXCL10 production by purified human tonsillar CD123+ PDC, stimulated for 22 h with IL-3 and hCD40L-transfected L cells, virus, or IL-3, hCD40L-transfected L cells, plus virus was measured by intracellular staining. Results shown are representative of five experiments performed.
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production by activated PDC (Fig. 7
). Indeed, CXCL9/MIG and IFN- were detected exclusively in the supernatant of PDC, which were incubated with virus and CD40L but not with virus or CD40L alone (P=0.034; Fig. 7A
). Intracellular staining showed that MIG/CXCL9 production is concomitant to IFN- secretion by CD40L + virus-activated CD123+ PDC (Fig. 7B) . We performed a kinetics analysis, which showed that the intracytoplasmic expression of IFN- and MIG/CXCL9 is optimal after 22 h of activation (of note, no IFN- was detected at 8 h), suggesting that the low percentage of detected IFN--producing cells is unlikely related to the selected time-points (not shown). This IFN- intracellular staining excludes the involvement of few contaminants and demonstrates formally that purified CD123+ PDC produce IFN- in response to CD40L+ virus. Of note, the percentage of intracellular IFN--expressing cells is in a comparable range with that of IFN--expressing cells at 22 h (Fig. 7B)
.
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Figure 7. Concomitant production of MIG/CXCL9 and IFN- by virus and CD40L-activated PDC. Purified human tonsillar PDC (5x105 cells/ml) were stimulated with IL-3 and hCD40L-L cells, virus, or a combination of IL-3, hCD40L-L cells, plus virus. (A) MIG/CXCL9 (open symbols) and IFN- contents (shaded symbols) were evaluated by ELISA after 60 h activation. Each symbol represents an independent experiment, and the same symbol is kept for a given donor. Horizontal bars represent the means. (B) MIG/CXCL9, IFN-, and IFN- production was measured by intracellular staining after 22 h activation. Results shown are representative of four experiments performed. (C) Mean fluorescence intensity (MFI) of CD80, CD86, and CCR7 in purified PDC was analyzed on fresh cells and after 48 h incubation by flow cytometry. Data are shown as means of two independent experiments ± range.
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, and IP-10/CXCL10) by virus + CD40L coactivation does not correlate to a higher percentage of activated PDC or a higher level of activation of PDC, as activation markers such as CD80, CD86, and CCR7 showed the same expression pattern in terms of percentage of positive cells and fluorescence intensity between IL-3 + CD40L– and IL-3 + CD40L + virus-stimulated PDC at 48 h, and virus-activated PDC express lower level of those molecules (Fig. 7C)
. Thus, synergistic activity of CD40L and virus is specific for Th1 chemokine/cytokine production.
Endogenous IFN- production is responsible for the synergistic activity of virus and CD40L on Th1 chemokine production by PDC
The role of type I and type II IFNs in the regulation of IP-10/CXCL10 and MIG/CXCL9 was evaluated by adding to parallel cultures IFN- and IFN- or antibodies that block type I IFN binding to IFN-/ßRII or IFN-. As previously mentioned (Fig. 3)
, exogenous IFN- induces MIG/CXCL9 and increased IP-10/CXCL10 by virus- or CD40L-activated tonsillar PDC. Conversely, addition of exogenous IFN- to IL-3/CD40L signal only moderately boosts production of IP-10/CXCL10 and MIP-1ß/CCL4 with a 1.7- and 1.5-fold increased level (not shown). Of note, in the absence of activation signals such as CD40L or virus, exogenous IFN- and IFN- enhanced the production of IP-10/CXCL10 (5.8 ng/ml and 4 ng/ml, respectively, vs. 0.8 ng/ml) without significantly inducing MIP-1ß/CCL4 production (<0.5 ng/ml) by IL-3-cultured tonsillar PDC, and only IFN- was able to induce MIG/CXCL9 (3.3 ng/ml). At last, IFN- alone, a survival factor that is produced in high quantities by virus-activated PDC, only marginally induced tonsillar PDC to secrete IP-10/CXCL10 (0.62±0.25 ng/ml, n=3) or MIP-1ß/CCL4 (<0.1 ng/ml, n=2). Thus, IFN-2b alone did induce some IP-10/CXCL10 production but not at the high levels seen following virus treatment.
As demonstrated in Figure 8
(upper panel), neutralization of type I IFNs, which are produced at high levels by virus-activated PDC, inhibited by 50% the production of IP-10/CXCL10 in those culture conditions, suggesting that IP-10/CXCL10 production by virus-activated PDC is partially dependent on endogenous type I IFNs. Furthermore, in response to combined CD40L and virus, blocking IFN- abrogates the synergistic activity on IP-10/CXCL10 production, and an identical level is observed in the presence of virus versus virus + CD40L + anti-IFN-. Finally, neutralization of endogenous IFN- completely abolished MIG/CXCL9 production, and anti-type I IFN receptor antibody has no effect (Fig. 8
, lower panel). Collectively, these results showed that virus-induced type I IFNs trigger IP-10/CXCL10 production, and MIG/CXCL9 production is under the dependence of endogenous IFN- triggered through the synergistic action of virus + CD40L.
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Figure 8. Endogenous type I and type II IFNs are involved in IP-10/CXCL10 and MIG/CXCL9 production by activated PDC. Purified human tonsillar PDC (5x105 cells/ml) were stimulated with IL-3 plus virus or a combination of IL-3, virus plus hCD40L-L cells alone, or with anti-human IFN-/ßRII, anti-IFN-, or both. As IFN- is an endogenous survival factor produced in response to virus, we added IL-3 to the virus group to circumvent the effect of neutralization of type I IFN on PDC death. IP-10/CXCL10 and MIG/CXCL9 production was evaluated by ELISA following 60 h activation with IL-3 + virus (hatched bars) or IL-3 + CD40L + virus (solid bars). Data, given as mean of duplicate wells ± SD, are representative of three experiments performed independently (*, P<0.02; **, P<0.005; ***, P<0.001).
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and overrides the CD40L-mediated Th2 profile.
CD40L- but not virus-activated tonsillar PDC express a high level of MDC/CCL22, in agreement with previous observations [33
] and significant levels of TARC/CCL17, in contrast to blood PDC [34
]. Of note, unlike myeloid DC ([34
, 40
] and our observations), neither CCL17 nor CCL22 transcripts and proteins were detectable in freshly isolated PDC. Conversely, the Th1 chemokines IP-10/CXCL10 and MIP-1ß/CCL4 are secreted at high levels by virus- but not CD40L-activated PDC. This is consistent with previous studies showing that these chemokines, as well as RANTES/CCL5 and IL-8/CXCL8, are secreted by activated PDC and myeloid DC [34
35
36
]. Although virus (or CD40L) alone does not induce MIG/CXCL9 expression in human PDC, we demonstrate that it synergizes with IFN- to further induce MIG/CXCL9 production. This differential pattern of chemokine expression was also observed on leukemic PDC, extending the similarities between normal and leukemic PDC [12
, 13
].
By comparing polarizing activation conditions, IL-3 + CD40L with virus + IFN-, we observed dominant recruitment of memory CD4 T cells and IFN--activated NK cells, respectively. MDC/CCL22 appeared to be the major chemokine present in the CD40L-activated PDC supernatant responsible for the recruitment of CD4+ CD45RO+ T cells as demonstrated by the use of a neutralizing anti-MDC/CCL22 mAb. Of interest, the responding CD4 memory T cells comprise a significant proportion of CD25+ T cells, which could possibly represent regulatory T cells [41
]. In addition to PDC, mature monocyte-derived DC have also been shown to attract regulatory CD4 T cells by secretion of CCR4 ligands [30
]. Together with the recently described capacity of PDC to induce regulatory T cell development [23
24
25
26
27
, 42
], this observation may argue for a specific role of PDC in eliciting a tolerogenic environment under certain conditions.
Furthermore, we have observed that supernatants of Herpes simplex virus type 1 (HSV1)-activated PDC attracted preferentially CXCR3+ CD4 T cells, which secreted IFN- upon activation, indicating that virus-activated PDC may recruit mostly Th1 cells (our unpublished observation). In this same line, it has been reported that supernatants of HSV-stimulated PDC induced chemotaxis of phytohemagglutinin + IL-2-activated CD4 T cells through IP-10/CXCL10 and MIP-1ß/CCL4, and naive CD4 T cells were poor responders [36
]. This preferential migration of memory CD4 T cells is linked to their expression of high levels of CCR4, CCR5, and CXCR3 [30
, 43
44
45
], which endow them to respond to their respective ligands produced by activated PDC, in contrast to naive T cells. In contrast to fresh cells, IFN--treated NK cells migrate in response to virus-activated, PDC-derived supernatant. CXCR3 ligands induce moderate migration of NK cells [46
], and IP-10/CXCL10 and MIP-1ß/CCL4 have been shown to be involved in PDC-induced NK cell chemotaxis [36
]. However, although supernatants of virus and IFN--activated PDC contained high levels of IP-10/CXCL10 and MIG/CXCL9, these CXCR3 ligands were only marginally involved in attracting IFN--treated NK cells. As CXCR3 expression was selectively up-regulated by IFN- on NK cells (not shown), experiments are ongoing to assess the involvement of I-TAC/CXCL11 (the third CXCR3 ligand) in the migration of IFN--treated NK cells. A cross-talk between DC and NK cells has been demonstrated in human [47
] and in mouse models, in vitro [48
] and in vivo, upon infection with murine cytomegalovirus [15
]. We further extend this notion by demonstrating that virus-induced production of IFN- is required to sensitize NK cells, which may occur in the blood, and in a second stage, chemokines secreted by virus-activated PDC could recruit activated NK cells to migrate from the periphery to the tissues. At last, as PDC migrate in response to CXCR3 ligands [49
, 50
], virus-activated PDC may participate toward an amplification loop of PDC recruitment [33
, 51
].
It is important that a combination of virus and CD40 synergized to induce the secretion of high levels of IFN- by PDC. We cannot rule out that the percentage of intracellular IFN--expressing cells detected by flow cytometry is underestimated. We are currently working on the optimization of the intracytoplasmic detection (time of brefeldin, in particular) and are extending this observation to other TLR and T cell signals. It is the first demonstration that human DC could produce IFN-, whereas mouse myeloid DC and more recently, mouse PDC have previously been reported to produce IFN- [52
53
54
]. Thus, IFN- production may be critical in the described, PDC-induced Th1 orientation [18
, 19
, 55
], in as much as the IFN- receptor has recently been shown to colocalize with the T cell receptor in the T cell immunological synapse [56
], suggesting a key role of IFN- production by the antigen-presenting cells in the early phase of a T cell commitment.
IFN- gene transcription is dependent on numerous transcription factor families such as nuclear factor (NF)-
B, activated protein (AP)-1, NF of activated T cells, Ying Yang 1, signal transducer and activator of transcription-4, c-jun, GATA-1, and T-bet activation. Our study shows that IFN- production by PDC is strictly dependent on the cooperative action of the virus and CD40 signaling pathways. Virus, likely engaging TLR7, and CD40L are known activators of NF-B via tumor necrosis factor receptor (TNFR)-associated factor (TRAF)6, a signaling adaptor molecule common to the IL-1R/TLR (e.g., TLR7) and TNFR (e.g., CD40) superfamilies [57
], suggesting that this synergy is not related to the common use of TRAF6. Synergy between TRAF6-engaging molecules such as CD40L and IL-1 has been described previously [58
, 59
] and might be linked to alternative signaling pathways (other TRAFs, mitogen-activated protein kinase, Src kinases, phosphatidylinositol-3 kinase). In addition, induction/activation of transcription factors of the IFN regulatory factor (IRF) family is a likely candidate to explain the synergy between virus/TLR7 and CD40 activation for IFN- up-regulation. In particular, IRF-7 is constitutively expressed by PDC [60
61
62
63
], and it has been recently shown that TLR7 activation forms a complex with MyD88, TRAF6, and IRF-7, which results in IRF-7 activation [64
, 65
]. Moreover, IRF-1 and IFN consensus sequence-binding protein/IRF-8 are expressed in human PDC [62
]. The different downstream pathways "used" through CD40- or TLR/MyD88-activated TRAF6 may lead to preferential activation of specific NF-B and AP-1 components, which will synergize with IRF members to activate the transcription of a new set of genes, such as IFN-, which are not activated when TLR and CD40 agonists are used separately. It remains to be determined if this synergistic induction of IFN- is also observed with other TLRs.
Concomitantly to IFN- induction, CD40L and virus synergized to enhance the secretion of most of the virus-induced Th1-attracting chemokines (RANTES/CCL5, IL-8/CXCL8, and IP-10/CXCL10) and to induce the production of another Th1-attracting chemokine MIG/CXCL9. Enhanced IP-10/CXCL10 production and induced MIG/CXCL9 production were mediated by an IFN- autocrine loop. In contrast, IP-10/CXCL10, but not MIP-1ß/CCL4 production by virus-activated PDC, is clearly mediated by type I IFNs. However, our experiments showed that IFN- is necessary but suboptimal alone for this virus-induced IP-10/CXCL10 production by activated PDC, in agreement with observations in monocytes [66
] and HSV-stimulated blood PDC [36
]. Indeed, addition of exogenous IFN- only moderately promotes IP-10/CXCL10 production by PDC, and blocking type I IFN signaling does not completely abolish IP-10/CXCL10 production by virus-activated PDC. Furthermore, CD40L can trigger IP-10/CXCL10 production in the absence of detectable type I IFN. In contrast, type 2 chemokines and MIP-1ß/CCL4 production were not affected by combining CD40L and virus. Collectively, these synergistic activities between virus and CD40L demonstrate that virus will override the propensity of CD40L-activated PDC to produce Th2 mediators.
Our observation is consistent with recent literature reporting that the concomitant delivery of TLR and CD40 agonists enhanced immune responses. Indeed, PDC required the virus and CD40 signaling to induce the generation of plasma cells [67 ] as well as TLR9 agonists (oligodeoxynucleotide CpG 2006) and CD40 to produce bioactive IL-12p70 [55 ]. In vivo, bacterial/viral-derived stimuli, which are TLR agonists, synergized with CD40 stimulation to induce IL-12 production [68 ] and to stimulate CD8+ T cell responses [69 ] including antitumor cytolytic T lymphocyte responses [70 ].
This synergistic activity likely reproduces physiological conditions, where DC encounter the TLR signal at the site of infection and receive a CD40 stimulus from antigen-specific T cells at the draining lymph node for a primary immune response or at the site of infection during memory response. Under such condition, PDC will polarize the response toward Th1 through T cell commitment, as previously reported [17 18 19 20 21 ], and recruitment of Th1 effectors. However, the delayed production of MDC/CCL22 and TARC/CCL17 may participate through the recruitment of regulatory T cells in a negative feedback controlling the intensity of the response.
3 Current address: Dynavax Technologies, Berkeley, CA.
4 Current address: Department of Immunology and Center for Cancer Immunology Research, University of Texas, MD Anderson Cancer Center, Houston, TX.
Received July 1, 2004; accepted June 21, 2005.
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/ß in plasmacytoid dendritic cells Eur. J. Immunol. 31,2154-2163[CrossRef][Medline]/ß J. Exp. Med. 197,885-898/ß-producing cells link innate and adaptive immunity J. Exp. Med. 192,219-226+ B220+ dendritic cells endowed with type 1 interferon production capacity and tolerogenic potential Blood 100,383-390+ DC, with a plasmacytoid phenotype, induce differentiation and support function of T cells with regulatory properties Immunology 108,481-492[CrossRef][Medline] production by CD8+ lymphoid dendritic cells J. Exp. Med. 189,1981-1986, and IFN- by mouse dendritic cell subsets J. Immunol. 166,5448-5455 production involves p38 MAPK-dependent STAT1 phosphorylation in human plasmacytoid dendritic cell precursors J. Leukoc. Biol. 72,1011-1019 expression in human plasmacytoid and monocyte-derived dendritic cells J. Leukoc. Biol. 74,1125-1138 production by enveloped virus and lipopolysaccharide in human plasmacytoid dendritic cells J. Immunol. 173,1535-1548 induction through Toll-like receptors involves a direct interaction of IRF7 with MyD88 and TRAF6 Nat. Immunol. 5,1061-1068[CrossRef][Medline]-inducible protein-10 production is regulated by plasmacytoid dendritic cell-derived IFN- J. Immunol. 170,4061-4068This article has been cited by other articles:
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