Plasmacytoid Dendritic Cells Take Up Opsonized Antigen Leading to CD4 + and CD8 + Activation In Vivo

This information is current as Pia Björck, Andreas Beilhack, Edward I. Herman, Robert S. of September 29, 2021. Negrin and Edgar G. Engleman J Immunol 2008; 181:3811-3817; ; doi: 10.4049/jimmunol.181.6.3811 http://www.jimmunol.org/content/181/6/3811 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Plasmacytoid Dendritic Cells Take Up Opsonized Antigen Leading to CD4؉ and CD8؉ T Cell Activation In Vivo1

Pia Bjo¨rck,2* Andreas Beilhack,3† Edward I. Herman,† Robert S. Negrin,† and Edgar G. Engleman*

Plasmacytoid dendritic cells (pDC) are the body’s main source of IFN-␣, but, unlike classical myeloid DC (myDC), they lack phagocytic activity and are generally perceived as playing only a minor role in Ag processing and presentation. We show that murine pDC, as well as myDC, express Fc␥ receptors (CD16/CD32) and can use these receptors to acquire Ag from immune complexes (IC), resulting in the induction of robust Ag-specific CD4؉ and CD8؉ T cell responses. IC-loaded pDC stimulate CD4؉ .␥-T cells to proliferate and secrete a mixture of IL-4 and IFN-␥, and they induce CD8؉ T cells to secrete IL-10 as well as IFN In contrast, IC-loaded myDC induce both CD4؉ and CD8؉ T cells to secrete mainly IFN-␥. These results indicate that pDC can shape an immune response by acquiring and processing opsonized Ag, leading to a predominantly Th2 response. The Journal of Downloaded from Immunology, 2008, 181: 3811–3817.

lasmacytoid dendritic cells (pDC)4 are the body’s first line pDC in innate immunity, their ability to acquire and process op- of defense against invading pathogens, and can efficiently sonized Ags is unknown. This led us to ask whether pDC, which P fight viruses through their high and immediate production express Fc␥R (CD16/CD32, Fc␥RIII/Fc␥RII), can take up exog- of IFN-␣ (1). In their resting state, pDC do not resemble classical enous Ags via this , and, if so, whether such Ags would be http://www.jimmunol.org/ myeloid dendritic cells (myDC), with long dendritic protrusions, processed in a manner that promotes Ag-specific T cell activation. but rather have a rounded morphology more like lymphocytes (2, 3). Unlike myDC, pDC do not phagocytose or take up exogenous Materials and Methods Ags efficiently, mainly because they lack DEC-205 and Mice mannose receptors (4). In addition, they express low levels of Female or male C57BL/6, OT-I, and OT-II OVA-TCR transgenic (OVA- MHC Ags and accessory molecules (1–3). In turn, this leads to TCR Tg) mice were obtained from The Jackson Laboratory and used at weak activation of naive Ag-specific T cells. However, endoge- 8–12 wk of age. OT-I and OT-II mice crossed onto RAGϪ/Ϫ background nous Ags in the form of self-Ags or viral products may be effi- were purchased from Taconic Farms and bred in-house. All experiments ciently presented by pDC, suggesting a role for these cells in both were performed under institutional guidelines according to an approved by guest on September 29, 2021 protocol. viral defense and maintenance of tolerance in the steady state (5, 6). Moreover, pDC can activate Ag-experienced and memory T Reagents cells (7), further indicating a role for these cells in shaping the All fluorochrome-labeled Abs were purchased from BD Pharmingen. adaptive immune response. IFN-␣ was detected using a from R&D Systems, according to the man- In addition to acquiring Ags via and pinocytosis, ufacturer’s instructions. IL-6 and TNF-␣ were detected using Abs pur- , , and myDC express and take up Ags in chased from eBioscience. Mouse anti-chicken OVA (IgG1) was obtained immune complexes (IC) via their Fc␥R (8), resulting in Ag pro- as ascites from Sigma-Aldrich. The Ab was dialyzed and filtered before the titer was confirmed in direct OVA ELISA. Sterile aliquots were stored at cessing and presentation to T cells. Such opsonized Ags were de- –20°C until used. DQ-OVA, a self-quenched dye emitting green fluores- scribed more than a century ago (9), and opsonization is known to cence upon degradation, and CFSE were purchased from Invitrogen. LPS, play a central role in the induction of innate immune responses to PMA, ionomycin, and brefeldin A were all purchased from Sigma-Aldrich. various pathogens. However, despite the well-established role of OVA323–339 (ISQAVHAAHAEINEAGR) and OVA257–264 (SIINFEKL) were synthesized at the University of Pittsburgh and purified using HPLC. OVA/Anti-OVA-IC were prepared by incubating DQ-OVA (100 ␮g/ml) *Department of Pathology/Stanford Blood Center and †Department of Medicine, Di- with mouse-anti-OVA (titer 1:20) for1hat37°C. The complexes were vision of Blood and Marrow Transplantation, Stanford University, Palo Alto, CA used at a final concentration of 10 ␮g/ml DQ-OVA and 1/200 dilution of 94304 the Ab. Received for publication January 7, 2008. Accepted for publication July 11, 2008. Uptake of IC in vivo The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance To determine uptake of IC by dendritic cells (DC) in vivo, mice were with 18 U.S.C. Section 1734 solely to indicate this fact. injected s.c. in the footpad with 50 ␮l of DQ-OVA-containing IC. Five to 1 This work was supported by Grants AR051748 (to E.G.E.) and CA80006 (to 24 h later, mice were sacrificed, and draining and nondraining lymph nodes R.S.N.). were harvested. Single-cell suspensions were prepared in the presence of collagenase IV (Worthington Biochemical), washed, and stained with di- 2 Address correspondence and reprint requests to Dr. Pia Bjo¨rck, Stanford University, Department of Pathology/Stanford Blood Center, 3373 Hillview Avenue, Palo Alto, rectly conjugated Abs. Cells were analyzed by four-color flow cytometry CA 94304. E-mail address: [email protected] using anti-CD11c allophycocyanin, anti-CD19 PE, anti-CD3 PE, and IC 3 measured in the FITC channel. B220 PE-Cy5 and CD11b PE-Cy5 were Current address: Department of Medicine II, Wu¨rzburg University, Wu¨rzburg, used to detect pDC and myDC, respectively. Germany. 4 Abbreviations used in this paper: pDC, plasmacytoid ; DC, dendritic Cell preparation and flow cytometry cell; IC, ; myDC, myeloid DC; Tg, transgenic. Bone marrow-derived DC were prepared, as described by Gilliet et al. (10). Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 Briefly, bone marrow cells were obtained by flushing the femur and tibia www.jimmunol.org 3812 pDC PRESENT Ag IN VIVO using a 23G needle. After red cell lysis, total leukocytes were cultured in HEPES-buffered RPMI 1640 supplemented with 5% FCS, antibiotics, 2 Ϫ5 mM L-glutamine, 5 ϫ 10 M 2-ME, nonessential amino acids, Na-pyru- vate, and 100 ng/ml human rFlt3 ligand. Cultures were performed at 1 ϫ 106 cells/ml in six-well plates (Costar). After 10 days, DC were harvested, and after washing, directly conjugated anti-B220 allophycocyanin and anti- CD11b allophycocyanin-Cy7 Abs were added and cells were further incu- bated on ice for 15 min. FcR-blocking Abs were deliberately excluded from the cell preparation protocol, because they would have interfered with our subsequent studies. After final washing, cells were sorted on a FACSVantage (BD Biosciences). Purity exceeded 96% for both pDC and mDC. Postsort analyses showed all cells expressed the mouse DC marker CD11c. OVA-specific CD4ϩ T cells were isolated from the spleens of OT-II (C57BL/6) mice. Spleens were dispersed into single-cell suspensions and, after lysis of red cells, anti-CD4 Ab-conjugated magnetic beads (Miltenyi Biotec) were added and cells were further incubated for 20 min at 4°C. Purity was Ͼ90%. T cells were labeled with CFSE (10 ␮M) for 10 min at 37°C, washed, and resuspended in HEPES-buffered RPMI 1640 supplemented with 5% FCS, antibiotics, and 2 mM L-glutamine. A total of 5 ϫ 104 DC and 2 ϫ 105 T cells was cultured in duplicate wells in 96-well round-bottom plates at a final volume of 200 ␮l. Cultures were supplemented with either OVA-IC or DQ-OVA. Control cultures containing OVA peptide were set up in parallel. Downloaded from In vivo T cell activation Sorted DC subsets were incubated together with OVA-IC overnight at 37°C. After washing in PBS, 1–2 ϫ 106 DC pulsed with IC were injected s.c. in the footpad. OVA Tg, splenic T cells were isolated using either anti-CD4- or anti-CD8-conjugated magnetic beads (Miltenyi Biotec) and labeled with CFSE (1 ␮M). A total of 8–10 ϫ 106 T cells was administered http://www.jimmunol.org/ by i.v. injection in the tail. Three to 4 days later, mice were sacrificed, and draining popliteal and superficial inguinal nodes were harvested and CD4ϩ or CD8ϩ T cells were reisolated using MACS beads, stained with directly fluorochrome-labeled Abs, and analyzed by flow cytometry. CTL assay For determination of CTL activity, EG7-OVA were labeled with a high concentration of CFSE (1 ␮M/ml), and control EL-4 tumor cells were labeled with a lower CFSE concentration (0.1 ␮M/ml) (11), mixed in equal parts, and used as target cells. Graded numbers of effector OT-I T cells, by guest on September 29, 2021 either unstimulated or cultured for 6 days with pDC-IC or myDC-IC, were added to the labeled target cells and incubated overnight at 37°C. Percent- FIGURE 1. pDC and myDC express Fc␥R (CD16/CD32) and take up age of specific lysis of target cells was determined using FACS according IC in vivo. a and b, Expression of Fc␥R (CD16/CD32) on DC subsets ϭ low high to the following formula: ratio (percentage of CFSE /CFSE ); per- isolated from spleen (a) or lymph nodes (b). c, IC containing fluorescent centage of specific lysis ϭ (1 Ϫ (ratio unprimed/ratio primed)) ϫ 100. DQ-OVA was injected into the left footpads of mice and, after 8 h, draining ELISA for OVA-IC popliteal and superficial inguinal lymph nodes were isolated. Contralateral (nondraining) popliteal lymph nodes served as control. d, Uptake of IC Mouse anti-chicken OVA (Sigma-Aldrich) was dialyzed against PBS, ster- OVA-IC at 5 h postinjection. e, Uptake of DQ-OVA 8 h postinjection. IC ile filtered, and stored at –20°C. ELISA plates (Costar) were coated with or DQ-OVA uptake was analyzed by four-color flow cytometry. Gates DQ-OVA (10 ␮g/ml) in PBS at 100 ␮l/well and incubated overnight at ϩ were set on CD11c DC, excluding cells in the PE channel, dead cells, and 4°C. The plates were blocked for1hat37°C using 3% BSA in PBS-Tween 20, and washed, and serial dilutions of mouse-anti-OVA mAb were added debris. IC uptake was measured in the FITC channel, and pDC or myDC to duplicate wells. After incubation overnight at 4°C, the plates were were detected using PE-Cy5-conjugated mAb to B220 and CD11b, respec- Ј washed and biotinylated F(ab )2 goat anti-mouse IgG (Jackson Immuno- tively. Data are representative of three independent experiments. Research Laboratories) was added. After 1-h incubation at room temper- ature, the plate was washed and extravidin-peroxidase (Sigma-Aldrich) was added. After an additional 1-h incubation and washing, the plates were developed using tetramethylbenzidine substrate (Kirkegaard & Perry Lab- popliteal and superficial inguinal lymph nodes were harvested at oratories). Ab titer was determined as highest OD450 (typically 1:10,000). different time points (5–24 h postinjection), and single-cell sus- pensions were stained and analyzed by four-color flow cytometry. Statistical analysis As shown in Fig. 1c, both pDC and myDC acquired opsonized Ag. For comparisons between groups, Student’s t test was performed. Uptake was detectable at 5 h, peaked at 8 h, and declined markedly at 12–24 h postinjection (Fig. 1d). No staining of pDC or myDC Results was seen in the contralateral lymph nodes of IC-injected mice (Fig. ␥ pDC express Fc R and capture IC in vivo 1c), nor was any staining detected in pDC in mice injected with As shown in Fig. 1a, freshly isolated splenic pDC and myDC DQ-OVA alone (Fig. 1e). express Fc␥R (CD16/CD32) at low levels (Fig. 1a). DC from ␥ pDC exposed to IC in vitro take up Ag and trigger Ag-specific lymph nodes expressed somewhat higher levels of Fc R (Fig. 1b). ϩ To determine whether pDC can use their Fc␥R to take up opso- CD4 T cell activation nized Ag, we injected one footpad of mice with IC containing To evaluate the effects of IC on pDC, we purified pDC and myDC anti-OVA Ab and DQ-OVA, a self-quenched fluorescent OVA from bone marrow cells cultured for 10 days in medium containing conjugate exhibiting green fluorescence after proteolytic degrada- Flt3 ligand. Fc␥R expression was higher on these DC than on DC tion. Control mice were injected with DQ-OVA alone. Draining analyzed immediately after their isolation from spleen or lymph The Journal of Immunology 3813

FIGURE 2. Exposure of pDC to IC leads to Ag uptake in vitro. a, Expression of Fc␥R (CD16/ CD32) on bone marrow-derived, FACS-sorted DC subsets. b, FACS-sorted pDC and myDC were in- cubated overnight together with OVA-IC consisting of DQ-OVA and mouse IgG1 anti-OVA Ab, DQ- OVA, or medium alone. Cells were washed and stained for I-Ab, CD80, and CD40. Data are repre- sentative of at least five independent experiments. c, IC do not induce IFN-␣, IL-6, or TNF-␣ production in pDC in the absence of a TLR agonist (CpG 2336). Downloaded from A total of 1 ϫ 105 pDC was cultured either in me- dium alone, or together with IC-OVA, CpG, or IC- OVA plus CpG for 48 h. Culture supernatants were harvested and analyzed by ELISA. http://www.jimmunol.org/

nodes (Fig. 2a). We incubated each population with either DQ- proliferation. Fig. 3b shows that a proportion of the CD4ϩ T cells by guest on September 29, 2021 OVA or OVA-IC for 24 h, after which the cells were examined by cultured with pDC-IC or myDC-IC produced IFN-␥. Fewer T cells flow cytometry for evidence of Ag uptake and for changes in se- produced IL-4 and IL-10, but such cells were more frequent in lected surface markers. Ag uptake was clearly demonstrated in pDC-IC/T cell cultures than myDC-IC/T cell cultures. Both pDC as well as myDC, although uptake was greater in myDC (Fig. pDC-IC and myDC-IC promoted T cell proliferation in these cul- 2b). For both DC subsets, Ag uptake was accompanied by cellular tures, with the number of cell divisions indicated by progressive activation, based on up-regulation of MHC class II (I-Ab), CD80, reduction in intracellular CFSE content. and CD40 (Fig. 2b). Some Ag uptake and cellular activation of pDC occurred when OVA was provided as free protein; however, ϩ more Ag acquisition and cellular stimulation were seen when the pDC-IC promote Ag-specific CD4 T cell proliferation and pDC were exposed to OVA-IC. We also examined IFN-␣ secre- production in vivo tion, the hallmark of pDC activation. pDC were cultured with We extended our investigations to examine whether opsonized Ag OVA-IC alone or with both OVA-IC and a TLR agonist, CpG processed by pDC could also induce T cell activation in vivo. 2336. In addition, we examined IL-6 and TNF-␣, two proinflam- Sorted pDC and myDC from C57BL/6 mice were incubated with matory produced by pDC. As shown in Fig. 2c,inthe OVA-IC overnight at 37°C. After washing, pDC-IC or myDC-IC absence of TLR agonists, OVA-IC did not induce IFN-␣ produc- (1–2 ϫ 106) were injected s.c. in the footpads of syngeneic ani- tion, not did they produce IL-6 or TNF-␣. mals. Immediately thereafter, CFSE-labeled, MACS-isolated To determine whether Ag uptake by pDC leads to processing splenic CD4ϩ T cells (10 ϫ 106) from OT-II (OVA-TCR Tg) mice and presentation of antigenic peptides, we cultured sorted pDC or were injected i.v. in the tail. Three to 4 days after T cell admin- myDC together with CFSE-labeled CD4ϩ OT-II T cells in the istration, draining popliteal and superficial inguinal lymph nodes presence of OVA-IC, DQ-OVA, or an immunodominant I-Ab/d were harvested, stained, and analyzed using multicolor flow cy-

OVA-peptide (OVA323–339). After 4 days, the T cells were stained tometry. As shown in Fig. 4, both pDC-IC and myDC-IC induced for CD25 and analyzed by flow cytometry for evidence of prolif- T cell activation in vivo, as indicated by up-regulation of CCR7, as eration. Fig. 3a shows that CD4ϩ T cells proliferated in response well as T cell proliferation. T cells from control animals given PBS to both pDC and myDC pulsed with IC (pDC-IC and myDC-IC). did not proliferate. To examine cytokine production by T cells pDC incubated with OVA alone induced poor T cell stimulation, activated in vivo with DC-IC, we injected mice with pDC-IC or whereas OVA-pulsed myDC induced T cell activation similar to myDC-IC, as above. After 4 days, draining lymph nodes were that generated by myDC-IC. Addition of an Fc␥R-blocking Ab at harvested and CD4ϩ T cells were isolated and stimulated with the time pDC were pulsed with IC resulted in inhibition of pDC- PMA and ionomycin before undergoing analysis for intracellular IC-, but not myDC-IC-induced T cell proliferation. As expected, cytokines. The results show that T cells exposed to pDC-IC in vivo pDC or myDC pulsed with OVA323–339 strongly promoted T cell displayed a predominantly Th2 cytokine profile, with more cells 3814 pDC PRESENT Ag IN VIVO Downloaded from

FIGURE 4. Fc␥R-mediated Ag uptake by pDC leads to CD4ϩ T cell activation and cytokine production in vivo. A total of 1 ϫ 106 FACS-sorted http://www.jimmunol.org/ DC subsets incubated with OVA-IC overnight was injected into the foot- pad, and CFSE-labeled, CD4ϩ OVA-Tg T cells (10 ϫ 106) were injected i.v. in the base of the tail. a, After 3–4 days, draining lymph nodes were harvested and stained for CCR7. Fine line indicates T cell control. b, Lymph node T cells were analyzed for their production of cytokines after stimulation with PMA and ionomycin in vitro. OVA-Tg CD4ϩ T cells were gated based on forward and side scatter and TCR V␣2 expression, and IFN-␥- and IL-4-secreting cells were identified by intracellular stain- Indicates p Ͻ 0.05. Results are representative of three independent ,ء .ing

experiments. by guest on September 29, 2021

both cases, the responding T cells had a CD44ϩ memory pheno- type. Although T cell proliferation was greater with myDC-IC than pDC-IC on a per cell basis, a substantial proportion of the CD8ϩ T cells underwent division when cultured with pDC-IC, as com- FIGURE 3. a, Exposure of pDC to IC leads to T cell activation in vitro. pared with CD8ϩ T cells cultured with pDC and OVA alone. 4 A total of 5 ϫ 10 FACS-sorted pDC or myDC was cultured for 4 days pDC-IC also induced cytokine production by cocultured CD8ϩ T with OVA-IC, DQ-OVA, or OVA peptide, together with 1 ϫ 105 323–339 cells. Both IFN-␥- and IL-10-producing cells were generated, with CFSE-labeled CD4ϩ OVA-Tg T cells (OT-II). Cells were stained for CD25 approximately twice as many cells producing IFN-␥ than IL-10 and analyzed using a FACSCalibur. Numbers indicate percentage of cells ϩ not dividing. Lower panel, OVA-IC and Fc␥R-blocking Ab (2.4G2, 25 (Fig. 5b). Although a larger proportion of CD8 T cells cultured ϩ ␥ ␮g/ml) or isotype control. b, Four-day cultures of CD4 T cells and DC-IC with myDC-IC produced IFN- , there were few, if any, IL-10- were examined for IFN-␥, IL-4, and IL-10 production by intracellular producing cells in the myDC-IC cultures. To determine whether staining with cytokine-specific Abs. Cellular divisions were determined OT-I T cells activated by pDC-IC could kill OVA-transfected tu- using CFSE. Data shown are representative of three independent mor cells, we cultured OT-I T cells together with pDC-IC or experiments. myDC-IC for 6 days. After washing, the T cells were incubated overnight with CFSE-labeled EL-4 cells or EL-4 cells expressing OVA (EG7-OVA) to analyze their cytotoxic activity. As shown in producing IL-4 than IFN-␥, whereas T cells from animals given Fig. 5c, myDC-IC induced CTL capable of lysing EG7-OVA cells, myDC-IC displayed a predominantly Th1 response (Fig. 4b). whereas pDC-IC did not. In the presence of LPS, pDC-IC, as well

ϩ as myDC-IC, induced CTL in a dose-dependent manner, and, at pDC can cross-present OVA to CD8 T cells ratios of 5:1 or greater, CTL induction by pDC-IC was only Cross-presentation of Ag to CD8ϩ T cells is an important property slightly less efficient than that induced by myDC-IC. of myDC, particularly CD8␣␣ϩ myDC (12, 13). To examine We also examined the ability of pDC-IC and myDC-IC to cross- whether pDC can cross-present opsonized Ag, we incubated present Ag in vivo by injecting CFSE-labeled OT-I T cells into pDC-IC with CFSE-labeled, MACS-enriched, CD8ϩ OT-I T cells mice that had received one or the other DC-IC subset. Two days for 4–5 days, as above. As shown in Fig. 5a, OT-I T cell prolif- after T cell administration, total CD8ϩ T cells were isolated from eration was induced by pDC-IC, but not by pDC that had been draining lymph nodes and analyzed for evidence of proliferation cultured with OVA alone. In contrast, myDC cultured with either by CFSE staining and for intracellular IFN-␥. As shown in Fig. 6a, OVA-IC or OVA alone stimulated equivalent OT-I activation. In both pDC-IC and myDC-IC induced CD8ϩ T cell activation. The Journal of Immunology 3815

FIGURE 5. pDC can cross-present IC-derived Ag to CD8ϩ T cells in vitro. a, FACS-sorted DC subsets (5 ϫ 104) were cultured in the presence of OVA-IC (consisting of DQ-OVA plus anti-OVA), DQ-OVA, or medium alone together with CFSE- labeled CD8ϩ OVA-Tg (OT-I) T cells (1 ϫ 105) for 4 days. CD8ϩ cells were analyzed using a FACSCalibur. b, OT-I T cells cultured with DC-IC were examined for IFN-␥ and IL-10 production by Indicates p Ͻ 0.05. Data ,ء .intracellular staining shown are representative of three independent ex- periments. c, OT-I T cells alone, or stimulated for 6 days with either pDC-IC or myDC-IC, were cul- tured at increasing numbers together with a fixed number of target EG7-OVA and EL4 tumor cells labeled with high and low concentrations of CFSE, respectively, in the absence or presence of LPS. Af- ter overnight incubation, the percentage of lysis was Downloaded from determined by FACS, as described in Materials and Methods. Data shown are representative of three in- dependent experiments. http://www.jimmunol.org/

When stimulated in vitro with PMA and ionomycin, the CD8ϩ T type cytokines from CD4ϩ T cells, these data suggest that under cells from mice that had received pDC-IC produced mainly IL-10, noninflammatory conditions, pDC-IC mainly promote a Th2/ whereas the CD8ϩ T cells from mice that had received myDC-IC tolerogenic environment in vivo. produced mainly IFN-␥ (Fig. 6b). In combination with our results (Fig. 4b) showing that pDC-IC also stimulated production of Th2- Discussion We initiated this study with the objective of evaluating the Ag- presenting capacity of pDC. The extraordinary ability of myDC to by guest on September 29, 2021 present and cross-present Ags is well established (14, 15). How- ever, evidence for exogenous Ag presentation by pDC has been limited. Our data show that pDC, like myDC (16), are capable of ac- quiring opsonized Ags through their IgG1-binding FcR, leading to Ag presentation and cross-presentation in vivo. Recent reports de- scribe a new type of lectin-binding receptor, Siglec-H, which is specifically expressed on pDC and can function in Ag uptake, lead- ing to Ag presentation and cross-presentation (17, 18). It seems likely that Siglec-H and Fc␥R mediate the uptake of different types of Ags, thus providing pDC with multiple mechanisms for acquir- ing Ag and modulating the immune response. Expression of Fc␥R was higher on pDC derived from cultured bone marrow than on freshly prepared spleen or lymph node cells, and this may partly explain why Fc␥R on murine pDC has not been described previously. In addition, we deliberately avoided the use of FcR-blocking Ab (clone 2.4G2) in our FACS-staining pro- tocols, which would have prevented detection of FcR. Moreover, we focused our study on stimulating Fc␥RIII/Fc␥RII by using an Ab, 2.4G2, that recognizes an epitope shared by CD16 and CD32. FIGURE 6. pDC can cross-present IC-derived Ag to CD8ϩ T cells in This receptor mainly binds Abs of the IgG1, IgG2a, and IgG2b vivo. FACS-sorted DC subsets (1–2 ϫ 106) incubated with OVA-IC over- subclasses contained in IC (19). We used an IgG1 anti-OVA Ab to ϩ night were injected in the footpad, and CFSE-labeled, CD8 OVA-Tg T prepare OVA plus anti-OVA-IC, in part, because the IgG1 Abs ϫ 6 cells (8–10 10 ) were injected i.v. in the base of the tail. a, After 2 days, consistently bind and stimulate other cells in vivo (20). Although draining lymph nodes were harvested and CD8ϩ (OT-I) T cell activation both pDC and mDC express CD64 (Fc␥RI) at low levels, this was determined. b, Lymph node T cells were analyzed for their production of cytokines after restimulation with PMA and ionomycin. OVA-Tg CD8ϩ receptor does not bind IgG1 Abs well in the mouse, in contrast to T cells were gated based on forward and side scatter and TCR V␣2 ex- human CD64, which binds IgG1 with high affinity (19). Exposure Indi- of pDC to IC not only resulted in Ag uptake, but also cellular ,ء .pression, and IFN-␥- and IL-10-producing cells were determined cates p Ͻ 0.05. Data shown are representative of five independent activation, as indicated by up-regulation of MHC class II and ac- experiments. cessory molecules. Such activated pDC-IC, in turn, induced the 3816 pDC PRESENT Ag IN VIVO growth and activation of Ag-specific CD4ϩ and CD8ϩ T cells. pus erythematosus have circulating IC containing small nuclear myDC, like pDC, acquired opsonized Ag, became activated, and RNA and anti-small nuclear RNA autoantibody; recent studies stimulated Ag-specific T cells, confirming a previous report (16). have shown that pDC can acquire such IC via their Fc␥R, resulting MyDC also acquired and processed free protein relatively effi- in stimulation of TLR7 and 8 and production of IFN-␣ (30). Be- ciently. By contrast, pDC that had been pulsed with free protein cause IFN-␣ is thought to play a fundamental role in the patho- failed to stimulate T cell responses. This is probably explained genesis of systemic erythematosus, IC-mediated activation both by the reduced ability of pDC to take up free Ag as opposed of pDC may be a key initiation factor in this disease. Thus, the to opsonized Ag, and by their reduced cellular activation relative consequences of IC uptake by pDC appear to depend on the nature to myDC-IC. The mechanism responsible for IC-induced activa- of the Ag as well as the presence or absence of other factors (e.g., tion of pDC remains to be explored. TLR ligands) in the environment that help determine whether these Surprisingly, the cytokine profile of pDC-IC-activated CD4ϩ T cells induce a proinflammatory or anti-inflammatory/tolerogenic cells varied, depending on whether the cells were activated in vitro response. or in vivo. In vitro, pDC-IC induced mainly IFN-␥-producing Th1 cells, whereas in vivo pDC-IC induced mainly IL-4-producing Th2 Acknowledgments cells. By contrast, myDC-IC promoted mainly Th1 cells in vitro We thank Drs. R. G. Morris and R. de Waal Malefyt for critical reading of and in vivo. The reason for this discrepancy is not clear from our the manuscript. data, but it is apparent that cells and soluble factors in addition to those present in our in vitro culture system can modulate the effects Disclosures of pDC-IC in vivo. The absence of Th1-biasing factors could also The authors have no financial conflict of interest. Downloaded from favor the generation of a Th2 response by pDC-IC. In this regard, we were not able to detect IFN-␣ in supernatants from pDC cul- References tured in the presence of IC (Fig. 2c), and pDC are more likely to 1. Liu, Y.-J. 2005. IPC: professional type 1 interferon-producing cells and plasma- cytoid dendritic cell precursors. Annu. Rev. 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