Fcγ Receptor IIb Strongly Regulates Fcγ Receptor-Facilitated T Cell Activation by Dendritic Cells

This information is current as Nadine van Montfoort, Peter A. C. 't Hoen, Sara M. of October 2, 2021. Mangsbo, Marcel G. M. Camps, Peter Boross, Cornelis J. M. Melief, Ferry Ossendorp and J. Sjef Verbeek J Immunol 2012; 189:92-101; Prepublished online 30 May 2012; doi: 10.4049/jimmunol.1103703 http://www.jimmunol.org/content/189/1/92 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 © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Fcg Receptor IIb Strongly Regulates Fcg Receptor- Facilitated T Cell Activation by Dendritic Cells

Nadine van Montfoort,*,1 Peter A. C. ’t Hoen,† Sara M. Mangsbo,*,2 Marcel G. M. Camps,* Peter Boross,†,3 Cornelis J. M. Melief,* Ferry Ossendorp,*,4 and J. Sjef Verbeek†,4

FcgR ligation by Ag–Ab immune complexes (IC) not only mediates effective Ag uptake, but also strongly initiates (DC) maturation, a requirement for effective T cell activation. Besides the activating FcgRI, FcgRIII, and FcgRIV, the inhibitory FcgRIIb is expressed on DCs. It is unclear how the ratio between signals from the activating FcgR and the inhibitory FcgRIIb determines the outcome of FcgR ligation on DCs. By microarray analysis, we compared the transcriptomes of steady state and IC- activated bone marrow-derived wild-type (WT) DCs expressing all FcgR or DCs expressing only activating FcgR (FcgRIIb knockout [KO]) or only the inhibitory FcgRIIb (FcR g-chain KO). In WT DCs, we observed a expression profile associated with effective T cell activation, which was absent in FcR g-chain KO, but strikingly more pronounced in FcgRIIb KO bone marrow- Downloaded from derived DCs. These microarray results, confirmed at the level for many cytokines and other immunological relevant , demonstrate that the transcriptome of IC-activated DCs is dependent on the presence of the activating FcgR and that the mod- ulation of the expression of the majority of the genes was strongly regulated by FcgRIIb. Our data suggest that FcgRIIb-deficient DCs have an improved capacity to activate naive T lymphocytes. This was confirmed by their enhanced FcgR-dependent Ag presentation and in vivo induction of CD8+ T cell expansion compared with WT DCs. Our findings underscore the potency of FcgR ligation on DCs for the effective induction of T cell immunity by ICs and the strong regulatory role of FcgRIIb. The Journal http://www.jimmunol.org/ of Immunology, 2012, 189: 92–101.

ntigen-presenting cells activate naive T lymphocytes by molecules and components of the Ag presentation machinery; presenting antigenic peptides in their MHC molecules, production of cytokines, chemokines, and growth factors; and in- A leading to clonal expansion of Ag-specific T cells. For the creased migration capacity. These characteristics endow DCs to development of an effective T cell response, extra stimuli are optimally induce and coordinate the Ag-specific T cell response at required that can be provided by dendritic cells (DCs) after their the sites where interaction with naive T cells can take place, the maturation (1). lymph nodes. by guest on October 2, 2021 DC maturation is the result of cellular differentiation upon DCs express a large repertoire of pathogen-recognition recep- encounters with pathogen- or self-derived danger signals and is tors (2–5), including TLR; nucleotide oligomerization domain- phenotypically characterized by upregulation of costimulatory like receptors; cytoplasmic caspase-recruiting domain helicases, such as retinoic acid-inducible gene I; receptors of the C-type lectin family; and receptors for endogenous inflammatory signals *Department of Immunohematology and Blood Transfusion, Leiden University Med- ical Center, Leiden 2333 ZA, The Netherlands; and †Department of Human Genetics, that are produced by the local tissue in response to encounter with Leiden University Medical Center, Leiden 2333 ZA, The Netherlands pathogens (6). These endogenous danger signals can substitute 1Current address: Department of Gastroenterology and Hepatology, Erasmus MC, direct pathogen-recognition receptor ligation on DCs, at least Rotterdam, The Netherlands. in vitro. 2 Current address: Department of Immunology, Genetics, and Pathology, Uppsala A separate class of molecules that activates DCs are Ag–Ab University, Uppsala, Sweden. immune complexes (IC) (7). IgG IC are natural ligands for FcgR. 3Current address: Department of Immunology, Immunotherapy Lab, University Med- ical Center Utrecht, Utrecht, The Netherlands. The FcgR family is functionally divided in two classes, the acti- vating receptors and the inhibitory receptor FcgRIIb (8). In mice, 4F.O. and J.S.V. contributed equally to this manuscript. these classes are also structurally different; activating receptors Received for publication December 19, 2011. Accepted for publication April 27, 2012. are multimeric receptors composed of a ligand-binding a-chain This work was supported by Dutch Cancer Society Grant UL 2004-3008 (to N.v.M. and a dimer of the signal-transducing g-chain, containing an and M.G.M.C.), European Union Integrated Project Cancer Immunotherapy LSHC- ITAM. The inhibitory receptor is a single-chain receptor con- CT-2006-518234 (to N.v.M.), and STW Project 10412 (to S.M.M.). taining an ITIM in its cytoplasmic domain. Both functionally The array data presented in this article have been submitted to distinct types of FcRg are commonly coexpressed on the same Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE33718. cell. This coexpression functions as a threshold for the IC-medi- Address correspondence and reprint requests to Dr. J. Sjef Verbeek, Department of ated activation of the cell (9, 10). Human Genetics, Leiden University Medical Center, Leiden 2333 ZA, The Nether- lands. E-mail address: [email protected] The ITAM in the cytoplasmic tail of the g-chain is crucial for The online version of this article contains supplemental material. the initiation of the downstream signal transduction pathways. Abbreviations used in this article: AvFC, average fold change; BM-DC, bone The critical event after cross-linking of FcgR is phosphorylation marrow-derived dendritic cell; DC, dendritic cell; FcRn, neonatal FcR; FDR, false of the tyrosines in the ITAM by protein tyrosine kinases, followed discovery rate; IC, immune complex; KO, knockout; pDC, plasmacytoid DC; WT, by Syk phosphorylation within minutes. It has been demonstrated wild-type. in mouse DCs that Syk is indispensible for FcgR-mediated DC Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 maturation (11). Downstream cascades ultimately lead to nuclear www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103703 The Journal of Immunology 93 translocation of NF-kB, activation of MAPK, and activation of IgG subclasses and their IgG binds to all mouse FcgR. The used batch of calcium signaling pathways (12). Calcium signaling ultimately OVA was selected based on the following functional assay: different leads to activation of NFAT and calcium-regulated transcription batches of OVA were titrated up to 100-fold the concentration used in this study. After 48 h of incubation with the different titrated batches of OVA, factors, such as CREB. The pathways downstream of FcgR liga- IL-12p40 production was measured in the supernatants of DCs. No IL- tion have been primarily studied in mast cells and macrophages. 12p40 production by DCs was observed at all concentrations for the batch Although the intracellular signal transduction module might be obtained from Worthington, whereas some other batches did induce IL- similar between different cell types, the outcome of the signaling 12p40 production at higher concentrations. This functional assay indicated that the used batch of OVA did not contain contaminants such as endo- is the activation of cell type-specific effector mechanisms. toxins that could significantly influence our experiments. Ag–Ab ICs targeted to FcgR on DCs induce Ag presentation and DC maturation, characterized by phenotypical changes in RNA preparation, processing, and microarray hybridization membrane expression of costimulatory molecules and production To study the effects of FcgR ligation in DCs, the panel of cells was in- of cytokines (7, 13). Because both activating and inhibiting FcR cubated with either ICs (stimulated) or medium (unstimulated), and tran- are expressed on DCs, the specific contribution of each class of scriptomes were analyzed by microarray. After 4 h of incubation, RNA was 2 receptors to the induction of DC maturation and Ag presentation is isolated using the RNeasy from Qiagen. RNA was stored at 80˚C. RNA purity and concentration were assessed by using Nanodrop (Ther- not completely understood. moScientific). RNA integrity was assessed using Lab on a chip on an In this study, we analyzed the genes downstream of FcgR li- Agilent 2100 Bioanalyzer (Agilent Technologies). A total of 2 mgRNA gation by a full-genome microarray screening of DCs incubated was amplified using the Illumina RNA Amplification kit, which included with their natural ligands, Ag–Ab complexes. Importantly, we incorporation of biotin. Subsequently, the samples were hybridized on dissected the roles of the inhibitory receptor FcgRIIb versus the Illumina mouse Sentrix-6 v1.1 expression Beadchips and stained with Cy3.

Finally, the BeadChips were scanned on the Illumina BeadArray 500 GX Downloaded from activating FcgRs in the initiation of FcgR-mediated DC matura- scanner. All samples were processed and handled by the same person. tion and Ag presentation in a unique set of DCs from either wild- type (WT) mice, or FcgRIIb knockout (KO) mice (activating Microarray data analysis FcgRs only), or FcR g-chain KO mice (inhibitory FcgR only). Raw array data were exported by Illumina Beadstudio software version 3.0 Both KO strains have been generated by gene targeting in C57BL/ and subsequently imported and stored in Rosetta Resolver software. Pre- 6 embryonic stem cells (14, 15) and therefore lack 129-derived processing and normalization were performed using a customized pre-

processing pipeline for Illumina arrays. Rosetta Resolver was used to http://www.jimmunol.org/ flanking genomic regions, which we recently have shown to analyze the data. The data have been deposited in National Center for strongly influence the immune system if present in a C57BL/6 Biotechnology Information’s Gene Expression Omnibus (van Montfoort background (14). et al., 2011) and are accessible through Gene Expression Omnibus Series By performing microarray-basedgeneexpressionprofiling, accession number GSE33718 (http://www.ncbi.nlm.nih.gov/geo/query/ multiplex cytokine production assays, and flow cytometry with acc.cgi?acc=GSE33718). Baseline gene expression intensities of unstimulated BM-DCs derived DCs derived from a unique panel of C57BL/6 FcgR KO mice from the three different mouse strains were compared by error-weighted incubated with well-defined ICs, we demonstrate that FcgR liga- ANOVA, including Benjamin Hochberg false discovery rate (FDR) cor- tion modulates the expression of many genes associated with T rection, followed by Student-Newman-Keuls post hoc test. cell response induction, which is strongly regulated by FcgRIIb. To identify genes with a relevant change in expression after IC stimu- lation, the fold change for each gene was calculated by dividing the log by guest on October 2, 2021 Moreover, by studying FcgR-mediated Ag-specific expansion of intensity of the values obtained from stimulated DCs with the log inten- CD8 T cells in a new DC-specific FcgRIIb KO mouse, we could sity of the values obtained from unstimulated DCs for each sample indi- confirm this regulatory role of FcgRIIb on DCs in vivo. vidually. To compare gene expression between mouse strains, the average fold change (AvFC) per mouse strain was assessed by calculating the mean of the log ratios of the four individual samples. An AvFC of 2 was used as Materials and Methods a limit to discriminate differentially expressed genes. Gene names are Mice written in italics, whereas names of are written in normal font. C57BL/6 Jico mice were obtained from Charles River. FcR g-chain KO Assessment of cytokine production after stimulation by ICs mice on C57BL/6 background were provided by T. Saito (Laboratory for Cell Signaling, RIKEN Research Center for Allergy and Immunology, Supernatants were harvested at 4, 8, 24, or 48 h after stimulation with ICs Yokohama, Japan) (15) and bred in our institute. FcgRIIb KO mice on or 24 h after stimulation with culture medium (background) and stored at C57BL/6 background were recently generated in our institute (14). DC- 220˚C. Cytokine production was assessed using a Luminex mouse 23-plex specific FcgRIIb KO mice were generated by crossing CD11cCre mice cytokine panel on Bioplex suspension array system (Bio-Rad). Data were (16), provided by B. Reizis (Department of Microbiology and Immunol- analyzed using Bioplex Manager software. ogy, Columbia University, New York, NY), with C57BL/6 mice with floxed FcgRIIb alleles (FcgRIIbfl/fl) generated in our laboratory (14). The MHC class I-restricted Ag presentation assay mice were used at 8–12 wk of age. All animal experiments were approved BM-DCs were incubated with IgG–OVA complexes or medium for 24 h, as by the institution’s animal welfare committee. described above. A total of 5 3 104 BM-DCs was cocultured with an equal DC generation and incubation with Ag–Ab complexes amount of the T cell hybridoma B3Z, which is specific for the OVA CTL epitope SIINFEKL that binds to MHC class I H-2Kb (18). B3Z T cells Immature DCs were derived from bone marrow harvested from the femurs express a b-galactosidase reporter construct driven by elements of the IL-2 and tibia from two female and two male mice of each mouse strain. The cells promotor. After 24 h of coculture, T cell activation was measured by of all 12 mice were cultured separately in IMDM supplemented with GM- a colorimetric assay on B3Z lysates using chlorophenol red-b-D-gal- CSF containing supernatant in petri dishes, as described (17). Nonadherent actopyranoside as substrate. and semiadherent cells were transferred to new dishes and fresh medium on days 4 and 7. On day 10, the cells were harvested and the percentage of Flow cytometry immature CD11c+ DCs was determined by flow cytometry, which was g typically .85%. Bone marrow-derived DCs (BM-DCs) were transferred to Phenotype and Fc R expression of the BM-DCs were analyzed by flow new culture dishes at a density of 1 million BM-DCs per 2 ml medium. cytometry 48 h after incubation with ICs or medium. Data acquisition was BM-DCs were incubated with either culture medium (unstimulated) or performed on a BD Biosciences FACScan. For data analysis, CellQuest IgG-OVA ICs (IC stimulated) containing 1 mg/ml OVA (Worthington) and software was used. 50 mg/ml rabbit anti-OVA IgG (ICN Biomedicals) at 37˚C for time In vivo T cell priming intervals, as indicated. Importantly, we used a ratio of OVA and anti-OVA IgG that was previously shown to be optimal for the induction of DC Recipient mice received CD8-enriched CFSE-labeled cells i.v. from OT-1 maturation (7). We used rabbit IgG anti-OVA because rabbits do not have mice containing ∼3 3 106 Va2+/CD8+ cells. Purity of CD8 T cells after 94 FcgR-DEPENDENT DC GENE EXPRESSION PROFILE negative selection was determined to 90%. One day later, 150 mg rabbit differentially expressed genes in the FcgRIIb KO DCs compared anti-OVA IgG was injected to half of the mice in each strain, and, 4 h with WT DCs. To get insight in the biological relevance of the postAb injection, 2.5 mg OVA per mouse was administrated to all mice. modulated genes and the signature of the DC after FcRg ligation, Three days after the injection of the Ag, spleen was collected and proliferation of Thy1.1 CFSE-labeled T lymphocytes was analyzed by flow immunologically relevant genes were selected and grouped cytometry on a BD Biosciences LSRII. according to their function (Fig. 1, Table I). FcgR ligation induces production of cytokines, chemokines, Results and growth factors The goal of this study was to assess the downstream effects of Expression levels of most cytokine genes were low in unstimulated FcgR ligation on DCs. To dissect the role of the three activating DCs (Table I, second column). The cytokine genes Il2, Il6, Il10, FcgR versus the inhibiting FcgRIIb, a unique panel of three dif- Il15, Il23a, Il27, and Ifnb1 were upregulated in both WT BM-DCs ferent C57BL/6 mouse strains was used: WT, expressing all four and FcgRIIb KO BM-DCs, but not in FcR g-chain KO BM-DCs FcgRs; FcgRIIb KO, lacking the inhibiting FcgRIIb; and the FcR after incubation with ICs (Fig. 1, Table I). Fold changes were g-chain KO, lacking functional expression of all three activating higher in FcgRIIb KO BM-DCs than in WT BM-DCs. In contrast, FcgR. Flow cytometry analysis of cell surface FcgR expression the suppressive cytokine Tgfb3, constitutively expressed at high confirmed the genetic deficiency of the respective FcgRs (data not levels, was significantly downregulated in both WT and FcgRIIb shown). KO BM-DCs upon incubation with ICs. No major differences in steady state gene expression between Biologically active IL-12, IL-23, IL-27, and IL-35, directly the three genotypes involved in T cell activation, are heterodimers that share some Downloaded from To assess the differences in gene expression at steady state, the subunits. Active IL-12 (IL-12p70) is a heterodimer of IL-12p35 transcriptomes of unstimulated DCs from the three different mouse (Il12a) and IL-12p40 (Il12b). However, IL-12p40 can also form strains were compared by microarray analysis. Comparison of a heterodimer with IL-23p19 (Il23a), generating active IL-23. unstimulated WT and FcR g-chain KO BM-DCs revealed 194 Alternatively, IL-12p35 can pair with IL-27b (Ebi3) to form IL- differentially expressed genes (FDR ,0.1). Top 10 up- and down- 35. Ebi3 was constitutively expressed at high levels and further regulated genes are shown in Supplemental Table I. As expected, upregulated in both WT and FcgRIIb KO BM-DCs, but not up- expression of FcR g-chain and the NeoR, the gene replacing regulated in FcR g-chain KO BM-DCs upon incubation with ICs. http://www.jimmunol.org/ FcR g-chain in the FcR g-chain KO mouse, represented the most Remarkably, Il12a and Il12b were hardly upregulated in any of striking differences between WT and FcR g-chain KO DCs (Supplemental Table Ia). The expression of the a-chains of all FcgR genes, including FcgRIIb, was not influenced by the ab- sence of the FcR g-chain (data not shown). Comparison of WT and FcgRIIb KO BM-DCs revealed 64 differentially expressed genes, of which most differences were relatively small (Supple-

mental Table Ib). The expression of the a-chains of the three by guest on October 2, 2021 activating FcgR genes was not influenced by the absence of FcgRIIb. Remarkably, steady state expression of Fcgr2b (FcgRIIb) was not different between WT and FcgRIIb KO BM-DCs. This can be explained by the probe used in the microarray: the probe is spe- cific for a part of the gene that is not deleted in the FcgRIIb KO. However, the remaining EC1 and C2 encoding parts of FcgRIIb gene do not lead to functional expression of FcgRIIb on the cell surface (data not shown). From these observations, we concluded that the DCs from the three different genotypes mainly differ in expression of FcRg family members, whereas the steady state levels of other genes were almost identical. Differential gene expression in DCs after incubation with ICs is mainly dependent on FcR g-chain and strongly regulated by FcgRIIb To study the effects of FcgR ligation in DCs, BM-DCs derived from the three different mouse strains were incubated with ICs (stimulated) or medium (unstimulated) and transcriptomes were analyzed by microarray (see Materials and Methods). In WT BM- DCs stimulated with IC, 963 genes were upregulated and 1017 genes were downregulated by AvFC of 2 or more. In FcgRIIb KO BM-DCs stimulated with ICs, 1383 genes were upregulated and 2088 genes were downregulated. In FcR g-chain KO BM-DCs stimulated with IC, 310 genes were upregulated and 218 genes were downregulated. Taken together, the combined microarray data strongly indicate that the interaction of ICs with DCs mod- FIGURE 1. Differentially expressed immunologically relevant genes as ulates the expression of a large number of genes in a FcR g-chain– determined with microarray. The AvFC of IC versus medium is depicted for dependent manner, whereas FcgRIIb acts as a strong threshold for WT, FcgRIIb KO, and FcR g-chain KO BM-DC. n = 4 DC cultures per activation, as reflected by the 75% increase in the number of genotype. Dotted line indicates threshold fold change of 22and2. The Journal of Immunology 95

Table I. Differentially regulated genes per category

AvFC

Gene Baseline Intensity WT FcgRII KO FcR g-Chain KO Cytokines Il2 6.7 .100.0 .100.0 1.6 Il10 8.5 57.9 91.3 1.2 Il27 16.3 16.0 37.2 1.5 Il23a 23.1 17.5 47.1 1.5 Il6 52.7 8.5 11.9 1.7 Ifnb1 13.2 5.4 14.2 1.9 Ebi3 (IL27b) 1,601.5 4.9 5.6 1.9 Il15 56.5 4.7 7.0 1.2 Tgfb3 345.4 24.3 25.5 21.3 IL-1 family Il1a 78.0 70.0 89.6 28.1 Il1b 3,430.0 12.8 23.4 8.7 Il1rn 432.8 7.8 18.5 4.8 Il1r2 1,822.8 1.8 2.3 1.4 Il18bp 88.8 2.2 4.0 1.0 Growth factors

Csf1 (M-CSF) 227.5 4.6 6.3 1.9 Downloaded from Csf2 (GM-CSF) 16.4 5.5 10.3 1.2 Csf3 (G-CSF) 16.2 14.0 19.4 2.2 Chemokines Ccl1 30.7 82.1 .100.0 12.7 Ccl2 1,368.3 3.6 4.2 2.3 Ccl3 (Mip-1a) 3,319.0 6.4 11.0 3.7 Ccl4 (Mip-1b) 3,630.5 8.6 16.4 3.0 http://www.jimmunol.org/ Ccl7 7,705.3 2.5 3.1 1.9 Ccl9 6,498.6 2.8 3.8 1.7 Ccl12 978.3 2.5 4.3 2.5 Ccl17 8,518.5 4.2 5.3 4.1 Ccl22 2,689.8 12.5 14.2 2.5 Cxcl1 587.6 12.7 19.1 3.6 Cxcl2 146.5 36.2 41.3 17.7 Cxcl9 38.9 10.4 20.0 3.7 Cxcl10 16.8 58.3 .100.0 25.8 Adhesion molecules and

Cdh1 (cadherin 1) 441.7 23.0 25.2 21.2 by guest on October 2, 2021 Itgae (CD103) 346.4 22.1 23.1 21.2 Itgb7 ( b7) 2,570.7 22.5 22.5 21.7 Edn1 (endothelin 1) 77.4 9.2 22.8 2.4 Icam1 (ICAM-1) 6,720.4 3.6 2.7 2.3 Itga5 (integrin a5) 58.3 3.4 5.3 1.9 Costimulatory and inhibitory molecules Cd40 393.8 14.2 19.6 4.7 Cd83 2,509.8 3.0 4.1 1.7 Cd86 5,117.8 2.2 2.0 1.6 Cd274 (PD-) 6,718.8 2.9 3.2 1.9 Havcr2 (Tim-3) 285.9 2.3 3.2 21.4 Lgals9 (Galectin-9) 435.1 2.4 3.2 1.0 TNF receptors and ligands Tnf 605.5 11.2 13.2 3.9 Cd70 149.1 24.8 28.4 3.5 Tnfrsf9 (4-1BB) 370.8 2.6 2.9 2.5 Tnfsf9 (4-1BBL) 119.6 5.9 6.6 21.0 Tnfsf4 (Ox-40L) 236.7 3.7 4.0 1.5 Tnfsf10 (Trail) 22.8 4.2 8.7 1.3 Ag presentation H2-K1 683.6 2.2 2.3 2.2 H2-Q5 821.0 2.1 2.6 1.9 H2-Q7 232.4 2.1 3.4 1.9 H2-T22 1,116.2 2.1 3.5 1.2 H2-T9 916.6 2.1 4.0 1.1 Tap1 444.6 3.3 4.4 1.6 Tapbp (Tapasin) 441.5 2.0 3.1 1.3 Tapbpl 463.8 2.3 3.2 1.6 H2-DMa (HLA-DM) 14,441.4 22.2 22.2 21.3 Pathogen and scavenger receptors Tlr2 2,457.1 4.4 3.3 3.5 Tlr5 133.6 23.7 26.3 21.5 Tlr6 118.2 2.6 3.7 1.1 Tlr7 238.7 2.5 2.9 1.2 Cd14 740.6 7.7 9.0 4.1 (Table continues) 96 FcgR-DEPENDENT DC GENE EXPRESSION PROFILE

Table I. (Continued)

AvFC

Gene Baseline Intensity WT FcgRII KO FcR g-Chain KO Nod1 164.6 3.2 4.2 1.7 Ddx58 (RIG-I) 148.8 2.8 1.3 2.3 Clec4e (Mincle) 250.5 20.3 17.6 9.8 Clec7a (Dectin-1) 1,302.1 2.2 2.4 1.7 Cd209a (DC-SIGN) 284.8 25.4 27.9 22.4 Cd207 () 587.9 11.7 6.9 16.3 Fcgrt (FcRn) 5,097.9 23.4 23.8 22.0 Marco 50.8 6.8 9.4 5.8 Miscellaneous C3 1,717.4 2.6 2.7 3.0 Adam17 (TACE) 618.5 2.4 2.4 1.8 Lox (lysyl oxidase) 18.1 23.2 28.8 2.5 Second column represents steady state expression in unstimulated WT BM-DCs. Columns 3–5 represent AvFC of ICs versus medium-incubated DCs (n = 4) per mouse strain. Downloaded from the DCs tested, whereas the dimerization partners were upregu- Taken together, the RNA expression data and the cytokine lated in DCs that expressed the activating FcgRs. production data indicate that incubation of DCs with ICs induced The IL-1 family is a family of proinflammatory cytokines with a the production of a broad range of cytokines that was largely pleiotropic effect on several immune cells (19). IL-1b is generated dependent on the activating FcgRs and tightly regulated by as pro–IL-1b that requires cleavage by caspase-1 (Casp1)tobe- FcgRIIb. come biologically active. Il1b (Table I) and Casp1 (data not shown) Besides the production of cytokines, BM-DCs also upregulated were constitutively highly expressed in BM-DCs derived from all the expression of growth factors and chemokines upon IC stim- http://www.jimmunol.org/ three mouse strains. Il1a and Il1b were both upregulated in DCs ulation: WT and FcgRIIb KO BM-DCs strongly upregulated the derived from all three mouse strains after incubation with IC. In- expression of Csf1 (M-CSF), Csf2 (GM-CSF), and Csf3 (G-CSF), terestingly, Il1rn (IL-1R antagonist) and Il18bp (IL-18–binding cytokines driving the differentiation of hematopoietic stem cells protein), both potent regulatory members of the IL-1 family that are toward macrophages, DCs, and granulocytes, respectively. Thir- used to treat patients with autoimmune disease (19), were upregu- teen different chemokines of both the CC and the CXC families lated in parallel. Upregulation of Il1a, Il1b,andIl1rn was stronger were upregulated in WT and FcgRIIb KO BM-DCs (Fig. 1, Table in FcgRIIb KO DCs compared with WT BM-DCs, whereas it was I). No chemokine genes were downregulated. Upregulation of

less pronounced in FcR g-chain KO BM-DCs. growth factors and chemokines was higher in FcgRIIb KO BM- by guest on October 2, 2021 The effector cytokine tnf (TNF-a), member of the TNF family, DCs compared with WT BM-DCs, whereas upregulation was sig- was constitutively expressed by immature WT BM-DCs and was nificantly lower or absent in FcR g-chain KO BM-DCs upon IC upregulated in DCs derived from all three mouse strains after incubation. Strong upregulation was, among others, observed for incubation with ICs (Fig. 1, Table I). TNF-a is a trans-membrane Cxcl9 and Cxcl10. CXCL9 and CXCL10 are ligands for CXCR3 molecule that needs cleavage by Adam17 before it is released into that is expressed by activated and circulating T cells, NK cells, the extracellular space (20). Adam17 was constitutively expressed and B cells (21). CXCR3 is associated with Th1 responses. In line and slightly upregulated after IC incubation in WT and FcgRIIb with the RNA expression data, Mip-1a (Ccl3) and Mip-1b (Ccl4) KO BM-DCs (Table I). were detected in the supernatants of WT and FcgRIIb KO BM- To determine whether changes in expression at the mRNA level DCs after incubation with ICs (Fig. 2). In contrast to the RNA reflected changes in production of cytokines, the latter was de- expression data, chemokines Mip-1a and Mip-1b could hardly be termined in supernatants of DCs from the three different strains detected in the supernatants of FcR g-chain KO BM-DCs incu- using a mouse Luminex array. The supernatants were harvested at bated with ICs (Fig. 2). Taken together, these results indicate that 4, 8, 24, or 48 h after incubation with ICs or medium. Kinetics the incubation of DCs with ICs induced the expression of a variety varied between cytokines (Fig. 2). In contrast to the other cyto- of chemokines. The upregulation of chemokines was largely de- kines that reached their maximum levels after 24–48 h, the levels pendent on the activating FcgRs and tightly regulated by FcgRIIb. of TNF-a and IL-10 peaked at 4–8 h. Both WT and FcgRIIb KO BM-DCs produced the cytokines IL-2, IL-6, IL-10, IL-1a, IL-1b, FcgR ligation induces migratory DC phenotype and TNF-a after incubation with ICs (Fig. 2). Cytokine levels in Integrins Itgae (CD103) and Itgb7 (integrin b7) and the adhesion supernatants of FcgRIIb KO BM-DCs were higher compared with molecule Cdh1 (cadherin 1) were downregulated in WT BM-DCs the cytokine levels in supernatants of WT BM-DCs. In contrast, and FcgRIIb KO BM-DCs upon IC stimulation (Fig. 1, Table I). cytokines were hardly detectable in the supernatants of FcR Downregulation of cadherin 1 is associated with increased cell g-chain KO BM-DCs. This is in line with RNA expression data. motility and invasion of DCs into surrounding tissues. Cadherin 1 Although Il12b (IL-12p40) and Il12a (IL-12p35) were not notably is also important for DC cluster formation, which is inversely upregulated at RNA level at 4 h after IC incubation, high levels associated with DC maturation (22). Icam1 (ICAM-1), important of IL-12p40 and IL-12p70 were measured in the supernatants of for endothelial transmigration and interaction with T lymphocytes WT and FcgRIIb KO BM-DCs 24 h after IC incubation. The delay (23), was constitutively expressed at high levels and upregulated in peak production of IL-12p40 and IL-12p70 compared with in all three genotypes. In addition, Itga5 (integrin a5) was up- the other cytokines may indicate that measuring changes in RNA regulated in WT and FcgRIIb KO DCs, whereas Edn1 (endothelin expression at 4 h was too early for Il12b and Il12a. 1) was strongly upregulated in WT and FgcRIIb KO DCs after The Journal of Immunology 97 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 2. FcgR-dependent cytokine and chemokine production upon IC incubation. BM-DCs of WT (closed square), FcgRIIb KO (open circle), or FcR g-chain KO (closed triangle) were incubated with 1 mg/ml IC. Supernatants were collected after 0, 4, 8, 24, or 48 h, and levels of the indicated cytokines or chemokines were measured by Luminex bead array.

IC stimulation. Differential expression of integrins and adhesion BM-DCs. Together with CD70, these molecules are important for molecules after IC stimulation was less pronounced in WT DCs the progression of T cell responses. They regulate the frequency of compared with FcgRIIb KO DCs and lower or absent in FcR effector and memory T lymphocytes by providing proliferative g-chain KO DCs. These data indicate that FcgR ligation by ICs and survival signals and promote cytokine production by T lym- initiates the migration of DCs out of the periphery toward the phocytes (24). CD27, the receptor for CD70, is constitutively lymph nodes. IC-induced migration is mainly dependent on acti- expressed by T cells. These results indicate that FcgR ligation on vating FcgRs and regulated by FcgRIIb. DCs regulates the expression of both costimulatory receptors and costimulatory receptor ligands (25). FcgR ligation induces upregulation of costimulatory molecules In contrast, the expression of Tnfrsf9 (4-1BB) was upregulated Incubation of DCs with ICs induced upregulation of several genes at a similar level in DCs from all three genotypes (Table I) after belonging to B7 and TNF(R) families of costimulatory molecules: incubation with IC, suggesting a FcgR-independent activation Cd70, Cd40, Cd83,andCd86 were significantly upregulated in both mechanism. Data obtained from 4-1BB–deficient mice suggest WT and FcgIIb KO BM-DCs. Cd40 and Cd70 upregulation was less that 4-1BB on DCs is important for longevity of DCs and DC– pronounced in FcR g-chain KO BM-DCs compared with their T cell interactions. upregulation in WT and FcgRIIb KO BM-DCs (Fig. 1, Table I). Changes in cell surface expression of CD40 and CD86 were also FcgR ligation enhances MHC class I presentation, but not analyzed by flow cytometry at 48 h after IC stimulation (Fig. 3). MHC class II presentation Both CD40 and CD86 were upregulated in WT and FcgRIIB KO The effect of FcgR ligation by ICs on both MHC class I and MHC BM-DCs, but not in FcR g-chain KO BM-DCs. Interestingly, in FcR class II Ag presentation pathways was studied. Five different g-chain KO BM-DCs, the upregulation of CD40 at RNA level does genes of MHC class I loci were upregulated after incubation with not correlate with an increase in cell surface expression of CD40. ICs in both WT and FcgRIIb KO BM-DCs (Table I). Moreover, TNFR family members Tnfsf4 (OX-40L) and Tnfsf9 (4-1BBL) Tap1 (TAP1), Tapbp (tapasin, component of the MHC class I- were upregulated upon IC stimulation in WT and FcgRIIb KO loading complex), and Tapbpl (tap-binding protein like) were 98 FcgR-DEPENDENT DC GENE EXPRESSION PROFILE

FIGURE 4. MHC class I-restricted presentation of the OVA Ag upon OVA-IC incubation. Ag presentation by WT, FcgRIIb KO, and FcR g-chain KO BM-DCs to the OVA-specific B3Z CD8+ T cell hybridoma was measured using a colorimetric assay. Mean and SD are displayed. This experiment was performed twice with similar results. Downloaded from Furthermore, we assessed the regulatory role of FcgRIIb on CD8 T cell activation in vivo after FcgR ligation by in vivo formed ICs. The expansion of OVA-specific CD8 T cells upon in vivo formation of OVA-IgG ICs was compared between WT and FcgRIIb KO recipients and recipients that selectively lacked fl/fl FcgRIIb expression on DCs (CD11cCre X FcgRIIb ). In both http://www.jimmunol.org/ FIGURE 3. FcgR-dependent upregulation of CD40 and CD86 upon IC FcgRIIb KO and DC-specific FcgRIIb KO, the OVA-specific CD8 incubation. BM-DCs from WT, FcgRIIb KO, or FcR g-chain KO mice were stained with mAb specific for CD40 (y-axis) and CD86 (x-axis) after T cell expansion was significantly higher than in WT mice (Fig. 48 h of incubation with medium (left panel)or1mg/ml ICs (right panel) 5). These results indicate that FcgRIIb has a regulatory role on and analyzed by flow cytometry. CD8 T cell priming after targeting Ag to FcgRs in vivo. Moreover, by making use of the DC-specific KO, we showed that FcgRIIb regulates downstream effects of FcgR ligation on DCs under upregulated. In contrast, we did not observe changes in expression physiological conditions in vivo. These results indicate that the of genes coding for MHC class II a- and b-chains that were ratio between the activating FcgR and the inhibitory FcgRIIb on by guest on October 2, 2021 constitutively expressed at a high level (data not shown). MHC II accessory molecule HLA-DM (H2-Dma, Table I) that regulates peptide loading in MHC class II was downregulated in WT and FcgRIIb KO BM-DCs. These data might indicate that FcgR li- gation enhances MHC class I presentation capacity, but not MHC class II presentation. Alternative explanations for the observed striking differences in MHC class I and MHC class II gene ex- pression might be that MHC class II presentation does not require de novo synthesis of MHC class II complexes or that we chose a too late time point for measuring MHC class II expression, because it has been shown that MHC class II expression already peaks at 1 h after incubation with LPS (26). To analyze the functional consequences of the observed striking differences in gene expression between the DCs from the different genotypes, we compared the functional MHC class I Ag presentation capacity after incubation with OVA-containing ICs between the DCs from the three different genotypes. Ag pre- sentation was measured using a reporter T cell hybridoma that becomes activated upon recognition of the OVA-derived CTL epitope SIINFEKL in the MHC class I molecule Kb (Fig. 4). Ag FIGURE 5. OVA-IC–induced in vivo MHC class I-restricted OVA- presentation was observed with WT DCs, although absent with specific T cell expansion. Thy1.2+ recipient WT, FcgRIIb KO, and DC- FcR g-chain KO BM-DCs, as previously described (Fig. 4) (17), specific FcgRIIb KO (CD11c Cre/FcgRIIb KO) mice were adoptively + showing that presentation of ICs is completely dependent on ac- transferred with purified OVA-specific Thy1.1 CD8 T cells. One day later, mice received rabbit IgG anti-OVA, followed by i.v. injection with OVA tivating FcRs. Ag presentation by FcgRIIb KO BM-DCs was 2 (Ab+), or a single injection with OVA (Ab ). After another 3 d, the spleens much higher than Ag presentation by WT DCs (Fig. 4), indicating were isolated and the percentage of proliferating Thy1.1+ cells within the that FcgR-facilitated Ag presentation is strongly regulated by total population of CD8+ cells in the live gate was determined. Mean FcgRIIb. Higher Ag presentation by FcgRIIb KO BM-DCs cannot percentage as well as the minimum and maximum (whiskers) per group (5 be attributed to higher uptake of ICs, because uptake of ICs is mice/group) are depicted. Groups are compared by Student unpaired t test: similar between DCs from WT and FcgRIIb KO mice (Supple- p , 0.05 (*) or p , 0.001 (***). This experiment was performed twice mental Fig. 1). with similar results. The Journal of Immunology 99

DCs in vivo determines the extent of DC maturation and T cell in Table I for FcRn. Remarkably, in our experiments, FcRn ex- activation after FcgR ligation. pression was downregulated after incubation with ICs in all three types of BM-DCs, although less pronounced in FcR g-chain KO Discussion DCs (Table I), suggesting that after IC incubation the cross- DCs play a crucial role in initiating and steering T cell immunity. presentation capacity of these cells is regulated independent of The extent of T cell activation, proliferation, and survival after TCR activating FcgR ligation. ligation is not only dependent on the strength of TCR stimula- For cytokines, chemokines, and some costimulatory molecules, tion, but also on the availability of prosurvival cytokines and the changes in expression after stimulation with ICs were monitored presence of costimulatory signals. We and others have shown that both on mRNA and protein level. The two methods correlated very FcgR ligation by optimal Ag–Ab ICs is one of the most vigorous well for the majority of the genes tested. One of the exceptions approaches to induce DC maturation and activation. Binding of was the high level of IL-12p40 in the supernatant of DCs incubated ICs to FcgR on DCs induces both CD4+ and CD8+ T cell with ICs (Fig. 2), whereas changes in mRNA expression were responses in vitro and in vivo (7). DCs are one of the few cell absent in these cells. This can be explained by the timing of our types that express all four known mouse FcgRs, functionally di- analysis. IL-12p40 is a relatively late gene with a peak RNA level vided in two categories: the (three) activating FcgRs and the (one) at 9–10 h after DC ligation (32). Another remarkable discrepancy inhibitory FcgR (8). To analyze the specific contributions of the is the observation that in FcR g-chain KO BM-DCs, some genes, activating FcgRs and the regulatory role of the inhibiting FcgRon including CD40, CD86, Mip-1a, and Mip-1b, were upregulated at DCs, we used a unique panel of BM-DCs, all having the same the mRNA level after incubation with ICs, whereas, at the protein C57BL/6 genetic background, but lacking either FgRIIb (activa- level, no significant increase could be detected by flow cytometry. tory FcgR only) or FcR g-chain (inhibitory FcgR only). We ex- This suggests that posttranscriptional or posttranslational modifi- Downloaded from tensively examined the effects of FcgR ligation by ICs on these cation may exist regulated by FcR g-chain–mediated mechanisms. BM-DCs by using microarrays, flow cytometry, and multiplex A combined approach of genomics and proteomics should reveal cytokine production assays. Our results show that ICs induce the significance of these modifications after FcgR ligation (33). a gene expression profile in DCs that is associated with cell Previous research has shown that FcgR-dependent maturation maturation and effective T cell activation, which was largely de- of human DCs by FcgRIIb blockade in the presence of Igs was pendent on the activating FcgRs and required signaling through associated with a distinct IFN response signature (34). In line with http://www.jimmunol.org/ the FcR g-chain. This gene expression signature was strikingly these observations, our list of genes that were upregulated in more pronounced in the FcgRIIb KO BM-DCs compared with mouse DCs after incubation with ICs contained many IFN- WT BM-DCs, indicating that FcgRIIb is an important modulator responsive genes, such as several IFN-a–inducible proteins (ifi), of the expression of the majority of these genes after FcgR liga- signal transducer and activator of transcription (stat1 as well as tion. This suggests that FcgRIIb KO BM-DCs have an improved stat5), immune-responsive gene1 (Irg1), IFN regulatory factor 7 capacity to induce naive T lymphocytes and to uphold ongoing (irf7), and myxovirus resistance 2 (Mx2). The upregulation of T cell responses. Indeed, we observed in vitro enhanced FcgR- these IFN-inducible genes was accompanied by enhanced upreg- dependent Ag presentation to CD8+ T cells by FcgRIIb KO DCs ulation of IFN-b, but not IFN-a mRNA, in FcgRIIb KO BM-DCs. by guest on October 2, 2021 compared with WT DCs (Fig. 4) (10). Moreover, also in vivo, Ab- IFN-b upregulation in FcR g-chain KO BM-DCs was decreased mediated OVA-specific T cell proliferation was higher in mice that compared with the upregulation in WT BM-DCs. However, the were deficient for FcgRIIb (Fig. 5). Most importantly, enhanced difference in upregulation of IFN-b between the different geno- OVA-specific T cell proliferation was also observed in mice that types could not be confirmed at the protein level (data not shown). selectively lacked FcgRIIb on DCs, confirming our conclusion Because all observations pointed to an effective activation of from microarray, flow cytometry, and multiplex cytokine produc- T cell immunity after ligation of activating FcgR on DCs by ICs, it tion assays with BM-DCs that FcgRIIb regulates FcgR-facilitated is surprising that the expression of some genes associated with Ag presentation by DCs (Fig. 5). Our findings with FcgRIIb-de- inhibition of T cell function was also increased. Cd274 (PD-L1) ficient mouse DCs are in agreement with previous observations (Table I), described to dampen the T cell response through an that selective Ab-mediated blockade of FcgRIIb on human DCs in interaction with PD-1 on T lymphocytes (35), was upregulated in the presence of activating Ig ligands leads to DC maturation and WT and FcgRIIb KO BM-DCs, whereas its steady state expres- can enhance immunogenicity of DCs in vitro after incubation with sion was already relatively high on all three types of BM-DCs ICs (27). (Table I). In addition, expression of Havcr2 (Tim-3) (36) and its In our analysis of FcgR function on DCs, we used BM-DCs. ligand Lgals9 (Galactin-9) was upregulated in WT and FcgRIIb KO BM-DCs take up ICs very efficiently, as was also observed for BM-DCs (Table I). However, Tim-3 has a divergent role on dif- plasmacytoid DCs (pDCs) and CD8+/CD11b2 and CD82/CD11b+ ferent cell types and may have a proinflammatory role on DCs (37). splenic DCs in vivo (28). However, pDCs lack expression of ac- Our results suggest that the ratio between the activating FcgR tivating FcgR and only express FcgRIIb. Consequently, uptake of and the inhibitory FcgRIIb on DCs in vivo determines the strength ICs by pDCs does not lead to DC maturation and Ag presentation of DC maturation and T cell activation after FcgR ligation. In the to T lymphocytes (29). Cross-presentation of IC-derived peptides present study, we analyzed the regulatory role of FcgRIIb by using by CD82 splenic DCs is FcR g-chain dependent, whereas, at high mice deficient for FcgRIIb either on all cells or specifically on DCs. concentration of ICs, splenic CD8+ DCs can cross-present IC- Because this is an artificial condition, we analyzed whether also derived peptides in the absence of FcgR (30). Similar to CD82 under natural conditions the ratio between the expression of the splenic DCs, BM-DCs can cross-present low doses of Ag captured activating FcgR and the inhibitory FcgRIIb can vary substantially. in ICs. This capacity is attributed to synergism between FcgR and We determined the changes in expression of the activating and in- the neonatal FcR (FcRn) (31) in which FcgR, expressed at the cell hibitory FcgR on mouse BM-DCs after incubation with different surface, is involved in uptake of the ICs, whereas subsequently, cytokines and LPS. We found that the expression of FcgRIIb was FcRn, expressed in intracellular acidic compartments, shuttles the upregulated by IL-4, IL-10, and LPS, with IL-4 giving the strongest Ag toward cross-presentation compartments (31). Both receptors effect, whereas the receptor was downregulated by IFN-g (data not are expressed by CD82 splenic DCs (31) and BM-DCs, as shown shown). Interestingly, all activating FcgR were upregulated by 100 FcgR-DEPENDENT DC GENE EXPRESSION PROFILE

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BM-DCs were incubated for one hour with medium (filled histogram) or 1 µg/ml IC (unfilled histogram) and analyzed by flow cytometry. The percentage of Alexa488-positive cells was assessed (IC-Alexa488).

Supplementary Table I: Comparison of Steady State Gene expression levels in DCs of different genotypes

Top 10 genes differentially expressed between unstimulated WT and FcR γ-chain KO BM- DCs (a) and WT and FcγRIIb KO BM-DCs (b) assessed by error weighted ANOVA using Benjamin Hochberg FDR correction and Student-Newman-Keuls post-hoc test.

Ia: Genes expressed at a higher level in FcR γ-chain KO compared to WT. Top 10 of 194 genes. Gene name Alias Fold higher in FcR γ-chain KO NeoR Neomycine Resistent cassette >20 Gbp1 Guanulate binding protein 1 7.50 Cd209a DC SIGN 4.55 Slco2b1 Solute carrier family 22, member 9 4.26 Cd209c SIGNR2 2.95 Tspan8 8 2.82 Fcrls IgSR 2.80 Myo5a Myosin 5 a 2.77 LOC268730 Predicted sequence (withdrawn from refseq) 2.70 LOC383125 Predicted sequence 2.70

Genes expressed at a lower level in FcR γ-chain KO compared to WT. Top 10 of 194 genes. Gene name Alias Fold higher in WT Fcer1g FcR γ-chain >10 AI480556 serine arginine-rich pre-mRNA splicing factor SR-A1 4.36 Saa3 Serum Amyloid A3 4.16 Anpep alanyl (membrane) amino peptidase 3.97 Gpr84 G-protein coupled receptor 84 3.38 Foxp4 Forkhead box P4 3.18 A530032J19Rik Unknown sequence 3.18 Tgfb1 Transforming growth factor β 3.07 Ap2a1 Adaptor protein complex AP-2, α 1 subunit 2.93 Fmnl1 Lymphocyte specific formin related protein 2.74

Ib: Genes expressed at a higher level in FcγRIIb KO compared to WT Top 10 of 64 genes. Gene name Alias Fold higher in FcγRIIb KO Wdfy1 WD repeat and FYVE domain containing 1 3.41 Anpep alanyl (membrane) amino peptidase 2.14 Bcat2 branched chain aminotransferase 2,mitochondrial 1.96 Thy1 CD90 1.93 Nt5dc2 5'-nucleotidase domain containing 2 1.70 Dgat2 diacylglycerol O-acyltransferase 2 1.69 Tmem104 trans 104 1.67 Aldoa fructose-bisphosphate aldolase A 1.55 Fcnb Ficolin B 1.54 Slc7a8 Solute carrier family 7, member 8 1.54

Genes expressed at a lower level in FcγRIIb KO compared to WT. Top 10 of 64 genes. Gene name Alias Fold higher in WT Enah enabled homolog 3.16 Slco2b1 solute carrier organic anion transporter family member 2b1 2.45 Tm6sf1 trans membrane 6 superfamily member 1 1.91 Usp47 ubiquitin specific peptidase 47 1.74 0610010K06Rik hypothetical protein LOC71678 1.48 Hmgcs1 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 1.48 6330403M23Rik RIKEN cDNA 6330403M23 gene 1.41 A930005I04Rik RIKEN cDNA A930005I04 gene 1.41 Flnb Filamin β 1.38 Camk1d calcium/calmodulin-dependent protein kinase 1.38