FcγRIIb on B Cells and Myeloid Cells Modulates Activation and Autoantibody Responses via Different but Synergistic Pathways in -Prone Yaa This information is current as Mice of September 23, 2021. Qingshun Lin, Mareki Ohtsuji, Hirofumi Amano, Hiromichi Tsurui, Norihiro Tada, Ryota Sato, Hidehiro Fukuyama, Hiroyuki Nishimura, J. Sjef Verbeek and Sachiko Hirose J Immunol published online 29 October 2018 Downloaded from http://www.jimmunol.org/content/early/2018/10/28/jimmun ol.1701487 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2018/10/29/jimmunol.170148 Material 7.DCSupplemental

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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 © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published October 29, 2018, doi:10.4049/jimmunol.1701487 The Journal of Immunology

FcgRIIb on B Cells and Myeloid Cells Modulates B Cell Activation and Autoantibody Responses via Different but Synergistic Pathways in Lupus-Prone Yaa Mice

Qingshun Lin,*,1 Mareki Ohtsuji,† Hirofumi Amano,‡ Hiromichi Tsurui,* Norihiro Tada,x Ryota Sato,{ Hidehiro Fukuyama,{ Hiroyuki Nishimura,† J. Sjef Verbeek,‖,1 and Sachiko Hirose*,1

C57BL/6 (B6).FcgRIIb2/2.Yaa mice spontaneously develop lethal lupus nephritis. To define the cell type–specific role of FcgRIIb in Yaa-associated lupus, we established B cell– (CD19Cre.Yaa), myeloid cell– (C/EBPaCre.Yaa), and dendritic cell– (DC) (CD11cCre.Yaa) specific FcgRIIb-deficient B6.Yaa mouse strains. CD19Cre.Yaa mice developed milder lupus than B6.FcgRIIb2/2. Yaa mice, indicating that FcgRIIb deficiency on B cells is not sufficient for the development of severe disease. Surprisingly, Downloaded from C/EBPaCre.Yaa mice also showed autoantibody production and mild lupus similar to that in CD19Cre.Yaa mice, whereas CD11cCre.Yaa mice stayed disease free. These observations indicate that FcgRIIb deficiency in B cells and myeloid cells, but not DCs, contributes to the severe disease in B6.FcgRIIb2/2.Yaa mice. Flow cytometric analysis showed that the frequency of peripheral Gr-12 but not Gr-1+ was increased in B6.FcgRIIb2/2.Yaa and C/EBPaCre.Yaa but not CD19Cre.Yaa mice, suggesting a link between FcgRIIb deficiency on myeloid cells and the high frequency of Gr-12 . RNA sequencing revealed that compared with Gr-1+ monocytes, Gr-12 monocytes expressed higher levels of the B cell–stimulating cytokines http://www.jimmunol.org/ BSF-3, IL-10, and IL-1b, the DC markers CD11c, CD83, and Adamdec1, and the antiapoptotic factors Bcl2 and Bcl6. In conclusion, in Yaa-associated lupus nephritis, FcgRIIb on B cells and myeloid cells modulates B cell activation via different but synergistic pathways. Gr-12 monocytes are the most likely candidate myeloid cells involved. The Journal of Immunology, 2018, 201: 000–000.

ice have four types of IgG Fc receptors, FcgRI, Lupus nephritis, the characteristic feature of systemic lupus FcgRIII, FcgRIV, and FcgRIIb (1, 2). The former three erythematosus (SLE), is an IC-mediated renal glomerular and vascular

M are activating receptors composed of a ligand-binding inflammatory disease. FcgRIIb encoding gene is identified as a by guest on September 23, 2021 a-chain and a dimer of the FcR g-chain (FcR g) that mediates ac- susceptibility locus for SLE both in mice and humans (3–5). We tivation signals. FcgRIIb is a single-chain receptor, which inhibits previously found that the Fcgr2b gene is polymorphic and that cell activation upon coengagement with the activating FcgRby SLE-prone strains, such as NZB, BXSB, and MRL all share immune complexes (ICs). The balance of stimulatory and inhib- deletion polymorphism in the Fcgr2b promoter region (3). This itory signals determines the outcome of FcgR signaling in mye- causes downregulation of FcgRIIb expression particularly on loid effector cells, which controls IC-mediated cellular responses activated B cells, which results in increased IgG Ab produc- such as Ab-dependent cell-mediated cytotoxicity, Ab-dependent tion (6, 7). The SLE-prone BXSB strain contains not only the cellular , and release of inflammatory mediators. autoimmune-type Fcgr2b locus but also the Y – On B cells, coengagement of FcgRIIb and the BCR downregulates linked autoimmune acceleration gene (Yaa) mutation. The etio- the production of Abs. logic basis of Yaa is a duplication of the TLR7 gene due to a

*Department of Pathology, Juntendo University School of Medicine, Tokyo 113-8421, The sequences presented in this article have been submitted to the National Center Japan; †Toin Human Science and Technology Center, Department of Biomedical En- for Biotechnical Information’s Gene Expression Omnibus (https://www.ncbi.nlm.nih. gineering, Toin University of Yokohama, Yokohama 225-8502, Japan; ‡Department of gov/geo/query/acc.cgi?acc=GSE116757) under accession number GSE116757. Internal Medicine and Rheumatology, Juntendo University School of Medicine, Tokyo x Address correspondence and reprint requests to Dr. Sachiko Hirose at the current 113-8421, Japan; Atopy Research Center, Juntendo University School of Medicine, { address: Department of Biomedical Engineering, Toin University of Yokohama, 1614 Tokyo 113-8421, Japan; Laboratory for Lymphocyte Differentiation, RIKEN Center ‖ Kurogane-cho, Aoba-ku, Yokohama 225-8502, Japan. E-mail address: sacchi@toin. for Integrative Medical Sciences, Yokohama 230-0045, Japan; and Department of ac.jp Human Genetics, Leiden University Medical Center, 2333ZA Leiden, the Netherlands The online version of this article contains supplemental material. 1Current address: Toin Human Science and Technology Center, Department of Biomedical Engineering, Toin University of Yokohama, Yokohama, Japan. Abbreviations used in this article: ACR, albumin/creatinine ratio; B6, C57BL/6; BSF-3, B cell–stimulating factor-3; DC, dendritic cell; FcR g, FcR g-chain; FPKM, ORCIDs: 0000-0002-6457-0630 (H.F.); 0000-0003-0934-6924 (J.S.V.). fragment per kilobase of exon per million reads; GC, germinal center; GO, gene Received for publication October 25, 2017. Accepted for publication September 22, ontology; GSEA, Gene Set Enrichment Analysis; IC, ; MOMA-1, 2018. metallophilic macrophage Ab; PANTHER, analysis through evolutionary relationships; PAS, periodic acid–Schiff; PNA, peanut agglutinin; RNP, ribonucleo- This work was supported by grants from the Ministry of Education, Culture, Science, protein; SLAM, signaling lymphocytic activation molecule; SLE, systemic lupus Sports, Science and Technology of Japan (26460493 and 15K08432) and a grant from erythematosus; Yaa, Y chromosome–linked autoimmune accelerating gene. the Ministry of Health, Labour and Welfare of Japan (16ek0109019s0703). Q.L., H.F., J.S.V., and S.H. designed, conducted, and analyzed experiments and wrote Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$37.50 the manuscript; M.O., H.T., N.T., and R.S. conducted and analyzed experiments and approved the manuscript; H.A. and H.N. designed and interpreted experiments and approved the manuscript.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1701487 2 FcgRIIb ON B CELLS AND MYELOID CELLS translocation of the TLR7-containing region in the X chromosome B cells, monocytes, neutrophils, macrophages, and DCs were examined by to the Y chromosome (8, 9). Because the SLE phenotype in BXSB staining PBLs or spleen cells with fluorescent-conjugated mAbs against male mice was almost completely reversed by substitution of the B220 (RA3-6B2), CD11b (M1/70), CD11c (HL3), and Gr-1 (RB6-8C5), and with biotin-conjugated anti-FcgRIIb (Ly17.2) mAb followed by autoimmune-type Fcgr2b with C57BL/6 (B6)-type Fcgr2b (10), the streptavidin-allophycocyanin. For spleen cell subset analysis, cells were autoimmune-type Fcgr2b is a crucial susceptibility factor for SLE stained with fluorescent-conjugated mAbs against CD3 (17A2), CD4 in BXSB male mice. However, BXSB female mice carrying the (RM4-5), B220, ICOS (398.4A), Foxp3 (FJK-16s), CD25 (PC61), CD138 autoimmune-type Fcgr2b do not develop SLE because of the lack of (281-2), CD11b, and CD11c and peanut agglutinin (PNA). For staining of the intracellular marker Foxp3, cells were surface stained with mAbs against TLR7 duplication due to the X chromosome inactivation mechanisms CD4 and CD25 followed by intracellular staining with mAb against Foxp3 that equalize gene expression, indicating that the autoimmune-type using the FOXP3 Fix/Perm Buffer Set and protocol (BioLegend). For PBL Fcgr2b locus contributes to SLE susceptibility through a strong ep- subset analysis, cells were stained with fluorescent-conjugated mAbs against b istatic interaction with the Yaa locus. B220, CD11b, Gr-1, CD115 (AFS98), I-A (K25-8.7), NK1.1 (PK136), In addition to the polymorphic Fcgr2b gene, the downstream FcgRIV (9E9), CCR2 (475301), CD11c (N418), and polyclonal anti- CX3CR1 Ab. Stained cells were analyzed using a FACSAria cytometer signaling lymphocytic activation molecule (SLAM) family genes and FlowJo software (Tree Star). are also polymorphic with two known haplotypes. Haplotype 1 is found in B6 and related strains, whereas haplotype 2 is found in all Serum levels of autoantibodies autoimmune-prone mouse strains and also in 129 strain (11). In- Autoantibody levels were measured using ELISA. Levels of Abs against triguingly, it has been shown that FcgRIIb2/2 mice generated dsDNA and chromatin were expressed in units, referring to a standard curve 3 using 129-derived embryonic stem cells and backcrossed into B6 obtained by the serial dilution of a pooled serum from (NZB NZW) F1 mice over 8 mo of age, containing 1000 U/ml (22). Levels of anti- background spontaneously develop lupus nephritis (12); however, ribonucleoprotein (RNP) Abs were measured by a commercially avail- Downloaded from 2/2 FcgRIIb mice generated using B6-derived embryonic stem able (a Diagnostic) and expressed as relative units according to the 2 2 cells do not (13). The addition of Yaa locus to B6.FcgRIIb / manufacturer’s instructions. mice results in the development of lupus nephritis with 30% Urinary albumin/creatinine ratio penetrance at the age of 10–12 mo (13). Thus, the deletion of FcgRIIb, the presence of SLAM haplotype 2, and the Yaa locus all Renal function was monitored by measurement of urinary albumin/creatinine ratio (ACR) using Siemens DCAVantage according to the manufacturer’s contribute to the development of severe autoimmunity (14, 15). http://www.jimmunol.org/ instruction. FcgRIIb is a major negative regulator of B cell activation; however, FcgRIIb is expressed not only on B cells but also on a Histopathology and tissue immunofluorescence wide variety of myeloid cell types (1, 2). In previous studies using Paraffin kidney sections were stained with periodic acid–Schiff (PAS) and cell type–specific FcgRIIb-deficient mouse strains, it has been hematoxylin (PAS staining). For immunohistochemical studies, frozen suggested that FcgRIIb expression in distinct cell populations kidney sections were stained with FITC-labeled goat Abs to IgG or to C3 contributes to the development of the autoimmune disease via (ICN Pharmaceuticals). Frozen spleen sections were three-color stained with Alexa 488–labeled anti-CD4 and -CD8 mAbs, Alexa 647–labeled different mechanisms in different models (16–18). In the current anti-B220 mAb, and Alexa 546–labeled PNA, or two-color stained with study, we examined the cell type–specific contribution of FcgRIIb Alexa 546–labeled PNA and rat anti–metallophilic macrophage Ab to the development of Yaa-associated lupus nephritis. Because a (MOMA-1) (BMA Biomedicals) followed by Alexa 488–conjugated goat by guest on September 23, 2021 synergistic interaction was also reported between SLAM haplo- anti-rat IgG (Jackson ImmunoResearch Laboratories). Color images type 2 and Yaa (8), we generated B cell–, myeloid cell–, and were obtained using laser scanning microscopy (Zeiss LSM 510). dendritic cell– (DC) specific FcgRIIb-deficient mice on pure B6 Estimation of the severity of the glomerular lesion background and introduced the Yaa locus to avoid the synergistic Histological score of kidney pathology was performed with PAS-stained effect of SLAM haplotype 2. Our analyses suggest that FcgRIIb kidney tissue sections. The severity of lesion was evaluated on a 0–4 deficiency on B cells and monocytes control B cell activation and score as follows: score 0, normal; score 1, mild glomerular cell prolifer- autoantibody responses via different but synergistic pathways in ation; score 2, moderate cell proliferation with increased mesangial matrix; the autoimmune-prone B6.FcgRIIb2/2.Yaa mice. score 3, severe proliferative changes with or without segmental glomer- ulosclerosis; score 4, glomerular damage with crescent formation and sclerosis. The average score of individual mice was calculated with scores Materials and Methods of 20 glomeruli. Mice Cell sorting To examine the effect of FcgRIIb deficiency on pure B6 genetic background, mice with floxed Fcgr2b alleles (FcgRIIbfl/fl) and with FcgRIIb knockout Gr-1+ and Gr-12 monocyte subsets were sorted using an autoMACS alleles (FcgRIIb2/2) were generated using a B6-derived BAC clone and Pro Separator (Miltenyi Biotec) and a BD FACSAria III cell sorter B6-derived embryonic stem cells, as described previously (13). Through (BD Biosciences). PBLs were stained with FITC-labeled anti-CD3 and crossing the FcgRIIbfl/fl mice with transgenic mice expressing Cre under -B220 mAbs and incubated with anti-FITC MicroBeads to remove control of the CD19 promoter (kindly provided by A. Waisman, University T cells and B cells using MACS Separators. The negatively selected cells Medical Center Mainz, Mainz, Germany) (19), the C/EBPa promoter were stained with FITC-labeled anti-CD11b and PE-labeled anti–Gr-1 mAbs, (kindly provided by I. Touw, Erasmus University Medical Center, Rotterdam, washed, resuspended in RPMI 1640 medium with 5% FCS and 1 mg/ml Netherlands) (20), or the CD11c promoter (kindly provided by B. Reizis, DAPI, and sorted using DAPI2 living cells. Columbia University, New York, NY) (21), B cell– (CD19Cre), myeloid cell– (C/EBPaCre), and DC- (CD11cCre)specificFcgRIIb-deficient mouse RNA sequencing strains on B6 background were generated. The FcgRIIbfl/fl,FcgRIIb2/2,and By sorting peripheral blood, four Gr-1+ monocyte subsets were obtained the above three cell type–specific conditional knockout strains were crossed 2 2 from two FcgRIIb / .Yaa mice (1 and 2), one CD19Cre.Yaa mouse, and with B6.Yaa mice to introduce the Yaa locus and tentatively designated as Cre 2 fl/fl 2/2 Cre Cre one C/EBPa .Yaa mouse, and five Gr-1 monocyte subsets from one FcgRIIb .Yaa,FcgRIIb .Yaa, CD19 .Yaa,C/EBPa .Yaa,and 2/2 Cre Cre g Yaa CD11c .Yaa, respectively. Experiments were conducted in accordance mouse of each of the following mouse strains: Fc RIIb . , CD19 . Yaa,C/EBPaCre.Yaa,CD11cCre.Yaa,andFcgRIIbfl/fl.Yaa. Sorted cells with approvals by the Institutional Animal Care and Use Committee at ∼ 3 5 Juntendo University School of Medicine. ( 1 10 ) were centrifuged and resuspended in the TRIzol reagent. cDNA libraries were generated using SMART-Seq v4 Ultra Low Input Flow cytometric analysis RNA Kit. Sequencing was performed on a HiSeq 1500 sequencer (Illumina) in a 50-bp single-end read mode. Data with the fragments per Single-cell suspensions were obtained from peripheral blood or spleen after kilobase of exon per million reads (FPKM) were used for further analysis the lysis of RBCs with ACK lysing buffer. FcgRIIb expression levels on after mapping of the sequence reads. RNA sequencing data analysis was The Journal of Immunology 3

2 2 2 performed as follows: nine (4 Gr-1+ and 5 Gr-1 ) datasets were generated compared with FcgRIIb / .Yaa mice at the age of 8 mo (Fig. 2D). + 2 from sorted Gr-1 and Gr-1 monocyte subsets as described above and Comparison of histopathological scores of the glomerular lesion subjected to open-source software Gene Set Enrichment Analysis (GSEA) between mouse strains (Fig. 2E) reflected the extent of ACR. On the version 2.2.2 (23, 24). We obtained detailed -ordered gene list (13,322 genes) in the analysis by comparing four Gr-1+ and 5 Gr-12 basis of the disease parameters mentioned above, the following three datasets. We cut off genes with the rank score below or equal to 1.5 and mouse strains were lupus prone in order of disease severity from above or equal to 21.5. Sets of genes left were subjected to high to low: FcgRIIb2/2.Yaa,CD19Cre.Yaa,andC/EBPaCre.Yaa, (GO) studies using protein analysis through evolutionary relationships whereas CD11cCre.Yaa and FcgRIIbfl/fl.Yaa mice were disease free. (PANTHER) list analysis (25, 26). RNA sequencing data have been deposited in the National Center for Biotechnical Information’s Gene Serum autoantibody levels, germinal center reaction, Expression Omnibus database (https://www.ncbi.nlm.nih.gov/geo/query/ acc.cgi?acc=GSE116757). and lymphocyte activation status In vitro cytokine production The serum levels of autoantibodies against dsDNA, chromatin, and RNP were highest in FcgRIIb2/2.Yaa mice. In CD19Cre.Yaa mice, 5 + 2 A total of 10 sorted Gr-1 or Gr-1 monocytes per well of 96-well round- these autoantibody levels were significantly higher than in m 2 2 bottom plates in 200 l of RPMI supplemented with 10% FCS was cul- FcgRIIbfl/fl.Yaa mice but lower than in FcgRIIb / .Yaa mice. tured with or without 3 mg/ml R837 (TLR7 agonist) or LPS 1 mg/ml Cre overnight, and production of IL-10, IL-1b, and B cell–stimulating Intriguingly, C/EBPa .Yaa mice showed autoantibody titers factor-3 (BSF-3) in culture supernatants was measured using commercially comparable to those in CD19Cre.Yaa mice. CD11cCre.Yaa mice available ELISA kits (IL-10: Abcam; ab46103, IL-1b: Abcam; ab100704, showed the same low levels of autoantibodies as observed in BSF-3: Lifespan Bioscience; LS-F8396). control FcgRIIbfl/fl.Yaa mice (Fig. 3A).

Statistical analysis Immunohistochemistry of spleen sections showed large clusters Downloaded from of PNA+ B cells in spleens from lupus-prone strains (Fig. 3B); Statistical analysis was performed using the statistical software package + StatView version 5.0. The significance of differences was examined using however, intriguingly, the distribution patterns of PNA B cells Cre Kaplan–Meier method for the incidence of proteinuria and survival rate. The was different between mouse strains. In C/EBPa .Yaa mice, + correlation was assessed using Spearman rank correlation (rs) analysis. Stu- PNA B cells were typically located in germinal centers (GCs) dent t test was used to compare two groups. When multiple groups were located in B cell follicles near the T cell zone. These GCs were compared, data were analyzed by one-way ANOVAwith the post hoc Tukey– located inside the lining of metallophilic macrophages positively http://www.jimmunol.org/ Kramer test. A p value , 0.05 was considered statistically significant. stained with anti–MOMA-1. In contrast, in FcgRIIb2/2.Yaa and CD19Cre.Yaa mice, PNA+ B cells were located not only in B cell Results area but also in T cell area around central arterioles and even Cell type–specific deficiency of FcgRIIb expression outside the lining of metallophilic macrophages. Both GC B cells The cell type–specific FcgRIIb deficiency was analyzed by flow and extrafollicular B cells contribute to pathogenic autoantibody 2/2 cytometry. Fig. 1 shows the histogram profiles of FcgRIIb ex- production (28–30). It has been shown that B6.FcgRIIb mice pression on blood B cells, monocytes, and neutrophils and splenic have increased spontaneous GC B cell responses (15) and that macrophages and DCs of the CD19Cre.Yaa, C/EBPaCre.Yaa, and B cell–intrinsic TLR7 signal contributes to GC development (31). CD11cCre.Yaa mouse strains. Profiles of the same cell types in Thus, it is feasible that the combined effect of Yaa mutation and by guest on September 23, 2021 FcgRIIb2/2.Yaa and in FcgRIIbfl/fl.Yaa mice were used as nega- the lack of FcgRIIb expression on B cells contributes to the for- tive and positive control, respectively. In CD19Cre.Yaa mice, mation of extrafollicular sites in addition to the GC formation. FcgRIIb was exclusively absent on B cells. In C/EBPaCre.Yaa Because it was surprising that FcgRIIb deficiency on myeloid Cre mice, FcgRIIb was absent on monocytes, neutrophils, macro- cells in C/EBPa .Yaa mice also induced B cell activation with phages and a majority of DCs. In CD11cCre.Yaa mice, a majority GC formation, we explored further the underlying molecular and of DCs lacked FcgRIIb expression, whereas expression on B cells, cellular mechanisms of this undefined regulatory pathway. monocytes, neutrophils, and macrophages was unaffected. First of all, we evaluated spleen cell populations from FcgRIIb2/2.Yaa mice at the age of 8 mo and from the four other Kidney disease and survival rate mouse strains at the age of 12 mo. In the three lupus-prone strains, We evaluated proteinuria by examining the urinary ACR. a marked splenomegaly was observed (Table I), and absolute FcgRIIb2/2.Yaa mice showed high ACR already at 6 mo of age numbers of B cells, T cells, and myeloid lineage cells were all (Fig. 2A). An ACR of 100 or more was regarded as positive increased compared with the disease-free strains (Supplemental proteinuria (27). The cumulative incidence of proteinuria in Table I). Consistent with the previous report in TLR7 transgenic FcgRIIb2/2.Yaa mice was increased up to 90% with a mortality lupus-prone mice (32), the B cell frequencies were decreased in rate of 86% at the age of 10 mo (Fig. 2B, 2C). In contrast, the three lupus-prone strains compared with the disease-free CD19Cre.Yaa mice developed proteinuria with later onset and strains, probably due to the relatively stronger increase of the lower incidence and showed lower mortality rate compared with myeloid cells (Table I). The frequencies of macrophages and DCs FcgRIIb2/2.Yaa mice. However, when compared with control of both B2202 and B220+ subsets were significantly increased in FcgRIIbfl/fl.Yaa mice, ACR was higher at 12 mo of age, and the all three lupus-prone strains, suggesting that the myeloid cell cumulative incidence of proteinuria was higher after 10 mo of expansion was not a myeloid cell–intrinsic effect of FcgRIIb de- age. Unexpectedly, C/EBPaCre.Yaa mice also showed proteinuria ficiency. The frequencies of PNA+-activated B cells, CD138+ with an incidence comparable to that in CD19Cre.Yaa mice, and plasma cells, and ICOS+CD4+-activated T cells were significantly ACRs were higher than in FcgRIIbfl/fl.Yaa mice at 12 mo of age increased in all three lupus-prone strains compared with control (Fig. 2A, 2B). CD11cCre.Yaa did not develop proteinuria by the FcgRIIbfl/fl.Yaa mice (Table I). age of 12 mo. To identify the possible cytokines involved in the B cell acti- At the age of 8 mo, FcgRIIb2/2.Yaa mice showed severe lupus vation in C/EBPaCre.Yaa mice, we further examined expression nephritis with marked cellular proliferation, matrix expansion, and levels of these cytokines using mRNA extracted from the whole thickening of capillary loops of glomeruli with marked IgG and C3 spleen. The result showed that IL-6 expression was upregulated in depositions. At the age of 12 mo, CD19Cre.Yaa and C/EBPaCre.Yaa FcgRIIb2/2.Yaa and CD19Cre.Yaa mice; however, there was no mice showed milder matrix expansion and IgG and C3 depositions increase in cytokines that could explain the activation of B cells in 4 FcgRIIb ON B CELLS AND MYELOID CELLS Downloaded from http://www.jimmunol.org/

FIGURE 1. Histogram showing the expression levels of FcgRIIb on indicated cell subsets in each strain of mice. FcgRIIb expression levels were analyzed at 6 mo of age using specific mAb against FcgRIIb (Ly17.2) in B220+ B cells, CD11b+Gr-12∼+ monocytes, and CD11b+Gr-1high neutrophils in by guest on September 23, 2021 peripheral blood and CD11b+ macrophages and CD11c+ DCs in the spleen. Shadow area bordered by the dotted line shows negative expression profile obtained in FcgRIIb2/2.Yaa mice. One representative experiment out of three is shown.

C/EBPaCre.Yaa mice (Supplemental Fig. 1). The heterogeneous C/EBPaCre.Yaa mice compared with CD19Cre.Yaa mice (Fig. 4B). populations in whole spleen may be not suitable to detect differ- These observations show that there is a positive association between ences in the levels of relevant cytokines in particular cell types. FcgRIIb deficiency on monocytes and the increased number of Thus, additional studies were required to explore the mechanism Gr-12 monocytes in the blood. of B cell activation in C/EBPaCre.Yaa mice. Detailed flow cytometric analysis revealed that Gr-12 but not Gr-1+ monocytes express the activating FcgRIV (Fig. 4C), sug- Peripheral leukocyte subsets gesting that Gr-12 monocytes are in more active stage than Gr-1+ We next examined the effect of myeloid cell–specific FcgRIIb de- monocytes. Moreover, Gr-1+ and Gr-12 monocytes were CCR2+ ficiency on the frequencies of myeloid cells in peripheral blood. and CCR22, respectively, and Gr-1+ monocytes and the majority PBLs from 8-mo-old mice were stained with mAbs against Gr-1 of Gr-12 monocytes were CX3CR1dull and CX3CR1+, respec- and CD11b and were analyzed by flow cytometry. In all strains, tively. These observations indicate that the Gr-1+ and Gr-12 whereas neutrophils showed the highest Gr-1 expression, mono- monocyte subset correspond to the classical and nonclassical + 2 cytes were divided into two populations, Gr-1 and Gr-1 subsets monocyte subset, respectively (34). (Fig. 4A, upper figures). To distinguish monocytes from neutrophils, To determine if the increased numbers of Gr-12 monocytes play a we further stained PBLs with mAb against M-CSF receptor CD115 role in B cell activation, the correlations between peripheral Gr-12 + and examined the CD11b/Gr-1 expression profiles of CD115 - monocyte frequencies and splenic PNA+-activated B cell frequencies gated cells (Fig. 4A, lower figures). Virtually all monocytes of were examined. Interestingly, we observed a statistically significant + 2 + both Gr-1 and Gr-1 subsets were included in these CD115 - positive correlation in FcgRIIb2/2.Yaa and C/EBPaCre.Yaa mice but gated cells as reported (33), and these gated cells did not include not in CD19Cre.Yaa mice (Fig. 5). No correlation was observed be- + + + b B220 B cells, NK1.1 NK cells, and MHC class II (I-A ) DCs tween the frequencies of peripheral Gr-1+ monocytes and splenic (Supplemental Fig. 2). No strain difference was observed in the PNA+ B cells in the three lupus-prone strains. Thus, it seems likely neutrophil frequencies, whereas the monocyte frequencies were that FcgRIIb deficiency in monocytes contributes to both an increase significantly higher in all three lupus-prone strains compared with in the number of Gr-12 monocytes and the activation of B cells. + disease-free strains (Fig. 4B). Intriguingly, although Gr-1 2 monocyte frequency was significantly higher in CD19Cre.Yaa mice Gr-1 monocytes have unique features for B cell activation compared with FcgRIIb2/2.Yaa and C/EBPaCre.Yaa mice, the Gr-12 To investigate the possible role of Gr-12 monocytes in B cell monocyte frequency was significantly higher in FcgRIIb2/2.Yaa and activation, we decided to compare the gene expression profiles The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 2. Development of severe lupus nephritis in FcgRIIb2/2.Yaa mice and the mild form of lupus nephritis in CD19Cre.Yaa and C/EBPaCre.Yaa mice. (A) Urinary ACR at indicated months of age in each strain of mice. ACR 100 was the cutoff point for positive proteinuria. The ratio of each mouse is plotted with mean and SEM. Statistical significance is shown. (B) Comparisons of cumulative incidence of proteinuria between the indicated strains of mice. The number of mice examined is shown in the parenthesis. The incidence in FcgRIIb2/2.Yaa mice was significantly higher than that in CD19Cre.Yaa mice after 6 mo of age, and the incidences in CD19Cre.Yaa and C/EBPaCre.Yaa mice were significantly higher than the incidence in FcgRIIbfl/fl.Yaa mice after 10 mo of age and at 12 mo of age, respectively. (C) Comparisons of survival rate in the indicated strains of mice. The number of mice examined is shown in the parenthesis. FcgRIIb2/2.Yaa mice showed statistically significant lower survival rate compared with that in CD19Cre.Yaa mice after 9 mo of age. (D) Representative histopathological findings of glomeruli with PAS staining and immunofluo- rescent staining for IgG and C3 deposition in the indicated strains of mice at 8 or 12 mo of age. Results are representative of those obtained from six mice in each strain. Scale bars, 50 mm. (E) The extent of glomerular lesions was evaluated using the arbitrary pathological score from 0 to 4 based on observations in PAS-stained tissue sections. The score of each mouse is plotted with the mean value. Statistical significance is shown. *p , 0.05, **p , 0.01, ***p , 0.001. between Gr-1+ and Gr-12 peripheral monocyte subsets from score below 21.5 considered as upregulated in Gr-12 monocytes mixed genotypes to look for consistent alternations across the (Supplemental Fig. 3). Based on these two gene sets, we per- different genotypes. We extracted total RNA from sorted over 95% formed PANTHER list analysis (25, 26) and obtained a list of pure Gr-1+ and Gr-12 peripheral monocyte subsets (Fig. 6A) and GO terms with fold enrichment score above 5 (Table II). Among compared the transcriptomes of each subset by RNA sequencing. As these terms, to search for genes potentially contributing to describedintheMaterials and Methods section, Gr-1+ monocyte B cell activation, we selected the term “positive regulation of subsets were obtained from two FcgRIIb2/2.Yaa mice (1 and 2), one cell activation” with a statistically significant value of p =0. CD19Cre.Yaa mouse, and one C/EBPaCre.Yaa mouse, four in total, and 0000342. From the list of this term (Table III), we found that the Gr-12 monocyte subsets from one mouse of each of the following expression levels of BSF-3, IL-10, and IL-1b, all of which have mouse strains: FcgRIIb2/2.Yaa,CD19Cre.Yaa,C/EBPaCre.Yaa, the potential to activate B cells, were upregulated predomi- CD11cCre.Yaa,andFcgRIIbfl/fl.Yaa, five in total. Using open-source nantly in Gr-12 monocytes (Fig. 6B, Table III). In addition, software GSEA (23, 24), we ranked 13,322 genes with a rank antiapoptotic Bcl2 and Bcl6 and DC markers (CD11c, CD83, order score by comparing the 4 Gr-1+ and 5 Gr-12 monocyte gene and Adamdec 1) were also upregulated in Gr-12 monocytes expression datasets. We then analyzed two distinct gene sets of the (Fig. 6B, Table III). ranked 13,322 genes: 1) 363 genes (2.7%) with GSEA rank order To confirm the upregulated expression of B cell–stimulating score above 1.5 considered as being upregulated expression in cytokines in Gr-12 monocytes on the protein level, sorted Gr-1+ monocytes and 2) 292 genes (2.2%) with GSEA rank order peripheral Gr-1+ and Gr-12 monocytes from 6- to 8-mo-old 6 FcgRIIb ON B CELLS AND MYELOID CELLS Downloaded from http://www.jimmunol.org/

FIGURE 3. Comparisons of serum levels of autoantibodies and histological findings of spleen between the indicated strains of mice. (A) Serum levels of autoantibodies were examined by ELISA at 8 mo. Measurement of IgG anti-dsDNA and anti-chromatin Abs was performed using anti-mouse g-chain– specific second Abs. Anti-RNP Abs were examined using commercially available ELISA kits, which detect total Ig classes. The number of mice examined is shown in the parenthesis. Mean and SEM are shown with statistical significance. *p , 0.05, **p , 0.01, ***p , 0.001. (B) Representative immu- nofluorescent staining of the spleen. Frozen spleen sections from FcgRIIb2/2.Yaa mice at 8 mo of age and the other strains of mice at 12 mo of age were by guest on September 23, 2021 triple-stained with a mixture of anti-CD4 and anti-CD8 mAbs, anti-B220 mAb, and PNA, or doubly stained with anti–MOMA-1 mAb and PNA. Results are representative of those obtained from six mice in each strain. Scale bars, 100 mm.

FcgRIIb2/2.Yaa mice were cultured for 24 h, and the cytokine stimulation was not sufficient to induce IL-1b secretioninour levels in culture supernatants were examined (Fig. 6C). With- system. Gr-12 monocytes produced a higher amount of IL-1b out stimulation, Gr-12 monocytes produced a higher amount of compared with Gr-1+ monocytes when stimulated with a com- BSF-3comparedwithGr-1+ monocytes; however, there were bination of R837 and LPS probably due to the increased stim- no detectable levels of IL-10 and IL-1b in both monocyte ulation of inflammasomes for the processing and secretion of subsets. When stimulated with TLR7 agonist R837, a higher IL-1b (35), although LPS stimulation alone was not suffi- amount of IL-10 was produced by Gr-12 monocytes, but this cient (Fig. 6C). Flow cytometric analysis showed that Gr-12

Table I. Comparisons of the spleen size and the frequencies of spleen cell subsets examined by flow cytometric analysis

Strain FcgRIIb2/2.Yaa CD19Cre.Yaa C/EBPaCre.Yaa CD11cCre.Yaa FcgRIIbfl/fl.Yaa Age (no. of mice) 8 mo (n =6) 12mo(n = 11) 12 mo (n =8) 12mo(n =7) 12mo(n =6) Spleen weight (g) 0.58 6 0.09a/a 0.48 6 0.06a/a 0.43 6 0.11a/a 0.12 6 0.01 0.14 6 0.01 Cell counts (3108) 5.35 6 1.4b/b 4.48 6 0.5b/b 4.32 6 0.9a/a 1.35 6 0.2 1.42 6 0.1 Frequencies (%) B220+ B cells/total cells 33.8 6 5.4a/b 35.7 6 2.8b/c 40.6 6 5.9a/b 52.1 6 3.5 60.3 6 2.3 PNA+ B/total B cells 2.6 6 1.12/a 3.7 6 1.62/a 5.6 6 1.4a/b 0.9 6 0.3 0.3 6 0.1 CD138+ plasma/total cells 2.9 6 0.5a/a 2.2 6 0.22/a 2.1 6 0.42/a 1.0 6 0.2 0.9 6 0.2 CD3+ T cells/total cells 17.4 6 0.5 16.5 6 1.2 21.5 6 2.6 17.2 6 1.6 22.8 6 1.2 ICOS+CD4+ T cells/total CD4+ T cells 30.2 6 2.8a/b 31.5 6 4.9a/b 36.6 6 4.4b/c 18.5 6 2.0 12.9 6 1.7 Foxp3+CD25+CD4+Treg/CD4+T cells 19.8 6 4.4 21.8 6 7.1 17.4 6 3.5 23.1 6 1.6 20.3 6 2.4 CD11b+ CD11c2 macrophages/total cells 23.9 6 3.6b/b 28.3 6 3.7c/c 17.3 6 2.32/a 9.8 6 1.2 6.7 6 0.8 CD11c+B2202 DCs/total cells 7.8 6 2.6c/c 5.3 6 0.9c/b 5.8 6 0.7c/b 1.5 6 0.3 2.0 6 0.4 CD11c+B220+ DCs/total cells 2.3 6 0.6a/a 2.8 6 0.7c/b 3.8 6 0.4c/c 0.7 6 0.1 0.8 6 0.3 Values are the mean 6 SEM. The age and the number of mice examined are shown. Boldface indicates a statistically significant difference compared with the finding in CD11cCre.Yaa or FcgRIIbfl/fl.Yaa mice in ANOVA (versus CD11cCre.Yaa/versus FcgRIIbfl/fl.Yaa: ap , 0.05, bp , 0.01, cp , 0.001. 2, NS. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 4. Comparisons of phenotypes and frequencies of peripheral neutrophils and monocytes between the indicated strains of mice at 8 mo of age. (A) Upper figures show the representative flow cytometric profiles for CD11b/Gr-1 expression in PBLs. CD11b+ cells are composed of three populations, namely Gr-1high neutrophils and Gr-1+ and Gr-12 monocyte subsets. Lower figures depict the CD11b/Gr-1 expression profiles of gated CD115+ monocytes, clearly showing CD11b+Gr-1+ and CD11b+Gr-12 subsets. Results are representative of those obtained from 8 to 11 mice in each strain. (B) The frequencies of neutrophils, monocytes, and Gr-1+ and Gr-12 monocyte subsets per total cells are compared between the indicated strains. The number of mice examined is shown in the parenthesis. Mean and SEM are shown with statistical significance. *p , 0.05, **p , 0.01, ***p , 0.001. (C) Representative flow cytometric profiles for Gr-1/FcgRIV, Gr-1/CCR2, and Gr-1/CX3CR1 expressions in gated CD115+CD11b+ monocytes. Results are representative of those obtained from 8 to 11 mice in each strain. 8 FcgRIIb ON B CELLS AND MYELOID CELLS

FIGURE 5. Correlation between the frequencies of peripheral Gr-1+ or Gr-12 monocytes and the frequencies of splenic PNA+ B cells in 8-mo-old Downloaded from 2/2 Cre Cre FcgRIIb .Yaa mice and 12-mo-old CD19 .Yaa and C/EBPa .Yaa mice. Spearman rank correlation coefficient (rs) analysis was used for statistical analysis, and a significant association is shown by p value. but not Gr-1+ monocytes expressed CD11c (Fig. 6D). These monocytes, Gr-12 monocytes might contribute to B cell activation/ observations suggest that Gr-1+ and Gr-12 monocytes differ- differentiation through the higher expression of B cell–stimulating ently contribute to immune regulation. Compared with Gr-1+ cytokines. http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 6. Upregulated expressions of transcriptomes and in Gr-12 monocyte subset compared with Gr-1+ subsets at 6–8 mo of age. (A)CD11b/Gr-1 expression profile of over 95% pure sorted Gr-1+ and Gr-12 monocyte subsets. Representative data obtained using peripheral blood from 8-mo-old C/EBPaCre.Yaa mice are shown. (B) Comparisons of gene expression levels (FPKM) of relevant immune regulatory molecules identified in PANTHER list analysis and in GSEA (Table III) between four Gr-1+ (white bars: from the top, FcgRIIb2/2.Yaa no. 1, FcgRIIb2/2.Yaa no. 2, CD19Cre.Yaa,C/EBPaCre.Yaa)andfiveGr-12 (black bars: from the top, FcgRIIb2/2.Yaa,CD19Cre.Yaa, C/EBPaCre.Yaa,CD11cCre.Yaa,FcgRIIbfl/fl.Yaa) monocyte gene expression datasets. (C) Levels of in vitro production of BSF-3, IL-10, and IL-1b were compared between Gr-1+ and Gr-12 monocyte subsets. Sorted Gr-1+ and Gr-12 subsets from 6- to 8-mo- old FcgRIIb2/2.Yaa mice were cultured for 24 h with or without indicated stimulations, and levels were examined using ELISA. Data (mean and SEM of triplicates) are from one out of three independent experiments with similar results. Statistical significances are shown. *p , 0.05, **p , 0.01. (D)Histograms showing the expression of CD11c on Gr-12 but not Gr-1+ monocytes in 6-mo-old FcgRIIb2/2.Yaa mice. ND, not detectable. The Journal of Immunology 9

Table II. GO terms with fold enrichment score over five in biologic processes of PANTHER list analysis

Term Count Fold Enrichment p Value Gr-1+ monocyte subset GO:0002685 regulation of leukocyte migration 14 5.35 0.00479 GO:0098840 protein transport along microtubule 7 11.11 0.0337 GO:0043902 positive regulation of multiorganism process 12 5.28 0.034 Gr-12 monocyte subset GO:0050670 regulation of lymphocyte proliferation 17 6.29 0.000027 GO:0050671 positive regulation of lymphocyte proliferation 14 8.16 0.0000276 GO:0032944 regulation of mononuclear cell proliferation 17 6.2 0.0000311 GO:0032946 positive regulation of mononuclear cell proliferation 14 8.03 0.0000334 GO:0050867 positive regulation of cell activation 20 5.13 0.0000342 GO:0070663 regulation of leukocyte proliferation 17 6 0.0000543 GO:0070665 positive regulation of leukocyte proliferation 14 7.68 0.0000582 GO:0050727 regulation of inflammatory response 19 5.19 0.0000695 GO:1903037 regulation of leukocyte cell–cell adhesion 19 5.1 0.0000923 GO:0032649 regulation of IFN-g production 12 9.11 0.000111 GO:1903039 positive regulation of leukocyte cell–cell adhesion 15 6.55 0.000135 GO:0022409 positive regulation of cell–cell adhesion 16 5.95 0.000168 GO:0051251 positive regulation of lymphocyte activation 17 5.15 0.000476 GO:0050866 negative regulation of cell activation 14 6.42 0.000526 Downloaded from GO:0050777 negative regulation of immune response 12 7.27 0.00123 GO:0050870 positive regulation of T cell activation 13 5.99 0.00331 GO:0042102 positive regulation of T cell proliferation 10 8.08 0.00527 GO:0002695 negative regulation of leukocyte activation 12 6.18 0.00677 GO:0051250 negative regulation of lymphocyte activation 11 6.61 0.0101 GO:0043393 regulation of protein binding 13 5.4 0.0105 GO:0002757 immune response-activation signal transduction 12 5.71 0.0152 http://www.jimmunol.org/ GO:0043112 receptor metabolic process 9 7.87 0.0237 GO:2001239 regulation of extrinsic pathway 7 11.19 0.0318 GO:0002764 immune response-regulating signaling pathway 12 5.31 0.0318 GO:0032729 positive regulation of IFN-g production 8 8.71 0.0404

Discussion the observation in adoptive transfer experiments that Gr-12 but + Yaa autoimmune-prone B6 mice deficient for FcgRIIb spontane- not Gr-1 monocytes have the capacity to home to noninflamed ously develop lethal lupus nephritis. In this study, we showed that tissues, including splenic marginal zone, and that a fraction of 2 FcgRIIb deficiency exclusively on the B cells of these mice causes Gr-1 monocytes can acquire a DC phenotype (45). In Yaa mice, by guest on September 23, 2021 only mild disease, indicating that another FcgRIIb-deficient cell is duplication of TLR7 renders B cells hyper-responsive to RNA- required for lethal disease. By using DC-specific FcgRIIb- containing endogenous ligands (50). Consistently, the introduction deficient mice, we could exclude DCs because these mice of Yaa into nonautoimmune B6 mice induces autoantibody pro- stayed disease free. Surprisingly, panmyeloid-specific FcgRIIb- duction, although the titer is too low for the development of lupus deficient Yaa mice developed mild lupus comparable to that in nephritis (13, 51). With our findings in this study, we propose the B cell–specific FcgRIIb-deficient mice, suggesting that FcgRIIb following hypothesis: in Yaa mice lacking FcgRIIb expression on on another myeloid cell than the mature DC modulates B cell myeloid cells (C/EBPaCre.Yaa mice), the subtle amount of ICs tolerance in Yaa mice. composed of RNA-containing autoantigens and autoantibodies Although our current experiments do not provide direct evi- induces FcR g–mediated maturation of monocytes from the Gr-1+ 2 2 dence, based on our own data presented in this paper and a large to the Gr-1 subset. Subsequently, a fraction of activated Gr-1 amount of consistent data from the literature, we hypothesize that monocytes migrates to the marginal zone in the spleen, activating the Gr-12 nonclassical monocyte subset is the most promising B cells through the production of B cell–stimulating cytokines. candidate FcgRIIb-expressing myeloid cell to modulate B cell However, we could not confirm the upregulation of IL-10 and tolerance. In Yaa-associated lupus-prone mice, the development IL-1b expression in the RT-PCR analysis of total spleen of of lupus nephritis is associated with monocytosis, an FcR g– C/EBPaCre.Yaa mice. RT-PCR analysis on sorted splenocytes dependent expansion of the monocytic compartment consisting might overcome that problem. Moreover, further studies are 2 mainly of Gr-12 monocytes (36–38). By RNA sequencing anal- required to confirm the migratory capacity of Gr-1 monocytes ysis of sorted peripheral blood monocytes, we could show that into the spleen in our model. three genes encoding the cytokines BSF-3, IL-10, and IL-1b, CD19Cre.Yaa and C/EBPaCre.Yaa mice showed milder disease 2 2 which potentially can activate B cells (39–44), were upregulated compared with FcgRIIb / .Yaa mice. Besides the synergistic ef- in Gr-12 monocytes compared with the classical Gr-1+ mono- fect of B cell– and myeloid cell–specific FcgRIIb deficiency in the cytes. This was confirmed at the protein level by culturing isolated development of high autoantibody titers and severe lupus in 2 2 monocytes in vitro. Our data show that the antiapoptotic factors FcgRIIb / .Yaa mice, we cannot exclude that liver sinusoidal Bcl2 and Bcl6 were upregulated in Gr-12 monocytes in keeping endothelial cell– and mesangial cell–specific FcgRIIb deficiency with the observations that Gr-1+ monocytes are short-lived, may also contribute to the severe pathology as suggested in pre- whereas Gr-12 monocytes have a longer half-life (45). These vious reports. FcgRIIb on liver sinusoidal endothelial cells can data also show that Gr-12 monocytes are committed to differen- clear small ICs from circulation to prevent systemic IC-mediated tiate into DCs because they showed higher expression of the inflammation (52). Mesangial cells secrete several inflammatory following markers known to be expressed by DCs: CD11c, CD83 chemokines, including MCP-1, upon binding of IC to activating (46, 47), and Adamdec1 (48, 49). This notion is consistent with FcgR on these cells. This is augmented in the absence of FcgRIIb 10

Table III. List of genes of GO term “positive regulation of cell activation” in PANTHER list analysis and of genes of interests in GSEA

Gene Expression Levels (FPKM)

2 2 2 2 2 2 Rank FcgRIIb / .Yaa (#1) FcgRIIb / .Yaa (#2) CD19Cre .Yaa C/EBPaCre .Yaa FcgRIIb / .Yaa CD19Cre .Yaa C/EBPaCre .Yaa CD11cCre .Yaa FcgRIIbfl/fl .Yaa 2 2 2 2 2 Gene Score Gr-1+ Monocytes Gr-1+ Monocytes Gr-1+ Monocytes Gr-1+ Monocytes Gr-1 Monocytes Gr-1 Monocytes Gr-1 Monocytes Gr-1 Monocytes Gr-1 Monocytes GO:0050867 positive regulation of cell activation Il10 21.583 0.364883 1.70289 0 0.461777 13.0253 11.6279 7.22919 1.85181 11.5267 Nck2 21.585 0.729915 1.24093 1.15489 0.554245 2.21752 1.97058 2.1036 2.63205 1.647 Sox15 21.605 0.143502 0 0 0.108965 1.08992 2.07382 1.36967 0.804941 0.991642 Cd81 21.626 2.34615 4.42735 10.9049 2.63352 32.1523 70.7594 36.068 37.6006 25.0742 Hsp90aa1 21.687 28.3184 39.9201 24.1752 35.4087 54.5401 57.1228 85.2978 73.4759 60.5371 Il1b 21.718 45.8317 176.129 32.6455 101.957 516.528 226.088 615.368 518.757 406.494 Card11 21.814 0.377305 0.0480236 0.528554 0.260453 2.00598 1.53793 2.23217 2.22635 0.812665 Itgal (CD11a) 21.838 91.6238 88.3686 99.4485 116.402 213.834 321.379 370.572 397.798 200.147 Havcr2 21.974 5.72176 9.48499 8.57173 9.09876 25.571 52.1458 33.3925 41.6736 23.111 Blm 22.04 0.702299 0.321852 0.675757 0.556423 1.78375 1.51479 1.49558 1.87555 1.08417 Bcl6 22.048 27.1048 30.1522 33.982 20.6475 63.3787 74.2401 78.9934 61.7777 56.3069 Rasgrp1 22.108 4.40488 7.6613 4.13564 4.71442 26.32 40.2866 37.9124 33.5791 14.6208 Pdgfb 22.111 0.832268 0.137088 0.141451 2.08177 6.02373 3.86406 13.3811 9.29762 13.5807 Zbtb16 22.147 2.03288 2.05647 0.438348 2.8637 10.796 6.34148 13.9488 11.4071 8.61533 Hes1 22.424 9.02887 15.651 8.28159 14.1198 42.2066 58.9078 77.1306 94.4866 85.2059

Clcf1 (BSF-3) 22.561 0.218273 1.52801 0 1.32593 7.79174 5.58085 11.6478 8.39559 10.3429 Fcg Spn 22.822 32.2679 27.4359 9.21182 25.6624 112.064 93.8872 156.022 112.626 121.348 Pdcd1lg2(PD-L2) 23.285 0.166982 0.23379 0.120615 0.158493 6.40469 7.47151 11.7723 6.95798 6.34646 CELLS MYELOID AND CELLS B ON RIIb Bcl2 23.422 3.74033 7.22683 2.77483 2.59596 42.3839 28.4635 31.1264 38.8734 40.7786 Cd274 (PD-) 23.556 4.96108 11.4022 7.86196 5.38029 64.8313 60.2782 70.2581 71.5748 46.2129 GSEA Itgax (CD11c) 20.792 1.56112 4.10127 1.0305 1.57166 36.1704 37.9001 65.9558 300.851 33.8354 Cd83 21.279 1.45021 2.80392 1.13066 1.678 14.0043 2.7125 38.2596 50.3574 29.3144

Adamdec1 22.333 0.364246 0.42498 0 0.230485 30.0165 8.59324 16.4612 17.9019 21.5748

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2 2 (53). Comparison of the disease severity between FcgRIIb / .Yaa 13. Boross, P., V. L. Arandhara, J. Martin-Ramirez, M. L. Santiago-Raber, Cre aCre Yaa F. Carlucci, R. Flierman, J. van der Kaa, C. Breukel, J. W. Claassens, M. Camps, and double CD19 /C/EBP . mice will uncover the role of et al. 2011. The inhibiting Fc receptor for IgG, FcgRIIB, is a modifier of au- these additional cell type–specific FcgRIIb deficiencies in the toimmune susceptibility. J. Immunol. 187: 1304–1313. development of severe lupus in FcgRIIb2/2.Yaa mice. 14. Kanari, Y., A. Sugahara-Tobinai, H. Takahashi, M. Inui, A. Nakamura, S. Hirose, and T. Takai. 2014. Dichotomy in FcgRIIB deficiency and autoimmune-prone TLR7 is expressed on B cells, plasmacytoid DCs, and con- SLAM haplotype clarifies the roles of the Fc receptor in development of auto- ventional DCs (9, 54, 55). Using the Cre recombinase system in and glomerulonephritis. BMC Immunol. 15: 47. the lupus model of the floxed TLR7 transgenic B6 mice on SLAM 15. Soni, C., P. P. Domeier, E. B. Wong, Shwetank, T. N. Khan, M. J. Elias, S. L. Schell, A. E. Lukacher, T. K. Cooper, and Z. S. Rahman. 2015. Distinct and haplotype 2 background, it has been shown that normalization of synergistic roles of FcRIIB deficiency and 129 strain-derived SLAM family TLR7 on conventional DCs, rather than on B cells and plasma- proteins in the development of spontaneous germinal centers and autoimmunity. cytoid DCs, is effective to reduce the severe lupus nephritis (32, J. Autoimmun. 63: 31–46. 16. Sharp, P. E., J. Martin-Ramirez, S. M. Mangsbo, P. Boross, C. D. Pusey, 55). This indicates that in this lupus model, the immunological I. P. Touw, H. T. Cook, J. S. Verbeek, and R. M. Tarzi. 2013. FcgRIIb on myeloid tolerance breakdown induced by SLAM haplotype 2 is modified cells and intrinsic renal cells rather than B cells protects from nephrotoxic ne- mainly by the upregulated TLR7 signal in conventional DCs (55). phritis. J. Immunol. 190: 340–348. 17. Li, F., P. Smith, and J. V. Ravetch. 2014. Inhibitory Fcg receptor is required for the It would be interesting to analyze the cell type–specific contri- maintenance of tolerance through distinct mechanisms. J. Immunol. 192: 3021–3028. bution of TLR7 signaling in the development of lupus nephritis 18. Yilmaz-Elis, A. S., J. M. Ramirez, P. Asmawidjaja, J. van der Kaa, A. M. Mus, observed in our lupus-prone mice. M. D. Brem, J. W. Claassens, C. Breukel, C. Brouwers, S. M. Mangsbo, et al. 2 2014. FcgRIIb on myeloid cells rather than on B cells protects from collagen- Like mouse Gr-1 monocytes, the human counterpart induced arthritis. J. Immunol. 192: 5540–5547. CD14lowCD16+ monocytes secrete high amounts of IL-1b in a 19. Rickert, R. C., J. Roes, and K. Rajewsky. 1997. B lymphocyte-specific, Cre- mediated mutagenesis in mice. Nucleic Acids Res. 25: 1317–1318. Downloaded from TLR7-TLR8-MyD88–dependent manner (56). In addition, serum 20. Wo¨lfler, A., A. A. Danen-van Oorschot, J. R. Haanstra, M. Valkhof, C. Bodner, levels of anti-dsDNA Abs highly correlate with the percentage of E. Vroegindeweij, P. van Strien, A. Novak, T. Cupedo, and I. P. Touw. 2010. nonclassical monocytes in patients with SLE (57). Further studies Lineage-instructive function of C/EBPa in multipotent hematopoietic cells and early thymic progenitors. Blood 116: 4116–4125. are required to shed on the role of monocytes in the patho- 21. Caton, M. L., M. R. Smith-Raska, and B. Reizis. 2007. Notch-RBP-J signaling genesis of human SLE to clarify whether they are potential thera- controls the homeostasis of CD8- dendritic cells in the spleen. J. Exp. Med. 204: peutic targets in this disease. 1653–1664. 22. Zhang, D., K. Fujio, Y. Jiang, J. Zhao, N. Tada, K. Sudo, H. Tsurui,

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Supplemental Table 1 Comparisons of cell numbers of spleen cell subsets estimated by flow cytometric analysis

Strain FcγRIIb-/-.Yaa CD19Cre.Yaa C/EBPαCre.Yaa CD11cCre.Yaa FcγRIIbfl/fl.Yaa

age (No. of mice) 8M (n=6) 12M (n=11) 12M (n=8) 12M (n=7) 12M (n=6) Cell number (x106) Total B220+ B cells 157.5 ± 22.8*/* 148.9 ± 15.4*/* 156.2 ± 16.0**/** 67.4 ± 8.5 85.6 ± 7.1 PNA+B cells 3.4 ± 1.3*/* 6.6± 3.2*/* 8.3 ± 2.8*/* 0.6 ± 0.2 1.2 ± 0.6 CD138+ plasma cells 14.2 ± 2.7*/* 15.1 ± 3.4**/** 11.6 ± 3.8*/* 1.1 ± 0.3 1.8 ± 0.3 Total CD3+ T cells 96.1 ± 26.1**/** 71.6 ± 8.4**/* 95.0 ± 20.5**/** 21.3 ± 2.9 33.8 ± 1.4 ICOS+CD4+ T cells 15.0 ± 2.7*/* 15.0 ± 4.3*/* 15.5 ± 5.1*/* 2.3 ± 0.2 3.2 ± 0.8 Foxp3+CD25+CD4+Treg cells 10.6 ± 2.2*/* 11.6 ± 2.0*/* 11.8 ± 1.9*/* 3.5 ± 0.3 4.7 ± 0.7 CD11b+ CD11c- macrophages 107.6 ± 19.3***/*** 116.6 ± 16.7***/*** 87.5 ± 10.8*/* 13.2 ± 1.6 9.5 ± 1.2 CD11c+B220- DCs 40.5 ± 12.0***/*** 24.7 ± 4.1**/** 25.8 ± 4.2**/** 2.1 ± 0.4 2.8 ± 0.6 CD11c+B220+ DCs 12.2 ± 2.7**/** 12.5 ± 3.0**/** 16.4 ± 1.7***/*** 0.9 ± 0.2 1.2 ± 0.4

Values are the mean ± SEM. The age and the number of mice examined are shown in the upper row. Boldface represents the statistically significant difference compared to the finding in CD11cCre.Yaa or FcγRIIbfl/fl.Yaa mice in ANOVA (vs. CD11cCre.Yaa/vs. FcγRIIbfl/fl.Yaa in right-upper: * P < 0.05, ** P < 0.01, *** P < 0.001).

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Supplemental Figure 1 Quantitative real-time PCR (qRT-PCR) analysis of cytokine mRNA expression levels in spleen from FcγRIIb-/-.Yaa mice at 8 months of age and the other strains of mice at 12 months of age. Total RNA was isolated from spleen and first-stranded cDNA was synthesized using an oligo(dT)-primer with Superscript II First-Strand Synthesis kit (Invitrogen). The cDNA product was used for each qRT-PCR sample. The data were normalized to β-actin reference. Primer pairs used are as follows (forward and reverse); BAFF (5’-GGCAGGTACTACGACCATCTC-3', 5'-TGGGCCTTTTCTCACAGAAGT-3’), IL-1β (5’-GCCCATCCTCTGTGACTCAT-3’, 5’-AGGCCACAGGTATTTTGTCG-3’), IL-4 (5’-CCTCACAGCAACGAAGAACA-3’, 5’-AAGTTAAAGCATGGTGGCTCA-3’), IL-6 (5’-GACAAAGCCAGAGTCCTTCAGAGAG-3’, 5’-CTAGGTTTGCCGAGTAGATCTC-3’), IL-10 (5’-CCAAGCCTTATCGGAAATGA-3’, 5’-TGGCCTTGTAGACACCTTGG-3’), IL-21 (5’-CAGGGTTTGATGGCTTGAGT-3’, 5’-GTCCCAAACTACCGAACTCA-3’), IFNα (5’-ATGGCTAGRCTCTGTGCTTTCCT-3’, 5’-AGGGCTCTCCAGAYTTCTGCTCTG-3’), IFNγ (5’-AAGACAATCAGGCCATCAGC-3’, 5’-ATCAGCAGCGACTCCTTTTC-3’), and β-actin (5’-TGGGTATGGAATCCTGTGG-3’, 5’-GTACTTGCGCTCAGGAGGAG-3’). The -∆∆CT quantity was normalized using the formula of the 2 method. Value of FcγRIIbfl/fl.Yaa mice was designated as 1, and values of other strains of mice were evaluated as fold change compared with the values in FcγRIIbfl/fl.Yaa mice. Mean and SEM of 6 mice in each strain are shown. The IL-6 expression level was significantly higher in FcγRIIb-/-.Yaa and CD19Cre.Yaa mice compared to FcγRIIbfl/fl.Yaa mice (*P < 0.05).

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Supplemental Figure 2 Phenotypes of peripheral monocytes in FcγRIIb-/-.Yaa mice at 8 months of age. Monocytes were highly positive for CD115 expression. These CD115+ cells were gated and examined the expression levels of Gr-1, B220, NK1.1, and I-Ab molecules. The CD115+ gated monocytes are divided into Gr-1- and Gr-1+ subsets and do not express B cell-, NK cell-, and DC-specific markers.

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Supplemental Figure 3 Gene expression plots and selected gene population of Gr-1+ and Gr-1- monocyte datasets. GSEA rank score in y-axis and gene expression value in FPKM of individual gene is plotted. Data were obtained from sorted Gr-1+ (filled) and Gr-1- (open or cross) monocytes of FcγRIIb-/-.Yaa (black circle), CD19Cre.Yaa (orange triangle), C/EBPαCre.Yaa (red diamond), CD11cCre.Yaa (gray rectangle), and FcγRIIbfl/fl.Yaa (cross) mice. Rank scores reflect how different Gr-1+ and Gr-1- monocyte gene expression levels of a particular gene are. Take an example: GAB1 gene contains 9 plots (red thin lines) aligning at the rank score of 4.58879 (dotted black line). In the alignment of 9 plots, 5 Gr-1- datasets are fairly separated from 4 Gr-1+ datasets. In this regard, orange-color dotted area in the graph contains Gr-1+ plots with higher gene expression values. On the other hand, green-color dotted area contains Gr-1- plots with lower gene expression values. In contrast, blue- and red-color dotted areas are reversed in negative GSEA rank score. We draw 2 red bold lines as cut-offs in GSEA rank score. Sets of genes with GSEA rank score above 1.5 and that below -1.5 are selected and grouped into ‘up-regulated in Gr-1+ monocytes’ and ‘up-regulated in Gr-1- monocytes’ gene sets, respectively. These sets of genes are subjected to PANTHER list analysis.

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