Type II but Not Type I IFN Signaling Is Indispensable for TLR7-Promoted Development of Autoreactive B Cells and Systemic Autoimmunity This information is current as of September 26, 2021. Sathi Babu Chodisetti, Adam J. Fike, Phillip P. Domeier, Harinder Singh, Nicholas M. Choi, Chelsea Corradetti, Yuka Imamura Kawasawa, Timothy K. Cooper, Roberto Caricchio and Ziaur S. M. Rahman

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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 © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published January 3, 2020, doi:10.4049/jimmunol.1901175 The Journal of Immunology

Type II but Not Type I IFN Signaling Is Indispensable for TLR7-Promoted Development of Autoreactive B Cells and Systemic Autoimmunity

Sathi Babu Chodisetti,* Adam J. Fike,* Phillip P. Domeier,*,1 Harinder Singh,† Nicholas M. Choi,* Chelsea Corradetti,‡ Yuka Imamura Kawasawa,x,{ Timothy K. Cooper,‖,2 Roberto Caricchio,‡ and Ziaur S. M. Rahman*

TLR7 is associated with development of systemic lupus erythematosus (SLE), but the underlying mechanisms are incompletely under- stood. Although TLRs are known to activate type I IFN (T1IFN) signaling, the role of T1IFN and IFN-g signaling in differential regulation of TLR7-mediated Ab-forming cell (AFC) and germinal center (GC) responses, and SLE development has never been directly investigated. Using TLR7-induced and TLR7 overexpression models of SLE, we report in this study a previously unrecognized Downloaded from indispensable role of TLR7-induced IFN-g signaling in promoting AFC and GC responses, leading to autoreactive B cell and SLE development. T1IFN signaling in contrast, only modestly contributed to autoimmune responses and the disease process in these mice. TLR7 ligand imiquimod treated IFN-g reporter mice show that CD4+ effector T cells including follicular helper T (Tfh) cells are the major producers of TLR7-induced IFN-g. Transcriptomic analysis of splenic tissues from imiquimod-treated autoimmune-prone B6.Sle1b mice sufficient and deficient for IFN-gR indicates that TLR7-induced IFN-g activates multiple signaling pathways to regulate TLR7-promoted SLE. Conditional deletion of Ifngr1 gene in peripheral B cells further demonstrates that TLR7-driven autoimmune http://www.jimmunol.org/ AFC, GC and Tfh responses and SLE development are dependent on IFN-g signaling in B cells. Finally, we show crucial B cell- intrinsic roles of STAT1 and T-bet in TLR7-driven GC, Tfh and plasma cell differentiation. Altogether, we uncover a nonredundant role for IFN-g and its downstream signaling molecules STAT1 and T-bet in B cells in promoting TLR7-driven AFC, GC, and SLE development whereas T1IFN signaling moderately contributes to these processes. The Journal of Immunology, 2020, 204: 000–000.

ystemic lupus erythematosus (SLE) is a debilitating au- Ab-forming cell (AFC) and follicular germinal center (GC) toimmune disease mediated by Abs reactive to DNA- and responses are traditionally thought to be involved mainly in anti- S RNA-associated self-antigens. Although extrafollicular pathogen Ab responses, AFCs and GCs can also develop spon- by guest on September 26, 2021 taneously in the absence of purposeful immunization or overt *Department of Microbiology and Immunology, Pennsylvania State University infection (1, 2). Dysregulation in spontaneous AFC (Spt-AFC) and College of Medicine, Hershey, PA 17033; †J. Craig Venter Institute, Rockville, Spt-GC responses in SLE patients and SLE-prone mice promotes MD 20850; ‡Rheumatology Division, Temple University, Philadelphia, PA 19122; xDepartment of Pharmacology, Institute for Personalized Medicine, Pennsylvania State autoreactive B cell survival and pathogenic autoantibody pro- University College of Medicine, Hershey, PA 17033; {Department of Biochemistry and duction (3–8). However, the mechanisms that drive Spt-AFC and Molecular Biology, Institute for Personalized Medicine, Pennsylvania State University spontaneous GC (Spt-GC) responses which can lead to the de- College of Medicine, Hershey, PA 17033; and ‖Department of Comparative Medicine, Pennsylvania State University College of Medicine, Hershey, PA 17033 velopment of autoreactive B cells, autoantibodies and SLE are 1Current address: Immunology Program, Benaroya Research Institute, Seattle, WA. incompletely understood. Genome-wide association studies revealed multiple risk alleles 2Current address: Integrated Research Facility, National Institute of Allergy and Infectious Diseases, Frederick, MD. in the TLR pathway that are strongly associated with SLE de- ORCIDs: 0000-0003-4801-4973 (N.M.C.); 0000-0002-8638-6738 (Y.I.K.); 0000- velopment (9, 10). Overexpression and overactivity of TLR7 are 0002-1379-1118 (R.C.); 0000-0001-8431-9681 (Z.S.M.R.). associated with development of SLE in humans and in mouse Received for publication September 25, 2019. Accepted for publication November models (11–13). Recent collaborative works also have highlighted 18, 2019. the significance of TLR7 in promoting extrafollicular double This work was supported by National Institutes of Health RO1AI091670 to Z.S.M.R., negative B cell populations involved in SLE pathogenesis (8). Lupus Research Alliance Award 548931 to Z.S.M.R., and the Finkelstein Memorial Award to S.B.C. TLR7 gene escape from X chromosome inactivation enhances the B cell response to TLR7 stimulation (14). Studies in the MRL-lpr The sequences presented in this article have been submitted to the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE141433) under model highlight the opposing inflammatory and regulatory roles accession number GSE141433. for TLR7 and TLR9, respectively, in extrafollicular autoreactive Address correspondence and reprint requests to Dr. Ziaur S.M. Rahman, Pennsylvania B cell responses and SLE development (15). Using a variety of State University College of Medicine, 500 University Drive, Hershey, PA 17033-0850. SLE mouse models, we and others discovered that TLR7 signaling E-mail address: [email protected] specifically in B cells promoted SLE development through for- The online version of this article contains supplemental material. mation of Spt-AFCs and Spt-GCs (11, 16, 17). These data high- Abbreviations used in this article: AFC, Ab-forming cell; ANA, anti-nuclear Ab; GC, germinal center; GN, glomerulonephritis; IMQ, imiquimod; PAS, periodic light the importance of TLR7 signaling in promoting autoimmune acid-Schiff; RNA-seq, RNA sequencing; SA, streptavidin; SLE, systemic lupus AFC and GC responses, leading to the development of autoreac- erythematosus; SmRNP, Smith/ribonucleoprotein; Spt-AFC, spontaneous AFC; tive B cells, autoantibody production and SLE; however, mecha- Spt-GC, spontaneous GC; Tfh, follicular helper T; T1IFN, type I IFN. nisms of TLR7-mediated autoimmunity and SLE development are Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 not clearly defined.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1901175 2 REGULATION OF TLR7-DRIVEN AUTOREACTIVE B CELL DEVELOPMENT

Type I IFN (T1IFN) and type II (IFN-g) IFN signaling are both studies were conducted at Penn State Hershey Medical Center in ac- implicated in SLE (9, 18). An IFN-a signature is associated with cordance with the guidelines approved by our Institutional Animal human SLE (19, 20). Lupus-prone mice deficient in IFN-aR have Care and Use Committee. Animals were housed in a specific pathogen- free barrier facility. reduced anti-nuclear Abs (ANAs) and renal disease (21). Exoge- nous IFN-a treatment accelerates the SLE phenotype in female Imiquimod treatment 3 (NZW NZB) F1 mice with an early development of anti- For epicutaneous imiquimod (IMQ) treatment, 5% IMQ cream (Glenmark dsDNA Abs and nephritis (22). The administration of IFN-a in Pharmaceuticals) was applied on the ears of mice 3 times weekly for lupus-prone mice also leads to enhanced short-lived AFC and GC 4–12 wk, depending on the experiments as previously described (38, 39). responses (23), and an altered B cell selection in the GCs (24). We For PCR array and RNA-seq sample preparation, mice were treated for 4 wk. To study the systemic autoimmune responses, such as AFC, GC, and others recently showed a B cell–intrinsic role of T1IFN sig- follicular helper T (Tfh), and Ab responses, mice were treated for 8 wk. To naling in loss of tolerance and autoreactive B cell development in evaluate immune complex deposition and kidney pathology, mice were the AFC and GC pathways in lupus-prone mice (25, 26). SLE treated for 12 wk (three times a wk for 8 wk, followed by once a week for patients also showed an increased production of IFN-g (27, 28), another 4 wk). and blockade of IFN-g normalizes IFN regulated gene expression Flow cytometry in SLE patients (29). MRL/lpr and NZW/NZBF1 lupus-prone mice deficient in IFN-g have reduced autoantibody production Flow cytometric analysis of total mouse splenocytes was performed using the following Abs: B220-BV605 (RA3-6B2), CD4-AF700 (RMP4-5), CD44- and renal disease (30–35). We and others discovered a B cell– allophycocyanin (IM7), CD62L-PECy7 (MEK-14), PD1-PE (29F.1A12), intrinsic requirement of IFN-g signaling in Spt-AFC and Spt-GC NK1.1-biotin (PK136), Ly6G-PB (1A8), and MHCII-PE-Cy7 (M5/114.15.2) responses and SLE development (4, 36). These data indicate that (BioLegend); GL7-FITC (GL-7), CD95- PeCy7, CXCR5-biotin (2G8), Downloaded from both T1IFN and IFN-g signaling contribute to autoreactive B cell IFN-gR-biotin (GR20), STAT1-PE (1/STAT1), CD11b-AF700 (M1/70), and CD11c-FITC (HL3) (BD Biosciences); Ly6C-allophycocyanin (HK1.4), development in the AFC and GC pathways. However, the mech- PDC-PE (eBio927), and CD8a (clone 53–6.7) (eBioscience). All cells were anisms and relative contribution of T1IFN and IFN-g signaling in stained with fixable viability dye-eFluor780 (Invitrogen) prior to surface TLR7-mediated AFC, GC, and autoimmunity development have staining. Stained cells were analyzed using the BD LSR II flow cytometer never been directly investigated using a TLR7-induced model, (BD Biosciences). Data were acquired using FACSDiva software (BD Bio- sciences) and analyzed using FlowJo software (Tree Star). especially when T1IFN signaling is believed to be strongly as- http://www.jimmunol.org/ sociated with TLR signaling and SLE development. Immunofluorescence and ANA staining T1IFN and IFN-g signaling mediate signals through STAT1. Mouse spleens or kidneys were embedded in OCT compound and snap Interestingly, we previously reported a B cell–intrinsic require- frozen over liquid nitrogen. Five-micrometer-thin sections were cut on a ment of STAT1 in the Spt-GC responses (36) which we also cryostat, mounted on ColorFrost Plus microscope slides (Thermo Fisher showed dependent on TLR7 signaling in B cells (16). However, Scientific), and fixed in cold acetone for 20 min. The following Abs and how STAT1 downstream of T1IFN and IFN-g signaling in B cells reagents were used for immunofluorescence staining of mouse spleen sections for GCs: PE–anti-CD4 (GK1.5; BioLegend), FITC-GL7 (RA3- controls TLR7-mediated AFC and GC responses, promoting de- 6B2; BD Biosciences), and allophycocyanin–anti-IgD (11-26c2a; BD velopment of autoreactive B cells and SLE, remain unanswered. Biosciences). Kidney sections were stained for C3 using FITC–anti-C3 In this study, we used TLR7-induced and -overexpression models (ICL Laboratories) or Biotin–anti-IgG (Jackson ImmunoResearch), by guest on September 26, 2021 of SLE to dissect the relative contribution of T1IFN and IFN-g followed by streptavidin (SA)–PE. ANA reactivity was detected by signaling in driving autoimmune AFC and GC responses, pro- indirect immunofluorescence staining of HEp-2 cell slides using sera from indicated mice at a 1:50 dilution and probed with FITC-rat anti- moting development of autoreactive B cells, autoantibody pro- mouse k (H139-52.1). The images of stained spleen and kidney sec- duction, and SLE. We found that IFN-g and STAT1 signaling in tions were captured using the Leica DM4000 fluorescence microscope B cells played an indispensable role in TLR7-promoted AFC and and analyzed using a Leica application suite–advanced fluorescence GC responses and SLE development, whereas global T1IFN sig- software (LAS-AF; Leica Microsystems). For the measurement of the GC area, a total of at least 10 randomly selected GCs (GL-7+)per naling only moderately contributed to these processes. Using a mouse spleen section from at least five mice of each genotype were TLR7-induced SLE model we show that TLR7 not only increased measured for total area (square micrometers) using the LAS-AF expression of T1IFN genes but also the IFN-g gene in splenic quantitation tool. tissue compared with untreated mice. Our data from IFN-g re- Kidney histopathology porter mice and RNA sequencing (RNA-seq) analysis indicate that TLR7-driven and T cell–produced IFN-g activates multiple sig- Kidneys from 6-mo-old mice were fixed in 10% neutral buffered for- malinandembeddedinparaffin.Kidneysectionswerecutat3-and naling pathways to regulate TLR7-promoted SLE. Together, our 6-mm thickness for periodic acid-Schiff (PAS) and H&E staining, re- data show that TLR7 drives autoreactive B cell development in the spectively. All images were obtained with an Olympus BX51 micro- AFC and GC pathways and promotes SLE by activating T1IFN scope and DP71 digital camera using cellSens Standard 1.12 imaging and IFN-g signaling in which IFN-g signaling plays an indis- software (Olympus America, Center Valley, PA). Two pathologists pensable role, which cannot be overcome by the activation of blinded to the genotype of the mice examined kidney sections. One kidney section per mouse was evaluated: each glomerulus was exam- other downstream pathways of TLR7 signaling. ined at 4003 magnification and scored from 0 (normal) to 4 (severe) based on glomerular size and lobulation, presence of karyorrhectic Materials and Methods nuclear debris, capillary basement membrane thickening, and the de- gree of mesangial matrix expansion and mesangial cell proliferation Mice (40). H&E sections were scored for overall glomerular damage and C57BL/6J (B6), B6.129S7-Ifngr1tm1Agt/J (IFN-gR12/2), B6(Cg)-Ifnar1tm1.2Ees/J interstitial inflammation as described (41). 2/2 tm3.1Lky (IFN-aR1 ), C.129S4(B6)-Ifng /J mice (IFN-g eYFP), ELISPOT assay B6.129S(Cg)-Stat1tm1Dlv/J (STAT12/2), B6.129S-Stat1tm1Mam/Mmjax (STAT1fl/fl), C57BL/6N-Ifngr1tm1.1Rds/J (IFN-gRfl/fl), B6.129-Tbx21tm2Srnr/J ELISPOT assays were performed as previously described (25, 36). Briefly, (T-betfl/fl), and B6.SB-Yaa/J(B6.yaa) mice were originally purchased from splenocytes in RPMI 1640 containing 10% FBS were plated at a con- The Jackson Laboratory and bred in house. The B6.Sle1b mice (congenic for centration of 1 3 106 cells per well onto multiscreen 96-well filtration the Sle1b sublocus) were described previously (37). B6.Sle1bYaa mice were plates (Millipore) coated with salmon sperm dsDNA (Invitrogen), nucle- generatedbycrossingB6.YaamicetoB6.Sle1bmice.B6.CD23Cre osome (histone from Sigma Aldrich plated on a layer of dsDNA), or (B6.Cg-Tg(Fcer2a-cre)5Mbu, provided by Dr. Meinrad Busslinger, Smith/ribonucleoprotein (SmRNP; Arotec Diagnostics). Serially diluted were crossed to B6.Sle1b autoimmune mice in house. All animal (1:2) cells were incubated for 12 h at 37˚C. dsDNA-, nucleosome-, and The Journal of Immunology 3 smRNP-specific AFCs were detected by biotinylated anti-k Ab (Invi- B6.Sle1b mice (Sle1b-IMQ) displayed more splenocytes com- trogen) and SA–alkaline phosphatase (Vector Laboratories) or alkaline pared with untreated control counterparts (Supplemental Fig. 1A). phosphatase-conjugated anti-mouse IgG (Molecular Probes). Plates Overall percentage of B220+ BcellsandCD4+ T cells was not were developed using the Vector Blue Alkaline phosphatase Substrate Kit III (Vector Laboratories). ELISPOTs were enumerated and ana- significantly different in untreated and treated B6 and B6.Sle1b lyzed using a computerized ELISPOT plate imaging/analysis system mice (Supplemental Fig. 1B, 1C), but the numbers of B cells and (Cellular Technology). CD4+ T cells were higher in Sle1b-IMQ mice than three other ELISA groups (Supplemental Fig. 1D, 1E). In addition, Sle1b-IMQ mice had much higher percentage of splenic B220+GL7hiCD95hi GC Serum autoantibodies were measured using standard ELISA protocols as B cells and CD4+CD44hiPD-1hiCXCR5hi Tfh cells than untreated described (3). Briefly, total IgG autoantibody titers were measured in ELISA plates coated with salmon sperm dsDNA, nucleosome or smRNP B6.Sle1b mice (Fig. 1A, 1B). Flow cytometry gating strategy for and detected with biotinylated secondary Ab followed by SA–alkaline GC B cells and Tfh cells is shown (Supplemental Fig. 1F). Ele- phosphatase (Vector Laboratories). The plates were developed using vated GC and Tfh responses in Sle1b-IMQ mice were strongly p-nitrophenyl phosphate, disodium salt (Thermo Fisher Scientific) associated with increased numbers of dsDNA- and nucleosome- l substrates for alkaline phosphatase and read at 405 nm on Synergy H1 specific splenic AFCs, serum ANA reactivity, and autoantibody (BioTek Instruments). titers (Fig. 1C–F). Treated B6 mice (B6-IMQ) had a 2-fold RNA-seq higher GC and Tfh responses than untreated B6 mice but no RNA was extracted from spleen tissues by TRIzol as per the manufacturer’s autoantibody production. We then treated 8-wk-old mice with instructions (Ambion). OD values of extracted RNA were measured using IMQ for 12 wk and found that Sle1b-IMQ mice had more severe

NanoDrop to confirm an A260:A280 ratio above 1.9. RNA integrity nephritis than B6.Sle1b untreated mice and B6-treated mice Downloaded from number was measured using BioAnalyzer (Agilent Technologies). RNA (Fig. 1G, 1H). These elevated responses in Sle1b-IMQ mice 6000 Pico Kit to confirm RIN above 7. The cDNA libraries were pre- pared using the Ovation RNA-Seq System V2 (NuGEN Technologies, San were accompanied by an increased expression of IFN gene sig- Carlos, CA) as per the manufacturer’s instructions. The unique barcode natures in the splenic tissue (Fig. 1I), and elevated IFN-aRand sequences were incorporated in the adaptors for multiplexed high- IFN-gR expression on B cells upon TLR7 stimulation in vitro throughput sequencing. The final product was assessed for its size dis- (Fig. 1J). Thus, Sle1b-IMQ mice represent a strong TLR7-driven tribution and concentration using BioAnalyzer High Sensitivity DNA Kit systemic autoimmunity model with autoimmune phenotypes http://www.jimmunol.org/ (Agilent Technologies) and then loaded onto TruSeq SR v3 flow cells on an Illumina HiSeq 2500 (Illumina) and run for 100 cycles using a paired- (elevated AFC, GC, Tfh, ANA, and nephritis) closely resembling read recipe (TruSeq SBS Kit v3; Illumina) according to the manufacturer’s human SLE. instructions. Illumina CASAVA pipeline (released version 1.8; Illumina) was used to obtain demultiplexed sequencing reads (fastq files) passed the IFN-g, but not T1IFN, signaling is critical for TLR7-driven default purify filter. Additional quality filtering used FASTX-Toolkit development of autoreactive B cells and SLE pathogenesis (http://hannonlab.cshl.edu/fastx_toolkit) to keep only reads that have at least 80% of bases with a quality score of 20 or more (conducted by fastq_ Although the induction of T1IFNs by TLR signaling and their roles quality_filter function) and reads left with 10 bases or longer after being in SLE pathogenesis have been well described (44–46), we know end trimmed with a base quality score of 20 (conducted by fastq_quality_ little about how IFN-g signaling may contribute to TLR-mediated trimmer function). A bowtie2 index was built for the mouse reference SLE autoimmunity. Therefore, using the TLR7-induced Sle1b- by guest on September 26, 2021 genome (GRCm38) using bowtie version 2.1.0. The RNA-seq reads were mapped using Tophat version 2.0.9 (42) supplied by Ensembl annotation IMQ model, in this study we determined the relative contribu- file; GRCm38.78.gtf. Gene expression values were computed using tion of T1IFN and IFN-g signaling in TLR7-mediated SLE fragments per kilo base per million mapped reads values. Differential autoimmune responses. Although IMQ-treated B6.Sle1b mice gene expression was determined using Cuffdiff tool, which is available in deficient in IFN-aR(Sle1b.IFN-aR2/2-IMQ)hadlowerGC Cufflinks version 2.2.1 supplied by GRCm38.78.gtf. Normalization was performed via the median of the geometric means of fragment counts B cell and Tfh cell responses than Sle1b-IMQ mice, these re- across all libraries, as described (43). Statistical significance was sponses were diminished in treated B6.Sle1b mice deficient in 2 2 assessed using a false discovery rate threshold of 0.05 to determine the IFN-gR(Sle1b.IFN-gR / -IMQ) (Fig. 2A, 2B). The frequency differentially abundant genes. Genes that were expressed at $1.5-fold of GCs between Sle1b-IMQ and Sle1b.IFN-aR2/2-IMQ mice was differences between control and test samples were selected for fur- similar, but GC sizes were smaller in Sle1b.IFN-aR2/2-IMQ mice ther analysis. The heatmaps of selected genes were generated via the than Sle1b-IMQ control mice, whereas a negligible number of very pheatmap package in R using Euclidean distance and Ward clustering 2 2 method. small GCs was observed in Sle1b.IFN-gR / -IMQ mice (Fig. 2C, The accession number for the RNA-seq data reported in this paper is in 2D). In addition, the number of dsDNA- and nucleosome-specific the Gene Expression Omnibus: GSE141433 (https://www.ncbi.nlm.nih. splenic AFCs (Fig. 2E), serum dsDNA-, nucleosome- and SmRNP- gov/geo/query/acc.cgi?acc=GSE141433). specific Abs (Fig. 2F), and serum ANA reactivity (Fig. 2G) were Statistical analysis moderately reduced in Sle1b.IFN-aR2/2-IMQ mice, whereas they 2/2 The p values were calculated using unpaired, nonparametric, Mann– were diminished in Sle1b.IFN-gR -IMQ mice compared with Whitney, Student t test (for comparison of two groups), or one-way or Sle1b-IMQ control mice. Consistent with systemic autoimmune two-way ANOVA, with a follow-up Tukey multiple-comparison test B and T cell responses and serum ANA reactivity, immune (for comparison of more than two groups) or two-way ANOVA, with a complex (IC) deposition (IgG and C3) and severe glomerulo- follow-up Sidak multiple-comparison test (for comparison of two groups). , , , nephritis (GN) in B6.Sle1b-IMQ mice were moderately reduced The p values are as follows: *p 0.05, **p 0.01, ***p 0.001, and 2/2 ****p , 0.0001. GraphPad Prism 6 software was used. in Sle1b.IFN-aR -IMQ mice, whereas they were completely absent in Sle1b.IFN-gR2/2-IMQ mice (Fig. 2H–J). Next, we determined the role of IFN-g signaling in SLE Results autoimmunity development in B6.Sle1b mice overexpressing TLR7-driven AFC, GC, and systemic autoimmunity model TLR7 by breeding Sle1b.IFN-gR2/2 mice to B6.Yaa mice that To identify the mechanisms and determine the role of T1IFN and carry an extra copy of TLR7 to generate Sle1bYaaIFN-gR2/2 IFN-g signaling in differential regulation of TLR7-mediated au- mice. B6.Sle1b male mice expressing Yaa (Sle1bYaa) developed toimmunity, we have used a TLR7-induced SLE model in which splenomegaly, which was reversed to a level observed in B6.Sle1b we treated 8 wk old female mice with TLR7 agonist IMQ as males in the absence of IFN-gR (Fig. 3A). Elevated GC B cell described (38). IMQ-treated B6 (B6-IMQ) and autoimmune-prone (Fig. 3B, 3D, 3E) and Tfh cell (Fig. 3C) responses in Sle1bYaa 4 REGULATION OF TLR7-DRIVEN AUTOREACTIVE B CELL DEVELOPMENT

FIGURE 1. IMQ-induced mouse model of SLE. Eight- to ten-week-old C57BL/6 (B6) and B6.Sle1b mice were epicutaneously treated on the ears with TLR7 ligand IMQ for 8 wk. The per- centages of splenic B220+GL7hiCD95hi GC B cells (A) and CD4+CD44hiPD- 1hiCXCR5hi Tfh cells (B) from these mice were obtained through flow cyto- metric analysis. (C and D) The number of dsDNA- and nucleosome-specific AFCs in the spleens was quantified by ELISPOT assay. These data represent Downloaded from four mice of each group. ANA reactiv- ity (E) and serum titers of dsDNA-, nucleosome-, and SmRNP-specific Abs (F) in these mice (six mice of each group) are shown. Representative images of kidney sections stained with PAS (G)andGNscore(H)areshown.(I) http://www.jimmunol.org/ B6.Sle1b mice were untreated or treated with IMQ for 4 wk, and the spleen tis- sues were processed for IFNs and IFN receptors PCR array. Arrows in the scatter plots represent upregulated genes upon IMQ treatment. (J) Expression of IFN-gRandIFN-aR on B cells before and after stimulation with R848 for 48 h. These data represent two to three ex- periments and each symbol indicates an by guest on September 26, 2021 individual mouse (A, B,andJ)ora glomerulus (H). Statistical analysis was performed by one-way (A, B,and H)ortwo-way(C, D,andF)ANOVA, with a follow-up Tukey multiple- comparison test or unpaired, nonpara- metric Mann-Whitney Student t test (J). Error bars in (A), (B), and (H) rep- resent mean 6 SD, and error bars in (F), (C), and (D) represent mean 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. ns, not significant.

mice were significantly reduced in Sle1bYaaIFN-gR2/2 mice. pathogenesis, whereas T1IFN signaling only moderately contrib- Similarly, the number of dsDNA- and nucleosome-specific splenic utes to these responses. AFCs (Fig. 3F), serum dsDNA-, nucleosome- and SmRNP- specific Abs (Fig. 3G), and serum ANA reactivity (Fig. 3H) IFN-g regulates gene expression of several cellular pathways were significantly reduced in Sle1bYaaIFN-gR2/2 mice compared to promote AFC and GC responses in TLR7-accelerated SLE with Sle1bYaa control mice. Finally, IC deposition in the kidney To investigate how IFN-g may promote autoreactive B cell and the developed GN in Sle1bYaa mice were also diminished in development into the AFC and GC pathways in TLR7- Sle1bYaaIFN-gR2/2 mice (Fig. 3I–K). Our data from TLR7- accelerated SLE, we analyzed the gene transcripts in spleens induced and -overexpression SLE models indicate that IFN-g of IMQ-treated B6.Sle1b and Sle1b.IFN-gR2/2 mice by RNA- signaling plays a nonredundant role in TLR7-promoted AFC and seq. Clustering of differentially expressed genes showed a GC responses and subsequent autoantibody production and SLE distinct transcriptional profile in Sle1b.IFN-gR2/2-IMQ mice The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 2. Differential roles of type I and II IFNs in TLR7-mediated AFC, GC, and Tfh responses and SLE development. Representative flow cytometry plots (A) and percentage (B) of B220+GL7hiCD95hi GC B cells and CD4+CD44hiPD-1hiCXCR5hi Tfh cells in IMQ treated B6.Sle1b, Sle1b.IFN-aR2/2 and Sle1b.IFN-gR2/2 female mice. (C) Representative spleen sections stained with GC B cell marker GL7 (green), naive B cell marker IgD (blue), and T cell marker CD4 (red). (D) Number of GCs per 53 field (top panel) and GC areas (bottom) were measured in 10 mice of each genotype. (E) Numbers of dsDNA- and nucleosome-specific splenic AFCs in these mice were quantified by ELISPOT assay. dsDNA-, nucleosome- and (Figure legend continues) 6 REGULATION OF TLR7-DRIVEN AUTOREACTIVE B CELL DEVELOPMENT compared with B6.Sle1b-IMQ control mice. We found that 130 role of IFN-g signaling in B cells in TLR7-mediated AFC, GC, and and 213 genes were up and downregulated, respectively, in Tfh responses and the development of autoreactive B cells, auto- Sle1b.IFN-gR2/2-IMQ mice (p , 0.05; fold change $1.5; antibodies, and lupus nephritis. Fig. 4A). Functional classification of the differentially expressed Crucial B cell–intrinsic roles of STAT1 and T-bet in TLR7- genes with gene ontology analysis revealed many biologi- driven GC, Tfh, and Ab responses, which cannot be overcome cal processes or the pathways that were downregulated (Fig. 4B) by potential activation of the other TLR7 pathways or upregulated (Fig. 4C) in the absence of IFN-gR signal- ing. Genes differentially regulated between B6.Sle1b-IMQ and We next asked how much of a role STAT1 that functions down- B6.Sle1b.IFN-gR2/2-IMQ splenic tissues were associated with stream of IFN-g signaling played in TLR7-driven GC, Tfh, and gene transcription regulation (Fig. 4D), ubiquitination/proteasome IgG Ab responses under a scenario in which several pathways complex (Fig. 4E), cell proliferation and migration (Fig. 4F), (NF-kB, IRF5/7, and P38/MAPK) can be potentially activated by autoimmunity (Fig. 4G), inflammation (Fig. 4H), cellular and the strong in vivo TLR7 stimulation. We first treated B6 and 2/2 oxidative stress (Fig. 4I), and cellular metabolism (Fig. 4J). These B6.STAT1 mice with IMQ and found that STAT1 is absolutely results indicate that IFN-g signaling promotes TLR7-mediated required for the development of TLR7-driven GC B cell, Tfh cell, autoimmunity by regulating multiple biological processes. and plasma cell responses (Fig. 7A–C). Next, by using conditional knockout mice with peripheral B cells deficient in STAT1 + CD4 T cells including Tfh produce TLR7-induced IFN-g (STAT1fl/flCD23Cre+), we found the requirement of B cell–intrinsic Considering the increased expression of the Ifn-g gene in splenic STAT1 in TLR7-mediated GC, Tfh, plasma cell, and IgG Ab re- tissue (Fig. 1I) and regulation of multiple signaling pathways by sponses (Fig. 7D–F). Consistent with these data, B cells stimu- Downloaded from IFN-g signaling (Fig. 4) in IMQ-treated mice, we next determined lated with TLR7 ligand had much higher STAT1 expression than the cellular source of IFN-g in TLR7-mediated autoimmunity. We unstimulated B cells (Fig. 7G). Similar to the increased STAT1 treated IFN-g–eYFP reporter mice with IMQ for 6 wk and found expression, T-bet downstream of STAT1 was also upregulated in that CD4+ T cells were the major producers of IFN-g, although B cells stimulated with TLR7 ligand (Fig. 7H). To solidify the CD8+ T cells and NK cells produced this cytokine to a certain importance of B cell–intrinsic T-bet in TLR7 driven GC, Tfh, and + hi fl/fl Cre+ extent (Fig. 5A). Of the CD4 CD44 effector T cells, a higher Ab responses, we treated T-bet CD23 mice where peripheral http://www.jimmunol.org/ frequency of pre-Tfh and Tfh cells produced IFN-g than non-Tfh B cells are deficient in T-bet. We observed a crucial B cell– cells upon IMQ treatment (Fig. 5B–D). Dendritic cells, macro- intrinsic role of T-bet in TLR7-mediated GC, Tfh, and IgG Ab phages, eosinophils, monocytes, and neutrophils did not produce responses (Fig. 7I–K). These data indicate that STAT1 and T-bet IFN-g in these mice (Fig. 5E). These data together with increased downstream of TLR7 and IFN-g signaling play a significant role IFN-gR expression on B cells upon TLR7 stimulation ex vivo in TLR7-driven GC, Tfh, and IgG Ab responses, which cannot (Fig. 1J) point to an important role for TLR7 in regulating the be overcome by other pathways potentially activated by TLR7 IFN-g–IFN-gR pathway in B cells by inducing T cell IFN-g stimulation. production and upregulating IFN-gR on B cells.

Discussion by guest on September 26, 2021 Absolute requirement of IFN-g signaling in B cells in Cellular and molecular mechanisms by which TLR7 over- regulating TLR7-mediated AFC, GC, and lupus expression or overactivity promotes autoreactive B cell develop- nephritis development ment in the extrafollicular AFC and follicular GC pathways that are To determine how TLR7-induced and T cell–produced IFN-g activated in SLE remain largely unknown. In this study, using the may signal in B cells for regulating autoimmune AFC, GC, and TLR7-induced and TLR7-overexpression models of SLE, as well Tfh responses and promoting SLE development, we condi- as an IFN-g reporter mouse model, we demonstrate that TLR7 tionally deleted the Ifngr1 gene in peripheral B cells in overactivation drives development of autoreactive B cells in both B6.Sle1b mice (designated Sle1bIFN-gRfl/fl.CD23Cre+). IMQ- the AFC and GC pathways and promotes SLE by driving pre- treated Sle1bIFN-gRfl/fl.CD23Cre+ mice did not show spleno- dominantly IFN-g production by T cells and mediating IFN-g megaly that was observed in IMQ-treated Sle1bIFN-gRfl/fl signaling in B cells. TLR7-driven and T cell–produced IFN-g control mice (Fig. 6A, 6B). Sle1bIFN-gRfl/fl.CD23Cre+ mice controls gene transcription in multiple cellular pathways im- also had markedly reduced B220+GL-7hiCD95hi GC B cell and portant for immune cell survival, proliferation, metabolism, and CD4+CD44hiCXCR5hiPD-1hi Tfh cell responses, including re- autoimmunity. duced frequency and size of GCs, compared with Sle1bIFN-gRfl/fl Several groups, including ours, previously identified a B cell– mice (Fig. 6C–G). Sle1bIFN-gRfl/fl.CD23Cre+ mice also had di- intrinsic role of TLR7-MyD88 signaling in promoting autoim- minished serum ANA reactivity (Fig. 6H), reduced serum auto- mune AFC and GC responses, leading to SLE pathogenesis antibody titers (Fig. 6I), and splenic autoantibody-producing in the mouse models (5, 11, 16, 17, 47). We and others also AFCs (Fig. 6J). Dampened AFC, GC, and Tfh responses in reported B cell–intrinsic roles of T1IFN and IFN-g signaling in Sle1bIFN-gRfl/fl.CD23Cre+ mice were strongly associated with di- regulating SLE-associated AFC, GC, and autoantibody re- minished IC deposition in the kidney and with significantly im- sponses in SLE-prone mice (4, 25, 36). Although TLR7 signal- proved kidney inflammation such as GN, interstitial nephritis, and ing is believed to drive T1IFN production and activation of vasculitis score (Fig. 6K–M). These data highlight the nonredundant T1IFN signaling in SLE (19, 45, 48, 49), how TLR7-driven

SmRNP-specific serum IgG titers (F) and serum ANA reactivity (G) were measured by ELISA and HEp-2 assays, respectively. Data in (F) and (G) represent six mice of each genotype. (H) Representative kidney sections from these mice were stained with anti-C3 (green) and anti-mouse IgG (red). Representative PAS-stained kidney sections (I) and GN score (J) from all three genotypes are shown. These data represent three experiments and each symbol indicates an individual mouse (B and E) or a GC (D) or a glomerulus (J). Statistical analysis was performed by one-way (B, D, E, and J) or two-way (F) ANOVA, with a follow-up Tukey multiple-comparison test. Error bars in (B), (D), (E), and (J) represent mean 6 SD, and error bars in (F) represent mean 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. ns, not significant. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 3. Essential role of IFN-g in the Yaa mouse model of SLE. Representative images of spleen size (A), the percentages of B220+GL7hiCD95hi GC B cells (B), and CD4+CD44hiPD-1hiCXCR5hi Tfh cells (C) are shown. Histological images of splenic GCs (D) stained for GC B cell marker, GL7 (green), mature B cell marker, IgD (blue), and T cell marker, CD4 (red) and GC areas (E) are shown. Number of splenic dsDNA- and nucleosome-specific AFCs (F), serum Ab titers against dsDNA, nucleosome and SmRNP (G), and serum ANA reactivity (H) are illustrated. Data in (G) and (H) represent 7–10 mice of each group. Representative images of kidney sections stained with anti-C3 (green) and anti-IgG (red) (I). Representative images of PAS stained kidney sections (J) and GN score (K). These data were derived from 6- to 8-mo-old B6.Sle1b, Sle1bYaa and SlebYaaIFN-gR2/2 mice and represent two to three experiments. Each symbol represents a mouse (B–F) or a glomerulus (K). Statistical analysis was performed by one-way (B, C, E, F, and K) or two-way (G) ANOVA, with a follow-up Tukey multiple-comparison test. Error bars in (B), (C), (E), (F), and (K) represent mean 6 SD, and in error bars in (G) represent mean 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001.

IFN-g production and activation of IFN-g signaling in B cells may kidney pathology. Using IFN-g reporter mice, we further estab- promote TLR7-mediated autoimmune AFC and GC responses and lished that TLR7 induced IFN-g production primarily by CD4+ SLE development has previously not been addressed. Using the T cells, including pre-Tfh and Tfh. Together, our data demonstrate TLR7-induced SLE model and B cell–specific conditional dele- that TLR7-driven and T cell–produced IFN-g act nonredundantly tion of the Ifngr1 gene, we found that IFN-g signaling in B cells to activate IFN-g signaling in B cells, promoting development of was essential for TLR7-mediated AFC, GC, and Tfh responses autoreactive B cells and SLE pathogenesis. and GN development, whereas global T1IFN signaling deficiency Although MRL/lpr and NZW/NZBF1 lupus-prone mice defi- moderately reduced these responses and was associated with less cient in IFN-g or treated with anti–IFN-g blocking therapy had 8 REGULATION OF TLR7-DRIVEN AUTOREACTIVE B CELL DEVELOPMENT Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 4. Transcriptome and gene ontology analysis of IMQ-treated Sle1b.IFN-gR2/2 and B6.Sle1b splenic tissue RNA. B6.Sle1b and Sle1b.IFN-gR2/2 mice were treated with IMQ for 4 wk and the splenic tissues were processed for RNA-seq. (A) Heat map showing the relative expression pattern of the top

343 differentially expressed genes between the groups. Fold-change values were visualized as log10 (FC). Gene ontology and pathway analysis for the genes that were downregulated (B) or upregulated (C) in Sle1b.IFN-gR2/2-IMQ mice (FC . 2 and p value ,0.05). (D–J) Heat maps display differentially expressed genes that were categorized based on their functions in particular biological processes (FC . 1.5 and p value ,0.05). Hierarchical-clustering analysis was conducted for the transcript levels of the differentially expressed genes (Ward clustering algorithm and Euclidean distance measure; n =3, each sample was pooled from four mice of each genotype). reduced autoantibody production and renal disease (30–35), the autoreactive B cell expansion in the GC pathway was not directly role of IFN-g signaling in autoreactive B cell development in tested in these studies using a TLR7-induced SLE mouse model either the extrafollicular or the GC pathways in these mice was not (4, 36). In this study, we find a significantly reduced number of tested. David Rawlings’ and our groups previously discovered the autoantibody-producing AFCs and dampened GC responses in role of IFN-g signaling in regulating SLE-associated GC re- the spleens of TLR7-induced SLE-prone mice. Our current data sponses; however, the link between TLR7 and IFN-g signaling in highlight the importance of B cell–intrinsic IFN-g signaling The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 5. CD4+ T cells are the major IFN-g producers upon TLR7 stimulation. Three- to four-month-old B6 (non-eYFP) and eYFP-IFN-g reporter (B6.eYFP) mice were left untreated (UT) or treated with IMQ for 4 wk, and splenocytes were analyzed for eYFP expressing cells by flow cytometry. (A) Bar diagrams show the percentage of eYFP+ (IFN-g+) total CD4+, CD8+, and NK1.1+ cells. These data represent six mice per group. (B) Flow cytometry gating strategy for CD4+CD44hi effector T cells that are subdivided into CD4+PD-1loCXCR5lo non-Tfh, CD4+PD-1intCXCR5int pre-Tfh, and CD4+PD-1hiCXCR5hi Tfh. (C) Representative flow cytometry dot plots from indicated mouse strains show frequency of eYFP-expressing (IFN-g+) cells in non-Tfh, pre-Tfh, Tfh, and CD4 effector cells. (D) Bar diagrams show the percentage of IFN-g+ cells in non-Tfh, pre-Tfh, and Tfh cells in UT and IMQ- treated B6.eYFP mice. These data represent five mice of each group. (E) Flow cytometry contour plots represent eYFP expression in the indicated myeloid cell populations. moMPs, monocyte-derived macrophages. Statistical analysis was performed by unpaired, nonparametric Mann-Whitney Student t test or two-way ANOVA, with a follow-up Sidak multiple-comparison test. Error bars in (A) and (D) represent mean 6 SEM. **p , 0.01, ***p , 0.001, ****p , 0.0001. ns, not significant. downstream of TLR7 signaling in regulating autoreactive B cell been described (50). IFN-g–STAT1 signaling was found to aid development in both the extrafollicular and GC pathways, leading TLR-mediated inflammatory cytokine production by regulating to TLR7-promoted autoantibody production and SLE development. cellular metabolism (51) and gene transcription through chromatin Synergy between TLR-MyD88 and IFN-g–STAT1 signaling in remodeling of the inflammatory gene promoter and enhancer re- potentiating inflammatory responses in macrophages has previously gions (52). MyD88 was also shown to directly interact with IFN-gR 10 REGULATION OF TLR7-DRIVEN AUTOREACTIVE B CELL DEVELOPMENT Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 6. B cell–intrinsic IFN-g signaling is necessary for TLR7-mediated autoimmune responses and lupus nephritis development. (A–M) Sle1bIFN-gRfl/fl and Sle1bIFN-gRfl/fl.CD23Cre+ mice were treated with IMQ for 12 wk. Spleen pictures (A) and their weight (B) are shown. Repre- sentative spleen sections were stained for GCs (C) with GL7 (green), CD4 (red), and IgD (blue); and the areas (D) and the frequency of GCs (E)were measured. The percentage of B220+GL7hiCD95hi GC B cells (F)andCD4+CD44hiPD-1hiCXCR5hi Tfh cells (G) were measured by flow cytometry. Serum ANA reactivity (H) and autoantibody titers (I) against dsDNA, nucleosome, and SmRNP are shown. Data in (H)and(I) represent six mice of each genotype. Numbers of splenic dsDNA-, nucleosome-, and SmRNP-specific AFCs were quantified by ELISPOT assay (J). (K) Representative kidney sections were stained with anti-C3 (green) and anti-IgG (red). Representative kidney sections were stained with PAS (L)orH&E(M) and scored for GN, interstitial nephritis, or inflammation in the vessels. These data represent two to three experiments,and each symbol indicates an individual mouse (B, F, G, J,andM)oraGC(D and E) or a glomerulus (L). Statistical analysis was performed by unpaired, nonparametric Mann-Whitney Student t test or two-way ANOVA, with a follow-up Sidak multiple-comparison test (I). Error bars in (B), (D)–(G), (J), (L), and (M)representmean6 SD, and error bars in (I)representmean6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. The Journal of Immunology 11 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 7. B cell–intrinsic roles of STAT1 and T-bet in TLR7-mediated GC, Tfh, and Ab responses. (A) Representative histological images of splenic GCs in B6 and B6.STAT12/2 mice treated with IMQ for 8 wk. Spleen sections were stained for GC B cell marker, GL7 (green), mature B cell marker, IgD (blue), and T cell marker, CD4 (red). (B) Flow cytometry gating strategy and (C) the percentage of GC B cells, Tfh, and plasma cells in B6 and B6.STAT12/2 mice are shown. (D and E)B6.STAT1fl/fl control mice and STAT1fl/flCD23Cre+ conditional knockout mice were treated with IMQ for 8 wk before performing similar analyses as in (B)and(C). (F) Serum IgG and IgG2c Abs from mice described in (D)and(E) were measured by ELISA. STAT1 (G) and T-bet (H) expression in unstimulated (media) and TLR7 stimulated B cells were measured by intracellular staining (ICS) and flow cytometry analysis. (I) Representative histological images of splenic GCs, (J) percentage of GC B cells and Tfh, and (K) total serum IgG and IgG2c Abs in IMQ-treated B6.STAT1fl/fl and STAT1fl/flCD23Cre+ mice were analyzed as in (A)–(C)and(F). Statistical analysis was performed by unpaired, nonparametric Mann-Whitney Student t test (C, E, G, H,andJ) or two-way ANOVA, with a follow-up Sidak multiple-comparison test (F and K). Error bars in (C), (E), (G), (H), and (J)representmean6 SD, and error bars in (F)and(K)representmean6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. 12 REGULATION OF TLR7-DRIVEN AUTOREACTIVE B CELL DEVELOPMENT to stabilize the IFN-g–induced cytokine and chemokine mRNA for the transcription factor (XBP-1) and is required for plasma cell (53). In addition, STAT1 was shown to interact with cell cycle differentiation and autoimmune responses (69, 70), is downregu- regulators cyclin D1 and Cdk4 (54). Consistent with these previ- lated in these mice. IL-6 signaling is involved in autoimmune ous reports, we found that global and B cell–specific deficiency GC formation and autoimmune disease progression likely by in- of STAT1 resulted in reduced GC B cell and Tfh responses upon creasing the production of IFN-g (71, 72). Interestingly, il6r 2/2 treatment with a TLR7 ligand, highlighting the importance of gene expression is downregulated in Sle1b.IFN-gR1 mice. In STAT1inBcellsinTLR7-mediatedGCandTfhresponses. addition, the relationship between IL-6 and IFN-g signaling in Published data and our current findings collectively suggest that autoimmunity is further supported by the upregulation of the TLR7-MyD88 and IFN-g–STAT1 signaling may synergize to ac- Smyd2_TSS3427 gene in Sle1b.IFN-gR2/2 mice, a gene that en- tivate multiple biological processes in B cells that are important codes a histone methyltransferase that negatively regulates IL-6 for mediating SLE-associated AFC, GC, and Tfh responses in production (73). Finally, genes encoding chemokine receptor TLR7-accelerated SLE autoimmunity. Cxcr3 and its ligand Cxcl9 that play an important role in auto- Although T-bet has been associated with age-associated B cells immunity development (74–76) are downregulated in the absence and class switching to the IgG2a/c isotype (55–57), we found of IFN-gR signaling. Overall, our data indicate that TLR7 stim- significantly reduced TLR7-driven GC and Tfh responses in B cell ulation induces expression of genes that are associated with au- conditional T-bet–deficient mice. Rubtsova et al. (58) recently toimmune responses in an IFN-gR signaling dependent manner. showed a B cell–intrinsic role of T-bet in B cell differentiation into In summary, our data define a previously unrecognized in- age-associated B cells, Spt-GC B cells, and plasma cells and dispensable role for IFN-g signaling and its downstream tran- which subsequently increased kidney pathology. In addition, scription factors STAT1 and T-bet in B cells in promoting Downloaded from CD11chiT-bet+ B cells have recently been described in SLE pa- TLR7-accelerated AFC and GC responses and SLE autoimmunity. tients (59). An earlier report showed that TLR7 stimulation of In contrast, we find moderate effects of T1IFN signaling on these B cells upregulated T-bet and that T-bet expression was elevated in processes. Our current T1IFN-signaling data reflect the moderate 564Igi autoreactive B cells (60). Rubtsova et al. (61) similarly efficacy of the clinical trials for anti–IFN-a or anti–IFN-aR found that BCR and TLR7 signaling synergize with IFN-g sig- therapy in SLE patients. Future treatment approaches could aim to

naling to drive T-bet expression in B cells. The Cancro group reduce TLR7 signaling in combination with T1IFN or IFN-g http://www.jimmunol.org/ recently reported that T-bet expression is induced in B cells by signaling to a threshold that can prevent or treat SLE patients and TLR signaling but not to BCR and/or anti-CD40 stimulation alone maintain protection against pathogenic infection. (62). Published data and our current observation together suggest that TLR and IFN-g–STAT1 signaling cooperate to drive T-bet Acknowledgments expression in B cells, which is important for GC, Tfh, and Th1- We thank the Pennsylvania State University Hershey Medical Center flow biased IgG2c Ab responses. Consistent with this idea, we found cytometry core facility for assistance. We thank the Pennsylvania State upregulation of STAT1 and T-bet in TLR7 stimulated B cells, University Hershey Medical Center Department of Comparative Medi- implicating the involvement of both T-bet and STAT1 in TLR7- cine for animal housing and care. We thank Dr. Meinrad Busslinger for mediated AFC, GC, and SLE autoimmune responses. providing CD23Cre mice. by guest on September 26, 2021 TLR7 can potentially activate multiple signaling pathways (e.g., NF-kB, IRF5/7, and p38/MAPK) to promote autoimmunity (63). Disclosures Both canonical and noncanonical NF-kB pathways have been The authors have no financial conflicts of interest. shown to be important for foreign Ag-driven AFC and GC re- sponses (64, 65), although the role of NF-kB in spontaneous or TLR7-mediated AFC and GC responses in SLE is unclear. Al- References tered NF-kBactivitywaspreviouslyreportedinBcellsand 1. Luzina,I.G.,S.P.Atamas,C.E.Storrer,L. C. daSilva, G. Kelsoe, J. C. Papadimitriou, and B. S. Handwerger. 2001. 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Figure S1. Analysis of spleen cells including B, GC B and CD4 T cells during TLR7-mediated autoimmune responses. B6 and B6.Sle1b mice were left untreated or treated epicutaneously with imiquimod (IMQ) for 8 wks. The number of splenocytes (A), and the percentage (B, C) and number (D, E) of B220+ B cells and CD4+ T cells in these mice are shown. (F) Gating strategy for defining B220+GL- 7hiCD95hi GC B cells and CD4+CXCR5hiPD-1hi Tfh cells. Statistical analysis was performed by unpaired, nonparametric Mann-Whitney Student’s t-test or two-way ANOVA, with a follow-up Sidak multiple- comparison test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Error bars in A, B, C, D and E represent mean ± SD.