Transcriptomes in Lymph Node and Follicular Dendritic Cell

Lymphotoxin-β Receptor-Dependent Genes in Lymph Node and Follicular Dendritic Cell Transcriptomes

This information is current as Christoph Huber, Caroline Thielen, Harald Seeger, Petra of September 28, 2021. Schwarz, Fabio Montrasio, Mark R. Wilson, Ernst Heinen, Yang-Xin Fu, Gino Miele and Adriano Aguzzi J Immunol 2005; 174:5526-5536; ; doi: 10.4049/jimmunol.174.9.5526

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Supplementary http://www.jimmunol.org/content/suppl/2005/04/18/174.9.5526.DC1 Material http://www.jimmunol.org/ References This article cites 52 articles, 25 of which you can access for free at: http://www.jimmunol.org/content/174/9/5526.full#ref-list-1

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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 © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Lymphotoxin-␤ Receptor-Dependent Genes in Lymph Node and Follicular Dendritic Cell Transcriptomes1

Christoph Huber,* Caroline Thielen,† Harald Seeger,* Petra Schwarz,* Fabio Montrasio,* Mark R. Wilson,‡ Ernst Heinen,† Yang-Xin Fu,§ Gino Miele,2* and Adriano Aguzzi2*

Afﬁnity maturation and Ab class switches occur in lymphoid germinal centers (GCs), in which differentiation and maintenance depend on lymphotoxin (LT) signaling and include differentiation of follicular dendritic cells (FDCs). The events leading to FDC and GC maturation are poorly deﬁned. Using several approaches of functional genomics, we enumerated transcripts affected in mice by suppressing LT ␤ receptor (LT␤R) signaling and/or overrepresented in FDC-enriched GC isolates. Protein expression analysis of 3 of 12 genes both enriched in FDCs and down-regulated by LT␤R signaling suppression validated them as FDC markers. Functional analysis of one of these three, clusterin, suggests a role as an FDC-derived trophic factor for GC B cells.

Hence, the set of genes presented in this study includes markers emanating from LT␤R signaling and transcripts relevant to GC Downloaded from and FDC function. The Journal of Immunology, 2005, 174: 5526–5536.

ignaling through the lymphotoxin (LT)3 ␤ receptor 4, 7, 8), impaired affinity maturation (LT␤RϪ/Ϫ) (1), and compro- (LT␤R) and the TNFR1 is crucial for organogenesis and mised B cell memory (LT␣Ϫ/Ϫ,LT␤RϪ/Ϫ, and TNFR1Ϫ/Ϫ) (1, 8, S maintenance of the structural integrity of secondary lym- 9). FDCs may represent a key player in promoting and regulating phoid organs (1, 2). These organs provide the compartmentalized these events occurring during the GC reaction. In vitro experi- http://www.jimmunol.org/ microenvironment essential for mounting efficient humoral im- ments have shown that FDCs and FDC-conditioned medium exert mune responses. chemotaxis on B cells and CD4ϩ T cells (10). FDCs can promote Mice deficient for LT␤R, TNFR1, or their ligands suffer from survival and proliferation of GC B cells (11, 12), and coculture complex and partially overlapping pathologic phenotypes of the with FDCs can enhance Ig secretion by B cells (13). lymphoreticular system. TNF-␣ mice and TNFR1-deficient Given the complex nature of GCs, the identification of mole- (TNFR1Ϫ/Ϫ) mice develop lymph nodes, but TNFR1Ϫ/Ϫ mice cules that act downstream of LT and TNF signaling and that may show hypoplastic Peyer’s patches (3–5). LT␣-deficient (LT␣Ϫ/Ϫ) control these phenomena has proven difficult. Also, the specific and LT␤-deficient (LT␤Ϫ/Ϫ) mice lack Peyer’s patches and most molecular contribution of FDCs to these processes has been elu- lymph nodes, except for mesenteric and cervical lymph nodes (6, sive, as it has proven technically impossible to isolate mature by guest on September 28, 2021 7). LT␤RϪ/Ϫ mice display the severest phenotype: they lack Pey- FDCs to purity without the concomitant loss of markers that au- er’s patches and all lymph nodes (1). thenticate their mature state (FDC-M1) and functionality (CD21/ All these mutant mice develop a spleen, but show varying de- CD35 and FDC-M2). grees of disturbance in white pulp compartmentalization, as well as To gain insights into FDC-associated functions controlled by impaired formation of germinal center (GC) and follicular den- LT␤R signaling, many of which may contribute to the GC reac- dritic cell (FDC) networks after challenge with antigenic sub- tion, we pursued two complementary strategies aimed at defining stances. These deficits are reflected in reduced isotype switching the transcriptional profile associated with the presence of GC and (LT␣Ϫ/Ϫ,LT␤Ϫ/Ϫ,LT␤RϪ/Ϫ, TNF-␣Ϫ/Ϫ, and TNFR1Ϫ/Ϫ) (1, 3, FDC networks. First, we used high-density oligonucleotide microarrays to identify transcripts affected in vivo by administration of a soluble LT␤R-Ig, which blocks LT␤R signaling. Second, we *Institute of Neuropathology, University Hospital of Zu¨rich, Zu¨rich, Switzerland; screened for transcripts overrepresented in cell preparations en- † ‡ Institute of Human Histology, University of Lie`ge, Lie`ge, Belgium; School of Bi- riched for FDCs relative to non-FDC splenic cell types by both ological Sciences, University of Wollongong, Wollongong, New South Wales, Aus- tralia; and §Department of Pathology, University of Chicago, Chicago, IL 60637 microarrays and suppression subtractive hybridization. As might Received for publication October 27, 2004. Accepted for publication February be expected, some of the transcripts were found to overlap in both 14, 2005. populations. This allowed us to identify LT␤R signaling-depen- The costs of publication of this article were defrayed in part by the payment of page dent genes preferentially expressed in GCs in association charges. This article must therefore be hereby marked advertisement in accordance with FDCs. with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by grants of the Bundesamt fu¨r Bildung und Wissenschaft (EU) and the Swiss National Foundation, the U.S. National Prion Research Program, Materials and Methods and the NCCR on Neural Plasticity and Repair (to A.A.), and by the Verein zur Enrichment of FDC clusters Fo¨rderung des Akademischen Nachwuchses and the Stiftung fu¨r Biomedizinische Forschung (to C.H.) and Functional Genomics Centre, Zu¨rich. FDCs were prepared by adapting a procedure of Thielen et al. (14). Eight- ␮ 2 Address correspondence and reprint requests to Drs. Adriano Aguzzi or Gino Miele, to 10-wk-old female C57BL/6 mice were injected with 200 g of OVA in Institute of Neuropathology, University Hospital of Zu¨rich, Schmelzbergstrasse 12, alum i.p. to increase the amount of FDCs present in secondary lymphoid CH-8091 Zu¨rich, Switzerland. E-mail address: [email protected] or tissues. Spleens were harvested 3 days after injection and cut in small [email protected] pieces using a tissue chopper. The spleen pieces were digested two times 3 Abbreviations used in this paper: LT, lymphotoxin; SAM, significance analysis of for 20 min at 37°C in RPMI 1640 medium containing 1 mg/ml collagenase microarray; PNA, peanut agglutinin; SSH, suppression subtractive hybridization; A (Boehringer Mannheim), 0.5 mg/ml dispase (type II; Boehringer Mann- DIG, digoxigenin; GC, germinal center; FDC, follicular dendritic cell; ECM, extra- heim), 0.04 mg/ml DNase (type I; Boehringer Mannheim), and 0.4% BSA cellular matrix; LPA, lysophosphatidic acid; MFG, milk fat globule. (Sigma-Aldrich). The supernatants of the two digestions were pooled and

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 The Journal of Immunology 5527 the cells were collected by centrifugation. The pelleted cells were resus- using the algorithms provided by the dChip software. For the correlation pended in PBS containing 0.4% BSA and layered over FCS for four re- analysis of the genes represented on the MOE430A chips, cluster A was peated sedimentations at1gat4°C. The cells contained in the supernatant divided into two smaller subclusters exhibiting expression minima at days of the first sedimentation were collected and remaining FDCs were re- 3 or 27 postinjection. The average expression pattern of the two subclusters moved using FDC-M1 coupled magnetic beads (Dynal Biotech). The re- was used to calculate the correlation coefficients. For genes represented on sulting cell fraction was frozen and used as the FDC-depleted sample for the MOE430B chips the entire cluster A of this chip set was used for RNA isolation. To remove contaminating macrophages, the FDC-enriched calculation of the correlation coefficients. Functional annotation of genes cell clusters obtained after the four repetitive sedimentations were incu- was performed using information provided by Affymetrix NetAffx (͗www. bated in a plastic culture dish for 60 min with RPMI 1640 containing 10% affymetrix.com/analysis/index.affx͘), LocusLink (͗www.ncbi.nlm.nih.gov/͘), ͗ ͘ FCS at 37°C, 5% CO2. Nonadherent cell clusters were frozen and used as and PubMed ( www.ncbi.nlm.nih.gov/entrez/query.fcgi ) databases. All the FDC-enriched cell fraction for immunofluorescence staining and RNA microarray data sets are available at ͗www.ncbi.nlm.nih.gov/geo͘. Acces- isolation. sion numbers are GSE2123 (FDC-E vs FDC-D) and GSE2124 (soluble LT␤R-Ig-treated mesenteric lymph nodes). Treatment of C57BL/6 mice with soluble LT␤R-Ig Immunohistochemistry of cytospin FDC clusters Ten-wk-old female C57BL/6 mice were injected with 100 ␮g of soluble LT␤R-Ig i.v. As a control, identical mice were injected with the same FDC clusters were isolated and cytospun onto glass slides. Slides were volume of carrier only (PBS). The experimental mice were sacrificed 1, 2, fixed with acetone on ice, air dried and stored frozen at Ϫ80°C until used 3, 27, and 35 days after injection; the control mice were sacrificed 3 days for staining. For immunostaining slides were air dried, refixed with ace- after administration of PBS. Mesenteric lymph nodes and spleens were tone, and redried. After rehydration in PBS, blocking was achieved with harvested for further processing. 2% goat serum in PBS. Then clusters were stained with FDC-M1 (1/50; Ј ϩ BD Pharmingen) and FITC goat F(ab )2 anti-rat IgG (H L) (1/200; Bio- Preparation of labeled cRNA and microarray hybridization Source International) and after thorough washing were fixed with 4% para- Downloaded from Total RNA was extracted from all samples (FDC-enriched or -depleted cell formaldehyde in PBS. Staining was then continued with CD4-Bio (1/100; fractions and mesenteric lymph nodes) using TRIzol (Invitrogen Life Tech- R&D Systems), CD8-Bio (1/100; R&D Systems), CD68-Bio (1/200; Ino- nologies) according to the manufacturer’s instructions. RNA was subjected tech), CD11c-Bio (1/100; BD Pharmingen), and streptavidin-Alexa 594 to a clean up step using RNeasy mini kit (Qiagen). RNA quality was as- (1/500; Molecular Probes). All Abs were diluted in 2% goat serum in PBS. sessed by agarose gel electrophoresis or Agilent 2100 Bioanalyzer. Pro- Photographs were taken with a Leica SP2 confocal laser scanning cessing of FDC-enriched and depleted cell fractions used: 15 ␮g of total microscope. RNA was subjected to cDNA synthesis using a cDNA Synthesis kit (cat. Immunohistochemistry on cryosections no. 11917-010; Invitrogen Life Technologies) and primer 5Ј-GGCCAGT http://www.jimmunol.org/ GAATTGTAATACG ACTCACTATAGGGAGGCGG(dT)24–3Ј. Biotin- FDC-M1, CD21/CD35, peanut agglutinin (PNA), and B220 stainings for labeled cRNA was synthesized, using Enzo BioArray HighYield RNA light microscopy were performed as previously described (17). For double transcript labeling kit (T7) (Enzo Biochem), cleaned with the RNeasy mini staining with milk fat globule-epidermal growth factor 8 (MFG-E8) and kit, quantified by spectrophotometry, and quality was assessed again by prion protein (PrP), 5 ␮M cryosections were blocked with 1% goat serum agarose gel electrophoresis or Agilent 2100 Bioanalyzer. Typical yields and 0.5% BSA in PBS. Washes were performed with PBS and incubations ranged from 60 to 90 ␮g of labeled cRNA. Labeled cRNA (15 ␮g) was with primary and secondary Abs with 1% goat serum and 0.5% BSA in fragmented in 40 mM Tris-acetate, 100 mM KOAc, 30 mM MgOAc, pH PBS at room temperature. Abs used were: anti-MFG-E8 (1/1000; kind gift 8.1, at 95°C for 35 min. After fragmentation an aliquot of one represen- from Dr. S. Nagata, Osaka University Medical School, Osaka, Japan), anti- tative of the replicates of each sample was hybridized to Affymetrix Test PrP XN (1/1000), B220 (1/400; BD Pharmingen), F4-80 (1/50; Inotech), Ј Ј

3 arrays to determine the quality of the probes, reflected by the 3 to 5 FDC-M1 (1/50; BD Pharmingen), FDC-M2 (1/50; Immunokontakt), Alexa by guest on September 28, 2021 ratio, and to test all buffers. The 3Ј to 5Ј ratio of the cRNAs used was Fluor 594 goat anti-rat IgG (HϩL) (1/100; Molecular Probes), FITC Ͻ Ј always 2. The samples were then hybridized to Affymetrix MGU74vA2, F(ab )2 goat anti-hamster IgG (1/100; Inotech), and Alexa 546 goat anti- Bv2, and Cv2 chips. All hybridizations were conducted for 16 h at 45°C at rabbit IgG (HϩL) (1/500; Molecular Probes). 60 rpm. After hybridization chips were washed and conjugated with For double staining with clusterin, endogenous peroxidase was streptavidin-PE according to the manufacturer’s instructions on the Af- quenched with iView inhibitor (Ventana). Then staining of clusterin was fymetrix GeneChip Fluidics Station 450 with and scanned using the Af- performed with the Tyramide Signal Amplification fluorescein system fymetrix GS Scanner 2500 in conjunction with Affymetrix Microarray (TSA; PerkinElmer), using anti-clusterin Ab (1/500; Santa Cruz Biotech- Suite 5.0 software. Processing of mesenteric lymph nodes samples showed: nology) as a primary and HRP donkey anti-goat IgG (1/200; Jackson Im- equal amounts of high quality total RNA from the individual mesenteric munoResearch Laboratories) as a secondary Ab according to the manu- lymph node preparations were combined to obtain three individual pools of facturer’s instructions. Double staining with FDC-M1, B220, and F4-80 15 ␮g of RNA per time point. Complementary DNA and RNA synthesis were performed as described. Photographs were taken with an Axiovert were conducted as described. Labeled cRNA was hybridized to Affymetrix 200 M microscope (Zeiss). MOE430A and B chips. Quantitative real-time PCR Microarray data analysis Snap frozen mesenteric lymph nodes or spleens were homogenized in Intensity values of all chips were normalized with dChip software (͗www. TRIzol reagent (Invitrogen Life Technologies) with a T18 Basic Disperser dchip.org͘) (15) and, applying the model-based expression analysis algo- (Ika Works), and total RNA was extracted according to the manufacturer’s rithm (PM-only model), the expression values were calculated. instructions. Before cDNA synthesis, residual genomic DNA was removed FDC-enriched (three biological replicates per chip set) and FDC-de- by the DNA-free kit (Ambion). Total RNA (5 ␮g) was converted into pleted (two biological replicates per chip set) cell samples were compared cDNA using the first strand cDNA synthesis kit (Amersham Pharmacia applying a 2-fold change (using the lower 90% confidence boundary of Biotech). Successful cDNA synthesis and contamination of total RNA with fold change) as the lower limit. In the case of the MGU74Bv2 chip set, two genomic DNA was tested by PCR with primers specific for Actb. Quanti- instead of three biological replicates for the FDC-enriched samples were tative real-time PCR was performed using the SYBR Green PCR Master included in the analysis due to contamination of one of the replicates. Only Mix (Applied Biosystems) on an ABI PRISM 7700 Sequence Detector those genes that were flagged present in all FDC-enriched samples were (PerkinElmer). The following primer pairs were used: clusterin Clu,5Ј- considered. To increase the stringency a 4-fold change including the same CCAGTTCCCAGACGTTGATT-3Ј and 5Ј-AGCAGGGATGAGGTGTT criteria as mentioned was applied as the lower limit. GAG-3Ј; cochlear Coch,5Ј-CGTGCAAGGGGATCTAATGT-3Ј and 5Ј- Significantly modulated genes by soluble LT␤R-Ig treatment were de- GCTTACCTATCCACTTGAATGC-3Ј; ectonucleotide pyrophosphatase/ termined by significance analysis of microarrays (SAM) (16). A total of phosphodiesterase 2 Enpp2,5Ј-TGGCTTACGTGACATTGAGG-3Ј and 100 random permutations of the data set were applied to calculate back- 5Ј-AAATCCAAACCGGTGAGATG-3Ј; glycoprotein GpM6b,5Ј-AA ground changes and the false discovery rates used in SAM. Thresholds for GAGCTGCACGGTGAGTTT-3Ј and 5Ј-GCACAAGCCACAATGAA significant changes were set to 0.38285 for analysis of MOE430A chips CAGG-3Ј; glycodelin A Gda,5Ј-GTTGTGGGCCTAGTCCATGT-3Ј and (lower cutoff 0.90741) and to 0.20248 for MOE430B chips (lower cutoff 5Ј-GATACCGAGGGGAAGGATGT-3Ј; lysozyme Lyzs,5Ј-TCCCTTGT 0.90741). The random number seed was set to 1234567. Of the signifi- CAGTCAGCACAG-3Ј and 5Ј-CTGTGCCCTCAGAAACCTTC-3Ј; cantly changed genes, those considered for further analysis were flagged in Serpina1a,5Ј-TCCAGATCCATATCCCCAGA-3Ј and 5Ј-AGGAACG two of three replicates of at least one sampling time point. Unsupervised GCTTCAAAGACTG-3Ј; glycosylation-dependent cell adhesion molecule hierarchical clustering of genes and correlation analysis was performed 1 Glycam1,5Ј-GCATTGATGGGCTCAGATTT-3Ј and 5Ј-ACTTCAAC 5528 FDC TRANSCRIPTOME

CCCAGGAAAACC-3Ј; RIKEN cDNA C030033F14Rik, 5Ј-AAAGTA In situ hybridization AACACCGAAGGGACA-3Ј and 5Ј-TCGGGAAAGCAACAATCAAT3Ј; Mfge8,5Ј-CCCTTCTCTCAGGCATTCTG-3Ј and 5Ј-AACCTGTCAACCA Plasmids containing the differentially expressed cDNAs were linearized by CCCAGAG-3Ј; prion protein Prnp,5Ј-GCTGGCCCTCTTTGTGACTA-3Ј restriction digest with NotIorSalI and isolated by gel purification and 5Ј-CTGGGCTTGTTCCACTGATT-3Ј; Cxcl13,5Ј-TCGTGCCAAATG (QIAquick gel purification kit). Digoxigenin (DIG)-labeled antisense and GTTACAAA-3Ј and 5Ј-ACAAGGATGTGGGTTGGGTA-3Ј; actin Actb, sense riboprobes were synthesized using the SP6/T7 DIG labeling kit ␮ 5Ј-GACGGCCAGGTCATCACTAT-3Ј and 5Ј-ACATCTGCTGGAAGG (Roche). Thick cryosections (5 M) of spleens were directly fixed with 4% TGGAC-3Ј. paraformaldehyde in PBS on ice for 30 min and washed in PBS. Slides were then pretreated with 0.1 M HCl for 5 min and after washing acetylated Isolation of GC B cells with acetic anhydride for 5 min. After further washing with PBS the sections were dehydrated with ethanol (EtOH, 50, 70, 80, and 95%) and Eight-wk-old C57BL/6 mice were immunized as described for FDC en- air-dried.

richment, and spleens were isolated. RBC were lysed with 155 mM NH4Cl, After prehybridization at 37°C for 3 h with prehybridization solution ϫ 10 mM KHCO3, and 0.1 mM EDTA. The spleen cells were then incubated (50% formamide, 2.5 Denhardt’s, 50 mM EDTA, 50 mM Tris-HCl pH with FITC anti-IgD Ab (1/100; BD Pharmingen) and GC B cells were 7.6, 0.25 mg/ml yeast total RNA, 20 mM NaCl), the slides were incubated thereafter negatively selected with magnetic beads directed against FITC, with denatured probe diluted in hybridization solution (50% formamide, CD43, Thy1.2, Mac-1, and CD11c (all Miltenyi Biotec) using an au- 10% dextransulfate, 0.33 M NaCl, 20 mM Tris-HCl pH 7.5, 1 mM EDTA, toMACS sorter (Miltenyi Biotec). Alternatively, GC B cells were posi- 1ϫ Denhardt’s, 0.5 mg/ml yeast tRNA) at 45–55°C in a humidiﬁed cham- tively selected by incubation with FITC anti-GL7 Ab (1/100; BD Pharm- ber overnight. The slides were then washed three times for 30 min each at ingen) and subsequent autoMACS sorting with anti-FITC magnetic beads. the temperature used for hybridization with 0.2ϫ SSC, 0.1ϫ SSC, 50% Purity of the GC B cells was assessed by FACS: 1 ϫ 105 cells taken before formamide, 0.2ϫ SSC, and then at room temperature with 0.2ϫ SSC and and after MACS were resuspended in FACS buffer (20 mM EDTA, 2% 0.1 M Tris-HCl pH 7.5, 150 mM NaCl. Blocking was performed with

FCS, 0.05% NaN3) and stained with the Abs B220-allophycocyanin (1/ blocking reagent (Roche) according to the manufacturer’s instructions. Downloaded from 100; BD Pharmingen) and IgMb-PE (1/100; BD Pharmingen). FACS was DIG labels were then detected with anti-DIG Fab (Roche) at a dilution of performed on a four-color FACSCalibur ﬂow cytometer (BD Biosciences). 1/100 and a color reaction with nitroblue tetrazolium/5-bromo-4-chloro-3- Purity of B220ϩIgMbϩIgDϪ negatively selected GC B cells was Ͼ86% indolyl phosphate. and purity of B220ϩGL7ϩ positively selected cells was Ͼ93% after MACS. Results Cell viability assay ␤

Transient blockade of LT R signaling in mesenteric lymph http://www.jimmunol.org/ MACS purified GC B cells were cultured in IMDM medium (Sigma-Al- nodes by soluble LT␤R-Ig drich) supplemented with 1% penicillin/streptomycin (Invitrogen Life LT␤R signaling can be transiently inhibited in adult mice by ad- Technologies), 1.5% FCS (Sigma-Aldrich), and 2 mM L-glutamine (In- vitrogen Life Technologies) in 96-well plates at a density of 1.5 ϫ 105 ministration of the soluble decoy receptor, soluble LT␤R-Ig (19– cells/ml. Untreated cells were cultured in medium only, to treated cells the 21). We elected to analyze the transcriptional profile of mesenteric following reagents were added: anti-CD40 mAb (FGK 45.5, 10 ␮g/ml), lymph nodes associated with the blockade of LT␤R signaling. We ␮ human clusterin (50 g/ml) purified as previously described (18), lyso- chose mesenteric lymph nodes because we expected that, based on phosphatidic acid (LPA, 1 ␮M; Sigma-Aldrich), and human ␣1-antitrypsin (1 mg/ml; Sigma-Aldrich). Boiled human clusterin was incubated at 95°C their histologic structure, the contribution of GC and FDC to the for 15 min. Viability of the cells was assessed at the time points indicated total tissue transcriptome would be far greater than in spleen. by trypan blue exclusion in triplicates. Although the effects of soluble LT␤R-Ig treatment on FDCs in by guest on September 28, 2021 splenic follicles have been previously documented (19), the con- Construction of subtracted cDNA libraries using suppression sequences of this treatment for mesenteric lymph nodes have not subtractive hybridization (SSH) technique been described. We first defined the time course of events occur- FDC clusters and FDC-depleted cells isolated from 200 C57BL/6 mice as ring after soluble LT␤R-Ig injection on FDCs in mesenteric lymph described were resuspended in buffer RLT of the RNeasy Midi kit (Qiagen) nodes. A single i.v. dose of 100 ␮g of soluble LT␤R-Ig was ad- and homogenized with a T18 Basic Disperser (Ika Works). Total RNA was extracted according to the manufacturer’s instructions. Poly(Aϩ) mRNA ministered to C57/BL6 mice, and general mesenteric lymph node was isolated from the total RNA using Oligotex mRNA spin columns (Qia- follicular microarchitecture was analyzed by immunohistochemis- gen) following the manufacturer’s instructions. Total RNA was DNase try at defined time points following treatment. In contrast to car- treated using the DNA-free kit (Ambion). rier-only injections (PBS), FDC markers FDC-M1 and CD21/ ϩ ␮ Poly(A ) mRNA (0.3 g) from FDC-enriched cells was used as the CD35 completely disappeared from the lymph follicles of “tester” or the population whose up-regulated transcripts were to be identified, and an equal amount of poly(Aϩ) mRNA from FDC-depleted cells mesenteric lymph nodes within 3 days. However, both markers served as the “driver” or the population whose transcripts served as a ref- reappeared 27–35 days later (Fig. 1). PNA staining of GC B cells erence for cDNA subtraction (forward SSH library). The construction of disappeared with slightly delayed kinetics relative to the FDCs, as the forward and reverse libraries was performed as per the SSH procedure residual PNA positivity was still detectable at day 3 postinjection. using the PCR select cDNA subtraction kit (BD Clontech). Subtracted target cDNAs were ligated with the pGEM-T easy plasmid vector Reappearance of PNA staining 27 and 35 days after administration (Promega). of soluble LT␤R-Ig occurred in parallel with the FDC-marker FDC-M1 (Fig. 1). Treatment with soluble LT␤R-Ig had no mor- Inverted Northern blot phologically discernible effects on the localization of non-FDC Plasmid (300 ng) was digested with 10 U EcoRIfor3hat37°C in 97-well cells or on the expression of non-FDC markers within mesenteric plate in a GeneAmp PCR System 9700 PCR machine (Applied Biosys- lymph nodes during the period of analysis, as assessed by staining tems). The digested product was divided into two halves and electropho- with the B cell marker B220 (Fig. 1), the macrophage marker resed through a 1% agarose gel in duplicates. The DNA was then trans- ϩ F4-80, the dendritic cell marker CD11c, and the T cell markers ferred to a Hybond-N membrane (Amersham Pharmacia Biotech). For hybridization 50 ng of FDC-E and FDC-D cDNA were labeled with CD4 and CD8 (data not shown). [␣-32P]dCTP using random hexamer primers (Stratagene). After hybridization the membranes were exposed to BAS-MS imaging plates (FUJIFILM) overnight and the plates were scanned using a BAS-1800 II Transcriptional patterns in mesenteric lymph nodes after PhosphorImager (FUJIFILM). The signals were quantified with AIDA 2.41 transient soluble LT␤R-Ig-mediated FDC depletion image analyzer (Raytest). Hybridization signals obtained for each clone were normalized to the background signal originating from the vector in To identify gene expression patterns resulting in mesenteric lymph the same lane. Normalized signals for each clone were then compared nodes from transient depletion of FDCs, we profiled the transcrip- between the two hybridizations. tome of mesenteric lymph nodes harvested 1, 2, 3, 27, and 35 days The Journal of Immunology 5529

FIGURE 1. Immunohistochemical analysis of the temporal effects of soluble LT␤R-Ig treatment on mesenteric lymph nodes. Stains were performed on cryostat sections of mesenteric lymph nodes harvested form C57BL/6 mice at the indicated time points post administration of soluble LT␤R-Ig or carrier only (PBS). Sections were counterstained by hematoxylin. Treatment with soluble LT␤R-Ig leads to complete disappearance of FDC markers (FDC- M1, CD21) and reactive GCs (PNA) form the follicular area within 3 days. Reformation of FDC networks and PNA-positive GC can be detected at days 27–35 postinjection. Localization and expression of markers specific for B cells (B220) were not affected by the treatment. Scale bar represents 200 ␮m. Downloaded from after injection of soluble LT␤R-Ig or PBS. Three biological rep- categories as cluster C, but the majority (63.0%) could not be licates per time point were hybridized to Affymetrix oligonucleo- functionally annotated (Fig. 2B). These results suggested that clus- tide microarray sets MOE430A and MOE430B. Genes whose ex- ters A and B might contain genes coding for specific biological pression was significantly changed by the blockade of LT␤R functions attributable to LT␤R signaling, whereas the changes in signaling were identified by SAM (16). When highly stringent expression of genes in clusters C and D reflected more general

thresholds for significant changes were set, SAM identified 235 changes in basic cellular activity, possibly reflecting secondary http://www.jimmunol.org/ probe sets with a false discovery rate of 0.35% for chip A and 145 effects downstream of the blockade of LT␤R signaling. probe sets with a false discovery rate of 3.11% for chip B. Unsupervised hierarchical clustering (dCHIP software) (15) A collection of transcripts predominantly expressed by FDCs with these genes revealed four major expression patterns (Fig. 2A) As the expression pattern of cluster A correlated well with the Two of these, clusters A and B, contained genes reflecting short kinetics of disappearance and reappearance of PNAϩ GC and FDC response latency to the blocking of the LT␤R signaling pathway. networks from soluble LT␤R-Ig-treated mesenteric lymph node These transcripts showed an early change (decrease or increase) in cryosections (Fig. 1), we analyzed this cluster in more detail. The expression with a peak of maximal change at day 2, 3, or 27, and highly stringent filtering criteria applied in SAM yielded an initial returned to near-baseline levels at day 35 (Fig. 2A). The remaining set of 21 genes for cluster A. To avoid excluding potentially valid by guest on September 28, 2021 two clusters, C and D, showed longer response latency with little candidates from further analysis, we chose to perform a correlation change during the first two days of the treatment. Only at day 3 did analysis using the dCHIP software. We calculated correlation co- the expression levels of the genes within these clusters start to be efficients for the association of each gene expression pattern with affected, with a maximal change at day 35 relative to control the average expression pattern of cluster A. We then selected genes treatment. with a correlation coefficient Ͼ0.9 and a greater than 0.85-fold The rapid response to the treatment of the genes contained in down-regulation ( p Ͻ 0.05) at the time of minimal expression clusters A and B suggested that LT␤R signaling may transcrip- relative to control treatment. We identified a total of 80 unique tionally regulate some of these genes. Hence they may reflect bi- transcripts whose expression patterns correlated with cluster A, 42 ological functions that are directly controlled by this pathway. In of which could be annotated to known genes. Eleven of these contrast, the longer response latency of the genes in clusters C and genes are known to be expressed by stromal cells within secondary D could reflect secondary and/or indirect effects of the treatment. lymphoid organs, and most of them have been shown to be ex- To further assess these questions we functionally annotated the pressed by FDCs or to form part of subcellular structures, such as known genes identified by SAM. For those genes with a known desmosomes that have been assigned to FDCs by electron micros- function, the four predominant clusters showed a bias toward sep- copy (22). These include cell adhesion molecules (Coch, Dsc3, arate functional categories (Fig. 2B). Genes of the clusters A and Dsg2, Glycam1, and Madcam1), chemokines (Cxcl13 and Ccl19), B were primarily annotated to the functional categories: (I) mem- and cell surface molecules (BstI, Prnp, Cr2, and Plxnb1). Genes brane-bound surface molecules; (II) chemokines, cytokines, im- typically associated with the macrophage/monocyte lineage were munomodulatory and other secreted molecules; (III) immune ef- also affected (Lyzs, Mfge8, and Faah), which is consistent with the fector and apoptosis-related molecules; and (IV) signal expression of LT␤R on macrophages (23). transduction. Genes of the clusters C and D fell mostly into the categories (VII) DNA binding, transcription, translation; (VIII) Selection of a set of GC/FDC-associated genes metabolism; and (IX) transport (Fig. 2B). In cluster A the genes To complement the first approach, and to identify transcripts that related to cell-cell adhesion (category I, 30.4%) and chemotaxis are overrepresented in cells associated with FDC networks in GCs, (category II, 13.0%) were particularly striking. Cluster B was en- we purified FDC cell clusters and characterized their transcriptome riched in surface receptors (category I, 25.0%) and cytokines and relative to FDC-depleted spleen cells. Immunohistochemical anal- chemokines (33.3%). Cluster C showed an enrichment of genes ysis showed that the FDC clusters basically represented small encoding ribosomal subunits, proteasome subunits, or ubiquitin- functional units of GC light zone. Double staining with FDC-M1, conjugating enzymes, transcription factors, and proteins involved B220, CD4, CD68, and CD11c revealed the presence of B cells, in energy metabolism, representing a total of 23.3% of all genes in Th cells, macrophages, and dendritic cells adherent to FDCs in this cluster. The genes of cluster D reflected similar functional these clusters (Fig. 3A). 5530 FDC TRANSCRIPTOME

Labeled cRNA from FDC-enriched cell clusters and FDC-depleted spleen cells was hybridized to the entire chip sets (A, B, and C) of Affymetrix MGU74v2 chips. Using 2-fold changes as the lower limit and further filtering by selecting those probe sets with high significance (lower 90% confidence boundary of fold change), we found that 960 probe sets were significantly overrepresented in the FDC-enriched vs the FDC-depleted specimen. To reduce background noise, we increased the stringency of our criteria by selecting only genes with a Ͼ4-fold change for functional annotation. This filter was passed by 478 probe sets. The majority of annotated genes encoded membrane proteins (category I, 18.2%) and secreted proteins, such as immunomodulatory cytokines and chemokines (category II, 7.1%; Fig. 3B). Among the membrane proteins, the cell adhesion molecules ICAM-1, VCAM-1, MCAM, GlyCAM-1, and E-selectin were overexpressed in FDC-enriched cell clusters. Jagged-1 and Del- ta-1, two ligands of the Notch-2 receptor, were overrepresented in the FDC associated over the FDC-depleted cell fraction. Within

the class of secreted proteins (category II) we identified six che- Downloaded from mokines that attract lymphocytes and macrophages (CCL2, FIGURE 2. Four predominant expression patterns are induced in mesenteric lymph node by soluble LT␤R-Ig treatment, which represent sepa- CXCL12, and CXCL13), T cells and NK cells (CXCL10), and rate functional categories. A, Unsupervised hierarchical clustering of genes neutrophils (CXCL1 and CXCL7). Of the cytokines that were significantly changed (as detected by SAM) during the sampled time points overrepresented in FDC-enriched cell clusters, IL-1␣ and IL-6 represented on MOE430A and MOE430B chips. The normalized expres- have been shown to act on B cells. Three other cytokines exert sion values of each gene were standardized to have a mean of 0 and a SD their effects mainly on the stromal compartment (platelet-derived http://www.jimmunol.org/ of 1. Each expression measurement displayed represents an average of growth factor (PDGF)A, connective tissue growth factor (CTGF), three biological replicates. Rows represent the standardized expression lev- bone morphogenetic protein (BMP)-2). They can induce prolifer- els during the time course of the individual probe sets and columns rep- ation, migration or differentiation of mesenchymal cells and lead to resent sampling time points. Red reflects expression levels above mean production of extracellular matrix (ECM) components (24–26). In expression of a probe set across all time points, green reflects expression accordance with the up-regulation of these cytokines, there was a lower than the mean, black reflects mean expression. Gene clusters (A, B, C, and D) with similar expression patterns in response to soluble LT␤R-Ig clear overrepresentation of genes encoding ECM components in treatment are indicated. The color-scale indicates the color code applied to the FDC clusters vs the remainder of spleen cells, such as Col1a1, display standardized expression values between Ϫ3.0 and 3.0. B, The Col1a2, Col3a1, Col4a1, Col4a2, Col5a2, Col14a1, Col18a1, genes of each of the four clusters (A, B, C, and D) were annotated to the Tnxb, Coch, Bgn, and Mglap (category X, 6.3%). by guest on September 28, 2021 functional categories: (I) membrane-bound surface molecules; (II) chemokines, cytokines, immunomodulatory and other secreted molecules; (III) immune effector molecules and apoptosis related; (IV) signal transduction GC/FDC-associated genes identified by SSH related; (V) cytoskeleton related; (VI) cell cycle and stress; (VII) DNA In a further experiment we used SSH to identify transcripts asso- binding, transcription, and translation; (VIII) metabolism; (IX) transport; (X) ECM components; (XI) other functions; and (XII) unknown functions. ciated with GC and FDC networks. SSH is PCR-based and non- The percentage of the total genes represented by each functional category biased: it allowed us to generate a library of cDNAs overrepre- is indicated for the four clusters identified by hierarchical clustering. sented in FDC-enriched vs FDC-depleted cell clusters without

FIGURE 3. Immunoﬂuorescence characteriza- tion of FDC-enriched cell clusters and functional annotation of genes overrepresented in such preparations. A, FDC-enriched cell clusters were cytospun onto glass slides and stained with FITC-labeled Abs directed against FDCs (FDC-M1) and costained with Alexa 594-coupled Abs for the presence of B cells (B220), Th cells (CD4), dendritic cells (CD11c), or macrophages (CD68). Most of the cells, adherent to FDCs, were B cells followed by Th cells. Only very few dendritic cells could be detected. This indicated that the major cell types taking part during a GC reaction are present in isolated FDC clusters. Scale bar represents 40 ␮m. B, The genes Ͼ4-fold overrepresented in FDC-enriched cell clusters were annotated to the 12 categories described in Fig 3. The percentage of the total genes represented by each functional category is indicated. The Journal of Immunology 5531

FIGURE 4. Identification of genes overexpressed in association with FDCs by SSH. A, The 17 genes identified by SSH with their corresponding proteins are listed. Genes identified by both SSH and microarray analysis are shown in gray. LocusLink numbers and the fold overexpression in FDC-enriched (FDC-E) compared with FDC-depleted (FDC-D) specimens as determined by inverted Northern blot are indicated. B, In situ hybridization of two representative SSH candidates. Spleen cryosections from C57BL/6 mice were hybridized with

DIG-labeled antisense riboprobes for Mfge8 and Ig- Downloaded from fbp3. To control for hybridization speciﬁcity sense riboprobes were incubated in parallel. The images are representative of two independent experiments. http://www.jimmunol.org/

prior knowledge of their sequence. The FDC-enriched cDNA li- FDC cell clusters by quantitative real-time PCR. All 12 genes were brary was screened for FDC/GC-associated transcripts by inverted consistently down-regulated in soluble LT␤R-Ig-treated relative to Northern blot. Seventeen clones from the library were found to be control treated mesenteric lymph nodes and were up-regulated in by guest on September 28, 2021 overexpressed in GC and FDC networks Ͼ2-fold and were se- purified FDC clusters relative to non-FDC associated spleen cells quenced (Fig. 4A). Six of 16 genes (Clu, Cyr61, Igfbp3, Mfge8, (Fig. 5C). The fold down-regulation (between 2- and 20-fold) and Serpina1, and Sparc) were also identified as overexpressed by the fold overexpression (between 1.5- and 283-fold) measured by filters set in the corresponding microarray analysis (Fig. 4A). In quantitative PCR reproduced the microarray experiments, although situ hybridization with two of the candidates, Igfbp3 and Mfge8, the magnitude of changes was generally underestimated by the revealed that they were both expressed in the follicular areas of DNA chips. spleen (Fig. 4B). Although Mfge8 expression appeared confined to the follicles, Igfbp3 showed additional intense labeling of marginal Immunohistochemical validation of transcripts identified by zone cells. These data confirm the concept that novel genes pref- microarray analysis erentially expressed in association with FDCs can be identified by To correlate the transcriptional analysis with protein expression, comparing FDC-enriched and FDC-depleted splenic cell fractions. we performed immunohistochemistry of spleen cryosections of mice treated with PBS or soluble LT␤R-Ig with fluorescent-la- ␤ A core set of LT R responsive transcripts in FDC-enriched cell beled Abs directed against PrPC, which has been previously iden- clusters tified as being expressed by FDCs (21, 27) and two of the novel By combining the two sample preparation approaches we selected candidate proteins. Two-color immunostaining with Abs directed a core set of genes that were not only associated with the presence against FDCs (FDC-M1), B cells (B220), and macrophages (F4- of FDCs in enriched cell clusters but were also dependent on sig- 80) showed that clusterin, MFG-E8, and PrPC were expressed in naling through LT␤R (Fig. 5A). The intersection of the 80 genes the B cell area of splenic follicles (Fig. 6). Whereas clusterin im- associated with FDC disappearance upon blockade of LT␤R sig- munoreactivity was also found at low levels in the marginal zone naling and the 960 genes enriched in FDC cell clusters yielded a and red pulp (Fig. 6A), most of the MFG-E8 and PrPC signal was core set of 12 genes (Fig. 5B), three of which were also identified confined to the follicular area (Fig. 6, B and C). High power im- by SSH (Clu, Mfge8, and Serpina1). Two of the proteins encoded ages of double-stained spleen cryosections with each of the Abs by the 12 genes are related to cell adhesion (GlyCAM-1 and MFG- directed against the candidate proteins and FDC-M1 or FDC-M2 E8), three have enzymatic activity (ENPP2, GDA, and LYSZ), one showed a high degree of colocalization of all three candidate pro- has chemotactic activity (CXCL13), one is related to apoptosis teins with these two markers on cells with morphology typical of (clusterin), one to proteolysis (serpin a1a), one is an ECM com- FDCs localized on FDCs (Fig. 7). In the case of MFG-E8 staining ponent (cochlin), and for three the functions are not clear at the was also observed on round cells lacking the typical shape of time (prion protein, glycoprotein m6b, RIKEN C030033F14Rik). FDCs in addition to FDCs (Fig. 7, C and D). These cells may We verified the down-regulation of the core set genes by block- represent tingible-body macrophages. As Hanayama et al. (28) ade of LT␤R signaling and the overrepresentation of these genes in have previously shown by costaining of MFG-E8 with CD68, 5532 FDC TRANSCRIPTOME Downloaded from

FIGURE 5. Identification and confirmation of genes blocked by soluble LT␤R-Ig treatment and overexpressed in FDC clusters. A, Venn diagram http://www.jimmunol.org/ illustrating the approach used to identify genes blocked by soluble LT␤R-Ig treatment and overexpressed in FDC clusters. Twelve core set genes were found in common between 80 genes correlating with expression pattern A in mesenteric lymph nodes upon soluble LT␤R-Ig treatment (correlate with cluster A) and the 960 genes overrepresented in FDC-enriched cell clusters (FDC-E). B, The 12 core set genes with their corresponding proteins are listed. NCBI accession numbers are indicated in column 4 (footnote 1). Maximal fold down-regulation relative to control treatment as detected by microarray analysis is indicated in column 5 (footnote 2). Fold overrepresentation in FDC-enriched cell clusters (FDC-E) relative to FDC-depleted (FDC-D) spleen cells are indicated. C, Down-regulation of the 12 core set genes by soluble LT␤R-Ig treatment and enrichment in FDC-cell clusters was confirmed by quantitative real-time PCR. Soluble LT␤R-Ig treatment shown as values of fold-reduction relative to control treatment (PBS) at the day of maximal effect (soluble LT␤R-Ig treatment). FDC enrichment shown as values expressed as fold-enrichment in FDC-enriched cell clusters relative to non-FDC spleen cells. The experiment was done in triplicate and SEM is indicated with error bars. by guest on September 28, 2021

FIGURE 6. Analysis of the localization and expression of a subset of the core candidates by im- munoﬂuorescence double staining. To determine whether the candidate proteins were localized in splenic GCs (right) and localized in FDCs (left)ina LT␤R-dependent fashion, cryostat sections of spleens from control-treated mice (PBS) or mice treated with soluble LT␤R-Ig for 2 days, were stained with Abs or antisera directed against clusterin (A), MFG-E8 (B) and PrPC (C) (all green) and Abs to FDCs (FDC-M1), B cells (B220), and macrophages (F4-80), respectively (red). Reactivity against the candidate proteins colocalized mainly with FDC networks in control-treated mice, whereas it was mostly absent 2 days after administration of soluble LT␤R-Ig (left). Costaining with B cell and macrophage markers demonstrated that expression of the candidate proteins was conﬁned to the follicular B cell areas and that treatment with soluble LT␤R-Ig did not lead to down-regulation of the B220 and F4-80 epitopes (right). Exposure times of images with the same immunostainings were kept constant to ensure comparability of the images. The images are representative of two independent experiments. Scale bars represent 200 ␮m. The Journal of Immunology 5533

set genes and no signiﬁcant change in the remaining nine genes (Fig. 8A).

Clusterin acts as a survival factor for GC B cells in vitro Experiments with FDC cultures have suggested that FDCs provide Ag-independent support for B cell follicles and GC reactions (10– 13). We therefore investigated whether clusterin, human ␣1-antitrypsin (the human homologue of Serpina1a), and LPA, which is produced by ENPP2 (31), could support survival of GC B cells in vitro. In two independent experiments GC B cells were isolated from Ova-immunized C57BL/6 mice by positive or negative (data not shown) magnetic bead sorting. Viability of GC B cells placed in culture under low serum conditions (1.5% FCS) was increased from 36.8 to 60.4% after 24 h, from 18.9 to 42.4% after 48 h, and from 9.9 to 25.6% after 72 h and in 35 to 57% after 24 h, from 8 to 29% after 48 h, and from 2 to 19% after 72 h of culture with purified human clusterin relative to untreated controls (Fig. 8B). Treatment with anti-CD40 Ab, a potent stimulator preventing GC Downloaded from B cell apoptosis (32, 33), increased survival to a similar degree as did clusterin (Fig. 8A) with 59.6% viability at 24 h, 48.1% at 48 h, and 33.9% at 72 h. To exclude the possibility that contaminating endotoxins may have contributed to the survival effect observed in FIGURE 7. High power images depicting colocalization of clusterin, the clusterin treated samples, we included boiled clusterin as a MFG-E8, and PrPC with FDC markers FDC-M1 and FDC-M2. Cryostat control. This treatment abrogated the GC B cell survival effect of http://www.jimmunol.org/ sections of spleens were costained with Abs or antisera directed against clusterin (Fig. 8D) (54% viability at 24 h, 30% at 48 h, and 20% C clusterin (A and B), MFG-E8 (C and D), and PrP (E and F) (all green) and at 72 h). Addition of human ␣1-antitrypsin had little effect during the FDC markers FDC-M1 (A, C, and E) or FDC-M2 (B, D, and F) (red). the first 48 h of culture but increased viability of GC B cells to ϫ Original magnification of the images is 200. 20% at 72 h of culture (Fig. 7D). Viability of cultured cells was not increased by addition of LPA or ␣1-antitrypsin to the medium (Fig. 8, E and F), which speaks against a survival function of these these non-FDC cells are most likely tingible-body macrophages (Fig. 7C). molecules for LPA in the GC reaction. Both, positive and negative We then tested whether blockage of LT␤R-signaling by soluble sorting (data not shown) of GC B cells yielded identical results and by guest on September 28, 2021 LT␤R-Ig affected protein expression levels of the three candidates. it is therefore unlikely that the survival results from activation of Costaining of spleen cryosections taken from mice treated with the cells during isolation. soluble LT␤R-Ig for 2 days showed that protein expression of clusterin, MFG-E8 and PrPC and localization within the GC were all critically dependent on intact LT␤R signaling (Fig. 6). Discussion Although much is known about the developmental and morpho- Expression of the core set genes in mice lacking GC and FDC genetic events governed by LT␤R signaling, the LT␤R-dependent networks molecular cascades that control GC and FDC network function are Having defined a core set of genes whose expression was associ- ill defined. To circumvent the inherent difficulties in analyzing ated with functional GC and FDC networks, we assessed whether these structures, we used two complementary approaches to char- expression of these genes is uniquely down-regulated by pharma- acterize the transcriptional profiles associated with GC and FDC cologic suppression of LT␤R signaling, or whether they would networks and to identify genes that may functionally contribute to also be down-regulated in various mouse models with genetic dis- the events occurring therein. turbances of these structures. We therefore measured the expres- In a first approach, we took advantage of the transient depletion sion levels of each of the 12 genes by quantitative real-time PCR of GCs and FDCs that occurs upon interference with the LT sig- in spleens of LT␤RϪ/Ϫ, TNFR1Ϫ/Ϫ, and TNFR2Ϫ/Ϫ mice, and naling axis by administration of soluble LT␤R-Ig. We reasoned compared them with expression in wild-type mice (C57BL/6). that any gene that might experience down-regulation under these In LT␤RϪ/Ϫ mice, eight of 12 genes were significantly down- conditions would represent at least a candidate marker for GCs and regulated (Fig. 8A). Particularly strong was the down-regulation of FDCs, and that some of them might be involved in GC or FDC Cxcl13 (97.0-fold), Clu (8.1-fold), Enpp2 (15.3-fold), Mfge8 function. (22.8-fold), and Serpina1a (34.7-fold). Mice deficient in TNFR1 Transient blockade of the LT␤R pathway led to the identifica- signaling showed a similar down-regulation in seven of the eight tion of a total of 80 differentially expressed transcripts that exhib- genes down-regulated in LT␤RϪ/Ϫ mice (Fig. 8A). However, the ited rapid and reversible changes in gene expression after admin- fold-reduction was not as pronounced (Cxcl13, 3.3-fold; Clu, 2.4- istration of soluble LT␤R-Ig. These closely followed the sequence fold; Enpp2, 2.4-fold; Mfge8, 2.4-fold; and Serpina1a, 9.4-fold). of histologic disappearance and reappearance of FDCs, GCs, and This is consistent with the observation that the lymphoid architec- their markers from mesenteric lymph nodes and may be directly ture of TNFR1Ϫ/Ϫ mice is less dramatically impaired than that of regulated by LT␤R signaling. The proteins encoded by these genes LT␤RϪ/Ϫ mice. TNFR2Ϫ/Ϫ mice do not have any impairment in were predominantly assigned to four key categories (I-IV). This formation of GC and FDC networks (29, 30). Accordingly, expands previous reports that LT␤R signaling controls proinflam- TNFR2Ϫ/Ϫ mice showed marginal reduction in three of the 12 core matory molecules, chemokines, and cytokines (34). The fact that 5534 FDC TRANSCRIPTOME

FIGURE 8. Expression of the core set genes cor- relates with presence of functional GC. A, Real-time quantitative PCR was performed on cDNAs derived from spleens of C57BL/6, LT␤RϪ/Ϫ, TNFR1Ϫ/Ϫ, and TNFR2Ϫ/Ϫ mice. Fold down-regulation was calculated relative to C57BL/6 mice after normaliza- tion to the ␤-actin signal. Error bars indicate SEM and are representative of three biological replicates. Puriﬁed murine GC B cells were cultured in the presence or absence of anti-CD40 Ab FGK45.5 (10 Downloaded from ␮g/ml) (B), puriﬁed human serum clusterin (50 ␮g/ ml) (C), boiled human clusterin (50 ␮g/ml) (D), LPA (1 ␮M) (E), or human ␣1-antitrypsin (1 mg/ml) (F). Viability was assessed by trypan blue exclusion at the start of the cultures and after 1, 2, and 3 days. The experiment was performed in triplicate plus http://www.jimmunol.org/ SEM. SD is indicated by error bars. by guest on September 28, 2021

25% of these genes are normally associated with the stromal com- appreciated yet. Ablation of VCAM-1 expression had identified a partment of secondary lymphoid organs emphasizes the impor- crucial role for this molecule in mounting of efficient secondary tance of this compartment in executing functions controlled by IgG1 Ab responses (41). GlyCAM-1 has been shown to stimulate LT␤R signaling. adherence of lymphocytes to ICAM-1 expressing high endothelial Our second approach consisted of analyzing cell clusters highly venules via activation of L-selectin (42). Local synthesis of Gly- enriched for FDCs. These clusters represented small functional CAM-1 suggests that FDCs may actively stimulate B cells, via units of GCs and allowed us to define a set of genes overexpressed activation of L-selectins, to attach to ICAM-1 molecules present in such structures. Particularly remarkable was the identification of on FDCs. Additional experiments will need to be devised to clarify genes of the functional categories (I) surface receptors and cell the contribution to FDC function of the additional cell adhesion adhesion molecules, (II) chemokines and cytokines, and (X) ECM molecules identified in this study. components. In accordance with elevated expression of cytokines exerting We have identified a specific set of chemokines that are ex- their effects mainly on the stromal compartment (PDGFA, CTGF, pressed in association with FDCs. These are able to attract the BMP-2), we detected a marked increase in transcripts encoding typical cell types of hemopoietic origin, which cluster during a GC several types of collagens and other ECM proteins. It is likely that reaction in FDC networks, such as B cells (CXCL13) (35), Th cells their increased deposition in the vicinity of FDCs facilitates mi- (CXCL10, CXCL12) (36, 37), and macrophages/monocytes gration and adherence of immune cells into this area, for example, (CCL2, CXCL12) (37, 38). Their high level expression within via interactions with integrins on these cells. They may also have FDC networks explains the chemotactic activities for B cells and proliferative effects, as was shown for biglycan, which stimulates Th cells previously identified in FDC conditioned medium (10). proliferation of pre-B cells by bone marrow stromal cells (43); and The overrepresentation of several cell adhesion molecules in may enhance clustering of B lymphocytes in this area, as is the FDC cell clusters reflects the intensive intercellular contacts oc- case for tenascin in an LFA-1-dependent fashion (44). curring between FDCs and cognate cells of hemopoietic origin. By intersection of the two analyses we selected a core set of ICAM-1 and VCAM-1 are both highly expressed on FDCs (39), genes that is not only tightly associated with FDCs and is highly and similarly E-selectin has been detected on FDCs present in expressed within GC, but is also dependent on signaling through inflamed thyroid glands of patients with Hashimoto’s thyroiditis the LT␤R. We demonstrated the validity of this approach by pro- (40). Expression of GlyCAM-1 and MCAM by FDCs had not been tein expression analysis of PrPC, clusterin, and MFG-E8. The Journal of Immunology 5535

Expression analysis in spleens of LT␤RϪ/Ϫ, TNFR1Ϫ/Ϫ, and instances of spongiform encephalopathies, including scrapie (50) TNFR2Ϫ/Ϫ mice uncovered that most core set genes are down- and variant (51) as well as sporadic (52) Creutzfeldt-Jakob disease. regulated whenever induction of GC structures and FDC network Whenever the localization of prions within the lymphoreticular formation is defective (LT␤RϪ/Ϫ and TNFR1Ϫ/Ϫ mice) (1, 2, 30), system was studied, they were traced to FDCs. but are normally expressed when normal induction is possible, as It is not implausible to expect that pharmacologic ablation of in wild-type or TNFR2Ϫ/Ϫ mice (29). Expression of three of the FDCs may be beneficial in these diseases, as it would remove the core set genes (Cxcl13, Mfge8, and Clu) has been found to be sanctuaries of pathogen persistence. If the regimen is initiated diminished also in LT␣Ϫ/Ϫ mice (45), supporting the concept that early after exposure, depletion of FDCs by soluble LT␤R can expression of these genes is linked to the presence of functional largely protect mice from the clinical consequences of exposure to GC and FDC networks. The fact that seven of eight genes down- infectious scrapie prions (21). The set of molecules identified in regulated in LT␤RϪ/Ϫ spleens were also down-regulated in the present work may therefore encompass potential therapeutic TNFR1Ϫ/Ϫ spleens suggests that the two pathways exert partially target genes, whose manipulations in the conditions described redundant functions. Given that the two receptors share down- above may prove clinically beneficial. stream signaling adaptors, such as NF-␬B (46), it is not surprising that an overlapping set of genes is down-regulated in mice lacking Acknowledgments the respective receptors. We thank Rita Moos for excellent technical assistance and Bernhard Odermatt Our core set of genes indicates that LT␤R signaling participates for discussion. at several stages in the GC reaction by controlling gene expression associated with FDC networks: secretion of chemokines may at- Disclosures Downloaded from tract subsets of activated cells of hemopoietic origin into the fol- The authors have no financial conflict of interest. licular B cell areas. For example, Ag-specific activated B cells are attracted by CXCL13 (35). FDCs and stromal cells present in this References area provide a specific extracellular environment by secretion of 1. Futterer, A., K. Mink, A. Luz, M. H. Kosco-Vilbois, and K. Pfeffer. 1998. The particular ECM components and presentation of cell adhesion mol- lymphotoxin ␤ receptor controls organogenesis and affinity maturation in peripheral lymphoid tissues. Immunity 9:59. ecules, which are permissive for migration, adherence, interaction 2. Pasparakis, M., L. Alexopoulou, M. Grell, K. Pfizenmaier, H. Bluethmann, and http://www.jimmunol.org/ and survival of the infiltrating cells. LT␤R signaling may foster G. Kollias. 1997. Peyer’s patch organogenesis is intact yet formation of B lym- adherence by enhancing expression of molecules such as Gly- phocyte follicles is defective in peripheral lymphoid organs of mice deficient for tumor necrosis factor and its 55-kDa receptor. Proc. Natl. Acad. Sci. USA CAM-1 and cochlin. Activated B cells that have migrated into the 94:6319. GC area differentiate into centroblasts and expand rapidly in an 3. Pfeffer, K., T. Matsuyama, T. M. Kundig, A. Wakeham, K. Kishihara, Ag-independent fashion to undergo somatic hypermutation and Ab A. Shahinian, K. Wiegmann, P. S. Ohashi, M. Kronke, and T. W. Mak. 1993. Mice deficient for the 55 kd tumor necrosis factor receptor are resistant to en- class switch. ␣1-Antitrypsin, the human homologue to Serpina1a, dotoxic shock, yet succumb to L. monocytogenes infection. 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