CD83 Modulates Activation and Germinal Center Responses Lena Krzyzak, Christine Seitz, Anne Urbat, Stefan Hutzler, Christian Ostalecki, Joachim Gläsner, Andreas Hiergeist, This information is current as André Gessner, Thomas H. Winkler, Alexander of September 26, 2021. Steinkasserer and Lars Nitschke J Immunol published online 16 March 2016 http://www.jimmunol.org/content/early/2016/03/16/jimmun

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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 © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published March 16, 2016, doi:10.4049/jimmunol.1502163 The Journal of Immunology

CD83 Modulates B Cell Activation and Germinal Center Responses

Lena Krzyzak,* Christine Seitz,* Anne Urbat,† Stefan Hutzler,† Christian Ostalecki,‡ Joachim Gla¨sner,x Andreas Hiergeist,x Andre´ Gessner,x Thomas H. Winkler,{ Alexander Steinkasserer,*,1 and Lars Nitschke†,1

CD83 is a maturation marker for dendritic cells. In the B cell lineage, CD83 is expressed especially on activated B cells and on zone B cells during the germinal center (GC) reaction. The function of CD83 during GC responses is unclear. CD832/2 mice have a strong reduction of CD4+ T cells, which makes it difficult to analyze a functional role of CD83 on B cells during GC responses. Therefore, in the present study we generated a B cell–specific CD83 conditional knockout (CD83 B-cKO) model. CD83 B-cKO B cells show defective upregulation of MHC class II and CD86 expression and impaired proliferation after different stimuli. Analyses of GC responses after immunization with various Ags revealed a characteristic shift in dark zone and light zone B cell Downloaded from numbers, with an increase of B cells in the dark zone of CD83 B-cKO mice. This effect was not accompanied by alterations in the level of IgG immune responses or by major differences in affinity maturation. However, an enhanced IgE response was observed in CD83 B-cKO mice. Additionally, we observed a strong competitive disadvantage of CD83-cKO B cells in GC responses in mixed bone marrow chimeras. Furthermore, infection of mice with Borrelia burgdorferi revealed a defect in bacterial clearance of CD83 B-cKO mice with a shift toward a Th2 response, indicated by a strong increase in IgE titers. Taken together, our results show that CD83 is important for B cell activation and modulates GC composition and IgE Ab responses in vivo. The Journal of Immu- http://www.jimmunol.org/ nology, 2016, 196: 000–000.

he CD83 was introduced in 1992 as an Ig-like is also expressed on a variety of other cell types, including thymus molecule that is expressed on Langerhans cells and ac- epithelial cells (4), T cells (5), and especially regulatory T cells (6, tivated lymphocytes (1). Even though CD83 was mainly 7) and B cells (8). Interestingly, it was shown that CD83 expression T + used as a selective marker for mature dendritic cells (DCs) (2, 3), it on thymus epithelial cells is essential for CD4 T cell development (4). The detailed mechanism for this observation is still unknown, but CD832/2 animals show a strong reduction in the CD4+ Tcell by guest on September 26, 2021 compartment in the thymus as well as in the periphery (4). *Department of Immune Modulation, University Hospital Erlangen, 91052 Erlangen, On developing B lymphocytes CD83 expression is detectable Germany; †Division of Genetics, Department of Biology, University of Erlangen, 91058 Erlangen, Germany; ‡Department of Dermatology, University Hospital Erlan- beyond the pre–B cell stages once they express a functional BCR gen, 91052 Erlangen, Germany; xInstitute for Medical Microbiology and Hygiene, { (9, 10). Naive B cells only express low levels of CD83. In contrast, University of Regensburg, 93053 Regensburg, Germany; and Division of Genetics, after stimulation with anti-IgM or TLR ligands, CD83 is rapidly Nikolaus Fiebiger Center for Molecular Medicine, University of Erlangen, 91058 Erlangen, Germany upregulated on B lymphocytes in vitro and also upon immuniza- 1A.S. and L.N. contributed equally to this work. tion in vivo (10, 11). By generation of CD83-deficient mice, ORCIDs: 0000-0002-5030-1720 (L.K.); 0000-0002-6676-0497 (C.O.); 0000-0002- Fujimoto and colleagues (10) showed that CD83 expression is not 3154-0638 (A.H.); 0000-0001-7187-6729 (A.S.). essential for B cell development or tissue localization, but it is 2/2 Received for publication October 6, 2015. Accepted for publication February 18, required for B cell longevity. When stimulating CD83 B cells 2016. with LPS and anti-IgM in vitro, an impaired CD86 and MHC class This work was supported by Deutsche Forschungsgemeinschaft Grants GK 1660 and II (MHC II) upregulation was observed, whereas no differences SFB1181 Projects B03 and B06. regarding their proliferative potential were detected (4, 12). In The sequences presented in this article have been submitted the National Center for contrast to studies using CD832/2 mice, animals overexpressing Biotechnology Information SRA database (http://www.ncbi.nlm.nih.gov/sra) under accession number PRJNA310174. CD83 under the control of an MHC class I (MHC I) Address correspondence and reprint requests to Prof. Lars Nitschke or Prof. Alexander (CD83 transgenic [CD83tg] mice) show increased CD86 and Steinkasserer, Division of Genetics, Department of Biology, University of Erlangen, MHC II expression as well as higher amounts of IL-10 in super- 91058 Erlangen, Germany (L.N.) or Department of Immune Modulation at the natants of LPS-stimulated B cells (8). In general, the CD83tg mice Department of Dermatology, University Hospital Erlangen, 91052 Erlangen, Germany (A.S.). E-mail addresses: [email protected] (L.N.) or alexander.steinkasserer@ showed stronger B cell phenotypes in vivo compared with the 2 2 uk-erlangen.de (A.S.) CD83 / mice. These phenotypes included B cell maturation 2+ The online version of this article contains supplemental material. defects, defects in B cell Ca signaling, suppression of serum Ig Abbreviations used in this article: alum, aluminum hydroxide; CD83 B-cKO, B cell– levels, reduced humoral responses to thymus-dependent and specific CD83 conditional knockout; CD83tg, CD83 transgenic; CGG, chicken thymus-independent Ags, and reduced Ig responses during in- g-globulin; DC, ; DZ, dark zone; ES, embryonic stem; GC, germinal center; KLH, keyhole limpet hemocyanin; KO, knockout; LZ, light zone; MHC I, fections. Only some of these phenotypes were shown to be B cell MHC class I; MHC II, MHC class II; MID, multiplex identifier; MZ, marginal zone; intrinsic by mixed bone marrow chimeras. The functional role of NGS, next generation sequencing; NP, 4-hydroxy-3-nitrophenylacetyl; sCD83, solu- CD83 on B cells during humoral immune responses to thymus- ble CD83; SHM, somatic hypermutation; T , T follicular helper; WT, wild-type. FH dependent Ags could not be studied in CD832/2 mice owing to + Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 their strong Th cell defect caused by decreased CD4 T cell

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1502163 2 ROLE OF CD83 DURING GERMINAL CENTER REACTION numbers. No influence of a B cell CD83 deficiency on the humoral plifying the CD83 fragments: short arm, 59-TGCGGCCGCATGTCCAG- responses was detected when transfer experiments with CD832/2 TAAGACAAAC-39 and 59-TGGATCCTGAAACCCAGGGCTGTG-39; bone marrow were performed (13). exon 1 and 2 fragment, 59-TCTCGAGGAGCTGCCCACCCTATC-39 and 59-TGTCGACGAGCCAATAGCGAGCCT-39, long arm, 59-TGTCGACTGC- Expression of CD83 on germinal center (GC) lymphocytes was first CACCTTGCTTCTTCATG-39 and 59-TCTCGAGTTCCAGGCAGTGA- shownin1992(1),andsince2012ithasbeenusedasamarkerfor CAGAACC-39. All primer contained restriction sites at their 59 end. light zone (LZ) B cells during GC reactions (14). GC formation takes JM8A3 embryonic stem (ES) cells were electroporated with the linearized place after Ag stimulation in lymphoid organs (15). GCs are the site targeting vector. Four positive ES cell clones were identified and injected into blastocysts. of somatic hypermutation (SHM) and class switch recombination Blastocysts were transferred into pseudopregnant females, and resulting leading to the production of high-affinity Ab-secreting plasma and offspring was analyzed with PCR to identify correct germline mutations. memory B cells (16). One anatomical feature of the GC is its division Those were bred with Flp mice to deplete the neo-cassette and then bred to into dark zone (DZ) and LZ. The DZ is the site where B cells homozygosity. For B cell–specific depletion of the cd83 , floxed CD83 strongly proliferate and SHM occurs, whereas the LZ is the zone animals were crossed with CD19-cre mice (CD83 B-cKO) (25). All mice were on a C57BL/6 background. Age-matched control and B cell–specific where selection for B cells with high-affinity BCRs with the help of CD832/2 mice of 8–14 wk of age were used for analysis, except for follicular DCs and T follicular helper (TFH) cells takes place (15). adoptive transfer experiments where 20-wk-old recipient mice were used. Victora et al. (14) showed a distinct upregulation of CD83 specif- RNA preparation and PCR ically in LZ B cells, whereas DZ B cells express high CXCR4 levels. Additionally, a recent study analyzing secondary GCs on a tran- RNA from single-cell suspension was prepared using an RNeasy Plus mini scriptional level revealed CD83 expression in two of four individual (Qiagen) according to the manufacturer’s instructions. RNA yield was measured using a spectrophotometer (NanoDrop 2000c, Thermo Scien- clusters of class-switched B cells in the LZ. CD83 as an LZ marker tific). For cDNA synthesis, 1 mg RNA was transcribed using a first-strand Downloaded from and the DNA polymerase Polh as a DZ marker segregated the sec- cDNA synthesis kit (Fermentas) as specified by the manufacturer. CD83 ondary GC transcriptional program into four stages that regulated was determined using PCR, and PCR products were an- divergent mechanisms of memory BCR evolution. Distinct changes alyzed using gel electrophoresis. in expression pattern including CD83 may be important for dis- Flow cytometry tinguishing reentry into the DZ from ongoing BCR rediversification Single-cell suspensions from spleen, bone marrow, peritoneal cavity, and (17). CD83 on LZ B cells is thought to be involved in Ag pre- http://www.jimmunol.org/ blood were stained in PBS. Staining was done in ice-cold PBS containing sentation to TFH cells, a process that is important for selection (18). Live/Dead fixable aqua dead cell stain (Life Technologies) using the fol- CD83 expression is not only important as a transmembrane lowing Abs (conjugated with biotin, FITC, PE, PE-Cy7, allophycocyanin, protein on different immune cells, but also a soluble CD83 (sCD83) PerCP, allophycocyanin-Cy7, BV421, and Pacific Blue): anti-B220 (RA3- molecule can be found. sCD83 is generated from the extracellular 6B2; BD Biosciences), anti-CXCR4 (2B11; eBioscience), anti-Fas (15A7; eBioscience), anti-GL7 (GL7; BioLegend), anti-IgM (RMM1; BioLegend), domain of the membrane-bound CD83 and can be detected in anti–MHC II (I-A/I-E; M5/114; BD Biosciences), anti-CD4 (RM4-5; BD human serum (19). This form of sCD83 possesses immunomod- Biosciences), anti-CD5 (53-7-3; BD Biosciences), anti-CD8 (53-6.7; BD ulatory effects, as recombinant sCD83 inhibits DC maturation and Biosciences), anti-CD19 (6D5; BioLegend) anti-CD21 (7E9; BioLegend), subsequently DC-mediated T cell stimulation (20). Application of anti-CD23, anti-CD45.1 (104; BioLegend), anti-CD45.2, anti-CD69 sCD83 into mice reduces severity of autoimmune diseases and (H1.2F3; BioLegend), anti-CD83 (Michel-19; BD Biosciences), and anti- by guest on September 26, 2021 CD86 (Gl-1; BD Biosciences). Biotinylated Abs were further stained with induces prolonged graft survival (21–23). streptavidin-FITC or PerCP-Cy5.5. Detection of cell surface marker ex- However, knowledge regarding distinct mechanisms of the pression was performed with a flow cytometer and analyzed with FlowJo CD83 molecules expressed on different cell types remains still (Tree Star). Living lymphocytes were gated for further analyses. elusive. Previous mouse models were hampered by the fact that in TLR ligand stimulation and cytometric bead array analysis the reported CD83tg mice, a broad CD83 overexpression was obtained on almost all cell types due to the fact that CD83 ex- Splenic cells were sorted with anti-CD19 beads (Miltenyi Biotec), and obtained B cells were cultivated at a density of 106 cells/ml for FACS pression was driven under the control of an MHC I promoter (11). analyses or 5 3 105 cells/ml for cytokine analysis. Cells were stimulated Even in bone marrow chimeras it cannot be excluded that strong for 24–72 h with LPS, CpG, R848, and anti-IgM. Cells were then har- B cell phenotypes are also caused by increasing sCD83 levels in vested for further FACS analyses and supernatants were used for mea- the mice. In vivo B cell functions are hard to study in CD832/2 suring cytokine levels using cytometric bead array (BD Biosciences) mice owing to the strong CD4+ T cell defect that affects humoral according to the manufacturer’s instructions. Ig responses. To gain deeper insights in the role of CD83 on Proliferation assay B cells, we therefore generated B cell–specific CD83 knockout B cells were purified from RBC-lysed splenic single-cell suspensions by (KO) animals. In these mice, we observed characteristic B cell magnetic separation with anti-CD19 beads (Miltenyi Biotec), labeled developmental blocks affecting both B1 cells as well as marginal with CellTrace Violet dye (Molecular Probes/Thermo Fisher Scientific) zone (MZ) B cells. In vitro, CD832/2 B cells showed a strong according to the manufacturer’s instructions and cultivated at a density of 5 impact of loss of CD83 on CD86 and MHC II expression after 10 cells/ml. After 72, 96, and 120 h of stimulation with different con- centrations of LPS, R848, and CpG (InvivoGen), proliferation was ana- stimulation. Additionally, during GC responses, changes in GC lyzed using a flow cytometer and FlowJo (Tree Star). B cell numbers, as well as a characteristic shift of DZ and LZ B cell numbers, were observed in B cell–specific CD83 KO ani- In vitro class switch analysis mals. Furthermore, infection of mice with Borrelia burgdorferi Splenic cell were sorted with anti-CD19 beads (Miltenyi Biotec), and obtained revealed a defect in bacterial clearance of B cell–specific CD83 B cells were cultivated at a density of 106 cells/ml with 10 mg/ml LPS and 10 conditional KO (CD83 B-cKO) mice with a shift toward a Th2 ng/ml IL-4 for 6 d. Supernatants were analyzed for Ig secretion using ELISA. response, underlined by a strong increase in IgE titers. Immunizations and ELISA Materials and Methods CD83fl/fl 3 CD19cre/+ and control mice were i.p. immunized with 100 mg Generation of B cell CD83-deficient mice 4-hydroxy-3-nitrophenylacetyl (NP)–keyhole limpet hemocyanin (KLH) or 50 mg NP– chicken g-globulin (CGG) in aluminum hydroxide (alum) A targeting vector containing floxed exons 1 and 2 of the cd83 gene was with PBS for analysis of T cell–dependent immune responses. Blood was generated by PCR cloning. Three DNA fragments were cloned into the taken at days 0, 7, 14, 21, and 42. At day 63 after first immunization, mice pRAPIDflirt vector (24). The following primes were used for PCR am- were immunized again with 100 mg NP-KLH and blood was taken at days The Journal of Immunology 3

70, 77, and 84. For analysis of GC responses against SRBCs, mice were 9 Results injected i.p. with 10 SRBCs/mouse. Animals were sacrificed 6, 10, or 12 d Generation of B cell–specific CD83 KO animals after immunization (as indicated in figures). Ig serum titers were measured using standard ELISA methods (26). In brief, MaxiSorp plates (Nunc) To delete CD83 in specific cell types, floxed CD83 mice have been were coated with Ag (5 mg/ml NP-BSA) for detection of NP-specific Igs in generated. Therefore, exons 1 and 2 of the five exons of the CD83 sera of immunized mice. The sera were added in serial dilutions. gene were flanked with two loxP sites (floxed) and inserted into the Adoptive transfer genome of ES cells. Positive ES cell clones were injected into embryonic blastocysts and chimeras were generated (Supplemental For bone marrow reconstitutions, a 1:1 mixture of purified wild-type (WT; CD45.1+) and CD83fl/fl 3 CD19cre/+ (CD45.2+) bone marrow cells was i.v. Fig. 1). After germline transmission and deletion of the neomycin injected into lethally irradiated (11 Gy) Rag12/2 mice. Successful re- resistance cassette, floxed mice were used for further matings with constitution of lymphocytes was analyzed 5 wk after transfer using blood CD19-Cre mice for B cell–specific deletion of CD83 (Fig. 1A). The samples by flow cytometry. Seven weeks after transfer, mice were im- successful KO of CD83 in B cells was examined using mRNA and munized with SRBCs, and at days 6 and 12 following immunization, FACS analysis. Because CD83 is weakly expressed on naive B cells, mice were sacrificed and the contribution of different donor cells in several lymphocyte populations was analyzed using anti-CD45.1 and anti- but upregulated on activated B cells (9), LPS-activated B cells were + CD45.2 staining in combination with other markers. examined. The CD83 mRNA of isolated LPS-stimulated CD19 BcellsofCD83fl/fl 3 CD19Cre/+ (CD83 B-cKO) mice was strongly Next generation sequencing and SHM analyses reduced. Quantification of the band intensity using the Bio1D To prepare samples for next generation sequencing (NGS), synthesized software revealed a 30% level of the CD83 cDNA in CD83 B-cKO cDNA was used in a seminested PCR approach. As a forward primer, animals compared with CD83fl/fl control animals (Fig. 1B). FACS Downloaded from VH558 primers (59-GRGCCTGGGRCTTCAGTGAAG-39) containing analysis by Ab staining after LPS stimulation of CD19+ B cells multiplex identifier (MID) tags for individual mice and the A-Key for 454 sequencing were used. In the first PCR round, a Cg1 outer reverse showed loss of CD83 surface expression on B cells of CD83 B- primer (59-GGAAGGTGTGCACACCGCTGGAC-39) and in the second cKO mice, whereas floxed CD83 mice showed comparable upreg- round a Cg1 inner primer (59- GGCTCAGGGAAATAGCCCTTGAC-39) ulationofCD83toWTanimals(Fig.1C).CD83floxedmicewere with the B-Key for 454 sequencing were used. Protocols were estab- used as controls throughout all experiments, as it is known that lished as previously described (27, 28). For purification of PCR products, CD19Cre/+ mice are phenotypically normal (25, 34). The remaining a QiaQuick PCR purification kit (Qiagen) was used according to the http://www.jimmunol.org/ manufacturer’s instructions. DNA yield was assessed using Qubit CD83 mRNA in CD83 B-cKO mice could be due to a noncomplete + (Invitrogen) according to the manufacturer’s instructions. The 454 se- deletionofCD83inCD19 B cells by CD19-Cre, however the quencing was performed on a Roche 454 platform. After splitting for CD83 surface expression was lost. MID tags for the different samples, individual FASTA files for each individual sample containing 4500–8000 high-quality reads were ana- Reduced splenic MZ B cell and reduced B1a cell numbers in lyzed on the IMGT/HighV-QUEST platform (29). Sequences using the peritoneal cavity of CD83 B-cKO mice canonical V186.2 gene (resembling the VH1-72 gene according to the IMGT nomenclature) were subjected to a Bayesian estimation of Ag- First, B cell development was examined in CD83 B-cKO mice. In the driven selection using the BASELINe algorithm (30). bone marrow, no changes in pro–B, pre–B immature, or mature B cells were detected (not shown). However, in the spleen we observed Infection with B. burgdorferi reduced transitional B cells type 2 and MZ B cell numbers in CD83 by guest on September 26, 2021 Infection of mice with B. burgdorferi as well as measurement of ankle B-cKO mice compared with control animals (Fig. 2A). In contrast, swelling, B. burgdorferi–specific Ab response, and bacterial load in organs mature follicular zone B cell numbers were normal in the spleen were performed as previously described (31). Cytokine production was (Fig. 2B). The subpopulations of B-1 cells were found in normal assessed using a LEGENDplex assay (BioLegend) following the manu- numbers in the spleen (not shown), but in the peritoneal lavage of facturer’s instructions. CD83 B-cKO mice a significant decrease in cell numbers of B1a and Immunofluorescence B2 cells could be detected compared with control mice (Fig. 2C). Thus, B1 or MZ B cells were affected by the loss of CD83, whereas mature Immunofluorescence images of cryosections of the spleen have been generated using the multi-epitope ligand cartography technique. Sample follicular B cells were observed in normal numbers in the spleen. preparations from tissue, data generation, and analysis were performed as Defective upregulation of MHC II and CD86 on activated described previously (22). ImageJ software was used to determine the size 2/2 of DZ and LZ within the GC. CD83 B cells in vitro Statistical analysis We studied B cell signaling in CD83 B-cKO mice, because it was reported that the level of CD83 on B cells affects BCR signaling, Statistical analysis was carried out using the Mann–Whitney U test or the including Ca2+ signaling (8). When we stimulated B cells of CD83 Student t test. B-cKO mice with anti-IgM, we did not detect any changes in Ca2+ Calcium mobilization assay in primary mouse cells signaling (Supplemental Fig. 2A). As it was shown previously that Splenic cells were loaded with Indo-1 as described (32). Cells were stained CD83 expression affects the expression of the B cell activation with anti-CD5 and anti-B220 and analyzed using an LSR II (Becton markers MHC II and CD86 on B cells (11, 12), we separated + Dickinson). Cells were stimulated with anti-IgM [F(ab9)2] (Jackson splenic CD19 cells using MACS technology and analyzed the ImmunoResearch Laboratories). expression of MHC II, CD86, and CD69 after stimulation with Ag presentation assay different stimuli, including the TLR ligands LPS, CpG, R848, or anti-IgM. In this study, a significant defective upregulation of B cells were purified from RBC-lysed single-cell suspensions by magnetic MHC II and CD86 on CD83 B-cKO B cells 24 after stimulation separation with anti-CD19 beads (Miltenyi Biotec), and OT-II CD4+ Tcells were purified with a CD4 purification kit (Miltenyi Biotec) according to the independent of used stimuli could be detected (Fig. 3A). This manufacturer’s instructions. The assay was performed as described (33). In observation was specific for MHC II and CD86, as CD69 ex- brief, B cells were stained with anti-IgM F(ab9)2-biotin and subsequently pression was not affected. Additionally, the observed effect was with OVA Ag delivery agent (Miltenyi Biotec), containing an anti-biotin Ab still consistent after 48 h (not shown) and 72 h after stimulation conjugated to OVA and FITC, to deliver Ag via the BCR. Ag-loaded B cells + (Fig. 3B), independent of the trigger for B cell stimulation. were cocultured with OT-II CD4 T cells, and T cell activation was assessed + via the IL-2 production in the supernatants of cocultures using cytometric CD19 B cells were stimulated with LPS and IL-4 for 6 d bead array (BD Biosciences). and supernatants were examined for IgM and IgG1 levels using 4 ROLE OF CD83 DURING GERMINAL CENTER REACTION Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 1. Successful conditional depletion of CD83 on CD19+ B cells. KO strategy (A) for generation of tissue-specific KO animals. The WT CD83 genome with its five exons is depicted. pRAPIDflirt-CD83 is the targeting vector, which contains the neomycin cassette (Neo) surrounded by flippase recognition target sites (d), the thymidine kinase (tk), and the two loxP sites (<). After mating with Flp mice, the neomycin casette is depleted (CD83flox). Mating with CD19cre mice leads to deletion of flanked exons and thus to specific deletion of CD83 in CD19+ B cells (CD83del). (B) cDNA analysis of cd83 gene expression in CD19+ B cells. The arf gene was used as a control. (C) FACS analysis for CD83 surface expression of LPS-stimulated activated B cells (B220+MHC IIhigh).

ELISA. Addition of IL-4 to B cell culture will lead to a class B cells compared with controls, whereas IL-6 and TNF-a pro- switchandtoashifttoIgG1Absecretioncomparedwithonly duction was not influenced (Fig. 4A). Not only is the cytokine stimulation with LPS. We could detect slightly reduced levels of secretion affected by loss of CD83, but also the proliferative IgM in supernatants of CD83 B-cKO B cells after LPS stimula- capacity of B cells after stimulation with LPS, R848, and CpG at tion, but no changes in IgG1 levels after stimulation with both different concentrations and different time points was signifi- LPS and IL-4 (Fig. 3C). Additionally, serum Ig levels of the different cantly increased (Fig. 4B, 4C). Ig classes were unchanged in CD83 B-cKO mice (Supplemental Increased GC B cells and characteristic shift of DZ and LZ Fig. 2B). B cells numbers in CD83fl/fl 3 CD19cre/+ mice after Higher IL-10 secretion and increased proliferative capacity of immunization with SRBCs fl/fl 3 cre/+ TLR ligand–stimulated B cells of CD83 CD19 mice Previous work has shown that CD83 is an important marker for compared with control B cells LZ B cells within the GC reactions (14). This led us to hy- The loss of CD83 on B cells may also affect cytokine secretion and pothesize that CD83 itself might have a specific function during proliferation after TLR stimulation. Therefore, sorted splenic the GC reaction. To study a potential effect of B cell–specific CD19+ B cells were analyzed after 72 h for secreted cytokines loss of CD83 during the GC reaction, mice were immunized with in their supernatants and the proliferation was measured using SRBCs and at days 6 and 10, splenocytes were analyzed using CellTrace Violet proliferation dye. Interestingly, we could show flow cytometry. Remarkably, we could observe a significant in- increased IL-10 secretion by CpG-stimulated CD83 B-cKO crease in GC B cell numbers of CD83 B-cKO mice compared The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 2. Reduced MZ B cells and reduced B1a cells in peritoneal cavity of CD83fl/fl 3 CD19cre/+ mice. (A) Analysis of splenic B cell subsets. FACS staining of MZ (B220+CD21+CD23low) and follicular zone (FZ) B cells (B220+CD21medCD23high). (B) Staining of follicular mature (FM) cells (B220+ CD21medIgMmed), transitional B cells type 1 (T1; B220+CD21lowIgMhigh), T2-MZ transitional B cells type 2 (T2-MZ), and MZ B cells (B220+CD21high IgMhigh). Further gating on CD23 distinguishes between MZ (CD232) and type 2 (T2) B cells (CD23+). (C) Peritoneal lavage cells were analyzed using FACS for expression of CD19, CD5, and CD43. All results are shown from at least three independent experiments with three to five animals per group. Statistical analyses was performed using a Mann–Whitney U test. *p , 0.05, **p , 0.01. with controls at day 6 (Fig. 5A, 5B). Moreover, a more detailed After immunization with NP-KLH, CD83 B-cKO mice show investigation of the different zones within the GC revealed a normal IgG but increased IgE responses and a characteristic relative increase in DZ B cells (CXCR4+) in CD83 B-cKO an- shift of DZ and LZ B cells numbers + imals, whereas LZ B cells (CD86 ) are relatively decreased at To study GC formation after NP-KLH immunization, animals day 6 (Fig. 5C–E). This abnormality is in line with a significant were immunized with NP-KLH in alum and sera were tested for increase in the DZ/LZ ratio. These changes were restricted to anti-NP–specific IgM and IgG1 by ELISA. Additionally, animals B cell populations within the GCs, whereas TFH cells were not were boosted with NP-KLH at day 63 after first immunization influenced (not shown). GCs were also analyzed at day 10 after and sera were analyzed by ELISA to study secondary immune SRBC immunization, and the results of a shifted DZ/LZ ratio responses. No alterations in the Ag-specific IgM or IgG1 re- were similar to day 6 (Fig. 5H–J). Histology of the spleen con- sponse of CD83 B-cKO mice compared with control mice could firmed these findings. In spleen sections of SRBC-immunized be observed at the indicated time points. Also, no significant mice, the DZ of the GC was identified by the marker GL-7high, differences could be detected in the secondary response after whereas the LZ was identified as CD21highGL-7low (Fig. 5F). boost at day 63 (Fig. 6A). Interestingly, total IgE titers and NP- When the size of the DZ and the LZ area was quantified with specific IgE titers (Supplemental Fig. 4A) of CD83 B-cKO ImageJ software, a characteristic shift in the ratio of DZ/ LZ size mice after NP-KLH were significantly increased at indicated time in CD83 B-cKO mice was detected as well (Fig. 5G). points and a general higher IgE response could be observed. 6 ROLE OF CD83 DURING GERMINAL CENTER REACTION Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 3. Defective upregulation of MHC II and CD86 on CD83fl/fl 3 CD19cre/+ B cells. Splenic B cells were purified by magnetic separation for CD19 and stimulated with indicated stimuli for 24 (A) and 72 h (B). Summary of mean fluorescence intensity (MFI) of respective activation markers (n =3) of one independent experiment. (C) ELISA analysis of supernatants of stimulated CD19+ B cells. B cells were stimulated with LPS and LPS and IL-4 for 6 d. All results are shown from at least three independent experiments with three to four animals per group. Statistical analyses were performed using a Student t test. *p , 0.05, **p , 0.01, ***p , 0.001. MFI, mean fluorescence intensity.

SRBC immunization leads to a very strong GC response, so the To study this, an Ag presentation assay was performed. B cells of question arose whether immunization with a thymus-dependent Ag CD83 B-cKO and control mice were loaded via the BCR with will lead to similar characteristic changes GC composition. To OVA and the response of OVA-specific OT-II T cells was mea- investigate this issue, mice were immunized with NP-CGG in alum sured. However, no difference was detected in IL-2 production or and analyzed at days 6 (data not shown) and 12 after immunization. in T cell proliferation after Ag presentation by CD83-deficient or After immunization, GC B cell populations were characterized. control B cells (Supplemental Fig. 3). FACS analysis studying the distribution of DZ and LZ B cells exposed similar findings in line with immunization with SRBCs. Normal Ag-driven selection of CD83 B-cKO GC B cells after Again, DZ B cells are relatively increased and so is the DZ/LZ ratio immunization with NP-CGG (Fig. 6B). We also observed an impaired MHC II expression on To understand whether differences in DZ and LZ B cell numbers GC B cells of CD83 B-cKO mice (Fig. 6C). The impaired MHC II have an influence on the Ag-driven selection within the GC, we expression may affect Ag presentation of CD83-deficient B cells. analyzed the Ab VDJ repertoire of sorted GC cells using NGS. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 4. Higher IL-10 secretion and increased proliferative capacity of TLR ligand–stimulated B cells of CD83fl/fl 3 CD19cre/+ mice compared with control B cells. (A) Splenic B cells were purified by magnetic separation for CD19 and stimulated with indicated stimuli for 72 h. Supernatants were used for cytometric bead array analysis to determine cytokine production. Data represent typical results of two pooled experiments with n = 4 animals/group. Statistical analysis was performed using a Mann–Whitney U test. (B) CD19+ splenic B cells were cultured for 72, 96, and 120 h in vitro. Proliferation was measured by CellTrace Violet dilution. A representative histogram of LPS (20 mg/ml)-stimulated B cells after 72 h is shown. Numbers on top of gate show percentage of the gated population. Mean fluorescence intensity (MFI) for gated population is given. (C) Summary of percentage of divided cells after 72, 96, and 120 h, gated as in (B). Graphs show triplicate samples of one typical experiment as an example of two experiments performed. Statistical analyses were performed using a Student t test. *p , 0.05, **p , 0.01, ***p , 0.001. 8 ROLE OF CD83 DURING GERMINAL CENTER REACTION Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 5. Increased GC B cells and characteristic shift of DZ and LZ B cell numbers in CD83fl/fl 3 CD19cre/+ mice after immunization with SRBCs. Analysis of GC formation 6 and 10 d after immunization with SRBCs is shown. (A) Representative FACS plot of GC B cells at day 6 after immunization. (B) Statistical analysis of one independent experiment with n = 4 mice/group. (C) Representative FACS analysis of DZ (CXCR4+) and LZ (CD86+) B cells at day 6 after immunization. Summary and statistical analysis of DZ/LZ ratio (D) and percentage of DZ and LZ B cells (E) at day 6 after immunization are shown. All results shown are from at least four independent experiments with three to five animals per group. (F) Histological analyses of splenic sections (original magnification 3100) at day 6 after immunization. Staining of tissue sections included IgD (red), CD21 (green), GL-7 (pink), and CD4 (blue). DZ and LZ areas of GCs are marked (white). (G) Statistical analyses of compared size of DZ area to LZ area. Data represent at least three evaluated GCs of one spleen section from three mice of each genotype. (H) Statistical analysis of GC B cells at day 10 after immunization of one independent experiment with n = 4 mice/group. (I) Representative FACS analysis of DZ (CXCR4+) and LZ (CD86+) B cells at day 10 after immunization. Summary and statistical analysis of the DZ/LZ ratio are shown in (J). Statistical analyses was performed using a Mann–Whitney U test. *p , 0.05, **p , 0.01, ***p , 0.001.

CD83 B-cKO and control mice were immunized with NP-CGG and NGS sequence data are deposited at the National Center for 12 d after immunization, NP binding GC B cells were sorted. After Biotechnology Information SRA database (http://www.ncbi.nlm. RNA extraction and cDNA synthesis, PCR using a specific primer nih.gov/sra, accession no. PRJNA310174). The obtained results for the J558 VH gene family and Cg1 primer was performed. The were analyzed using IMGT/V-QUEST software (29). We focused The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 6. Normal thymus-dependent immune response in CD83fl/fl 3 CD19cre/+ mice after immunization with NP-KLH, but increase in total IgE titers and characteristic shift of DZ and LZ B cells numbers with comparable Ab affinity. (A) Analysis of specific Ab levels at indicated time points after immunization with NP-KLH. Anti-NP IgM and IgG1 responses as well as total IgE titers were measured by ELISA. (B) Analysis of GC formation 12–13 d after immunization with NP-CGG. Representative FACS analysis of DZ (CXCR4+)andLZ(CD86+) B cells of immunized CD83fl/fl,CD83fl/fl 3 CD19cre/+,andnonimmunized CD83fl/fl mice as control. Summary and statistical analysis of the DZ/LZ ratio of one independent experiment with n = 4 mice/group are shown. (C)MHCII expression quantified from B220+,GL7+Fas+ cells of NP-KLH immunized mice (day 12). All results (mean 6 SEM) are shown from at least two independent ex- fl/fl periments with four to five animals per group. (D) Frequency of W33→LmutationsintheVH186.2 gene. NP-binding GC B cells were sorted from CD83 3 cre/+ CD19 andcontrolmice12dafterimmunizationwithNP-CGGandNGS was performed to analyze Ab affinity. Percentage of W→33L mutations of the VH186.2 gene was calculated and summary (mean 6 SEM) of the total of five mice per group is shown in a scatter plot. (E) Analysis of selection (Figure legend continues) 10 ROLE OF CD83 DURING GERMINAL CENTER REACTION

our analysis on productive sequences of the VH186.2 gene for with B. burgdorferi and the clinical manifestation, bacterial clear- which a characteristic high-affinity anti-NP W33L mutation has ance, and B cell responses were analyzed. Although CD83 B-cKO been described repeatedly (35). On average, ∼60% of all sequences animals show comparable relative ankle swelling and thus no on day 12 GC B cells from five control and five CD83 B-cKO mice worsening in disease symptoms (Fig. 8A), their ability to control the contained the W33L mutation, suggesting a similar affinity matu- pathogen is significantly impaired in the infected (i.e., right) tibio- ration in both types of mice (Fig. 6D). For a more detailed analysis, tarsal joint and by tendency is impaired in the heart (Fig. 8B). FACS we subjected all VH186.2 sequences to a quantitative analysis of analyses of GCs in the spleen 45 d postinfection revealed again a Ag-driven selection recently described (30). As shown in Fig. 6E, characteristic shift in the DZ/LZ ratio (Fig. 8C). Splenocytes, which cumulative analysis of all sequences from individual mice clearly were taken from infected mice at day 45 postinfection, were shows positive selection in the CDR regions as indicated by positive restimulated with Con A and supernatants were analyzed for cy- selection strength (S) values and negative or no selection in the tokine secretion. Interestingly, a specific reduction of INF-g was framework regions. As the S values were comparable in control and observed, whereas IL-6 or TNF-a, for example, was not altered CD83 B-cKO mice, Ag-driven selection is apparently not affected (Fig. 8D). Induction of IL-4 or IL-5 could not be detected. Con- by the altered DZ/LZ ratio. comitant with decreased INF-g production, analysis of sera of 2 2 B. burgdorferi–infected mice using ELISA showed a strong increase Competitive disadvantage of CD83 / B cells after bone 2 2 in total IgE titers in CD83 B-cKO mice compared with controls at marrow transfer in RAG / animals all eight time points after infection was observed (Fig. 8E). In Despite the absence of significantly altered Ag-driven selection in contrast, specific IgG1 and IgG2c levels were comparable between the GCs of CD83 B-cKO mice, we wanted to gain further insight CD83 B-cKO and control mice (Supplemental Fig. 4B). Downloaded from into the role of CD83 during GC reactions in a competitive setting 2/2 between CD83 and WT B cells. Thus, BM chimeras were Discussion generated by mixing BM cells from both CD83 B-cKO and Ly5.1 CD83 B-cKO animals showed an overall normal B cell development control mice. CD83-cKO cells are positive for the congenic in the bone marrow and normal B cell numbers in the periphery. marker CD45.2, whereas Ly5.1 cells are positive for CD45.1. CD83 is only weakly expressed on the surface of naive B cells (9) but

Bone marrow cells from both groups were mixed in a 50:50 ratio http://www.jimmunol.org/ 2 2 is strongly upregulated on the surface of activated B cells, and and injected i.v. in RAG / recipients. Five weeks after cell therefore no strong changes on B cell maturation were expected transfer, blood was taken and reconstitution of lymphocyte pop- upon the loss of CD83. Nevertheless, the reduction of MZ and B1a ulations was analyzed using FACS. Interestingly, even in un- cell numbers has so far not been observed in mice with a total CD83 challenged, naive mice, a competitive disadvantage of CD83-cKO KO (10). However, mice carrying a point mutation in the CD83 B cells in the blood could be observed. In contrast, T cell pop- gene, which affects CD83 expression, also show decreased MZ ulations, that is, CD4+ and CD8+ T cells of the KO bone marrow, B cell numbers, whereas transgenic mice overexpressing CD83 did not show reduced reconstitution (Fig. 7A). To examine show increased MZ numbers (9). Thus, the number of MZ B cells whether this effect would persist after challenge by immunization, seems to be directly affected by the expression level of CD83. reconstituted bone marrow chimeras were immunized with SRBC, by guest on September 26, 2021 MZ B cells belong to the innate-like B cells that produce nat- and B cell populations, as well as the GC reaction, were analyzed ural IgM and are also involved in thymus-independent type 2 6 and 12 d after immunization by flow cytometry. Strikingly, a responses. Immunizations of CD83 B-cKO mice with thymus- disadvantage for CD83-cKO B cells could still be observed and independent type 2 Ags did not lead to changes in specific Ab the effect was even stronger within the GC B cell population responses (results not shown). Additionally, B1 cells from the (Fig. 7B). A strong reduction of CD83-cKO B cells within the GC spleen and peritoneal cavity also possess pronounced innate B cell population was also found on day 12 after immunization, functional features and also cooperate with MZ B cells (37). whereas the percentages of B220+ B cells are unaltered at the later Similar to MZ B cells, B1 cells produce natural Abs and they time point (Fig. 7C). Within the GC B cells, there is also a strong are the main producers of natural IgM (38). Despite the re- decrease in both LZ as well as DZ B cells from CD83-cKO cells. duced numbers of MZ B cells and peritoneal B1a cells, we did A clear shift in the DZ/LZ ratio could not be observed. Only at not observe decreased levels of natural IgM in the serum of day 12 could marginal differences be detected, with a tendency of CD83 B-cKO mice. Apparently the normal numbers of B1 cells more CD83-cKO B cells being present in the DZ compared with in spleen and other organs produce unchanged IgM levels. WT B cells (Fig. 7D). CD83 B-cKO bone marrow–derived CD4+ It has been reported in CD83tg mice that higher expression of and CD8+ T cells in the spleen are significantly increased both at CD83 leads to impaired BCR signaling with impaired proximal day 6 and day 12 postimmunization. tyrosine kinase signaling and decreased Ca2+ mobilization (11, 39). CD83 B-cKO mice show normal clearance of B. burgdorferi This led to the hypothesis that CD83 might be a negative regulator infection with a shift toward a Th2 response of BCR signaling. Our demonstration of normal Ca2+ signaling in To obtain insight in the role of CD83 on B lymphocytes during a B cells of CD83 B-cKO mice does not support this hypothesis. bacterial infection in which Abs play an important role for pro- One possibility to explain these different results is the fact that an tection, CD83 B-cKO mice were analyzed in the murine model of MHC I promoter was used for the CD83tg mice. Overexpression Lyme arthritis. Because B. burgdorferi elimination is completely on other cell types may influence B cell functions or general dependent on adequate Ab production and because bacterial cells overexpression of CD83 may lead to increased levels of sCD83, are strong B cell mitogens, this infection model is ideal for the which is immunomodulatory (20, 23) study of B cell activation, GC reactions, and especially Ab re- The defective upregulation of CD86 and MHC II after TLR sponses (36). CD83 B-cKO and control animals were infected stimulation in CD83 B-cKO mice was similarly observed in total

strength by subjection of sequences to BASELINe mutation analysis software. Statistical analyses were performed using a Student t test or Mann–Whitney U test. *p , 0.05, **p , 0.01. The Journal of Immunology 11 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 7. Competitive disadvantage of CD832/2 B cells after bone marrow transfer in RAG2/2 animals. Bone marrow cells of CD83fl/fl 3 CD19cre/+ mice and Ly5.1 control mice were mixed in a 50:50 ratio and injected i.v. in RAG2/2 mice. Seven weeks after transfer of bone marrow cells, mice were immunized with SRBCs and 6 or 12 d postimmunization (p.i.), spleens were analyzed using FACS. For statistical analysis, the percentage of CD45.1+ cells is compared with the percentage CD45.2+ cells after gating on respective cell type (e.g., gating on B220+, CD4+, or CD8+ cells). (A) Five weeks after transfer of bone marrow cells, blood was taken and reconstitution of lymphocyte populations was analyzed using FACS. Representative FACS plots of blood of bone marrow chimeras and summary (mean 6 SEM) of n = 8 reconstituted mice. (B and C) Summary of B cell and T cell distribution in spleens 6 and 12 d p.i. Analysis of B cells and GC B cells at day 6 (B) and day 12 (C) p.i. is shown. (D) Statistical analysis of the ratio of DZ (CXCR4+) to LZ B cells (CD86+) at day 10 after immunization. Statistical analyses were performed using a Student t test. *p , 0.05, **p , 0.01, ***p , 0.001.

CD83 KO mice (4, 12, 18), and CD83tg mice showed increased not observed in B cells of total CD83 KO mice (4). We conclude CD86 and MHC II expressions on LPS-stimulated B cells (8). A that B cells of CD83 B-cKO mice have a B cell activation defect potential role of CD83 on MARCH1-dependent ubiquitination of after BCR or TLR stimulation, resulting in impaired upregula- MHC II and CD86 has been reported. In this study, the trans- tion of activation markers, a cytokine deviation but proliferation membrane domain of CD83 enhances MHC II and CD86 ex- advantage. pression by blocking association of MHC II with MARCH1 (18). CD83 is used as a marker for LZ B cells during GC reactions This mechanism could be an explanation for accelerated cell (14). CD83-cKO mice showed a characteristic shift in the DZ/ LZ surface turnover of CD86 and MHC II in CD83 KO cells (12). As ratio not only after SRBC immunization, but also when a soluble CD83 is important for stabilizing MHC II and CD86 on the cell Ag such as NP-CGG was used or during B. burgdorferi infection. surface, its loss would lead to diminished MHC II and CD86. As the function of CD83 on LZ GC B cells is unknown, it can only TLR stimulation not only affected upregulation of activation be speculated about the mechanism for the relative expansion of markers, but it also led to increased proliferation and increased IL- DZ cells in GCs from CD83 B-cKO mice. Because a role of CD83 10 secretion. Changes in IL-10 levels were also observed in CD83tg in Ag presentation has been detected (18) and because CD83 is mice, because LPS-stimulated B cells of these mice secrete higher highly expressed on LZ GC B cells, CD83 may be involved in Ag amounts of IL-10 (8). In our CD83 B-cKO B cells, LPS-induced presentation to TFH cells in the LZ and thereby in the selection cytokine secretion was normal, whereas CpG-induced IL-10 was process of GC B cells. Surprisingly, Ag presentation was normal, found in higher levels. Surprisingly, an increased proliferation was even though not only CD83 is missing, but also MHC II is 12 ROLE OF CD83 DURING GERMINAL CENTER REACTION Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 8. CD83 B-cKO mice show impaired clearance of B. burgdorferi infection with a shift toward a Th2 response. Control and CD83 B-cKO were infected s.c. with B. burgdorferi into the right hind foot pad. (A) The clinical manifestation of the disease was monitored via the relative ankle swelling of the right compared with the left tibiotarsal joint. (B) The bacterial burden was assessed at day 45 postinfection in the right tibiotarsal joint and in the heart by quantitative PCR using extracted DNA. Copy numbers of borrelial flagellin B gene (flaB) gene were normalized to mouse nidogen-1 gene. (C)AnalysisofGC formation by 45 d of infection with B. burgdorferi. FACS analysis of DZ (CXCR4+) and LZ (CD86+) B cells of infected CD83fl/fl and CD83fl/fl 3 CD19cre/+ mice. Summary and statistical analysis of the DZ/LZ ratio are shown. (D) Cytokine secretion of restimulated splenocytes derived from mice at day 45 of infection with B. burgdorferi. Splenocytes were stimulated with Con A for 48 h and supernatants were analyzed using LEGENDPlex assay. (E) Total IgE Ab levels in sera of B. burgdorferi infected mice. Total IgE was detected by ELISA at indicated time points. All results are shown from a summary (mean 6 SEM) of 10 animals per group. Statistical analyses were performed using a Mann–Whitney U test. *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 13 expressed at lower levels on CD832/2 GC B cells. It is possible implicated that it could not only be FAS but also other molecules that the in vitro Ag-presenting assay is either not sensitive enough that are important for the emergence of the described rogue GC for subtle alterations in the Ag-presenting capacity of B cells or B cells, and one of those molecules could potentially be CD83. might not reflect the interaction of GC B cells and TFH cells in the During infection with B. burgdorferi, an adaptive immune re- LZ of the GC. As it has been described that B cell division in the sponse is controlled by both T cells and B cells. Lipoproteins of DZ is directly proportional to Ag presentation and selection by B. burgdorferi are strong inducers of humoral immune responses (47, + TFH cells in the LZ (40), the higher number of DZ B cells in CD83 48). B cell responses are enhanced by CD4 T cells, which secrete B-cKO mice is unexpected. This finding would indicate a more high INF-g levels (49) and which are also located in the inflamed efficient selection in the LZ by TFH cells, that is, a negative role of joint (50, 51). Additionally, in the inflamed synovial tissue high CD83 in this process. Clearly, we expected that an accumulation levels of Borrelia-specific Abs are detected, which are essential of B cells in the DZ of CD83 B-cKO mice would affect the rate of for bacterial elimination and of some importance for arthritis SHM and possibly also the magnitude of the Ab response. resolution (52–54). Reduction of INF-g production along with a It was reported before that total CD83 KO mice showed impaired strong increase in IgE titers in CD83 B-cKO mice revealed a Ab responses after immunization with the thymus-dependent Ag change in the immune response toward a Th2 response. However, DNP-KLH (4). The reduced Ig response in total CD83 KO animals Th2 cytokines such as IL-4 are generally very hard to detect in can be explained by a strong reduction of CD4+ Th cells, which B. burgdorferi infections in C57BL/6 mice, and the Th1/Th2 cyto- normally trigger Ig class switch in T cell–dependent immune re- kine ratio is mouse strain specific (55, 56). B. burgdorferi induces sponses. This is in accordance with findings from bone marrow an expansion of MZ B cells and peritoneal B1 cells in WT mice chimeras using transfer of total CD83 KO bone marrow in WT (57). These B cells, which were found in lower abundance in Downloaded from animals, which show no influence on the humoral responses (13). In CD83 B-cKO mice, might be activated via TLR2 by lipoproteins contrast, overexpression of CD83 in CD83tg mice led to reduced of B. burgdorferi. Secreted natural Abs are assumed to contribute humoral responses to thymus-dependent and thymus-independent to reduction of early bacterial replication (38, 58). Thus, the defect Ags (13). Thus, Abs were not affected in CD83 B-cKO mice. in bacterial clearance observed in CD83 B-cKO could be a cu- Despite the shift in DZ and LZ numbers of CD83 B-cKO mice, mulative result of imperfect GC B cell as well as innate B cell

we could not find differences in the frequency of tryptophan to activation and proliferation. http://www.jimmunol.org/ leucine exchange at position 33, which is the most frequently In summary, we could show that CD83 on B cells is an activation observed somatic mutation in VH186.2 leading to a 10-fold higher marker, which is important for B cell activation. B cells lacking affinity after NP-CGG immunizations (35). Furthermore, the de- CD83 fail to upregulate specific costimulatory molecules on the tailed quantification of positive selection using the BASELINe B cell surface, but they show enhanced proliferation. CD83 B-cKO algorithm did also not reveal any significant difference between mice show a characteristic shift in composition of the GC with WT and CD83 B-cKO mice. These results are surprising, but we higher B cell numbers in the DZ and lower numbers in the LZ. cannot exclude that a different Ag dose or analysis of other im- Although this shift does not drastically impair the amount and munization time points may have a stronger effect on affinity affinity of Ag-specific IgG, it leads to increased IgE responses. maturation. We observed a strong disadvantage of CD832/2 Furthermore, a characteristic shift to Th2 responses with higher by guest on September 26, 2021 B cells in participation of the GC response in a competitive cel- IgE production accompanied by impeded bacterial clearance is lular setting of mixed bone marrow chimaeras. Because CD832/2 observed during B. burgdorferi infections in CD83 B-cKO mice. B cells have an activation defect, detected by defective upregu- lation of activation markers needed for B–T cell interactions, this Acknowledgments may lead to strong competitive disadvantage compared with WT We thank S. Angermuller€ and C. Draßner for technical help. B cells in the induction or maintenance of GCs. Interestingly, IgE responses were significantly increased in Disclosures CD83 B-cKO animals compared with controls during an immu- The authors have no financial conflicts of interest. nization with both NP-KLH and B. burgdorferi. Class switching to IgE can be direct or subsequent to IgG1 class switching, as revealed recently by IgE reporter mice (41–43). These studies References showed that IgE-producing plasma cells derived from IgE+ GC 1. Zhou, L. J., R. Schwarting, H. M. Smith, and T. F. Tedder. 1992. A novel cell- surface molecule expressed by human interdigitating reticulum cells, Langerhans B cells are of a more transient nature compared with plasma cells cells, and activated lymphocytes is a new member of the Ig superfamily. J. + derived from IgG1 GC B cells and leave the GC after fewer Immunol. 149: 735–742. selection rounds (41, 43, 44). Thus, when GC B cells of CD83 2. Berchtold, S., P. Muhl-Z€ urbes,€ C. Heufler, P. Winklehner, G. Schuler, and A. Steinkasserer. 1999. Cloning, recombinant expression and biochemical B-cKO mice are present to a higher extent in the DZ of GCs, a characterization of the murine CD83 molecule which is specifically upregulated result of the GC reaction could be increased differentiation of IgE+ during dendritic cell maturation. FEBS Lett. 461: 211–216. GC B cells into plasma cells, with the result of higher IgE titers. In 3. Zhou, L. J., and T. F. Tedder. 1995. Human blood dendritic cells selectively express CD83, a member of the immunoglobulin superfamily. J. Immunol. 154: support of this, a shift of the LZ/DZ ratio was observed in GFP 3821–3835. reporter mice for IgE+ GC B cells. In these studies, IgE+ GC 4. Fujimoto, Y., L. Tu, A. S. Miller, C. Bock, M. Fujimoto, C. Doyle, D. A. Steeber, and T. F. Tedder. 2002. CD83 expression influences CD4+ T cell development in B cells showed an increased proportion of DZ GC cells compared the thymus. 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