CD83 Modulates B Cell 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 ol.1502163 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2016/03/16/jimmunol.150216 Material 3.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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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 light 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 protein 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) promoter 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, dendritic cell; 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 gene, 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.