Endogenous CD83 Expression in CD4+ Conventional T Cells Controls Inflammatory Immune Responses

This information is current as Katarina Liedtke, Christina Alter, Anne Günther, Nadine of September 28, 2021. Hövelmeyer, Robert Klopfleisch, Ronald Naumann, F. Thomas Wunderlich, Jan Buer, Astrid M. Westendorf and Wiebke Hansen J Immunol published online 27 April 2020

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published April 27, 2020, doi:10.4049/jimmunol.2000042 The Journal of Immunology

Endogenous CD83 Expression in CD4+ Conventional T Cells Controls Inﬂammatory Immune Responses

Katarina Liedtke,* Christina Alter,* Anne Gu¨nther,* Nadine Ho¨velmeyer,† Robert Klopﬂeisch,‡ Ronald Naumann,x F. Thomas Wunderlich,{,‖ Jan Buer,* Astrid M. Westendorf,* and Wiebke Hansen*

The glycoprotein CD83 is known to be expressed by different immune cells including activated CD4+Foxp3+ regulatory T cells (Tregs) and CD4+Foxp32 conventional T cells. However, the physiological function of endogenous CD83 in CD4+ T cell subsets is still unclear. In this study, we have generated a new CD83flox mouse line on BALB/c background, allowing for specific ablation of CD83 in T cells upon breeding with CD4-cre mice. Tregs from CD83flox/flox/CD4-cretg/wt mice had similar suppressive activity as Tregs from CD83flox/flox/CD4-crewt/wt wild-type littermates, suggesting that endogenous CD83 expression is dispensable for the inhibitory capacity of Tregs. However, CD83-deficient CD4+ conventional T cells showed elevated proliferation and IFN-g Downloaded from secretion as well as an enhanced capacity to differentiate into Th1 cells and Th17 cells upon stimulation in vitro. T cell–specific ablation of CD83 expression resulted in aggravated contact hypersensitivity reaction accompanied by enhanced CD4+ T cell activation. Moreover, adoptive transfer of CD4+CD45RBhigh T cells from CD83flox/flox/CD4-cretg/wt mice into Rag2-deficient mice elicited more severe colitis associated with increased serum concentrations of IL-12 and elevated CD40 expression on CD11c+ dendritic cells (DCs). Strikingly, DCs from BALB/c mice cocultured with CD83-deficient CD4+ conventional T cells showed enhanced CD40 expression and IL-12 secretion compared with DCs cocultured with CD4+ conventional T cells from http://www.jimmunol.org/ CD83flox/flox/CD4-crewt/wt wild-type mice. In summary, these results indicate that endogenous CD83 expression in CD4+ conventional T cells plays a crucial role in controlling CD4+ T cell responses, at least in part, by regulating the activity of CD11c+ DCs. The Journal of Immunology, 2020, 204: 000–000.

he glycoprotein CD83 is a type 1 transmembrane protein In addition to the full-length membrane-bound form of CD83, a and member of the Ig superfamily (1). Genetic and soluble form of the protein exists (soluble CD83 [sCD83]) (8). The structural analysis revealed that CD83 is well conserved soluble protein was detected in the blood of healthy donors and

T by guest on September 28, 2021 among mammalian species. The murine cd83 gene consists of five elevated sCD83 levels in patients suffering from hematological exons, and the protein shares 63% sequence homology to the malignancies and rheumatoid arthritis (9, 10). Moreover, sCD83 human protein (2). CD83 has been described as one of the most was described to be released from activated DCs, B cells (8), and prominent marker molecules to identify mature dendritic cells stimulated CD83+ T cells (11). Several studies provide evidence (DCs) (3, 4). However, other immune cells including activated for sCD83 having immunosuppressive functions. Treatment of B cells and T cells have been shown to express CD83 (5–7). DCs or monocytes with sCD83 resulted in impaired maturation and T cell stimulatory function in vitro (12–15). Strikingly, application of sCD83 to mice ameliorates experimental colitis (16), *Institute of Medical Microbiology, University Hospital Essen, University Duisburg- Essen, 45147 Essen, Germany; †Institute for Medical Medicine, University Medical prevents the development of experimental autoimmune enceph- Center of the Johannes-Gutenberg University Mainz, 55131 Mainz, Germany; alitis (EAE) (13), alleviates experimental autoimmune uveitis ‡Institute of Veterinary Pathology, Free University of Berlin, 14163 Berlin, Germany; x (17), and has protective effects in different transplantation settings Transgenic Core Facility, Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany; {Max Planck Institute for Metabolism Research, (18, 19). 2 2 Center for Endocrinology, Diabetes and Preventive Medicine, University Hospital of / ‖ Analysis of CD83 mice and B cell–specific CD83-deficient Cologne, University of Cologne, 50931 Cologne, Germany; and Excellence Cluster mice revealed that CD83 is involved in B cell activation. Loss of on Cellular Stress Responses in Aging Associated Diseases, University of Cologne, 50931 Cologne, Germany CD83 expression in B cells resulted in downregulation of MHC ORCIDs: 0000-0002-6308-0568 (R.K.); 0000-0003-2084-5150 (R.N.). class II (MHC-II) and CD86 as well as decreased proliferation Received for publication January 13, 2020. Accepted for publication April 10, 2020. upon stimulation (20, 21). Stabilization of MHC-II and CD86 by CD83 was also reported for thymus cortical epithelial cells (22) This work was supported by the Deutsche Forschungsgemeinschaft (HA5774/3) to W.H. and DCs (23). These results suggest that CD83 is involved in + Address correspondence and reprint requests to Prof. Wiebke Hansen, Institute of CD4 T cell development and priming by modulating the function 2 2 Medical Microbiology, University Hospital Essen, University Duisburg-Essen, of APCs in the thymus and periphery. Indeed, CD83 / mice Hufelandstraße 55, 45147 Essen, Germany. E-mail address: wiebke.hansen@uk- showed tremendously reduced frequencies of peripheral CD4+ essen.de T cells (24), and downregulation of CD83 on human DCs resulted The online version of this article contains supplemental material. in impaired T cell stimulation in an allogenic MLR (25). Abbreviations used in this article: CD83cKO, CD83flox/flox/CD4-cretg/wt; CD83cWT, CD83flox/flox/CD4-crewt/wt; CHS, contact hypersensitivity; DC, dendritic cell; DNFB, It is well established that regulatory T cells (Tregs) play an 2,4-dinitrofluorobenzene; EAE, experimental autoimmune encephalitis; MHC-II, important role in the maintenance of immune homeostasis and MHC class II; mLN, mesenteric lymph node; sCD83, soluble CD83; Treg, regulatory control of immune responses against pathogens and tumor cells T cell; WT, wild-type. because of their immunosuppressive function (26–28). We iden- + Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 tified CD83 to be highly expressed by Foxp3 Tregs in comparison

www.jimmunol.org/cgi/doi/10.4049/jimmunol.2000042 2 ENDOGENOUS CD83 EXPRESSION REGULATES T CELL RESPONSES

2 with Foxp3 T cells (5). In accordance, CD83+ T cells from Scientific) following the manufacturer’s instructions and stimulated with 1 mg/ml anti-CD3 in the presence of 3 3 105 irradiated splenocytes from CD83-eGFP reporter mice have been shown to express Treg- + associated molecules and to have inhibitory activity in vitro as Thy1.1 BALB/c mice as APCs for 72 h. Proliferation was assessed as loss of eFluo670 on gated CD4+Thy1.2+ T cells by flow cytometry. To deter- well as during experimental colitis in vivo (11). Overexpression of mine the suppressive capacity CD4+CD25+ Tregs were isolated by cell murine CD83 by retroviral gene transfer conferred a suppressive sorting and cocultured with Cell Proliferation Dye eFluor 670–labeled 2 phenotype to CD4+CD252 T cells in vitro and adoptive transfer of CD4+CD25 responder T cells from Thy1.1 mice in the presence of these CD83-overexpressing T cells ameliorated the development 1 mg/ml anti-CD3 and irradiated splenocytes for 3 d. Proliferation was assessed as loss of eFluor 670 on gated CD4+Thy1.1+ responder T cells by of EAE and contact hypersensitivity (CHS) reaction in vivo (5). flow cytometry. Most recently, similar results were described for T cells from bacterial artificial chromosome transgenic mice expressing human Differentiation of Th1 and induced Tregs + 2 CD83. CD4 T cells from these mice exhibited reduced prolifer- A total of 1 3 106 sorted CD4+CD25 T cells were stimulated with ative activity, and transgenic mice recover faster from EAE than 5 mg/ml anti-CD3 (BD Biosciences) plate-bound and 1 mg/ml anti-CD28 wild-type (WT) mice (29). These studies indicate that enforced (BD Biosciences) (Th0) either in the presence of 100 U/ml IL-2 (Thermo Fisher Scientific) and 5 ng/ml rTGF-b (R&D Systems) for CD83 expression modulates the function of CD4+ T cells. + 3 d (induced Treg), in the presence of 20 ng/ml rIL-12 (R&D Systems) and However, the impact of endogenous CD83 on the CD4 Tcell 200 ng/ml anti–IL-4 (Thermo Fisher Scientific) for 5 d (Th1) or in the 2 phenotype, in particular on CD4+Foxp3 conventional T cells, presence of 2 ng/ml rTGF-b, 50 ng/ml rIL-6, 20 ng/ml rIL-1b, 100 ng/ml still remains elusive. CD832/2 mice are not suitable for functional rIL-21, 20 ng/ml rIL-23, and 200 ng/ml anti–IFN-g (all R&D Systems) and analysis of CD83-deficient T cells because of the defect in CD4+ 200 ng/ml anti–IL-4 and 200 ng/ml anti–IL-2 (all Thermo Fisher Scien- tific) for 6 d (Th17). For Th1 and Th17 differentiation, stimulated cells T cell development. Therefore, we have generated a new trans- were split in a ratio 1:1 at day 3 or day 5, respectively. Additionally, for Downloaded from flox genic CD83 mouse line allowing for ablation of endogenous Th17 differentiation, the medium was exchanged at day 3. CD83 expression specifically in the T cell compartment upon DC/T cell coculture breeding with CD4-cre transgenic mice. Interestingly, our study revealed that CD83 expression in CD4+ conventional A total of 5 3 104 MACS purified CD11c+ DCs from WT BALB/c mice 3 5 + 2 T cells controls their activationmostlikelybyinterferingwith were cocultured with 2.5 10 sorted CD4 CD25 T cells isolated from CD83cKO mice or CD83cWT littermates in the absence (control) or presence the activity of APCs. of 1 mg/ml anti-CD3 and 1 mg/ml LPS for 16 h. http://www.jimmunol.org/ Flow cytometry Materials and Methods Mice Flow cytometric analyses were performed using anti-CD3, anti-CD4, anti- CD8, anti-CD25, anti-CD44, anti-CD69, anti-CD62L, anti-CD90.1, anti- To generate CD83flox/+ mice, three DNA fragments containing CD83 in- CD11c, anti-CD40, anti-CD86, anti-CD80, anti-CD19, and anti-CD83 tron 2 (short arm), CD83 exon 3 (exon 3 fragment), or CD83 intron 3–4, (Michel19) (all BD Biosciences) and anti-CD45RB and anti–I-A/I-E exon 4, intron 4–5, and exon 5 (long arm) were amplified from genomic (BioLegend) as allophycocyanin, FITC, PE, Pacific Blue, Brilliant Violett DNA by PCR and inserted into the pRAPIDflirt vector (30). After se- 421 and 510, BD Horizon V450, Alexa Fluor 488, or PE/Cy7 conjugates. quence verification, linearization and purification the targeting construct Dead cells were excluded from analysis by using the Fixable Viability Dye was introduced into BALB/c-derived embryonic stem cells. Embryonic eFluor 780 (eBioscience). Intracellular staining was performed using by guest on September 28, 2021 stem cell clones were selected by G418-positive selection and ganciclovir- the Foxp3 Staining Kit from eBioscience with anti-Foxp3 or anti-CD83 negative selection. Positive clones with homologous integration were (Michel19; BD Biosciences) according to the manufacturer’s recommen- identified by Southern blot and injected into blastocysts to generate chi- dations. For determining the expression level of p–ZAP-70, splenocytes meras. Germline transmission was obtained after crossing with BALB/c from CD83cKO mice and CD83cWT littermates were stimulated with mice. To remove the neomycin resistance-encoding cassette transgenic 1 mg/ml anti-CD3 and 1 mg/ml anti-CD28 for 5, 10, and 20 min or left mice were bred with Rosa-Flp mice on BALB/c background. Successful unstimulated as control. Afterwards, cells were stained with anti-CD4, deletion of the neomycin cassette was verified by PCR. For T cell–specific permeabilized with Cytofix/Cytoperm buffer (BD Biosciences), washed deletion of CD83, homozygous floxed mice were crossed with CD4-cre with permeabilization buffer (eBioscience), and stained with anti-p– mice (CD83flox/flox/CD4-cretg/wt [CD83cKO]). BALB/c mice (Envigo), ZAP-70 (Tyr319; Cell Signaling Technology). Thy1.1 and Rag22/2 mice (both BALB/c background) were bred and IFN-g and IL-17 expression was analyzed after stimulation with maintained under specific pathogen-free conditions at the local Animal 10 ng/ml PMA (Sigma-Aldrich) and 100 mg/ml ionomycin (Sigma- Facility of the University Hospital Essen. Female and male mice were used Aldrich) in the presence of 5 mg/ml brefeldin A (Sigma-Aldrich) for for all experiments. All animal experiments were performed in accordance 4 h, treatment with 2% paraformaldehyde and 0.1% IGEPAL-630 (Sigma- with the guidelines of the German Animal Protection Law and approved by Aldrich), and staining with anti-mouse IFN-g (BD Biosciences) or anti- the State Authority for Nature, Environment, and Customer Protection, North mouse IL-17 Abs (BioLegend). All flow cytometric analyses were done on Rhine-Westphalia, Germany. an LSR II with Diva software (BD Biosciences). Cell isolation Cytokine and sCD83 release Splenocytes were isolated from the whole organ by rinsing with erythrocyte Cytokine concentrations in serum samples or supernatants were quan- lysis buffer and washing with PBS supplemented with 2% FCS and 2 mM tified by polystyrene bead–based Luminex technology (R&D Systems) EDTA. Lymph nodes were removed and crushed through a 100-mm nylon according to the manufacturer’s instructions. The assay was measured mash in PBS supplemented with 2% FCS and 2 mM EDTA. CD4+ T cells on a Luminex AtheNA Multi-Lyte System (Promega) using Luminex IS and CD11c+ DCs were isolated from splenocytes by using the CD4+ T Cell Software. For determining sCD83 serum concentration, the ELISA Kit Isolation Kit or CD11c MicroBeads (both Miltenyi Biotec), respectively for Cluster of Differentiation 83 (CD83) from Cloud-Clone was used according to the manufacturers recommendations. Alternatively, CD4+ according to the manufacturer’s recommendations. The ELISA plate T cells were further purified by cell sorting using an FACSAria II (BD was measured on a Sunrise absorbance microplate reader with Magellan Biosciences). For isolation of CD4+CD25+ and CD4+CD252 T cells, Tracker V7.2 software (Tecan). CD4+ T cells were enriched from splenocytes using the CD4+ TCell Isolation Kit (Miltenyi Biotec), stained with fluorochrome-labeled anti- PCR CD4 and anti-CD25 Abs and separated by using an FACSAria II (BD MACS-sorted CD4+ T cells were stimulated with anti-CD3 for 24 h. Biosciences). Subsequently, total RNA was isolated by using the RNeasy Mini Kit Proliferation (QIAGEN) according to the manufacturer’s recommendation. For cDNA synthesis, 1 mg RNA was reverse transcribed using M-MLV Reverse For assessing proliferative activity 1 3 105 sorted CD4+CD252 T cells Transcriptase (Promega) in the presence of dNTPs, oligo(dT), and random from CD83cKO and CD83flox/flox/CD4-crewt/wt (CD83cWT) littermates hexamer primer (Thermo Fisher Scientific) at 42˚C for 60 min. Semi- were stained with Cell Proliferation Dye eFluor 670 (Thermo Fisher quantitative PCR was performed with GoTaq Hot Start Polymerase and The Journal of Immunology 3 specific primers for CD83 (59-TCGAGGCCCCCAGGAGAA-39 and 59- between CD83cKO and CD83cWT mice (data not shown). TTGCAGGTGAAAATGATGAGTGTC-39) and RPS9 as housekeeping However, T cell–specific ablation of CD83 resulted in a slightly gene (59-CTGGACGAGGGCAAGATGAAGC-39 and 59-TGACGTTGG- increased percentage and absolute number of CD4+Foxp3+ Tregs CGGATGAGCACA-39). within the spleen, whereas the number of CD4+Foxp32 conven- Contact hypersensitivity tional T cells was reduced compared with CD83cWT controls + 2 Mice were sensitized to 0.5% 2,4-dinitrofluorobenzene (DNFB) on the (Fig. 2A). In addition, sorted CD4 CD25 T cells from CD83cKO shaved back at day 0. Five days later, CHS response was provoked by and CD83cWT mice showed the same capacity to differentiate applying 0.3% DNFB on the right ear. At day 7, ear swelling was deter- into Foxp3+-induced Tregs upon stimulation in the presence of mined as measure of CHS response, and splenocytes and draining lymph IL-2 and TGF-b (Fig. 2B). To investigate whether endogenous node cells were isolated for flow cytometric analysis. CD83 is involved in the inhibitory activity of Tregs, we sorted Adoptive transfer CD4+CD25+ Tregs from spleen of naive CD83cKO mice and A total of 1 3 106 sorted CD4+CD45RBhigh splenocytes from CD83cKO naive CD83cWT littermates. Flow cytometry analysis revealed + + mice or CD83cWT controls were injected i.v. into Rag22/2 mice. Mice that ∼95% of CD4 CD25 Tregs express Foxp3 with no differ- were monitored weekly for wasting disease. Seven weeks after reconstitution, ences between CD83-deficient Tregs and Tregs isolated from spleen and mesenteric lymph nodes (mLN) were removed and analyzed CD83cWT mice (Fig. 2C). We cocultured sorted CD4+CD25+ by flow cytometry. Serum samples were assessed for cytokine and sCD83 Tregs from CD83cKO mice and CD83cWT littermates, together concentration. Colons were prepared as swiss rolls, stored in 4% parafor- + 2 maldehyde, and embedded in paraffin. Colon sections (4 mm) were scored for with CD4 CD25 responder cells at different ratios in the pres- inflammatory cell infiltrates, epithelial damage, hyperplasia, and crypt ence of irradiated splenocytes and anti-CD3. The proliferation abscesses (no change, 0; mild change, 1; moderate change, 2; and pro- of responder cells was determined at day 3 by flow cytometry. Downloaded from found change, 3) of the oral, middle, and rectal parts in a blinded manner As depicted in Fig. 2D, CD83-deficient Tregs and Tregs from and summarized as histological score. CD83cWT mice showed similar suppressive capacities in vitro Statistical analysis (Fig. 2D). These results indicate that endogenous CD83 expres- + Statistical analyses were performed with Student t test for parametric and sion in CD4 T cells has no impact on the induction and inhibitory + Mann–Whitney U test or Kruskal–Wallis test for nonparametric distributed activity of Foxp3 Tregs. , , data as indicated with significance set at the levels of *p 0.05, **p 0.01, http://www.jimmunol.org/ + and ***p , 0.001. Normality was tested using the D’Agostino–Pearson CD83-deficient CD4 T cells exhibit elevated proliferation and omnibus test. All analyses were calculated with GraphPad Prism 7.0 IFN-g production Software (GraphPad Software). To gain insights into the function of endogenous CD83 in CD4+ conventional T cells, we analyzed the expression of CD69 and Results CD62L on T cells isolated from naive mice by flow cytometry. Ablation of CD83 expression in T cells from Interestingly, the frequency of CD69-expressing CD4+Foxp32 flox/flox CD83 /CD4-cre mice T cells was significantly elevated in spleen of CD83cKO mice CD83-deficient mice are not suitable to examine the role of en- compared with CD83cWT controls. Well in line, the percentage of

+ 2 by guest on September 28, 2021 dogenous CD83 on T cell function because of a defect in the T cell CD62L-expressing CD4 Foxp3 T cells was significantly reduced + development (22). Therefore, we have generated a new transgenic in naive CD83cKO mice, indicating that CD4 conventional CD83flox mouse line allowing for T cell–specific ablation of CD83 T cells from T cell–specific CD83-deficient mice are more activated (Fig. 1A). We isolated CD4+ T cells from spleens of CD83cKO already under homeostatic conditions (Fig. 3A). Next, we deter- + 2 mice and CD83cWT littermates and stimulated them with anti- mined the proliferative activity of CD83-deficient CD4 CD25 + 2 CD3 in vitro. Whereas we detected CD83 mRNA expression of T cells in comparison with CD4 CD25 T cells from CD83cWT + 2 stimulated cells from CD83cWT mice by PCR, CD4+ T cells mice. For this purpose, sorted CD4 CD25 T cells from CD83cKO from CD83cKO mice did not express CD83 mRNA (Fig. 1B). mice and CD83cWT littermates were stimulated with anti-CD3 or Moreover, we performed flow cytometric analysis to determine left unstimulated as control in the presence of irradiated spleno- + 2 CD83 protein expression. As depicted in Fig. 1C, CD4+ T cells cytes. CD83-deficient CD4 CD25 T cells exhibited significantly from CD83cWT mice significantly upregulated CD83 upon enhanced proliferative activity (Fig. 3B) and secreted more IFN-g + 2 activation as expected. Strikingly, we detected almost no CD83- and IL-2 (Fig. 3C) than CD4 CD25 T cells from CD83cWT expressing CD4+ T cells from CD83cKO mice even after stimulation controls. Based on this finding, we next examined the capacity of + 2 (Fig.1C).Next,weseparatedCD4+Foxp3+ Tregs from CD4+Foxp32 CD83-deficient CD4 CD25 T cells to differentiate into Th1 cells. + 2 conventional T cells and again determined the amount of CD83- Sorted CD4 CD25 T cells from CD83cKO mice and CD83cWT expressing cells upon stimulation by flow cytometry (Fig. 1D). controls were stimulated with anti-CD3 and anti-CD28 (control, Both CD4+ T cell subsets isolated from CD83cWT mice significantly Th0) and in addition under Th1-polarizing conditions (Th1). upregulated CD83 expression in contrast to CD4+Foxp3+ Tregs and After 5 d of culture, CD83-deficient T cells expressed twice CD4+Foxp32 T cells from CD83cKO mice (Fig. 1E, 1F). CD83 as much IFN-g as T cells from CD83cWT mice (Fig. 3D). protein expression of activated CD8+ T cells was also significantly To clarify whether endogenous CD83 has also an impact on + 2 reduced in CD83cKO mice because of CD4 promotor activity during the capacity of CD4 CD25 T cells to differentiate into + 2 the double-positive stage of thymocytes in the course of T cell Th17 cells, we stimulated sorted CD4 CD25 Tcellsfrom development (Supplemental Fig. 1). In contrast, we did not detect CD83cKO mice and CD83cWT controls with anti-CD3 and any differences in the frequency of CD83-expressing CD19+ Bcells anti-CD28 under Th17-polarizing conditions for 6 d. Deficiency and CD11c+ DCs from CD83cKO mice compared with CD83cWT in CD83 expression resulted in a significant increase in IL-17– + mice upon stimulation with LPS in vitro (Supplemental Fig. 1). expressing CD4 T cells (Fig. 3D). These results indicate a more pronounced T cell activation in the absence of endogenous CD83 T cell–specific CD83 deficiency has no impact on the function expression. To gain further insights, we analyzed the expression of of Tregs an early molecule of the TCR signaling pathway and detected Next, we determined the frequencies of CD4+ and CD8+ T cells in elevated phospho–ZAP-70 expression in CD83-deficient CD4+ thymus, mLN, and spleen and did not observe any differences conventional T cells compared with CD83cWT controls after 4 ENDOGENOUS CD83 EXPRESSION REGULATES T CELL RESPONSES

FIGURE 1. T cell–specific deletion of CD83 expression. (A)Strategyforthe generation of conditional CD83-deficient mice. The WT CD83 consists of five exons. pRAPIDflirt-CD83 was used as targeting vector containing an FRT site (gray circles) flanked neomycin resistance gene (neoR), two loxP sites (white triangles), and the thymidine kinase (TK). After breeding with Rosa-Flp mice, which express the Flp recom- binase, the neomycin resistance cassette is deleted (CD83flox). Crossing CD83flox mice with CD4-cre mice results in dele- tionofCD83exon3(CD83del). (B)PCR Downloaded from analysis of CD83 mRNA expression of sorted, anti-CD3–stimulated CD4+ T cells from CD83cKO mice and CD83cWT littermates. CD83 protein expression was analyzed in unstimulated (0 h) and anti- CD3–stimulated (24 h and 48 h) spleno- http://www.jimmunol.org/ cytes (C)gatedonCD4+ Tcells,(E)gated on CD4+Foxp3+ Tregs, and (F)ongated CD4+Foxp32 T cells from CD83cKO and CD83cWT mice by flow cytometry. (D) Representative dot blots of Foxp3 and CD83 expression as well as Foxp3+ and Foxp32 gating strategy. Data from two independent experiments with n =7–9 mice in total are shown as mean 6 SEM.

Mann–Whitney U test were used for sta- by guest on September 28, 2021 tistical analysis. ***p , 0.001.

10 and 20 min of stimulation with anti-CD3 and anti-CD28 CD83 deficiency aggravates T cell transfer colitis (Fig. 3E). In CD83cKO mice, CD83 expression is ablated in both CD4+Foxp3+ + 2 Exacerbated CHS reaction in T cell–specific TregsaswellasCD4Foxp3 conventional T cells and also + CD83-deficient mice significantly reduced in CD8 T cells, which might have an impact on CHS response in CD83cKO mice. To dissect the Our results provide evidence for CD83 to be involved in the ac- + + function of endogenous CD83 expression, especially in CD4 tivation of CD4 conventional T cells at least in vitro. To evaluate conventional T cells, we sorted CD4+CD45RBhigh naive T cells whether endogenous CD83 expression has an impact on T cell from CD83cKO mice and CD83cWT littermates and adoptively function during ongoing immune responses in vivo, we pro- transferred them into Rag22/2 mice to induce colitis. Control voked and analyzed a CHS reaction in CD83cKO mice and Rag2-deficient mice did not receive any cells. As depicted in CD83cWT control mice. For this purpose, we sensitized mice to Fig. 5A, mice adoptively transferred with CD4+CD45RBhigh DNFB on the shaved back and elicited the CHS reaction by ap- naive T cells loose body weight over time compared with plication of DNFB on the right ear 5 d later. T cell–specific CD83- controls (Fig. 5A). We did not observe significant differences deficient mice developed a significantly more severe CHS reaction in body weight loss between Rag22/2 mice that received than CD83cWT mice, as assessed by measuring the ear swelling at CD83-deficient T cells in comparison with mice transferred day 7 (Fig. 4A). Accordingly, the relative numbers of CD69- and with T cells from CD83cWT mice (Fig. 5A). However, Rag22/2 CD44-expressing CD4+ T cells (Fig. 4B) as well as the frequency of mice that received T cells from CD83cKO mice exhibited sig- IFN-g– and IL-17–producing CD4+ T cells (Fig. 4C) were signifi- nificantly enhanced colon weight to length ratio (Fig. 5B) and, cantly elevated in CD83cKO mice compared with CD83cWT con- importantly, an increased histological score (Fig. 5C), indicating a trols. Hence, endogenous CD83 expression controls the activation of more severe disease. Accordingly, a higher percentage of reiso- CD4+ T cells during the CHS reaction. lated CD83-deficent T cells from spleen and mLN of diseased The Journal of Immunology 5

FIGURE 2. CD83-deficient Tregs exhibit similar suppressive capacity as WT Tregs. (A) The frequency and absolute numbers of CD4+Foxp3+ Tregs and CD4+Foxp3+ conventional T cells from CD83cKO and CD83cWT mice (n = 14–28 in total) were analyzed by flow cytometry. (B) Sorted CD4+CD252 from T cell–specific CD83-deficient mice (CD83cKO) and control littermates (CD83cWT) were stimulated with anti-CD3 and anti-CD28 in the absence (Th0) or presence of IL-2 and TGF-b (induced Treg). After 3 d of culture, the percentage of Foxp3-expressing CD4+ Tregs was analyzed by flow cytometry. (C) CD4+CD25+ Tregs were sorted from naive CD83cKO mice and CD83cWT controls and analyzed for Foxp3 expression (representative dot blots are shown). (D) Sorted Tregs were cocultured with proliferation dye Downloaded from eFluor 670–labeled CD4+CD252 Tcells from Thy1.1 mice (responder cells) at different ratios, as indicated in the presence of anti-CD3 and irradiated splenocytes from BALB/c mice, as control responder cells were stimulated without Tregs. Proliferation http://www.jimmunol.org/ was assessed as loss of eFluor 670 on gated CD4+Thy1.1+ responder cells, and data from each independent experiment were calculated as relative proliferation (percentage of responder cell proliferation [1/mean fluorescence intensity (MFI)]). Data from two to three independent experiments with n =4–7 mice are shown as mean 6 SEM. Statistical analysis were performed by using Student t test and Mann–Whitney U test. *p , 0.05. by guest on September 28, 2021

Rag22/2 mice expressed CD69 and CD44 (Fig. 5D). Interestingly, CD83cWT controls and cocultured them together with CD11c+ DCs the frequency of Foxp3+ Tregs was also increased in mLN, but not from spleen of WT BALB/c mice in the presence or absence of LPS spleen, of Rag22/2 mice that received CD83-deficient T cells and anti-CD3. Strikingly, CD11c+MHC-II+ DCs expressed more compared with mice adoptively transferred with CD4+CD45RBhigh CD40 (Fig. 6A), showed no differences in expression of CD80 T cells from CD83cWT mice (Fig. 5D). Moreover, we detected (Fig. 6B), and produced higher amounts of IL-12 (Fig. 6C) upon significantly less sCD83 in sera of mice that received T cells from coculture with CD83-deficient CD4+CD252 T cells in contrast to CD83cKO mice in comparison with mice adoptively transferred coculture with T cells from CD83cWT mice. Thus, endogenous with T cells from CD83cWT mice (Fig. 5E). Interestingly, serum CD83 expression of CD4+ conventional T cells modulates the concentrations of IL-12 were elevated in Rag22/2 mice adoptively phenotype of CD11c+MHC-II+ DCs. transferred with CD83-deficient T cells compared with mice that received T cells from CD83cWT mice (Fig. 5E). Because IL-12 is Discussion mainly produced by DCs, we examined CD11c+MHC-II+ DCs in The glycoprotein CD83 is known to be expressed by a variety of + + more detail. The relative number as well as the CD40 and CD86 immune cells, including activated CD4 T cells and Foxp3 Tregs expression of CD11c+MHC-II+ DCs were enhanced in Rag22/2 (5, 11, 31). However, the functional impact of endogenous CD83 + + mice upon transfer of CD4+CD45RBhigh T cells from CD83cKO expression in CD4 T cells, in particular in CD4 conventional mice in comparison with mice that received T cells from CD83cWT T cells, still remains elusive. Ubiquitous deletion of CD83 ex- littermates (Fig. 5F). These results indicate that CD83 expressed by pression in mice resulted in tremendously reduced peripheral + CD4+ conventional T cells control immune responses during T cell CD4 T cells because of a defect in T cell development (22, 24). 2/2 transfer colitis. Hence, CD83 mice are not suitable to analyze the impact of + endogenous CD83 expression on the phenotype and function of T cell–expressed CD83 modulate CD11c DC activation CD4+ T cells. Therefore, we have generated a new CD83flox Results from our colitis experiments revealed that T cell–expressed transgenic mouse line on BALB/c background and crossed them CD83 has an impact on DC activation during an ongoing immune to CD4-cre mice to carefully dissect the functional impact of response in vivo. Therefore, we wondered whether T cell–derived endogenous CD83 expression in CD4+ T cells. Relative numbers CD83 has a direct effect on DCs or whether this is a more indirect of CD4+ T cells and CD8+ T cells in spleen and mLN were un- consequence due to an altered inflammatory milieu in Rag2-deficient changed in naive CD83cKO compared with CD83cWT mice, in- mice that received CD4+CD45RBhigh T cells from CD83cKO mice. dicating that T cell–specific ablation of CD83 expression has no We sorted CD4+CD252 T cells from either CD83cKO mice or impact on T cell development, as expected. 6 ENDOGENOUS CD83 EXPRESSION REGULATES T CELL RESPONSES Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 3. CD83 expression of CD4+CD252 T cells interferes with proliferative activity, Th1 and Th17 differentiation, and TCR signaling. (A) Ex- pression of CD69 and CD62L on gated CD4+Foxp32 conventional T cells isolated from spleen of naive CD83cKO mice and CD83cWT controls was analyzed by flow cytometry. (B) Sorted CD4+CD252 T cells from CD83cKO mice and CD83cWT littermates were labeled with the proliferation dye eFluor 670 and stimulated with anti-CD3 in the presence of irradiated splenocytes from Thy1.1 mice for 72 h. Proliferation was assessed as loss of eFluor 670 on gated CD4+Thy1.2+ T cells by flow cytometry. Proliferation (1/mean fluorescence intensity [MFI]) of stimulated WT CD4+ T cells from each independent experiment was set as 100%. (C) Supernatants of stimulated cells from (B) were analyzed for IFN-g and IL-2 by Luminex technology. Results of each individual experiment are calculated as percentage of WT mean. (D) Sorted CD4+CD252 from T cell–specific CD83-deficient mice (CD83cKO) and control littermates (CD83cWT) were stimulated with anti-CD3 and anti-CD28 in the absence (Th0) or presence of IL-12 and anti–IL-4 (Th1) for 5 d (left) or under Th17-polarizing conditions for 6 d (right). The percentage of IFN-g–expressing cells or IL-17–producing cells was determined by flow cytometry. (E) Splenocytes isolated from CD83cKO mice and control CD83cWT mice were left untreated or stimulated with anti-CD3 for 5, 10, or 20 min. The frequency of p–ZAP-70+ CD4+ T cells was analyzed by flow cytometry and depicted as percentage of unstimulated cells (0 h) for each independent experiment. Results from three to five independent experiments with n = 4–15 mice in total were summarized as mean 6 SEM. Student t test and Mann– Whitney U test were used for statistical analysis. *p , 0.05, **p , 0.01.

Our functional data revealed that CD83-deficient Tregs isolated by Doebbeler et al. (31) that has shown CD83 expression to be from spleen of naive CD83cKO mice exhibit the same inhibitory important for Treg differentiation. However, for their Treg dif- activity as Tregs isolated from naive CD83cWT mice. In accor- ferentiation experiments, they used CD4+ conventional T cells dance, Doebbeler et al. (31) have shown most recently that Tregs from mice with Treg-specific CD83 deficiency that are still able from Treg-specific CD83-deficient mice (CD83flox/flox/Foxp3-cre to upregulate CD83 expression upon activation. In our approach, mice) still have immunosuppressive capacity comparable to WT we used CD83-deficient CD4+CD252 conventional T cells iso- Tregs in vitro and during T cell transfer-mediated colitis in vivo. lated from CD83cKO mice that produced elevated levels of IL-2 These results suggest that although CD83 is highly expressed by upon stimulation in vitro. IL-2 is well known as a key cytokine activated Tregs, endogenous CD83 expression is dispensable for for the generation and maintenance of Tregs (32). Hence, one the suppressive activity of Foxp3+ Tregs. Interestingly, we de- might speculate about enhanced IL-2 secretion of activated CD83- tected no differences in the ability of CD4+CD252 conventional deficient CD4+CD252 conventional T cells that overrule the impact T cells from CD83cWT mice and CD83cKO littermates to dif- of endogenous CD83 expression for the generation and stability of ferentiate into Foxp3+ Tregs. This is in contrast to the recent study induced Tregs in our experimental setting. The Journal of Immunology 7

FIGURE 4. T cell–expressed CD83 controls CHS reaction. CD83cKO mice and control littermates (CD83cWT) were sensitized to DNFB in the shaved back. At day 5, CHS reaction was elicited by DNFB application on the right ear and analyzed 2 d later. (A) Ear swelling of CD83cKO mice and CD83cWT littermates. (B)The percentage of CD69- and CD44-expressing, as well as (C)IFN-g– and IL-17–producing CD4+ T cells, in spleen and/or draining lymph nodes (dLN) of diseased mice was

analyzed by ﬂow cytometry. Results from Downloaded from two to ﬁve independent experiments, with n = 10–24 mice in total, are depicted as mean 6 SEM. Student t test and Mann– Whitney U test were used for statistical analysis. *p , 0.05, **p , 0.01. http://www.jimmunol.org/

In addition, Treg-specific CD83-deficient mice exhibited re- endogenous CD83 expression controls T cell activation and cy- duced frequencies of Foxp3+ Tregs compared with control mice tokine production. In good agreement, enforced expression of by guest on September 28, 2021 (31), whereas we detected slightly elevated numbers of Tregs in CD83 has been described to result in the opposite phenotype. We spleen of CD83cKO mice that are deficient for CD83 expression not have shown that overexpression of CD83 in CD4+ T cells inter- only in Tregs but also in CD4+CD252 conventional T cells. CD83- feres with the proliferative activity in vitro (5). Adoptive transfer deficient CD4+ conventional T cells showed a more activated phe- of CD83-overexpressing cells ameliorated clinical signs of CHS as notype already under homeostatic conditions in comparison with well as EAE, and reisolated cells produced less IFN-g and IL-17 T cells from CD83cWT mice. In accordance, diseased Rag22/2mice upon Ag-specific restimulation in vitro (5). Most recently, Zinser that received CD83-deficient CD4+CD45high T cells exhibited higher et al. (29) demonstrated decreased IFN-g and IL-17 production of frequencies of Tregs together with elevated T cell activation and allogenic stimulated T cells from a transgenic mouse line that colitis severity than mice adoptively transferred with CD4+CD45high expresses human CD83 compared with controls. These studies T cells isolated from CD83cWT mice. This increase of Tregs might further underline the impact of CD83 expression on the functional be the consequence of the inflammatory environment triggered phenotype of CD4+ conventional T cells. by the activated phenotype of CD83-deficient CD4+ conven- Although Th1 and Th17 CD4+ T cells are well established for tional T cells to counterbalance overwhelming immune re- eliciting CHS responses (33), we could not exclude that CD83 sponses. However, further detailed comparative analysis is deficiency in Foxp3+ Tregs or in CD8+ T cells have an impact on needed to clarify the specific impact of endogenous CD83 ex- the immune reaction in CD83cKO mice. Therefore, we performed pression for the development, stability, and function of thymus- adoptive T cell transfer colitis experiments with sorted naive derived Tregs and peripherally induced Tregs under homeostasis CD4+ conventional T cells. Well in line with results from our CHS and inflammation. experiments, endogenous CD83 deficiency in CD4+ conventional Besides Tregs, CD83 expression is also upregulated on CD4+ T cells aggravated the development of colitis, associated with an conventional T cells upon activation. In this study, we demon- increased activation status of reisolated T cells. Interestingly, we strated that CD4+CD252 T cells from CD83cKO mice exhibit detected significantly reduced levels of sCD83 in serum of Rag2- enhanced proliferation and elevated TCR signaling and IFN-g deficient mice that received CD4+ T cells from CD83cKO mice, secretion upon stimulation compared with T cells from CD83cWT which negatively correlates with disease severity. One might controls. Activation of CD83-deficient CD4+ conventional T cells speculate about secretion of sCD83 from transferred T cells upon under Th1- or Th17-polarizing conditions induced significant activation in Rag2-deficient mice. Release of sCD83 was de- higher percentages of IFN-g– or IL-17–producing cells than CD4+ scribed for sorted CD4+CD83+eGFP+ T cells from CD83-eGFP T cells from CD83cWT mice, respectively. Moreover, CD83cKO reporter mice (11). However, in these mice, more than 50% of mice developed an exacerbated CHS response accompanied CD4+CD83+eGFP+ T cells are Foxp3+ Tregs, suggesting that by enhanced expression of activation markers and production of mainly Tregs are able to secrete sCD83. In our hands, sCD83 IFN-g and IL-17 in CD4+ conventional T cells, indicating that production of activated CD4+ conventional T cells isolated from 8 ENDOGENOUS CD83 EXPRESSION REGULATES T CELL RESPONSES

FIGURE 5. T cell–specific CD83 deficiency exacerbates T cell transfer colitis. Sorted CD4+CD45RBhigh cells from CD83cKO mice and CD83cWT controls (ctrl) were adoptively transferred to Rag22/2 mice. Rag22/2 mice without cell transfer served as negative ctrl. (A) Weight loss of mice was monitored over time. At day 37 post- transfer, Rag22/2 mice that received no cells (ctrl) or T cells from CD83cKO and CD83cWT mice were sacrificed, and (B) colon weight to length ratio and (C) histological score of colon sections were determined. Results from (A and B) seven or (C) two independent experiments with (A and B) n = 4 (ctrl) and n = 33 mice or (C) n =4 (ctrl) and n = 9 mice in total were summarized as mean 6 SEM. (D) The fre- + quencies of CD69 and CD44-expressing Downloaded from as well as Foxp3-expressing CD4+ T cells in spleen and mLN were analyzed by flow cytometry. (E) The concentration of sCD83 and IL-12 in sera of diseased mice was analyzed by ELISA or Luminex technology, respectively. (F) The percentages of + + CD11c MHC-II DCs and the expression http://www.jimmunol.org/ (mean fluorescence intensity [MFI]) of CD40 andCD86ongatedCD11c+MHC-II+ DCs in spleen of Rag22/2 mice that received T cells from CD83cKO or CD83cWT mice were determined by flow cytometry. Results from four to six independent experiments with n = 11–25 mice are shown as mean 6 SEM. Kruskal–Wallis test with Dunn multiple comparisons (A–C) or Student t by guest on September 28, 2021 test (D–F) were used for statistical analysis. *p , 0.05, **p , 0.01, ***p , 0.001.

CD83cWT mice was below the detection limit of the commer- and/or anti-CD3 to induce CD83 expression on T cells in vitro. cially available CD83 ELISA. sCD83 might also derive from in- Strikingly, the absence of endogenous CD83 expression in con- nate immune cells in diseased Rag2-deficient mice, including ventional CD4+ T cells resulted in elevated CD40 expression activated DCs, which have already been demonstrated to secrete of CD11c+MHC-II+ DCs accompanied by increased produc- sCD83 (8). However, we detected less activation of CD11c+MHC- tion of IL-12, suggesting that T cell–derived CD83 dampen DC II+ DCs but enhanced serum concentration of sCD83 in Rag2- activation. This process might be mediated via a direct or an in- deficient mice that received CD4+ T cells from CD83cWT mice. direct CD83-dependent mechanism. We demonstrated that CD83- Hence, increase in sCD83 serum level due to enhanced DC acti- deficient CD4+CD252 T cells showed a more activated phenotype vation seems to be rather unlikely. and secreted elevated levels of IFN-g upon stimulation in vitro. Strikingly, IL-12 concentration was elevated in serum of Rag2- The proinflammatory cytokine IFN-g has been previously de- deficient mice that received CD4+ T cells from CD83cKO mice scribed as inducer of CD40 expression and IL-12 production compared with mice adoptively transferred with CD4+ T cells of DCs (38–40). Hence, enhanced IFN-g production of CD83- from CD83cWT mice. IL-12 is well known to play a crucial deficient CD4+CD252 T cells might trigger activation of CD11c+ role in the pathogenesis of colitis. Elevated IL-12 production was DCs in our experiments. correlated to disease activity in patients suffering from Crohn However, CD83, in particular sCD83, is well established to disease (34, 35), and inflammation was reversed by the treatment directly interfere with DC maturation and function (14, 41). Al- with anti–IL-12 Abs in different colitis models (36, 37). though the concentration of sCD83 in supernatants of cocultures In addition to elevated IL-12 production, the expression of was below the detection limit, we could not exclude secretion of costimulatory molecules CD86 and CD40 was enhanced on low amounts of sCD83 from T cells of CD83cWT mice that CD11c+MCH-II+ DCs from Rag22/2 mice that received CD83- modulate the phenotype of CD11c+ DCs. Beside sCD83, mCD83 deficient T cells in contrast to mice adoptively transferred with also affects DCs. Recently, it has been demonstrated that incu- T cells from CD83cWT mice. From these results, we hypothesize bation of DCs with CD83-expressing Chinese hamster ovary cells that T cell–expressed CD83 interferes with DC activation and or CD83–Fc resulted in significantly less IL-12 production (42), function. To test this in more detail, we cocultured CD11c+ DCs similar to that in our coculture experiments. The nature of one or from WT mice with sorted CD4+CD252 T cells from either more CD83 ligands is still a matter of debate. Evidence of CD83 CD83cKO mice or CD83cWT controls in the presence of LPS binding partners have been described on B cells, CD8+ T cells, The Journal of Immunology 9

of CD83 on T cells might be an attractive approach to boost in- effective immune responses. Acknowledgments We thank Ralph Waldschu¨tz for providing Rosa-Flp mice, Sina Luppus for excellent technical assistance, and Witold Bartosik and Christian Fehring for cell sorting. Disclosures The authors have no ﬁnancial conﬂicts of interest.

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