The Analysis of CD83 Expression on Human Immune Cells Identifies a Unique CD83 + -Activated T Cell Population

This information is current as Xinsheng Ju, Pablo A. Silveira, Wei-Hsun Hsu, Zehra of October 1, 2021. Elgundi, Renz Alingcastre, Nirupama D. Verma, Phillip D. Fromm, Jennifer L. Hsu, Christian Bryant, Ziduo Li, Fiona Kupresanin, Tsun-Ho Lo, Candice Clarke, Kenneth Lee, Helen McGuire, Barbara Fazekas de St. Groth, Stephen R. Larsen, John Gibson, Kenneth F. Bradstock, Georgina J.

Clark and Derek N. J. Hart Downloaded from J Immunol 2016; 197:4613-4625; Prepublished online 11 November 2016; doi: 10.4049/jimmunol.1600339 http://www.jimmunol.org/content/197/12/4613 http://www.jimmunol.org/

Supplementary http://www.jimmunol.org/content/suppl/2016/11/11/jimmunol.160033 Material 9.DCSupplemental References This article cites 50 articles, 24 of which you can access for free at: http://www.jimmunol.org/content/197/12/4613.full#ref-list-1 by guest on October 1, 2021

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The Analysis of CD83 Expression on Human Immune Cells Identifies a Unique CD83+-Activated T Cell Population

Xinsheng Ju,* Pablo A. Silveira,*,† Wei-Hsun Hsu,*,† Zehra Elgundi,* Renz Alingcastre,* Nirupama D. Verma,* Phillip D. Fromm,*,† Jennifer L. Hsu,*,‡ Christian Bryant,*,‡ Ziduo Li,*,† Fiona Kupresanin,* Tsun-Ho Lo,*,† Candice Clarke,x Kenneth Lee,†,x Helen McGuire,{ Barbara Fazekas de St. Groth,{ Stephen R. Larsen,†,‡ John Gibson,†,‡ Kenneth F. Bradstock,*,† Georgina J. Clark,*,† and Derek N. J. Hart*,†

CD83 is a member of the Ig superfamily, first identified in activated lymphocytes. Since then, CD83 has become an important marker for defining activated human dendritic cells (DC). Several potential CD83 mRNA isoforms have been described, including a soluble form detected in human serum, which may have an immunosuppressive function. To further understand the biology of CD83, we examined its expression in different human immune cell types before and after activation using a panel of mouse Downloaded from and human anti-human CD83 mAb. The mouse anti-human CD83 mAbs, HB15a and HB15e, and the human anti-human CD83 mAb, 3C12C, were selected to examine cytoplasmic and surface CD83 expression, based on their different binding charac- teristics. Glycosylation of CD83, the CD83 mRNA isoforms, and soluble CD83 released differed among blood DC, , and -derived DC, and other immune cell types. A small T cell population expressing surface CD83 was identified upon T cell stimulation and during allogeneic MLR. This subpopulation appeared specifically during viral Ag challenge. We did not observe human CD83 on unstimulated human natural regulatory T cells (Treg), in contrast to reports describing expression of CD83 on http://www.jimmunol.org/ mouse Treg. CD83 expression was increased on CD4+, CD8+ T, and Treg cells in association with clinical acute graft-versus-host disease in allogeneic hematopoietic cell transplant recipients. The differential expression and function of CD83 on human immune cells reveal potential new roles for this molecule as a target of therapeutic manipulation in transplantation, inflammation, and autoimmune diseases. The Journal of Immunology, 2016, 197: 4613–4625.

D83 is a member of the Ig superfamily of receptors, human CD83 (hCD83) are 63% identical, but the mouse consisting of a single variable-like Ig domain, a trans- lacks a 10-aa portion of the extracellular Ig domain. How this by guest on October 1, 2021 C membrane domain, and C-terminal cytoplasmic domain influences the expression or function of mCD83 is unknown, but (1). The amino acid sequences of mouse CD83 (mCD83) and differences between the species might be expected (1, 2). hCD83 is expressed both as membrane-bound and soluble CD83 (sCD83) protein (3), but data on naturally derived sCD83 in mice are lacking. *ANZAC Research Institute, Concord Repatriation General Hospital, Sydney, New Likewise, CD83 mRNA splice variants are present in human cells, South Wales 2139, Australia; †Sydney Medical School, University of Sydney, Syd- ney, New South Wales 2006, Australia; ‡Institute of Haematology, Royal Prince but the status of their translation remains unknown and similar splice Alfred Hospital, University of Sydney, Sydney, New South Wales 2050, Australia; variants were not very evident in mice (4, 5). xAnatomical Pathology Department, Concord Repatriation General Hospital, Sydney, New South Wales 2139, Australia; and {Centenary Institute, Royal Prince Alfred CD83 on thymic epithelial cells enables mouse T cells to develop + Hospital, Sydney, New South Wales 2050, Australia from double-negative to mature naive CD4 T cells ORCIDs: 0000-0001-9755-9908 (X.J.); 0000-0001-5074-8824 (P.A.S.); 0000- (6), and its engagement on mouse B cells modulates their function 0002-6902-4719 (W.-H.H.); 0000-0002-4456-9368 (R.A.); 0000-0001-7047- (7). Cell surface CD83 is induced on mouse activated dendritic 1113 (P.D.F.); 0000-0001-9920-0720 (J.L.H.); 0000-0002-4173-6394 (Z.L.); 0000-0003-0229-6004 (T.-H.L.); 0000-0001-8700-5581 (K.L.); 0000-0003- cells (DC) (8) and macrophages (9). Recently, it was described on 2047-6543 (H.M.); 0000-0001-6817-9690 (B.F.d.S.G.); 0000-0001-7894- unactivated mouse regulatory T cells (Treg) (10, 11). Surface 232X (G.J.C.); 0000-0003-2620-1612 (D.N.J.H.). CD83 expression is upregulated upon human DC activation (1) Received for publication March 2, 2016. Accepted for publication October 10, 2016. and is commonly used to identify DC maturation/activation. In This work was supported by Australian Government National Health Medical Research addition, it is induced on activated human B cells (12, 13), mono- Council Program Grant 543727 (to D.N.J.H. and K.F.B.) and Cancer Institute New cytes (14), macrophages (14), neutrophils (15), and a regulatory South Wales Translational Program Grant 11/TPG/3-02 (to D.N.J.H. and K.F.B.). The Cooperative Research Centre for Biomarker Translation also provided research support. subset of NK cells (16). We and others described CD83 on activated Address correspondence and reprint requests to Prof. Derek N.J. Hart, ANZAC human T cells (6, 10, 17), but its expression on specific T cell Research Institute, Concord Repatriation General Hospital, Gate 3, Hospital Road, subsets has not been well scrutinized (18). Sydney, NSW 2139, Australia. E-mail address: [email protected] We previously identified sCD83 in human serum, described its The online version of this article contains supplemental material. release from activated DC and B lymphocytes (19), and suggested Abbreviations used in this article: AF, AlexaFluor; alloHCT, allogenic hematopoietic that sCD83 was produced by the shedding of cell surface CD83 cell transplant; AlloPC, allophycocyanin; BV, Brilliant Violet; CEF, PepMix CMV, EBV, influenza A; Cy, cyanine; DC, ; GVHD, graft-versus-host disease; molecules, although translation from a specific CD83 splice var- hCD83, human CD83; mCD83, mouse CD83; Mo-DC, monocyte-derived DC; pDC, iant remains a formal possibility (3). Recombinant sCD83-Fc plasmacytoid DC; sCD83, soluble CD83; TGN, trans–Golgi network 46; Treg, reg- fusion have been shown to inhibit T cell proliferation ulatory T cell. (4, 20) and DC maturation (21), suppress chronic inflammatory Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 diseases (22), and prolong allogeneic corneal and renal transplant www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600339 4614 CD83 EXPRESSION ON HUMAN IMMUNE CELLS survival in murine models (23, 24). Increased serum levels of lated by MACS positive selection using CD14 microbeads (Miltenyi sCD83 have been reported in patients with hematopoietic malig- Biotec). T cells were isolated with an EasySep human T cell isolation nancies and rheumatoid arthritis (25, 26). (STEMCELL Technologies). The CD304 (BDCA-4) microbead kit (Mil- tenyi Biotec) was used to deplete plasmacytoid DC (pDC) from PBMC. In Investigations in CD83-deficient, mutant, and transgenic re- the experiments indicated, DC, monocytes, B cells, NK cells, or T cells porter mice (6, 7, 27) have suggested both direct and indirect roles were sorted using an Influx Cell Sorter System (BD Biosciences), and the for CD83 in T cell development and function. CD83-deficient gating strategy is shown (Supplemental Fig. 1). + mice have reduced numbers of CD4 T cells, attributed to de- Abs fects in thymic T cell selection secondary to altered Ag presen- tation by thymic epithelial cells (6). The CD83 transmembrane The anti-human Ab conjugates used included the following: CD3 (SP34-2; Brilliant Violet [BV]450 or AlexaFluor [AF]700), CD4 (L200; PerCP- region has been found to enhance mouse MHC class II and CD86 cyanine [Cy]5.5), CD8 (SK1; allophycocyanin [AlloPC]-H7, PerCP- expression by preventing their ubiquitination by the ubiquitin li- Cy5.5), CD11c (S-HCL-3, AF700), CD1c (AD5-8E7, AlloPC), CD14 gases MARCH1 or MARCH8 (28–30). An association mediated (M5E2; PE or PerCP-Cy5.5 or PE-Cy7), CD16 (3G8; V500 or PerCP- by the cytoplasmic portion of hCD83 with GRASP55 in the Golgi Cy5.5), CD20 (2H7; PE or V450), CD34 (581; PE-CF594), CD56 (HCD56; BV450), IgD (IA6-2; AlloPC), CD69 (FN50; AlloPC), and HLA- processing pathway has been described to regulate CD83 gly- DR (L243; AlloPC-H7 or AlloPC-Cy7); gd-TCR (B1; BV650) obtained cosylation and surface expression (31). CD83 on mouse Treg from BD Biosciences; CD25 (BC96; V421), CD27 (O323; BV650), CD56 contributes to their immunosuppressive function in vivo (10). (HCD56; Pacific Blue), CD86 (IT2.2; BV650), and CD127 (AO19D5; PE- An intriguing report proposed that CD83 acts as its own ligand, Cy7) obtained from BioLegend; and CD141 (AD5-14H12; PE) and CD304 with homotypic interactions mediating a suppressive effect, (17F6; AlloPC) obtained from Miltenyi Biotec. Labeled mouse anti- hCD83 Ab HB15a (FITC) was obtained from Beckman Coulter, and including blocking the differentiation of monocyte-derived DC HB15e (FITC) from BD Biosciences. Purified anti-CD83 Abs HB15a Downloaded from (Mo-DC) (21). (Beckman and Coulter), HB15e (STEMCELL Technologies), and F5 Despite the wide use of CD83 as a marker of DC activation in (Santa Cruz Biotechnology) were used, as indicated. The 3C12C is a fully humans, the biology of hCD83 is poorly understood and differences human IgG1 anti-hCD83 mAb selected from a phage display library (36, 37) and further engineered by L chain shuffling to increase its affinity (34). to mCD83 biology are very likely. The additional 10 aa in the The isotype control Abs mouse IgG1 k FITC and mouse IgG2b k FITC extracellular domain of hCD83 might be related to an alternative were obtained from BD Biosciences, and human IgG1 k from Sigma- function. So too might the potential for human cells to express Aldrich. http://www.jimmunol.org/ different CD83 mRNA isoforms either as proteins or regulatory Bio-layer interferometry analysis RNAs. The disease linkage of sCD83 in humans, for example, increased levels in hematological cancers such as Hodgkin lym- HB15a, HB15e, and 3C12C were assessed for affinity to CD83 using a BLItz System (forte´bio; Pall Life Sciences). CD83-Ig recombinant protein phoma (32), also argues for a more complex set of CD83 immune was immobilized to a human IgG Fc capture sensor. A five-point dilution cell interactions. We used rabbit polyclonal anti-hCD83 Ab to series of each mAb was tested for association and dissociation. Kinetic rate deplete activated DC to prevent graft-versus-host disease (GVHD) constants were estimated using BLItz Pro software with a global fitting 1:1 and preserve human T cell responses after human PBMC trans- binding model. plantation in SCID mice (33). To develop this concept of specific Flow cytometry analysis immunosuppression, we generated a human anti-hCD83 mAb, by guest on October 1, 2021 3C12C (34). Despite our earlier observation that CD83 was pre- Cell preparations were incubated initially with TruStain FcX Fc Block (BioLegend) for 10 min at room temperature and then with specific or sent on activated human T cells (17), the human memory antiviral isotype control Abs for 20 min at 4˚C. To discriminate between live and T cell responses and human natural Treg were retained after anti- dead cell events, samples were further stained with the cell viability dyes CD83 mAb treatment (34). These results led us to undertake a DAPI (Thermo Fisher Scientific) or the fixable Zombie Aqua dye (Bio- comprehensive study of the expression of CD83 on immune cells Legend). For intracellular staining, cells were fixed and permeabilized with a Foxp3 transcription factor staining kit (eBiocience). Cell data were using several different mouse and human mAbs to hCD83. The collected using the Influx Cell Sorter System (BD Biosciences), and the human cells examined expressed distinct CD83 epitopes, differed data were analyzed with FlowJo software. in cytoplasmic and surface CD83 expression, and had very dif- ferent capacities to release sCD83. Activation of human T cells Mo-DC generation in vitro and in vivo during allogeneic hematopoietic cell trans- To generate Mo-DC, CD14+ monocytes were cultured with 800 U/ml GM-CSF plants (alloHCT) induced transient transcription and surface (Miltenyi Biotec) and 1000 U/ml IL-4 (Life Technologies) supplemented expression of CD83 on subpopulations of responding T cells. every 2 d. On day 5, 1 mg/ml LPS (Escherichia coli K-12; InvivoGen) or a cytokine mixture containing 5 ng/ml IL-1b, 150 ng/ml IL-6, 5 ng/ml TNF-a These distinct expression profiles of CD83 isoforms in different (R&D Systems), and 1 mg/ml PGE2 (Sigma-Aldrich) was added to mature human immune cells are likely to contribute to immune regu- DC for 48 h. latory pathways. In vitro functional assays Materials and Methods KM-H2 cells (38) were cultured in complete RPMI 1640 medium con- Human blood cell isolation and culture taining 10% FCS (Thermo Fisher Scientific). To stimulate cells, PBMC were cultured with 1 mg/ml LPS or 1 mg/ml CpG-B ODN-2006 (Invivo- Healthy donor and patient venous blood was collected in Concord Repa- Gen) to activate B cells or 5 mg/ml CpG-A ODN-2216 (InvivoGen) to triation General Hospital and Royal Prince Alfred Hospital with informed activate pDC. CD14+ monocytes were stimulated with 1000 U/ml human consent approved by the Sydney Local Health District Human Research rIFN-a2a (Miltenyi Biotec). T cells were stimulated by culturing with Ethics Committee in accordance with the Declaration of Helsinki. Patients T cell activation/expansion microbeads (anti-CD2, anti-CD3, and anti- receiving alloHCTwith prophylactic immunosuppressive therapy to prevent CD28; Miltenyi Biotec) at a 1:2 ratio or with 10 mg/ml PHA (Sigma- GVHD at the Royal Prince Alfred Hospital were monitored weekly. Grading Aldrich). To investigate the potential of the sCD83 effect, T cells were of acute GVHD was performed using the International Bone Marrow labeled with CFSE (Sigma-Aldrich), stimulated with CD2/CD3/CD28 Transplant Registry severity index (35). Human PBMC were isolated by microbeads, and cultured with 25% supernatant of Mo-DC or LPS- centrifugation on Ficoll-Paque-PLUS (GE Healthcare) and cultured in activated Mo-DC in the presence of 3C12C (5 mg/ml) for 5 d. Specific complete RPMI 1640 medium containing 10% human AB serum, 2 mM peptide stimulation was performed using either 1 mg/ml CMV pp65 glutaMAX, 100 U/ml penicillin, 100 mg/ml streptomycin, 1 mM sodium PepTivator (Miltenyi Biotec) or 10 mg/ml PepMix CMV, EBV, influenza A pyruvate, 10 mM HEPES, and 10 mM 2-ME (Thermo Fisher Scientific) at (CEF) peptide pool (Innovative Peptide Solution). Stimulations were + 37˚C, in 5% CO2. CD1c blood DC were isolated from PBMC by MACS performed in either 6-well flat-bottom cell culture plates or 96-well using the BDCA-1 isolation kit (Miltenyi Biotec). Monocytes were iso- U-bottom cell culture plates, and cells were harvested between 1 and 72 h The Journal of Immunology 4615 for analysis. For two-way allogeneic MLRs, 2 3 106 PBMC from each of (surface + intracellular) permeabilization and fixation using the Foxp3 two blood donors were cultured together for up to 6 h. In Transwell ex- transcription factor staining buffer set (eBioscience). All cells were also periments, purified T cells were added to the bottom chamber, and T cell– stained with an anti-Foxp3 Ab (FJK-16s, AlloPC; eBioscience) after per- depleted PBMC were added to the upper chamber of Transwell plate with a meabilization and fixation to identify Treg. Flow cytometric analysis was 0.4-mm–pore polyester membrane Transwell insert (Corning Incorpora- performed as above. tion). CD2/CD3/CD28 microbeads were added to both chambers at 1:2 ratio for 6 h. Immunofluorescence staining Deglycosylation study with tunicamycin treatment Human CD1c+ DC or FACS-purified CD3+ T cells isolated from PBMC were cultured in complete RPMI 1640 with 1 mg/ml LPS (Invivogen) for PBMC were incubated for 20 h in complete RPMI 1640 at 37˚C and 5% 24 h or PHA for 6 h, after which the cells were harvested, washed in PBS/ 5 CO2 in the presence of 1 mg/ml LPS with or without 1 mg/ml tunicamycin 0.5% BSA, and resuspended at a concentration of 5 3 10 cells/ml. Aliquots (Sigma-Aldrich). Cells were subsequently stained with anti-CD83 Abs or (200 ml) of the prepared cell suspension were cytospun onto glass slides and matched isotype control Abs. CD1c+ DC and CD14+ monocytes were fixed in acetone for 10 min at 220˚C. This was followed by two cycles of gated by flow cytometry to analyze CD83 expression. rehydration in PBS/1% BSA for 30 min at 4˚C, followed by blocking with 10% goat serum. Cells were stained with the following Abs: HB15a or Western blots HB15e CD83 Abs, rabbit polyclonal anti-human trans–Golgi network 46 Cells were lysed in mammalian protein extraction reagent (Thermo Fisher (TGN) Ab (Abcam), biotinylated anti–HLA-DR Ab (iCyt Mission Technol- Scientific), and equal amounts of protein (as determined by bicinchoninic ogy), AF647 goat anti-mouse or AF546 goat anti-rabbit Ab, or streptavidin- acid protein assay; Bio-Rad) were separated by electrophoresis on a 4– AF555 conjugate (Thermo Fisher Scientific). Nuclear staining was achieved by a final incubation in 45 mM DAPI solution. Alternatively, human CD1c+ 12% Bis-Tris Plus gel (Thermo Fisher Scientific) with or without anti- DC were stained with anti-CD83 and HLA-DR Abs and the corresponding oxidant and transferred to nitrocellulose membrane using the iBlot system (Thermo Fisher Scientific). Membranes were blocked with 5% secondary Abs described above, then cytospun and fixed in 4% paraformal- BSA in TBST and incubated with anti-CD83 Abs (HB15a, HB15e, or dehyde and stained with DAPI. Cells were visualized using a laser-scanning Downloaded from 3C12C) or anti–b-actin Ab (Abcam), followed by HRP goat anti-mouse confocal microscope (Leica SP8), and composite images were produced using IgG secondary Ab for HB15a, HB15e, and anti–b-actin Ab or HRP goat ImageJ (National Institutes of Health). anti-human IgG (Sigma-Aldrich) for 3C12C. Signal was detected with an Immunohistology staining ECL reagent (Clarity ECL kit; Bio-Rad) and analyzed using the Chemidoc image system (Bio-Rad). Immunohistochemical double staining was performed on 3 mm sections of formalin-fixed paraffin-embedded tissue of human lymph node and sCD83 ELISA tonsil. The primary Abs used were CD83 (F5; Santa Cruz Biotechnol- http://www.jimmunol.org/ Human sCD83 was detected with an ELISA kit (Sino Biological), which has ogy) and CD3 (Leica Biosysytems). Double staining was performed on a a detection limit of 3.9 pg/ml. A 96-well plate was precoated overnight with Leica Bond III Autostainer (Leica Biosystems) using a Bond Polymer 9 the supplied CD83 capture mAb. Culture supernatant or a recombinant Refine Detection kit for visualization with 3,3 -diaminobenzidine and a Bond Polymer Refine Red Detection kit for visualization with alkaline CD83-Fc standard was incubated for 2 h, and sCD83 was detected using a phosphatase. mouse anti-hCD83 HRP for 1 h. The amount of secondary Ab bound was visualized using tetramethylbenzidine (Sigma-Aldrich) substrate solution, Statistical analysis which was read at 450 nM on a microplate reader (PerkinElmer). Data are shown as mean and SEM, unless otherwise stated. Student t test RT-PCR and real-time PCR analysis of sorted PBMC was performed using Prism 6.01 (GraphPad Software, La Jolla, CA); a populations p value ,0.05 was considered significant. by guest on October 1, 2021 RNA was extracted with TRIzol (Life Technologies), and cDNA was transcribed from 100 ng total RNA using SuperScript III First-Strand Results Synthesis kit and random hexamers primer (Thermo Fisher Scientific), CD83 expression by resting and stimulated immune cells following the manufacturer’s protocol. To detect CD83 splice variants, cDNA from the specified immune populations was amplified by PCR using We undertook preliminary testing using the commercially available the exon 2 forward primer 59-AGGTTCCCTACACGGTCTCC-39 and mouse (HB15a, HB15e) and in-house human (3C12C) anti-hCD83 exon 5 reverse primer 59-AAGATACTCTGTAGCCGTGCAAAC-39. Pri- mAbs. The affinity for HB15a (4.167–33.33 nM), HB15e (4.167– mers to the GAPDH housekeeping gene 59-ATGGGGAAGGTGAAG- 33.33 nM), and 3C12C (3–100 nM) was ,10212 M, 1.192 3 1029 M, GTCGGA-39 (forward) and 59-AGGGGCCATCCACAGTCTTCTG-39 2 (reverse) were used as a reference. Amplified fragments were separated and 6.1 3 10 9 M, respectively. To test for related binding epi- on 3% MetaPhor (Lonza)/1% agarose (Thermo Fisher Scientific) gel. topes, we preincubated the CD83+ KM-H2 cell line with selected Quantitative PCR was performed using the SYBR Green real-time PCR unconjugated (blocking) mAbs and then added a subsaturating master mix kit (Thermo Fisher Scientific) and run on the 7500 Fast Real-Time System (Applied Biosystems). The primers used for real- concentration of conjugated (test) Ab prior to detection by flow time PCR were as follows: CD83, forward 59-TCCTGAGCTGCGCC- cytometry. Blocking with 3C12C, even at very low concentrations, TACAG-39 and reverse 59-GCAGGGCAAGTCCACATCTT-39;HPRT, inhibited .80% HB15a and HB15e binding. In contrast, HB15a forward 59-AATTATGGACAGGACTGAACGTCTTGCT-39 and reverse only partially blocked the binding of HB15e and 3C12C, suggesting 59-TCCAGCAGGTCAGCAAAGAATTTATAGC-39. Amplification efficiency that 3C12C and HB15a bind to epitopes that partially overlap or are of both sets of primers was .95%. The level was calculated as fold change = 22ΔΔCt (39). in close proximity, resulting in steric hindrance (Supplemental Fig. 2). Likewise, the failure of HB15a and HB15e to fully block each CD83 expression on mouse Treg other indicated that they probably recognize different epitopes. All mice were housed at the ANZAC Research Institute under specific Therefore, the HB15a, HB15e, and 3C12C mAbs were each used to pathogen-free conditions, and all mouse experiments were approved by the study CD83 expression on human immune cells. CD83 was not Sydney Local Health District Animal Ethics Committee. Single-cell sus- detected on the surface of any of the nonstimulated (resting) im- pensions were made from C57BL/6 mouse spleens by mechanical dis- mune cells tested, including DC, monocytes, B cells, NK cells, and ruption through 70 mm cell strainers, and red cells were removed using 13 red cell lysis buffer (eBioscience). Splenocytes (2 3 105) were cultured for T cells (Fig. 1A), despite previous reports of CD83 expression on 5 6 h at 37˚C and 5% CO2 in complete RPMI 1640 with 1 3 10 mouse T monocytes, macrophages, and DC (14). We then studied intracel- activator (CD3/CD28) microbeads (Dynal) or for 6 h at 4˚C. T cells within lular CD83 expression in resting immune cells by flow cytometry samples were distinguished through staining with anti-mouse Abs: TCR (Fig. 1A). HB15e and 3C12C detected cytoplasmic CD83 within (H57-597, BV421; Australian Biosearch), CD4 (RM4-5, BV650; BD Biosciences), CD25 (PC61.5, PE; eBioscience), and CD8 (53-6.7, PerCP- DC, monocytes, B cells, T cells, and NK cells. In contrast, HB15a Cy5.5; Jomar). Cells were also stained with the rat anti-mCD83 detected cytoplasmic CD83 only in monocytes and NK cells. The mAb (Michel-19; FITC) either before (surface only) or before and after different published CD83 mRNA splice variants [full-length CD83 4616 CD83 EXPRESSION ON HUMAN IMMUNE CELLS Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 1. CD83 expression on human immune cells. (A) Surface and intracellular CD83 expression on CD1c+ DC, pDC, monocytes, B cells, NK cells, and T cells gated from fresh isolated human PBMC stained with HB15a, HB15e, or 3C12C mAbs (unfilled histograms) or isotype controls (filled gray histograms) and analyzed with flow cytometry. Representative plots from one of three similar experiments shown. (B) Human PBMC were stimulated with LPS or CpG-A for 24 h before analysis of surface CD83 expression on gated CD1c+ DC, CD16+ DC, CD141+ DC, and pDC by flow cytometry with HB15a, HB15e, and 3C12C mAb. Representative histograms from one of four similar experiments are shown. (C) CD83 expression on monocytes gated from PBMC cultured alone or with LPS or CpG-A for 24 h was analyzed by flow cytometry (n = 4). (D) Highly purified CD14+ monocytes from PBMC were stimulated with IFN-a for 24 h before CD83 expression was analyzed by flow cytometry with HB15a, HB15e, and 3C12C (n = 3). (E) CD83 expression was analyzed on naive (IgD+CD272), switched memory (IgD2CD27+), and nonswitched memory (IgD+CD27+) B cells gated from PBMC treated with CpG-B for 24 h and stained with HB15a, HB15e, or 3C12C mAb. One representative of three similar experiments is shown.

(CD83TM) and the CD83 splice variants CD83a, CD83b, and recognized distinct CD83 epitopes, and that the form of intracellular CD83c (4)] were examined using a RT-PCR assay on highly puri- and cell membrane CD83 protein might differ between cell types. fied, sorted immune cells, including CD3+ Tcells,CD19+ B cells, Next, PBMC were stimulated with different TLR ligands (LPS, CD56+ NK cells, CD14+ monocytes, and Lineage2HLA-DR+ DC CpG-A, or CpG-B) or media alone for 24 h, and the expression of subpopulations. The CD83TM and CD83b variants predominated in CD83 was analyzed using HB15a, HB15e, and 3C12C. CD83 was most cell types (Supplemental Fig. 3A, 3B), but more restricted highly expressed by all activated DC, including the CD1c+, expression of the CD83c splice variant mRNA, which potentially CD141+, and CD16+ DC subsets (Fig. 1B). Interestingly, the encodes sCD83 protein (4), was seen in monocytes, CD141+ DC, CD141+ DC was the major myeloid DC subset to upregulate CD83 and B cells. These results again suggested that the different mAbs expression after 24-h culture in media alone. CpG-A induced CD83 The Journal of Immunology 4617 expression on pDC, as expected. Stimulation with LPS via TLR4 confocal microscopy. Both HB15a and HB15e stained surface induced moderate CD83 expression on classical CD14+CD162 CD83 on activated DC, and this colocalized with HLA-DR, but monocytes. However, considerable induction of CD83 on CD14+ membrane CD83 staining was more prominent using HB15a monocytes within cultured PBMC was also found upon CpG-A (Fig. 2C). These data suggest the different CD83 Abs could detect stimulation (Fig. 1C), although monocytes lack TLR9 (40). This distinct CD83 epitopes with a somewhat different distribution in seemed likely to be due to IFN-a release from pDC in the PBMC, cellular compartments. which then acted to stimulate monocytes. We confirmed that sorted CD83 protein expression, glycosylation, and sCD83 secretion monocytes upregulate CD83 in response to IFN-a (41), as shown (Fig. 1D), and demonstrated that CD83 expression was decreased Biochemical studies have described CD83 obtained from DC, on CpG-A–stimulated monocytes, when pDC were removed from macrophages, or cell lines as 22-, 37- to 40-, 45-, or 50- to 60-kDa PBMC with pDC-specific microbeads (data not shown). Upon proteins (14, 31, 42). We probed lysates made from PBMC or Mo- stimulation with CpG-B, all subsets increased surface CD83, DC with HB15a, HB15e, and 3C12C. HB15a and HB15e did not most notably naive B cells (Fig. 1E). detect CD83 under nonreducing conditions, whereas 3C12C de- tected several specific CD83 bands, that is, 45, 50 kDa (data not CD83 is present in defined surface and intracellular + shown). All three mAbs detected a 45-kDa protein in reduced compartments in activated CD1c DC PBMC (data not shown) and Mo-DC lysates (Fig. 3A), which may The ability of HB15e, but not HB15a, to detect intracellular CD83 represent the preformed CD83 identified intracellularly in most in most blood cells (Fig. 1A) was investigated further by confocal immune cells by 3C12C and HB15e and intracellularly in mono- microscopy of CD1c+ DC fixed and permeabilized with acetone cytes and NK cells by HB15a. The upregulation of the 50-kDa (Fig. 2A). We observed that HB15e detected little CD83 in non- protein on activated Mo-DC was also detected in reducing con- Downloaded from activated CD1c+ DC. In activated CD1c+ DC (Fig. 2B), colocal- ditions by 3C12C, but not HB15a and HB15e. In addition, a ization of MHC class II with CD83 was clearly detected by the 65-kDa protein induced on activated Mo-DC was detected by HB15a Ab. CD83 stained with HB15a within activated CD1c+ DC HB15a and HB15e (Fig. 3A). Interestingly, the CD83 protein colocalized with the TGN protein, which is part of a major se- levels detected on CD1c+ DC with 3C12C appeared much lower cretory pathway directing newly synthesized proteins to different than those on activated Mo-DC, especially the 50-kDa protein subcellular destinations. In contrast, HB15e staining did not (Fig. 3B). This suggests that different forms of CD83 are expressed http://www.jimmunol.org/ colocalize with TGN protein (Fig. 2B). As the fixation and per- on immune cells according to their state of activation. Together with meabilization can influence the Ab access to Ags on cell surface the blocking data, these data imply that the CD83 epitope detected and subcellular compartments, cells were fixed cells in 4% para- by 3C12C is a conformational epitope, whereas HB15a and formaldehyde, stained with HB15a or HB15e, and analyzed on HB15e bind the linear (reduced) form of CD83. The latter mAb by guest on October 1, 2021

FIGURE 2. CD83 colocalization with cellular compartments. Nonactivated CD1c+ DC (A) or LPS-activated CD1c+ DC (B) were cytospun onto slides, fixed with acetone, and then costained with either HB15a or HB15e CD83 Ab (green) and biotinylated HLA-DR (red), or TGN (red) Abs. DAPI (blue) was used to stain nuclei. Scale bars, 5 mm. (C) LPS-activated CD1c+ DC were stained first with either CD83 Ab (green), biotinylated HLA-DR (red), or the corresponding secondary Abs, then cytospun onto slides, and fixed with 4% paraformaldehyde. DAPI (blue) was used to stain nuclei. Scale bars, 5 mm. 4618 CD83 EXPRESSION ON HUMAN IMMUNE CELLS

CD83 expression induction on activated CD4+ and CD8+ T cells, we compared the results of PHA- and CD2/CD3/CD28-stimulated T cells with MLR activation (Fig. 5A, 5B). HB15a labeling showed that PHA stimulation resulted in peak surface CD83 ex- pression at 6 h, whereas CD2/CD3/CD28 microbead stimulation of surface CD83 expression peaked at 24 h. PHA and CD2/CD3/ CD28 microbeads resulted in a higher percentage of CD4+CD83+ cells, whereas MLRs generated more CD8+CD83+ T cells. The CD83+ T cells represented a small population of the total cells, Downloaded from

FIGURE 3. Detection of CD83 protein on DC and monocytes with CD83 Abs. (A) Western blot analysis of lysates from Mo-DC stimulated with or without cytokine mixtures (CC) (two donors) for 48 h probed with HB15a, HB15e, and 3C12C or actin. Equivalent amounts of protein (30 mg) were loaded in each lane. (B) Western blot analysis of lysates of http://www.jimmunol.org/ CD1c+ DC without or with LPS for 48 h, KMH2 cells, and Mo-DC acti- vated with LPS for 48 h probed with 3C12C or actin Abs. (C) CD83 surface expression on CD1c+ DC monocytes gated from LPS-activated PBMC treated with or without 1 mg/ml tunicamycin for 24 h was analyzed by flow cytometry. One representative of three experiments is shown. may detect an earlier epitope or additional isoform. The binding of HB15a and HB15e to LPS-activated DC was affected by tunica-

mycin, whereas binding to LPS-activated monocytes was unaf- by guest on October 1, 2021 fected, suggesting that glycosylation of CD83 on activated DC contributes to the pattern of mAb binding, or alternatively stabi- lizes CD83 expression (Fig. 3B, data not shown). We described the release of sCD83 from activated Mo-DC and tonsil B lymphocytes (19). Surface CD83 expression is more stable in LPS-activated Mo-DC than in monocytes and macrophages (14), so, to extend these findings, we assessed the sCD83 level in supernatant from DC or monocyte cultures after 24-h activation with LPS. The level of sCD83 increased in LPS-activated mono- cytes and more prominently after LPS activation of in vitro gen- erated Mo-DC. It was notable that very little sCD83 was released by blood CD1c+ DC even after LPS activation (Fig. 4A). As recombinant sCD83 has immune-inhibitory function (11, 20, 22), we added the supernatant of activated Mo-DC to cultures of acti- vated T cells and found it inhibited T cell proliferation compared with adding supernatant from unactivated Mo-DC or no supernatant (Fig. 4B, 4C, p = 0.017 and 0.0395, respectively). 3C12C partially abolished this inhibitory effect (Fig. 4B, 4C), suggesting that sCD83 and other factors in the supernatant of activated Mo-DC mediated the effect. There was no effect of 3C12C on T cell proliferation FIGURE 4. sCD83 released from Mo-DC inhibit T cell proliferation. (A) itself. The differential expression of CD83 molecular forms and in + particular the secretion of sCD83 are likely to affect immune cell sCD83 concentration in cell culture supernatant of CD1c DC (n =4), monocytes (n = 6), and Mo-DC (n = 5) cultured in LPS for 48 h was de- function. termined by ELISA. Data were normalized to the same cell concentration of CD83 upregulation on the surface of activated T cells 2 3 105/ml. (B) Purified T cells were labeled with CFSE and activated with CD2/CD3/CD28 microbeads overnight. Cells were subsequently cultured for We reported transient upregulation of surface CD83 on human 5 d without or with 25% supernatant (SN) of Mo-DC or LPS-activated Mo- T cells in MLR (17). Repeat MLR kinetic experiments demon- DC or in the presence of 3C12C (5 mg/ml). Proliferation index (PI) was strated that peak T cell expression occurred as early as 3 h analyzed on FlowJo software. One representative flow cytometry histogram + + (Fig. 5A). Both the CD4 and CD8 populations showed an in- is shown. Mean 6 SEM data of PI from four experiments were shown in (C). crease in surface CD83 after stimulation. To further investigate The p value was calculated by paired Student t test. The Journal of Immunology 4619 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 5. CD83 expression on activated T cells. (A)CD4+ and CD8+ T cells within PBMC stimulated with CD2/CD3/CD28 microbeads, PHA, or from a two-way MLR were analyzed by flow cytometry at different times after stimulation. CD83 surface expression was detected using the HB15a mAb. Mean 6 SEM of three to six experiments are shown. One representative experiment of CD83 surface expression on cultured nonstimulated T cells, stimulation with PHA 6 h, CD2/CD3/CD28 microbeads 6 h, or stimulation under MLR at 3 h are shown in (B). (C) Intracellular CD83 expression on CD4+ and CD8+ Tcells within PBMC stimulated with CD2/CD3/CD28 microbeads 6 h, or PHA 6 h was detected using HB15a and HB15e mAbs and flow cytometry. Data are from one representative of three experiments. (D) Purified CD3+ T cells activated with or without PHA for 6 h; cells were stained with HB15a or HB15e CD83 Ab (green) detected with AF647 goat anti mouse Ab. DAPI (blue) was used to stain nuclei. AF647 goat anti-mouse secondary Ab only used as control. Insert picture shows the enlarged image of one representative cell. Scale bars, 5 mm. (E) Tonsil biopsy sample stained with brown 3,39-diaminobenzidine detecting with CD3 Ab and/or red alkaline phosphatase detecting with CD83 Ab counterstained with H&E. Arrow indicates the CD3+CD83+ Tcells.

,8% by PHA stimulation, ,5% by CD2/CD3/CD28 microbeads, HB15e, but its expression dropped after 6 h PHA stimulation and and ,1% in MLR conditions. HB15e and 3C12C labeling of there was very little HB15a staining, which was mainly on the activated T cells showed similar trends, but with earlier kinetics surface. CD83 mRNA was also detectable before stimulation, in- and relatively lower peak levels of HB15e and 3C12C staining creased rapidly after PHA stimulation, and reached maximum compared with HB15a (Supplemental Fig. 4), once again em- levels at 1 h (Supplemental Fig. 3D, 3E) before decreasing. This phasizing the differences in epitope specificity of the mAbs. As indicates that CD83 expression on T cells from mRNA tran- described earlier, only HB15e and 3C12C detected intracellular scription to translation, expression in the cytoplasm, and expres- CD83 in resting T cells (Fig. 1A). After activation, intracellular sion on the cell surface are both fast and transient, although CD83 staining with HB15a mAb increased rapidly, whereas stimulus dependent. CD83 expression on activated T cells was HB15e staining dropped, again implying that these two CD83 further confirmed by histological staining on tonsil and normal mAb bind different forms of CD83 (Fig. 5C). This was further reactive lymph nodes (Fig. 5E, data not shown). Double staining confirmed by confocal cytoplasmic staining of highly purified showed CD3+CD83+ T cells in the interfollicular region of tonsil CD3+ T cells with HB15a and HB15e (Fig. 5D). Intracellular and lymph nodes. Occasional larger single-membrane–positive CD83 was detected in nonstimulated T cells by staining with CD83 cells were also present in among the T lymphocytes, presumed 4620 CD83 EXPRESSION ON HUMAN IMMUNE CELLS to represent APCs such as DC or activated B cells. T cells did not data not shown). However, there was still a clear increase of CD83 appear to secrete sCD83 (data not shown). expression on activated HLA-DRlo cells T cells. To further confirm

+ this finding, purified T cells were added to the bottom chamber of Specificity of CD83 T cell population in response to viral Transwell plates and cocultured with T cell–depleted PBMC in the stimulation upper insert chamber, stimulating both with CD2/CD3/CD28 To assess whether surface CD83 is specifically upregulated by T cells microbeadsfor6h;CD83expressiononpurifiedTcellswascom- responding to viral antigenic epitopes, we compared the response of pared with whole PBMC. CD83+CD69+ expression was increased on HLA-A*0201 CMV seropositive and seronegative donors in response T cells in Transwells stimulated without contact with T cell–depleted to CMV pp65 peptide restimulation. We found that CMV pp65 PBMC, but was again higher within stimulated PBMC, where the challenge increased CD83 expression on both CD4+ and CD8+ T cells T cells had contact with other potentially CD83-expressing DC/APC from CMV positive but not negative donors, and that the effect was (Fig. 6C). Similar results were obtained using the CEF peptide pool most apparent on HLA-DRhi cells (Fig. 6A). Because both HLA-DR to stimulate T cells separated from DC/APC in Transwell compared and CD83 expression on DC are much higher than on T cells with whole PBMC CEF peptide stimulations (data not shown). These (Supplemental Fig. 3C, data not shown), we investigated whether data suggested that induction of surface CD83 on HLA-DRlo T cells the CD83 on T cells was transferred by trogocytosis or other means was a TCR-dependent cell-intrinsic process, whereas trogocytosis of from DC or other cells. We purified CD3+ T cells (.99% purity) and DC membranes was a major contributor to the CD83+DR+ T cell stimulated the cells with the viral HLA-A,B,C–restricted CEF peptide population in mixed culture of T cells and DC. pool. The CD83+DR+ population represented a smaller proportion of T cells in the cultures containing purified T cells compared with CD83 expression on mouse and human Treg unsorted PBMC. Within PBMC, some of the CD83+DR+ cells were Mouse natural Treg have been shown to express CD83, which is Downloaded from also CD1c+, suggesting DC transfer of membrane molecules (Fig. 6B, further increased after activation (10, 11), as shown in this study http://www.jimmunol.org/

FIGURE 6. CD83+ T cells are viral specific. (A) PBMC, from serum CMV2 (n = 3) and CMV+ (n = 3) donors,

stimulated with or without CMV pep- by guest on October 1, 2021 tide pool for 6 h, were analyzed for CD83 expression on the CMV peptide– stimulated T cells. (B) PBMC and FACS-sorted CD3+ T cells from same donor were cultured with or without CEF peptide pool for 6 h; CD83 ex- pression on CEF-stimulated T cells was analyzed by flow cytometry (n = 3). (C) Highly purified T cells from PBMC were added to the bottom chamber of Transwell plate and cocultured with CD3-depleted PBMC in the top cham- ber with 0.4-mm–pore-size filter at the ratio same as whole PBMC. Cells from both chambers were stimulated with CD2/CD3/CD28 microbeads for 6 h. Whole PBMC stimulated with CD2/ CD3/CD28 microbeads 6 h and unsti- mulated T cells were used as control. CD83 and CD69 expression on CD4+ and CD8+ T cells was analyzed by flow cytometry (n = 4). Error bars represent mean 6 SEM. The p values were cal- culated by paired Student t test. The Journal of Immunology 4621

(Fig. 7A). About 4% of human peripheral blood CD4+ T cells cosylated molecules, to be expressed at differing levels among were Treg, and these nature Treg expressed low levels of CD83 leukocytes. These differences are most evident upon cellular ac- transcript, as did other T cells (Supplemental Fig. 3B); however, tivation. Our studies have reinforced important differences between we could not detect surface CD83 expression on unactivated hu- mCD83 and hCD83. Most importantly, mice differ from humans in man natural CD4+CD25hiCD1272 Treg (Fig. 7B). When T cells that they probably express only a single CD83 mRNA isoform (5), were activated through their TCR using anti-CD2/CD3/CD28 the mCD83 Ig domain lacks a significant amino acid sequence, microbeads, a small population (3–6% of CD4+CD25high and no soluble form of native murine sCD83 has been identified. CD1272Foxp3+) of Treg was CD83+ (Fig. 7B, data not shown). Murine sCD83 was detected by a commercial ELISA in CD83 No CD83 expression was observed on gd T cells (data not shown). knock-in mice (11), but we have not detected human sCD83 in hCD83 gene/mCD83-deleted mice (data not shown). The obser- CD83 expression on in vivo activated human T cells in clinical vation that immunomodulatory sCD83 is secreted by activated alloHCT patients human monocytes and Mo-DC, but in low amounts only by blood Peripheral blood T cells from six patients receiving alloHCT for DC, is new and significant. It suggests a paradigm in which human hematological malignancy and prophylactic immunosuppression DC membrane CD83 contributes to stimulation, whereas monocytic were monitored weekly by flow cytometry for CD83 expression sCD83 is likely to be suppressive. Our work has also established using HB15a. Three patients with biopsy-proven acute GVHD had that surface CD83 is not expressed by circulating human natural + significant elevation of surface CD83 on peripheral CD4 and Treg in the steady state, in contrast to the mouse, again emphasizing + + + lo CD8 T cells as well as Treg (CD4 CD25 CD127 ) cells, which the major species differences in CD83. Finally, our data showing reduced after treatment with steroids (Fig. 8). In contrast, low that CD83 is transiently upregulated by TCR-dependent T cell levels of CD83 on the same T cell populations were observed on activation and during the alloimmune response associated with Downloaded from three patients without acute GVHD. clinical GVHD in alloHCT recipients raise important questions as to how CD83 might regulate further T cell proliferation and Discussion differentiation. CD83 is a member of the Ig superfamily, and reports to date would All the immune cells examined expressed the CD83TM transcript, suggest that it has, as expected, an important immune function. The and most the CD83b splicing variant. The CD83a variant was also experiments described in this work highlight the broad expression widespread, but the putative CD83c isoform less so. There is no http://www.jimmunol.org/ of the different spliced hCD83 mRNA isoforms and the potential evidence as yet that the CD83 protein detected within the cells is any for different CD83 protein isoforms, including alternatively gly- other form than the full-length protein described in the original by guest on October 1, 2021

FIGURE 7. Detection of CD83 expression on mouse and human Treg. (A) Splenocytes isolated from C57BL/6 mice were cultured with or without mouse anti-CD3/CD28 T cell activator microbeads for 6 h. Cells were stained with mAbs specific for TCR, CD4, and CD25, and fixed and permeabilized for intracellular staining with anti-Foxp3 mAb. CD83 expression detected by the anti-mCD83 mAb, Michel19, was analyzed on CD25+ Foxp3+ mouse Treg (n =2). (B) Human PBMC were stimulated with or without CD2/CD3/CD28 microbeads for 6 h, and the Treg (CD25hiCD1272) and conventional T cell (Tcon) pop- ulation were analyzed for CD83+CD69+ expression by flow cytometry (n = 3). The last dot plot shows the CD69+CD83+CD4+ T cells backgated onto Treg and Tcon cell population. 4622 CD83 EXPRESSION ON HUMAN IMMUNE CELLS Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 8. CD83 expression on human T cells in alloHCT patients. PBMC from alloHCT recipients were analyzed by flow cytometry for surface CD83 (using HB15a) on CD3+ T cells by examining the CD4+, CD8+, and CD25high CD127low Treg subsets weekly for 100 d. Three patients, who developed GVHD (upper panel), which resolved with steroids, and three patients without GVHD (lower panel) are shown. The biopsy diagnosis of acute GVHD in each case is indicated by an arrow. studies (31). Transient expression of CD83a has been reported, and variants and translated CD83 protein epitopes will require the the shorter CD83c has also been expressed (4), but the latter product generation of mAb specific for individual splice variants. The pu- does not correlate with the known human sCD83 m.w. or cell tative sCD83 protein shown by others to be coded from the smallest derivation. Cytoplasmic staining revealed the surprising new ob- splicing variant CD83c (4) has not been identified to date as a servation that CD83 is widely expressed by immune cells in a cy- product of primary human cells. However, we did detect high levels toplasmic form. The preformed CD83 cytoplasmic protein is readily of sCD83 secretion from human KM-H2 cells, which do not express detected with HB15e, although this mAb did not label the surface of the CD83c splice variant, suggesting that the sCD83 produced nonactivated immune cells. The universal expression of the 45-kDa physiologically was the product of the full-length (CD83TM) CD83 protein in immune cells matches the pattern of intracellular mRNA and was derived via proteolytic cleavage of the full-length staining by HB15e (positive in all immune cells), but the HB15a transmembrane CD83 protein (data not shown). epitope was only found within monocytes and NK cells (Figs. 1A, Surface CD83 expression was increased after stimulation with 3A). To establish the relationship between CD83 mRNA splice different TLR ligands. Compared with DC, the surface expression The Journal of Immunology 4623 of CD83 on activated monocytes and T cells was transient, and the (within hours) and decreased quickly. More CD83 was detected on stability of surface CD83 appears to relate to the glycosylation of CD4+ T cells than CD8+ T cells in response to PHA and CD2/ CD83 protein (8). Our data show that glycosylation affects the CD3/CD28 microbead stimulation, whereas the reverse was true detection of CD83 surface expression on activated DC, but not on after allogenic stimulation. Peak levels of CD83 also differed LPS-activated monocytes (14, 31). Thus, there are distinct CD83 according to the stimulation conditions, emphasizing that distinct glycosylation patterns on the surface of different immune cell signaling pathways, including NF-kB [as demonstrated for EBV- types. activated B cells (47)], are involved in upregulating CD83 CD83 molecules are known to interact with other surface expression. Our experiments stimulating purified T cells, and molecules. In the mouse, interaction with CD83 increases MHC particularly our Transwell experiments, suggest that some of the class II and CD86 on the DC surface by blocking their ubiq- surface CD83 detected on T cells activated in the presence of uitination and degradation by membrane-associated ring finger DC is derived from DC membrane, together with HLA-DR and protein, MARCH1 (28). CD83 expression influences the turnover CD1c. What function the CD83 synthesized by T cells performs of MHC-class II on mouse B cells (43). Our data showed that the remains to be determined. surface expression of CD83 correlated with upregulation of sur- To examine the clinical relevance of T cell CD83 expression, we face HLA-DR on activated human DC. Other functions have been used specific viral Ags to test the upregulation of CD83 on human attributed to CD83. CD83 binds to Golgi reassembly-stacking viral-specific T cells. HLA-A*0201–restricted peptide challenge protein, GRASP 55, important in determining the structure of increased CD83 expression on T cells (CD83 expression on CD4+ the Golgi apparatus (31). It also seems likely that CD83 plays a T cells might be due to indirect effect of induced cytokines, e.g., role in stabilizing MHC II in endosomes. Although the original IFN-a), and the majority of Ag-specific CD83+ T cells expressed a suggestions of a ligand for CD83 have not been confirmed by us or memory phenotype (data not shown). In humans, CD83 was not Downloaded from others, more recent data have raised the possibility that mCD83 readily induced in Treg, although we did previously show that may act as its own ligand to mediate negative intracellular signals sCD83 released by mature DC following CMV infection was able (44). It is now important to determine whether the different iso- to inhibit the ongoing T cell response (20). PHA or anti-CD2/3/ forms of hCD83 deliver similar signals. 28–activated T cells did not produce sCD83, but Ag-specific We described human sCD83 originally (26), and, given the wide stimulated T cells (particularly CD8+ T cells) that express CD83 expression of intracellular CD83 in immune cells, we studied the may also be induced by virus to cleave or release sCD83, thereby http://www.jimmunol.org/ physiological production of sCD83 by different human leukocyte inhibiting the antiviral T cell response. T cells coexpressing CD83 populations. Interestingly, blood CD1c+ DC secrete very low were identified in human tonsil and lymph node. We also inves- levels of sCD83 compared with in vitro generated Mo-DC; how- tigated CD83 induction in human in vivo during the alloimmune ever, it remains possible that DC produce sCD83 in response to response associated with GVHD in alloHCT recipients. The pre- alternative stimuli. Recombinant sCD83-Fc has been shown to sumed immune activation (through conditioning regimens) was inhibit DC maturation and DC-mediated T cell proliferation (44). associated with upregulation of CD83 on a small subpopulation Our study shows that supernatant of LPS-activated Mo-DC, which within each of human T cell subsets. This upregulation resolved contains high levels of natural sCD83, inhibits T cell proliferation. with effective steroid therapy and was not seen in the patients by guest on October 1, 2021 Our CD83 Ab 3C12C partially abolishes the inhibition, although without GVHD. These data support our conclusion that T cell not significantly, and other soluble factors in the supernatant of activation and expression of CD83 are an important part of the Mo-DC are likely to contribute to the inhibitory effect seen. IL-10 human immune response, and monitoring a larger cohort of transcript and protein are known to be produced from Mo-DC in alloHCT recipients may be able to relate this to clinical outcomes. response to LPS (45). No sCD83 secretion by activated T cells Our findings have implications for how one might use hCD83 to was detected at the detection levels of our ELISA, leading to the manipulate the immune system in patients. First, the exact form probable conclusion that the rapid downregulation of surface (protein identity and glycosylation) of sCD83 produced in hu- CD83 expression is due to the very transient CD83 mRNA tran- mans needs to be established. This will impact on which form of scription, rather than shedding from the T cell surface. rCD83 might be used for immunosuppressive purposes and the CD83 expression on mouse thymic epithelial cells is known to type of immune (or other) cells it might target. DC are essential influence T cell development (6), and, in mouse adoptive transfer for mounting an effective immune response, and CD83 on acti- experiments, the rapid and transient induction of CD83 on acti- vated DC is certainly a potential target for therapeutics (48, 49). vated B cells and T cells is required for lymphocyte longevity Depending on the CD83 epitope bound, mAbs may target dif- (46). Our studies emphasize that there are major species differ- ferent cell types and their ultimate therapeutic effects could vary ences in the expression of CD83 in T cells. Reporter studies (50). Our previous data showed that anti-hCD83 Abs depleted performed in mice have not revealed the widespread cytoplasmic activated DC and were immunosuppressive, preventing T cell expression of CD83 demonstrated by our studies of human responses in MLR and GVHD in a xenogeneic model (33, 34). PBMC. Most importantly, it is clear that CD83 is expressed on This work, showing that CD83 is neither present nor expressed to mouse natural Treg in the steady state and increases with acti- any great extent on unactivated human natural Treg, suggests vation, implying it may have a regulatory role (11). It has been that anti-CD83 mAb therapy could encourage Treg alloimmune claimed recently that part of the regulatory function of Treg can be regulation after transplantation. attributed to the production of sCD83. As indicated above, this In summary, to our knowledge, this is the first comprehensive seems unlikely in humans, as our extensive analyses failed to study to characterize the expression profile and distribution of identify CD83 on the vast majority of human natural Treg. hCD83 across the different subsets of DC, monocytes, NK, B, and Our new data extend our previous findings that activated T cells T cells, before and after activation. The widespread expression of upregulate surface CD83 expression transiently (17). We have now cytoplasmic CD83 in immune cells is a novel finding that has shown that both human CD4+ and CD8+ T cells increase surface many implications. Our data regarding the different CD83 mRNA CD83 expression in vitro after nonspecific PHA stimulation, isoforms and potential CD83 molecular forms, including its gly- specific CD2/CD3/CD28 activation, or in response to allogeneic cosylation, suggest that there are multiple possible mechanisms stimulation in a MLR. CD83 expression occurred relatively early for the molecule to interact either with itself or other potential 4624 CD83 EXPRESSION ON HUMAN IMMUNE CELLS functional partners. The fact that activated blood DC express 19. Hock, B. D., M. Kato, J. L. McKenzie, and D. N. Hart. 2001. A soluble form of CD83 is released from activated dendritic cells and B lymphocytes, and is de- stable surface CD83, whereas activated monocytes and their de- tectable in normal human sera. Int. Immunol. 13: 959–967. rivative Mo-DC produce sCD83, emphasizes, once again, the 20. Se´ne´chal, B., A. M. Boruchov, J. L. Reagan, D. N. Hart, and J. W. Young. 2004. fundamental difference between these cell types. Expression of Infection of mature monocyte-derived dendritic cells with human cytomegalo- virus inhibits stimulation of T-cell proliferation via the release of soluble CD83. surface CD83 on activated human T lymphocytes, but not natural Blood 103: 4207–4215. Treg, raises further questions as to how CD83 contributes to T cell 21. Bates, J. M., K. Flanagan, L. Mo, N. Ota, J. Ding, S. Ho, S. Liu, M. Roose- function and differences between mice and humans. The upreg- Girma, S. Warming, and L. Diehl. 2015. Dendritic cell CD83 homotypic inter- actions regulate inflammation and promote mucosal homeostasis. Mucosal ulation of CD83 on human T cells may become a useful marker in Immunol. 8: 414–428. clinical alloHCT and other diseases. Ultimately, the further clar- 22. Eckhardt, J., S. Kreiser, M. Do¨bbeler, C. Nicolette, M. A. DeBenedette, € ification of some of these important aspects of CD83 biology will I. Y. Tcherepanova, C. Ostalecki, A. J. Pommer, C. Becker, C. Gunther, et al. 2014. Soluble CD83 ameliorates experimental colitis in mice. Mucosal Immunol. require better definition of epitopes and, in particular, which iso- 7: 1006–1018. forms or post-translationally modified CD83 molecular forms are 23. Bock, F., S. Ro¨ssner, J. Onderka, M. Lechmann, M. T. Pallotta, F. Fallarino, L. Boon, C. Nicolette, M. A. DeBenedette, I. Y. Tcherepanova, et al. 2013. expressed by which cell types and in which subcellular location. 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