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International Immunology 2007 19(8):977-992; doi:10.1093/intimm/dxm067
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© The Japanese Society for Immunology. 2007. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

CD83 influences cell-surface MHC class II expression on B cells and other antigen-presenting cells

Yoshihiro Kuwano1,2,*, Charlene M. Prazma3,*, Norihito Yazawa1,3, Rei Watanabe1,2, Nobuko Ishiura1,2, Atsushi Kumanogoh4, Hitoshi Okochi2, Kunihiko Tamaki1, Manabu Fujimoto1,2,5 and Thomas F. Tedder3

1 Department of Dermatology, Faculty of Medicine, University of Tokyo, Tokyo, Japan
2 Department of Regenerative Medicine, Research Institute, International Medical Center of Japan, Tokyo, Japan
3 Department of Immunology, Duke University Medical Center, Durham, NC 27710, USA
4 Department of Immunopathology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
5 Department of Dermatology, Kanazawa University Graduate School of Medical Science, Ishikawa, Japan

Correspondence to: T. F. Tedder; E-mail: thomas.tedder{at}duke.edu


   Abstract
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 Abstract
Introduction
 Methods
 Results
 Discussion
 Funding
 References
 
CD83 is a member of the Ig superfamily expressed primarily by mature dendritic cells (DCs). In mice, CD83 expression by thymic stromal cells regulates CD4+ T cell development, with CD83–/– mice demonstrating dramatic reductions in both thymus and peripheral CD4+ T cells. In this study, CD83 expression was also found to affect MHC class II antigen expression within the thymus and periphery. CD83 deficiency reduced cell-surface class II antigen expression by 25–50% on splenic B cells and DCs, thymic epithelial cells and peritoneal macrophages. Reduced class II expression was a stable and intrinsic property that resulted from increased internalization of class II from the surface of CD83–/– B cells. Otherwise, class II antigen transcription, intracellular expression, heterodimer structure, antigen processing and antigen presentation were normal. Reduced class II antigen expression was not the primary cause of the CD83–/– phenotype since thymocyte and peripheral T cell development was normal in class II+/– mice. Comparable blocks in CD4+ thymocyte development were also observed in CD83–/– and CD83–/–class II+/– littermates. TCR and CD69 expression patterns in CD83–/– mice further suggested that double-positive thymocytes proceed through the class II-dependent stages of positive selection in the absence of CD83. These studies further emphasize a role for CD83 in lymphocyte development and immune regulation and reveal an unexpected role for CD83 expression in influencing cell-surface MHC class II turnover.

Keywords: CD83, MHC class II, turnover, cell surface expression, B cell


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Funding
 References
 
CD83 is a 45-kDa type-1 membrane glycoprotein of the Ig superfamily (1–4). Cell-surface CD83 is expressed by most dendritic cells (DCs), including thymic DCs, skin Langerhan's cells, circulating DCs, interdigitating reticulum cells present in the T cell zones of lymphoid organs and monocyte-derived DCs (1, 2, 5, 6). CD83 is therefore considered a marker for mature DCs (1). Human CD83 is also expressed at low levels by some germinal center cells and is induced in vitro on mitogen-activated lymphocytes (1, 7). Transcription factor nuclear factor-{kappa}B (NF-{kappa}B) and post-transcriptional regulation control inducible CD83 gene expression (8–11). In mouse, thymic epithelial cells also express CD83, which is required for CD4+ thymocyte development (12, 13). As a consequence, CD83–/– mice have a 68% reduction in CD4 single-positive thymocytes and a 75–90% reduction in peripheral CD4+ T cells (12). The residual CD4 single-positive thymocytes in CD83–/– mice are predominantly CD4+CD8low transitional cells that have not committed to the CD4+ lineage. There is also a dramatic absence of naive CD4+ T cells in CD83–/– mice, explaining their modest contact hypersensitivity responses and primary humoral immune responses (12, 13).

A role for CD83 in human T cell function was first suggested by its limited expression patterns within the immune system (1–4). Down-modulation of CD83 expression on mature DCs also correlates with impaired T cell stimulatory capacity of DCs during in vitro mixed leukocyte reaction (MLR) assays (9). In addition, studies using recombinant human and mouse CD83 extracellular domains suggest a role for CD83 in immune responses (14–20). In these studies, recombinant CD83 completely abrogates the DC-mediated stimulation of T cells in MLR assays in a dose-dependent manner and interferes with DC maturation (16). Treatment of mice with soluble CD83 extracellular domain also prevents experimental autoimmune encephalomyelitis in mice (21). CD83 ligand activity has been reported on activated CD8+ T cells, DCs and/or B cells (14, 16, 22, 23). Moreover, elasmobranch and teleost fish express CD83 homologs, suggesting a role for CD83 in cell-mediated immunity that has been conserved for >450 million years of vertebrate evolution (24).

Unexpectedly, splenic B cells from CD83–/– mice express cell-surface MHC class II antigens at ~50% lower levels than their wild-type littermates and class II expression remains 3- to 4-fold below normal levels following mitogen activation in vitro (12). Whether this is due to decreased class II antigen transcription or secondary to the deficiency in peripheral CD4+ T cells is unknown. MHC class I and class II molecules are crucial for T cell development, and consequently, class I-deficient (class I–/–) (25, 26) or class II-deficient (class II–/–) mice (27, 28) produce relatively few CD8+ or CD4+ T cells, respectively. Class II antigen expression is tightly controlled and restricted to thymic epithelial cells and professional antigen-presenting cells (APCs) (B cells, DCs and macrophages), with CD83 similarly expressed by thymic epithelial cells and professional APCs. Mice deficient in molecules required for class II processing of antigens (e.g. H2-M–/– mice and Ii–/– mice) also have compromised CD4+ T cell development as well as inhibited class II maturation, expression and/or antigen presentation (28–32). CD83 and class II molecules also co-localize in endocytic vesicles of mature human DCs, while CD83 is present in the Golgi compartment and in recycling endosomes of immature and mature human DCs (33). Since both CD83 and MHC class II molecules are important for CD4+ thymocyte and T cell development, we have assessed the relationship between CD83 deficiency and class II antigen expression in CD83–/– mice.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Funding
 References
 
Mice
CD83–/– mice were generated as described (12) and were backcrossed 6 times or more with C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME, USA). MHC class II–/– mice were purchased from Taconic Farms, Inc. (Germantown, NY, USA). CD83–/–class II–/– and CD83–/–class II+/– mice were generated by crossing mice with homozygous single deficiencies. All mice were 2–3 months of age when used and were housed in a specific pathogen-free barrier facility. Control age-matched wild-type mice were generated from breedings of heterozygous CD83+/– mice. OT-II transgenic mice were purchased from Jackson Laboratory. All studies and procedures were approved by the Animal Care and Use Committees of the International Medical Center of Japan and Duke University.

Antibodies
Antibodies used included FITC-, PE- or PE–Cy5-conjugated anti-B220 (clone RA3-6B2), FITC-conjugated CD8 (clone 53-6.7), PE-conjugated CD4 (clone RM4-5), biotinylated CD69 (clone H1.2F3) and FITC-conjugated CD86 (clone GL1) mAbs from BD PharMingen (San Diego, CA, USA) or unconjugated, FITC- or PE-conjugated anti-MHC class II I-A/I-E (clone M5/114.15.2) and PE-conjugated anti-MHC class I (clone 28-14-8) mAbs from eBioscience (San Diego, CA, USA). PE-conjugated anti-rat IgG2b secondary antibody was used to reveal unlabeled M5/114 staining (Southern Biotechnology, Inc., Birmingham, AL, USA). The following mAbs were used to distinguish MHC class II structural epitopes: BP107.2.2 (anti-I-Ab, ATCC TIB154), Y3P (anti-I-Ab, ATCC HB183) and anti-murine class II-associated Ii peptide (CLIP):I-Ab complex (clone 15G4; a kind gift of A. Rudensky, University of Washington School of Medicine, Seattle, WA, USA) mAbs. FITC-conjugated rat anti-mouse CD83 mAb (Michel17; Biocarta, San Diego, CA, USA) and biotinylated mouse anti-mouse CD83 mAb (clone MB83-1; generated in our laboratory; C. M. Prazma, preparation manuscript submitted) were used to assess CD83 expression. PE-conjugated streptavidin (Fischer Scientific, Pittsburgh, PA, USA; Southern Biotechnology Associates) was used to reveal biotinylated mAb staining. The viability probe, 7-amino-actinomycin D (7-AAD, BD PharMingen), was used in conjunction with light scatter properties to measure cell death in MHC class II internalization studies.

Cell preparation and immunofluorescence staining
Splenic B cells were purified from single-cell suspensions by removing T cells with anti-Thy1.2 mAb-coated magnetic beads (Dynal, Inc., Lake Success, NY, USA). Splenic DCs were isolated from single-cell suspensions using CD11c antibody-coated magnetic microbeads (Miltenyi Biotec, Auburn, CA, USA). Thymic epithelial cells were isolated from day 14 or 15 fetal thymi and cultured as described (34, 35). After 6–7 days, CD45+ cells were depleted from the cultured cells using antibody-coated magnetic beads (Miltenyi Biotec). Macrophages were flushed from the peritoneal cavity using 10 ml of sterile RPMI 1640 medium and were kept at 4°C after isolation. Blood was obtained by retroorbital venous plexus puncture.

Single-cell suspensions were stained for two- or three-color immunofluorescence analysis at 4°C for 20 min using predetermined optimal concentrations of antibodies as described (36). Blood erythrocytes were lysed after staining using the Whole Blood Immuno-Lyse kit (Beckman Coulter, Miami, FL, USA). Intracellular MHC class II antigen levels were determined as described (37). Briefly, cells were first incubated with saturating concentrations of unconjugated anti-MHC class II mAb (M5/114) for 45 min at 4°C, fixed for 30 min with 2% PFA and then permeabilized for 30 min with PBS containing 0.5% saponin, 5% FCS and 10 mM HEPES buffer. The cells were then stained with saturating concentrations of FITC-conjugated anti-MHC class II mAb. Positive and negative populations of cells were determined using non-reactive isotype-matched antibodies (Southern Biotech) as controls for background staining. To measure cell viability, 7-AAD was added to cells immediately prior to FACS analysis, following the manufacturer's instructions. Immunofluorescence staining was analyzed using an Epics Altra (Beckman Coulter) or FACScan (BD Immunocytometry Systems, San Diego, CA, USA) flow cytometer, with fluorescence intensities shown on a four-decade log scale. Fluorescence contours are shown as 50% log density plots.

B cell stimulation
Purified B cells or whole single-cell splenocyte preparations were stimulated with LPS (10 µg ml–1, Escherichia coli 0111:B4; Sigma, St Louis, MO, USA) or F(ab')2 anti-mouse IgM antibody (40 µg ml–1, Cappel, Durham, NC, USA) in RPMI 1640 medium containing 5% heat-inactivated FCS, with flow cytometry or biochemical analysis at the indicated times. For extracellular signal-regulated kinase (ERK) activation studies, the cells were solubilized in lysis buffer (pH 8.0) containing 1% NP-40, 150 mM NaCl, 50 mM Tris–HCl, 1 mM Na orthovanadate, 2 mM EDTA, 50 mM NaF and protease inhibitors. The lysates were subjected to SDS–PAGE, transferred onto membranes, incubated with anti-active mitogen-activated protein kinase antibody (Promega, Madison, WI, USA) followed by HRP-conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and developed using an enhanced chemiluminescence kit (Pierce, Rockford, IL, USA). NF-{kappa}B activation was assessed using a TransAMTM NF-{kappa}B p65 Transcription Factor Assay Kit (Active Motif, Carlsbad, CA, USA). Briefly, whole-cell lysates in binding buffer were added to microplate wells for 1 h at room temperature. Wells were washed three times; primary antibody (1:1000 dilution) was added to each well, with incubation for 1 h at room temperature. After washing three times, HRP-conjugated secondary antibody (1:1000 dilution) was added to each well and incubated for an additional 1 h at room temperature. Developing solution was added after four washes and the 450-nm absorbance was determined using a microplate reader (Bio-Rad, Hercules, CA, USA).

Radiolabeling and immunoprecipitation
MHC class II {alpha}ß dimer formation was analyzed as described (38). Briefly, splenic B cells from wild-type and CD83–/– littermates were enriched by T cell depletion using Thy1.2 mAb-coated magnetic beads (Dynal, Inc.) and washed with DMEM (Invitrogen, Carlsbad, CA, USA) containing 2% FCS. Splenocytes were suspended (2 x 107 cells ml–1) in DMEM without L-glutamine, sodium pyruvate, L-methionine and L-cystine (Invitrogen) and containing 5% FCS. After culture for 1 h at 37°C, [35S]-L-methionine (125 µCi ml–1; ICN Biomedicals, Irvine, CA, USA) was added to the culture medium for an additional 1 h. The labeled cells were then washed once with chase medium (DMEM containing 15% FCS and 2 mM methionine), cultured for 3 h in 10 ml of chase medium at 37°C, pelleted, washed twice with ice cold PBS and solubilized in lysis buffer, with the cell extracts cleared of nuclei by centrifugation (15 000 r.p.m.) in a microcentrifuge for 20 min at 4°C. Before immunoprecipitations, the cell lysates were pre-cleared twice with rat IgG2b mAb (10 µg, clone KLH/G2b1-2; Southern Biotechnology) for 1 h at 4°C followed by precipitation with protein G-Sepharose beads (50 µl; Amersham Pharmacia Biotech, Piscataway, NJ, USA) for 2 h at 4°C. Pre-cleared lysates were transferred to new tubes and were incubated with 10 µg of M5/114 or KLH/G2b1-2 mAbs for 2 h at 4°C and then precipitated with protein G-Sepharose beads (50 µl) overnight at 4°C. The beads were washed four times with RIPA lysis buffer and solubilized in 100 µl Laemmli buffer [2% SDS and 2-mercaptoethanol (2-ME)] for 60 min at room temperature or incubated in a boiling water bath for 10 min. Samples and [14C]-labeled molecular weight standards (Amersham Pharmacia, UK) were subjected to SDS–PAGE analysis, with label detected by autoradiography.

For analyzing MHC class II ubiquitination, 3 x 107 splenic B cells were purified from wild-type and CD83–/– mice and were solubilized in lysis buffer. Cell lysates were incubated with the M5/114 mAb and protein G beads for 2 h at 4°C. The immunoprecipitates were subjected to SDS–PAGE, transferred to nitrocellulose membranes, blotted with anti-ubiquitin mAb (clone P4D1; Covance) followed by incubation with HRP-conjugated secondary antibody and developed using an enhanced chemiluminescence kit. The stripped membrane was re-probed with KL295 mAb (anti-I-Ab, ATCC CRL-1996) to detect class II ß chain.

Measurement of [Ca2+]i
Spleen cells were isolated at room temperature, washed, suspended at 107 ml–1 in RPMI 1640 medium containing 5% FCS and 10 mM HEPES buffer and loaded with 1 µM indo-1-AM ester (Molecular Probes, Eugene, OR, USA) at 37°C for 30 min. The cells were washed and stained with FITC-conjugated anti-B220 mAb for 15 min at room temperature, washed and suspended at 2 x 106 cells ml–1 for analysis. The fluorescence ratio (488/407 nm) of B220+ cells (1.5 x 106 cells in 0.75 ml samples) was determined by flow cytometry. Baseline fluorescence ratios were collected for 1 min before the addition of F(ab')2 anti-mouse IgM antibodies (40 µg ml–1) were added with fluorescence ratios collected for 7 min. Results were plotted as the fluorescence ratio at 20 s intervals with the background subtracted. Increased fluorescence ratios indicate increased [Ca2+]i.

B cell co-culture and adoptive transfer assays
Purified CD83–/– or wild-type B cells were labeled with VybrantTM CFDA SE [carboxyfluorescein succinimidyl ester (CFSE); Molecular Probes, Carlsbad, CA, USA] for 10 min at 37°C, were mixed 1:1 and co-cultured without stimulus or with LPS (10 µg ml–1) or F(ab')2 anti-mouse IgM mAb (40 µg ml–1) in RPMI 1640 medium supplemented with 10% heat-inactivated FCS. After 24 h, the cells were stained with PE-conjugated M5/114 mAb and assessed by two-color flow cytometry. For adoptive transfer experiments, wild-type and CD83–/– splenocytes were labeled with 0.1 and 1 µM CFSE (Molecular Probes), respectively, according to the manufacturer's instructions. After labeling, wild-type (CFSElow) and CD83–/– (CFSEhigh) splenocytes were suspended in PBS at 108 cells ml–1 and mixed in equal volumes, with 4 x 107 mixed cells injected intravenously into the tail vein of CD83–/– and wild-type littermates. Spleens and peripheral lymph nodes were harvested 1 week after transfer, stained with PE-conjugated M5/114 and PE–Cy5-labeled B220 mAbs and analyzed by flow cytometry. For B cell activation, purified B cells or whole splenocytes were cultured without stimulus or with 10 µg ml–1 LPS or LPS and 100 ng ml–1 recombinant mouse IL-4 (eBioscience) for 24 h in RPMI 1640 medium containing 10% heat-inactivated FCS. MHC class II expression on B220+ cells was determined by immunofluorescence staining as described above.

MHC class II internalization
For assessing MHC class II transport, wild-type and CD83–/– splenocytes were cultured in RPMI 1640 medium containing 10% FCS (2 x 106 ml–1) and stimulated for 6–10 h at 37°C with 10 µg ml–1 LPS or LPS and cycloheximide (CHX) (2.5 µg ml–1; Sigma). After washing twice, the splenocytes were stained with PE-conjugated M5/114 and PE–Cy5-conjugated anti-B220 mAbs and assessed by flow cytometry.

Class II internalization by wild-type and CD83–/– spleen B cells was quantified by measuring the disappearance of anti-class II mAb Fab fragments from the cell surface during cell culture at 37°C. Fab fragments of the M5/114 mAb were generated using an ImmunoPure Fab Preparation Kit and then biotinylated using EZ-Link Sulfo-NHS-LC-Biotin according to the manufacturer's instructions (Pierce). Splenic B220+ cells (107 cells ml–1) were incubated at 4°C for 1.5 h with M5/114 mAb Fab fragments (15 µg ml–1) to saturate cell-surface MHC class II molecules and then washed twice in cold medium. Half of the cells were cultured at 37°C for measuring class II internalization, while the other half was kept at 4°C as controls. Cells (106) were removed from each fraction at different times and placed immediately on ice, with the biotinylated mAb remaining on the cell surface detected using PE-conjugated streptavidin. The extent of cell-surface Fab fragment labeling was quantified by flow cytometry.

Class II internalization was also measured as described (39), with modifications for measuring molecule turnover. Purified B cells were incubated at 4°C for 45 min with saturating concentrations of unlabeled M5/114 mAb, washed and incubated at 37°C for 90 min. Nascent, unlabeled, class II molecule expression on the cell surface during the 90-min culture period was detected with PE-conjugated M5/114 mAb. Saturation of surface class II with unlabeled mAb was confirmed by the lack of staining with PE-conjugated mAb after incubation of the cells on ice for 90 min. Baseline class II expression on purified B cells was measured on B cells that were cultured at 4°C for 90 min and then stained with PE-conjugated M5/114 mAb and assessed by flow cytometry. Total surface class II expression remained constant during incubations at 4 and 37°C as there was minimal change in mean fluorescence intensity on cells stained with PE-conjugated M5/114 mAb after 90 min incubation. Furthermore, there was no net loss of cell-surface class II expression during incubations since comparable staining was observed for cells labeled with PE-conjugated anti-rat IgG2b secondary antibody immediately after M5/114 mAb saturation and cells incubated on ice for 90 min before staining with M5/114 mAb and labeled secondary antibody.

Antigen processing and presentation
CD4+ T cells were purified from the pooled spleens of OT-II transgenic mice using a CD4+ T cell isolation kit following the manufacturer's instructions (Miltenyi Biotec) and re-suspended in proliferation media (2 x 106 ml–1; RPMI 1640 containing 100 U ml–1 penicillin/streptomycin, 2 mM L-glutamine, 10 mM HEPES, 100 µM MEM non-essential amino acids and 55 µM 2-ME). Splenic CD11c+ and B220+ cells were isolated using CD11c and B220 antibody-coated magnetic microbeads following the manufacturer's instructions (Miltenyi Biotec). CD11c+ DCs were re-suspended in proliferation media (4 x 105 ml–1 or diluted). B220+ cells (107 ml–1) were incubated with 50 µg ml–1 mitomycin C for 30 min at 37°C, washed three times and re-suspended in proliferation media (4 x 106 ml–1 or diluted). DCs or B220+ cells (50 µl per well) were added to triplicate wells of 96-well flat-bottom tissue culture plates containing CD4+ T cells (2 x 105) from OT-II mice. Ovalbumin (OVA) peptide (final concentration of 10 µM; OVA323—339; American Peptide Company, Sunnyvale, CA, USA) was added to wells (200 µl total volume) before culture for 3 days at 37°C. To quantify protein processing, DCs (2 x 104) or mitomycin C-treated B220+ cells (2 x 105) were cultured with CD4+ T cells (2 x 105) from OT-II+ mice and OVA (Sigma) was added to each culture. [3H]-thymidine (1.0 µCi per well) was added to each well during the last 16 h of culture. The cells were harvested onto glass fiber filtermats using a Tomtec Mach IIIW automatic harvester, with radiation quantified using a MicroBeta TriLux scintillation counter (all from Perkin Elmer Life Sciences, Boston, MA, USA).

Statistical analysis
All data are shown as mean values ± SEMs. Comparisons between groups were made using the Student's t-test.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Funding
 References
 
Cell-surface MHC class II antigen densities in CD83–/– mice
MHC class II expression is decreased on B cells from CD83–/– mice (12). Therefore, class II expression was analyzed on additional APC populations, including B cells, to determine if CD83 influences class II antigen densities similarly on all APC populations. Splenic B220+ B cells from CD83–/– mice expressed class II antigens at 50 ± 2% lower densities than B cells from wild-type littermates (Fig. 1A and B). Likewise, thymic epithelial cells, splenic DCs and peritoneal macrophages from CD83–/– mice expressed class II antigens at 30 ± 5, 25 ± 3 and 34 ± 4% lower densities than their wild-type counterparts, respectively (Fig. 1B and C). In contrast, there was no alteration in MHC class I antigen expression by any of these cell types isolated from CD83–/– mice (Fig. 1B). The absence of CD83 primarily affected cell-surface class II antigen expression since cytoplasmic class II antigen densities were normal in B cells from CD83–/– mice (Fig. 1D and E). There were no significant differences in class II {alpha} or ß chain mRNA levels between B cells from CD83–/– and wild-type littermates as determined using semi-quantitative reverse transcription–PCR assays (data not shown). Cell-surface class II antigen expression increased on CD83–/– and wild-type B220+ splenocytes following LPS and F(ab')2 anti-IgM antibody activation, yet class II densities on CD83–/– B220+ cells remained below wild-type levels (Fig. 1D and E). Thus, CD83 deficiency selectively affected the cell-surface density of class II molecules on B cells and other APCs.


Figure 1
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  		Fig. 1. Decreased MHC class II antigen expression by B cells and APCs from CD83–/– mice. (A) Representative class II (MHC II; M5/114 mAb) and class I (MHC I) antigen expression by splenic B cells from CD83–/– and wild-type littermates as assessed by direct immunofluorescence staining with flow cytometry analysis (four-decade log scale). Vertical dashed lines were added to indicate relative positions within histograms for comparative purposes. (B) Summary of class I and class II antigen expression by splenic B220+ B cells, thymic epithelial cells (TEC; CD205+CD11c–), splenic DCs (CD11c+) and peritoneal macrophages (M{Phi}, CD11b+B220–) from CD83–/– mice. Values (±SEM) represent class I or class II antigen mean fluorescence intensities as assessed in (A and C) relative to comparable cells from wild-type littermates (100%) and represent results from 3 or more littermate pairs. (C) Representative class II antigen expression on thymic epithelial cells (TEC), DCs and macrophages (M{Phi}) from wild-type and CD83–/– mice. Background staining of PE-conjugated isotype-matched control mAbs is shown. Histograms are representative of the results is shown in (B). (D) Representative cell-surface and intracellular class II antigen expression by B cells from CD83–/– and wild-type littermates. Splenic B cells were cultured alone (media) or with LPS or F(ab')2 anti-IgM antibody for 24 h. Intracellular class II antigen expression was assessed by first saturating cell-surface class II with unlabeled anti-class II mAb to block cell-surface staining. The cells were then treated with saponin, followed by staining with PE-conjugated anti-class II mAb (M5/114 mAb) with flow cytometry analysis. Background staining of PE-conjugated isotype-matched control mAbs is shown. (E) Summary of cell-surface and intracellular class II expression. Values (±SEM) represent mean fluorescence intensities of cells as shown in (D) relative to comparable cells from wild-type littermates without stimulation (100%) and represent results from triplicate cultures of four independent experiments. (B and E) Asterisks indicate significantly different sample means between littermate pairs, P < 0.05.

 
Reduced MHC class II antigen expression on CD83–/– B cells is intrinsic
To determine whether decreased MHC class II antigen expression by B cells from CD83–/– mice was intrinsic or due to extrinsic factors, CD83–/– and wild-type B cells were cultured alone or co-cultured in vitro with CFSE-labeled wild-type or CD83–/– B cells, respectively. Additionally, CFSE-labeled CD83–/– or wild-type B cells were cultured with mitogens. Following 24 h of stimulation and in all conditions tested, class II antigen expression by CD83–/– B cells remained reduced relative to wild-type B cells (Fig. 2A). Furthermore, the addition of wild-type B cells to cultures did not induce an increase in class II antigen expression by CD83–/– B cells. Likewise, wild-type B cells expressed normal class II antigen densities when they were co-cultured with CD83–/– B cells. Although CD83–/– B cell activation with F(ab')2 anti-IgM antibody or LPS induced significantly higher class II antigen expression (P < 0.01), CD83–/– B cells expressed significantly lower densities of class II than wild-type B cells even when exogenous IL-4 was added to the cultures (Fig. 2A and B). Specifically, class II antigen expression on CD83–/– B220+ cells without activation was 55 ± 3% of wild-type levels, while class II density was only 71 ± 7 and 82 ± 5% of wild-type levels following LPS- or LPS + IL-4-induced activation. Thus, while class II transcription and expression were induced by exogenous stimuli in the absence of CD83 expression, class II expression was characteristically reduced on CD83–/– B cells.


Figure 2
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  		Fig. 2. Reduced MHC class II (MHC II) antigen expression by CD83–/– B cells is intrinsic. (A) Class II antigen expression by CD83–/– B cells cultured in vitro. Spleen B cells from CD83–/– or wild-type littermates were cultured for 24 h either alone or with CFSE-labeled wild-type or CD83–/– spleen B cells, respectively, before class II antigen expression was assessed by direct immunofluorescence staining with flow cytometry analysis. The cells were also activated using LPS or F(ab')2 anti-mouse IgM antibody as indicated. Values (±SEM) represent mean fluorescence intensities of CFSEnegB220+ cells relative to comparable cells from wild-type littermates (100%) and represent results from triplicate cultures of four independent experiments. (B) Increased class II antigen expression on CD83–/– and wild-type B cells following 24 h in vitro cultures with media (CTL), LPS or LPS plus IL-4. Class II antigen expression on B220+ splenocytes was determined by immunofluorescence staining with flow cytometry analysis. Values (±SEM) represent mean fluorescence intensities for CD83–/– B220+ cells relative to comparable cells from wild-type littermates in four independent experiments. (C–E) Class II antigen expression by CFSE-labeled CD83–/– and wild-type B cells transferred into wild-type and CD83–/– littermates, respectively. (C) Representative analysis of donor CFSE-labeled B220+ splenocytes from CD83–/– (CFSEhigh) and wild-type (WT, CFSElow) mice 1 week after transfer into CD83–/– or wild-type recipients as assessed by flow cytometry. Recipient CFSEnegB220+ cells served as internal controls (CTL) for class II antigen expression. (D) Class II antigen expression by adoptively transferred B220+ cells. Values represent mean fluorescence intensities (±SEM) of class II staining for CD83–/– (CFSEhigh) and wild-type (CFSElow) donor B220+ cells after transfer into CD83–/– and wild-type recipients as indicated. (E) Class II antigen expression by adoptively transferred B220+ cells relative to endogenous class II expression by recipient mouse B220+ lymphocytes. (D and E) Values represent mean (±SEM) results from independent experiments using three wild-type and three CD83–/– recipients. (A–E) * and **indicate significant differences between sample means P < 0.05 and P < 0.01, respectively.

 
To determine whether the presence of CD83 influences MHC class II expression in vivo, splenocytes from wild-type and CD83–/– mice were labeled with differing intensities of CFSE, mixed at a 1:1 ratio and adoptively transferred into CD83–/– and wild-type recipients. Peripheral B cells were harvested 1 week after transfer and class II expression on recipient (CFSEneg) B220+ cells, CD83–/– donor B220+ cells (CFSEhigh) and wild-type donor B220+ cells (CFSElow) was assessed by flow cytometry (Fig. 2C). Class II antigen expression on CD83–/– B cells did not recover following transfer into wild-type recipients (63 ± 4% of wild-type densities) or following wild-type and CD83–/– B cell transfers into CD83–/– recipients (51 ± 2% of wild-type densities, Fig. 2D). Moreover, class II antigen expression on donor CD83–/– B220+ cells was not significantly different from endogenous expression on CD83–/– recipient B cells (104 ± 2%). Likewise, class II antigen expression on wild-type donor B cells (CFSElowB220+) remained 205 ± 8% (P < 0.001) higher than that expressed on CD83–/– recipient B cells. In wild-type recipients, class II expression by CD83–/– and wild-type donor B cells was 65 ± 4% (P < 0.005) and 104 ± 1% of endogenous wild-type levels, respectively. Since the host genotype did not significantly affect class II antigen expression on adoptively transferred CD83–/– B cells, decreased class II expression is an intrinsic property of CD83–/– cells.

Impaired up-regulation of MHC class II expression in CD83–/– B cells
Wild-type resting B cells constitutively express MHC class II, with little CD83 expression detected by immunofluorescence staining with flow cytometry analysis (Fig. 3A). However, both MHC class II and CD83 expression increased following LPS or F(ab')2 anti-IgM antibody stimulation. Therefore, whether CD83 expression influenced the kinetics of class II antigen induction was assessed. Class II antigen expression by CD83–/– B cells remained significantly below wild-type levels following F(ab')2 anti-IgM antibody (6 h, 58 ± 14%; 24 h, 51 ± 1%, P < 0.01) and LPS (6 h, 52 ± 14%; 24 h, 28 ± 2%, P < 0.01) stimulation. Likewise, CD86 expression increased on CD83–/– and wild-type B cells following activation, but was ~68 ± 3% lower on CD83–/– B cells 24 h following LPS-induced or anti-IgM antibody-induced activation. In contrast, cell-surface CD69, intracellular adhesion molecule-1 (ICAM-1) and CD44 induction were comparable on CD83–/– and wild-type B cells following activation (Fig. 3A, data not shown). Impaired signal transduction did not explain reduced MHC class II induction on CD83–/– B cells since [Ca2+]i release, NF-{kappa}B activation and ERK activation were normal in CD83–/– B cells (Fig. 3B and D). LPS and anti-IgM antibody also induced normal proliferation in CD83–/– B cells, as previously described (12). Collectively, these results indicate that CD83 expression is required for optimal cell-surface class II expression, but not the induction of class II antigen expression following B cell activation.


Figure 3
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  		Fig. 3. Impaired MHC class II antigen expression, but normal signal transduction in CD83–/– B cells in vitro. (A) CD83 (FITC-labeled Michel17 mAb), class II (MHC II), CD86 and CD69 expression by CD83–/– and wild-type spleen B cells stimulated with LPS or F(ab')2 anti-mouse IgM antibody. Values represent average mean fluorescence intensities of antigen expression as determined by two-color immunofluorescence staining with flow cytometry analysis of B220+ cells from three CD83–/– and wild-type littermate pairs. (B) Intracellular Ca2+ responses of CD83–/– (open circles) and wild-type (closed squares) B220+ B cells following anti-IgM antibody stimulation. Splenocytes were loaded with 1 µM indo-1-AM ester, stained with FITC-labeled anti-B220 mAb and activated using F(ab')2 anti-mouse IgM antibody (arrow), with relative [Ca2+]i levels of B220+ cells assessed by flow cytometry. An increase in [Ca2+]i is shown as an increase in the ratio of indo-1 fluorescence. (C) NF-{kappa}B activation in purified CD83–/– and wild-type spleen B220+ B cells following LPS activation for the indicated times. Values represent mean OD450 nm values (±SEM) from triplicate B cell cultures as assessed by ELISA for NF-{kappa}B p65 transcription factor activity. (D) Representative ERK phosphorylation following CD83–/– or wild-type spleen B cell activation using F(ab')2 anti-IgM antibody at the times indicated (top panel). Cell lysates were subjected to SDS–PAGE and transferred to nitrocellulose membranes for anti-phosphoERK immunoblotting. A graphical summary of results is shown (lower panel). Values (±SEM) represent levels of ERK phosphorylation relative to wild type at the 5 min time point (100%). (B–D) All results represent those obtained in 3 or more experiments.

 
CD83 deficiency accelerates cell-surface MHC class II internalization
Since cell-surface MHC class II antigen densities were decreased on CD83–/– APCs while intracellular class II levels and signal transduction were normal in CD83–/– B cells, the process by which CD83 influences the dynamic movement of class II onto or from the cell surface was assessed. Wild-type and CD83–/– splenocytes were treated with CHX, as previously described (40, 41), to block nascent protein synthesis. Splenocytes from CD83–/– and wild-type littermates were cultured for 6–10 h with LPS or with LPS and CHX. The concentration and duration of CHX treatment did not alter cell viability as measured by trypan blue dye exclusion and staining with the viability probe 7-AAD which identifies dying cells (data not shown). LPS induced the up-regulation of class II on the cell surface of wild-type and CD83–/– B cells, although class II expression remained reduced in the absence of CD83 (Fig. 4A and B). Treatment with LPS and CHX reduced cell-surface class II expression on wild-type and CD83–/– splenocytes when compared with the respective splenocytes cultured with LPS alone. While wild-type B cells exhibited only a 10% reduction in cell-surface class II in the presence of CHX, there was a more significant reduction of class II expression on CD83–/– B cells (37 ± 4%, P < 0.01). LPS stimulation also induced rapid CD83 expression on the cell surface of wild-type B cells, which was inhibited by 46 ± 4% with CHX treatment, suggesting that intracellular stores of CD83 are depleted in the absence of de novo protein synthesis. There was no difference in the expression of B220, CD19, IgM, MHC class I and CD23 on wild-type and CD83–/– splenocytes after CHX treatment (Fig. 4A, data not shown). Thus, in the absence of CD83 expression, either the rate of intracellular class II transport to the cell surface was reduced or cell-surface class II internalization was accelerated.


Figure 4
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  		Fig. 4. Increased endocytosis of MHC class II (MHC II) on CD83–/– B cells. (A) Representative class II antigen, CD83 (biotin-labeled MB83-1 mAb) and B220 expression on CD83–/– and wild-type B220+ splenocytes after incubation for 6 h at 37°C with LPS (solid line) or LPS plus CHX (dotted line). Isotype-matched control mAb staining is also shown (dashed line). Staining of CD83–/– B cells with anti-CD83 mAb (MB83-1) (lower panels) was used as a control for CD83 mAb staining on wild-type B cells (upper panels). B220 expression is shown for whole-splenocyte populations. (B) Cell-surface class II antigen expression by CD83–/– and wild-type B220+ splenocytes as shown in (A). Values (±SEM) represent average mean fluorescence intensities from three independent experiments. (C) Class II antigen endocytosis as assessed by the presence of nascent class II expression. Baseline class II levels (dashed line) on splenic B cells were determined by staining B cells with PE-conjugated class II mAb after 90 min culture at 4°C. MHC II expression on splenic B cells from CD83–/–, wild-type and heterozygous class II+/– mice treated with unlabeled class II (M5/114) mAb, washed and kept at 4°C (thin line) or 37°C (thick line) for 90 min before staining with PE-conjugated class II (M5/114) mAb. Nascent cell-surface class II antigen was identified as the portion of class II molecules available for staining with PE-conjugated class II mAb and thus not blocked by previous treatment of cells with unlabeled class II mAb. (D) CD83–/– B cells express elevated levels of nascent cell-surface class II antigen as assessed by flow cytometry in (C). Values represent mean (±SEM) percentages of nascent class II antigen staining as a fraction of total cell-surface class II expression based on relative mean fluorescence intensities. Results were obtained from triplicate cultures and represent means obtained in four independent experiments. (E) Representative class II Fab expression on wild-type and CD83–/– B cells at 0 min (solid line) or after culture for 90 min (dotted line) at 4 or 37°C. (F) Endocytosis of surface class II antigen occurs more rapidly on CD83–/– B cells. Endocytosis of class II Fab fragments on wild-type (circle) and CD83–/– (square) B cells measured over a 90 min culture period at 4°C (unfilled) or 37°C (solid). The mean (±SEM) percent of control class II Fab remaining on the cell surface for wild-type and CD83–/– B cells at each time point is shown. (G) Representative MHC class II ubiquitination in CD83–/– and wild-type spleen B cells (left panel). Class II was immunoprecipitated from cell lysates (M5/114 mAb), subjected to SDS–PAGE and transferred to nitrocellulose membranes for anti-ubiquitin (P4D1 mAb) immunoblots. Membranes were stripped and re-probed with anti-MHC class II (KLH295 ß chain-specific mAb, right panels). Non-ubiquitinated MHC II was detected after short exposures (bottom panel) while detection of ubiquitinated class II required longer exposure times (top panel). The intensity of each band from wild-type B cell lysates was determined independently (100%) and the relative intensity of each corresponding band from CD83–/– B cell lysates was determined. The values in the bar graph represent mean (±SEM) relative intensities. (B, D, F and G) Results are representative of 3 or more independent experiments and asterisks indicate sample means are significantly different from wild-type sample mean, P < 0.05.

 
Nascent cell-surface MHC class II expression on wild-type, CD83–/– and class II+/– B cells was also assessed as previously described (39). Baseline cell-surface class II levels on B cells were determined by staining with PE-conjugated anti-class II mAb before and after incubation for 90 min at 4 or 37°C. The incubation period did not affect the amount of total cell-surface class II detected on wild-type, CD83–/– or MHC II+/– B cells (Fig. 4C, data not shown). To measure nascent class II antigen expression, B cells were first treated with saturating concentrations of unlabeled anti-class II mAb, washed and cultured at 37°C for 90 min before staining with PE-conjugated anti-class II mAb. Control experiments using wild-type and CD83–/– B cells verified that the class II-blocking mAb was able to completely inhibit binding of PE-conjugated class II mAb under the assay conditions used (data not shown). After culture, wild-type, CD83–/– and class II+/– B cells displayed newly expressed class II antigen on the cell surface, as measured by fluorescence staining with PE-conjugated anti-class II mAb. When assessed as a percentage of baseline cell-surface class II antigen expression, nascent class II antigen expression on CD83–/– B cells was 62% of baseline levels, while nascent class II on wild-type B cells was only 41% of baseline levels (Fig. 4D). Increased traffic of class II to the cell surface of CD83–/– B cells was not simply explained by reduced class II expression since nascent class II expression on class II+/– B cells was only 40% of baseline cell-surface levels. Likewise, the movement of cell-surface B220 and MHC class I antigen was equal for CD83–/– and wild-type B cells (data not shown).

Taken together, the above data indicate that MHC class II molecules either move to the cell surface or internalize from the cell surface at a significantly faster rate in the absence of CD83 expression. To differentiate between these possibilities, the disappearance of class II from the surface of wild-type and CD83–/– B cells was measured directly. Cell-surface class II was saturated with biotinylated anti-class II mAb Fab fragments before culturing splenocytes at 4 or 37°C for the indicated times. The cell-surface class II molecules remaining after incubation were visualized by staining with PE-conjugated streptavidin. After 90 min at 37°C, 50 ± 2% of cell-surface class II antigen had been internalized by CD83–/– B cells whereas only 34 ± 1% of class II molecules had been internalized from wild-type B cells. There was minimal change in class II staining for wild-type and CD83–/– B cells cultured at 4°C. Thus, in the absence of CD83 expression, class II internalization on B cells was significantly increased when compared with class II internalization by wild-type B cells (Fig. 4E and F).

Down-regulation of MHC class II endocytosis is critical for efficient antigen presentation and is a defining feature of DC maturation (42). Class II ß chain ubiquitination may play a key role in regulating class II endocytosis since decreased class II ß chain ubiquitination correlates with decreased endocytosis of class II on mature DCs (43–45). To determine whether ubiquitination of class II was altered in the absence of CD83 expression, class II was immunoprecipitated from wild-type and CD83–/– B cells. While there was a slight enhancement of class II ubiquitination when compared with wild-type levels in the absence of CD83, the difference was not significant (Fig. 4G, n ≥ 3 experiments). Nitrocellulose membranes re-probed with a mAb specific for class II ß chain (clone KL295) confirmed ubiquitination of class II ß chains of appropriate molecular weights and relative intensities (Fig. 4G, top panel). This further confirms that the levels of class II mRNA and total protein were similar between CD83–/– and wild-type B cells. Collectively, these data reveal a novel role for CD83 in stabilizing class II surface expression such that class II molecules turn over at a significantly faster rate in the absence of CD83. However, the increase in class II surface turnover in B cells does not appear to be due to alterations in class II cytoplasmic tail ubiquitination.

MHC class II heterodimers are stable and functional in the absence of CD83 expression
One factor that determines the stability and/or transport of MHC class II molecules to the plasma membrane is the nature of the peptide cargo loaded into the peptide-binding groove (46, 47). To assess total class II and class II associated with peptide, splenocytes from CD83–/– and wild-type mice were stained with mAbs specific for independent I-Ab epitopes, CLIP–I-Ab and non-CLIP–I-Ab (31, 48–51). Decreased total cell-surface class II expression on CD83–/– B220+ cells was verified using the M5/114 and Y3P mAbs and three-color flow cytometry analysis (Fig. 5A; Y3P 32 ± 6% and M5/114 40 ± 5%). CD83–/– B cells were also stained with the 15G4 mAb that recognizes CLIP–I-Ab and the BP107 mAb that recognizes non-CLIP–I-Ab complexes to determine whether class II molecules in CD83–/– B cells carry altered peptide content. CLIP–I-Ab and non-CLIP–I-Ab complexes were detected on CD83–/– B cells but at reduced levels due to the concomitant decrease in total surface class II expression (28 ± 5% of wild type and 34 ± 5% of wild type, respectively; n ≥ 4 experiments). Therefore, in the absence of CD83, H2-M appears to function normally in regulating the dissociation of CLIP from I-Ab and non-CLIP–I-Ab complexes can be loaded properly. Non-CLIP–I-Ab complexes are not detected on H2-M–/– peripheral B cells (29, 31, 52, 53).


Figure 5
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  		Fig. 5. Normal structure and function of MHC class II molecules expressed by CD83–/– splenic B cells (A) Splenocytes from CD83–/– (dotted line) and wild-type (solid line) littermates were stained using mAbs reactive with distinct structural conformations of class II molecules and analyzed by flow cytometry. (B) Splenocytes from CD83–/– and wild-type (WT) littermates were pulse labeled with [35S]-methionine for 1 h, followed by a 3-h chase in cold methionine, before detergent lysis. Class II complexes were immunoprecipitated using the M5/114 mAb or a control (CTL) rat IgG2b mAb (shown with wild-type mouse splenocytes) and solubilized at 100°C (B) or at room temperature (NB) before SDS–PAGE resolution. The relative migration of molecular weight markers and compact {alpha}ß heterodimers [C({alpha}ß)] and free {alpha} and ß chains are indicated. (A and B) Results represent those obtained in 4 or more independent experiments. (C) Normal peptide presentation in the absence of CD83. CD11c+ and B220+ splenic APCs from wild-type and CD83–/– mice present OVA323–339 peptide with equal efficiency to OT-II transgenic CD4+ T as measured by the proliferation of OT-II CD4+ T cells cultured in the presence of 10uM OVA323–339 for 3 days at 37°C. Proliferation was measured by [3H]-thymidine incorporation. (D) Equivalent antigen (OVA) uptake and processing by wild-type and CD83–/– splenic APCs. CD11c+ or B220+ cells purified from wild-type and CD83–/– spleens were cultured with 2 x 105 OT-II transgenic CD4+ T cells in the presence of the indicated concentration of OVA protein for 3 days at 37°C. OT-II CD4+ T cell proliferation was measured by [3H]-thymidine incorporation. (C and D) Results are representative of 3 or more independent experiments and asterisks indicate sample means are significantly different from wild-type sample mean, P < 0.05.

 
Mature MHC class II heterodimers with properly loaded antigenic peptides are stable in the presence of SDS, while CLIP–I-Ab complexes and other unstable class II–peptide complexes typically have reduced electrophoretic mobilities due to the presence of high-molecular weight class II aggregates that have been termed ‘floppy dimers’ (29, 30, 38, 54). To examine class II conformation in the absence of CD83, wild-type and CD83–/– B cells were biosynthetically labeled and evaluated for the expression of stable class II dimers. Enriched wild-type and CD83–/– splenic B cells were pulse labeled with [35S]-methionine and immunoprecipitated proteins from cell lysates were dissociated by boiling or by incubation at room temperature with SDS. Stable class II dimers were detected in both CD83–/– and wild-type B cells after 3 h of chase and were present at the expected relative frequency and molecular mass (~50 kDa) in non-boiled samples (Fig. 5B). Thus, in the absence of CD83, class II molecules formed SDS-stable, compact dimers. Collectively, these data demonstrate that the reduced surface expression of class II in CD83–/– B cells is not due to disrupted class II assembly or maturation.

Antigen presentation in the absence of CD83
To confirm that exogenous peptide was loaded properly onto class II molecules in the absence of CD83, the ability of wild-type and CD83–/– APCs to present exogenously added peptide was evaluated. Wild-type and CD83–/– APCs were cultured in vitro in the presence of OVA323–339 peptide and OT-II+ CD4+ T cells. OT-II CD4+ T cells proliferated to the same extent when wild-type or CD83–/– APCs were used as stimulators (Fig. 5C). These results demonstrate that peptide is loaded effectively in the absence of CD83 and that CD83-deficient APCs are efficient stimulators of T cell proliferation. Invariant chain-deficient mice present peptides efficiently, but cannot process or present whole proteins effectively (30, 31, 55). To determine whether CD83–/– APCs process and present peptides derived from intact proteins, antigen presentation assays were preformed using whole OVA protein. Wild-type, CD83–/– and MHC II+/– APCs were cultured at a fixed APC:T cell ratio with intact OVA and OT-II CD4+ T cells. When APCs were cultured with whole antigen, CD83–/– APCs were more effective in inducing T cell proliferation than wild-type APCs (Fig. 5D). Therefore, despite the increased turnover of cell-surface class II molecules on CD83–/– APCs, the level of class II expression is sufficient to induce normal T cell proliferation. Thus, CD83–/– APCs were able to efficiently process OVA protein, load peptide onto class II heterodimers and present stable class II–peptide complexes on the cell surface for recognition by OT-II+ CD4+ T cells.

Decreased MHC class II expression in CD83–/– mice does not affect thymocyte development
Since TCR and MHC interactions are essential for thymocyte development, the influence of reduced MHC class II antigen expression on T cell development in CD83–/– mice was assessed. Thymic epithelial cells from heterozygous class II+/– mice had a 51 ± 2% reduction in cell-surface class II antigen expression (Fig. 6A), but these mice maintained normal CD4+ thymocyte development (Fig. 6B; Table 1). Total class II deficiency resulted in mice with few CD4+ thymocytes, particularly the CD4 single-positive and CD4+CD8low subsets, as described (27, 28). Class II expression was reduced by 50 ± 2% in CD83–/–class II+/– mice when compared with CD83–/– littermates, with a 65 ± 1% reduction in class II expression when compared with wild-type littermates (Fig. 6A). CD4 single-positive and CD4+CD8low thymocyte development in CD83–/–class II+/– mice was not significantly different from thymocyte development in CD83–/– mice (Fig. 6B; Table 1). Likewise, CD4 single-positive and CD4+CD8low thymocyte development in CD83–/–class II–/– mice was similar, if not identical to thymocyte development in class II–/– mice.


Figure 6
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  		Fig. 6. CD4+ T cell development in mice differentially expressing CD83 and MHC class II (MHC II) antigens. (A) Class II antigen expression by thymic epithelial cells from MHC II+/–, MHC II–/–, CD83–/–, CD83–/–MHC II+/– and CD83–/–MHC II–/– mice and their wild-type littermates. Values represent mean (±SEM) class II antigen densities relative to wild-type littermates (100%) as determined by immunofluorescence staining with flow cytometry analysis. (B) Representative CD4 and CD8 expression by thymocytes from mice differentially expressing CD83 and class II antigen. Percentages of cells within each gated region are shown. (C) Numbers of circulating, spleen and lymph node CD4+ T cells from mice differentially expressing CD83 and class II antigens as determined by indirect immunofluorescence staining with flow cytometry analysis. Values (±SEM) represent those obtained in eight independent experiments. (D) Altered TCRß expression on thymocyte and splenic T cell subsets from CD83–/– mice (filled bars) and their wild-type littermates (open bars). (E) Altered CD5 expression on thymocyte and splenic T cell subsets from CD83–/– mice (filled bars) and their wild-type littermates (open bars). (D and E) Values represent average mean fluorescence intensities (±SEM), with 3–8 mice per group. (A and C–E) * and **indicate significant differences between sample means P < 0.05 and P < 0.01, respectively.

 

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  		Table 1. Thymocyte development in mice deficient in CD83 and/or MHC class II expressiona

 
The dosage of MHC class II antigen expression required for CD4+ T cell expansion in the periphery was also examined using CD83–/–class II+/– and CD83–/–class II–/– mice. Again, a 50% reduction in class II antigen expression did not significantly affect CD4+ T cell frequencies or numbers in the blood, spleen or lymph nodes of class II+/– mice, while class II deficiency dramatically reduced CD4+ T cell numbers in each tissue (Fig. 6C). Combining CD83 deficiency with reduced class II expression in class II+/– mice resulted in further significant reductions in peripheral CD4+ T cell numbers compared with either deficiency alone. However, combined CD83 and MHC class II deficiencies did not affect peripheral CD4+ T cell numbers beyond the effects of class II deficiency alone. This suggests that T cells exiting the thymus of CD83–/– mice are likely to require optimal class II antigen expression for their expansion or continued survival in the periphery. Consistent with a need for class II for expansion, the majority of peripheral CD4+ T cells in CD83–/– mice exhibits a memory phenotype (12).

The cell-surface expression of TCRß, CD69 and CD5 are influenced by interactions between MHC–peptide complexes and T cell receptors expressed on immature and mature T cells. This interaction leads to characteristic expression patterns of TCRß, CD69 and CD5 on double-positive thymocytes and thymic and peripheral CD4 single-positive T cells (51, 56–58). In class II–/– mice, the level of TCRß expressed on CD4+CD8+ double-positive thymocytes is increased by 2- to 3-fold, while there was only a 28 ± 1% increase in the absence of CD83 (Fig. 6D). Additionally, TCRß expression is decreased by 2-fold on thymic and peripheral CD4 single-positive T cells from class II–/– mice (28), while TCRß levels on thymic and peripheral CD4 single-positive T cells in CD83–/– mice were only decreased by 35–39%. In class II–/– mice, CD5 expression on CD4 single-positive thymocytes is decreased by ~30% (59), while CD5 expression was reduced by ~25% on CD4 single-positive thymocytes and peripheral CD4+ T cells in CD83–/– mice (Fig. 6E). The expression of TCRß and CD5 are normal on class II+/– thymocytes (60). More importantly, CD4+CD8+ thymocytes were able to transition through the initiation checkpoint of thymocyte development in CD83–/– mice since CD4+CD8+CD69+ double-positive thymocytes were present at the same frequencies in wild-type (4.9 ± 1%) and CD83–/– (5.0 ± 1%) littermates and CD69 was expressed at the same level on these populations (data not shown). This early phase of thymocyte development crucially requires MHC expression by thymic epithelial cells and thus indicates that the level of class II expressed on CD83–/– thymic epithelial cells was sufficient for early development (58, 61, 62). Collectively, these results imply that CD83 supports CD4 thymocyte development downstream of the MHC-dependent phase of thymocyte selection.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Funding
 References
 
The effect of CD83 deficiency on MHC class II antigen expression was assessed in the current study to determine whether reduced class II densities impaired CD4+ T cell development in CD83–/– mice. Cell-surface class II expression was reduced by 25–50% on tissue B cells, thymic epithelial cells, DCs and peritoneal macrophages in CD83–/– mice (Fig. 1B and C). B cells exhibited the most significant reductions in class II antigen expression, both before and after mitogen stimulation (Fig. 1D). Significantly reduced class II expression was limited to the cell surface, since intracellular class II densities were normal or slightly increased in CD83–/– B cells (Fig. 1D and E). Other cell-surface molecules found on B cells remained at wild-type levels. Reduced class II surface expression was not the result of abnormal protein folding or significant alterations in class II epitopes expressed on the cell surface (Fig. 5A and B), altered production of MHC class II transcripts (data not shown), autocrine factors (Fig. 2A), in vivo microenvironment effects (Fig. 2C and D) or altered B cell signal transduction (Fig. 3), but was, however, an intrinsic defect for CD83–/– B cells (Fig. 2C and D). Unexpectedly, decreased class II antigen expression resulted from significantly increased cell-surface class II turnover by CD83–/– B cells (Fig. 4). Significant differences in class II internalization between wild-type and CD83–/– B cells were obvious within 10 min of culture at 37°C (Fig. 4E). Thus, cell-surface class II molecules were rapidly internalized in the absence of CD83 expression resulting in their decreased numbers on the cell surface.

APCs and thymic epithelial cells from MHC class II+/– mice show decreases in MHC class II surface expression that were more significant than were observed in CD83–/– mice (Fig. 6A, data not shown). Despite their reductions in class II antigen expression, class II+/– mice had normal CD4+ thymocyte and T cell development (Fig. 6; Table 1), as previously described (27, 63). Thereby, reduced class II antigen expression does not explain impaired CD4+ T cell development in CD83–/– mice. Furthermore, CD83–/– mice that were also haploinsufficient for class II expression had more significant CD4+ T cell defects than mice with either genetic alteration alone. However, class II deficiency was dominant in that combined class II–CD83 deficiencies did not reduce T cell numbers below those found in class II–/– mice. Thus, optimal CD83 and class II antigen expression were both required for normal CD4+ T cell development. However, in the absence of class II expression, there are alterations in the expression of other cell-surface markers. Specifically, cell-surface TCRß levels on CD4+CD8+ double-positive thymocytes are increased by 2- to 3-fold and decreased by 2-fold on single-positive CD4 thymocytes and peripheral CD4+ T cells (28). In the absence of CD83, TCRß expression by CD4+CD8+ double-positive thymocytes was only moderately increased (~30%) and there was only a 35–39% decrease in TCRß expression by single-positive CD4 thymocytes and peripheral CD4+ T cells (Fig. 6D). In addition, CD5 and CD69 expression by CD4+CD8+ double-positive thymocytes from CD83–/– mice were also normal, suggesting normal TCR interactions with MHC–peptide complexes at this stage of development (Fig. 6E, data not shown). Moreover, antigen processing, MHC–peptide complex formation and antigen presentation by DCs and B cells were normal in the absence of CD83 expression (Fig. 5C and D). Thus, these collective data infer that the block in CD4+ thymocyte development in CD83–/– mice is not caused by an inability of CD83–/– thymic epithelial cells to present self-antigen to CD4+ T cells.

Genetic deficiency of molecules required for MHC class II processing of antigens results in compromised class II maturation, expression and antigen presentation, with resulting inhibition of CD4+ T cell development (28–32). However, the absence of floppy {alpha}ß dimers in CD83–/– B cells (Fig. 5B) suggest that improperly folded or improperly loaded MHC class II molecules are not produced (29, 30). Moreover, defects in the removal of CLIP from the peptide-binding groove of class II molecules were not observed (Fig. 5A). Additionally, an increase in CLIP–I-Ab complexes would generate an intermediate-sized band on SDS–PAGE, and no such band was present after the immunoprecipitation of CD83–/– B cell lysates with the M5/114 mAb (Fig. 5B). Further, non-CLIP–I-Ab complexes were found on the surface of CD83–/– B cells (Fig. 5A) and CD83–/– APCs processed and presented antigen efficiently to CD4+ T cells (Fig. 5C and D). Additionally, CD4+ T cells from H2-M–/– mice exhibit a heightened reactivity to syngeneic I-Ab APCs, while CD83–/– CD4 T cells had a normal response signifying that CD83–/– APCs produce class II molecules which display a diverse peptide repertoire (29, 31, 53). Moreover, defective B cell maturation is not observed in CD83–/– mice (12), but is apparent in Ii–/– and H2-M–/– mice (30). Thus, the absence of CD83 does not appear to disrupt class II folding, peptide antigen binding to class II molecules or presentation of antigen by class II. In fact, CD83–/– APCs induced T cell proliferation more effectively than wild-type APCs during in vitro assays. This may reflect an increase in functional engagements between APCs and T cells in the absence of CD83 as a result of increased class II surface turnover and/or possibly the generation or presentation of more immunogenic peptides. Since CD4+ transgenic T cells specific for OVA323–339 were used as responders in these experiments, the latter possibility is less likely. It is also remains possible that CD83 facilitates class II clustering on the cell surface or is directly involved in multivalent peptide–MHC complex engagement, which is required for the initiation of TCR signal transduction (64, 65). These possibilities fit with the current available data suggesting that CD83 plays an immunostimulatory role in immune responses. While many of the molecular interactions required for efficient antigen processing and presentation have been addressed in this study, it remains possible that one or more of the molecules involved in the production of class II–peptide complexes is altered by the absence of CD83. Regardless, the current study demonstrates that CD83 primarily regulates class II stability on the cell surface.

In agreement with the results of the current study (Fig. 5), APCs generated from CD83–/– mice induce normal syngeneic and allogeneic MLR responses (12, 13), while contrasting studies demonstrate that down-regulation of CD83 expression by viral infection correlates with decreased CD4+ T cell responses (9). The decreased T cell responses shown in these viral studies could be due to disrupted expression of a combination of molecules required for T cell stimulation since viral immune regulatory proteins also target other molecules important for the generation of immune responses, such as MHC class II and CD86. Also in contrast with the current studies, recombinant soluble CD83 blocks DC-induced T cell proliferation (16, 18, 21). These contrasting observations suggest that the ligand or ligands for CD83 are able to engage additional receptors. If this is indeed the case, then additional receptor–ligand interactions that are necessary for CD4+ T cell responses may be blocked by the binding of recombinant CD83 to its physiologic ligands shared by multiple receptors. Alternatively, CD83 may function as a negative regulator of T cell activation, whereby the engagement of CD83 ligands by CD83 fusion protein may generate inhibitory signals that diminish T cell responses. Regardless, molecular explanations for these disparate observations are yet to be identified.

In conclusion, CD83–/– mice reveal a role for CD83 in maintaining normal cell-surface MHC class II expression, in addition to its role in CD4+ thymocyte development. Similarly, class II expression is stated to be increased on B cells in a transgenic mouse that over-expresses CD83 (66). Since T cell engagement and maturation of DCs (and perhaps other APCs) rearrange class II transport to the cell surface (67, 68), it is feasible that CD83 interactions with its appropriate ligands contribute to this process by either inducing/stabilizing cellular interactions or inducing cellular activation/maturation. Additionally, low-level cytoplasmic CD83 expression by APC could be functionally important since CD83 is found in the perinuclear and endoplasmic reticulum regions of mature DCs and in a recycling compartment of immature human monocyte-derived DCs (69). Recent studies have demonstrated that endocytosis of CD86 and class II is increased by cytoplasmic tail ubiquitination (43, 70). However, CD83 deficiency did not appear to alter class II ubiquitination (Fig. 4G). CD83 deficiency may also have indirect effects on class II expression as a result of CD4+ T cell deficiency and systemic immunosuppression, although the adoptive transfer of CD83–/– B cells into wild-type recipients did not restore normal B cell class II expression levels (Fig. 2). Even though further studies will be required to determine the exact mechanisms explaining how CD83 regulates class II stability, these studies show that altered class II expression does not explain abnormal CD4+ T cell development in CD83–/– mice. Thus, the ability of CD83 to regulate class II surface expression in conjunction with its previously known effects on CD4 T cell development (12) and autoimmune disease (21) further confirms the importance of this molecule during normal and abnormal immune responses in vivo.


   Funding
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
References
 
National Institutes of Health (CA098492, CA96547, CA81776 and AI56363) to T.F.T. and Ministry of Education, Science and Culture of Japan, Takeda Science Foundation and Japanese Dermatological Association (Shiseido) to M.F.


   Acknowledgements
 
We thank A. Rudensky for providing reagents for these studies and S. Yoshitake for technical assistance.


   Abbreviations
 
APC, antigen-presenting cell
7-AAD, 7-amino-actinomycin D
CFSE, carboxyfluorescein succinimidyl ester
CHX, cycloheximide
CLIP, class II-associated Ii peptide
DC, dendritic cell
ERK, extracellular signal-regulated kinase
2-ME, 2-mercaptoethanol
MLR, mixed leukocyte reaction
NF-{kappa}B, nuclear factor-{kappa}B
OVA, ovalbumin
ICAM-1, intracellular adhesion molecule-1

   Notes
 
* These authors contributed equally to this study and share first authorship. Back

Transmitting editor: S. Koyasu

Received 6 March 2007, accepted 13 April 2007.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
 References
 

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