The transmembrane glycoprotein CD83 is an important regulator of both thymic T cell maturation and peripheral T cell response. Recent studies suggested that CD83 is also involved in the regulation of B cell maturation, activation, and homeostasis. In this study, we show that in vivo overexpression of CD83 dose dependently interfered with the Ig response to thymus-dependent and thymus-independent model Ag immunization. CD83 deficiency, in contrast, which was restricted to B cells in mixed bone marrow chimeras, led to unchanged or even slightly increased Ig responses. Strikingly, the engagement of CD83 that is naturally up-regulated on wild-type B cells by injection of anti-CD83 mAb in vivo induced a 100-fold increase in the IgG1 response to immunization. Kinetic analysis revealed that CD83 had to be engaged simultaneously or shortly after the B cell activation through injection of Ag, to modulate the IgG1 secretion. Furthermore, using mixed bone marrow chimeras in which either selectively the B cells or the dendritic cells were CD83 deficient, we demonstrate that anti-CD83 mAb mediated its biologic effect by engaging CD83 on B cells and not on CD11c+ dendritic cells. Taken together, we provide strong evidence that CD83 transduces regulatory signals into the very B cell on which it is expressed.

The transmembrane glycoprotein CD83 is a well-conserved protein that belongs to the Ig superfamily (1, 2, 3). Expression pattern and immunological functions of CD83 have been discussed controversially. Because it is strongly up-regulated on murine and human dendritic cells (DC)3 upon activation, CD83 used to be described as a highly specific maturation marker for DC (4, 5). Many other studies, however, report CD83 surface expression on various cell types in vitro and in vivo, such as activated T and B lymphocytes (1, 6, 7, 8, 9), regulatory T cells (10), activated macrophages (11, 12), and neutrophils (13, 14), as well as a regulatory subset of NK cells (15). There is also evidence for CD83 expression in the brain (2, 3) and on thymic epithelial cells (TEC) (16), thus demonstrating that CD83 expression is not DC specific.

Regarding CD83 function, the analysis of two independently generated CD83-deficient mouse strains clearly showed that CD83 expression on radio-resistant TEC is needed to allow the progression from double-positive thymocytes to single-CD4-positive thymocytes, and finally to mature naive CD4+ T cells in the peripheral circulation, whereas the maturation of CD8+ T cells was CD83 independent (16, 17). In line with these results, we showed that thymic maturation in the presence of a soluble CD83Ig fusion protein led to the maturation of CD4+ T cells in normal numbers, but with impaired function, whereas the function of CD8+ T cells was unchanged (18). Several lines of evidence also suggest an implication of CD83 in the regulation of peripheral T cell responses (19). Addition of soluble CD83 species has been shown to inhibit murine T cell activation in vitro (20, 21, 22, 23) and to act immunosuppressive in vivo (24, 25). Other groups, however, did not observe such suppressive effects in comparable systems (26).

Recent studies provided evidence that CD83 is also involved in the regulation of B cell maturation, homeostasis, and function (27). CD83 is expressed at low levels by B cells beyond the pre-B cell stage, i.e., once they express a functional BCR. Artificial overexpression of CD83 in CD83 transgenic (CD83tg) mice interfered with the splenic maturation of transitional to follicular B cells and with the peripheral survival of mature naive B cells, whereas CD83 deficiency on the B cells conferred a mild selection advantage in the periphery (28). Naive wild-type B cells rapidly up-regulated CD83 upon activation by BCR or TLR engagement in vitro (6) and in vivo (8, 9). The overexpression of CD83 in vivo strongly interfered with the humoral response to thymus-dependent (TD) and thymus-independent (TI) model Ag immunization and to infection. This impaired function was B cell specific because the protective T cell response to Leishmania major infection was unchanged in CD83tg mice. Impaired B cell function was due to CD83 overexpression on the B cells themselves and not on environmental cells because CD83tg B cells did not respond to immunization, whereas wild-type B cells within the very same mixed bone marrow chimera readily did (8). Furthermore, this phenotype was also visible in vitro: purified CD83tg B cells displayed reduced Ig responses and calcium signaling upon in vitro activation, whereas CD83-deficient B cells displayed the reciprocal phenotype (6). These findings had led us to hypothesize that activation-induced CD83 negatively regulates B cells (27), thus contributing to the various mechanisms of negative regulation by receptors such as CD22 (29, 30) and CD72 (31) that counteract BCR-mediated signal transduction (32).

In this study, we directly compare the in vivo Ig responses of CD83-deficient and CD83-overexpressing mice. Using two different CD83tg mouse strains, we demonstrate that the interference with the humoral response to TD and TI Ag immunization is directly correlated to the intensity of CD83 overexpression: the more CD83 being expressed within cells of CD83tg mice, the less Ig response is detectable. Using bone marrow chimeras that allow normal CD4+ Th cell development of CD83-deficient bone marrow in wild-type hosts, we show that CD83 deficiency selectively on hematopoietic cells induces a slight, albeit not significant, increase in the humoral response to model Ag immunization. Finally, we show that in vivo engagement of activation-induced wild-type CD83 by anti-CD83 mAb dramatically increases IgG1 responses to TI immunization. Using mixed bone marrow chimeras in which either selectively B cells or DC were CD83 deficient, we demonstrate that the anti-CD83 mAb-mediated effect was transduced through CD83 expressed on the B cells themselves. Taken together, we provide strong evidence that CD83 expressed by activated B cells regulates their further differentiation and function.

C57BL/6, CD83tg founder 1 (f1), CD83tg founder 2 (f2), CD83 mutant (CD83mu), and JHT mice were bred in the animal facilities of the Bernhard-Nocht-Institute for Tropical Medicine or in the University Hospital Hamburg-Eppendorf. CD83tg f1 and f2 mice were generated at the Bernhard-Nocht-Institute for Tropical Medicine (33). CD83mu mice (17) (termed LCD4.1 originally) were a gift from F. Ramsdell (Zymogenetics, Seattle, WA). JHT mice (34) were provided by K. Rajewsky and the MGC Foundation (Munich, Germany), and CD11c-diphtheria toxin (DT) receptor (DTR)-tg (35) mice were provided by A. Radbruch (Deutsches Rheumaforschungszentrum, Berlin, Germany). Animals were used at age 6–10 wk and were maintained in a pathogen-free environment. All animal experimentations performed were approved by the Amt für Gesundheit und Verbraucherschutz (Hamburg, Germany). mAbs were obtained from BD Pharmingen. The mAbs to mouse CD83 Michel-19, Michel-17, and 1D6 were generated at the Bernhard-Nocht-Institute by immunizing a rat with a CD83Ig fusion protein (20).

The FcRs of ex vivo prepared spleen cells were blocked with mouse serum (Sigma-Aldrich; 5% v/v) for 10 min on ice. Cells were stained with 1/100 dilutions of the indicated mAb for 20 min on ice and analyzed on a BD Biosciences FACSCalibur equipped with CellQuest Pro software. The competitive capacity of different anti-mouse CD83 mAb was analyzed by incubating 58αβ cells transfected with murine CD83 with 2, 10, or 20 μg/ml unlabeled anti-CD83 mAb (clone: M19, M17, 1D6) for 10 min on ice. Subsequently, 2 μg/ml FITC-labeled anti-mouse CD83 (either clone M19 or clone 1D6) was added for another 30 min, and competitive capacity was analyzed by FACS. mAb and reagents used were as follows: FITC-labeled, biotinylated, or unlabeled anti-mouse CD83, clone Michel-19; FITC-labeled or unlabeled anti-mouse CD83, clone 1D6; unlabeled anti-mouse CD83, clone Michel-17; FITC-labeled rat IgG1, clone R3-34; biotinylated rat IgG1, clone R3-34; allophycocyanin-labeled anti-mouse CD19, clone 1D3; allophycocyanin-labeled anti-mouse CD4, clone RM4-5; allophycocyanin-labeled rat IgG2a, clone R35-95; PE-labeled anti-mouse CD11c, clone HL3; PE-labeled Armenian hamster IgG1, clone G235-2356; FITC-labeled anti-mouse IgMa, clone DS-1; biotinylated anti-mouse IgMb, clone AF6-78; and allophycocyanin-streptavidin.

Sex- and age-matched C57BL/6 mice or fully reconstituted chimeras were immunized by i.p. injection of 200 μg of 4-hydroxy-3-iodo-5-nitrophenylacetyl (NIP) conjugated to Ficoll (NIP-Ficoll; Biosearch Technologies) in 200 μl of PBS. For immunization in the presence of anti-CD83 mAb, C57BL/6 mice were injected i.p. with indicated amounts of anti-CD83 mAb or control Ig 1 day prior to and 1 day post-NIP-Ficoll immunization. Alternatively, a single dose of 20 μg of Michel-19 was injected at different time points prior to and/or post-NIP-Ficoll immunization.

Immunized mice were bled by puncture of the tail vein. Blood samples were allowed to coagulate at room temperature for 1 h and centrifuged for 10 min at 15,200 × g. Serum was harvested from the supernatant and stored at −20°C for further analysis. For the detection of Ag-specific Ig, ELISA plates were coated overnight with 1 μg/ml NIP-BSA in PBS 01% BSA. Plates were washed, blocked by incubation with PBS 1% BSA for 2 h at room temperature, and incubated with serial dilutions of serum (1/100 to 1/102,400) in duplicates overnight at 4°C. Ag-specific Ig was detected by HRP-labeled anti-mouse IgM, IgG1, IgG2b (Zymed Laboratories; 1/1000), or IgG3 (Southern Biotechnology Associates; 1/1000). Titers were calculated by defining the highest serum dilution, resulting in an OD450 above the doubled background that was generally below OD450 0.15. For the detection of anti-CD83 mAb serum levels, ELISA plates were coated overnight at 4°C with 1 μg/ml murine CD83Ig fusion protein. Plates were washed, blocked, and incubated with serial dilutions of serum in duplicates overnight at 4°C. Anti-CD83 mAb in the serum was detected by HRP-labeled anti-rat Ig (Jackson ImmunoResearch Laboratories; 1/20,000); the concentration of serum anti-CD83 mAb was calculated by using purified anti-CD83 mAb as standard.

Mixed bone marrow chimeras were generated, as described (36). Briefly, recipient IgHa-congenic C57BL/6, C57BL/6, or JHT mice received 8 Gy of γ-irradiation from a cesium source. The next day, bone marrow was extracted from tibias and femurs of donor mice (JHT, C57BL/6, CD83tg, CD83mu, and CD11c-DTR-tg mice). Recipient mice received either 2 × 106 C57BL/6, CD83tg, or CD83mu bone marrow cells or a mixture of either 1.8 × 106 CD83mu or C57BL/6, and either 4.2 × 106 JHT or CD11c-DTR-tg bone marrow cells by i.v. injection. To allow reconstitution of the lymphoid system, chimeras were left for 8 wk before immunization. Chimeras were treated orally with 0.05% (v/v) Baytril (Bayer) in drinking water starting 1 wk before transfer until 4 wk after transfer. Reconstitution was analyzed in the peripheral blood and spleen by FACS analysis. Indicated CD11c-DTR-tg chimeras were injected i.p. with 8 ng of DT (Sigma-Aldrich) per gram body weight. DT was applied 1 day prior to injection and 1 and 3 days postinjection of anti-CD83 mAb or control Ig. Depletion was monitored in two mice per group that were sacrificed and analyzed for splenic cell composition at the day of immunization.

We have shown before that CD83 overexpression on B cells in CD83tg mice strongly interfered with the humoral response to model Ag immunization and infection (8). To further analyze the impact of CD83 in B cell activation, we used two independently generated strains of CD83tg mice, displaying an incremental increase in constitutive CD83 expression, CD83tg f1 and CD83tg f2 (28). Fig. 1 shows that overexpression of CD83 suppressed humoral response to both TI (Fig. 1, A–C) and TD (Fig. 1, D–F) model Ags, concerning all isotypes. Strikingly, the degree of suppression was directly correlated to the degree of constitutive CD83 overexpression (Fig. 1 G), thus demonstrating that CD83 interfered with humoral response in a dose-dependent manner.

FIGURE 1.

Interference of CD83 with the humoral response to TI and TD Ags is dose dependent. A–F, C57BL/6 (n = 4; □), CD83tg f1 (n = 4; ▪), and CD83tg f2 (n = 4; •) mice were immunized with either 200 μg of NIP-Ficoll (A–C) or 200 μg of DNP-KLH (D–F). NIP- and DNP-specific serum IgM (A and D), IgG1 (B and E), and IgG3 (C) or IgG2b (F) were analyzed by ELISA at the indicated time points. Results are presented as the mean titer; error bars show SD. G, Naive and LPS-activated C57BL/6 (black line), CD83tg f1 (light gray line), and CD83tg f2 (dark gray line) spleen cells were double stained for CD19 and either CD83 or isotype control (dotted line). A total of 2 × 104 CD19-positive cells was analyzed by FACS. These results are representative for at least three independent experiments.

FIGURE 1.

Interference of CD83 with the humoral response to TI and TD Ags is dose dependent. A–F, C57BL/6 (n = 4; □), CD83tg f1 (n = 4; ▪), and CD83tg f2 (n = 4; •) mice were immunized with either 200 μg of NIP-Ficoll (A–C) or 200 μg of DNP-KLH (D–F). NIP- and DNP-specific serum IgM (A and D), IgG1 (B and E), and IgG3 (C) or IgG2b (F) were analyzed by ELISA at the indicated time points. Results are presented as the mean titer; error bars show SD. G, Naive and LPS-activated C57BL/6 (black line), CD83tg f1 (light gray line), and CD83tg f2 (dark gray line) spleen cells were double stained for CD19 and either CD83 or isotype control (dotted line). A total of 2 × 104 CD19-positive cells was analyzed by FACS. These results are representative for at least three independent experiments.

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Next, we wanted to compare the humoral response to model Ag immunization in CD83-overexpressing and CD83-deficient mice. To this end, we used CD83mu mice that display a severe reduction in CD83 expression due to a missense mutation in the CD83 gene (17). Because CD83 expression on TEC is needed to allow normal maturation of CD4+ T cells (16), CD83mu mice show a dramatic reduction in the percentage of CD4+ T cells in the peripheral circulation (Supplementary Fig. 1A).4 To exclude an impact of the deficiency of Th cells on the outcome of experimental B cell responses, we generated bone marrow chimeras that allowed the thymic maturation of CD83mu thymocytes on wild-type TEC. Reconstitution of irradiated wild-type hosts with either CD83tg, CD83mu, or wild-type bone marrow resulted in chimeras consisting of CD83-overexpressing, CD83-deficient, or wild-type lymphocytes within a nonhematopoietic wild-type environment. In this study, the percentage of CD4+ T cells in the peripheral circulation was comparable in wild-type into wild-type and in CD83mu into wild-type chimeras (Supplementary Fig. 1B).4 CD83 overexpression on bone marrow-derived cells led to a severe reduction in humoral response to both TI and TD Ags in comparison with the wild-type into wild-type chimeras (Fig. 2, A–F). CD83 deficiency on bone marrow-derived cells, in contrast, did not induce the reduced humoral response observed in mice completely deficient for CD83 (16). In contrast, the response to TI and TD immunization was slightly, albeit not significantly, increased if selectively hematopoietic cells lacked CD83 (Fig. 2, G–L).

FIGURE 2.

Normal Ig response to TI and TD immunization in CD83mu/wild-type bone marrow chimeras. Lethally irradiated IgHa-congenic C57BL/6 mice were reconstituted with either 2 × 106 C57BL/6 (□), CD83tg (▪), or CD83mu (○) bone marrow cells. Eight weeks after engraftment, mice were immunized with either 200 μg of NIP-Ficoll (A, B, C, G, H, and I) or 200 μg of DNP-KLH (D, E, F, J, K, and L). NIP- and DNP-specific serum IgM (A, D, G, and J), IgG1 (B, E, H, and K), and NIP-specific IgG3 (C and I) or DNP-specific IgG2b (F and L) were analyzed by ELISA at the indicated time points. Results are presented as the mean titer of the groups (n = 5); error bars show SD. This result is representative for two independent experiments.

FIGURE 2.

Normal Ig response to TI and TD immunization in CD83mu/wild-type bone marrow chimeras. Lethally irradiated IgHa-congenic C57BL/6 mice were reconstituted with either 2 × 106 C57BL/6 (□), CD83tg (▪), or CD83mu (○) bone marrow cells. Eight weeks after engraftment, mice were immunized with either 200 μg of NIP-Ficoll (A, B, C, G, H, and I) or 200 μg of DNP-KLH (D, E, F, J, K, and L). NIP- and DNP-specific serum IgM (A, D, G, and J), IgG1 (B, E, H, and K), and NIP-specific IgG3 (C and I) or DNP-specific IgG2b (F and L) were analyzed by ELISA at the indicated time points. Results are presented as the mean titer of the groups (n = 5); error bars show SD. This result is representative for two independent experiments.

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The impact of CD83 expression levels on the humoral response within CD83-deficient and CD83-overexpressing mice that we have demonstrated above strongly suggests that CD83 regulates B cell functions. Because these results were generated using nonconditional tg and mu mice that artificially lack or overexpress CD83 throughout lymphocyte development, they may reflect artificial phenotypes. To circumvent this problem, we engaged wild-type CD83 in vivo by injection of anti-CD83 mAb during model Ag immunization. Fig. 3 shows that injection of anti-CD83 mAb Michel-19 induced a dramatic increase in the IgG1 response to NIP-Ficoll immunization, whereas the amount of NIP-specific IgM and IgG3 was not affected. The response to TD immunization was not changed by application of Michel-19 (data not shown). Titration of anti-CD83 mAb showed that as low amounts as 3.1 μg/mouse already induced a significant increase in NIP-specific IgG1. This response was further and dose dependently increased by anti-CD83, reaching a plateau upon application of 12.5 μg of anti-CD83 mAb per mouse (Fig. 3 A). This increased NIP-specific IgG1 detected in the serum of immunized mice was not due to artificial detection of the anti-CD83 mAb Michel-19 itself that is a rat IgG1 Ab, because 10 μg/ml purified mAb did not induce a signal in the NIP-specific IgG1 ELISA used (data not shown). Furthermore, treatment of mice with two independently generated anti-CD83 mAb 1D6 and Michel-17, displaying different affinities for CD83 and thus representing truly different reagents, induced the same increase specifically in IgG1 response to TI immunization that was observed upon treatment with Michel-19 (Supplementary Fig. 2 and Fig. 3).4

FIGURE 3.

Treatment with anti-CD83 mAb induces increased IgG1 response to TI immunization. A–C, C57BL/6 mice were injected i.p. with indicated amounts of anti-CD83 mAb (M19; filled symbols) or control Ig (□) 1 day prior to and 1 day post-NIP-Ficoll immunization. NIP-specific serum IgG1 (A), IgM (B), and IgG3 (C) were analyzed. This result is representative for five independent experiments. All results are presented as mean titer of each group (n = 4); error bars show SD.

FIGURE 3.

Treatment with anti-CD83 mAb induces increased IgG1 response to TI immunization. A–C, C57BL/6 mice were injected i.p. with indicated amounts of anti-CD83 mAb (M19; filled symbols) or control Ig (□) 1 day prior to and 1 day post-NIP-Ficoll immunization. NIP-specific serum IgG1 (A), IgM (B), and IgG3 (C) were analyzed. This result is representative for five independent experiments. All results are presented as mean titer of each group (n = 4); error bars show SD.

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Because these results were obtained by treating mice 1 day before and 1 day after model Ag immunization with the anti-CD83 mAb, we intended to define the exact time point in B cell activation that was sensitive to CD83 engagement. Fig. 4,A shows the increase in NIP-specific IgG1 response that was obtained by injecting a single dose of 20 μg of Michel-19 at different time points before and/or after the immunization with the model Ag NIP-Ficoll. Injection of anti-CD83 mAb 6 or 2 days before the immunization as well as 1 or 6 days after the immunization did not modulate the Ig response. Anti-CD83 mAb treatment, however, that was administered within a time window between 24 h before and 12 h after immunization induced the dramatic increase in NIP-specific Ig described above. Because injection of 20 μg of Michel-19 led to peak serum concentrations of 6.5–5.5 μg/ml anti-CD83 mAb 4–12 h postinjection and the mAb was detectable in the circulation for 2 days postinjection (Fig. 4 B), this result suggests that the biologic effect of anti-CD83 mAb in vivo is dependent on a high serum concentration of the mAb at the moment of NIP-Ficoll immunization.

FIGURE 4.

Kinetic analysis of the activity of anti-CD83 mAb in vivo. C57BL/6 (n = 3) were injected i.p. with 20 μg of M19 at the indicated time points prior to and/or post TI immunization with NIP-Ficoll. A, Titer of NIP-specific serum IgG1 was analyzed in serial dilutions at day 14 postimmunization by ELISA. Results are presented as fold increase in NIP-specific IgG1 titer in the M19-treated groups compared with the control group immunized with NIP-Ficoll only. B, Serum concentration of M19 was measured at the indicated time points by ELISA. The graph depicts the combined results of three independent experiments; error bars show SD.

FIGURE 4.

Kinetic analysis of the activity of anti-CD83 mAb in vivo. C57BL/6 (n = 3) were injected i.p. with 20 μg of M19 at the indicated time points prior to and/or post TI immunization with NIP-Ficoll. A, Titer of NIP-specific serum IgG1 was analyzed in serial dilutions at day 14 postimmunization by ELISA. Results are presented as fold increase in NIP-specific IgG1 titer in the M19-treated groups compared with the control group immunized with NIP-Ficoll only. B, Serum concentration of M19 was measured at the indicated time points by ELISA. The graph depicts the combined results of three independent experiments; error bars show SD.

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Having shown that CD83 overexpression specifically and dose dependently interfered with humoral response to model Ag immunization (Fig. 1) and that simultaneous engagement of CD83 and BCR in vivo led to an increased IgG1 response of wild-type B cells (Figs. 3 and 4), it is tempting to speculate that engagement of CD83 on the B cells themselves would modulate their function. However, because CD83 is not exclusively expressed by B cells, but by many activated leukocytes (27), it is likely that anti-CD83 mAb binds to all of these CD83-positive cell types in vivo. To identify the CD83-positive cell population responsible for the biologic effect of CD83 engagement, we generated mixed bone marrow chimeras in which selectively B cells (Fig. 5) or DC (Fig. 6) were CD83 deficient.

FIGURE 5.

Engagement of CD83 on B cells increases NIP-specific IgG1 response. A, The cartoon shows the experimental approach. Mixed bone marrow chimeras were generated by reconstituting lethally irradiated JHT mice with 4.2 × 106 JHT-derived bone marrow cells and either 1.8 × 106 CD83mu (chimera group (1))- or 1.8 × 106 C57BL/6-derived bone marrow cells (chimera group (2)). B, Eight weeks after bone marrow transfer, chimeras were analyzed for CD83 expression on CD11c+ splenic DC (upper panel) and on CD19+ splenic B cells that were either left naive (middle panel) or activated for 24 h with LPS (10 μg/ml; lower panel) by FACS analysis. The result is representative for two chimeras sacrificed for testing in each group. C, Chimeras were injected with either 20 μg of anti-CD83 mAb (M19) (filled symbols) or control Ig (open symbols) 1 day prior to and 1 day post-NIP-Ficoll immunization. NIP-specific serum IgG1 was analyzed at the indicated time points by ELISA. Results are presented as mean titer of the groups (n = 5); error bars show SD. This result is representative for two independent experiments.

FIGURE 5.

Engagement of CD83 on B cells increases NIP-specific IgG1 response. A, The cartoon shows the experimental approach. Mixed bone marrow chimeras were generated by reconstituting lethally irradiated JHT mice with 4.2 × 106 JHT-derived bone marrow cells and either 1.8 × 106 CD83mu (chimera group (1))- or 1.8 × 106 C57BL/6-derived bone marrow cells (chimera group (2)). B, Eight weeks after bone marrow transfer, chimeras were analyzed for CD83 expression on CD11c+ splenic DC (upper panel) and on CD19+ splenic B cells that were either left naive (middle panel) or activated for 24 h with LPS (10 μg/ml; lower panel) by FACS analysis. The result is representative for two chimeras sacrificed for testing in each group. C, Chimeras were injected with either 20 μg of anti-CD83 mAb (M19) (filled symbols) or control Ig (open symbols) 1 day prior to and 1 day post-NIP-Ficoll immunization. NIP-specific serum IgG1 was analyzed at the indicated time points by ELISA. Results are presented as mean titer of the groups (n = 5); error bars show SD. This result is representative for two independent experiments.

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FIGURE 6.

Engagement of CD83 on CD11c+ DC does not contribute to the increased IgG1 response. A, The cartoon shows the experimental approach. Mixed bone marrow chimeras were generated by reconstituting lethally irradiated C57BL/6 mice with 1.8 × 106 CD83mu-derived bone marrow cells and either 4.2 × 106 CD11c-DTR-tg- (chimera (1)) or 4.2 × 106 C57BL/6-derived bone marrow cells (chimera (2)). The application of DT to chimera (1) (+DT) resulted in the depletion of all CD11c-DTR-tg DCs. The remaining DCs (∼30% of normal) originated from CD83mu bone marrow, and therefore, lacked CD83. A second set of chimera group (1) did not receive DT (−DT). Here, all cell types of hematopoietic origin consisted of ∼30% CD83mu and ∼70% CD83 competent cells. B, Eight weeks after bone marrow transfer, reconstituted chimeras were analyzed for CD83 expression on CD11c+ splenic DC (upper panel) and on CD19+ splenic B cells that were either cultured in medium (middle panel) or for 24 h with LPS (10 μg/ml; lower panel) by FACS analysis. The result is representative for two chimeras sacrificed for testing in each group. C, In eight mice of chimera group (1), CD11c+ DC were depleted by DT application (squares), and eight mice were left untreated (circles). Eight mice of chimera group (2) were injected with DT (triangles). All chimeras received either 20 μg of Michel-19 (filled symbols) or control Ig (open symbols) 1 day prior to and 1 day post-NIP-Ficoll immunization, and NIP-specific serum IgG1 was analyzed at the indicated time points by ELISA. Results are presented as mean titer of the groups; error bars show SD.

FIGURE 6.

Engagement of CD83 on CD11c+ DC does not contribute to the increased IgG1 response. A, The cartoon shows the experimental approach. Mixed bone marrow chimeras were generated by reconstituting lethally irradiated C57BL/6 mice with 1.8 × 106 CD83mu-derived bone marrow cells and either 4.2 × 106 CD11c-DTR-tg- (chimera (1)) or 4.2 × 106 C57BL/6-derived bone marrow cells (chimera (2)). The application of DT to chimera (1) (+DT) resulted in the depletion of all CD11c-DTR-tg DCs. The remaining DCs (∼30% of normal) originated from CD83mu bone marrow, and therefore, lacked CD83. A second set of chimera group (1) did not receive DT (−DT). Here, all cell types of hematopoietic origin consisted of ∼30% CD83mu and ∼70% CD83 competent cells. B, Eight weeks after bone marrow transfer, reconstituted chimeras were analyzed for CD83 expression on CD11c+ splenic DC (upper panel) and on CD19+ splenic B cells that were either cultured in medium (middle panel) or for 24 h with LPS (10 μg/ml; lower panel) by FACS analysis. The result is representative for two chimeras sacrificed for testing in each group. C, In eight mice of chimera group (1), CD11c+ DC were depleted by DT application (squares), and eight mice were left untreated (circles). Eight mice of chimera group (2) were injected with DT (triangles). All chimeras received either 20 μg of Michel-19 (filled symbols) or control Ig (open symbols) 1 day prior to and 1 day post-NIP-Ficoll immunization, and NIP-specific serum IgG1 was analyzed at the indicated time points by ELISA. Results are presented as mean titer of the groups; error bars show SD.

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First, we reconstituted irradiated JHT mice (34) that lack B cells with a mixture of either JHT and wild-type or JHT and CD83mu bone marrow, as depicted in Fig. 5,A. Although the environment and somatic tissue in both chimeras were wild type with respect to CD83 expression, the B cells in the JHT/CD83mu chimera (Fig. 5,A chimera (1)) originated from the CD83mu bone marrow and were thus CD83 deficient. Other lymphocytes such as T cells and DC matured from both, CD83mu (30%) and JHT (70%) bone marrow graft, leading to a situation in which ∼70% of these cells were wild type with respect to CD83 expression. In contrast, B cells in the JHT/wild-type chimera matured selectively from the wild-type bone marrow and were thus capable of up-regulating CD83 upon activation (Fig. 5 A chimera (2)).

Fig. 5,B shows that activated splenic DC derived from both chimeras expressed CD83. Please note the mixture of CD83mu and JHT-derived DC in chimera (1) is reflected by the presence of two distinct cell populations, a CD11c+CD83low and a CD11c+CD83+, whereas the wild-type JHT chimera (2) selectively harbored CD11c+CD83+ splenic DC (Fig. 5,B). Analysis of B cells in these chimeras confirmed that no significant constitutive expression and no up-regulation of CD83 upon LPS activation were detectable in CD83mu/JHT chimera (1) whereas CD83 was expressed by activated B cells in wild-type/JHT chimera (2) (Fig. 5,B). Having thus constructed mice in which selectively the B cells lacked CD83, we immunized both chimera groups with NIP-Ficoll in the presence of either anti-CD83 mAb or isotype control. Comparison of normal IgG1 response to NIP-Ficoll immunization in the presence of isotype control revealed a slight increase in NIP-specific IgG1 in chimera (1) that lacked CD83 on the B cells compared with chimera (2) (Fig. 5,C, open symbols). This shows again that CD83 deficiency on B cells did not interfere with B cell function, but, in contrast, mildly enhanced IgG1 secretion. Interestingly, the NIP-specific IgG1 response in the presence or absence of anti-CD83 mAb was unchanged in chimera group (1), which lacked CD83 selectively on the B cells (Fig. 5,C, ○ and •). Control chimera group (2) that was capable of up-regulating CD83 on B cells again displayed a marked increase in IgG1 response upon anti-CD83 injection compared with isotype control (Fig. 5 C, □ and ▪). The Ag-specific IgG3 and IgM responses were unchanged in both chimeras in the presence of anti-CD83 (data not shown). These data strongly suggest that CD83 expression on B cells was necessary to mediate the biologic activity of anti-CD83 mAb in vivo that finally led to the observed phenotype of increased B cell activation.

Taking into account, however, that we compared chimeras that contained 100% CD83 wild-type CD11c+ DC (chimera group (2)) with chimeras that contained a mixture of CD83 wild-type CD11c+ DC and CD83mu CD11c+ DC (Fig. 5, A and B), it is possible that these reduced frequencies of CD83+ DC contributed to the lack of anti-CD83 mAb efficacy in this group. Although the mediators of Ig class switch in the response to TI-2 Ags such as NIP-Ficoll are still being investigated (37), CD11c+ DC have been shown to contribute to the Ig class switch in the absence of classical CD40-mediated T cell help (38). To finally exclude a contribution of CD83 expressed by CD11c+ DC to the biologic effect mediated by anti-CD83 treatment, we generated mice selectively lacking CD83 on DC. To this end, we used CD11cDTR mice that were engineered to coexpress the human DTR and CD11c. Although CD11c is not an exclusive marker for DC, all DC do express CD11c and will be transiently depleted by DT application together with other CD11c+ cells (35). Fig. 6,A shows our experimental approach. Irradiated hosts were reconstituted with either a mixture of CD83mu and CD11cDTR bone marrow (chimera group (1)) or CD83mu and wild-type bone marrow (chimera group (2)). After successful engraftment, 10 mice of chimera group (1) and chimera group (2) were treated with DT, which led to a transient depletion of CD11c+ cells in chimera group (1), but not in chimera group (2) that was generated to control for any side effects of the DT treatment on the humoral response. Fig. 6,B shows the CD83 expression on CD11c+ DC and B cells after DT application. In the DT-treated chimera (1), ∼70% of the CD11c+ cells originated from CD11cDTR bone marrow and were depleted by DT application. The remaining DC originated from the CD83mu bone marrow were insensitive to DT depletion, but did not express CD83 (Fig. 6,B, upper panel chimera (1) +DT). CD83 staining of CD11c+ cells in the untreated chimera group (1) showed that the DC population was still composed of a mixture of CD11cDTR-derived cells that expressed CD83 and CD83mu-derived cells that were CD83 negative (Fig. 6,B, upper panel chimera (1) −DT). Finally, the analysis of CD83 expression on CD11c+ cells in chimera (2) after DT treatment showed again the mixed CD11c+ population with some CD83 DC that originated from CD83mu bone marrow and some CD83+ cells that originated from wild-type bone marrow. Because both grafts were DT insensitive, the application of DT did not reduce the total amount of DC in this chimera group. Constitutive as well as LPS-induced CD83 expression on the B cells was comparable in all three groups, as expected (Fig. 6,B, lower panel). All groups were immunized with NIP-Ficoll in the presence of anti-CD83 mAb or isotype control. Fig. 6 C clearly shows that application of anti-CD83 increased the NIP-specific IgG1 response more than 10-fold in all chimera groups, i.e., in mice in which DC expressed CD83 and in mice lacking CD83+ DC at the moment of immunization and anti-CD83 treatment. This experiment clearly demonstrates that CD83 engagement on DC did not induce the observed increase in NIP-specific IgG1.

CD83 is rapidly up-regulated by activated B cells in vivo (8, 9) and in vitro (6). The tg overexpression of CD83 specifically on B cells was shown to interfere with B cell maturation, homeostasis (28), B cell activation, and Ig response to both model Ag immunization and infection (8). These findings had led us to hypothesize that CD83 negatively regulates B cells (27). To test our hypothesis, we asked whether CD83 deficiency induced a reciprocal increased activation that one may expect from B cells lacking a putative negative regulator. A direct comparison of B cell responses to model Ag immunization in vivo is not possible in CD83-deficient mice because they display severely reduced numbers of Th cells due to disturbed thymic maturation in the absence of CD83 on TEC (17). Indeed, CD83-deficient mice were shown to display a reduced Ig response to TD immunization (16) that could be due to CD83 deficiency, but may also be explained by the reduced numbers of Th cells in these mice. Using bone marrow chimeras that contained normal frequencies of Th cells and in which CD83 deficiency was restricted to hematopoietic cells (Fig. 2, J–L), we show that CD83 deficiency on bone marrow-derived cells did not interfere with the response to TD Ags. Moreover, CD83 deficiency that was restricted to the B cells even induced a mild increase in the early IgG1 response to TI model Ags, as we showed using mixed bone marrow chimeras (Fig. 5 C). In contrast, CD83 overexpression interfered with humoral response in a dose-dependent manner. These results are in line with our earlier findings, namely that purified CD83-deficient B cells displayed a slightly increased Ig secretion, whereas CD83-overexpressing B cells showed a reciprocally reduced Ig secretion in vitro (6), and thus consent with the hypothesis that CD83 would transduce negative signals into the very B cell on which it is expressed.

To further investigate the regulatory role of CD83 in B cell function in wild-type mice, we analyzed the impact of CD83 engagement in vivo. In line with our earlier results (8), we show that the application of anti-CD83 mAb induced an up to 100-fold increase in the IgG1 response to TI immunization. This increased IgG1 response was dependent on the dose of anti-CD83 mAb administered and was induced by all anti-CD83 mAb available for testing to date. Kinetic studies of efficiency further showed that anti-CD83 mAb application increased the humoral response optimally if the systemic anti-CD83 mAb concentration was high at the moment of immunization. Because engagement of CD83 2 days before the immunization did not alter the response, it is unlikely that CD83 engagement on B cells or other cells before the actual B cell activation is important for the outcome of the immunization. Also, the fact that the presence of anti-CD83 mAb has no dramatic effect on the B cell function 1 day postimmunization strongly suggests that modulation of already activated B cells by CD83 engagement is not possible. These results rather support the idea that CD83 engagement has to take place simultaneously with, or shortly after, the BCR engagement. Indeed, CD83 is maximally up-regulated on B cells 6 h postactivation in vitro (6) and in vivo (9). Therefore, CD83 induced on wild-type B cells by NIP-Ficoll immunization in vivo would be available for engagement by anti-CD83 mAb.

CD83, however, is also expressed by many activated murine leukocytes, such as DC (5, 7, 39), T effector cells (9, 20), regulatory T cells (10), TEC (16), and the periarteriolar lymphoid sheath in the white pulp (7). It is, therefore, likely that anti-CD83 mAb, upon injection, would bind to all of these CD83-positive cell types and tissues in vivo. Moreover, especially DC are important accessory cells that can induce Ig class switch in B cells upon TI immunization (38). Therefore, we had to identify through which cell population the CD83-mediated signal was transduced in vivo. Using mixed bone marrow chimeras in which either the B cells or the DC selectively lacked CD83, we showed that triggering of CD83 expressed by DC did not contribute to the biological activity of the anti-CD83 mAb treatment. In contrast, CD83 engagement on the B cells themselves mediated the increased IgG1 response observed, because anti-CD83 mAb treatment did not increase the IgG1 response anymore if selectively B cells were CD83 deficient.

Regarding the mechanism, it is conceivable that CD83 engagement on B cells induced positive signaling that selectively favored induction and expansion of IgG1-producing B cells. We consider this unlikely because in this case one would expect reduced IgG1 responses in mice deficient for CD83 on B cells, and hence lacking such CD83-mediated positive signal transduction into B cells. The IgG1 response in mice selectively lacking CD83 on B cells, however, was not reduced, but slightly increased in comparison with the IgG1 response of mice containing wild-type B cells (Fig. 5 C, ○, □). We therefore suggest that binding of anti-CD83 mAb to CD83 that is expressed by activated B cells in vivo prevents the transduction of a negative signal that otherwise would have dampened the Ig response.

One may argue that if prevention of putative negative signals by CD83 engagement increased the IgG1 response by two orders of magnitude, as we have shown (Figs. 3,A and 5,C), the complete CD83 deficiency on B cells in vivo as in the mixed JHT/CD83mu chimera should lead to a similar increase in IgG1. We did indeed observe an increase of IgG1 response to TI immunization in these mice (Fig. 5 C). However, this increased IgG1 response was admittedly much weaker than the increase induced by anti-CD83 mAb application. We argue that due to the lack of CD83 throughout the life of the B cells within CD83-deficient mice, other regulatory pathways may have compensated for the lack of negative regulation through CD83. Therefore, the sudden interruption of a negative feedback loop in wild-type mice by application of a putative neutralizing mAb would have a more pronounced effect on B cell activation than the lifelong deficiency as present in the CD83mu mice. In support of this hypothesis, CD83-deficient B cells were shown to display decreased expression of MHC-II and CD86, whereas CD83-overexpressing B cells displayed the reciprocal phenotype (6, 40). Decreased MHC-II expression will lead to a reduction of positive signals delivered by MHC-II directly into the B cell (41) and also result in an impaired interaction with the cognate Th cell, and thus, reduced costimulation via CD40 (42). CD86 has been shown to transduce positive signals into B cells, leading to increased activation and an increased IgG1 production (43, 44, 45). Again, the reduced expression of this costimulatory receptor in CD83-deficient mice may well reflect the compensation for the lack of the putative negative regulator CD83. We are currently generating inducible CD83-deficient and CD83tg mice to finally address this question.

The following remains to be elucidated: 1) why engagement of CD83 on B cells selectively enhances IgG1 responses to TI immunization; 2) how a putative regulatory signal may be transduced by CD83 that lacks a significant intracellular domain and does not contain intracellular tyrosine residues and thus no ITIM motifs (3); 3) whether transducing coreceptors are involved in CD83-mediated signal transduction; and 4) whether this putative CD83-mediated negative signal is transduced by tonic signaling or by engagement of a CD83 ligand that has not been identified to date. Despite the remaining open questions within this study, we have clearly shown that engagement of wild-type CD83 on wild-type B cells and not on DC modulates B cell function. Thereby, we have added further evidence for a regulatory function of CD83 on B cells.

We are indebted to Dr. Fred Ramsdell for sharing the CD83mu mice. We thank Dr. Andreas Radbruch for providing us with CD11c-DTR-tg mice, and Dr. Simon Fillatreau for the introduction into the technique of mixed bone marrow chimeras. We thank Dr. Anke Osterloh for help in preparation of figures.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by the Deutsche Forschungsgemeinschaft (FL 129/5-1, 5-2).

3

Abbreviations used in this paper: DC, dendritic cell; DT, diphtheria toxin; DTR, DT receptor; mu, mutant; NIP, 4-hydroxy-3-iodo-5-nitrophenylacetyl; TD, thymus dependent; TEC, thymic epithelial cell; tg, transgenic; TI, thymus independent; f1, founder 1; f2, founder 2.

4

The online version of this article contains supplementary material.

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