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CD83 regulates lymphocyte maturation, activation and homeostasis
Review
CD83 regulates lymphocyte maturation, activation and homeostasis Minka Breloer and Bernhard Fleischer Bernhard-Nocht-Institute for Tropical Medicine, 20359 Hamburg, Germany
The transmembrane CD83 molecule, a conserved member of the immunoglobulin superfamily, is known as one of the most characteristic cell surface markers for fully matured dendritic cells (DCs) in the peripheral circulation. An essential role for CD83 on murine DCs has not been found; however, evidence shows that its function primarily lies in the regulation of T- and B-lymphocyte maturation and in the regulation of their peripheral responses. Here, we review evidence for a role of CD83 in central lymphocyte maturation and novel, sometimes contradictory findings, regarding the function of CD83 in peripheral immune responses. Structure and expression pattern of CD83 CD83 used to be described as a highly specific marker for activated dendritic cells (DCs) in humans [1] and mice [2] (reviewed in [3–5]). However, many studies report CD83 surface expression on other cells in vitro and in vivo (Table 1) such as activated T and B lymphocytes [6–10], activated macrophages [11,12], a regulatory subset of NK cells [13], activated neutrophils [14,15], thymic epithelial cells [16] and uncharacterized cell population(s) in the brain [17,18]. Specific upregulation of CD83 was also observed in Foxp3+CD4+CD25+ natural regulatory T cells at the mRNA [19,20] and protein level (Hansen et al., unpublished data). The molecular and structural properties of CD83 have been reviewed elsewhere [5,21]. Briefly, CD83 is an evolutionarily well-conserved highly glycosylated type 1 transmembrane glycoprotein composed of 175 amino acids (AAs) in the mouse [18] and 186 in humans [6,17]. The cytoplasmic tail is predicted to consist of either of 39 [6] or 42 AAs [17], contains no tyrosine and thus does not give any clues as to possible direct signal transduction through immunoreceptor tyrosine-based inhibitory or activatory motifs (ITIM or ITAM). Studies using recombinant variants of the CD83 extracellular domain reported a disulfide-mediated homodimerization through cysteine 129, leaving four additional cysteine residues for formation of intramolecular disulfide bonds [22]. By contrast, there is no evidence for disulfide bond–mediated dimerization of transmembrane CD83, because nonreducing and reducing conditions led to an identical migration pattern in SDS– PAGE [6]. Release of CD83 from the plasma membrane into the supernatant was reported for in vitro cultured DCs and B Corresponding author: Breloer, M. ([email protected]).
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cells [23], as well as for virus-infected DCs [24]. Surface expression of CD83 is positively correlated to CD86 and major histocompatibility complex II (MHC-II) expression, because CD83-overexpressing B cells and DCs derived from CD83 transgenic (tg) mice display an increased surface expression of these molecules [9], whereas CD83deficient B cells and DCs showed a reciprocal reduction in CD86 and MHC-II surface expression [9,16,25]. Constitutive and activation-induced surface expression of other co-stimulatory molecules such as CD80, CD40 or MHC-I was unchanged in CD83tg- and CD83-deficient antigen presenting cells (APCs) [9]. In concordance with its nuclear factor-kB (NF-kB)–mediated gene regulation [2,26,27], CD83 is rapidly upregulated on murine B cells, reaching maximal expression 6 h after either toll-like receptor (TLR) engagement by lipopolysaccharide (LPS) or B cell receptor (BCR) ligation [9]. In vivo (e.g. in an ongoing infection), activated B cells are the dominant CD83positive cell population in the draining lymph nodes [8]. In contrast to some studies investigating human DCs and macrophages [11,28], no intracellular CD83 protein stores were detected in resting murine B cells or spleen cells [9], suggesting that the CD83 protein expressed by activated B cells is produced by de novo protein synthesis. The functional studies described below suggest that a ligand(s) for CD83 exists but thus far it has not been unequivocally identified. Data indicating possible CD83 ligand–expressing cell populations are controversial and have provided evidence for CD83 binding to human DCs [29], monocytes [30], human CD8+ T cells [30,31] and murine B2 cells [32]. Because all these cells also express CD83 after activation, a homotypic interaction cannot be excluded. CD83: central regulatory functions in the immune system CD83 is essential for the thymic maturation of CD4+ T cells The CD83 / mice described in 2002 [16] display dramatically reduced numbers of CD4 single positive (SP) thymocytes within the thymus (3% instead of 15% in the wild type) and equally reduced numbers of CD4+ T cells in the peripheral circulation (6% instead of 33% in the wild type). This phenotype was reproduced in CD83 / mice backcrossed to T cell receptor (TCR) transgenic mice. In elegant transfer experiments, the authors showed that, to allow normal maturation of CD4+ T cells, the CD83 molecule
1471-4906/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.it.2008.01.009 Available online 7 March 2008
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Table 1. CD83 expression in the immune system Human tissue and cells Various B and T cell lines, activated PBL Staphylococcus aureus Cowan (SAC) activated B cells, B cell lines, brain and lung tissue (no detection on activated T cell lines and tonsil derived T cells) B cell follicle: mantle zone and germinal center Peripheral blood–derived DCs (no detection on circulating B, T and NK cells) Peripheral blood–derived DCs Hodgkin’s lymphoma cells DC, B cells and Hodgkin’s lymphoma cells EBV-induced B cells PBL-derived mitogen stimulated T cells Activated neutrophils Activated macrophages Alveolar lung macrophages post lung transplantation DCs infiltrating in: Psoriatic lesions Colonic mucosa in Crohn’s disease Multiple sclerosis lesions Breast carcinoma Gallbladder carcinoma Gastric cancer Leprosy lesions NK cells Murine tissue and cells Brain and spleen tissue Bone marrow–derived DCs (no detection on B cell line A-20 and T cell lines 3DO and DO11.10) B cell lymhoma (A-20, Wehi 231, K46) and B cell myeloma X63 (no detection on the T cell hybridomas 3DO, DO11.10) J774, bone marrow–derived DCs Thymic epithelial cells and thymic DC but not double positive thymocytes Mature bone marrow–derived DCs Peri-arteriolar lymphoid sheath in the white pulp B cells in draining LN of L. major– and T. cruzi–infected mice Activated spleen-derived B cells in vitro LPS-activated spleenic DC, macrophages, activated spleenic T cells Regulatory T cells Other Shark: epigonal tissue Trout: MHC-II–positive tissue Sea bream: LPS-activated macrophages Chicken: tissue in proximity of B cells of all developmental stages (splenic B cell region, medulla of the bursa fabricii, splenic and tonsil germinal center, Harderian gland)
Detection method
Refs
Northern blot FACS (HB15a) Northern blot
[6] [17]
In situ hybridization FACS (HB15a,b,c) FACS (1G1; 4B5) (HB15a) Histology (HB 15a) ELISA (HB15a and rabbit anti-CD83AS); western blot (rabbit anti-CD83AS); FACS (HB15a) FACS (HB15a) Northern blot RT–PCR and FACS (HB15a) FACS (HB15e) Histology
[54] [26] [14] [15] [11] [12]
Histology (HB15a) Histology and FACS Immunhistochemistry Histology Histology Histology Histology (B10A-1) FACS (HB15a)
[55] [56] [57] [58] [59] [60] [61] [13]
Northern blot Northern blot
[18] [2]
FACS (Michel-19, Michel-17)
a
FACS (polyclonal rabbit anti-CD83 AS) In situ hybridisation and RT PCR FACS (Michel-17) Histology (Michel-17) FACS (Michel-19) FACS (Michel-19) western blot (polyclonal rabbit anti-mouse CD83 AS) FACS (Michel-19) Gene array FACS (Michel-19)
[32] [16] [7] [7] [8] [9]
RT–PCR
[62] [62] [63] [64]
RT–PCR Histology (polyclonal sheep anti-chicken CD83AS, mouse anti-chicken CD83mAb)
[17] [1] [51] [52] [53] [23]
a
[19,20], b
1G1, 4B5 and HB15a,b,c refer to the CD83 monoclonal antibody (mAb) clone. Abbreviations: DC, dendritic cell; EBV, Epstein-Barr virus; ELISA, enzyme-linked immunosorbent assay; FACS, fluorescence activated cell sorting; LPS, lipopolysaccharide; MHC II, major histocompatibility complex II; NK, natural killer; PBL, peripheral blood lymphocytes. a M.B. and B.F., unpublished. b Hansen et al., pers. commun.
needed to be expressed on radioresistant thymic epithelial cells (TECs) but not on the thymocytes themselves. These findings were confirmed in a mouse carrying a point mutation in the CD83 gene leading to dramatically reduced protein expression levels [33]. CD83 mutant (CD83mu) mice displayed the same reduction in CD4+ T cells in peripheral blood and CD4+ SP thymocytes as that observed in CD83 / mice. Because the numbers of CD4+ SP thymocytes returned to normal levels after transgenic expression of CD83 in CD83mu mice, CD83 deficiency was formally proven to be the cause of defective CD4+ T-cell maturation in CD83mu mice.
Interestingly, the 5% of CD4+ T cells that finally matured within CD83mu mice displayed an impaired phenotype, resulting in reduced proliferation and interleukin-2 (IL-2) secretion but increased IL-4 and IL-10 secretion as a result of in vitro stimulation and reduced delayed type hypersensitivity (DTH) responses in vivo [33]. This phenotype was acquired as a response to thymocyte maturation in a CD83-deficient thymus because CD83mu bone marrow transplanted into wildtype mice gave rise to CD4+ T cells that carried the mutated CD83 gene but responded normally to allogenic stimulation. 187
Review Transgenic mice producing a CD83 immunoglobulin fusion protein (CD83Igtg mice) have also yielded useful clues to the in vivo function of CD83. These mice produce CD83Ig under an MHC-II promoter, and the fusion protein is released into the serum and extracellular space. We observed that CD4+ T cells from CD83Igtg mice exhibited impaired functions similar to that seen in the knockout mice. Although thymocyte maturation in the presence of CD83Ig (10–15 ng/ml of serum) allowed development of normal frequencies of CD4+ T cells, the CD83Igtg mice were more susceptible to a Trypanosoma cruzi infection and showed reduced cytokine responses after in vitro stimulation [34]. It was shown that this defective phenotype was acquired upon thymic maturation of double positive (DP) thymocytes to CD4+ SP thymocytes and was not caused by the presence of CD83Ig in the periphery because of the following evidence: (i) The defective phenotype was restricted to CD4+ T cells; CD8+ T cells derived from CD83Igtg mice were as potent as wild-type T cells. (ii) Purified CD4+ CD83Igtg T cells displayed an impaired response to antigenic stimulation by wildtype APC in vitro even though they did not secrete the CD83Ig themselves. (iii) CD4+ SP thymocytes purified from the thymi of CD83Igtg mice (i.e. before they entered peripheral circulation) already displayed the impaired phenotype. (iv) In vivo application of recombinant CD83Ig did not revert the resistant phenotype of C56BL/6 mice to a
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sublethal T. cruzi infection to the highly susceptible phenotype of the CD83Igtg mice. Taken together, these three studies [16,33,34] highlight the central function of CD83 in the maturation of CD4+ T cells. CD83 expressed on TEC is needed to deliver a signal to the DP thymocytes that allows the further progression to CD4+ SP thymocytes (Figure 1a). Absence of this signaling by CD83 deficiency as in the knockout mice or the CD83mu mice results in the reduction of both CD4+ SP thymocytes and CD4+ T cells in the periphery (Figure 1b). Partial interference with this signal caused by the presence of competing CD83Ig fusion protein in the circulation, however, allows the development of CD4+ T cells in normal frequencies (Figure 1c). The CD4+ T cells that mature despite the disturbed thymic CD83 signaling in all of the three mouse models display an impaired phenotype in vivo [16,33,34] and in vitro [33,34] that reflects the incomplete CD83-mediated signaling during thymic development. By contrast, CD8+ T cells develop in normal numbers [16,33] and display normal function [34], thus further demonstrating that CD83 specifically regulates maturation of functional CD4+ T cells. CD83 is involved in B-cell maturation CD83 is expressed at low levels by immature B cells beyond the pre-B cell stage (i.e. once they express a functional BCR) [10]. The first hints that overexpression of CD83 may interfere with the late maturation of B cells were gained from the analysis of CD83tg mouse strains that displayed an increase in immature transitional B cells and a
Figure 1. Involvement of CD83 in T-cell thymic development. (a) Maturation of T cells in wild-type mice: Double positive (DP) thymocytes (CD4+8+) receive a signal from CD83 expressed on thymic epithelial cells (TECs), most likely through a putative CD83 ligand, that allows their further maturation to functional single positive (SP) CD4+ T cells. (b) Maturation of T cells in CD83mu (mutant) and CD83 / mice: in the absence of CD83 on thymic epithelium, the numbers of CD8+ T cells are unchanged, but the numbers of CD4+ SP thymocytes and peripheral CD4+ T cells are reduced. The function of the remaining CD4+ T cells is impaired, the function of CD8+ has not been investigated in this model. (c) Maturation of T cells in the presence of soluble CD83Ig: Soluble CD83Ig interferes with the physiological interaction of CD83 and its putative ligand in the thymus. SP thymocytes and CD4+ and CD8+ T cells develop in normal numbers. The function of the developing SP CD4+ thymocytes and peripheral CD4+ T cells is impaired, but the function of CD8+ T cells is unchanged.
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Table 2. Biological effects of soluble CD83 species in vitro and in vivo Construct E. coli–derived extracellular domain of human CD83
E. coli–derived extracellular domain of human CD83 with mutation on Cys129 (produces monomeric construct) Truncated variant of human CD83 lacking transmembrane and membrane proximal domain Human CD83Ig extracellular domain fused to human IgG1 and coated to beads Human CD83 released by HCMV infected monocyte-derived human DC Human CD83Ig Human CD83Ig Murine CD83Ig Murine CD83Ig Murine CD83Ig
Biological effect Inhibition of human MLR and human DC maturation in vitro Interference with veil formation in human DCs Prevention of EAE induction and cure of induced EAE in vivo Inhibition of human MLR and DC maturation in vitro Prevention of EAE induction in vivo
Concentration 5 mg/ml
Refs [29]
10 mg/ml 100 mg/mouse three doses
[65] [36]
10 mg/ml 100 mg/mouse three doses
[22] [37]
Inhibition of MLR in vitro
1.5 mg/ml
[66]
Inhibition of MLR in vitro
Beads coated with 0.5 mg/ml CD83Ig (number of beads not specified)
[29]
Suppression of MLR in vitro
1.5–3.5 ng/ml
[24]
Inhibition of P815 tumor rejection in vivo No biological effect on MLR in vitro No biological effect on course of EAE in vivo Partial inhibition of antigen-specific T-cell stimulation in vitro Moderate increase in mortality in sublethal T. cruzi infection Induction of delayed skin allograft rejection (4 days compared with untreated) in vivo
100 mg/mouse three doses 0.1–100 mg/ml 100 mg/mouse three doses 40 mg/ml
[43] [45] [32]
10–20 mg/ml serum level in vivo
[34]
500 mg/kg
[44]
Abbreviations: DC, dendritic cell; EAE, experimental autoimmune encephalomyelitis; MLR, mixed lymphocyte reactions.
reciprocal decrease in mature follicular B cells [8]. This interference with late B cell maturation was directly correlated to the CD83 expression level, as demonstrated by using CD83tg mouse strains with differential CD83 expression. Analysis of mixed bone marrow chimeras showed that the impaired B cell maturation was mediated by overexpression of CD83 on the developing B cells themselves (K. Lu¨thje et al., pers. commun.). CD83: regulatory functions in peripheral immune responses The initial reports that virus-mediated suppression of CD83 expression reduces the stimulatory capacity of human DCs [35] and that a soluble CD83Ig fusion protein inhibits murine antigen-specific T-cell stimulation in vitro [32] indicated that CD83 had an influence on T-cell activation. Several studies have tried to elucidate the function of CD83 by using soluble CD83 molecules or by changing
the expression of CD83 on cells of the immune system (summarized in Tables 2 and 3). Addition of soluble CD83 species The general rationale behind the addition of soluble CD83 species such as CD83Ig in vivo and in vitro is to interfere with the ‘normal’ interaction of CD83 with its putative ligand in the periphery (Figure 2a). The addition of soluble CD83 may competitively inhibit both engagement of the putative CD83 ligand and of CD83 as a possible signaling receptor itself (Figure 2b). Unfortunately, in this experimental setting, one cannot distinguish whether soluble CD83 engages a putative CD83 ligand in a way that prevents or induces signaling into the CD83 ligand positive cell. Because CD83 is also expressed on various cell populations, it is unclear which of the interacting cells, whether antigen presenting cells (APCs) or responding T cells, represent the putative CD83 ligand–positive or the
Table 3. Biological effects of manipulated CD83 expression levels in vitro and in vivo Overexpression in vivo: CD83tg mouse Overexpression in vitro: CD83tg mouse No expression: CD83 knockout mouse Strongly reduced expression: CD83mu mouse Increased CD83 expression: CD83 transfection Increased CD83 expression: CD83 mRNA transfection Reduced CD83 expression: CD83 si RNA transfection Reduced CD83 expression: CD83 siRNA transfection
APC phenotype Normal induction of protective Th1-like response to Leishmania major infection
Refs [8]
B cells: increased MHC-II and CD86 expression Bone marrow–derived DCs: normal stimulatory capacity to T cells in vitro B cells: decreased MHC-II and CD86 expression, normal stimulatory capacity to CD4+ T cells in vitro Bone marrow–derived DCs: normal stimulatory capacity in MLR B cells: reduced expression of CD86 and MHC-II CD11c+ splenic DC: normal stimulatory capacity in MLR Artificial APC (K562 expressing HLA-A0201 and CD80): increased priming of human CD8+ T cells
[9] [50] [16,25]
Human immature and mature blood-derived DCs: better stimulation of CD8+ T cell lines and allogenic T cells Impaired stimulation of human CD8+ T cell lines and allogenic T cells
[47] [47]
Impaired stimulation of allogenic human T cells
[48]
[9,33] [31]
Abbreviations: APC, antigen presenting cell; DC, dendritic cell; MHC II, major histocompatibility complex II; MLR, mixed lymphocyte reactions.
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Figure 2. Peripheral function of CD83. (a) ‘Normal’ signaling through a putative CD83 ligand (CD83L) and CD83. CD83 may be expressed by the antigen presenting cells (APCs) and engage a ligand on T cells (i) or CD83 is expressed on T cells and engages a ligand on APCs (ii). Also possible is the engagement of CD83 by a ligand in cis on T cells (iii) or APCs (iv). (b) Competitive inhibition of signaling through CD83L and/or through CD83 into T cells (I and iii) and/or APC (ii and iv) by addition of soluble CD83 species.
CD83-positive cells. The data available also do not exclude expression of CD83 and its putative ligand being on the same cell (Figure 2a, iii and iv). Most studies performed, as summarized in Table 2, reported an immune suppression after addition of either recombinant soluble extracellular domain of CD83 or CD83Ig fusion proteins in vivo and in vitro. Strikingly, Escherichia coli–expressed (and thus unglycosylated) recombinant extracellular domain of CD83 displayed the most impressive suppression of mixed lymphocyte reactions (MLRs) in vitro [29] and could even prevent experimental autoimmune encephalomyelitis (EAE) induction and reverse already established EAE in vivo [36]. In this model, the deletion of cysteine 129, which prevents dimerization, did not alter the biological activity of the recombinant CD83 in vitro [22] and in vivo [37]. Because the addition of recombinant soluble human CD83 readily suppressed an MLR using murine lymphocytes and EAE induction in mice, the underlying mechanism is obviously conserved between humans and mice. 190
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Immune suppression by more ‘naturally generated’ soluble CD83 species was also indirectly observed in a study identifying soluble CD83 that was released from the surface of human cytomegalovirus (HCMV) infected human DCs as the immunosuppressive agent present in the supernatant of these cells [24]. Release of surface CD83 in the supernatant was reported for human B cell lines and DCs, and soluble CD83 is also present in the serum of healthy human donors [23]. Because soluble CD83 together with soluble CD80 and CD86 [38,39] is present at strongly elevated concentrations in the serum of patients with several malignant diseases [40], one can speculate on the possible immunosuppressive activity of ‘naturally generated’ soluble CD83 in vivo. Indeed, both the virus-induced loss of surface CD83 either by shedding (HCMV) or by rapid degradation inside herpes simplex virus type 1 (HSV-1)–infected DC [41] and release of soluble CD83 by tumor cells might represent immune evasion strategies [5]. However, these data should be interpreted with caution, because the concentration of soluble CD83 measured in the sera of tumor patients (1– 10 ng/ml) [40] and the concentration of soluble CD83 in the supernatant of HCMV-infected DCs (2.5 ng/ml) [24] is several orders of magnitude below that used experimentally (>1 mg/ml; Table 2). Furthermore, soluble CD83 was also detected in the synovial fluid of patients with rheumatoid arthritis [42] (i.e. in the context of inflammation). A direct correlation between soluble CD83 released by tumors or virus-infected cells and immunosuppression therefore seems rather unlikely at the moment. Studies using CD83Ig fusion proteins have led to contradictory results (Table 2). Some authors reported immune suppression in vitro and in vivo in models of tumor rejection [43] and allotransplantation [44] that were comparable to those induced by application of the soluble extracellular domain of human CD83. Our own studies indicated that the addition of CD83Ig to T-cell stimulation assays induced only a partial suppression of antigenspecific cytokine release [32]. Another CD83Ig fusion protein construct completely failed to inhibit an MLR in vitro and EAE induction in vivo, although being thoroughly controlled for correct folding and glycosylation [45]. Taken together, it seems that the biological function of different soluble CD83 species varies with each construct and model of immune suppression used. Along these lines, anti-CD83 mAbs were added to in vitro T-cell stimulation cultures in several studies. Because information about the CD83 ligand is lacking, one cannot test whether an anti-CD83 mAb would be blocking or agonistic. None of the anti-CD83 mAbs described thus far have managed to inhibit human [1] or murine T-cell activation [10] (M.B. and B.F., unpublished data). Manipulation of CD83 expression Another important tool to investigate a possible function for CD83 in the regulation of peripheral immune responses is the generation of APCs and lymphocytes with manipulated CD83 expression levels (summarized in Table 3). Because CD83 is upregulated on DCs upon maturation, it is conceivable that it acts as a co-stimulatory receptor for T cells comparable to the activation-induced co-stimulatory
Review receptors CD80 and CD86 [46] (Figure 2a, i). The immunosuppressive activity of soluble CD83 described above would thus be interpreted as the consequence of competitive inhibition of an otherwise co-stimulatory signal to the T cell (Figure 2b, i). Studies using human cells support this hypothesis. Human leucocyte antigen (HLA)-A0201+ K562 tumor cells were able to prime and expand antigen-specific cytotoxic T lymphocytes (CTLs) in vitro only if co-expressing CD80 and CD83 [31]. The level of CD83 expression on DCs manipulated by mRNA or siRNA (small interfering RNA) transfection was positively correlated to the stimulatory capacity of the DCs [47,48]. All studies stated that the level of MHC-II and co-stimulatory receptors on the APCs was not changed upon manipulation of CD83 expression. Evidence for a co-stimulatory role of CD83 in the murine system was provided using artificial overexpression of CD83 on melanoma cells, where it induced enhanced T cell–mediated tumor rejection that was abrogated by in vivo application of CD83Ig [49]. APCs derived from CD83 / and CD83 transgenic mouse strains were compared for their respective stimulatory capacity. In strong contrast to the data presented above, the analysis of CD83-deficient DCs did not show a reduced stimulatory capacity compared with wild-type DCs [16]. Also CD83 transgenic bone marrow–derived DCs displayed the same capacity to present model antigens to CD4+ and CD8+ T cells as wild-type DCs [50]. Therefore, at least in the murine immune system, CD83 does not exhibit essential co-stimulatory functions for T-cell activation in the periphery. Apart from the species difference, the studies performed in the human system had to rely on cell lines or monocyte-derived DCs that are established models of the original tissue-dwelling DCs but may well differ in some features from the proper cell. In the murine system, this obstacle can be overcome by sorting CD83-transgenic and CD83-deficient CD11c+ DCs directly from the spleen, but this again did not show a correlation of stimulatory capacity and CD83 expression levels [25,33]. Finally, the analysis of B220+ splenic B cells derived either from CD83 / mice [25] or CD83-overexpressing mice (our unpublished observations) showed no dramatic difference in their capacity to stimulate antigenspecific CD4+ T cells despite the positive correlation of CD83 expression to MHC-II and CD86 expression [9,16,25]. In summary, CD83 on TECs is needed for maturation of CD4+ but not CD8+ T cells. Its blockade by endogenously produced CD83Ig leads to maturation of functionally impaired CD4+ cells in normal numbers. On murine APCs, CD83 deficiency or overexpression has no effect in vitro, whereas on human APCs, it seems to act as a co-stimulus in vitro. The effects of various soluble CD83 species in the mouse in vivo are still not well defined. CD83 as a regulator of peripheral B-cell function and homeostasis During an ongoing infection with Leishmania major or T. cruzi, B lymphocytes and not DCs are the dominant CD83positive cell population in the lymph nodes draining the site of infection [8]. The CD83 expression on activated B cells was specifically correlated with the kinetics of infection because it stayed at peak levels in the susceptible
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BALB/c mice but returned to background expression in the resistant C56BL/6 mice when infection was cleared. Similarly upregulation of CD83 on B cells in vivo upon engagement of a transgenic hen egg lysozyme (HEL)-specific BCR was reported [10]. Moreover, transgenic overexpression of CD83 in vivo resulted in a dramatically reduced B-cell response, whereas the T-cell response was normal. Using adoptive transfer and a mixed bone marrow chimera, it was shown that the defective humoral response was caused by CD83 overexpression on B cells themselves and not on other cells [8]. Furthermore, purified CD83-overexpressing B cells responded to in vitro stimulation with reduced Ig secretion but reciprocally increased IL-10 secretion, whereas CD83-deficient B cells displayed slightly increased Ig secretion and reduced IL-10 secretion [9]. Taken together, these findings show that CD83 is expressed by activated B cells and is involved in the modulation of BCR signaling, B cell differentiation to Igsecreting plasma cells and modulation of IL-10 secretion by B cells (Figure 3). Interpreting these facts, we suggested that the activation-induced upregulation of CD83 on B cells may render them susceptible to the reception of negative signals and thus represents an additional regulatory mechanism to prevent over stimulation of the B-cell population [8]. That CD83-deficient mice did not display an increased humoral response but showed a reduced Ig response to immunization in vivo can be explained by the compensatory effect of a reduced number of T helper cells caused by the defective thymic maturation in CD83-deficient mice [16,33]. In accord with this, in bone marrow chimeras of CD83mu bone marrow transplanted into wild-type hosts (that allow normal maturation of T helper cells), Ig responses to T cell–dependent antigen immunization were equal or slightly increased compared with wild-type into wild-type chimeras (our unpublished observations). Overexpression of CD83 was also shown to interfere specifically with the homoeostatic survival of naı¨ve B cells within mixed bone marrow chimeras, whereas CD83 deficiency conferred a mild selection advantage to B cells (K. Lu¨thje et al., pers. commun.). Because peripheral B cell survival depends on BCR-mediated signals, this altered homoeostasis can be interpreted as the consequence of increased or absent negative co-regulation. The impact of CD83 on lymphocyte homeostasis was B cell specific, because the survival of CD83-overexpressing T cells was slightly improved. Along these lines, Prazma et al. [10] showed that CD83 / T cells displayed a reduced survival in the periphery. Using mixed bone marrow chimeras, we showed that overexpression of CD83 did not interfere with the function of wild-type B cells within the same mouse, thereby excluding that soluble CD83 released by CD83-overexpressing cells would inhibit B cell function. By contrast, the data accumulated thus far strongly suggest that transmembrane CD83 confers negative signals to the B cell on which it is actually expressed. How these putative signals may be transduced by CD83 lacking a significant intracellular domain and/or ITIM as discussed above remains unclear. It is, however, conceivable that CD83 in association with other transmembrane molecules induces signaling into the 191
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Figure 3. Hypothesis on the role of CD83 in the regulation of B-cell function. (a) (i) Resting wild-type B cells express only background levels of CD83. (ii) Upregulation of CD83 upon activation by toll-like receptor ligands or B cell receptor signals in vivo and in vitro. (iii) CD83 expression may render B cells susceptible for the reception of negative signals (tonic or ligand-induced signaling). Negative signals contribute to regulatory mechanisms preventing overstimulation of the B cell population. (b) (i) CD83tg B cells overexpress CD83 constitutively, and major histocompatibility complex II (MHC-II) and CD86 expression is increased. (ii) Premature CD83 expression may lead to the premature and increased reception of negative signals. (iii) As a consequence, Ig secretion in vivo and in vitro is reduced, interleukin-10 secretion is increased and B-cell survival in vivo is decreased. (c) (i) Resting CD83 mutant (CD83mu) B cells display no CD83 expression, and MHC-II and CD86 expression is reduced. (ii) CD83 mutant B cells show strongly reduced CD83 upregulation of CD83 upon activation, rendering them less susceptible to the reception of CD83-mediated negative signals. (iii) As a consequence, they display normal to slightly increased Ig and reduced interleukin-10 secretion. B-cell survival in vivo is increased. This suggests that CD83 may function as a negative regulator of B cell responses.
B cell either by engaging a ligand in cis or trans or by tonic signaling. Because the correlation of defective B-cell function and altered CD83 expression was obtained using nonconditional CD83 transgenic and mutant models, it cannot be completely excluded that altered CD83 expression during maturation within the CD83tg and CD83mu mice results in the generation of dysfunctional B cells. It is, however, important to note that treatment of wild-type C57BL/6 mice with anti-CD83 mAb in vivo induced a dose-dependent ten-fold increase in the antigen-specific IgG1 response to T cell–independent antigen immunization [8]. This is the very first report of a biological activity of antiCD83mAb and thereby demonstrates a genuine role for naturally induced CD83 in the regulation of peripheral Bcell function. Within the hypothesis depicted in Figure 3, the increased Ig response in the presence of anti-CD83 Ab in vivo would be explained as the result of increased B-cell activation caused by the neutralization of CD83-mediated negative signaling to activated wild-type B cells. Concluding remarks Fifteen years after its initial description, CD83 still remains enigmatic. Whereas CD4+ T-cell maturation is clearly triggered by engagement of a putative CD83 ligand on DP thymocytes, B-cell maturation and homeostasis are modulated by CD83 expressed on the B cells themselves. Although the data accumulated thus far does not lead to a final model of peripheral CD83 function, it is clear that 192
CD83 is not expressed exclusively on activated DCs but on various cell types and does not exclusively deliver costimulatory signals to T cells but has a complex impact on B- and T-cell activation, survival and function in the periphery. Acknowledgements The authors thank Anke Osterloh for preparation of figures and Katja Lu¨thje and Birte Kretschmer for discussions.
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Review 9 Kretschmer, B. et al. (2007) CD83 modulates B Cell function in vitro: increased IL-10 and reduced Ig secretion by CD83Tg B cells. PLoS ONE 2, e755 10 Prazma, C.M. et al. (2007) CD83 expression is a sensitive marker of activation required for B cell and CD4+ T cell longevity in vivo. J. Immunol. 179, 4550–4562 11 Cao, W. et al. (2005) CD83 is preformed inside monocytes, macrophages and dendritic cells, but it is only stably expressed on activated dendritic cells. Biochem. J. 385, 85–93 12 Nicod, L.P. et al. (2005) Upregulation of CD40, CD80, CD83 or CD86 on alveolar macrophages after lung transplantation. J. Heart Lung Transplant. 24, 1067–1075 13 Mailliard, R.B. et al. (2005) IL-18-induced CD83+CCR7+ NK helper cells. J. Exp. Med. 202, 941–953 14 Yamashiro, S. et al. (2000) Expression of CCR6 and CD83 by cytokine-activated human neutrophils. Blood 96, 3958– 3963 15 Iking-Konert, C. et al. (2002) Up-regulation of the dendritic cell marker CD83 on polymorphonuclear neutrophils (PMN): divergent expression in acute bacterial infections and chronic inflammatory disease. Clin. Exp. Immunol. 130, 501–508 16 Fujimoto, Y. et al. (2002) CD83 expression influences CD4+ T cell development in the thymus. Cell 108, 755–767 17 Kozlow, E.J. et al. (1993) Subtractive cDNA cloning of a novel member of the Ig gene superfamily expressed at high levels in activated B lymphocytes. Blood 81, 454–461 18 Twist, C.J. et al. (1998) The mouse Cd83 gene: structure, domain organization, and chromosome localization. Immunogenetics 48, 383–393 19 Gavin, M.A. et al. (2007) Foxp3-dependent programme of regulatory Tcell differentiation. Nature 445, 771–775 20 Fontenot, J.D. et al. (2005) Regulatory T cell lineage specification by the forkhead transcription factor foxp3. Immunity 22, 329– 341 21 Prazma, C.M. and Tedder, T.F. (2008) Dendritic cell CD83: A therapeutic target or innocent bystander? Immunol. Lett. 115, 1–8 22 Lechmann, M. et al. (2005) CD83 is a dimer: Comparative analysis of monomeric and dimeric isoforms. Biochem. Biophys. Res. Commun. 329, 132–139 23 Hock, B.D. et al. (2001) A soluble form of CD83 is released from activated dendritic cells and B lymphocytes, and is detectable in normal human sera. Int. Immunol. 13, 959–967 24 Senechal, B. et al. (2004) Infection of mature monocyte-derived dendritic cells with human cytomegalovirus inhibits stimulation of T-cell proliferation via the release of soluble CD83. Blood 103, 4207– 4215 25 Kuwano, Y. et al. (2007) CD83 influences cell-surface MHC class II expression on B cells and other antigen-presenting cells. Int. Immunol. 19, 977–992 26 McKinsey, T.A. et al. (2000) Transcription factor NF-kappaB regulates inducible CD83 gene expression in activated T lymphocytes. Mol. Immunol. 37, 783–788 27 Berchtold, S. et al. (2002) Cloning and characterization of the promoter region of the human CD83 gene. Immunobiology 205, 231– 246 28 Klein, E. et al. (2005) CD83 localization in a recycling compartment of immature human monocyte-derived dendritic cells. Int. Immunol. 17, 477–487 29 Lechmann, M. et al. (2001) The extracellular domain of CD83 inhibits dendritic cell-mediated T cell stimulation and binds to a ligand on dendritic cells. J. Exp. Med. 194, 1813–1821 30 Scholler, N. et al. (2001) CD83 is a sialic acid-binding Ig-like lectin (Siglec) adhesion receptor that binds monocytes and a subset of activated CD8+ T cells. J. Immunol. 166, 3865–3872 31 Hirano, N. et al. (2006) Engagement of CD83 ligand induces prolonged expansion of CD8+ T cells and preferential enrichment for antigen specificity. Blood 107, 1528–1536 32 Cramer, S.O. et al. (2000) Activation-induced expression of murine CD83 on T cells and identification of a specific CD83 ligand on murine B cells. Int. Immunol. 12, 1347–1351 33 Garcia-Martinez, L.F. et al. (2004) A novel mutation in CD83 results in the development of a unique population of CD4+ T cells. J. Immunol. 173, 2995–3001
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34 Luthje, K. et al. (2006) Transgenic expression of a CD83immunoglobulin fusion protein impairs the development of immunecompetent CD4-positive T cells. Eur. J. Immunol. 36, 2035–2045 35 Kruse, M. et al. (2000) Inhibition of CD83 cell surface expression during dendritic cell maturation by interference with nuclear export of CD83 mRNA. J. Exp. Med. 191, 1581–1590 36 Zinser, E. et al. (2004) Prevention and treatment of experimental autoimmune encephalomyelitis by soluble CD83. J. Exp. Med. 200, 345–351 37 Zinser, E. et al. (2006) Determination of the inhibitory activity and biological half-live of soluble CD83: comparison of wild type and mutant isoforms. Immunobiology 211, 449–453 38 Hock, B.D. et al. (2003) The clinical significance of soluble CD86 levels in patients with acute myeloid leukemia and myelodysplastic syndrome. Cancer 98, 1681–1688 39 Hock, B.D. et al. (2004) Identification of a circulating soluble form of CD80: levels in patients with hematological malignancies. Leuk. Lymphoma 45, 2111–2118 40 Hock, B.D. et al. (2004) The soluble form of CD83 is present at elevated levels in a number of hematological malignancies. Leuk. Res. 28, 237– 241 41 Kruse, M. et al. (2000) Mature dendritic cells infected with herpes simplex virus type 1 exhibit inhibited T-cell stimulatory capacity. J. Virol. 74, 7127–7136 42 Hock, B.D. et al. (2006) Levels of the soluble forms of CD80, CD86, and CD83 are elevated in the synovial fluid of rheumatoid arthritis patients. Tissue Antigens 67, 57–60 43 Scholler, N. et al. (2002) Cutting edge: CD83 regulates the development of cellular immunity. J. Immunol. 168, 2599–2602 44 Xu, J.F. et al. (2007) A limited course of soluble CD83 delays acute cellular rejection of MHC-mismatched mouse skin allografts. Transpl. Int. 20, 266–276 45 Pashine, A. et al. (2008) Failed efficacy of soluble human CD83-Ig in allogeneic mixed lymphocyte reactions and experimental autoimmune encephalomyelitis: Implications for a lack of therapeutic potential? Immunol. Lett. 115, 9–15 46 Sharpe, A.H. and Freeman, G.J. (2002) The B7-CD28 superfamily. Nat. Rev. Immunol. 2, 116–126 47 Aerts-Toegaert, C. et al. (2007) CD83 expression on dendritic cells and T cells: correlation with effective immune responses. Eur. J. Immunol. 37, 686–695 48 Prechtel, A.T. et al. (2007) CD83 Knockdown in Monocyte-Derived Dendritic Cells by Small Interfering RNA Leads to a Diminished T Cell Stimulation. J. Immunol. 178, 5454–5464 49 Yang, S. et al. (2004) Melanoma cells transfected to express CD83 induce antitumor immunity that can be increased by also engaging CD137. Proc. Natl. Acad. Sci. U. S. A. 101, 4990–4995 50 Wolenski, M. et al. (2003) Enhanced activation of CD83-positive T cells. Scand. J. Immunol. 58, 306–311 51 Lechmann, M. et al. (2002) Overexpression, purification, and biochemical characterization of the extracellular human CD83 domain and generation of monoclonal antibodies. Protein Expr. Purif. 24, 445–452 52 Zhou, L.J. and Tedder, T.F. (1996) CD14+ blood monocytes can differentiate into functionally mature CD83+ dendritic cells. Proc. Natl. Acad. Sci. U. S. A. 93, 2588–2592 53 Sorg, U.R. et al. (1997) Hodgkin’s cells express CD83, a dendritic cell lineage associated antigen. Pathology 29, 294–299 54 Dudziak, D. et al. (2003) Latent membrane protein 1 of Epstein-Barr virus induces CD83 by the NF-kappaB signaling pathway. J. Virol. 77, 8290–8298 55 Koga, T. et al. (2002) In situ localization of CD83-positive dendritic cells in psoriatic lesions. Dermatology 204, 100–103 56 te Velde, A.A. et al. (2003) Increased expression of DC-SIGN+IL-12+IL18+ and CD83+IL-12-IL-18- dendritic cell populations in the colonic mucosa of patients with Crohn’s disease. Eur. J. Immunol. 33, 143–151 57 Plumb, J. et al. (2003) CD83-positive dendritic cells are present in occasional perivascular cuffs in multiple sclerosis lesions. Mult. Scler. 9, 142–147 58 Iwamoto, M. et al. (2003) Prognostic value of tumor-infiltrating dendritic cells expressing CD83 in human breast carcinomas. Int. J. Cancer 104, 92–97
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59 Furihata, M. et al. (2005) Prognostic significance of CD83 positive, mature dendritic cells in the gallbladder carcinoma. Oncol. Rep. 14, 353–356 60 Tsukayama, S. et al. (2005) Prognostic value of CD83-positive mature dendritic cells and their relation to vascular endothelial growth factor in advanced human gastric cancer. Oncol. Rep. 14, 369–375 61 Sieling, P.A. et al. (1999) CD1 expression by dendritic cells in human leprosy lesions: correlation with effective host immunity. J. Immunol. 162, 1851–1858 62 Ohta, Y. et al. (2004) Homologs of CD83 from elasmobranch and teleost fish. J. Immunol. 173, 4553–4560
63 Donate, C. et al. (2007) CD83 expression in sea bream macrophages is a marker for the LPS-induced inflammatory response. Fish Shellfish Immunol. 23, 877–885 64 Hansell, C. et al. (2007) Unique features and distribution of the chicken CD83+ cell. J. Immunol. 179, 5117–5125 65 Kotzor, N. et al. (2004) The soluble form of CD83 dramatically changes the cytoskeleton of dendritic cells. Immunobiology 209, 129–140 66 Dudziak, D. et al. (2005) Alternative splicing generates putative soluble CD83 proteins that inhibit T cell proliferation. J. Immunol. 174, 6672–6676
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CD83 regulates lymphocyte maturation, activation and homeostasis Minka Breloer and Bernhard Fleischer Bernhard-Nocht-Institute for Tropical Medicine, 20359 Hamburg, Germany
The transmembrane CD83 molecule, a conserved member of the immunoglobulin superfamily, is known as one of the most characteristic cell surface markers for fully matured dendritic cells (DCs) in the peripheral circulation. An essential role for CD83 on murine DCs has not been found; however, evidence shows that its function primarily lies in the regulation of T- and B-lymphocyte maturation and in the regulation of their peripheral responses. Here, we review evidence for a role of CD83 in central lymphocyte maturation and novel, sometimes contradictory findings, regarding the function of CD83 in peripheral immune responses. Structure and expression pattern of CD83 CD83 used to be described as a highly specific marker for activated dendritic cells (DCs) in humans [1] and mice [2] (reviewed in [3–5]). However, many studies report CD83 surface expression on other cells in vitro and in vivo (Table 1) such as activated T and B lymphocytes [6–10], activated macrophages [11,12], a regulatory subset of NK cells [13], activated neutrophils [14,15], thymic epithelial cells [16] and uncharacterized cell population(s) in the brain [17,18]. Specific upregulation of CD83 was also observed in Foxp3+CD4+CD25+ natural regulatory T cells at the mRNA [19,20] and protein level (Hansen et al., unpublished data). The molecular and structural properties of CD83 have been reviewed elsewhere [5,21]. Briefly, CD83 is an evolutionarily well-conserved highly glycosylated type 1 transmembrane glycoprotein composed of 175 amino acids (AAs) in the mouse [18] and 186 in humans [6,17]. The cytoplasmic tail is predicted to consist of either of 39 [6] or 42 AAs [17], contains no tyrosine and thus does not give any clues as to possible direct signal transduction through immunoreceptor tyrosine-based inhibitory or activatory motifs (ITIM or ITAM). Studies using recombinant variants of the CD83 extracellular domain reported a disulfide-mediated homodimerization through cysteine 129, leaving four additional cysteine residues for formation of intramolecular disulfide bonds [22]. By contrast, there is no evidence for disulfide bond–mediated dimerization of transmembrane CD83, because nonreducing and reducing conditions led to an identical migration pattern in SDS– PAGE [6]. Release of CD83 from the plasma membrane into the supernatant was reported for in vitro cultured DCs and B Corresponding author: Breloer, M. ([email protected]).
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cells [23], as well as for virus-infected DCs [24]. Surface expression of CD83 is positively correlated to CD86 and major histocompatibility complex II (MHC-II) expression, because CD83-overexpressing B cells and DCs derived from CD83 transgenic (tg) mice display an increased surface expression of these molecules [9], whereas CD83deficient B cells and DCs showed a reciprocal reduction in CD86 and MHC-II surface expression [9,16,25]. Constitutive and activation-induced surface expression of other co-stimulatory molecules such as CD80, CD40 or MHC-I was unchanged in CD83tg- and CD83-deficient antigen presenting cells (APCs) [9]. In concordance with its nuclear factor-kB (NF-kB)–mediated gene regulation [2,26,27], CD83 is rapidly upregulated on murine B cells, reaching maximal expression 6 h after either toll-like receptor (TLR) engagement by lipopolysaccharide (LPS) or B cell receptor (BCR) ligation [9]. In vivo (e.g. in an ongoing infection), activated B cells are the dominant CD83positive cell population in the draining lymph nodes [8]. In contrast to some studies investigating human DCs and macrophages [11,28], no intracellular CD83 protein stores were detected in resting murine B cells or spleen cells [9], suggesting that the CD83 protein expressed by activated B cells is produced by de novo protein synthesis. The functional studies described below suggest that a ligand(s) for CD83 exists but thus far it has not been unequivocally identified. Data indicating possible CD83 ligand–expressing cell populations are controversial and have provided evidence for CD83 binding to human DCs [29], monocytes [30], human CD8+ T cells [30,31] and murine B2 cells [32]. Because all these cells also express CD83 after activation, a homotypic interaction cannot be excluded. CD83: central regulatory functions in the immune system CD83 is essential for the thymic maturation of CD4+ T cells The CD83 / mice described in 2002 [16] display dramatically reduced numbers of CD4 single positive (SP) thymocytes within the thymus (3% instead of 15% in the wild type) and equally reduced numbers of CD4+ T cells in the peripheral circulation (6% instead of 33% in the wild type). This phenotype was reproduced in CD83 / mice backcrossed to T cell receptor (TCR) transgenic mice. In elegant transfer experiments, the authors showed that, to allow normal maturation of CD4+ T cells, the CD83 molecule
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Table 1. CD83 expression in the immune system Human tissue and cells Various B and T cell lines, activated PBL Staphylococcus aureus Cowan (SAC) activated B cells, B cell lines, brain and lung tissue (no detection on activated T cell lines and tonsil derived T cells) B cell follicle: mantle zone and germinal center Peripheral blood–derived DCs (no detection on circulating B, T and NK cells) Peripheral blood–derived DCs Hodgkin’s lymphoma cells DC, B cells and Hodgkin’s lymphoma cells EBV-induced B cells PBL-derived mitogen stimulated T cells Activated neutrophils Activated macrophages Alveolar lung macrophages post lung transplantation DCs infiltrating in: Psoriatic lesions Colonic mucosa in Crohn’s disease Multiple sclerosis lesions Breast carcinoma Gallbladder carcinoma Gastric cancer Leprosy lesions NK cells Murine tissue and cells Brain and spleen tissue Bone marrow–derived DCs (no detection on B cell line A-20 and T cell lines 3DO and DO11.10) B cell lymhoma (A-20, Wehi 231, K46) and B cell myeloma X63 (no detection on the T cell hybridomas 3DO, DO11.10) J774, bone marrow–derived DCs Thymic epithelial cells and thymic DC but not double positive thymocytes Mature bone marrow–derived DCs Peri-arteriolar lymphoid sheath in the white pulp B cells in draining LN of L. major– and T. cruzi–infected mice Activated spleen-derived B cells in vitro LPS-activated spleenic DC, macrophages, activated spleenic T cells Regulatory T cells Other Shark: epigonal tissue Trout: MHC-II–positive tissue Sea bream: LPS-activated macrophages Chicken: tissue in proximity of B cells of all developmental stages (splenic B cell region, medulla of the bursa fabricii, splenic and tonsil germinal center, Harderian gland)
Detection method
Refs
Northern blot FACS (HB15a) Northern blot
[6] [17]
In situ hybridization FACS (HB15a,b,c) FACS (1G1; 4B5) (HB15a) Histology (HB 15a) ELISA (HB15a and rabbit anti-CD83AS); western blot (rabbit anti-CD83AS); FACS (HB15a) FACS (HB15a) Northern blot RT–PCR and FACS (HB15a) FACS (HB15e) Histology
[54] [26] [14] [15] [11] [12]
Histology (HB15a) Histology and FACS Immunhistochemistry Histology Histology Histology Histology (B10A-1) FACS (HB15a)
[55] [56] [57] [58] [59] [60] [61] [13]
Northern blot Northern blot
[18] [2]
FACS (Michel-19, Michel-17)
a
FACS (polyclonal rabbit anti-CD83 AS) In situ hybridisation and RT PCR FACS (Michel-17) Histology (Michel-17) FACS (Michel-19) FACS (Michel-19) western blot (polyclonal rabbit anti-mouse CD83 AS) FACS (Michel-19) Gene array FACS (Michel-19)
[32] [16] [7] [7] [8] [9]
RT–PCR
[62] [62] [63] [64]
RT–PCR Histology (polyclonal sheep anti-chicken CD83AS, mouse anti-chicken CD83mAb)
[17] [1] [51] [52] [53] [23]
a
[19,20], b
1G1, 4B5 and HB15a,b,c refer to the CD83 monoclonal antibody (mAb) clone. Abbreviations: DC, dendritic cell; EBV, Epstein-Barr virus; ELISA, enzyme-linked immunosorbent assay; FACS, fluorescence activated cell sorting; LPS, lipopolysaccharide; MHC II, major histocompatibility complex II; NK, natural killer; PBL, peripheral blood lymphocytes. a M.B. and B.F., unpublished. b Hansen et al., pers. commun.
needed to be expressed on radioresistant thymic epithelial cells (TECs) but not on the thymocytes themselves. These findings were confirmed in a mouse carrying a point mutation in the CD83 gene leading to dramatically reduced protein expression levels [33]. CD83 mutant (CD83mu) mice displayed the same reduction in CD4+ T cells in peripheral blood and CD4+ SP thymocytes as that observed in CD83 / mice. Because the numbers of CD4+ SP thymocytes returned to normal levels after transgenic expression of CD83 in CD83mu mice, CD83 deficiency was formally proven to be the cause of defective CD4+ T-cell maturation in CD83mu mice.
Interestingly, the 5% of CD4+ T cells that finally matured within CD83mu mice displayed an impaired phenotype, resulting in reduced proliferation and interleukin-2 (IL-2) secretion but increased IL-4 and IL-10 secretion as a result of in vitro stimulation and reduced delayed type hypersensitivity (DTH) responses in vivo [33]. This phenotype was acquired as a response to thymocyte maturation in a CD83-deficient thymus because CD83mu bone marrow transplanted into wildtype mice gave rise to CD4+ T cells that carried the mutated CD83 gene but responded normally to allogenic stimulation. 187
Review Transgenic mice producing a CD83 immunoglobulin fusion protein (CD83Igtg mice) have also yielded useful clues to the in vivo function of CD83. These mice produce CD83Ig under an MHC-II promoter, and the fusion protein is released into the serum and extracellular space. We observed that CD4+ T cells from CD83Igtg mice exhibited impaired functions similar to that seen in the knockout mice. Although thymocyte maturation in the presence of CD83Ig (10–15 ng/ml of serum) allowed development of normal frequencies of CD4+ T cells, the CD83Igtg mice were more susceptible to a Trypanosoma cruzi infection and showed reduced cytokine responses after in vitro stimulation [34]. It was shown that this defective phenotype was acquired upon thymic maturation of double positive (DP) thymocytes to CD4+ SP thymocytes and was not caused by the presence of CD83Ig in the periphery because of the following evidence: (i) The defective phenotype was restricted to CD4+ T cells; CD8+ T cells derived from CD83Igtg mice were as potent as wild-type T cells. (ii) Purified CD4+ CD83Igtg T cells displayed an impaired response to antigenic stimulation by wildtype APC in vitro even though they did not secrete the CD83Ig themselves. (iii) CD4+ SP thymocytes purified from the thymi of CD83Igtg mice (i.e. before they entered peripheral circulation) already displayed the impaired phenotype. (iv) In vivo application of recombinant CD83Ig did not revert the resistant phenotype of C56BL/6 mice to a
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sublethal T. cruzi infection to the highly susceptible phenotype of the CD83Igtg mice. Taken together, these three studies [16,33,34] highlight the central function of CD83 in the maturation of CD4+ T cells. CD83 expressed on TEC is needed to deliver a signal to the DP thymocytes that allows the further progression to CD4+ SP thymocytes (Figure 1a). Absence of this signaling by CD83 deficiency as in the knockout mice or the CD83mu mice results in the reduction of both CD4+ SP thymocytes and CD4+ T cells in the periphery (Figure 1b). Partial interference with this signal caused by the presence of competing CD83Ig fusion protein in the circulation, however, allows the development of CD4+ T cells in normal frequencies (Figure 1c). The CD4+ T cells that mature despite the disturbed thymic CD83 signaling in all of the three mouse models display an impaired phenotype in vivo [16,33,34] and in vitro [33,34] that reflects the incomplete CD83-mediated signaling during thymic development. By contrast, CD8+ T cells develop in normal numbers [16,33] and display normal function [34], thus further demonstrating that CD83 specifically regulates maturation of functional CD4+ T cells. CD83 is involved in B-cell maturation CD83 is expressed at low levels by immature B cells beyond the pre-B cell stage (i.e. once they express a functional BCR) [10]. The first hints that overexpression of CD83 may interfere with the late maturation of B cells were gained from the analysis of CD83tg mouse strains that displayed an increase in immature transitional B cells and a
Figure 1. Involvement of CD83 in T-cell thymic development. (a) Maturation of T cells in wild-type mice: Double positive (DP) thymocytes (CD4+8+) receive a signal from CD83 expressed on thymic epithelial cells (TECs), most likely through a putative CD83 ligand, that allows their further maturation to functional single positive (SP) CD4+ T cells. (b) Maturation of T cells in CD83mu (mutant) and CD83 / mice: in the absence of CD83 on thymic epithelium, the numbers of CD8+ T cells are unchanged, but the numbers of CD4+ SP thymocytes and peripheral CD4+ T cells are reduced. The function of the remaining CD4+ T cells is impaired, the function of CD8+ has not been investigated in this model. (c) Maturation of T cells in the presence of soluble CD83Ig: Soluble CD83Ig interferes with the physiological interaction of CD83 and its putative ligand in the thymus. SP thymocytes and CD4+ and CD8+ T cells develop in normal numbers. The function of the developing SP CD4+ thymocytes and peripheral CD4+ T cells is impaired, but the function of CD8+ T cells is unchanged.
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Table 2. Biological effects of soluble CD83 species in vitro and in vivo Construct E. coli–derived extracellular domain of human CD83
E. coli–derived extracellular domain of human CD83 with mutation on Cys129 (produces monomeric construct) Truncated variant of human CD83 lacking transmembrane and membrane proximal domain Human CD83Ig extracellular domain fused to human IgG1 and coated to beads Human CD83 released by HCMV infected monocyte-derived human DC Human CD83Ig Human CD83Ig Murine CD83Ig Murine CD83Ig Murine CD83Ig
Biological effect Inhibition of human MLR and human DC maturation in vitro Interference with veil formation in human DCs Prevention of EAE induction and cure of induced EAE in vivo Inhibition of human MLR and DC maturation in vitro Prevention of EAE induction in vivo
Concentration 5 mg/ml
Refs [29]
10 mg/ml 100 mg/mouse three doses
[65] [36]
10 mg/ml 100 mg/mouse three doses
[22] [37]
Inhibition of MLR in vitro
1.5 mg/ml
[66]
Inhibition of MLR in vitro
Beads coated with 0.5 mg/ml CD83Ig (number of beads not specified)
[29]
Suppression of MLR in vitro
1.5–3.5 ng/ml
[24]
Inhibition of P815 tumor rejection in vivo No biological effect on MLR in vitro No biological effect on course of EAE in vivo Partial inhibition of antigen-specific T-cell stimulation in vitro Moderate increase in mortality in sublethal T. cruzi infection Induction of delayed skin allograft rejection (4 days compared with untreated) in vivo
100 mg/mouse three doses 0.1–100 mg/ml 100 mg/mouse three doses 40 mg/ml
[43] [45] [32]
10–20 mg/ml serum level in vivo
[34]
500 mg/kg
[44]
Abbreviations: DC, dendritic cell; EAE, experimental autoimmune encephalomyelitis; MLR, mixed lymphocyte reactions.
reciprocal decrease in mature follicular B cells [8]. This interference with late B cell maturation was directly correlated to the CD83 expression level, as demonstrated by using CD83tg mouse strains with differential CD83 expression. Analysis of mixed bone marrow chimeras showed that the impaired B cell maturation was mediated by overexpression of CD83 on the developing B cells themselves (K. Lu¨thje et al., pers. commun.). CD83: regulatory functions in peripheral immune responses The initial reports that virus-mediated suppression of CD83 expression reduces the stimulatory capacity of human DCs [35] and that a soluble CD83Ig fusion protein inhibits murine antigen-specific T-cell stimulation in vitro [32] indicated that CD83 had an influence on T-cell activation. Several studies have tried to elucidate the function of CD83 by using soluble CD83 molecules or by changing
the expression of CD83 on cells of the immune system (summarized in Tables 2 and 3). Addition of soluble CD83 species The general rationale behind the addition of soluble CD83 species such as CD83Ig in vivo and in vitro is to interfere with the ‘normal’ interaction of CD83 with its putative ligand in the periphery (Figure 2a). The addition of soluble CD83 may competitively inhibit both engagement of the putative CD83 ligand and of CD83 as a possible signaling receptor itself (Figure 2b). Unfortunately, in this experimental setting, one cannot distinguish whether soluble CD83 engages a putative CD83 ligand in a way that prevents or induces signaling into the CD83 ligand positive cell. Because CD83 is also expressed on various cell populations, it is unclear which of the interacting cells, whether antigen presenting cells (APCs) or responding T cells, represent the putative CD83 ligand–positive or the
Table 3. Biological effects of manipulated CD83 expression levels in vitro and in vivo Overexpression in vivo: CD83tg mouse Overexpression in vitro: CD83tg mouse No expression: CD83 knockout mouse Strongly reduced expression: CD83mu mouse Increased CD83 expression: CD83 transfection Increased CD83 expression: CD83 mRNA transfection Reduced CD83 expression: CD83 si RNA transfection Reduced CD83 expression: CD83 siRNA transfection
APC phenotype Normal induction of protective Th1-like response to Leishmania major infection
Refs [8]
B cells: increased MHC-II and CD86 expression Bone marrow–derived DCs: normal stimulatory capacity to T cells in vitro B cells: decreased MHC-II and CD86 expression, normal stimulatory capacity to CD4+ T cells in vitro Bone marrow–derived DCs: normal stimulatory capacity in MLR B cells: reduced expression of CD86 and MHC-II CD11c+ splenic DC: normal stimulatory capacity in MLR Artificial APC (K562 expressing HLA-A0201 and CD80): increased priming of human CD8+ T cells
[9] [50] [16,25]
Human immature and mature blood-derived DCs: better stimulation of CD8+ T cell lines and allogenic T cells Impaired stimulation of human CD8+ T cell lines and allogenic T cells
[47] [47]
Impaired stimulation of allogenic human T cells
[48]
[9,33] [31]
Abbreviations: APC, antigen presenting cell; DC, dendritic cell; MHC II, major histocompatibility complex II; MLR, mixed lymphocyte reactions.
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Figure 2. Peripheral function of CD83. (a) ‘Normal’ signaling through a putative CD83 ligand (CD83L) and CD83. CD83 may be expressed by the antigen presenting cells (APCs) and engage a ligand on T cells (i) or CD83 is expressed on T cells and engages a ligand on APCs (ii). Also possible is the engagement of CD83 by a ligand in cis on T cells (iii) or APCs (iv). (b) Competitive inhibition of signaling through CD83L and/or through CD83 into T cells (I and iii) and/or APC (ii and iv) by addition of soluble CD83 species.
CD83-positive cells. The data available also do not exclude expression of CD83 and its putative ligand being on the same cell (Figure 2a, iii and iv). Most studies performed, as summarized in Table 2, reported an immune suppression after addition of either recombinant soluble extracellular domain of CD83 or CD83Ig fusion proteins in vivo and in vitro. Strikingly, Escherichia coli–expressed (and thus unglycosylated) recombinant extracellular domain of CD83 displayed the most impressive suppression of mixed lymphocyte reactions (MLRs) in vitro [29] and could even prevent experimental autoimmune encephalomyelitis (EAE) induction and reverse already established EAE in vivo [36]. In this model, the deletion of cysteine 129, which prevents dimerization, did not alter the biological activity of the recombinant CD83 in vitro [22] and in vivo [37]. Because the addition of recombinant soluble human CD83 readily suppressed an MLR using murine lymphocytes and EAE induction in mice, the underlying mechanism is obviously conserved between humans and mice. 190
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Immune suppression by more ‘naturally generated’ soluble CD83 species was also indirectly observed in a study identifying soluble CD83 that was released from the surface of human cytomegalovirus (HCMV) infected human DCs as the immunosuppressive agent present in the supernatant of these cells [24]. Release of surface CD83 in the supernatant was reported for human B cell lines and DCs, and soluble CD83 is also present in the serum of healthy human donors [23]. Because soluble CD83 together with soluble CD80 and CD86 [38,39] is present at strongly elevated concentrations in the serum of patients with several malignant diseases [40], one can speculate on the possible immunosuppressive activity of ‘naturally generated’ soluble CD83 in vivo. Indeed, both the virus-induced loss of surface CD83 either by shedding (HCMV) or by rapid degradation inside herpes simplex virus type 1 (HSV-1)–infected DC [41] and release of soluble CD83 by tumor cells might represent immune evasion strategies [5]. However, these data should be interpreted with caution, because the concentration of soluble CD83 measured in the sera of tumor patients (1– 10 ng/ml) [40] and the concentration of soluble CD83 in the supernatant of HCMV-infected DCs (2.5 ng/ml) [24] is several orders of magnitude below that used experimentally (>1 mg/ml; Table 2). Furthermore, soluble CD83 was also detected in the synovial fluid of patients with rheumatoid arthritis [42] (i.e. in the context of inflammation). A direct correlation between soluble CD83 released by tumors or virus-infected cells and immunosuppression therefore seems rather unlikely at the moment. Studies using CD83Ig fusion proteins have led to contradictory results (Table 2). Some authors reported immune suppression in vitro and in vivo in models of tumor rejection [43] and allotransplantation [44] that were comparable to those induced by application of the soluble extracellular domain of human CD83. Our own studies indicated that the addition of CD83Ig to T-cell stimulation assays induced only a partial suppression of antigenspecific cytokine release [32]. Another CD83Ig fusion protein construct completely failed to inhibit an MLR in vitro and EAE induction in vivo, although being thoroughly controlled for correct folding and glycosylation [45]. Taken together, it seems that the biological function of different soluble CD83 species varies with each construct and model of immune suppression used. Along these lines, anti-CD83 mAbs were added to in vitro T-cell stimulation cultures in several studies. Because information about the CD83 ligand is lacking, one cannot test whether an anti-CD83 mAb would be blocking or agonistic. None of the anti-CD83 mAbs described thus far have managed to inhibit human [1] or murine T-cell activation [10] (M.B. and B.F., unpublished data). Manipulation of CD83 expression Another important tool to investigate a possible function for CD83 in the regulation of peripheral immune responses is the generation of APCs and lymphocytes with manipulated CD83 expression levels (summarized in Table 3). Because CD83 is upregulated on DCs upon maturation, it is conceivable that it acts as a co-stimulatory receptor for T cells comparable to the activation-induced co-stimulatory
Review receptors CD80 and CD86 [46] (Figure 2a, i). The immunosuppressive activity of soluble CD83 described above would thus be interpreted as the consequence of competitive inhibition of an otherwise co-stimulatory signal to the T cell (Figure 2b, i). Studies using human cells support this hypothesis. Human leucocyte antigen (HLA)-A0201+ K562 tumor cells were able to prime and expand antigen-specific cytotoxic T lymphocytes (CTLs) in vitro only if co-expressing CD80 and CD83 [31]. The level of CD83 expression on DCs manipulated by mRNA or siRNA (small interfering RNA) transfection was positively correlated to the stimulatory capacity of the DCs [47,48]. All studies stated that the level of MHC-II and co-stimulatory receptors on the APCs was not changed upon manipulation of CD83 expression. Evidence for a co-stimulatory role of CD83 in the murine system was provided using artificial overexpression of CD83 on melanoma cells, where it induced enhanced T cell–mediated tumor rejection that was abrogated by in vivo application of CD83Ig [49]. APCs derived from CD83 / and CD83 transgenic mouse strains were compared for their respective stimulatory capacity. In strong contrast to the data presented above, the analysis of CD83-deficient DCs did not show a reduced stimulatory capacity compared with wild-type DCs [16]. Also CD83 transgenic bone marrow–derived DCs displayed the same capacity to present model antigens to CD4+ and CD8+ T cells as wild-type DCs [50]. Therefore, at least in the murine immune system, CD83 does not exhibit essential co-stimulatory functions for T-cell activation in the periphery. Apart from the species difference, the studies performed in the human system had to rely on cell lines or monocyte-derived DCs that are established models of the original tissue-dwelling DCs but may well differ in some features from the proper cell. In the murine system, this obstacle can be overcome by sorting CD83-transgenic and CD83-deficient CD11c+ DCs directly from the spleen, but this again did not show a correlation of stimulatory capacity and CD83 expression levels [25,33]. Finally, the analysis of B220+ splenic B cells derived either from CD83 / mice [25] or CD83-overexpressing mice (our unpublished observations) showed no dramatic difference in their capacity to stimulate antigenspecific CD4+ T cells despite the positive correlation of CD83 expression to MHC-II and CD86 expression [9,16,25]. In summary, CD83 on TECs is needed for maturation of CD4+ but not CD8+ T cells. Its blockade by endogenously produced CD83Ig leads to maturation of functionally impaired CD4+ cells in normal numbers. On murine APCs, CD83 deficiency or overexpression has no effect in vitro, whereas on human APCs, it seems to act as a co-stimulus in vitro. The effects of various soluble CD83 species in the mouse in vivo are still not well defined. CD83 as a regulator of peripheral B-cell function and homeostasis During an ongoing infection with Leishmania major or T. cruzi, B lymphocytes and not DCs are the dominant CD83positive cell population in the lymph nodes draining the site of infection [8]. The CD83 expression on activated B cells was specifically correlated with the kinetics of infection because it stayed at peak levels in the susceptible
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BALB/c mice but returned to background expression in the resistant C56BL/6 mice when infection was cleared. Similarly upregulation of CD83 on B cells in vivo upon engagement of a transgenic hen egg lysozyme (HEL)-specific BCR was reported [10]. Moreover, transgenic overexpression of CD83 in vivo resulted in a dramatically reduced B-cell response, whereas the T-cell response was normal. Using adoptive transfer and a mixed bone marrow chimera, it was shown that the defective humoral response was caused by CD83 overexpression on B cells themselves and not on other cells [8]. Furthermore, purified CD83-overexpressing B cells responded to in vitro stimulation with reduced Ig secretion but reciprocally increased IL-10 secretion, whereas CD83-deficient B cells displayed slightly increased Ig secretion and reduced IL-10 secretion [9]. Taken together, these findings show that CD83 is expressed by activated B cells and is involved in the modulation of BCR signaling, B cell differentiation to Igsecreting plasma cells and modulation of IL-10 secretion by B cells (Figure 3). Interpreting these facts, we suggested that the activation-induced upregulation of CD83 on B cells may render them susceptible to the reception of negative signals and thus represents an additional regulatory mechanism to prevent over stimulation of the B-cell population [8]. That CD83-deficient mice did not display an increased humoral response but showed a reduced Ig response to immunization in vivo can be explained by the compensatory effect of a reduced number of T helper cells caused by the defective thymic maturation in CD83-deficient mice [16,33]. In accord with this, in bone marrow chimeras of CD83mu bone marrow transplanted into wild-type hosts (that allow normal maturation of T helper cells), Ig responses to T cell–dependent antigen immunization were equal or slightly increased compared with wild-type into wild-type chimeras (our unpublished observations). Overexpression of CD83 was also shown to interfere specifically with the homoeostatic survival of naı¨ve B cells within mixed bone marrow chimeras, whereas CD83 deficiency conferred a mild selection advantage to B cells (K. Lu¨thje et al., pers. commun.). Because peripheral B cell survival depends on BCR-mediated signals, this altered homoeostasis can be interpreted as the consequence of increased or absent negative co-regulation. The impact of CD83 on lymphocyte homeostasis was B cell specific, because the survival of CD83-overexpressing T cells was slightly improved. Along these lines, Prazma et al. [10] showed that CD83 / T cells displayed a reduced survival in the periphery. Using mixed bone marrow chimeras, we showed that overexpression of CD83 did not interfere with the function of wild-type B cells within the same mouse, thereby excluding that soluble CD83 released by CD83-overexpressing cells would inhibit B cell function. By contrast, the data accumulated thus far strongly suggest that transmembrane CD83 confers negative signals to the B cell on which it is actually expressed. How these putative signals may be transduced by CD83 lacking a significant intracellular domain and/or ITIM as discussed above remains unclear. It is, however, conceivable that CD83 in association with other transmembrane molecules induces signaling into the 191
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Figure 3. Hypothesis on the role of CD83 in the regulation of B-cell function. (a) (i) Resting wild-type B cells express only background levels of CD83. (ii) Upregulation of CD83 upon activation by toll-like receptor ligands or B cell receptor signals in vivo and in vitro. (iii) CD83 expression may render B cells susceptible for the reception of negative signals (tonic or ligand-induced signaling). Negative signals contribute to regulatory mechanisms preventing overstimulation of the B cell population. (b) (i) CD83tg B cells overexpress CD83 constitutively, and major histocompatibility complex II (MHC-II) and CD86 expression is increased. (ii) Premature CD83 expression may lead to the premature and increased reception of negative signals. (iii) As a consequence, Ig secretion in vivo and in vitro is reduced, interleukin-10 secretion is increased and B-cell survival in vivo is decreased. (c) (i) Resting CD83 mutant (CD83mu) B cells display no CD83 expression, and MHC-II and CD86 expression is reduced. (ii) CD83 mutant B cells show strongly reduced CD83 upregulation of CD83 upon activation, rendering them less susceptible to the reception of CD83-mediated negative signals. (iii) As a consequence, they display normal to slightly increased Ig and reduced interleukin-10 secretion. B-cell survival in vivo is increased. This suggests that CD83 may function as a negative regulator of B cell responses.
B cell either by engaging a ligand in cis or trans or by tonic signaling. Because the correlation of defective B-cell function and altered CD83 expression was obtained using nonconditional CD83 transgenic and mutant models, it cannot be completely excluded that altered CD83 expression during maturation within the CD83tg and CD83mu mice results in the generation of dysfunctional B cells. It is, however, important to note that treatment of wild-type C57BL/6 mice with anti-CD83 mAb in vivo induced a dose-dependent ten-fold increase in the antigen-specific IgG1 response to T cell–independent antigen immunization [8]. This is the very first report of a biological activity of antiCD83mAb and thereby demonstrates a genuine role for naturally induced CD83 in the regulation of peripheral Bcell function. Within the hypothesis depicted in Figure 3, the increased Ig response in the presence of anti-CD83 Ab in vivo would be explained as the result of increased B-cell activation caused by the neutralization of CD83-mediated negative signaling to activated wild-type B cells. Concluding remarks Fifteen years after its initial description, CD83 still remains enigmatic. Whereas CD4+ T-cell maturation is clearly triggered by engagement of a putative CD83 ligand on DP thymocytes, B-cell maturation and homeostasis are modulated by CD83 expressed on the B cells themselves. Although the data accumulated thus far does not lead to a final model of peripheral CD83 function, it is clear that 192
CD83 is not expressed exclusively on activated DCs but on various cell types and does not exclusively deliver costimulatory signals to T cells but has a complex impact on B- and T-cell activation, survival and function in the periphery. Acknowledgements The authors thank Anke Osterloh for preparation of figures and Katja Lu¨thje and Birte Kretschmer for discussions.
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