The CD83 Molecule - An Important Immune Checkpoint.

The CD83 molecule has been identified to be expressed on numerous activated immune cells, including B and T lymphocytes, monocytes, dendritic cells, microglia, and neutrophils. Both isoforms of CD83, the membrane-bound as well as its soluble form are topic of intensive research investigations. Several studies revealed that CD83 is not a typical co-stimulatory molecule, but rather plays a critical role in controlling and resolving immune responses. Moreover, CD83 is an essential factor during the differentiation of T and B lymphocytes, and the development and maintenance of tolerance. The identification of its interaction partners as well as signaling pathways have been an enigma for the last decades. Here, we report the latest data on the expression, structure, and the signaling partners of CD83. In addition, we review the regulatory functions of CD83, including its striking modulatory potential to maintain the balance between tolerance versus inflammation during homeostasis or pathologies. These immunomodulatory properties of CD83 emphasize its exceptional therapeutic potential, which has been documented in specific preclinical disease models.

CD83 expression is essential for Treg cell differentiation and stability.

Foxp3-positive regulatory T cells (Tregs) are crucial for the maintenance of immune homeostasis and keep immune responses in check. Upon activation, Tregs are transferred into an effector state expressing transcripts essential for their suppressive activity, migration, and survival. However, it is not completely understood how different intrinsic and environmental factors control differentiation. Here, we present for the first time to our knowledge data suggesting that Treg-intrinsic expression of CD83 is essential for Treg differentiation upon activation. Interestingly, mice with Treg-intrinsic CD83 deficiency are characterized by a proinflammatory phenotype. Furthermore, the loss of CD83 expression by Tregs leads to the downregulation of Treg-specific differentiation markers and the induction of an inflammatory profile. In addition, Treg-specific conditional knockout mice showed aggravated autoimmunity and an impaired resolution of inflammation. Altogether, our results show that CD83 expression in Tregs is an essential factor for the development and function of effector Tregs upon activation. Since Tregs play a crucial role in the maintenance of immune tolerance and thus prevention of autoimmune disorders, our findings are also clinically relevant.

FAM13A is associated with non-small cell lung cancer (NSCLC) progression and controls tumor cell proliferation and survival.

Genome-wide association studies (GWAS) associated Family with sequence similarity 13, member A (FAM13A) with non-small cell lung cancer (NSCLC) occurrence. Here, we found increased numbers of FAM13A protein expressing cells in the tumoral region of lung tissues from a cohort of patients with NSCLC. Moreover, FAM13A inversely correlated with CTLA4 but directly correlated with HIF1α levels in the control region of these patients. Consistently, FAM13A RhoGAP was found to be associated with T cell effector molecules like HIF1α and Tbet and was downregulated in immunosuppressive CD4+CD25+Foxp3+CTLA4+ T cells. TGFß, a tumor suppressor factor, as well as siRNA to FAM13A, suppressed both isoforms of FAM13A and inhibited tumor cell proliferation. RNA-Seq analysis confirmed this finding. Moreover, siRNA to FAM13A induced TGFß levels. Finally, in experimental tumor cell migration, FAM13A was induced and TGFß accelerated this process by inducing cell migration, HIF1α, and the FAM13A RhoGAP isoform. Furthermore, siRNA to FAM13A inhibited tumor cell proliferation and induced cell migration without affecting HIF1α. In conclusion, FAM13A is involved in tumor cell proliferation and downstream of TGFß and HIF1α, FAM13A RhoGAP is associated with Th1 gene expression and lung tumor cell migration. These findings identify FAM13A as key regulator of NSCLC growth and progression.

Murine CD83-positive T cells mediate suppressor functions in vitro and in vivo.

The CD83 molecule (CD83) is a well-known surface marker present on mature dendritic cells (mDC). In this study, we show that CD83 is also expressed on a subset of T cells which mediate regulatory T cell (Treg)-like suppressor functions in vitro and in vivo. Treg-associated molecules including CD25, cytotoxic T lymphocyte antigen-4 (CTLA-4), glucocorticoid-induced TNFR family-related gene (GITR), Helios and neuropilin-1 (NRP-1) as well as forkhead box protein 3 (FOXP3) were specifically expressed by these CD83(+) T cells. In contrast, CD83(-) T cells showed a naive T cell phenotype with effector T cell properties upon activation. Noteworthy, CD83(-) T cells were not able to upregulate CD83 despite activation. Furthermore, CD83(+) T cells suppressed the proliferation and inflammatory cytokine release of CD83(-) T cells in vitro. Strikingly, stimulated CD83(+) T cells released soluble CD83 (sCD83), which has been reported to possess immunosuppressive properties. In vivo, using the murine transfer colitis model we could show that CD83(+) T cells were able to suppress colitis symptoms while CD83(-) T cells possessed effector functions. In addition, this CD83 expression is also conserved on expanded human Treg. Thus, from these studies we conclude that CD83(+) T cells share important features with regulatory T cells, identifying CD83 as a novel lineage marker to discriminate between different T cell populations.

Topical application of soluble CD83 induces IDO-mediated immune modulation, increases Foxp3+ T cells, and prolongs allogeneic corneal graft survival.

Modulation of immune responses is one of the main research aims in transplant immunology. In this study, we investigate the local immunomodulatory properties of soluble CD83 (sCD83) at the graft-host interface using the high-risk corneal transplantation model. In this model, which mimics the inflammatory status and the preexisting vascularization of high-risk patients undergoing corneal transplantation, allogeneic donor corneas are transplanted onto sCD83-treated recipient animals. This model allows the direct and precise application of the immune modulator at the transplantation side. Interestingly, sCD83 was able to prolong graft survival after systemic application as well as after topical application, which is therapeutically more relevant. The therapeutic effect was accompanied by an increase in the frequency of regulatory T cells and was mediated by the immune-regulatory enzyme IDO and TGF-ß. In vitro, sCD83 induced long-term IDO expression in both conventional and plasmacytoid dendritic cells via autocrine or paracrine production of TGF-ß, a cytokine previously shown to be an essential mediator of IDO-dependent, long-term tolerance. These findings open new treatment avenues for local immune modulation after organ and tissue transplantation.

Murine whole-organ immune cell populations revealed by multi-epitope-ligand cartography.

Multi-epitope-ligand cartography (MELC) is an innovative high-throughput fluorescence microscopy-based method. A tissue section is analyzed through a repeated cycling of (1) incubation with a fluorophore-labeled antibody, (2) fluorescence imaging, and (3) soft bleaching. This method allows staining of the same tissue section with up to 100 fluorescent markers and to analyze their toponomic expression using further image processing and pixel-precise overlay of the corresponding images. In this study, we adapted this method to identify a large panel of murine leukocyte subpopulations in a whole frozen section of a peripheral lymph node. Using the resulting antibody library, we examined non-inflamed versus inflamed tissues of brain and spinal cord in the experimental autoimmune encephalomyelitis (EAE) model. The presence and activity of specific leukocyte subpopulations (different T cell subpopulations, dendritic cells, macrophages, etc.) could be assessed and the cellular localizations and the corresponding activation status in situ were investigated. The results were then correlated with quantitative RT-PCR.

Thymic stromal lymphopoetin-induced expression of the endogenous inhibitory enzyme SLPI mediates recovery from colonic inflammation.

Thymic stromal lymphopoetin (TSLP) influences numerous immune functions, including those in the colonic mucosa. Here we report that TSLP-deficient (Tslp(-/-)) mice did not exhibit increased inflammation during dextran sodium sulfate (DSS)-induced colitis but failed to recover from disease, resulting in death. Increased localized neutrophil elastase (NE) activity during overt inflammation was observed in Tslp(-/-) mice and was paralleled by reduced expression of an endogenous inhibitor, secretory leukocyte peptidase inhibitor (SLPI). Pharmacological inhibition of NE or treatment with rSLPI reduced DSS-induced mortality in Tslp(-/-) mice. Signaling through TSLPR on nonhematopoietic cells was sufficient for recovery from DSS-induced colitis. Expression of the receptor occurred on intestinal epithelial cells (IEC), with stimulation inducing SLPI expression. Therefore, TSLP is critical in mediating mucosal healing after insult and functions in a nonredundant capacity that is independent of restraining T helper 1 (Th1) and Th17 cell cytokine production.

Nucleotide oligomerization domain-containing proteins instruct T cell helper type 2 immunity through stromal activation.

Although a number of studies have examined the development of T-helper cell type 2 (Th2) immunity in different settings, the mechanisms underlying the initiation of this arm of adaptive immunity are not well understood. We exploited the fact that immunization with antigen plus either nucleotide-binding oligomerization domain-containing proteins 1 (Nod1) or 2 (Nod2) agonists drives Th2 induction to understand how these pattern-recognition receptors mediate the development of systemic Th2 immune responses. Here, we show in bone-marrow chimeric mice that Nod1 and Nod2 expression within the stromal compartment is necessary for priming of effector CD4(+) Th2 responses and specific IgG1 antibodies. In contrast, sensing of these ligands by dendritic cells was not sufficient to induce Th2 immunity, although these cells contribute to the response. Moreover, we determined that CD11c(+) cells were the critical antigen-presenting cells, whereas basophils and B cells did not affect the capacity of Nod ligands to induce CD4(+) Th2 effector function. Finally, we found that full Th2 induction upon Nod1 and Nod2 activation was dependent on both thymic stromal lymphopoietin production by the stromal cells and the up-regulation of the costimulatory molecule, OX40 ligand, on dendritic cells. This study provides in vivo evidence of how systemic Th2 immunity is induced in the context of Nod stimulation. Such understanding will influence the rational design of therapeutics that could reprogram the immune system during an active Th1-mediated disease, such as Crohn's disease.

The CD83 reporter mouse elucidates the activity of the CD83 promoter in B, T, and dendritic cell populations in vivo.

CD83 is the major surface marker identifying mature dendritic cells (DCs). In this study, we report the generation of reporter mice expressing EGFP under the control of the CD83 promoter. We have used these mice to characterize CD83 expression by various immune system cell types both in vivo and ex vivo and under steady-state conditions and in response to stimulation with a Toll-like receptor (TLR) ligand. With those mice we could prove in vivo that the CD83 promoter is highly active in all DCs and B cells in lymphoid organs. Interestingly, this promoter activity in B cells mainly depended on the stage of development, is up-regulated in the late pre-B cell stage, and was maintained on a high level in all peripheral B cells. We also confirmed that CD83 in those cells is mainly intracellular but is up-regulated after TLR stimulation. Otherwise, CD83 promoter activity in T cells seemed to depend on stimulation and could be found mainly in CD4(+)CD25(+) and CD8(+)CD25(+) T cells and in CD4(+) and CD8(+) memory cells. In addition, we identified the murine homologues of the human CD83 splice variants. In contrast to those in human, those extremely rare short transcripts were never found without the expression of the highly dominant full-length form. So, the murine CD83 surface expression is mainly regulated posttranslationally in vivo. Our CD83 reporter mice represent a useful mouse model for monitoring the activation status and migration of DCs and lymphocytes under various conditions, and our results provide much needed clarification of the true nature of CD83 promoter activity.

Aptamer-facilitated biomarker discovery (AptaBiD).

Here we introduce a technology for biomarker discovery in which (i) DNA aptamers to biomarkers differentially expressed on the surfaces of cells being in different states are selected; (ii) aptamers are used to isolate biomarkers from the cells; and (iii) the isolated biomarkers are identified by means of mass spectrometry. The technology is termed aptamer-facilitated biomarker discovery (AptaBiD). AptaBiD was used to discover surface biomarkers that distinguish live mature and immature dendritic cells. We selected in vitro two DNA aptamer pools that specifically bind to mature and immature dendritic cells with a difference in strength of approximately 100 times. The aptamer pools were proven to be highly efficient in flow- and magnetic-bead-assisted separation of mature cells from immature cells. The two aptamer pools were then used to isolate biomarkers from the cells. The subsequent mass spectrometry analysis of the isolated proteins revealed unknown biomarkers of immature and mature dendritic cells.

Herpes simplex virus type 1 induces CD83 degradation in mature dendritic cells with immediate-early kinetics via the cellular proteasome.

Mature dendritic cells (DCs) are the most potent antigen-presenting cells within the human immune system. However, Herpes simplex virus type 1 (HSV-1) is able to interfere with DC biology and to establish latency in infected individuals. In this study, we provide new insights into the mechanism by which HSV-1 disarms DCs by the manipulation of CD83, a functionally important molecule for DC activation. Fluorescence-activated cell sorter (FACS) analyses revealed a rapid downmodulation of CD83 surface expression within 6 to 8 h after HSV-1 infection, in a manner strictly dependent on viral gene expression. Soluble CD83 enzyme-linked immunosorbent assays, together with Western blot analysis, demonstrated that CD83 rapidly disappears from the cell surface after contact with HSV-1 by a mechanism that involves protein degradation rather than shedding of CD83 from the cell surface into the medium. Infection experiments with an ICP0 deletion mutant demonstrated an important role for this viral immediate-early protein during CD83 degradation, since this particular mutant strain leads to strongly reduced CD83 degradation. This hypothesis was further strengthened by cotransfection of plasmids expressing CD83 and ICP0 into 293T cells, which led to significantly reduced accumulation of CD83. In strong contrast, transfection of plasmids expressing CD83 and a mutant ICP0 defective in its RING finger-mediated E3 ubiquitin ligase function did not reduce CD83 expression. Inhibition of the proteasome, the cellular protein degradation machinery, almost completely restored CD83 surface expression during HSV-1 infection, indicating that proteasome-mediated degradation and HSV-1 ICP0 play crucial roles in this novel viral immune escape mechanism.

Determination of the inhibitory activity and biological half-live of soluble CD83: comparison of wild type and mutant isoforms.

A soluble form of CD83 ("sCD83") has been shown to block DC-mediated T cell stimulation in vitro and an immunosuppressive role has also been shown in vivo using an experimental-autoimmune-encephalomyelits (EAE) model. Using recombinant mutational analyses, recently, we could show that sCD83 forms a homo-dimer, whereby four cysteines are involved in the intra-molecular disulfide bonds and the fifth cysteine is responsible for the inter-molecular bridging of the two molecules. Further studies revealed that the two CD83-isoforms, i.e. the dimer and the monomer, have a similar inhibitory capacity when tested in vitro. Here we show that the biological (in vivo) half-life of the two sCD83 isoforms is comparable and was between 2 and 3h. In addition, using the EAE-model, we were able to show that a monomeric-mutant isoform of soluble CD83 has a similar inhibitory activity in vivo when compared with a dimeric-wildtype isoform.

CD83 is a dimer: Comparative analysis of monomeric and dimeric isoforms.

Recently, we reported that soluble CD83 has a strong immunosuppressive activity in vitro as well as in vivo. Sequence alignment of CD83 between different species revealed the presence of five cysteines in the extracellular Ig-domain of the protein. This opens up the possibility that four cysteines are involved in the formation of two intramolecular disulfide bonds and a possible involvement of the remaining fifth cysteine in the formation of an intermolecular covalent disulfide bond, leading to the dimerization of the extracellular protein domains. Using recombinant mutational analyses, where the fifth cytosine at amino acid position 129 was mutated to a serine, we could prove that the fifth cysteine residue was indeed necessary for the dimerization. Functional analyses revealed that the mutant protein inhibited almost completely the upregulation of CD83-expression during DC maturation. Furthermore, the functional activity of the mutant protein was investigated using MLR assays and we could show that the mutant soluble CD83 protein inhibited DC-mediated allogeneic T-cell stimulation in vitro.

The soluble form of CD83 dramatically changes the cytoskeleton of dendritic cells.

CD83 is the best-known surface marker for mature dendritic cells (DC) and recently we could show that a soluble form of CD83 inhibits DC maturation. In addition, this soluble form inhibits DC-mediated T cell proliferation in vitro and in vivo. Furthermore, several viruses induce CD83 degradation or shedding in infected DC. A soluble form of CD83 was also found in plasma and serum of healthy individuals and interestingly at highly elevated levels in a number of haematological malignancies. Thus, CD83 also has functional implications for the immune response. However, the molecular mechanism is not well defined. Here we describe for the first time that soluble CD83 completely changed the cytoskeleton (analysed using phalloidin-, tubulin- and fascin-specific antibodies) when administered at a concentration of 10 microg/ml to mature DC. The cells rounded off and had only short, truncated, or no veils at all. Furthermore, soluble CD83-treated cells were completely inhibited in their ability to form clusters with T cells, an absolute prerequisite in order to stimulate T cells.

Prevention and treatment of experimental autoimmune encephalomyelitis by soluble CD83.

CD83 is up-regulated on the surface of dendritic cells (DCs) during maturation and has been widely used as a marker for mature DCs. Recently, we reported the recombinant expression of the extracellular immunoglobulin domain of human CD83 (hCD83ext). Using this soluble form of CD83, allogeneic as well as specific cytotoxic T lymphocyte proliferation could be blocked in vitro. Here we report the functional analysis of soluble CD83 in vivo, using murine experimental autoimmune encephalomyelitis (EAE) as a model. Strikingly, only three injections of soluble CD83 prevented the paralysis associated with EAE almost completely. In addition, even when the EAE was induced a second time, CD83-treated mice were protected, indicating a long-lasting suppressive effect. Furthermore, soluble CD83 strongly reduced the paralysis in different therapeutic settings. Most important, even when the treatment was delayed until the disease symptoms were fully established, soluble CD83 clearly reduced the paralyses. In addition, also when EAE was induced a second time, soluble CD83-treated animals showed reduced disease symptoms. Finally, hCD83ext treatment almost completely reduced leukocyte infiltration in the brain and in the spinal cord. In summary, this work strongly supports an immunosuppressive role of soluble CD83, thereby indicating its therapeutic potential in the regulation of immune disorders in vivo.

Role of CD83 in the immunomodulation of dendritic cells.

Glycoprotein CD83 is one of the best-known maturation markers for human dendritic cells (DCs). The fact that CD83 is strongly upregulated together with co-stimulatory molecules such as CD80 and CD86 during DC maturation suggests it plays an important role in the induction of immune responses. Infection studies with herpes simplex virus type 1 (HSV-1) and the inhibition of the CD83 mRNA specific transport from the nucleus to the cytoplasm suggested a possible functional role for CD83. The first clear proof that CD83 is indeed important for DC biology came from recently performed studies using a soluble form of the extracellular CD83 domain. DC-mediated T cell proliferation could be completely inhibited using this recombinant molecule. Additional studies elucidated immunostimulatory as well as regulatory effects of the CD83 molecule. Furthermore, CD83-/- knockout mice revealed a block in CD4+ T cell generation, a new possible immunomodulatory function of CD83.

CD83 on dendritic cells: more than just a marker for maturation.

CD83 has been known for a long time to be one of the best markers for mature dendritic cells (DCs). Studies with herpes simplex virus type 1 (HSV-1)-infected DCs, whereby the viral infection leads to the degradation of CD83, as well as investigations inhibiting CD83 mRNA transport, have provided evidence that CD83 might also be important for DC biology. Recently, we have shown that the soluble extracellular CD83 domain inhibits DC-mediated T-cell proliferation, representing the first report describing a functional role for CD83.

Overexpression, purification, and biochemical characterization of the extracellular human CD83 domain and generation of monoclonal antibodies.

CD83 is a 45-kDa glycoprotein and member of the immunoglobulin (Ig) superfamily. It is the best known marker for mature dendritic cells. Although the precise function of CD83 is not known, its selective expression and upregulation together with the costimulators CD80 and CD86 suggests an important role of CD83 in the induction of immune responses. To perform functional studies and to elucidate its mode of action it is vital to obtain recombinant expressed and highly purified CD83 molecules. Therefore, the external Ig domain of human CD83 (hCD83ext) was expressed as a GST fusion protein (GST-hCD83ext) and the soluble protein was purified under native conditions. The fusion protein was purified using GSTrap columns followed by anion-exchange chromatography. GST-hCD83ext was then cleaved using thrombin and soluble hCD83ext was further purified using GSTrap columns and finally by a preparative gel filtration as a polishing step and used for further characterization. The purified GST-hCD83 fusion protein was also used to generate monoclonal anti-CD83 antibodies in a rat system. Two different monoclonal antibodies were generated. Using these antibodies, CD83 was specifically recognized in FACS and Western blot analyses. Furthermore, we showed that native CD83 is glycosylated and that this glycosylation influences the binding of the antibodies in Western blot analyses. Finally, the purified hCD83ext protein was analyzed by one-dimensional NMR and these analyses strongly indicate that hCD83ext is folded and could therefore be used for further structural and functional studies.