Role of the Cysteine 81 Residue of Macrophage Migration Inhibitory Factor as a Molecular Redox Switch.

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory and tumor-promoting cytokine that occurs in two redox-dependent immunologically distinct conformational isoforms. The disease-related structural isoform of MIF (oxMIF) can be specifically and predominantly detected in the circulation of patients with inflammatory diseases and in tumor tissue, whereas the ubiquitously expressed isoform of MIF (redMIF) is abundantly expressed in healthy and diseased subjects. In this article, we report that cysteine 81 within MIF serves as a "switch cysteine" for the conversion of redMIF to oxMIF. Modulating cysteine 81 by thiol reactive agents leads to significant structural rearrangements of the protein, resulting in a decreased ß-sheet content and an increased random coil content, but maintaining the trimeric quaternary structure. This conformational change in the MIF molecule enables binding of oxMIF-specific antibodies BaxB01 and BaxM159, which showed beneficial activity in animal models of inflammation and cancer. Crystal structure analysis of the MIF-derived EPCALCS peptide, bound in its oxMIF-like conformation by the Fab fragment of BaxB01, revealed that this peptide adopts a curved conformation, making the central thiol protein oxidoreductase motif competent to undergo disulfide shuffling. We conclude that redMIF might reflect a latent zymogenic form of MIF, and formation of oxMIF leads to a physiologically relevant, i.e., enzymatically active, state.

Selective Targeting of a Disease-Related Conformational Isoform of Macrophage Migration Inhibitory Factor Ameliorates Inflammatory Conditions.

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine and counterregulator of glucocorticoids, is a potential therapeutic target. MIF is markedly different from other cytokines because it is constitutively expressed, stored in the cytoplasm, and present in the circulation of healthy subjects. Thus, the concept of targeting MIF for therapeutic intervention is challenging because of the need to neutralize a ubiquitous protein. In this article, we report that MIF occurs in two redox-dependent conformational isoforms. We show that one of the two isoforms of MIF, that is, oxidized MIF (oxMIF), is specifically recognized by three mAbs directed against MIF. Surprisingly, oxMIF is selectively expressed in the plasma and on the cell surface of immune cells of patients with different inflammatory diseases. In patients with acute infections or chronic inflammation, oxMIF expression correlated with inflammatory flare-ups. In addition, anti-oxMIF mAbs alleviated disease severity in mouse models of acute and chronic enterocolitis and improved, in synergy with glucocorticoids, renal function in a rat model of crescentic glomerulonephritis. We conclude that oxMIF represents the disease-related isoform of MIF; oxMIF is therefore a new diagnostic marker for inflammation and a relevant target for anti-inflammatory therapy.

Optimization of an Antibody Light Chain Framework Enhances Expression, Biophysical Properties and Pharmacokinetics.

Efficacy, safety, and manufacturability of therapeutic antibodies are influenced by their biopharmaceutical and biophysical properties. These properties can be optimized by library approaches or rationale protein design. Here, we employed a protein engineering approach to modify the variable domain of the light chain (VL) framework of an oxidized macrophage migration inhibitory factor (oxMIF)-specific antibody. The amendment of the antibody sequence was based on homology to human germline VL genes. Three regions or positions were identified in the VL domain-L1-4, L66, L79-and mutated independently or in combination to match the closest germline V gene. None of the mutations altered oxMIF specificity or affinity, but some variants improved thermal stability, aggregation propensity, and resulted in up to five-fold higher expression. Importantly, the improved biopharmaceutical properties translated into a superior pharmacokinetic profile of the antibody. Thus, optimization of the V domain framework can ameliorate the biophysical qualities of a therapeutic antibody candidate, and as result its manufacturability, and also has the potential to improve pharmacokinetics.

Expression of langerin/CD207 reveals dendritic cell heterogeneity between inbred mouse strains.

Langerin/CD207 is expressed by a subset of dendritic cells (DC), the epithelial Langerhans cells. However, langerin is also detected among lymphoid tissue DC. Here, we describe striking differences in langerin-expressing cells between inbred mouse strains. While langerin+ cells are observed in comparable numbers and with comparable phenotypes in the epidermis, two distinct DC subsets bear langerin in peripheral, skin-draining lymph nodes of BALB/c mice (CD11c(high) CD8alpha(high) and CD11c(low) CD8alpha(low)), whereas only the latter subset is present in C57BL/6 mice. The CD11c(high) subset is detected in mesenteric lymph nodes and spleen of BALB/c mice, but is virtually absent from C57BL/6 mice. Similar differences are observed in other mouse strains. CD11c(low) langerin+ cells represent skin-derived Langerhans cells, as demonstrated by their high expression of DEC-205/CD205, maturation markers, and recruitment to skin-draining lymph nodes upon imiquimod-induced inflammation. It will be of interest to determine the role of lymphoid tissue-resident compared to skin-derived langerin+ DC.

Disruption of the langerin/CD207 gene abolishes Birbeck granules without a marked loss of Langerhans cell function.

Langerin is a C-type lectin expressed by a subset of dendritic leukocytes, the Langerhans cells (LC). Langerin is a cell surface receptor that induces the formation of an LC-specific organelle, the Birbeck granule (BG). We generated a langerin(-/-) mouse on a C57BL/6 background which did not display any macroscopic aberrant development. In the absence of langerin, LC were detected in normal numbers in the epidermis but the cells lacked BG. LC of langerin(-/-) mice did not present other phenotypic alterations compared to wild-type littermates. Functionally, the langerin(-/-) LC were able to capture antigen, to migrate towards skin draining lymph nodes, and to undergo phenotypic maturation. In addition, langerin(-/-) mice were not impaired in their capacity to process native OVA protein for I-A(b)-restricted presentation to CD4(+) T lymphocytes or for H-2K(b)-restricted cross-presentation to CD8(+) T lymphocytes. langerin(-/-) mice inoculated with mannosylated or skin-tropic microorganisms did not display an altered pathogen susceptibility. Finally, chemical mutagenesis resulted in a similar rate of skin tumor development in langerin(-/-) and wild-type mice. Overall, our data indicate that langerin and BG are dispensable for a number of LC functions. The langerin(-/-) C57BL/6 mouse should be a valuable model for further functional exploration of langerin and the role of BG.

Pharmacokinetics, disease-modifying activity, and safety of an experimental therapeutic targeting an immunological isoform of macrophage migration inhibitory factor, in rat glomerulonephritis.

New therapeutic agents are needed to overcome the toxicity and suboptimal efficacy observed in current treatment of glomerulonephritis (GN). BaxB01 is a fully human monoclonal antibody targeting a disease-related immunologically distinct isoform of Macrophage migration Inhibitory Factor (MIF), designated oxidized MIF (oxMIF) and locally expressed in inflammatory conditions. We report the pharmacokinetic profile of BaxB01, and its dose and exposure-related disease-modifying activity in experimentally induced rat GN. BaxB01 bound to rat oxMIF with high affinity and reduced rat macrophage migration in vitro. After intravenous administration in rats, BaxB01 demonstrated favorable pharmacokinetics, with a half-life of up to nine days. Disease modification was dose-related (≥ 10mg/kg) as demonstrated by significantly reduced proteinuria and diminished histopathological glomerular crescent formation. Importantly, a single dose was sufficient to establish an exposure-related, anti-inflammatory milieu via amelioration of glomerular cellular inflammation. Pharmacodynamic modeling corroborated these findings, consistently predicting plasma exposures that were effective in attenuating both anti-inflammatory activity and reducing loss of kidney function. This pharmacologic benefit on glomerular function and structure was sustained during established disease, while correlation analyses confirmed a link between the antibody's anti-inflammatory activity and reduced crescent formation in individual rats. Finally, safety assessment in rats showed that the experimental therapeutic was well tolerated without signs of systemic toxicity or negative impact on kidney function. These data define therapeutically relevant exposures correlated with mechanism-based activity in GN, while toxicological evaluation suggests a large therapeutic index and provides evidence for achieving safe and effective exposure to a MIF isoform-directed therapeutic in nephritis-associated disease.

Oxidized macrophage migration inhibitory factor is a potential new tissue marker and drug target in cancer.

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine, which was shown to be upregulated in cancers and to exhibit tumor promoting properties. Unlike other cytokines, MIF is ubiquitously present in the circulation and tissue of healthy subjects. We recently described a previously unrecognized, disease-related isoform of MIF, designated oxMIF, which is present in the circulation of patients with different inflammatory diseases. In this article, we report that oxMIF is also linked to different solid tumors as it is specifically expressed in tumor tissue from patients with colorectal, pancreatic, ovarian and lung cancer. Furthermore, oxMIF can be specifically targeted by a subset of phage display-derived fully human, monoclonal anti-MIF antibodies (mAbs) that were shown to neutralize pro-tumorigenic activities of MIF in vivo. We further demonstrate that anti-oxMIF mAbs sensitize human cancer cell lines (LNCaP, PC3, A2780 and A2780ADR) to the action of cytotoxic drugs (mitoxantrone, cisplatin and doxorubicin) in vitro and in an A2780 xenograft mouse model of ovarian cancer. We conclude that oxMIF is the disease related isoform of MIF in solid tumors and a potential new diagnostic marker and drug target in cancer.

Migratory Langerhans cells in mouse lymph nodes in steady state and inflammation.

Dendritic cells cells induce immunity or-in the steady state-maintain peripheral tolerance. Little is known in that regard about Langerhans cells. Therefore, we investigated migrating Langerhans cells in the steady-state versus inflammation. Increased numbers of Langerhans cells, as determined by immunostaining for Langerin/CD207, appeared in the lymph nodes in response to a contact allergen. Whereas a large proportion of Langerhans cells expressed CD86 in the steady state, CD40, and CD80 were found on a smaller percentage. During inflammation, more CD40(+), CD80(+), CD274/B7-H1/PD-L1(+), and CD273/B7-DC/PD-L2(+) Langerhans cells were found in the lymph nodes, and they expressed higher levels of these molecules. CD275/inducible T cell co-stimulator (ICOS) ligand was not detected. Langerhans cells in the nodes of contact allergen-treated mice produced more IL-12p40/70. This correlated with more interferon-gamma being produced by activated lymph node T cells. Epicutaneous immunization with ovalbumin under inflammatory conditions led to a more vigorous proliferation of antigen-specific CD4 T cells in vitro and in vivo as compared with immunization in the steady state. The latter modality, however did not induce strong CD4 T cell tolerance in this model. Thus, the overall phenotype of Langerhans cells is not an indicator for their immunogenic or tolerogenic potential.

Mouse lymphoid tissue contains distinct subsets of langerin/CD207 dendritic cells, only one of which represents epidermal-derived Langerhans cells.

Langerin/CD207 is a C-type lectin associated with formation of Birbeck granules (BG) in Langerhans cells (LC). Here, we describe a monoclonal antibody (mAb 205C1) recognizing the extracellular domain of mouse langerin. Cell-surface langerin was detected in all epidermal LC, which presented a uniform phenotype. Two subpopulations of langerin+ cells were identified in peripheral lymph nodes (LN). One population (subset 1) was CD11c(low/+)/CD8alpha(-/low)/CD11b+/CD40+/CD86+. The other population (subset 2) was CD11c(high)/CD8alpha+/CD11b(low), and lacked CD40 and CD86. Only subset 1 was fluorescein 5-isothiocyanate (FITC+) following painting onto epidermis, and the appearance of such FITC+ cells in draining LN was inhibited by pertussis toxin. Mesenteric LN, spleen, and thymus contained only a single population of langerin+ DC, corresponding to peripheral LN subset 2. Unexpectedly, BG were absent from spleen CD8alpha+ DC despite expression of langerin, and these organelles were not induced by mAb 205C1. Collectively, we demonstrate that two langerin+ DC populations (subsets 1 and 2) co-exist in mouse lymphoid tissue. Subset 1 unequivocally identifies epidermal LC-derived DC. The distribution of subset 2 indicates a non-LC origin of these langerin+ cells. These findings should facilitate our understanding of the role played by langerin in lymphoid organ DC subsets.

Visualization and characterization of migratory Langerhans cells in murine skin and lymph nodes by antibodies against Langerin/CD207.

Dendritic cells are professional antigen-presenting cells that initiate primary immunity. Migration from sites of antigen uptake to lymphoid organs is crucial for the generation of immune responses. We investigated the migratory pathways specifically of epidermal Langerhans cells by tracing them from the epidermis to the draining lymph nodes. This was possible with a new monoclonal antibody, directed against murine Langerin/CD207, a type II lectin specific for Langerhans cells. In situ, resident, and activated Langerhans cells express Langerin in the epidermis and on their way through dermal lymphatic vessels. Both emigrated and trypsinization-derived Langerhans cells expressed high levels of Langerin intracellularly but reduced it upon prolonged culture periods. Sizeable numbers of Langerin+ cells were found in skin draining lymph nodes but not in mesenteric nodes. Langerin+ cells localized to the T cells areas and rarely to B cell zones. Numbers of Langerin-expressing cells increased after application of a contact sensitizer. In the steady state, Langerhans cells in the skin-draining nodes expressed maturation markers, such as 2A1 and costimulatory molecules CD86 and CD40. These molecules, CD86 and CD40, were further upregulated upon inflammatory stimuli such as contact sensitization. Thus, the novel anti-Langerin monoclonal antibody permits the unequivocal visualization of migratory Langerhans cells in the lymph nodes for the first time and thereby allows to dissect the relative immunogenic or tolerogenic contributions of Langerhans cells and other types of dendritic cells.

Ontogeny of Langerin/CD207 expression in the epidermis of mice.

C-type lectin receptors help Langerhans cells (LC) to take up and process pathogens. Langerin/CD207 is a mannose-binding C-type lectin that is specifically expressed by LC. It is involved in antigen uptake in an as yet poorly defined way, and it is a major molecular constituent of Birbeck granules. We studied the emergence of Langerin expression in LC in epidermal sheets and cell suspensions during ontogeny. Langerin appears later than MHC II expression. Intracellular Langerin expression becomes apparent 2-3 d after birth. Only 10 days after birth all LC co-express Langerin. The intensity of Langerin expression reaches adult levels by 3 wk after birth. Early Langerin expression appears to correlate at least in part with the physical presence of Birbeck granules.

An essential role of ubiquitination in Cbl-mediated negative regulation of the Src-family kinase Fyn.

The Cbl family of ubiquitin ligases function as negative regulators of activated receptor tyrosine kinases by facilitating their ubiquitination and subsequent lysosomal targeting. Here, we have investigated the role of Cbl ubiquitin ligase activity in the negative regulation of a non-receptor tyrosine kinase, the Src-family kinase Fyn. Using primary embryonic fibroblasts from Cbl(+/+) and Cbl(-/-) mice, we demonstrate that endogenous Cbl mediates the ubiquitination of Fyn and dictates the rate of Fyn turnover. By analyzing CHO-TS20 cells with a temperature-sensitive ubiquitin activating enzyme, we demonstrate that intact cellular ubiquitin machinery is required for Cbl-induced degradation of Fyn. Analyses of Cbl mutants, with mutations in or near the RING finger domain, in 293T cells revealed that the ubiquitin ligase activity of Cbl is essential for Cbl-induced degradation of Fyn by the proteasome pathway. Finally, use of a SRE-luciferase reporter demonstrated that Cbl-dependent negative regulation of Fyn function requires the region of Cbl that mediates the ubiquitin ligase activity. Given the conservation of structure between various Src-family kinases and the ability of Cbl to interact with multiple members of this family, Cbl-dependent ubiquitination could serve a general role to negatively regulate activated Src-family kinases.

Human anti-macrophage migration inhibitory factor antibodies inhibit growth of human prostate cancer cells in vitro and in vivo.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine, originally discovered for its eponymous effect and now known for pleiotropic biologic properties in immunology and oncology. Circulating MIF levels are elevated in several types of human cancer including prostate cancer. MIF is released presumably by both stromal and tumor cells and enhances malignant growth and metastasis by diverse mechanisms, such as stimulating tumor cell proliferation, suppressing apoptotic death, facilitating invasion of the extracellular matrix, and promoting angiogenesis. Recently described fully human anti-MIF antibodies were tested in vitro and in vivo for their ability to influence growth rate and invasion of the human PC3 prostate cancer cell line. In vitro, the selected candidate antibodies BaxG03, BaxB01, and BaxM159 reduced cell growth and viability by inhibiting MIF-induced phosphorylation of the central kinases p44/42 mitogen-activated protein kinase [extracellular signal-regulated kinase-1 and -2 (ERK1/2)] and protein kinase B (AKT). Incubation of cells in the presence of the antibodies also promoted activation of caspase-3/7. The antibodies furthermore inhibited MIF-promoted invasion and chemotaxis as transmigration through Matrigel along a MIF gradient was impaired. In vivo, pharmacokinetic parameters (half-life, volume of distribution, and bioavailability) of the antibodies were determined and a proof-of-concept was obtained in a PC3-xenograft mouse model. Treatment with human anti-MIF antibodies blunted xenograft tumor growth in a dose-dependent manner. We therefore conclude that the anti-MIF antibodies described neutralize some of the key tumor-promoting activities of MIF and thus limit tumor growth in vivo.

Binding of Cbl to a phospholipase Cgamma1-docking site on platelet-derived growth factor receptor beta provides a dual mechanism of negative regulation.

Ubiquitin conjugation to receptor tyrosine kinases is a critical biochemical step in attenuating their signaling through lysosomal degradation. Our previous studies have established Cbl as an E3 ubiquitin ligase for ubiquitinylation and degradation of platelet-derived growth factor receptor (PDGFR) alpha and PDGFRbeta. However, the role of endogenous Cbl in PDGFR regulation and the molecular mechanisms of this regulation remain unclear. Here, we demonstrate that endogenous Cbl is essential for ligand-induced ubiquitinylation and degradation of PDGFRbeta; this involves the Cbl TKB domain binding to PDGFRbeta phosphotyrosine 1021, a known phospholipase C (PLC) gamma1 SH2 domain-binding site. Lack of Cbl or ablation of the Cbl-binding site on PDGFRbeta impedes receptor sorting to the lysosome. Cbl-deficient cells also show more PDGF-induced PLCgamma1 association with PDGFRbeta and enhanced PLC-mediated cell migration. Thus, Cbl-dependent negative regulation of PDGFRbeta involves a dual mechanism that concurrently promotes ubiquitin-dependent lysosomal sorting of the receptor and competitively reduces the recruitment of a positive mediator of receptor signaling.

Neutrophils rapidly migrate via lymphatics after Mycobacterium bovis BCG intradermal vaccination and shuttle live bacilli to the draining lymph nodes.

The early innate response after Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccination is poorly characterized but probably decisive for subsequent protective immunity against tuberculosis. Therefore, we vaccinated mice with fluorescent BCG strains in the ear dorsum, as a surrogate of intradermal vaccination in humans. During the first 3 days, we tracked BCG host cells migrating out of the dermis to the auricular draining lymph nodes (ADLNs). Resident skin dendritic cells (DCs) or macrophages did not play a predominant role in early BCG capture and transport to ADLNs. The main BCG host cells rapidly recruited both in the dermis and ADLNs were neutrophils. Fluorescent green or red BCG strains injected into nonoverlapping sites were essentially sheltered by distinct neutrophils in the ADLN capsule, indicating that neutrophils had captured bacilli in peripheral tissue and transported them to the lymphoid organ. Strikingly, we observed BCG-infected neutrophils in the lumen of lymphatic vessels by confocal microscopy on ear dermis. Fluorescence-labeled neutrophils injected into the ears accumulated exclusively into the ipsilateral ADLN capsule after BCG vaccination. Thus, we provide in vivo evidence that neutrophils, like DCs or inflammatory monocytes, migrate via afferent lymphatics to lymphoid tissue and can shuttle live microorganisms.

Dynamics and function of Langerhans cells in vivo: dermal dendritic cells colonize lymph node areas distinct from slower migrating Langerhans cells.

Langerhans cells (LCs) are prominent dendritic cells (DCs) in epithelia, but their role in immunity is poorly defined. To track and discriminate LCs from dermal DCs in vivo, we developed knockin mice expressing enhanced green fluorescent protein (EGFP) under the control of the langerin (CD207) gene. By using vital imaging, we showed that most EGFP(+) LCs were sessile under steady-state conditions, whereas skin inflammation induced LC motility and emigration to lymph nodes (LNs). After skin immunization, dermal DCs arrived in LNs first and colonized areas distinct from slower migrating LCs. LCs reaching LNs under steady-state or inflammatory conditions expressed similar levels of costimulatory molecules. Langerin and EGFP were also expressed on thymic DCs and on blood-derived, CD8alpha(+) DCs from all secondary lymphoid organs. By using a similar knockin strategy involving a diphtheria toxin receptor (DTR) fused to EGFP, we demonstrated that LCs were dispensable for triggering hapten-specific T cell effectors through skin immunization.

Negative regulation of Lck by Cbl ubiquitin ligase.

The Cbl-family ubiquitin ligases function as negative regulators of activated receptor tyrosine kinases by facilitating their ubiquitination and subsequent targeting to lysosomes. Cbl associates with the lymphoid-restricted nonreceptor tyrosine kinase Lck, but the functional relevance of this interaction remains unknown. Here, we demonstrate that T cell receptor and CD4 coligation on human T cells results in enhanced association between Cbl and Lck, together with Lck ubiquitination and degradation. A Cbl(-/-) T cell line showed a marked deficiency in Lck ubiquitination and increased levels of kinase-active Lck. Coexpression in 293T cells demonstrated that Lck kinase activity and Cbl ubiquitin ligase activity were essential for Lck ubiquitination and negative regulation of Lck-dependent serum response element-luciferase reporter activity. The Lck SH3 domain was pivotal for Cbl-Lck association and Cbl-mediated Lck degradation, with a smaller role for interactions mediated by the Cbl tyrosine kinase-binding domain. Finally, analysis of a ZAP-70-deficient T cell line revealed that Cbl inhibited Lck-dependent mitogen-activated protein kinase activation, and an intact Cbl RING finger domain was required for this functional effect. Our results demonstrate a direct, ubiquitination-dependent, negative regulatory role of Cbl for Lck in T cells, independent of Cbl-mediated regulation of ZAP-70.