A novel in vivo inducible dendritic cell ablation model in mice

Dendritic cells (DCs) are involved in T cell activation via their uptake and presentation of antigens. In vivo function of DCs was analyzed using transgenic mouse models that express diphtheria toxin receptor (DTR) or the diphtheria toxin-A subunit (DTA) under the control of the CD11c/Itgax promoter. However, CD11c⁺ cells are heterogeneous populations that contain several DC subsets. Thus, the in vivo function of each subset of DCs remains to be elucidated. Here, we describe a new inducible DC ablation model, in which DTR expression is induced under the CD11c/Itgax promoter after Cre-mediated excision of a stop cassette (CD11c-iDTR). Crossing of CD11c-iDTR mice with CAG-Cre transgenic mice, expressing Cre recombinase under control of the cytomegalovirus immediate early enhancer-chicken beta-actin hybrid promoter, led to the generation of mice, in which DTR was selectively expressed in CD11c⁺ cells (iDTRΔ mice). We successfully deleted CD11c⁺ cells in bone marrow-derived DCs in vitro and splenic CD11c⁺ cells in vivo after DT treatment in iDTRΔ mice. This mouse strain will be a useful tool for generating mice lacking a specific subset of DCs using a transgenic mouse strain, in which the Cre gene is expressed by a DC subset-specific promoter.     

Efficient B Cell Depletion via Diphtheria Toxin in CD19-Cre/iDTR Mice

B cells were first discovered as antibody producing cells, as B-1 B cells and finally as effector cells. In recent years their capacity to serve as antigen presenting cells is increasingly appreciated, and better tools are needed to study their function. We have previously described a new mouse model, the iDTR mice, that allow for the Cre-mediated expression of the diphtheria toxin receptor, thus rendering cells that express the Cre-recombinase sensitivity to diphtheria toxin. Herein we describe a new mouse line, the B-DTR mice, where the CD19-Cre was crossed to the iDTR mice. B-DTR allows for the efficient and cost-effective depletion of different B cell subpopulations, but only partially plasma cells. These mice can therefore be used to study the importance of B cells versus plasma cells in different immune responses and autoimmune diseases.     

Functionally relevant neutrophilia in CD11c diphtheria toxin receptor transgenic mice

Transgenic mice expressing the diphtheria toxin receptor (DTR) in specific cell types are key tools for functional studies in several biological systems. B6.FVB-Tg(Itgax-DTR/EGFP)57Lan/J (CD11c.DTR) and B6.Cg-Tg(Itgax-DTR/OVA/EGFP)1Gjh/Crl (CD11c.DOG) mice express the DTR in CD11c(+) cells, allowing conditional depletion of dendritic cells. We report that dendritic-cell depletion in these models caused polymorphonuclear neutrophil (PMN) release from the bone marrow, which caused chemokine-dependent neutrophilia after 6-24 h and increased bacterial clearance in a mouse pyelonephritis model. We present a transgenic mouse line, B6.Cg-Tg(Itgax-EGFP-CRE-DTR-LUC)2Gjh/Crl (CD11c.LuciDTR), which is unaffected by early neutrophilia. However, CD11c.LuciDTR and CD11c.DTR mice showed late neutrophilia 72 h after dendritic cell depletion, which was independent of PMN release and possibly resulted from increased granulopoiesis. Thus, the time point of dendritic cell depletion and the choice of DTR transgenic mouse line must be considered in experimental settings where neutrophils may be involved.     

Diphtheria toxin receptor-mediated conditional and targeted cell ablation in transgenic mice

Specific cell ablation is a useful method for analyzing the in vivo function of cells. We have developed a simple and sensitive method for conditional cell ablation in transgenic mice, called "toxin receptor-mediated cell knockout." We expressed the diphtheria toxin (DT) receptor in transgenic mice using a hepatocyte-specific promoter and found that injection of DT caused fulminant hepatitis. Three independently established transgenic lines demonstrated a good correlation between the sensitivity of hepatocytes to DT and the expression level of the DT receptors. Moreover, the degree of hepatocyte damage was easily controlled over a wide range of doses of injected DT without any obvious abnormalities in other cells or tissues. This system is useful for generating mouse models of disease and for studying the recovery or regeneration of tissues from cell damage or loss. As DT is a potent inhibitor of protein synthesis in both growing and non-growing cells, the method is applicable to a wide range of cells and tissues in mice or in other DT-insensitive animals.     

A diphtheria toxin receptor deficient in epidermal growth factor-like biological activity

Targeted cell ablation in animals is a powerful method for analyzing the physiological function of cell populations and generating various animal models of organ dysfunction. To achieve more specific and conditional ablation of target cells, we have developed a method termed Toxin Receptor mediated Cell Knockout (TRECK). A potential shortcoming of this method, however, is that overexpression of human heparin-binding epidermal growth factor-like growth factor (hHB-EGF) as a diphtheria toxin (DT) receptor in target cells or tissues may cause abnormalities in transgenic mice, since hHB-EGF is a member of the EGF growth factor family. To create novel DT receptors that are defective in growth factor activity and resistant to metalloprotease-cleavage, we mutated five amino acids in the extracellular EGF-like domain of hHB-EGF, which contains both DT-binding and protease-cleavage sites. Two of the resultant hHB-EGF mutants, I117A/L148V and I117V/L148V, possessed little growth factor activity but retained DT receptor activity. Furthermore, these mutants were resistant to metalloprotease-cleavage by 12-O-tetradecanoylphorbol-13-acetate stimulation, which is expected to enhance DT receptor activity. These novel DT receptors should be useful for the generation of transgenic mice by TRECK.     

Domain analysis of the tetraspanins: studies of CD9/CD63 chimeric molecules on subcellular localization and upregulation activity for diphtheria toxin binding

CD9 and CD63 belong to a tetramembrane-spanning glycoprotein family called tetraspanin, and are involved in a wide variety of cellular processes, but the structure-function relationship of this family of proteins has yet to be clarified. CD9 associates with diphtheria toxin receptor (DTR), which is identical to the membrane-anchored form of heparin-binding EGF-like growth factor (proHB-EGF). CD9 upregulates the diphtheria toxin (DT) binding activity of DTR/proHB-EGF, while CD63 does not upregulate the DT binding activity in spite of the fact that this protein also associates with DTR/proHB-EGF on the cell surface. CD9 molecules localize on the cell surface, while those of CD63 localize predominantly at lysosomes and intracellular compartments. We made CD9/CD63 chimeric molecules and then studied their intracellular localization and upregulation activities. The C-terminal regions of CD63, which includes the lysosome sorting motif, showed a strong inhibitory effect on the expression of the chimeric proteins at the cell surface, while mutants lacking the lysosome sorting motif delivered more efficiently on the cell surface, indicating that the lysosome sorting motif contributes to the inhibitory effect of the C-terminal region. However, the N-terminal half of this family of proteins containing the 1st to 3rd transmembrane domains also seems to influence the cell surface expression. For the upregulation of DT binding activity the large extracellular loop (EC2) of CD9 was essential, while the remaining regions influenced the upregulation activity by changing the efficiency of cell surface expression. From these results we discussed the structure-function relationship of this family of proteins.     

Transgenic mice expressing a fully nontoxic diphtheria toxin mutant, not CRM197 mutant, acquire immune tolerance against diphtheria toxin

We previously developed a method termed "toxin receptor-mediated cell knockout" (TRECK). By the TRECK method, a single or repeated shot(s) of diphtheria toxin (DT) conditionally ablates a specific cell population from transgenic mice expressing the DT receptor transgene under the control of a cell type-specific promoter. In some cases of TRECK, frequent and high-dose administration of DT is required, raising the concern that these frequent injections of DT could cause production of anti-DT antibody, which would neutralize further DT administration. To solve this problem, we aimed to generate transgenic mice genetically expressing a nontoxic DT mutant, with the expectation that they may naturally acquire immune tolerance to DT. Unexpectedly, the G52E DT mutant, which is well known as the nontoxic DT variant cross reacting material 197 (CRM197), exhibited cytotoxicity in yeast and mammalian cells. Cytotoxicity of CRM197 was abrogated in cells mutated for elongation factor 2 (EF-2), indicating that CRM197 exerts its toxic effects through EF-2, similar to wild-type DT. On the other hand, the K51E/E148K DT mutant exhibited no detectable cytotoxicity. This led us to successfully obtain DT gene transgenic mice, which exhibited no histological abnormalities, and indeed acquired immune tolerance to DT.     

Lineage-specific cell disruption in living mice by Cre-mediated expression of diphtheria toxin A chain

We have established a transgenic mouse line in which floxed neomycin resistant cassette was inserted between the CAG promoter and EGFP. When these transgenic mice were mated with Cre-expressing transgenic animals, the offspring obtained were fluorescent green. We then established a transgenic mouse line in which EGFP in the above construct was replaced by diphtheria toxin A chain (DT). When the latter transgenic mice were mated with mice expressing Cre restricted to germ cells, we obtained healthy but sterile offspring due to a disruption of germ line cells by DT expression. We predict that this strategy will be useful for the construction of new animal models for human diseases, featuring a variety of missing cell lineages produced by disruption with DT.     

An antibody that inhibits the binding of diphtheria toxin to cells revealed the association of a 27-kDa membrane protein with the diphtheria toxin receptor

A monoclonal antibody that blocks the binding of diphtheria toxin to Vero cells was isolated by immunizing mice with Vero cell membrane. The antibody inhibits the binding of diphtheria toxin and also CRM197, a mutant form of diphtheria toxin, to Vero cells, and consequently inhibits the cytotoxicity of diphtheria toxin. This antibody does not directly react with the receptor molecule of diphtheria toxin (DTR14.5). Immunoprecipitation and immunoblotting studies revealed that this antibody binds to a novel membrane protein of 27 kDa (DRAP27). When diphtheria toxin receptor was passed through an affinity column made with this antibody, the receptor was trapped only in the presence of DRAP27. These results indicate that DRAP27 and DTR14.5 closely associate in Vero cell membrane and that the inhibition of the binding of diphtheria toxin to the receptor is due to the binding of the antibody to the DRAP27 molecule. Binding studies using 125I-labeled antibody showed that there are many more molecules of DRAP27 on the cell surface than diphtheria toxin-binding sites. However, there is a correlation between the sensitivity of a cell line to diphtheria toxin and the number of DRAP27 molecules on the cell surface, suggesting that DRAP27 is involved in the entry of diphtheria toxin into the target cell.     

Dual Cre and Dre recombinases mediate synchronized lineage tracing and cell subset ablation in vivo

Genetic technology using site-specific recombinases, such as the Cre-loxP system, has been widely employed for labeling specific cell populations and for studying their functions in vivo. To enhance the precision of cell lineage tracing and functional study, a similar site-specific recombinase system termed Dre-rox has been recently used in combination with Cre-loxP. To enable more specific cell lineage tracing and ablation through dual recombinase activity, we generated two mouse lines that render Dre- or Dre+Cre-mediated recombination to excise a stop codon sequence that prevents the expression of diphtheria toxin receptor (DTR) knocked into the ubiquitously expressed and safe Rosa26 locus. Using different Dre- and Cre-expressing mouse lines, we showed that the surrogate gene reporters tdTomato and DTR were simultaneously expressed in target cells and in their descendants, and we observed efficient ablation of tdTomato⁺ cells after diphtheria toxin administration. These mouse lines were used to simultaneously trace and deplete the target cells of interest through the inducible expression of a reporter and DTR using dual Cre and Dre recombinases, allowing a more precise and efficient study of the role of specific cell subsets within a heterogeneous population in pathophysiological conditions in vivo.     

CD9 amino acids critical for upregulation of diphtheria toxin binding.

CD9 associates with a diphtheria toxin receptor (DTR) that is identical to the membrane-anchored form of heparin-binding EGF-like growth factor. We determined the region of CD9 important for upregulation activity. Human and monkey CD9 upregulates DT binding activity of DTR, while mouse CD9 has no upregulation activity. Transfection of chimeric constructs comprising monkey and mouse CD9s showed that the human sequence between Ala156 and Asp183 is essential for the upregulation activity. Studies of mutants, replacing a single amino acid within the region between Ala156 and Asp183 of monkey CD9 with the corresponding amino acid residue in mouse CD9, revealed that substitution of Gly158 is critical for the reduction of the upregulation activity and secondly for the substitution of Val159 and Thr175. These three amino acid residues were deduced to be located on the head domain of the second extracellular loop, suggesting that interactions of CD9 with DTR or DT at the domain containing these three amino acids were important for the upregulation of DT binding.     

Targeted ablation of oligodendrocytes triggers axonal damage

Glial dysfunction has been implicated in a number of neurodegenerative diseases. In this study we investigated the consequences of glial and oligodendrocyte ablation on neuronal integrity and survival in Drosophila and adult mice, respectively. Targeted genetic ablation of glia was achieved in the adult Drosophila nervous system using the GAL80-GAL4 system. In mice, oligodendrocytes were depleted by the injection of diphtheria toxin in MOGi-Cre/iDTR double transgenic animals. Acute depletion of oligodendrocytes induced axonal injury, but did not cause neuronal cell death in mice. Ablation of glia in adult flies triggered neuronal apoptosis and resulted in a marked reduction in motor performance and lifespan. Our study shows that the targeted depletion of glia triggers secondary neurotoxicity and underscores the central contribution of glia to neuronal homeostasis. The models used in this study provide valuable systems for the investigation of therapeutic strategies to prevent axonal or neuronal damage.     

Cell surface monkey CD9 antigen is a coreceptor that increases diphtheria toxin sensitivity and diphtheria toxin receptor affinity

Monkey (Mk) CD9 antigen has been shown previously to increase the diphtheria toxin (DT) sensitivity of cells when co-expressed with Mk proHB-EGF (DT receptor). We have elucidated here the mechanism whereby Mk CD9 influences Mk proHB-EGF and present evidence that Mk CD9 is a coreceptor for DT. We observed that Mk CD9 not only increased the DT sensitivity but also increased the DT receptor affinity of cells. Furthermore, the higher the Mk CD9/Mk proHB-EGF ratio, the higher the affinity. In contrast, mouse (Ms) CD9 did not increase the toxin sensitivity or receptor affinity of cells when co-expressed with Mk proHB-EGF. Using Mk/Ms chimeric CD9 molecules, we determined that the second extracellular domain of Mk CD9 is responsible for both increased sensitivity and receptor affinity. This domain of Mk CD9 also interacts with Mk proHB-EGF in a yeast two-hybrid system. Our findings thus suggest that Mk CD9 has a direct physical interaction with Mk proHB-EGF to form a DT receptor complex and that this contact may change the conformation of the receptor to increase DT binding affinity and consequently increase toxin sensitivity. We thus propose that Mk CD9 is a coreceptor for DT.     

A C terminus cysteine of diphtheria toxin B chain involved in immunotoxin cell penetration and cytotoxicity

The role of diphtheria toxin (DT) B-chain subdomains in DT cytotoxicity and immunotoxin mechanism of action has been investigated. OKT3 (mAb to the CD3 surface Ag of human T lymphocytes) was conjugated to DT or the DT mutant CRM 1001, which has a cys----tyr substitution at position 471 of the B chain. OKT3-CRM 1001 immunotoxin was about 1400-fold less cytotoxic for CD3 Jurkat cells than OKT3-DT and had a 12-fold slower kinetics of protein synthesis inactivation, CRM 1001 killed DT-sensitive Vero cells at a 5000-fold higher concentration than DT. Its cell surface-binding activity was comparable to DT. Based on kinetics of cell inactivation, toxicity determination at low extracellular pH and Triton X-114 distribution, it was concluded that CRM 1001 is defective in at least one crucial step of toxin penetration and is unable to cross cell membranes as efficiently as DT. The substituted cysteine appears to be important for DT translocating functions. Data on the function of DT B-chain subdomains are relevant for the study of whole toxin conjugates and their mechanism of action.     

Inducible ablation of mouse Langerhans cells diminishes but fails to abrogate contact hypersensitivity

Langerhans cells (LC) form a unique subset of dendritic cells (DC) in the epidermis but so far their in vivo functions in skin immunity and tolerance could not be determined, in particular in relation to dermal DC (dDC). Here, we exploit a novel diphtheria toxin (DT) receptor (DTR)/DT-based system to achieve inducible ablation of LC without affecting the skin environment. Within 24 h after intra-peritoneal injection of DT into Langerin-DTR mice LC are completely depleted from the epidermis and only begin to return 4 wk later. LC deletion occurs by apoptosis in the absence of inflammation and, in particular, the dDC compartment is not affected. In LC-depleted mice contact hypersensitivity (CHS) responses are significantly decreased, although ear swelling still occurs indicating that dDC can mediate CHS when necessary. Our results establish Langerin-DTR mice as a unique tool to study LC function in the steady state and to explore their relative importance compared with dDC in orchestrating skin immunity and tolerance.     

Interaction of diphtheria toxin B subunit with sensitive and insensitive mammalian cells

The recombinant fluorescent derivative of diphtheria toxin (EGFP-SbB) obtained by the replacement of toxin A subunit by enhanced green fluorescent protein (EGFP) has been used for visualization of the interaction of diphtheria toxin (DT) with sensitive and insensitive cells. It was shown that EGFP-SbB could interact with cell surface of both toxin-sensitive monkey cells (Vero cell line) and toxin-resistant mouse cells (3T3 cell line). The affinity of this protein for receptors of Vero cells was three times higher as compared with 3T3 cells. It was demonstrated that fluorescent derivate was able to interact with receptors of both cell lines and to internalize into these cells. Internalization of EGFP-SbB into the cells was inhibited by endocytosis inhibitor phenyl arsine oxide. We suppose that diverse sensitivity to DT of monkey and mouse cells can be explained not only by differences in their receptor affinity for DT but also by the processes that occur after internalization of the toxin into the cells.     

GPI-anchored diphtheria toxin receptor allows membrane translocation of the toxin without detectable ion channel activity.

We have investigated the role of the transmembrane and cytoplasmic domains of the diphtheria toxin (DT) receptor [heparin-binding epidermal growth factor (HB-EGF) precursor] in the intoxication pathway. Two mutants were constructed in which these domains were replaced by either a 37 amino acid sequence signalling membrane attachment via a glycosylphosphatidylinositol (GPI) anchor (DTR-GPI) or by the transmembrane and cytoplasmic domains of the human EGF receptor (DTR-EGFR). Similar amounts of DTA fragment were translocated through the plasma membrane of NIH 3T3 cells transfected with the wild-type receptor (DTR), DTR-GPI and DTR-EGFR, but translocation was about six times less efficient in the case of DTR-GPI and DTR-EGFR when taking into account the number of receptors expressed. Interestingly, DT-induced 22Na+ influx was weak in DTR-EGFR cells and not detectable in DTR-GPI cells. Whole cell patch-clamp analysis showed the DT at low pH induced depolarization and decreased input resistance in DTR cells (and to a lesser extent also in DTR-EGFR cells) but not in DTR-GPI cells. These results suggest that the transmembrane and cytoplasmic part of the receptor might be involved in channel activity and that translocation of the A fragment is independent of toxin-induced cation channel activity.     

Transgenic mice expressing the diphtheria toxin receptor are sensitive to the toxin

Whereas diphtheria and the mechanism of action of diphtheria toxin, the bacterial molecule that induces the disease, have been studied and understood for some time, the receptor that allows animal cells to bind the toxin escaped identification until recently. The receptor was identified by its ability to confer toxin-sensitivity to mouse cells, which are normally toxin-resistant. Although mice are also naturally resistant, we now demonstrate that transgenic mice expressing the diphtheria toxin receptor are as sensitive to the toxin as are humans and other toxin-sensitive animals. These transgenic mice provide a suitable model for studying modern antidotes for diphtheria.