NEDD9 Is a Novel and Modifiable Mediator of Platelet-Endothelial Adhesion in the Pulmonary Circulation.

Rationale: Data on the molecular mechanisms that regulate platelet-pulmonary endothelial adhesion under conditions of hypoxia are lacking, but may have important therapeutic implications. Objectives: To identify a hypoxia-sensitive, modifiable mediator of platelet-pulmonary artery endothelial cell adhesion and thrombotic remodeling. Methods: Network medicine was used to profile protein-protein interactions in hypoxia-treated human pulmonary artery endothelial cells. Data from liquid chromatography-mass spectrometry and microscale thermophoresis informed the development of a novel antibody (Ab) to inhibit platelet-endothelial adhesion, which was tested in cells from patients with chronic thromboembolic pulmonary hypertension (CTEPH) and three animal models in vivo. Measurements and Main Results: The protein NEDD9 was identified in the hypoxia thrombosome network in silico. Compared with normoxia, hypoxia (0.2% O2) for 24 hours increased HIF-1α (hypoxia-inducible factor-1α)-dependent NEDD9 upregulation in vitro. Increased NEDD9 was localized to the plasma-membrane surface of cells from control donors and patients with CTEPH. In endarterectomy specimens, NEDD9 colocalized with the platelet surface adhesion molecule P-selectin. Our custom-made anti-NEDD9 Ab targeted the NEDD9-P-selectin interaction and inhibited the adhesion of activated platelets to pulmonary artery endothelial cells from control donors in vitro and from patients with CTEPH ex vivo. Compared with control mice, platelet-pulmonary endothelial aggregates and pulmonary hypertension induced by ADP were decreased in NEDD9-/- mice or wild-type mice treated with the anti-NEDD9 Ab, which also decreased chronic pulmonary thromboembolic remodeling in vivo. Conclusions: The NEDD9-P-selectin protein-protein interaction is a modifiable target with which to inhibit platelet-pulmonary endothelial adhesion and thromboembolic vascular remodeling, with potential therapeutic implications for patients with disorders of increased hypoxia signaling pathways, including CTEPH.

CD160 inhibits activation of human CD4+ T cells through interaction with herpesvirus entry mediator.

CD160, a glycosylphosphatidylinositol-anchored member of the immunoglobulin superfamily, is expressed on both cytolytic lymphocytes and some unstimulated CD4+ T cells. Here we show that CD160 expression was increased after activation of human CD4+ T cells and that crosslinking CD160 with monoclonal antibody strongly inhibited CD3- and CD28-mediated activation. We found that herpesvirus entry mediator (HVEM) was a ligand of CD160 that acted as a 'bidirectional switch' for T cell activation, producing a positive or negative outcome depending on the engagement of HVEM by CD160 and known HVEM ligands such as B and T lymphocyte attenuator (BTLA) and the T lymphocyte receptor LIGHT. Inhibition of CD4+ T cell activation by HVEM-transfected cells was dependent on CD160 and BTLA; when the cysteine-rich domain 1 of HVEM was deleted, this inhibition was lost, resulting in strong T cell activation. CD160 thus serves as a negative regulator of CD4+ T cell activation through its interaction with HVEM.

Cross-reactivity of the BRAF VE1 antibody with epitopes in axonemal dyneins leads to staining of cilia.

Antibodies that recognize neo-epitopes in tumor cells are valuable tools in the evaluation of tissue biopsy or resection specimens. The VE1 antibody that recognizes the V600E-mutant BRAF protein is one such example. We have recently shown that the vast majority of papillary craniopharyngiomas-tumors that arise in the sellar or suprasellar regions of the brain-harbor BRAF V600E mutations. The VE1 antibody can be effective in discriminating papillary craniopharyngioma from adamantinomatous craniopharyngioma, which harbors mutations in CTNNB1 and not BRAF. While further characterizing the use of the VE1 antibody in the differential diagnosis of suprasellar lesions, we found that the VE1 antibody stains the epithelial cells lining Rathke's cleft cysts with very strong staining of the cilia of these cells. We used targeted sequencing to show that Rathke's cleft cysts do not harbor the BRAF V600E mutation. Moreover, we found that the VE1 antibody reacts strongly with cilia in various structures-the bronchial airways, the fallopian tubes, the nasopharynx, and the epididymis-as well as with the flagella of sperm. In addition, VE1 reacts strongly with the cilia of the ependymal lining of the brain and with the cilia-containing microlumens of ependymoma tumors. There is significant sequence homology between the synthetic peptide (amino acid 596-606 of BRAF V600E: GLATEKSRWSG) that was used to generate the VE1 antibody and regions of multiple axonemal dynein heavy chain proteins (eg, DNAH2, DNAH7, and DNAH12). These proteins are major components of the axonemes of cilia and flagella where they drive the sliding of microtubules. In ELISA assays, we show that the VE1 antibody recognizes epitopes from these proteins. A familiarity with the cross-reactivity of the VE1 antibody with epitopes of proteins in cilia is of value when evaluating tissues stained with this important clinical antibody.

Generating Monoclonal Antibodies.

Antibodies that are produced by hybridomas are known as monoclonal antibodies. Here we introduce methods for generating and screening monoclonal antibodies, including developing the screening procedure and producing hybridomas.

Determining the Class and Subclass of Monoclonal Antibodies Using Antibody Capture on Anti-Immunoglobulin Antibody-Coated Plates.

By definition, a monoclonal antibody should only be of a single class or subclass. Each class of antibody is associated with specific functions, and it can be useful to know the class/subclass of the monoclonal antibody produced by a specific hybridoma. In this protocol, class/subclass-specific antibodies are used to capture the monoclonal antibody from hybridoma supernatant. If the antibody is clonal, it will only be bound by one anti-heavy-chain capture antibody and one anti-light-chain antibody. No antigen is required.

Hybridoma Screening by Antigen Capture: Capture or Sandwich ELISA in 96-Well Plates.

In an antigen capture assay for hybridoma screening, the detection method identifies the presence of the antigen. Often this is achieved by labeling the antigen directly. In this assay, the polyvinyl chloride (PVC) wells of a high-binding-capacity ELISA plate are first coated with an affinity-purified rabbit anti-mouse immunoglobulin and then incubated with hybridoma tissue culture supernatant. Monoclonal antibodies in the supernatant are "captured" on the coated PVC surface and detected by screening with biotin- or histidine (His)-tagged antigen. The antigen can be labeled to a high specific activity and thus very little antigen is required for this procedure.

Hybridoma Screening by Antigen Capture: Reverse Dot Blot.

A dot blot is widely used to determine the productivity of a given hybridoma. This assay can also be used to screen a fusion or subclone plate for productive hybridoma clones. First, a nitrocellulose membrane is coated with an affinity-purified goat or rabbit anti-mouse immunoglobulin and then incubated with hybridoma tissue culture supernatant. Monoclonal antibodies in the supernatant are then "captured" on the coated nitrocellulose membrane surface and detected by screening with horseradish peroxidase (HRP).

Hybridoma Screening by Antigen Capture: Immunoprecipitation.

Immunoprecipitation is rarely used for screening hybridoma fusions because the assays are tedious and time-consuming. However, it can be useful when working with complex antigens because the precipitated antigen is normally detected after sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis and thus it is simple to discriminate between true and false positives. Furthermore, the assay provides information regarding the molecular weight of the antigen.

Hybridoma Screening by Antibody Capture: Detection of Cell-Surface Binding by Immunofluorescence.

If the antigen of interest is a cell-surface protein, immunofluorescence can be used to identify hybridomas secreting monoclonal antibodies to these proteins. They can be stained on microscope chamber slides, flat-bottomed cell culture plates (96, 48, or 24 well), or imaging plates (96 well). This protocol uses 96-well imaging plates.

Hybridoma Screening by Antibody Capture: Detection of Intracellular Binding by Immunofluorescence.

Hybridoma screening by immunofluorescence stainings of whole cells can be adapted to screen for antibodies to internal proteins by permeabilizing the cells before applying the hybridoma supernatants. In this protocol, cells are attached to a solid support (glass slides), which makes them easy to manipulate and transfer between different reagent solutions.

Immunizing Animals.

The traditional method for generating polyclonal and monoclonal antibodies requires the immunization of an animal. Selecting the best species of animal and getting that animal's immune system to respond to a target antigen with an antibody response are essential to obtaining good-quality antibodies and hybridomas. There are only a limited number of opportunities for a researcher to intervene to manipulate and tailor the response to a particular antigen. Here we present advice and methods for designing the way in which the antigen is presented to the immune system (i.e., the immunization protocol), including the choice of animal, the antigen dose, the use of adjuvants, the route and number of injections, and the period between injections.

Hybridoma Screening by Antibody Capture: Flow Cytometry/FACS with Permeabilized Cells to Detect Intracellular Binding.

Flow cytometry or fluorescence-activated cell sorting (FACS) can be used to identify hybridomas secreting monoclonal antibodies to internal cellular proteins, but the cells must be permeabilized before the hybridoma supernatants are applied. In using this technique, useful controls are positive and negative cell lines with primary and secondary antibodies as well as positive and negative cell lines with secondary antibody alone.

Hybridoma Screening by Antibody Capture: Flow Cytometry/FACS with Whole Cells to Detect Cell-Surface Binding.

If the antigen of interest is a cell-surface protein, flow cytometry or fluorescence-activated cell sorting (FACS) can be used to identify hybridomas secreting monoclonal antibodies to these proteins. Two alternative protocols are presented here-staining in individual tubes and staining in 96-well plates.

Determining the Class and Subclass of a Monoclonal Antibody by Ouchterlony Double-Diffusion Assays.

Originally, the Ouchterlony double-diffusion assays were the most common method for determining the class and subclass of a monoclonal antibody, and they still are useful, particularly when only a few assays will be performed. A sample of hybridoma tissue culture supernatant is placed in a well in a bed of agar, and class- and subclass-specific antisera are placed in other wells in a ring surrounding the test antibody. As the antibodies diffuse into the agar, they meet and multimeric immune complexes precipitate to form a visible "precipitin line."

Hybridoma Screening by Antibody Capture: Enzyme-Linked Detection (Indirect ELISA) in Polyvinyl Chloride Wells.

In this antibody capture assay for hybridoma screening, the antigen is immobilized on a solid substrate (the surface of the wells in a polyvinyl chloride [PVC] microtiter plate), and antibodies in the hybridoma tissue culture supernatant are incubated with the antigen. Unbound antibodies are removed by washing, and antibody-antigen complexes are detected by secondary antibody conjugated to alkaline phosphatase (AP), which catalyzes the conversion of a chromogenic substrate to a blue/green product. Alternative secondary antibodies are necessary for experiments in which immunoglobulin-fusion proteins have been used as immunogens or screening proteins.

Hybridoma Screening by Antibody Capture: Immunohistochemistry.

To determine the subcellular location of an antigen, hybridoma tissue culture supernatants can be screened using immunohistochemistry. For antibodies to have access to antigens in fixed and embedded tissue sections, the paraffin must be removed and the tissue must be rehydrated and digested before immunohistochemical staining.

Determining the Class and Subclass of Monoclonal Antibodies Using Antibody Capture on Antigen-Coated Plates.

The class or subclass of an antibody is defined by its heavy chain. There are five main classes of antibodies: M, G, A, E, and D. By definition, a monoclonal antibody should only be of a single class or subclass. Each class of antibody is associated with specific functions. The method of antibody purification will differ based on the class. In this protocol, antigen is used to capture antibodies reactive to it. Specific class and/or subclass secondary antibodies are then used to discern which class/subclass has been captured.

Antigen-specific regulatory T cells develop via the ICOS-ICOS-ligand pathway and inhibit allergen-induced airway hyperreactivity.

Asthma is caused by T-helper cell 2 (Th2)-driven immune responses, but the immunological mechanisms that protect against asthma development are poorly understood. T-cell tolerance, induced by respiratory exposure to allergen, can inhibit the development of airway hyperreactivity (AHR), a cardinal feature of asthma, and we show here that regulatory T (T(R)) cells can mediate this protective effect. Mature pulmonary dendritic cells in the bronchial lymph nodes of mice exposed to respiratory allergen induced the development of T(R) cells, in a process that required T-cell costimulation via the inducible costimulator (ICOS-ICOS-ligand pathway. The T(R) cells produced IL-10, and had potent inhibitory activity; when adoptively transferred into sensitized mice, T(R) cells blocked the development of AHR. Both the development and the inhibitory function of regulatory cells were dependent on the presence of IL-10 and on ICOS-ICOS-ligand interactions. These studies demonstrate that T(R) cells and the ICOS-ICOS-ligand signaling pathway are critically involved in respiratory tolerance and in downregulating pulmonary inflammation in asthma.

Induction of Ascites in Mice.

Smaller animals such as rats, mice, hamsters, and guinea pigs are usually poor choices for polyclonal antibody production because only small volumes of serum can be obtained. This problem can be reduced by inducing the formation of ascites in mice, which can provide up to 10 mL of ascites fluid from a single animal. Antibody titers in ascites fluids are almost as high as serum titers.

Inducing, Collecting, and Storing Ascites.

Ascitic fluid (also called ascites) is an intraperitoneal fluid extracted from mice that have developed a peritoneal tumor. For antibody production, the tumor is induced by injecting hybridoma cells into the peritoneum, which serves as a growth chamber for the cells. The hybridoma cells grow to high densities and continue to secrete the antibody of interest, thus creating a high-titered solution of antibodies for collection. A single mouse may yield as much as 10 mL of ascitic fluid or as little as 1 mL per batch. Antibody concentrations will typically be between 1 and 10 mg/mL. The most common problem encountered in storing ascites is contamination of these solutions with bacteria or fungi. This can be prevented by the addition of sodium azide.