Hybridoma Lines Research Articles

Assessing the Affinity Spectrum of the Antigen-Specific B Cell Repertoire via ImmunoSpot®.

The affinity distribution of the antigen-specific memory Bcell (Bmem) repertoire in the body is a critical variable that defines an individual's ability to rapidly generate high-affinity protective antibody specificities. Detailed measurement of antibody affinity so far has largely been confined to studies of monoclonal antibodies (mAbs) and are laborious since each individual mAb needs to be evaluated in isolation. Here, we introduce two variants of the Bcell ImmunoSpot® assay that are suitable for simultaneously assessing the affinity distribution of hundreds of individual B cells within a test sample at single-cell resolution using relatively little labor and with high-throughput capacity. First, we experimentally validated that both ImmunoSpot® assay variants are suitable for establishing functional affinity hierarchies using Bcell hybridoma lines as model antibody-secreting cells (ASC), each producing mAb with known affinity for a defined antigen. We then leveraged both ImmunoSpot® variants for characterizing the affinity distribution of SARS-CoV-2 Spike-specific ASC in PBMC following COVID-19 mRNA vaccination. Such ImmunoSpot® assays promise to offer tremendous value for future Bcell immune monitoring efforts, owing to their ease of implementation, applicability to essentially any antigenic system, economy of PBMC utilization, high-throughput capacity, and suitability for regulated testing.

NIH/3T3 Fibroblasts Selectively Activate T Cells Specific for Posttranslationally Modified Collagen Type II.

It has been shown that synovial fibroblasts (SF) play a key role in the initiation of inflammation and joint destruction, leading to arthritis progression. Fibroblasts may express major histocompatibility complex class II region (MHCII) molecules, and thus, they could be able to process and present antigens to immunocompetent cells. Here we examine whether different types of fibroblasts (synovial, dermal, and thymic murine fibroblasts, destructive LS48 fibroblasts, and noninvasive NIH/3T3 fibroblasts) may be involved in the initiation of rheumatoid arthritis (RA) pathogenesis and can process and present type II collagen (COL2)-an autoantigen associated with RA. Using a panel of MHCII/Aq-restricted T-cell hybridoma lines that specifically recognize an immunodominant COL2 epitope (COL2259-273), we found that NIH/3T3 fibroblasts activate several T-cell clones that recognize the posttranslationally glycosylated or hydroxylated COL2259-273 epitope. The HCQ.3 hybridoma, which is specific for the glycosylated immunodominant COL2 epitope 259-273 (Gal264), showed the strongest response. Interestingly, NIH/3T3 cells, but not destructive LS48 fibroblasts, synovial, dermal, or thymic fibroblasts, were able to stimulate the HCQ.3 hybridoma and other COL2-specific T-cell hybridomas. Our experiments revealed that NIH/3T3 fibroblasts are able to activate COL2-specific T-cell hybridomas even in the absence of COL2 or a posttranslationally modified COL2 peptide. The mechanism of this unusual activation is contact-dependent and involves the T-cell receptor (TCR) complex.

CRISPR/Cas9-based Engineering of Immunoglobulin Loci in Hybridoma Cells.

Development of the hybridoma technology by Köhler and Milstein (1975) has revolutionized the immunological field by enabling routine use of monoclonal antibodies (mAbs) in research and development efforts, resulting in their successful application in the clinic today. While recombinant good manufacturing practices production technologies are required to produce clinical grade mAbs, academic laboratories and biotechnology companies still rely on the original hybridoma lines to stably and effortlessly produce high antibody yields at a modest price. In our own work, we were confronted with a major issue when using hybridoma-derived mAbs: there was no control over the antibody format that was produced, a flexibility that recombinant production does allow. We set out to remove this hurdle by genetically engineering antibodies directly in the immunoglobulin (Ig) locus of hybridoma cells. We used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) and homology-directed repair (HDR) to modify antibody's format [mAb or antigen-binding fragment (Fab')] and isotype. This protocol describes a straightforward approach, with little hands-on time, leading to stable cell lines secreting high levels of engineered antibodies. Parental hybridoma cells are maintained in culture, transfected with a guide RNA (gRNA) targeting the site of interest in the Ig locus and an HDR template to knock in the desired insert and an antibiotic resistance gene. By applying antibiotic pressure, resistant clones are expanded and characterized at the genetic and protein level for their ability to produce modified mAbs instead of the parental protein. Finally, the modified antibody is characterized in functional assays. To demonstrate the versatility of our strategy, we illustrate this protocol with examples where we have (i) exchanged the constant heavy region of the antibody, creating chimeric mAb of a novel isotype, (ii) truncated the antibody to create an antigenic peptide-fused Fab' fragment to produce a dendritic cell-targeted vaccine, and (iii) modified both the constant heavy (CH)1 domain of the heavy chain (HC) and the constant kappa (Cκ) light chain (LC) to introduce site-selective modification tags for further derivatization of the purified protein. Only standard laboratory equipment is required, which facilitates its application across various labs. We hope that this protocol will further disseminate our technology and help other researchers. Graphical abstract.

Autoantigen specific T-cell receptor induces organ-specific autoimmunity by escaping T cell negative selection

Abstract Previously we reported a new model of spontaneous autoimmune uveitis in which mice with a hypomorphic mutation of Aire (AireGW/+) are combined with deletion of Lyn. The key autoantigen in this model is interphotoreceptor retinoid-binding protein (IRBP) as AireGW/+Lyn−/−IRBP−/− mice failed to develop uveitis. We performed single-cell RNA-seq with TCR sequencing for IRBP 271–290 (P2)-specific CD4+ T cells from eye-draining lymph nodes (LN) of these mice with and without uveitis. Both mice showed an expansion of these T cells in eye-draining LNs, but it was greater in mice with disease. A remarkable clonal diversity of TCRs was seen. Expansions of particular T cell clonotypes were more evident in LN of mice with disease, and even greater in the retinas of mice with disease. We reconstituted several P2–specific TCR clonotypes in a T cell hybridoma line and verified their specificity. One of the TCRs was expressed in thymocytes by retroviral transduction of bone marrow stem cells from Rag2−/− mice and transplanted into WT or AireGW/+ recipient mice. Remarkably, the small residual expression of IRBP in the thymus of AireGW/+ mice was sufficient to induce substantial negative selection of thymocytes with this TCR. In addition, we generated TCR transgenic mice with this TCR clonotype. The TCR transgenic mice spontaneously developed uveitis with expansion of P2-specific CD4+ T cells. Combined with AireGW/+ or IRBP−/−, there were more P2-specific CD4+ T cells in the thymus of TCR transgenic mice. Thus, suboptimal Aire function in AireGW/+Lyn−/− mice results in a partial defect in negative selection of T cells recognizing IRBP in the thymus, promoting development of uveitis. This disease is accelerated in mice expressing a TCR transgene recognizing IRBP. Supported by AI138479

Three sensitive and reliable serological assays for detection of potato virus A in potato plants

Vegetative propagation of seed potato often allows passaging of viruses to seed tubers, resulting in significant yield losses and reduction of potato tuber quality. Thus, virus detection approach is crucial for effective virus management programs and the production of virus-free seed potatoes. Among the reported potato-infecting viruses, potato virus A (PVA) is considered as one of the most important viruses in potato-growing regions worldwide. This study prepared four hybridoma lines secreting PVA-specific monoclonal antibodies (MAbs) (2D4, 8E11, 14A6 and 16H10) using purified PVA virions as an immunogen. Western blotting results indicated that all the four MAbs reacted strongly and specifically with the putative capsid protein of PVA. Using these four MAbs, this study developed antigen-coated plate enzyme-linked immunosorbent assay (ACP-ELISA), Dot-ELISA and Tissue print-ELISA for detection of PVA infection in potato plants. The results indicated that PVA can be detected in crude tissue extracts from infected potato plants diluted up to 1:327680 (w/v, g mL–1) by ACP-ELISA or up to 1:10240 by Dot-ELISA. The Tissue print-ELISA is the quickest and easiest approach among the three serological assays, and is more suitable for onsite large-scale potato screening programs. Further analyses of field-collected potato samples showed that the sensitivities and specificities of the three serological approaches were similar to those of RT-PCR in PVA detection and confirmed that PVA is currently widespread in Yunnan and Zhejiang provinces of China. Hence, the results strongly suggest that these highly sensitive serological approaches based on PVA-specific MAbs are useful and powerful for PVA-free seed potato production programs and PVA field surveys.

Anti-insulin B cell receptors display skewed immunoglobulin gene usage and limited mutation in pre-symptomatic type 1 diabetes donors

Abstract Antibodies targeting insulin and other islet antigens predict type 1 diabetes (T1D) onset. Insulin autoantibody (IAA)-positive individuals progress to diabetes more rapidly, yet little is known about how IAA-precursor anti-insulin B cells (AIBCs) recognize insulin. Complementarity-determining regions (CDRs) of the B cell receptor (BCR) are structurally diverse loops that constitute the antigen-binding site, and amino acid variability in BCRs increases the breadth of repertoire antigen-specificities. To investigate anti-insulin BCRs during the earliest detectable stages of T1D, we enrolled eight insulin therapy-naïve Type 1 Diabetes TrialNet Pathway to Prevention participants who were positive for ≥2 islet autoantibodies and thus at high risk for diabetes. We stimulated donor peripheral blood mononuclear cells to drive BCR secretion as antibody, screened for AIBC-containing wells by ELISA, and immortalized these cells as hybridomas. We validated 25 monoclonal anti-insulin hybridoma lines and sequenced 16 heavy chain (IgH) and 14 light chain (IgL) immunoglobulin genes. 25% of VH and 50% of VL sequences contained at least one mutation within a CDR compared to germline, with 13% of VH and 29% of VL sequences having ≥6 mutations within the V-region. We surveyed previously and currently reported AIBC VH (n=25 BCRs), which revealed skewed VH and JH gene use compared to the total polyclonal IgH repertoire (n= 8790 BCRs, p < 0.001 and p < 0.05, respectively, chi-squared test). These data expose unique features of insulin recognition among AIBCs isolated from pre-symptomatic T1D donors, including V and J gene usage bias and potential for germline IgH insulin recognition, providing genetic and molecular insight into AIBC origins.

T-cell receptor repertoire of mice with organ-specific autoimmunity resulting from a partial defect in T cell negative selection and dendritic cell enhancement

Abstract Previously, we reported a new genetic model of spontaneous autoimmune disease in which mice with a hypomorphic mutation of Aire (AireGW/+) combined with deletion of Lyn in dendritic cells develop autoimmune uveitis with a 50% penetrance. Here we demonstrate that a key autoantigen in this model is interphotoreceptor retinoid-binding protein (IRBP) as AireGW/+ Lyn−/− IRBP−/− mice failed to develop uveitis. The expansion of CD4+ T cells recognizing amino acids 271–290 (“P2”) of IRBP correlated strongly with autoimmune disease in mice with intact IRBP. To study the role of these T cells, we did single-cell RNA-seq with TCR sequencing for P2-specific CD4+ T cells isolated from eye-draining lymph nodes (LN) of AireGW/+ Lyn−/− mice with and without uveitis. Both mice showed an expansion of these T cells in eye-draining LNs, but it was greater in mice with disease. A remarkable clonal diversity of TCRs was seen, with some sharing of TCR Vα or Vβ sequences. Expansions of particular T cell clonotypes were more evident in LN of mice with disease, and even greater in the retinas of mice with disease. We reconstituted several P2–specific TCR clonotypes in a T cell hybridoma line and verified their specificity. One of the TCR clonotypes was expressed in developing thymocytes by retroviral transduction of bone marrow stem cells from Rag2−/− mice and transplanted into WT or AireGW/+ recipient mice. Remarkably, the small residual expression of IRBP in the thymus of AireGW/+ mice was sufficient to induce substantial negative selection of thymocytes with this TCR clonotype. Thus, deficiency of negative selection of P2 tetramer–specific TCR promotes the development of uveitis in AireGW/+ Lyn−/− mice, even if negative selection is only partly compromised.

Identification of a Novel RAMA/RON3 Rhoptry Protein Complex in Plasmodium falciparum Merozoites.

Malaria causes a half a million deaths annually. The parasite intraerythrocytic lifecycle in the human bloodstream is the major cause of morbidity and mortality. Apical organelles of merozoite stage parasites are involved in the invasion of erythrocytes. A limited number of apical organellar proteins have been identified and characterized for their roles during erythrocyte invasion or subsequent intraerythrocytic parasite development. To expand the repertoire of identified apical organellar proteins we generated a panel of monoclonal antibodies against Plasmodium falciparum schizont-rich parasites and screened the antibodies using immunofluorescence assays. Out of 164 hybridoma lines, 12 clones produced monoclonal antibodies yielding punctate immunofluorescence staining patterns in individual merozoites in late schizonts, suggesting recognition of merozoite apical organelles. Five of the monoclonal antibodies were used to immuno-affinity purify their target antigens and these antigens were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Two known apical organelle protein complexes were identified, the high-molecular mass rhoptry protein complex (PfRhopH1/Clags, PfRhopH2, and PfRhopH3) and the low-molecular mass rhoptry protein complex (rhoptry-associated proteins complex, PfRAP1, and PfRAP2). A novel complex was additionally identified by immunoprecipitation, composed of rhoptry-associated membrane antigen (PfRAMA) and rhoptry neck protein 3 (PfRON3) of P. falciparum. We further identified a region spanning amino acids Q221-E481 within the PfRAMA that may associate with PfRON3 in immature schizonts. Further investigation will be required as to whether PfRAMA and PfRON3 interact directly or indirectly.

Pine wood nematode (PWN)-secretory antigen 571 as a biomarker for the PWN and its monoclonal antibodies for detecting PWN and its infected pine tree

• PWN-SA571 is a biomarker for PWN. • Specific Mabs for PWN-SA571 were generated. • PWN-SA571 Mabs could detect PWN and PWNIPT by ELISA and dot blots. • PWN-SA571 Mabs could detect antigens by LFA. The biochemical characteristics of pine wood nematode (PWN)-secretory antigen 571 (PWN-SA571) identified from the PWN secretome and PWN-infected pine tree extracts (PWNIPT) were investigated. PWN-SA571 has homologs in various nematodes in diverse families and a signal peptide at the amino-terminal end. Since it was not possible to express the full-length PWN-SA571 protein, two peptides showing high antigenicity were synthesized and used as antigens for generating monoclonal antibodies (Mabs). Mab-secreting fusion hybridoma cell lines were selected based on immunoreactivity to free peptide and BSA-conjugated peptide (BSA pep) antigens by enzyme-linked immunosorbent assay for establishing Mab-secreting hybridoma lines. Ascites containing PWN-SA571-specific Mabs showed stronger immunoreactivity to PWNIPT than to healthy pine tree extracts. In addition, PWN-SA571-specific Mabs could recognize less than 20 ng of BSA pep in lateral flow assays. Furthermore, purified biotin-conjugated PWN-SA571-specific Mab IgG or IgM were able to detect BSA pep (<15 ng) and PWN extracts (<600 ng). Taken together, these results demonstrate that PWN-SA571-specific Mabs could detect PWN or PWNIPT extracts by ELISA, LFA, or dot blot analysis.

Recombinant Factor VIII Fc Inhibits B Cell Activation via Engagement of the FcγRIIB Receptor.

The development of neutralizing antibodies (inhibitors) against factor VIII (FVIII) is a major complication of hemophilia A treatment. The sole clinical therapy to restore FVIII tolerance in patients with inhibitors remains immune tolerance induction (ITI) which is expensive, difficult to administer and not always successful. Although not fully understood, the mechanism of ITI is thought to rely on inhibition of FVIII-specific B cells (1). Its efficacy might therefore be improved through more aggressive B cell suppression. FcγRIIB is an inhibitory Fc receptor that down-regulates B cell signaling when cross-linked with the B cell receptor (BCR). We sought to investigate if recombinant FVIII Fc (rFVIIIFc), an Fc fusion molecule composed of FVIII and the Fc region of immunoglobulin G1 (IgG1) (2), is able to inhibit B cell activation more readily than FVIII. rFVIIIFc was able to bind FVIII-exposed and naïve B cells from hemophilia A mice as well as a FVIII-specific murine B cell hybridoma line (413 cells). An anti-FcγRIIB antibody and FVIII inhibited binding, suggesting that rFVIIIFc is able to interact with both FcγRIIB and the BCR. Furthermore, incubation of B cells from FVIII-exposed mice and 413 cells with rFVIIIFc resulted in increased phosphorylation of SH-2 containing inositol 5-phosphatase (SHIP) when compared to FVIII. B cells from FVIII-exposed hemophilia A mice also exhibited decreased extracellular signal-regulated kinase (ERK) phosphorylation when exposed to rFVIIIFc. These differences were absent in B cells from naïve, non-FVIII exposed hemophilic mice suggesting an antigen-dependent effect. Finally, rFVIIIFc was able to inhibit B cell calcium flux induced by anti-Ig F(ab)2. Our results therefore indicate that rFVIIIFc is able to crosslink FcγRIIB and the BCR of FVIII-specific B cells, causing inhibitory signaling in these cells.

Isotyping antibodies produced from hybridoma A6G11C9

Hybridoma technology was discovered in 1975 to produce the monoclonal antibodies. By this way, we can produce a desired antibody in large amounts. From a single hybrid cell line, they grow and develop to produce a monoclonal antibody with large enough quantities to use for the research, treatment and diagnosis. The level of the antibody producing is depended on the cell density and the incubation period. The growth capacity of each hybrid cell line depends on the composition of the substances in the culture medium of animal cells. Among them, fetal bovine serum is the most commonly used serum-supplement for the in vitro cell culture. This is due to it having a very low level of antibodies and containing more growth factors. In the previous studies, the hybrid cell line (designed A6G11C9) was the best one secreting the highest anti-A monoclonal antibodies, whose specificity agglutinaned human red blood cells containing A antigen. This report showed the result of the study on the coditional culture and isotyping of the immunoglobulin. The hybrid cell line A6G11C9 was cultured in the DMEM medium with different level of fetal bovine serum. As the results, this hybridoma line grow in the DMEM medium containing 10% fetal bovine serum is five times faster than in the DMEM medium with 1% fetal bovine serum. The maximum of the cell density are 11.106 cells/ml after 50 hours innoculation. The maximum of the titer from culture supernatant are 1/1024 after 100 hours innoculation. The monoclonal antibodies derived from hybrid cell line A6G11C9 are IgM heavy chain and the kappa light chain.

Study on using the hybrid cell a6g11c9 to produce the anti-a monoclonal antibody that agglunating a antigen on the surface of red blood cells

Almost of the ABO blood grouping reagents is being trading derive from the monoclonal antibodies. There are two methods to produce the monoclonal antibodies from hybridoma lines, which were in vitro method (hybridoma cultured in the medium) and in vivo method (hybridoma cultured in the mice intra-abdominal). In Vietnam, Nguyen Thi Trung and co-authors was succesfully screened in hybridoma cell line A6G11C9 which generating of the anti A monoclonal antibody agglutinated A antigen on the surface of red blood cells. The fusion of mouse lymohocyte B generated anti-A antibody with mouse myeloma sp2/0 is formed that hybrid cell lines. The anti-A monoclonal antibody is produced from hybridoma cell line A6G11C9 have been highly intensive confirmed. It is capability of growth and anti B monoclonal antibody producing stability through the generations. In this study, the process to produce large amounts of monoclonal antibodies from B4D10C9 hybridoma by in vitro method are published. Firstly, hybridoma cells are stored in liquid nitrogen to wake by culture in medium. Then, First, hybrid cells are stored frozen in liquid nitrogen to wake cultured cells. Then, they were first inoculated to produce enough biomass to serve a larger scale. Cell biomass continues to be second inoculated into DMEM containing 10% fetal bovin serum for 10 days. The culture medium contained anti-A monoclonal antibodies were collected by centrifugation to remove cells. The anti-A monoclonal antibody levels in culture medium was concentrated and remove phenol red indicator by the precipitation with NH4SO4 50% saturated. The anti-A monoclonal antibody solution at 5 times concentrated have been better agglutinated with erythrocytes containing A antigen than monoclonal antibody solution non-concentration. 150 ml of concentrated antibodies were produced. Antibody titer of the anti-A monoclonal antibodies in the concentrated 5 times solution was 1/512. The intensity of the reaction anti-A monclonal antibody with red blood cell containing A antigen was 4+. Citation: Nguyen Thi Trung, Truong Nam Hai, 2018. Study on using the hybrid cell a6g11c9 to produce the anti-a monoclonal antibody that agglunating a antigen on the surface of red blood cells. Tap chi Sinh hoc, 40(1): x-xx. DOI: 10.15625/0866-7160/v40n1.9154. *Corresponding author: tnhai@ibt.ac.vn Received 12 January 2017, accepted 20 December 2017

ExpansinB3 as a marker for detecting pine wood nematode-infected pine trees

To develop a method for recognizing pine trees infected with pine wood nematodes (PWN), Bursaphelenchus xylophilus, we have constructed a monoclonal antibody (Mab) library for Expansin B3 (EXPB3), which is one of the known proteins that is secreted by PWN. We conducted a primary screening by using the EXPB3 peptides and selected 96 fusion cell lines with a high immunoreactivity. A secondary screening was performed using the PBS extracts of the PWN-infected pine trees (PWNIPTs). Thirteen clones, including 9 clones with high immunoreactivities, were selected, and limited dilutions were performed to establish Mab-secreting hybridoma lines. A total of 74 PWN infected pine trees collected from 9 PWN-affected areas in the Republic of Korea were analyzed by using EXPB3-Mab-3-1-H3-C11-E4 (EXPB3-C11-E4). EXPB3-C11-E4 recognized all of the infected pine trees with some variations. This Mab demonstrated a significantly higher reactivity to the black pines when compared to the red pines. This Mab recognized both PWN-infected red and black pine trees collected from different regions, but were able to displayed regional differences with the black pine trees. In addition, the EXPB3-C11-E4 immunoreactivity in black pine trees had a significant correlation regarding the degree of PWN infection and the perimeters of the trees. The results of this study reveal that EXPB3, an antigen secreted by PWN, and its specific Mabs may be used for the diagnosis of PWNIPTs.

A Humanized Single Chain Fragment Variable Constructed to Use in Breast Cancer Immunotherapy

The mouse B cell hybridoma line C3A8 was the starting material to generate monoclonal antibody to react specifically toward epidermal growth factor receptors (EGFR) of breast cancer cell line (MCF‐7). Phage display technologies were applied to produce molecular form of those IgG antibodies. The integrated cloning, screening, and phage display system allowed to use extended primers, which were incorporate the entire mouse VH and VL and their linker sequences collected in the Kabat database. The product was called single‐chain fragment variable (scFv) antibodies. The best candidate scFv sequences, based on preliminary enzyme‐linked immunosorbent assay (ELISA) screening data gave 100% identical sequences in genbank data base, while a band of 68kD was appeared in nitrocellose membrane in western blot test had used to characterize the specific reaction of scFv towards MCF‐7 breast cancer cells. Further, immuno‐fluorescence test clearly proved that the scFv recognized the MCF‐7 antigen epitopes which is localized in MCF‐7 nuclear. Also, 99% of the cells numbers were bind to scFv protein as measured by flow cytometry analysis. Herein the recombinant antibody technology is a rapid and effective approach to the development of the next generation of breast cancer diagnosis and immune‐therapy.

Metabolic requirements for cancer cell proliferation

BackgroundThe study of cancer metabolism has been largely dedicated to exploring the hypothesis that oncogenic transformation rewires cellular metabolism to sustain elevated rates of growth and division. Intense examination of tumors and cancer cell lines has confirmed that many cancer-associated metabolic phenotypes allow robust growth and survival; however, little attention has been given to explicitly identifying the biochemical requirements for cell proliferation in a rigorous manner in the context of cancer metabolism.ResultsUsing a well-studied hybridoma line as a model, we comprehensively and quantitatively enumerate the metabolic requirements for generating new biomass in mammalian cells; this indicated a large biosynthetic requirement for ATP, NADPH, NAD+, acetyl-CoA, and amino acids. Extension of this approach to serine/glycine and glutamine metabolic pathways suggested lower limits on serine and glycine catabolism to supply one-carbon unit synthesis and significant availability of glutamine-derived carbon for biosynthesis resulting from nitrogen demands alone, respectively. We integrated our biomass composition results into a flux balance analysis model, placing upper bounds on mitochondrial NADH oxidation to simulate metformin treatment; these simulations reproduced several empirically observed metabolic phenotypes, including increased reductive isocitrate dehydrogenase flux.ConclusionsOur analysis clarifies the differential needs for central carbon metabolism precursors, glutamine-derived nitrogen, and cofactors such as ATP, NADPH, and NAD+, while also providing justification for various extracellular nutrient uptake behaviors observed in tumors. Collectively, these results demonstrate how stoichiometric considerations alone can successfully predict empirically observed phenotypes and provide insight into biochemical dynamics that underlie responses to metabolic perturbations.Electronic supplementary materialThe online version of this article (doi:10.1186/s40170-016-0156-6) contains supplementary material, which is available to authorized users.

Transnuclear CD8 T cells specific for the immunodominant epitope Gra6 lower acute-phase Toxoplasma gondii burden.

SummaryWe generated a CD8 T‐cell receptor (TCR) transnuclear (TN) mouse specific to the Ld‐restricted immunodominant epitope of GRA6 from Toxoplasma gondii as a source of cells to facilitate further investigation into the CD8 T‐cell‐mediated response against this pathogen. The TN T cells bound Ld‐Gra6 tetramer and proliferated upon unspecific and peptide‐specific stimulation. The TCR beta sequence of the Gra6‐specific TN CD8 T cells is identical in its V‐ and J‐region to the TCR‐β harboured by a hybridoma line generated in response to Gra6 peptide. Adoptively transferred Gra6 TN CD8 T cells proliferated upon Toxoplasma infection in vivo and exhibited an activated phenotype similar to host CD8 T cells specific to Gra6. The brain of Toxoplasma‐infected mice carried Gra6 TN cells already at day 8 post‐infection. Both Gra6 TN mice as well as adoptively transferred Gra6 TN cells were able to significantly reduce the parasite burden in the acute phase of Toxoplasma infection. Overall, the Gra6 TN mouse represents a functional tool to study the protective and immunodominant specific CD8 T‐cell response to Toxoplasma in both the acute and the chronic phases of infection.

Non-covalent pomegranate (Punica granatum) hydrolyzable tannin-protein complexes modulate antigen uptake, processing and presentation by a T-cell hybridoma line co-cultured with murine peritoneal macrophages

In this work we characterize the interaction of pomegranate hydrolyzable tannins (HT) with hen egg-white lysozyme (HEL) and determine the effects of non-covalent tannin-protein complexes on macrophage endocytosis, processing and presentation of antigen. We isolated HT from pomegranate and complex to HEL, the resulting non-covalent tannin-protein complex was characterized by gel electrophoresis and MALDI-TOF MS. Finally, cell culture studies and confocal microscopy imaging were conducted on the non-covalent pomegranate HT-HEL protein complexes to evaluate its effect on macrophage antigen uptake, processing and presentation to T-cell hybridomas. Our results indicate that non-covalent pomegranate HT-HEL protein complexes modulate uptake, processing and antigen presentation by mouse peritoneal macrophages. After 4 h of pre-incubation, only trace amounts of IL-2 were detected in the co-cultures treated with HEL alone, whereas a non-covalent pomegranate HT-HEL complex had already reached maximum IL-2 expression. Pomegranate HT may increase rate of endocytose of HEL and subsequent expression of IL-2 by the T-cell hybridomas.

Screening of hybridoma cell lines secreting monoclonal antibodies specific for staphylococcal enterotoxin B (SEB) derived from Staphylococcus aureus

Staphylococcus aureus (S. aureus) produces 11 types of toxins and more than 20 different Staphylococcal enterotoxins (SEs), including SEA to SEE, SEG to SER and SEU. Among them, enterotoxin type B (Staphylococcal enterotoxin B - SEB) is quite heat stable and causes gastrointestinal diseases in food poisoning. The symptoms of SEB intoxication begin with the onset of sudden fever, about 40oC to 41oC, chills, headache, muscle aches and dry cough. Some patients feel shortness of breath and chest pain. Although SEB is not considered lethal, high level of exposure can lead to shock and death. Therefore, a nontoxic SEB recombinant antigen was produced to immunize mice to create B lymphocytes. Myeloma cells were fused with the B lymphocytes to generate hybridoma lines. The screening of monoclonal antibodies for the SEB antigen was determined by ELISA and Western blot tests. This study demonstrates that an SEB recombinant antigen can immunize a response against SEB in BALB/c mice. The production of monoclonal antibodies will be used to make a rapid detection strip for SEB based on immunochromatography.

Characterization of monoclonal antibodies against feline coronavirus accessory protein 7b

Feline coronaviruses (FCoVs) encode five accessory proteins termed 3a, 3b, 3c, 7a and 7b of unknown function. These proteins are dispensable for viral replication in vitro but are supposed to play a role in virulence. In the current study, we produced and characterized 7b-specific monoclonal antibodies (mAbs). A recombinant form of the 7b protein was expressed as a fusion protein in Escherichia coli, purified by immobilized metal affinity chromatography and used as immunogen. Two hybridoma lines, 5B6 and 14D8, were isolated that expressed mAbs that recognized 7b proteins of both FCoV serotypes. Using an extensive set of N- and C-terminally truncated 7b proteins expressed in E. coli and a synthetic peptide, the binding sites of mAbs 5B6 and 14D8 were mapped to an 18-residue region that comprises the only potential N-glycosylation site of the FCoV 7b protein. The two mAbs were suitable to detect a 24-kDa protein, which represents the nonglycosylated form of 7b in FCoV-infected cells. We speculate that glycosylation of 7b is part of the viral evasion strategy to prevent an immune response against this antigenic site.

Combination of Anti 􀀁-Gal-PpIX Conjugate and Photodynamic Therapy for the Diagnosis and Elimination of HeLa and CaSki Cells

The photodynamic therapy (PDT) is a specific and alternative treatment for premalignant and malignant diseases. The -Gal epitope is expressed in red blood cells and nucleated cells of placental mammals not primates, but absent in old world monkeys, apes and humans. -Gal epitope has been found in cervical biopsies HPV positive, in biopsies at different stages of cervical intraepithelial neoplasia and in cell lines such as HeLa and CaSki. The aim of this work was to obtain IgG anti a-Gal monoclonal antibody (mAb) and evaluate its usefulness in the photodynamic therapy in order to diagnosis and eliminate infected cells by HPV, types 16 and 18. From the 4C1F6D5G7B8 hybridoma line was obtained a producer hybridoma of IgG3, the mAb was conjugated with PpIX (IgG-PpIX). The IgG-PpIX conjugated was capable of identify the -Gal epitope present at the membrane of HeLa and CaSki cell lines by immunocitochemistry. The cell lines were exposed to IgG-PpIX and irradiated with a LED system at 635 nm at an energy dose of 64.3 J/cm2. The PDT using IgG anti Gal monoclonal antibody has effect on the viability of cervical carcinoma cells; the mortality results obtained were, in descendent order, HeLa (66.9 %) and CaSki (50.8 %). It was possible to conclude that PDT using the immunoconjugate IgG anti -Gal-PpIX is effective to diagnosis and eliminate cervical carcinoma cells.