Membrane immunoglobulin expressed by retroviral vector gene transfer mimics partial function of the B-cell receptor in vivo

Activation of B-cells is initiated by the ligation of B-cell receptors by its cognate antigen, inducing a series of signal cascades. Understanding the molecular mechanisms of these important events is a crucial goal for immunologists. Chimeric B cell receptors provide a powerful tool for analysis of B-cell signal function. However, this method can only be used in tool cells, but cannot be used for in vivo study. Here, we constructed a retroviral vector to encode both heavy chains and light chains of a membrane immunoglobulin, and expressed them in primary B-cells using retroviral gene transfer. Our results demonstrate that the membrane immunoglobulin expressed by retroviral vectors transfer can initiate B-cell receptor-mediated signaling, resulting in the phosphorylation of Syk and Erk1/2 proteins. The results showed that B-cells expressing membrane immunoglobulin can make proliferative responses to cognate antigen both in vitro and in vivo. Therefore, we provide a methodology for rapidly analyzing the downstream signals of B-cell receptors both in vitro and in vivo, which could expedite the identification of proteins involved in B-cell function.     

Expression of ST3GAL4 Leads to SLex Expression and Induces c-Met Activation and an Invasive Phenotype in Gastric Carcinoma Cells

Sialyl-Lewis X (SLe^x) is a sialylated glycan antigen expressed on the cell surface during malignant cell transformation and is associated with cancer progression and poor prognosis. The increased expression of sialylated glycans is associated with alterations in the expression of sialyltransferases (STs). In this study we determined the capacity of ST3GAL3 and ST3GAL4 sialyltransferases to synthesize the SLe^x antigen in MKN45 gastric carcinoma cells and evaluated the effect of SLe^x overexpression in cancer cell behavior both in vitro and in vivo using the chicken chorioallantoic membrane (CAM) model. The activation of tyrosine kinase receptors and their downstream molecular targets was also addressed. Our results showed that the expression of ST3GAL4 in MKN45 gastric cancer cells leads to the synthesis of SLe^x antigens and to an increased invasive phenotype both in vitro and in the in vivo CAM model. Analysis of phosphorylation of tyrosine kinase receptors showed a specific increase in c-Met activation. The characterization of downstream molecular targets of c-Met activation, involved in the invasive phenotype, revealed increased phosphorylation of FAK and Src proteins and activation of Cdc42, Rac1 and RhoA GTPases. Inhibition of c-Met and Src activation abolished the observed increased cell invasive phenotype. In conclusion, the expression of ST3GAL4 leads to SLe^x antigen expression in gastric cancer cells which in turn induces an increased invasive phenotype through the activation of c-Met, in association with Src, FAK and Cdc42, Rac1 and RhoA GTPases activation.     

Activated Brain Endothelial Cells Cross-Present Malaria Antigen

In the murine model of cerebral malaria caused by P. berghei ANKA (PbA), parasite-specific CD8⁺ T cells directly induce pathology and have long been hypothesized to kill brain endothelial cells that have internalized PbA antigen. We previously reported that brain microvessel fragments from infected mice cross-present PbA epitopes, using reporter cells transduced with epitope-specific T cell receptors. Here, we confirm that endothelial cells are the population responsible for cross-presentation in vivo, not pericytes or microglia. PbA antigen cross-presentation by primary brain endothelial cells in vitro confers susceptibility to killing by CD8⁺ T cells from infected mice. IFNγ stimulation is required for brain endothelial cross-presentation in vivo and in vitro, which occurs by a proteasome- and TAP-dependent mechanism. Parasite strains that do not induce cerebral malaria were phagocytosed and cross-presented less efficiently than PbA in vitro. The main source of antigen appears to be free merozoites, which were avidly phagocytosed. A human brain endothelial cell line also phagocytosed P. falciparum merozoites. Besides being the first demonstration of cross-presentation by brain endothelial cells, our results suggest that interfering with merozoite phagocytosis or antigen processing may be effective strategies for cerebral malaria intervention.     

Transmembrane Signaling in Chimeras of the Escherichia coli Aspartate and Serine Chemotaxis Receptors and Bacterial Class III Adenylyl Cyclases

The Escherichia coli chemoreceptors for serine (Tsr) and aspartate (Tar) and several bacterial class III adenylyl cyclases (ACs) share a common molecular architecture; that is, a membrane anchor that is linked via a cytoplasmic HAMP domain to a C-terminal signal output unit. Functionality of both proteins requires homodimerization. The chemotaxis receptors are well characterized, whereas the typical hexahelical membrane anchor (6TM) of class III ACs, suggested to operate as a channel or transporter, has no known function beyond a membrane anchor. We joined the intramolecular networks of Tsr or Tar and two bacterial ACs, Rv3645 from Mycobacterium tuberculosis and CyaG from Arthrospira platensis, across their signal transmission sites, connecting the chemotaxis receptors via different HAMP domains to the catalytic AC domains. AC activity in the chimeras was inhibited by micromolar concentrations of l-serine or l-aspartate in vitro and in vivo. Single point mutations known to abolish ligand binding in Tar (R69E or T154I) or Tsr (R69E or T156K) abrogated AC regulation. Co-expression of mutant pairs, which functionally complement each other, restored regulation in vitro and in vivo. Taken together, these studies demonstrate chemotaxis receptor-mediated regulation of chimeric bacterial ACs and connect chemical sensing and AC regulation.     

Immunosuppression by cobra factor: distribution, antigen-induced blast transformation and trapping of lymphocytes during in vivo complement depletion.

In vivo administration of cobra factor (CoF), the C3-activating protein of cobra venom, suppresses thymus-dependent antibody production. In a study of possible mechanisms for this effect binding of CoF to murine spleen cells in vitro was not detected, nor was there any effect on C3 or Fc receptors. The numbers of spleen cells bearing C3 receptors, Fc receptors, θ antigen or surface immunoglobulin were not altered by in vivo complement depletion of mice with CoF. The distribution and antigen-induced trapping of transferred ⁵¹Cr-labelled syngeneic spleen cells were unaffected by treatment of either donors or recipients with CoF. Furthermore, the antigen-induced generation, trapping and specific retention of immunospecific blast cells were normal in CoF-treated mice, despite profound suppression in these animals of IgG antibody production. The majority of these blast cells 3 days after immunisation were T cells, suggesting that complement depletion interferes with the process of T-dependent antibody production at a later stage than the activation of T cells by antigen.     

Antigenic characterization of chick erythrocytes and erythropoietic precursors: identification of several definitive populations during embryogenesis.

During ontogenesis of the chick, three normocyte populations can be resolved on the basis of their membrane antigens. Embryonic erythrocytes bear an embryonic antigen (E₁) which is not present on adult red cells. Conversely an adult antigen (A) is detected only on adult red cells. A third population bearing simultaneously both antigens develops transiently during the first month after hatching. These normocyte antigens were investigated at the level of erythrocytic stem cell identified by in vivo and in vitro assays. The antigens can be revealed only on erythrocytic precursors CFU-cE giving rise to erythrocytic clones in vitro in presence of avian erythropoietin. Three CFU-cE populations were defined therefrom. In marrow, all these populations are present in the embryo but only CFU-cE bearing A antigen subsist after hatching.     

Receptor-ligand interactions and tolerance induction in mature virgin B cells

The membrane events and the nature of the receptors involved in the induction of thymus-independent high-zone tolerance were investigated. Tolerance was induced in vitro by incubating cells for the 4 hr with DNP-polysaccharide conjugate. The degree of tolerance was estimated by determining the subsequent cellular response to antigenic challenge in vitro. Treatment with agents that inhibited energy metabolism, membrane fluidity, and movement of membrane receptors all inhibited the induction of tolerance. Agents which affect the integrity of cytoskeletal elements also interfered with tolerance induction. Taken together these results indicate that the induction of high-zone thymus-independent tolerance is an active process involving at least some aspects of antigen induced receptor modulation. The specific receptor involved appears to be IgM since tolerance could be induced by exposing cells first to subtolerogenic doses of antigen and then to antibodies specific for the IgM receptor.     

Nicotine Inhibits Memory CTL Programming

Nicotine is the main tobacco component responsible for tobacco addiction and is used extensively in smoking and smoking cessation therapies. However, little is known about its effects on the immune system. We confirmed that multiple nicotinic receptors are expressed on mouse and human cytotoxic T lymphocytes (CTLs) and demonstrated that nicotinic receptors on mouse CTLs are regulated during activation. Acute nicotine presence during activation increases primary CTL expansion in vitro, but impairs in vivo expansion after transfer and subsequent memory CTL differentiation, which reduces protection against subsequent pathogen challenges. Furthermore, nicotine abolishes the regulatory effect of rapamycin on memory CTL programming, which can be attributed to the fact that rapamycin enhances expression of nicotinic receptors. Interestingly, naïve CTLs from chronic nicotine-treated mice have normal memory programming, which is impaired by nicotine during activation in vitro. In conclusion, simultaneous exposure to nicotine and antigen during CTL activation negatively affects memory development.     

A Novel Ex Vivo Isolation and Expansion Procedure for Chimeric Antigen Receptor Engrafted Human T Cells

Genetically engineered T lymphocytes are a promising option for cancer therapy. Prior to adoptive transfer they have to be expanded in vitro to reach therapeutically sufficient numbers. So far, no universal method exists for selective in vitro expansion of engineered T lymphocytes. In order to overcome this problem and for proof of concept we incorporated a novel unique peptide sequence of ten amino acids as epitope (E-Tag) into the binding domains of two novel chimeric antigen receptors (ECARs) directed against either prostate stem cell antigen (PSCA) for the treatment of prostate cancer (PCa) or CD33 for the treatment of acute myeloide leukemia (AML). The epitope tag then was utilized for expanding ECAR engrafted T cells by triggering the modified T cells via a monoclonal antibody directed against the E-Tag (Emab). Moreover, the E-Tag served as an efficient selection epitope for immunomagnetic isolation of modified T cells to high purity. ECAR engrafted T cells were fully functional and mediated profound anti-tumor effects in the respective models of PCa or AML both in vitro and in vivo. The method can be integrated straightforward into clinical protocols to improve therapeutic efficiency of tumor treatment with CAR modified T lymphocytes.     

Thymus cell traffic induced by antigen stimulation

Spleens of adult mice immunized with either RSA or RGG responded in vitro to DNP-RSA or DNP-RGG, respectively, at a significantly higher rate than spleens of untreated mice. Stimulation in vitro could be achieved by short pulses of the antigen (5–15 min). It was found that thymectomy prior to injection of the carrier protein interfered with the subsequent response in vitro to the hapten-carrier conjugate, and that this was much more pronounced in 8- to 10-day-old mice than in older mice. It is suggested that antigen stimulation in vivo triggers thymus cell migration. Although this is by no means the only mechanism accountable for manifestation of the carrier effect, it may represent a device for amplification of the immune response in vivo.     

The in vitro induction of T cells which mediate delayed-type hypersensitivity toward horse red blood cells.

Humoral and cell-mediated immunity to the antigen horse red blood cells (HRBC) were induced in vitro. The type of immune response induced, however, was dependent on the concentration of antigen present in the culture. Whereas intermediate concentrations of HRBC induced antibody-forming cells, high and low concentrations of HRBC induced T cells which, on transfer, mediated delayed-type hypersensitivity reactions. The inverse relationship between humoral and cell-mediated immunity often observed in vivo is, therefore, also evident when lymphocytes are stimulated with antigen in vitro.     

Salmonella Uses Energy Taxis to Benefit from Intestinal Inflammation

Chemotaxis enhances the fitness of Salmonella enterica serotype Typhimurium (S. Typhimurium) during colitis. However, the chemotaxis receptors conferring this fitness advantage and their cognate signals generated during inflammation remain unknown. Here we identify respiratory electron acceptors that are generated in the intestinal lumen as by-products of the host inflammatory response as in vivo signals for methyl-accepting chemotaxis proteins (MCPs). Three MCPs, including Trg, Tsr and Aer, enhanced the fitness of S. Typhimurium in a mouse colitis model. Aer mediated chemotaxis towards electron acceptors (energy taxis) in vitro and required tetrathionate respiration to confer a fitness advantage in vivo. Tsr mediated energy taxis towards nitrate but not towards tetrathionate in vitro and required nitrate respiration to confer a fitness advantage in vivo. These data suggest that the energy taxis receptors Tsr and Aer respond to distinct in vivo signals to confer a fitness advantage upon S. Typhimurium during inflammation by enabling this facultative anaerobic pathogen to seek out favorable spatial niches containing host-derived electron acceptors that boost its luminal growth.     

Differential Expression of In Vivo and In Vitro Protein Profile of Outer Membrane of Acidovorax avenae Subsp. avenae

Outer membrane (OM) proteins play a significant role in bacterial pathogenesis. In this work, we examined and compared the expression of the OM proteins of the rice pathogen Acidovorax avenae subsp. avenae strain RS-1, a Gram-negative bacterium, both in an in vitro culture medium and in vivo rice plants. Global proteomic profiling of A. avenae subsp. avenae strain RS-1 comparing in vivo and in vitro conditions revealed the differential expression of proteins affecting the survival and pathogenicity of the rice pathogen in host plants. The shotgun proteomics analysis of OM proteins resulted in the identification of 97 proteins in vitro and 62 proteins in vivo by mass spectrometry. Among these OM proteins, there is a high number of porins, TonB-dependent receptors, lipoproteins of the NodT family, ABC transporters, flagellins, and proteins of unknown function expressed under both conditions. However, the major proteins such as phospholipase and OmpA domain containing proteins were expressed in vitro, while the proteins such as the surface anchored protein F, ATP-dependent Clp protease, OmpA and MotB domain containing proteins were expressed in vivo. This may indicate that these in vivo OM proteins have roles in the pathogenicity of A. avenae subsp. avenae strain RS-1. In addition, the LC-MS/MS identification of OmpA and MotB validated the in silico prediction of the existance of Type VI secretion system core components. To the best of our knowledge, this is the first study to reveal the in vitro and in vivo protein profiles, in combination with LC-MS/MS mass spectra, in silico OM proteome and in silico genome wide analysis, of pathogenicity or plant host required proteins of a plant pathogenic bacterium.     

Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

During development,axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation of the guidance receptors is the first step of the signaling mechanisms allowing axon tips, the growth cones, to respond to the ligands. As such, the modulation of their availability at the cell surface is one of the mechanisms that participate in setting the growth cone sensitivity. We describe here a method to precisely visualize the spatio-temporal cell surface dynamics of an axon guidance receptor both in vitro and in vivo in the developing chick spinal cord. We took advantage of the pH-dependent fluorescence property of a green fluorescent protein (GFP) variant to specifically detect the fraction of the axon guidance receptor that is addressed to the plasma membrane. We first describe the in vitro validation of such pH-dependent constructs and we further detail their use in vivo, in the chick spinal chord, to assess the spatio-temporal dynamics of the axon guidance receptor of interest.     

Nucleotide Insertions and Deletions Complement Point Mutations to Massively Expand the Diversity Created by Somatic Hypermutation of Antibodies

During somatic hypermutation (SHM), deamination of cytidine by activation-induced cytidine deaminase and subsequent DNA repair generates mutations within immunoglobulin V-regions. Nucleotide insertions and deletions (indels) have recently been shown to be critical for the evolution of antibody binding. Affinity maturation of 53 antibodies using in vitro SHM in a non-B cell context was compared with mutation patterns observed for SHM in vivo. The origin and frequency of indels seen during in vitro maturation were similar to that in vivo. Indels are localized to CDRs, and secondary mutations within insertions further optimize antigen binding. Structural determination of an antibody matured in vitro and comparison with human-derived antibodies containing insertions reveal conserved patterns of antibody maturation. These findings indicate that activation-induced cytidine deaminase acting on V-region sequences is sufficient to initiate authentic formation of indels in vitro and in vivo and that point mutations, indel formation, and clonal selection form a robust tripartite system for antibody evolution.     

The N-terminal amphipathic helix of Pex11p self-interacts to induce membrane remodelling during peroxisome fission

Pex11p plays a crucial role in peroxisome fission. Previously, it was shown that a conserved N-terminal amphipathic helix in Pex11p, termed Pex11-Amph, was necessary for peroxisomal fission in vivo while in vitro studies revealed that this region alone was sufficient to bring about tubulation of liposomes with a lipid consistency resembling the peroxisomal membrane. However, molecular details of how Pex11-Amph remodels the peroxisomal membrane remain unknown. Here we have combined in silico, in vitro and in vivo approaches to gain insights into the molecular mechanisms underlying Pex11-Amph activity. Using molecular dynamics simulations, we observe that Pex11-Amph peptides form linear aggregates on a model membrane. Furthermore, we identify mutations that disrupted this aggregation in silico, which also abolished the peptide's ability to remodel liposomes in vitro, establishing that Pex11p oligomerisation plays a direct role in membrane remodelling. In vivo studies revealed that these mutations resulted in a strong reduction in Pex11 protein levels, indicating that these residues are important for Pex11p function. Taken together, our data demonstrate the power of combining in silico techniques with experimental approaches to investigate the molecular mechanisms underlying Pex11p-dependent membrane remodelling.     

Transfer of cell-mediated hyperacute rejection reaction: The role of active sensitization

The transfer of lymph node cells draining graft sites of allogeneic murine skin results in adoptive immunization of syngeneic recipients, as per hyperacute rejection of allogeneic test skin grafts. The transfer of spleen cells from mice sensitized by i.p. injection of allogeneic cells does not have this result unless the cells undergo a secondary in vitro sensitization. The resultant hyperacute rejection is due in part to adoptive immunization via the spleen cells primed during the in vivo sensitization and rendered transfer effective by the in vitro exposure; in part it is due to active sensitization by carried-over antigen from the in vitro exposure. It follows that the transfer effect of spleen cells sensitized in vivo and in vitro is only in part abrogated by exposure to α-Thy. 1 serum and complement.     

The correlation between the activation state of B cells and their capacity for in vitro propagation of immunologic memory

The B-cell population responsible for in vitro antigen-mediated proliferation and expansion of the memory B-cell population is a large activated blast. Such cells predominate early after antigen priming and can be regenerated by adjuvant (Bordetella pertussis) stimulation in vivo. Although these cells are proliferating in vivo, additional stimuli are needed for expansion of the memory population in vitro. These triggering requirements include specific antigen (DNP-OVA) and the assistance of adherent accessory cells. Although T cells are present in the culture, their role in the propagation of memory is not completely clear. Using the unrelated antigen, sheep erythrocytes, we have shown that “bystander” T-cell help can mediate differentiation of these memory B-cell blasts to AFC, but it cannot induce expansion of the memory-cell population. However, the fact that the TI-2 antigen DNP-Ficoll is a relatively ineffective inducer of memory-cell propagation (inducing an expanded response which is less than 10% ofthat induced by the T-cell-dependent antigen, DNP-OVA) suggests that T cells may be involved, possibly via production of B-cell growth factor. Thus, the minimal requirements for triggering the propagation of B-cell memory include (i) a blastogenic signal which can be mediated by adjuvant, (ii) specific antigen, and (iii) adherent accessory-cell help.     

An anti-apoptotic peptide improves survival in lethal total body irradiation

Cell penetrating peptides (CPPs) have been used to deliver the anti-apoptotic Bcl-xL-derived BH4 peptide to prevent injury-induced apoptosis both in vitro and in vivo. Here we demonstrate that the nuclear localization sequence (NLS) from the SV40 large T antigen has favorable properties for BH4 domain delivery to lymphocytes compared to sequences based on the HIV-1 TAT sequence. While both TAT-BH4 and NLS-BH4 protected primary human mononuclear cells from radiation-induced apoptotic cell death, TAT-BH4 caused persistent membrane damage and even cell death at the highest concentrations tested (5-10 μM) and correlated with in vivo toxicity as intravenous administration of TAT-BH4 caused rapid death. The NLS-BH4 peptide has significantly attenuated toxicity compared to TAT-BH4 and we established a dosing regimen of NLS-BH4 that conferred a significant survival advantage in a post-exposure treatment model of LD90 total body irradiation.     

In vitro proliferation of rabbit bone marrow-derived and thymus-derived lymphocytes in response to vaccinia virus

Peripheral blood leukocytes from rabbits immunized with vaccinia virus were incubated in vitro with vaccinia antigen, and resultant lymphocyte proliferation was measured by incorporation of tritiated thymidine into acid-insoluble material. Significant lymphocyte stimulation was observed at a time when antiviral antibody was being synthesized in vivo. The extent of proliferation by bone marrow-derived lymphocytes after culture with viral antigen was determined by simultaneous detection of complement receptor lymphocytes (CRLs have been shown to be B cells) and uptake of tritiated thymidine in these CRLs by radioautography. The results indicate that both bone marrow-derived and thymus-derived lymphocytes participate in the in vitro proliferative response of rabbit peripheral blood lymphocytes to vaccinia antigen.