Specific binding of fibronectin--antifibronectin immune complexes to procollagen: a new pitfall in immunostaining

We observed intense intracellular immunofluorescence of rat lung fibroblasts stained with hybridoma culture supernatant containing monoclonal antibodies to human plasma fibronectin, but no pericellular matrix staining. Immunoprecipitation and absorption experiments revealed that this intracellular staining by hybridoma-conditioned medium was due to binding of fibronectin-antifibronectin immune complexes via the fibronectin to intracellular procollagen. The anomalous staining patterns we encountered were not revealed by the usual controls for immunohistochemical specificity, and also occurred in rat tissue sections. This general phenomena--binding of serum antigens present in hybridoma medium to cellular components--could in principle result in artifactual staining with monoclonal antibodies to other serum components, so investigators using monoclonal antibodies should be aware of this new artifact. Our results also demonstrate that fibronectin binds specifically to native procollagen. Monoclonal antibodies may be useful for studying fibronectin-procollagen and other macromolecular interactions.     

基于细胞自动机的实体肿瘤生长动态建模

基于细胞自动机方法,建立了一种简单的实体肿瘤免疫性系统模型,用来模拟实体肿瘤生长过程中的情况．我们基于二维随机、离散细胞自动机计算方法,在生物细胞水平上,建立了一种肿瘤免疫性系统生长模型．该模型考虑了免疫反应的宏观性质,描绘了癌细胞和机体免疫系统之间的细胞相互作用,表现了围绕在肿瘤机体周围,免疫系统细胞的免疫作用发展情况．我们研究了常规细胞自动机模型,设计出实体肿瘤模型随机演化生长规则．最终,构建离散细胞自动机肿瘤免疫性系统模型,并做了相应的数值仿真试验．通过对仿真模型不同参数的调整,最终的仿真结果表明,该模型能反映肿瘤免疫性系统基本的相关性质．     

A graph model for the evolution of specificity in humoral immunity

The immune system protects the body against health-threatening entities, known as antigens, through very complex interactions involving the antigens and the system's own entities. One remarkable feature resulting from such interactions is the immune system's ability to improve its capability to fight antigens commonly found in the individual's environment. This adaptation process is called the evolution of specificity. In this paper, we introduce a new mathematical model for the evolution of specificity in humoral immunity, based on Jerne's functional, or idiotypic, network. The evolution of specificity is modeled as the dynamic updating of connection weights in a dynamic graph whose nodes are related to the network's idiotypes. At the core of this weight-updating mechanism are the increase in specificity caused by clonal selection and the decrease in specificity due to the insertion of uncorrelated idiotypes by the bone marrow. As we demonstrate through numerous computer experiments, for appropriate choices of parameters the new model correctly reproduces, in qualitative terms, several immune functions.     

Liposome encapsulated tumor-associated antigens elicited humoral and cellular immune responses in mice bearing tumor

Chemically induced tumors in mice provide a system to investigate tumor-associated antigens (TAA). The cell surface glycoprotein antigens on such tumor cells have been identified as suitable targets for immune attack. The induction of immune responses against (TAA) in N-nitrosodiethylamine (DEN) exposed mice has been examined. In order to present antigens to the immune system, the liposome was used as vehicle to deliver the TAA. Liposomal-TAA formulation, elicited both humoral and the cellular immune responses, when administered intramuscularly in DEN-exposed mice. Presence of circulatory antibodies against TAA and the induction of cellular responses in immunized mice were monitored using ELISA and in vitro cell proliferation assay of lymphocytes respectively. Specificity of antibody against TAA in immune sera was analysed using immunoblotting technique. Based on these results, it is proposed that the liposome encapsulated TAA may successfully be used to induce humoral and cellular immune responses against tumor.     

Qualitative dynamics of a network model of regulation of the immune system: A rationale for the IgM to IgG switch

A mathematical model has been developed that simulates some of the main features of a network theory of regulation of the immune system. According to the network viewpoint, the V regions (idiotypes) on antibodies and lymphocytes are self-antigens, to which other lymphocytes of the system can respond specifically, just as they respond to foreign antigens. The resultant couplings between the lymphocytes are considered to be basic for the regulation of the system.The present mathematical model simulates the interactions between cells that recognize the antigen (“positive cells”), and “negative cells” that have receptors that specifically recognize the V regions of the positive cells. The mathematical model incorporates only the interactions that are postulated to be important in the four steady states of the theory, and includes neither the antigen nor any accessory (“A”) cells. The effects of both antigen-specific and anti-idiotypic T and B cells are included, as well as antigen-specific and anti-idiotypic T cell factors, and the two main classes of antibodies. The model is a first order autonomous ordinary differential equation in two variables. We describe a geometric technique that gives strong information on the model, without explicitly solving the ordinary differential equation. This technique proves to be powerful in permitting us to systematically scan the parameter space of the model. The detailed analysis leads to support for the idea that the model provides a rationale for the switch observed in the immune system from the production of one major class of antibody (IgM) to the other major class (IgG). The analysis also leads to a new, previously unsuspected possibility for the nature of the suppressed state within the context of the postulates of the symmetrical network theory.     

Natural and immune human antibodies reactive with antigens of virulent Neisseria gonorrhoeae: immunoglobulins G, M, And A

Natural and immune human antibodies reactive with heat-labile and heat-stable antigens of virulent Neisseria gonorrhoeae were studied by use of an indirect fluorescent-antibody (IFA) procedure. The immunoglobulin class of the reactive antibodies was identified by using fluorescein-conjugated antisera specific for human IgG, IgA, or IgM in the IFA procedure. The effects of heat and mercaptoethanol on IFA reactivities were also studied. It appeared that antibodies of the IgG, IgM, and IgA classes present in the sera of both infected persons (immune antibodies) and normal persons with no history of gonococcal infection (natural antibodies) react with heat-stable somatic antigens. Immune IgG antibodies, however, were distinguishable from natural IgG antibodies by their ability to recognize heat-labile surface antigens. The distinction between natural and immune IgM antibodies was less obvious. IgM antibodies from both infected and normal persons appeared to react with heat-labile antigens. Some, but not all, infected persons had immune IgA antibodies to heat-labile as well as to heat-stable antigens. Treatment of sera with mercaptoethanol had no effect on IgG antibodies. The IFA activity of IgM antibodies was decreased, but not abolished. The effects of mercaptoethanol on IgA antibodies were variable. Some sera showed a decrease in IgA titer, and others showed an increase in IgA activity to certain antigens. Immune IgG antibodies were more resistant to heating than were natural IgG antibodies. Natural and immune IgM antibodies appeared equally sensitive to heating. IgA activity, on the other hand, was increased by heating sera at 60 C, but was decreased at higher temperatures. Thus, it appears that natural and immune human IgG antibodies to N. gonorrhoeae may be distinguished by their interactions with heat-labile antigens and by their resistance to heating.     

Real-Time, label-free monitoring of tumor antigen and serum antibody interactions

Conventional techniques for the detection of biomolecular interactions can be limited by the need for exogenous labels, time- and labor-intensive protocols, as well as by poor sensitivity levels. A refractometer instrument has been reconfigured to detect biomolecular interactions through changes in surface plasmon resonance (SPR). The binding kinetics and affinity values of anti-NY-ESO-1 monoclonal antibody, ES121, to the cancer-testis antigen NY-ESO-1 were determined according to the surface heterogeneity model and resulted in K(D) values of 1.3x10(-9) and 2.1x10(-10) M. The reconfigured instrument was then used to measure the interaction between tumor antigens and serum antibodies against these antigens in preselected cancer patient sera samples. The tumor antigens assayed included NY-ESO-1, SSX2 and p53, all used as recombinant proteins containing polyhistidine tags. These results demonstrated that the instrument is capable of detecting the binding of serum antibodies from cancer patient sera to immobilized tumor antigens, consistent with those observed previously in ELISA-based experiments. These results demonstrate the potential of SPR technology for the rapid diagnosis and monitoring immune responses.     

Applying Mathematical Tools to Accelerate Vaccine Development: Modeling Shigella Immune Dynamics

We establish a mathematical framework for studying immune interactions with Shigella, a bacteria that kills over one million people worldwide every year. The long-term goal of this novel approach is to inform Shigella vaccine design by elucidating which immune components and bacterial targets are crucial for establishing Shigella immunity. Our delay differential equation model focuses on antibody and B cell responses directed against antigens like lipopolysaccharide in Shigella’s outer membrane. We find that antibody-based vaccines targeting only surface antigens cannot elicit sufficient immunity for protection. Additional boosting prior to infection would require a four-orders-of-magnitude increase in antibodies to sufficiently prevent epithelial invasion. However, boosting anti-LPS B memory can confer protection, which suggests these cells may correlate with immunity. We see that IgA antibodies are slightly more effective per molecule than IgG, but more total IgA is required due to spatial functionality. An extension of the model reveals that targeting both LPS and epithelial entry proteins is a promising avenue to advance vaccine development. This paper underscores the importance of multifaceted immune targeting in creating an effective Shigella vaccine. It introduces mathematical models to the Shigella vaccine development effort and lays a foundation for joint theoretical/experimental/clinical approaches to Shigella vaccine design.     

Granulomatous hypersensitivity to Schistosoma mansoni egg antigens in human schistosomiasis. II. In vitro granuloma modulation induced by polyclonal idiotypic antibodies

We have previously reported on Id/anti-Id-receptor interactions in clinical human schistosomiasis. These findings support a hypothesis that anti-SEA cross-reactive Id develop in some patients during the course of a chronic infection and participate in regulation of anti-SEA cellular immune responses. We report here on experiments that extend those observations to the regulation of granulomatous hypersensitivity measured by an in vitro granuloma model. T cells from chronic intestinal schistosomiasis patients were stimulated in vitro with anti-SEA Id and assayed in an autologous in vitro granuloma assay for modulation of granuloma formation. These anti-SEA Id-reactive T cells were capable of regulating autologous in vitro granuloma formation. Both CD4 and CD8 T cells could be activated to regulate granuloma formation. This regulatory activity, initiated with stimulatory anti-SEA idiotypic antibodies, was antigenically specific and was dependent on the presence of intact F(ab')2 Ig molecules. The ability to elicit this regulatory activity appears to be dose dependent and is more easily demonstrated in chronically infected intestinal patients or SEA-sensitized individuals. These data support the hypothesis that anti-SEA cross-reactive Id are important in regulating granulomatous hypersensitivity in chronic intestinal schistosomiasis patients and these cross-reactive Id appear to play a major role in cell-cell interactions that result in the regulation of anti-SEA cellular immune responses.     

Targeted inactivation of the tetraspanin CD37 impairs T-cell-dependent B-cell response under suboptimal costimulatory conditions

CD37 is a membrane protein of the tetraspanin superfamily, which includes CD9, CD53, CD63, CD81, and CD82. Many of these molecules are expressed on leukocytes and have been implicated in signal transduction, cell-cell interactions, and cellular activation and development. We generated and analyzed mice deficient for CD37. Despite the high expression of CD37 on cells of the immune system, no changes in development and cellular composition of lymphoid organs were observed in mice lacking CD37. Analyses of humoral immune responses revealed a reduced level of immunoglobulin G1 (IgG1) in the sera of nonimmunized mice and an alteration of responses to T-cell-dependent antigens. Antibody responses to model antigen administered in the absence of adjuvant and to viral infections were generally poor in CD37-deficient mice. These poor antibody responses could be overcome by the immunization of antigen together with adjuvant. These results suggest a role for CD37 in T-cell-B-cell interactions which manifests itself under suboptimal costimulatory conditions.     

Using lazy evaluation to simulate realistic-size repertoires in models of the immune system

We describe a method of implementing efficient computer simulations of immune systems that have a large number of unique B-and/or T-cell clones. The method uses an implementation technique called lazy evaluation to create the illusion that all clones are being simulated, while only actually simulating a much smaller number of clones that can respond to the antigens in the simulation. The method is effective because only 0.001–0.01% of clones can typically be stimulated by an antigen, and because many simulations involve only a small number of distinct antigens. A lazy simulation of a realistic number of clones and 10 distinct antigens is 1000 times faster and 10 000 times smaller than a conventional simulation—making simulations of immune systems with realistic-size repertoires computationally tractable.     

Assembly of human immunodeficiency virus (HIV) antigens on bacteriophage T4: a novel in vitro approach to construct multicomponent HIV vaccines

Bacteriophage T4 capsid is an elongated icosahedron decorated with 155 copies of Hoc, a nonessential highly antigenic outer capsid protein. One Hoc monomer is present in the center of each major capsid protein (gp23*) hexon. We describe an in vitro assembly system which allows display of HIV antigens, p24-gag, Nef, and an engineered gp41 C-peptide trimer, on phage T4 capsid surface through Hoc-capsid interactions. In-frame fusions were constructed by splicing the human immunodeficiency virus (HIV) genes to the 5' or 3' end of the Hoc gene. The Hoc fusion proteins were expressed, purified, and displayed on hoc(-) phage particles in a defined in vitro system. Single or multiple antigens were efficiently displayed, leading to saturation of all available capsid binding sites. The displayed p24 was highly immunogenic in mice in the absence of any external adjuvant, eliciting strong p24-specific antibodies, as well as Th1 and Th2 cellular responses with a bias toward the Th2 response. The phage T4 system offers new direction and insights for HIV vaccine development with the potential to increase the breadth of both cellular and humoral immune responses.     

Dense bodies of human cytomegalovirus induce both humoral and cellular immune responses in the absence of viral gene expression

Infection of fibroblast cell cultures with human cytomegalovirus (HCMV) leads to the production of significant amounts of defective enveloped particles, termed dense bodies (DB). These noninfectious structures contain major antigenic determinants which are responsible for induction of both the humoral and the cellular immune response against HCMV. We tested the hypothesis that, by virtue of their unique antigenic and structural properties, DB could induce a significant immune response in the absence of infectious virus. Mice were immunized with gradient-purified DB, which were either left untreated or subjected to sequential rounds of sonication and freeze-thawing to prevent cellular entry. Titers of neutralizing antibodies induced by DB were in a range comparable to levels present in convalescent human sera. The virus-neutralizing antibody response was surprisingly durable, with neutralizing antibodies detected 12 months following primary immunization. The HCMV-specific major histocompatibility complex class I-restricted cytolytic T-cell (CTL) response was assayed using mice transgenic for the human HLA-A2 molecule. Immunization with DB led to high levels of HCMV-specific CTL in the absence of de novo viral protein synthesis. Maximal total cytolytic activity in mice immunized with DB was nearly as efficient as the cytolytic activity induced by a standard immunization with murine cytomegalovirus. Furthermore, DB induced a typical T-helper 1 (Th1)-dominated immune response in mice, as determined by cytokine and immunoglobulin G isotype analysis. Induction of humoral and cellular immune responses was achieved without the concomitant use of adjuvant. We thus propose that DB can serve as a basis for the future development of a recombinant nonreplicating vaccine against HCMV. Finally, such particles could be engineered for efficient delivery of antigens from other pathogens to the immune system.     

Camouflaging endothelial cells: does it prolong graft survival?

Camouflaging antigens on the surface of cells seems an appealing way to prevent activation of the immune system. We explored the possibility of preventing hyperacute rejection by chemically camouflaging endothelial cells (EC). In vitro as well as in vivo experiments were performed. First, the ability of mPEG coating to prevent antibody-antigen interactions was evaluated. Second, we tested the degree to which mPEG coating prevents activation of EC by stimuli such as TNF-alpha and LPS. Third, in vivo experiments were performed to test the ability of mPEG coating to prolong xenograft survival. We demonstrate that binding of several antibodies to EC or serum proteins can be inhibited by mPEG. Furthermore, binding of TNF-alpha as well as LPS to EC is blocked since mPEG treatment of EC inhibits the subsequent up-regulation of E-selectin by these stimuli. However, in vivo experiments revealed that currently this method alone is not sufficient to prevent hyperacute rejection.     

An experimental protocol for the establishment of dogs with long-term cellular immune reactions to Leishmania antigens

Domestic dogs are considered to be the main reservoirs of zoonotic visceral leishmaniasis. In this work, we evaluated a protocol to induce Leishmania infantum/Leishmania chagasi-specific cellular and humoral immune responses in dogs, which consisted of two injections of Leishmania promastigote lysate followed by a subcutaneous inoculation of viable promastigotes. The primary objective was to establish a canine experimental model to provide positive controls for testing immune responses to Leishmania in laboratory conditions. After inoculation of viable promastigotes, specific proliferative responses of peripheral blood mononuclear cells (PBMCs) to either Leishmania lysate or recombinant proteins, the in vitro production of interferon-γ by antigen-stimulated PBMCs and a significant increase in circulating levels of anti-Leishmania antibodies were observed. The immunized dogs also displayed positive delayed-type hypersensitivity reactions to Leishmania crude antigens and to purified recombinant proteins. An important finding that supports the suitability of the dogs as positive controls is that they remained healthy for the entire observation period, i.e., more than seven years after infection. Following the Leishmania antigen lysate injections, the infection of dogs by the subcutaneous route appears to induce a sustained cellular immune response, leading to an asymptomatic infection. This provides a useful model for both the selection of immunogenic Leishmania antigens and for immunobiological studies on their possible immunoprotective activities.     

Engineering T lymphocyte antigen specificity.

Targeting of immune cells by bispecific antibodies has proven to be a powerful tool for the investigation of cellular cytotoxicity, lymphocyte activation and induction of cytokine production, as well as to represent an innovative form of immunotherapy for the treatment of cancer. The hallmark of this approach is the use of the specificity of monoclonal antibodies to join target and immune cells by virtue of the dual specificity of bispecific antibodies for the two entities. More precisely, the bispecific antibody has two different binding sites, which are capable of recognizing tumor associated antigens on the one hand and lymphocyte activation sites on the other. This process of crosslinking results in the activation of the lymphocyte and triggering of its lytic machinery, as well as lymphokine production. A major advantage of this therapeutic modality is, that use is made of the normal cellular immune defence system and therefore is only associated with minor toxicity. The distinct lymphocyte populations, which can be used for adoptive immunotherapy and the various bispecific antibody preparations, as well as the chimeric immunoglobulin/T cell receptor construction, are the major topics of this review.     

A cellular automata model of tumor-immune system interactions

We present a hybrid cellular automata-partial differential equation model of moderate complexity to describe the interactions between a growing tumor next to a nutrient source and the immune system of the host organism. The model allows both temporal and two-dimensional spatial evolution of the system under investigation and is comprised of biological cell metabolism rules derived from both the experimental and mathematical modeling literature. We present numerical simulations that display behaviors which are qualitatively similar to those exhibited in tumor-immune system interaction experiments. These include spherical tumor growth, stable and unstable oscillatory tumor growth, satellitosis and tumor infiltration by immune cells. Finally, the relationship between these different growth regimes and key system parameters is discussed.     

Nanobody-CD16 Catch Bond Reveals NK Cell Mechanosensitivity

Antibodies are key tools in biomedical research and medicine. Their binding properties are classically measured in solution and characterized by an affinity. However, in physiological conditions, antibodies can bridge an immune effector cell and an antigen-presenting cell, implying that mechanical forces may apply to the bonds. For example, in antibody-dependent cell cytotoxicity—a major mode of action of therapeutic monoclonal antibodies—the Fab domains bind the antigens on the target cell, whereas the Fc domain binds to the activating receptor CD16 (also known as FcgRIII) of an immune effector cell, in a quasi-bidimensional environment (2D). Therefore, there is a strong need to investigate antigen/antibody binding under force (2D) to better understand and predict antibody activity in vivo. We used two anti-CD16 nanobodies targeting two different epitopes and laminar flow chamber assay to measure the association and dissociation of single bonds formed between microsphere-bound CD16 antigens and surface-bound anti-CD16 nanobodies (or single-domain antibodies), simulating 2D encounters. The two nanobodies exhibit similar 2D association kinetics, characterized by a strong dependence on the molecular encounter duration. However, their 2D dissociation kinetics strongly differ as a function of applied force: one exhibits a slip bond behavior in which off rate increases with force, and the other exhibits a catch-bond behavior in which off rate decreases with force. This is the first time, to our knowledge, that catch-bond behavior was reported for antigen-antibody bond. Quantification of natural killer cells spreading on surfaces coated with the nanobodies provides a comparison between 2D and three-dimensional adhesion in a cellular context, supporting the hypothesis of natural killer cell mechanosensitivity. Our results may also have strong implications for the design of efficient bispecific antibodies for therapeutic applications.     

The impact of symbolism on immunogenetics: an application to HLA

The genes coding for the class I human lymphocyte antigens (HLA) are located on chromosome 6. These antigens are involved with the immunological interaction between cells. In some immunogenetic systems, such as HLA in humans, genes are defined by antibody/antigen reaction and are denoted by single symbolic identifiers. This symbolization assumes a one-to-one correspondence between antibodies, antigens and genes. Recent molecular studies, however, suggest that HLA antibody/antigen reaction is complex and most HLA class I specific antibodies may not uniquely identify a single allelic product. Where cross-reactivity is present in an immunogenetic system it is important to label each reagent with symbols corresponding to all genes coding for antigens with which the reagent will react. The problems of cross-reactive groups and unexplained linkage relations may be elucidated by the redefinition and clarification of certain HLA antigens. A computer program can suggest such labelling schemes using input given by phenotype reaction patterns with a panel of reagents. When this program was applied to data on the class I HLA antigens a genetic model was suggested that differs somewhat from the currently accepted model. The new model is able to predict what would appear as linkage relations in the accepted model. Our methodology can provide alternate models to guide in typing cloned genes in terms of the HLA locus and alleles.     

Stage-specific and common antigens of Brugia malayi identified with monoclonal antibodies

To identify parasite antigens that trigger protective, pathogenic, and allergic immune responses during filarial infections, we generated a series of monoclonal antibodies to infective larvae, adult worms, and microfilariae of Brugia malayi, a human pathogen. Quantitative and qualitative analysis of the reaction patterns of these monoclonal antibodies indicates the existence of stage-specific antigens of B. malayi, as well as of antigens shared by different stages of this parasite and by other related and unrelated helminths. These antibodies should provide invaluable tools for the analysis of host-parasite interactions in filariasis at the molecular level.