Thymus-dependent Antibody Responses Research Articles

Thermodynamic projection of the antibody interaction network: The fountain energy landscape of molecular interaction systems

The adaptive humoral immune system of vertebrates functions by evolving a huge repertoire of binding proteins, which target potentially all molecules that come into contact with developing B cells. The key to endowing these binders with immunological activity is the adjustment of antibody structure and affinity against molecular targets. As a result, antibodies with a wide range of affinities and specificities evolve during the lifetime of an individual. A recently developed a quantitative model for the description of antibody homeostasis suggests that a quantitative network can describe the dynamic antibody-antigen interaction space. Here, this molecular interaction space is projected onto an energy landscape defined by entropy and free energy of binding. I introduce the concept of binding fountain energy landscape, which allows the thermodynamic representation of binding events and evolution of binding paths of multiple interactions. I further show that the hypersurface of the binding fountain corresponds to the antibody-antigen interaction network. The binding energy landscape identifies unique properties of B1 cells and natural antibodies, and distinct patterns of thymus independent and thymus dependent antibody responses. Overall, the fountain energy landscape concept of molecular interactions allows a systems biological, thermodynamic perception and description of the functioning of the clonal humoral immune system and generally describes protein evolution in thermodynamic space.

Ex Vivo Culture Assay to Measure Human Follicular Helper T (Tfh) Cell-Mediated Human B Cell Proliferation and Differentiation.

Upon priming by antigens, B cells undergo activation, proliferation, and differentiation into antibody-secreting cells. During thymus-dependent (TD) antibody responses, the proliferation and differentiation of antigen-primed B cells essentially rely on the helper function from CD4+ T cells. Follicular helper T (Tfh) cells constitute a specialized Th subset that localizes in close proximity to B cells and supports B cell proliferation and differentiation through co-stimulatory receptors and cytokines. Impaired Tfh-mediated B cell proliferation and differentiation were observed in patients with immunodeficiency, while overactivation of this process may lead to dysregulated immune responses seen in autoimmune disorders. Here, we describe an ex vivo coculture assay using circulating Tfh cells and B cells isolated from human blood. This method can be used to examine the function of patients' B cells for proliferation, differentiation, and antibody secretion, mediated by the physiological help from Tfh cells.

Thermodynamic projection of the antibody interaction network: The fountain energy landscape of molecular interaction systems

The adaptive humoral immune system of vertebrates functions by evolving a huge repertoire of binding proteins, which target potentially all molecules that come into contact with developing B cells. The key to endowing these binders with immunological activity is the adjustment of antibody structure and affinity against molecular targets. As a result, antibodies with a wide range of affinities and specificities evolve during the lifetime of an individual. I recently developed a quantitative model for the description of antibody homeostasis and suggested that a quantitative network can describe the dynamic antibody-antigen interaction space. Here, I project this molecular interaction space onto an energy landscape defined by conformational entropy and free energy of binding. I introduce the concept of binding fountain energy landscape, which allows the thermodynamic representation of binding events and paths of multiple interactions. I further show that the hypersurface of the binding fountain corresponds to the antibody-antigen interaction network. I propose that thymus independent and thymus dependent antibody responses show distinct patterns of changes in the energy landscape. Overall, the fountain energy landscape concept of molecular interactions allows a systems biological, thermodynamic perception and description of the functioning of the clonal humoral immune system.

ICOS Co-Stimulation: Friend or Foe?

Over the last 15 years, the inducible T cell co-stimulator (ICOS) has been implicated in various immune outcomes, including the induction and regulation of Th1, Th2, and Th17 immunity. In addition to its role in directing effector T cell differentiation, ICOS has also been consistently linked with the induction of thymus-dependent (TD) antibody (Ab) responses and the germinal center (GC) reaction. ICOS co-stimulation, therefore, appears to play a complex role in dictating the course of adaptive immunity. In this article, we summarize the initial characterization of ICOS and its relationship with the related co-stimulatory molecule CD28. We then address the contribution of ICOS in directing an effector T cell response, and ultimately disease outcome, against various bacterial, viral, and parasitic infections. Next, we assess ICOS in the context of TD Ab responses, connecting ICOS signaling to follicular helper T cell differentiation and its role in the GC reaction. Finally, we address the link between ICOS and human autoimmune disorders and evaluate potential therapies aiming to mitigate disease progression by modulating ICOS signaling.

Polyanhydride Nanovaccines Induce Germinal Center B Cell Formation and Sustained Serum Antibody Responses.

Biodegradable polymeric nanoparticle-based subunit vaccines have shown promising characteristics by enhancing antigen presentation and inducing protective immune responses when compared with soluble protein. Specifically, polyanhydride nanoparticle-based vaccines (i.e., nanovaccines) have been shown to successfully encapsulate and release antigens, activate B and T cells, and induce both antibody- and cell-mediated immunity towards a variety of immunogens. One of the characteristics of strong thymus-dependent antibody responses is the formation of germinal centers (GC) and the generation of GC B cells, which is part of the T helper cell driven cellular response. In order to further understand the role of nanovaccines in the induction of antigen-specific immune responses, their ability to induce germinal center B cell formation and isotype switching and the effects thereof on serum antibody responses were investigated in these studies. Polyanhydride nanovaccines based on 1,6-bis(p-carboxyphenoxy)hexane and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane were used to subcutaneously administer a viral antigen. GC B cell formation and serum antibody responses induced by the nanovaccines were compared to that induced by alum-based vaccine formulations. It was demonstrated that a single dose of polyanhydride nanovaccines resulted in the formation of robust GCs and serum antibody in comparison to that induced by the alum-based formulation. This was attributed to the sustained release of antigen provided by the nanovaccines. When administered in a multiple dose regimen, the highest post-immunization titer and GC B cell number was enhanced, and the immune response induced by the nanovaccines was further sustained. These studies provide foundational information on the mechanism of action of polyanhydride nanovaccines.

Perceived Stress and Psychological Well-Being Are Associated With Antibody Status After Meningitis C Conjugate Vaccination

Objective Psychological stress has been associated with reduced immune response to a variety of vaccinations. This study is the first to examine antibody status after vaccination with a conjugate vaccine, in which a polysaccharide antigen is conjugated to a protein to elicit a thymus-dependent antibody response. Methods Sixty undergraduate students, who had received the meningitis C conjugate vaccine before recruitment, attended a single testing session. They provided a blood sample and completed a battery of questionnaires, including the Life Events Scale for Students, Perceived Stress Scale, and General Health Questionnaire (GHQ-28). Both meningitis C–specific antibody titer and the serum bactericidal assay titer to whole meningitis C bacteria were assayed. Results High perceived stress, but not life events stress, was associated with low antibody titers. Poor antibody titers were also predicted by relatively low levels of psychological well-being as measured by the GHQ-28. Of the GHQ-28 subscales, anxiety/insomnia and social dysfunction were associated with antibody status. No psychological variables emerged from bivariate analyses as predictive of the adequacy of bactericidal titer. Conclusions This study provides the first evidence that antibody status after a conjugate vaccination may be susceptible to psychological influences.

Induction of alloantigen-specific tolerance by B cells from CD40-deficient mice.

Interaction between CD40 on B cells and CD40 ligand molecules on T cells is pivotal for the generation of a thymus-dependent antibody response. Here we show that B cells deficient in CD40 expression are unable to elicit the proliferation of allogeneic T cells in vitro. More importantly, mice immunized with CD40-/- B cells become tolerant to allogeneic major histocompatibility complex (MHC) antigens as measured by a mixed lymphocyte reaction and cytotoxic T-cell assay. The failure of CD40-/- B cells to serve as antigen presenting cells in vitro was corrected by the addition of anti-CD28 mAb. Moreover, lipopolysaccharide stimulation, which upregulates B7 expression, reversed the inability of CD40-/- B cells to stimulate an alloresponse in vitro and abrogated the capacity of these B cells to induce tolerance in vivo. These results suggest that CD40 engagement by CD40 ligand expressed on antigen-activated T cells is critical for the upregulation of B7 molecules on antigen-presenting B cells that subsequently deliver the costimulatory signals necessary for T-cell proliferation and differentiation. Our experiments suggest a novel strategy for the induction of antigen-specific tolerance in vivo.

Thymic education curtailed: defective immune responses in nude rats reconstituted with immature thymocyte subsets.

We have studied the ability of thymocyte subsets from allotype marked donors to populate athymic nude rats with T cells and to restore immune responsiveness. Following adoptive transfer, CD4-CD8- double-negative (DN) thymocytes (lymphoid precursor cells) or the CD4+CD8+ double-positive (DP) subset (intermediate thymocytes) or CD4+CD8- single-positive (CD4 SP) cells (mature thymocytes) each generated a permanent population of CD4+ progeny in syngeneic nude recipients. DN and DP thymocytes also produced small numbers of CD8+ cells; there was no evidence of a CD4-CD8- or CD4+CD8+ donor cell population. CD4 SP thymocytes conferred T cell functions [graft-versus-host (GVH) responses, allograft rejection and thymus-dependent antibody responses] on nude rats that were almost indistinguishable from those conferred by mature peripheral recirculating CD4 T cells. Transfer of DP thymocytes extended the life-span of the immunoincompetent nudes and produced CD4+ progeny with near normal GVH responsiveness. However, DP-derived CD4+ cells were deficient at inducing allograft rejection and provided little or no help for antibody synthesis. The CD4+ progeny of DN thymocytes did not prolong the survival of nude recipients, gave reduced GVH reactivity, showed almost no capacity to initiate skin allograft rejection and failed to help B cells produce antibody. The results suggest that intrathymic development proceeds stepwise; each stage is accompanied by acquisition of additional properties that are reflected by T cell responses in the periphery. Thymic education does not become complete until the SP stage is reached when thymocytes become fully independent of the thymic microenvironment.

Inhibition of immunological memory and T‐independent humoral responses by monoclonal antibodies specific for murine complement receptors

The importance of the complement system for mounting an antibody response in vivo was investigated by down-regulating and blocking the complement receptors (CR) in mice with three different monoclonal rat antibodies (mAb): mAb 8C12 recognized the C3b-binding site of CR1, mAb 7G6 recognized another site of CR1 and the C3d-binding site of CR2 and mAb 7E9 again recognized other epitopes on CR1 and CR2. We have earlier shown that 7G6, is the only mAb that completely suppresses the primary antibody response to horse erythrocytes. This antibody was also shown to suppress induction of immunological memory and a secondary antibody response. In contrast to what seems to be the case for thymus-dependent antibody responses, all three mAb could inhibit the antibody response to a thymus-independent antigen, dextran B 1355S.

Quantitative and qualitative effects of m-AMSA (Amsacrine) on cellular immune components

The recently developed acridine derivative 4′-[(9-acridinyl)amino] methanesulphon- m-anisidine ( m-AMSA) has become one of the preferred agents in the management of acute leukaemia but little is known of its effects on cellular immune components. We evaluated the qualitative and quantitative effects of m-AMSA on immune cell numbers and function, during both the leucopenic and myelorestorative phases, following intermittent high-dose therapy. Cell-mediated and inflammatory responses were depressed in the treated animals during the leucopenic phase but restoration of immune capability paralleled the recovery of circulating leucocyte numbers. Additionally, m-AMSA displayed unexpected immunomodulatory features: thymus-dependent antibody and delayed-type hypersensitivity responses were actually increased, suggesting the potential of m-AMSA as an immunoregulator in clinical and experimental studies.

Editors' corner

This chapter discusses the analogous dual specificity of helper T cells cooperating in the generation of clonally restricted antibody responses. The mechanism by which clonally restricted antibody responses are generated remains poorly understood. The antibody produced in such responses usually has no special binding affinity for antigen so it is difficult to imagine that antigen selection of the best possible clone of precursor B lymphocytes has any significant role in clonal dominance. The thymus-dependent antibody response is analyzed to the antigen phosphorylcholine (PC) coupled to a variety of protein carriers. Results suggest that high levels of circulating idiotype are required for the expansion of the anti-idiotypic helper T cell subset during ontogeny. The anti-idiotypic T cells, which are present in large numbers in T15+ mice, are activated by antigen (carrier) and preferentially help B cells bearing the T15 idiotype during an anti-PC response. The chapter proposes that there are indeed two helper T cells involved in dominant idiotype production by B cells, but that these two sets of cells, in fact, are very similar to one another and arise by analogous pathways. The anti-MHC helper T cell can selectively activate B cells of a particular specificity only if the hapten is linked physically to the carrier, while the anti-idiotypic helper T cell can discriminate particular B cells directly via an antiidiotypic receptor. The ontogeny of anti-idiotypic helper T cells proposed here would account for the predominance of germ line idiotype production in certain situations by stating that helper T cells specific for these idiotypes were initially induced in the thymus by contact with other T cells bearing these same idiotypes.

Dendritic cells are critical accessory cells for thymus-dependent antibody responses in mouse and in man.

We report that dendritic cells (DC) are necessary and potent accessory cells for anti-sheep erythrocyte responses in both mouse and man. In mice, a small number of DC (0.3-1% of the culture) restores the response of B/T-lymphocyte mixtures to that observed in unfractionated spleen. An even lower dose (0.03-0.1% DC) is needed if the T cells have been primed to antigen. Responses are both antigen and T cell dependent. Selective depletion of DC from unfractionated spleen with the monoclonal antibody 33D1 and complement ablates the antibody response. In contrast to DC, purified spleen macrophages are weak or inactive stimulators. However, when mixed with DC, macrophages can increase the yield of antibody-secreting cells about 2-fold. In man, small numbers (0.3-1%) of blood DC stimulate antibody formation in vitro. Purified human monocytes do not stimulate but in low doses (1% of the culture) inhibit the antibody response. Likewise, selective removal of human monocytes with antibody and complement enhances or accelerates the development of antibody-secreting cells. We conclude that DC are required for the development of T-dependent antibody responses by mouse and human lymphocytes in vitro.

Restoration by cyclic guanosine monophosphate and extracellular calcium of butylated hydroxyanisole-suppressed primary murine thymus-dependent antibody response.

The effect of dibutyryl cyclic guanosine monophosphate (dbc-GMP) on butylated hydroxyanisole (BHA)-induced suppression of the primary in vitro thymus-dependent antibody response of BDF1 mouse spleen cultures was studied. When added at 0 hr relative to antigen addition, 1 mg of dbc-GMP (8 mM) restored by greater than 70% the BHA-inhibited primary immunoglobulin (Ig)M plaque-forming cell (PFC) response to sheep erythrocytes (SRBC). The suppression was not reversed by the addition of 50 microgram of dibutryl cyclic adenosine monophosphate (dbc-AMP), which is known to reverse suppressor T-cell activity. The addition of 10 mM extracellular calcium (Ca2+) at the same time as antigen to BHA-inhibited cultures resulted in more than 80% restoration of the anti-SRBC PFC response. Quantitation of c-GMP by radioimmunoassay demonstrated that BHA lowered by 58% the c-GMP content of splenic lymphocytes and abrogated the ability of lipopolysaccharide of E. coli (LPS) to elevate c-GMP levels in splenic lymphocytes. The data suggest that BHA exerts its immunosuppressive effect on the primary in vitro antibody response by inhibiting guanylate cyclase activity and effectively lowering c-GMP levels; exogenous dbc-GMP and Ca2+ can freely reverse the immunosuppressive effect of BHA.

Inverse relationship between immune interferon induction and mitogen effects on the maturation of the primary antibody response

Staphylococcal enterotoxin A (SEA) is both a potent inducer of immune interferon (IFN-γ) and suppressor of the murine primary in vitro plaque-forming cell (PFC) response to the thymus-dependent antigen sheep erythrocytes (SRBC). Staphylococcal enterotoxin D (SED), which is structurally related to but antigenically different from SEA, is, in contrast, a poor inducer of IFN-γ but a potent accelerator of PFC response maturation. SED added to cultures from 0.01 to 1.0 μg/ml induced profound enhancement of the PFC response on day 3 of culture. SEA caused no acceleration of PFC maturation and suppressed the day 5 PFC response over an equivalent dose range. At the same concentration, SED only poorly induced IFN-γ, while SEA was a potent IFN-γ inducer. SED induced DNA synthesis in C57B1/6 spleen cell cultures but not athymic nude (Nu/Nu) spleen cells, suggesting that SED is a T-cell mitogen. SED was most effective in accelerating PFC maturation and increasing the magnitude of the PFC response when added to cultures at the time of SRBC addition. SED was an equally effective adjuvant for SRBC of both high and low immunogenicity. Thus, two mitogens that are structurally related have diametrically opposite effects on the primary in vitro thymus-dependent antibody response that may be related to their relative abilities to induce IFN-γ. These effects could be related to differential activation of T-cell subpopulations.

Induction of clonally restricted thymus-dependent antibody responses in vitro using phosphorylcholine derivatized sheep erythrocytes

Induction of anti-phosphorylcholine responses in vitro using phosphorylcholine derivatized sheep erythrocytes (SRBC) is described. These responses are thymus dependent and restricted to expression of the TEPC 15 idiotype. Optimal B-cell concentrations in cultures were shown to be different for elicitation of direct anti-PC ∗ PFC responses than for elicitation of PFC specific for carrier (SRBC) determinants. Similarly, these responses differed in optimal immunogen concentration requirements. These differences presumably relate to the clonality of the respective responses. The methodology described provides an alternative to induction of clonally restricted thymus-dependent responses in vitro using PC derivatized soluble protein antigens. Induction of responses using PC-SRBC has the advantages of the relative ease of T-cell priming to SRBC and concurrent stimulation of monoclonal anti-PC responses and easily assayed polyclonal anti-SRBC responses.

Activation of murine B lymphocytes by anti-immunoglobulin is an inductive signal leading to immunoglobulin secretion.

Cultures of isolated mouse splenic B lymphocytes activated by the divalent F(ab')2 fragment of purified rabbit anti-mouse Fab or class-specific anti-mouse IgM antibodies can be driven on to high rate Ig secretion by the addition of the supernatant fluid of a 24-h culture of concanavalin A-activated spleen cells (SN). The polyclonal antibody response to anti-Ig pus SN is comparable in magnitude with the lipopolysaccharide response as measured in a reverse plaque assay. The addition of SN can be delayed for 24 h after addition of anti-Ig without changing the kinetics of the response. Addition at 48 h delays the response by 24 h. The response to F(ab')2 anti-Fab plus SN is sensitive to Fc-dependent inhibition because intact anti-Fab antibodies inhibit strongly at relatively low concentrations. The monovalent Fab' fragment fails to induce Ig secretion, indicating that cross-linkage of surface immunoglobulin is required. Although the production of active SN is T cell dependent, the response to anti-Ig plus SN is T independent. These findings are interpreted as a polyclonal model of a thymus-dependent antibody response. X

Experimental Malaria in the CBA/N Mouse

CBA/N mice carry an X-linked, recessive gene, which results in the absence of a B cell subset, and is expressed primarily as an inability to respond to a certain class of thymus-independent antigens. We have examined the responses of these mice to the malaria parasite Plasmodium yoelii and found that primary infections induced by this parasite are more severe and last longer in mice with X-linked defect than in normal controls. The decreased resistance of the defective mice is associated with a striking deficiency in their IgM antibody response. After recovery from a primary infection, defective mice resist reinfection with the homologous parasite as well as normal mice. Although as resistant as normal controls, B cells from defective mice transfer considerably less immunity to naive recipients than B cells from normal animals. Hence, two modes of thymus-dependent protective immunity may contribute to the host response to P. yoelii. Control of an acute primary infection appears to involve a thymus-dependent antibody response that CBA/N mice are deficient in. Resistance to reinfection may be mediated primarily by a different mechanism.

Myasthenia Gravis and Lymphoma

Myasthenia gravis and lymphoma rarely coexist, but the occurrence of myasthenia shortly after the treatment of a patient with poorly differentiated nodular lymphoma suggested that an immunological disorder may have contributed to the development of both diseases; the fundamental defects in this association may be impaired immunological surveillance and impaired regulation of immune responses to autoantigens. The finding of T-cell immunodeificiency, including profound T-cell lymphopenia, impaired delayed hypersensitivity responses, and failure to a thymus-dependent antibody response to Salmonella adelaide flagellin, is consistent with this hypothesis.

Properties of Murine Leukemia-Associated Suppressor Cells

Abstract The spleens of BALB/c mice bearing leukemia induced with Moloney leukemia virus contain cells capable of inhibiting the antibody response of normal immunocompetent lymphocytes in Marbrook culture vessels. The immunosuppression requires contact between the cells and appears to be mediated by leukemia-associated suppressor cells rather than by the leukemic cells themselves. Thymus-dependent antibody responses to sheep red blood cells (RBC), horse RBC or trinitrophenol coupled to ovalbumin (TNP-OVA) were inhibited proportionally to the number of suppressor cells added. In contrast, thymus-independent responses to Vibrio cholerae lipopolysaccharide and to TNP coupled to Ficoll were either unaffected or suppressed only with a high number of suppressor cells. The leukemia-associated suppressor cells from BALB/c mice (H-2d) inhibited the anti-sheep RBC response of syngeneic lymphocytes and of allogeneic DBA/2 lymphocytes bearing the H-2d haplotype but failed to inhibit lymphocytes from other allogeneic strains DBA/1 (H-2q), C57BR (H-2k), A (H-2a), and C57BL/10 (H-2b). Experiments with lymphocytes from congenic strains carrying various H-2 gene complexes on the B10 genetic background confirmed that the suppressor effect is restricted by genes in the H-2 complex. Further mapping suggested that the suppression requires identity in the K region between the suppressor and the responder cells. The inability to suppress the allogeneic, H-2 different lymphocytes was retained even when a majority of the leukemic, virus-producing cells were removed from the spleen cell suspension by using a specific cytotoxic antiserum. There was no correlation between permissivity of lymphocytes to infection by the Moloney leukemia virus in vitro and between their inhibition by the suppressor cells. Thus, the nonspecific (polyclonal) leukemia-associated suppressor cells preferentially inhibit helper T cells and the suppression involves a specific interaction with product(s) of K region genes on the surface of the responder cells.

Effect of Hexachlorobenzene on the Immune System of Rats Following Combined Pre- and Postnatal Exposure

ABSTRACTThe effect of HCB on the immune system was studied after combined pre- and postnatal exposure. Pregnant rats received diets containing 50 and 150 mg HCB/kg. Dosing continued during lactation and after weaning. At an age of 5 weeks the immune system was functionally assessed. in both treatment groups, the resistance to L.mono-cytogenes infection was suppressed, as was the resistance to an infection with T. spiralis (increased yield of muscle larvae). No effect was observed on allograft rejection or on mitogenic response of thymus and spleen cells. HCB enhanced the thymus-dependent antibody response to T.spiralis antigen and to tetanus toxoid (secondary IgG titers to tetanus toxoid were increased 16-fold in both test groups). No effect was found on the thymus-independent IgM response to LPS, on the passive cutaneous anaphylaxis and on the clearance of carbon particles and L.monocytogenes. HCB treatment caused morphologic changes in lymph nodes, lung and liver. It is concluded that HCB suppresses cel...