Suppression of asthma-like responses in different mouse strains by oral tolerance

In this study we examined the effect of oral antigen (Ag) administration on the development of experimental asthma in different mouse strains. We selected BALB/c, BP2, CBA/Ca interleukin (IL)-5 transgenic, and BALB/c T-cell receptor-delta-deficient mouse strains because they exhibit different aspects of the asthma syndrome. Mice exposed to 1% ovalbumin (OVA), dissolved in the drinking water for 5 consecutive days, became unresponsive to subsequent immunogenic OVA challenges. This regimen of OVA administration induced Ag-specific unresponsiveness in all mouse strains tested, including gammadelta-deficient mice that are said to be resistant to tolerance induction. The Ag-specific unresponsiveness was characterized by reduced (almost absent) airway eosinophilic inflammation, airway hyperreactivity, and mucus production; also by low levels of T helper (Th) 2-type cytokines in bronchoalveolar lavage fluid, and decreased immunoglobulin (Ig) G1 and IgE OVA-specific antibody production. The unresponsive state was not associated with increased levels of the suppressive cytokines IL-10 and transforming growth factor (TGF)-beta or with immune deviation toward the Th1 pathway due to increased levels of interferon-gamma and IL-12. Moreover, treatment with anti- TGF-beta antibodies did not abrogate oral tolerance. Oral Ag administration was quite effective in suppressing the development of key features of asthma when initiated after primary immunization (Day 0) or after booster (Day 7), but not after challenge (Day 14) when it increased allergic responses. Collectively, our findings show for the first time the beneficial and detrimental effects of oral Ag administration on the development of experimental asthma.     

Host-dependent control of early regulatory and effector T-cell differentiation underlies the genetic susceptibility of RAG2-deficient mouse strains to transfer colitis

De novo differentiation of CD4⁺Foxp3⁺ regulatory T cells (induced (i) Tregs) occurs preferentially in the gut-associated lymphoid tissues (GALT). We addressed the contribution of background genetic factors in affecting the balance of iTreg, T helper type 1 (Th1), and Th17 cell differentiation in GALT in vivo following the transfer of naive CD4⁺CD45RB^high T cells to strains of RAG2-deficient mice with differential susceptibility to inflammatory colitis. iTregs represented up to 5% of CD4⁺ T cells in mesenteric lymph nodes of less-susceptible C57BL/6 RAG2^−/− mice compared with <1% in highly susceptible C57BL/10 RAG2^−/− mice 2 weeks following T-cell transfer before the onset of colitis. Early Treg induction was correlated inversely with effector cell expansion and the severity of colitis development, was controlled primarily by host and not T-cell-dependent factors, and was strongly associated with interleukin-12 (IL-12)/23 production by host CD11c⁺CD103⁺ dendritic cells. These data highlight the importance of genetic factors regulating IL-12/23 production in controlling the balance between iTreg differentiation and effector-pathogenic CD4⁺ T-cell expansion in lymphopenic mice and indicate a direct role for iTregs in the regulation of colonic inflammation in vivo.     

Forward genetic dissection of innate response to infection in inbred mouse strains: selected success stories

Mouse genetics is a powerful tool for the dissection of genes, proteins, and pathways important in biological processes. Application of this approach to study the host response to infection has been a rich source of discoveries that have increased our understanding of the early innate pathways involved in responding to microbial infections. Here we review some of the key discoveries that have arisen from pinpointing the genetic defect in mouse strains with unusual or extreme response to infection and have led to insights into pathogen sensing pathways and downstream effector functions of the early innate immune response.     

Multivariate genetic analysis of atopy phenotypes in a selected sample of twins

BACKGROUND: Atopic traits often co-occur and this can potentially be caused by common aetiological relationships between traits, i.e. a common genetic or a common environmental background. OBJECTIVE: To estimate to what extent the same genetic and environmental factors influence wheeze, rhinitis, airway hyper-responsiveness (AHR), and positive skin prick test (posSPT) in a sample of adult twins. METHODS: Within a sampling frame of 21,162 twin subjects, 20-49 years of age, from the Danish Twin Registry, a total of 575 subjects (256 intact pairs and 63 single twins), who either themselves and/or their co-twins reported a history of asthma at a nationwide questionnaire survey, were clinically examined. Symptoms of wheeze and rhinitis were obtained by interview; airway responsiveness and skin test reactivity were measured using standard techniques. Correlations in liability between the different traits were estimated and latent factor models of genetic and environmental effects were fitted to the observed data using maximum likelihood methods. RESULTS: The various phenotypic correlations between wheeze, rhinitis, AHR and posSPT were all significant and ranged between 0.50 and 0.86. Traits that showed highest genetic correlations were wheeze-rhinitis (rho(A)=0.95), wheeze-AHR (rho(A)=0.85) and rhinitis-posSPT (rho(A)=0.92), whereas lower genetic correlations were observed for rhinitis-AHR (rho(A)=0.43) and AHR-posSPT (rho(A)=0.59). Traits with a high degree of environmental sharing were rhinitis-posSPT (rho(E)=0.92) and wheeze-posSPT (rho(E)=0.71), whereas a lower environmental correlation was seen for wheeze-rhinitis (rho(E)=0.25). The estimates were corrected for ascertainment and adjusted for age, sex, inhaled corticosteroids and smoking. CONCLUSIONS: Different atopic conditions share, to a large extent, a common genetic background. In particular, upper and lower respiratory symptoms seem to be different phenotypic expressions of a common set of genes. These results add new insight into the origins of clinical heterogeneity within atopy and should stimulate the search for pleiotropic genes of importance for these conditions.     

B- and T-cell determination in ALL.

The influence of different cytostatic treatment schemes on T- and B-cell behaviour has been studied in children with ALL and UAL. Under these treatment schemes the percentage of B-cells decreased, while the percentage of T-cells remained within normal limits. In long-term remissions, we found normal values. In the first attack of ALL before therapy, a different behaviour of lymphoid cells was found. One group of patients had no lymphoid cells with membrane-bound Ig and no rosette forming cells; a second group had mainly T-cells, and a third group showed normal values of lymphoid cells with membrane-bound Ig, but the ability of rosette formation was slightly diminished. Whether this membrane-bound Ig is really cell-produced or only attached to the cell-membrane (blocking antibodies?) is discussed. Capping and pitching behaviour was observed. There were no differences in cell-membrane fluidity as compared with the normal controls.     

Studies on B- and T-cell receptors for lysozyme.

BALB/c mice have been immunized by intravenous administration of native or reduced and alkylated lysozyme. Primary immune response to these antigens was studied at the humoral level (by the Farr assay) and at the cellular level (by the rosette and the plaque assays using lysozyme coupled to sheep or pigeon erythrocytes). Antibodies and theta-negative RFC were specific for the antigen used for immunization. Specific inhibition of theta-negative RFC after incubation with the soluble immunizing antigen confirmed this specificity. Conversely, most theta-positive RFC had double specificity both to native and denatured lysozyme and showed lower avidity for the immunizing antigen, as shown by inhibition studies with soluble antigen. These data suggest that T cells have a broader specificity for lysozyme than B cells and also that, in this particular system, cytophilic antibodies are probably not responsible for the formation of theta-positive rosettes.     

Breaking B-cell tolerance and CTL tolerance in three OVA-transgenic mouse strains expressing different levels of OVA

It is of major importance to overcome the immunological tolerance in attempts to generate efficient tumour vaccines. Here, we describe induction of autoantibodies and self-reactive CTL in three types of OVA-transgenic mouse strains, RIP-OVA^low, RIP-mOVA and RIP-OVA^HI exhibiting varying levels of OVA expression and tolerance. This was achieved by immunizing with DNA constructs where a foreign T-helper epitope, P30 from tetanus toxin, was inserted into the OVA sequence. OVA wild-type DNA as well as the P30-modified OVA DNA vaccines (OVA-P30) were constructed and used for immunization in the OVA-transgenic mouse strains as well as in control C57Bl/6 mice. The data show that insertion of a foreign T-helper peptide (P30) in OVA is sufficient for breaking B-cell tolerance in three different OVA-transgenic mice strain. This approach is sufficient for induction of self-reactive CTL in two of the three strains that expressed either a membrane-bound form of OVA or a low amount of soluble OVA. It was not possible to induce CTL but still possible to induce autoantibodies in the strain that expressed a higher level of soluble OVA.     

Epitope-specific T-cell responses and allergic phenotypes: implications for T-cell peptide therapy

In the 1990s, elucidation of the primary amino acid sequence of several major allergens using molecular cloning techniques opened the door to T-cell epitope mapping studies. Such analyses underscored the complexity of the allergen-specific T-cell repertoire and the challenges to using allergen-derived peptides to identify epitope-specific differences associated with allergic and nonallergic responses. This review highlights important factors that may influence the nature of epitope-specific T-cell responses observed in vitro. These include the properties of the allergen, genetics of the host and selection of patients with defined allergic phenotypes based on serum antibody profiles and skin test reactivity. By taking these factors into account, T-cell epitope-specific differences associated with distinct allergic phenotypes can be identified. Observations at the T-cell epitope level undermine the Th1/Th2 paradigm as a model for the development of allergic versus nonallergic responses. Instead, they support the mounting data that point to a network of interactions between T helper cells and regulatory T cells, which controls the allergic response. The ability of peptides that localize to polypeptide chain 2 of the major cat allergen, Fel d 1, to preferentially induce interleukin-10 and interferon-γ is discussed. Mechanisms whereby specific allergen-derived peptides may modify the T-cell repertoire and influence the immune outcome are also outlined. Further investigation of allergen-derived T-cell epitopes is warranted in order to optimize the design of peptide vaccines for the treatment of allergic disease.     

B- and T-cell autoantigens in pristane-induced arthritis.

Pristane-induced arthritis (PIA) is a murine disease resembling rheumatoid arthritis (RA) which is characterized by autoimmune responses to joint tissues. To identify the range of potential antigens targeted in PIA, proteins from arthritic or normal joint extracts were fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and systematically screened for the ability to react with either serum IgG, or cultured splenic T cells, obtained from healthy or arthritic mice. Extracts from both normal and arthritic animals contained multiple proteins that were capable of reacting with murine serum IgG in immunoblotting experiments. In healthy controls, more bands were identified in extracts prepared from 30-week-old mice than from 8-week-old animals, but the widest range of proteins bound were derived from arthritic joints. Furthermore, the sera from PIA-positive mice reacted with more bands from each of the extracts than did normal sera. Fractionated extracts prepared from healthy joints failed to stimulate the in vitro proliferation of splenic T cells from either normal or arthritic animals. When arthritic joint components were screened, T cells from healthy mice responded weakly to some fractions, but multiple fractions elicited strong proliferation by T cells from mice with PIA. A band of apparent molecular mass 60000 was the protein most commonly bound by serum IgG from arthritic mice, and the corresponding fraction stimulated the highest responses by T cells from PIA-positive animals. These results are consistent with the notion that the 60,000 MW mammalian heat-shock protein is an important antigen in PIA, but that the autoimmune response diversifies with the development of arthritis to target multiple joint components.     

Genetic control of oral tolerance to ovalbumin in mice.

We have investigated the genetic basis of oral tolerance to OVA in a number of inbred mouse strains. Our results emphasise the efficiency of the oral route for inducing tolerance and provide evidence for both MHC and non-MHC linked control of oral tolerance.     

Genetic lesions in T-cell tolerance and thresholds for autoimmunity

Summary: The cause of common organ‐specific autoimmune diseases is poorly understood because of genetic and cellular complexity in humans and animals. Recent advances in the understanding of the mechanisms of the defects underlying autoimmune disease in autoimmune polyendocrinopathy syndrome type 1 and non‐obese diabetic mice suggest that failures in central tolerance play a key role in predisposition towards organ‐specific autoimmunity. The lessons from such rare monogenic autoimmune disorders and well‐characterized polygenic traits demonstrate how subtle quantitative trait loci can result in large changes in the susceptibility to autoimmunity. These data allow us to propose a model relating efficiency of thymic deletion to T‐cell tolerance and susceptibility to autoimmunity.     

Effects of aging on early B- and T-cell development

Summary: Lymphocyte production in the bone marrow and the thymus is reduced during aging, but why this decline occurs has not been fully elucidated. The ability to isolate hematopoietic stem and progenitor cells using sophisticated flow cytometric strategies and to manipulate them in vitro and in vivo has provided insights into the effects of aging on primary lymphopoiesis. These analyses have showed that intrinsic changes in hematopoietic precursors that affect their proliferative potential are one factor that contributes to the age‐related decline in B‐ and T‐cell production. This and other age‐related defects may be exacerbated by changes in the lymphopoietic support potential of the bone marrow and thymic microenvironments as well as by age‐induced fluctuations in the production of various endocrine hormones. Particular attention with regard to the latter point has focused on changes in the production of sex steroids, growth hormone, and insulin‐like growth factor‐I. The present review summarizes recent studies of how age‐related perturbations affect primary lymphopoiesis and highlights how the data necessitate the reevaluation of a number of existing paradigms.     

Evidence for the genetic control of estradiol-regulated responses. Implications for variation in normal and pathological hormone-dependent phenotypes.

The ovarian steroid hormone estrogen (E2) elicits a multiplicity of both systemic and uterotropic responses in vivo. For example, the administration of E2 to ovariectomized (Ovx) and sexually immature rodents leads to uterine-specific inflammatory infiltrates. In this study, we quantitated the number of eosinophils and BM8+, Ia+, and CD4+ cells in uteri obtained from adult Ovx control and E2-treated C57BL/6J, C3H/HeJ, and (C57BL/6J x C3H/HeJ) (B6C3) F1 hybrid mice. All three strains exhibited a significant increase in the number of uterine eosinophils and BM8+ macrophages after E2 treatment. However, C57BL/6J and B6C3 F1 hybrid mice responded with a greater number of infiltrating eosinophils and macrophages as compared with C3H/HeJ. A similar analysis of Ia+ and CD4+ cells showed that E2 treatment either down-regulates or does not affect the number of such cells in all three strains. Genome exclusion mapping using a (C57BL/6J x C3H/HeJ) x C3H/HeJ backcross population localized Est1, the major locus controlling the number of eosinophils infiltrating the uterus after E2 treatment, to chromosome 4. In addition, suggestive linkage to marker loci on chromosomes 10 and 16 was detected and evidence for locus interaction is presented. Our results conclusively demonstrate that E2-regulated/ dependent responses can be genetically controlled, indicating that the phenotypic variation observed in both the normal and pathological effects of E2 may, in part, be due to a genetic component.     

Evidence for the genetic control of antibody affinity from breeding studies with inbred mouse strains producing high and low affinity antibody.

The amount (Abt) and relative affinity (KR) of antibody produced in response to protein antigens injected in saline has been measured in the parents, F1 hybrids and backcross offspring of inbred mice which produce high and low KR antibody to these antigens. The results obtained support the view that antibody affinity is under polygenic control. Furthermore, strain related variation in Abt is independent of KR and the breeding experiments indicate that these two parameters are under independent genetic control.     

Some new host-range phenotypes of T4D bacteriophage and their genetic control

Characteristics of some newly isolated host-range mutants of T4D, phenotypes h₁ and h₁h₃, are described. The genetic loci controlling these phenotypes are linked relatively closely to each other on the linkage group II, near the locus tu₄₁. The functions of these loci are discussed in respect to virus infection.     

T-cell tolerance and autoimmunity in transgenic models of central and peripheral tolerance

Experiments with transgenic mice expressing genes encoding both antigens in defined tissues and T-cell receptor genes of known specificities have enhanced our understanding of the mechanisms involved in the pathogenesis of autoimmune states. They have also shed light on the means by which potentially autoreactive cells may be prevented from exerting their autoaggressive potential. The value of the transgenic approach is that it can overcome the low frequency of peptide-specific T cells occurring in normal animals, and also provide a tissue-specific, cognate antigen that is absent in controls. These factors allow reactive T cells to be isolated or quantified by flow cytometry and their responses to antigen in vitro and in vivo be defined.     

Optimizing the germfree mouse model for in vivo evaluation of oral Vibrio cholerae vaccine and vector strains

The germfree mouse model of Vibrio cholerae infection can be used to judge immune responses to V. cholerae vaccine and vector strains. In the original model, a single oral inoculation was administered on day 0, a booster oral inoculation was administered on day 14, and immune responses were analyzed with samples collected on day 28. Unfortunately, immune responses in this model frequently were low level, and interanimal variability occurred. In order to improve this model, we evaluated various primary and booster V. cholerae inoculation schedules. The most prominent systemic and mucosal antibody responses were measured in mice that received a multiple primary inoculation series on days 0, 2, 4, and 6 and booster inoculations on days 28 and 42. These modifications result in improved preliminary evaluation of V. cholerae vaccine and vector strains in mice.     

Discovery and genetic localization of Down syndrome cerebellar phenotypes using the Ts65Dn mouse

Down syndrome (DS) is the most common genetic cause of mental retardation and affects many aspects of brain development. DS individuals exhibit an overall reduction in brain size with a disproportionately greater reduction in cerebellar volume. The Ts65Dn mouse is segmentally trisomic for the distal 12-15 Mb of mouse chromosome 16, a region that shows perfect conserved linkage with human chromosome 21, and therefore provides a genetic model for DS. In this study, high resolution magnetic resonance imaging and histological analysis demonstrate precise neuro- anatomical parallels between the DS and the Ts65Dn cerebellum. Cerebellar volume is significantly reduced in Ts65Dn mice due to reduction of both the internal granule layer and the molecular layer of the cerebellum. Granule cell number is further reduced by a decrease in cell density in the internal granule layer. Despite these changes in Ts65Dn cerebellar structure, motor deficits have not been detected in several tests. Reduction in granule cell density in Ts65Dn mice correctly predicts an analogous pathology in humans; a significant reduction in granule cell density in the DS cerebellum is reported here for the first time. The candidate region of genes on chromosome 21 affecting cerebellar development in DS is therefore delimited to the subset of genes whose orthologs are at dosage imbalance in Ts65Dn mice, providing the first localization of genes affecting a neuroanatomical phenotype in DS. The application of this model for analysis of developmental perturbations is extended by the accurate prediction of DS cerebellar phenotypes.     

A rice-based edible vaccine expressing multiple T-cell epitopes to induce oral tolerance and inhibit allergy

Plant pollens are the most common cause of seasonal allergic disease. The number of patients undergoing treatment for allergies to the pollen of Japanese cedar (major antigens, Cry j 1 and Cry j 2) has increased steadily each year. A rice seed-based edible vaccine has been shown to be effective for treating Japanese cedar pollinosis. Rice seeds containing the major T-cell epitopes derived from cedar pollen allergens were orally administrated to mice before systemic challenge with total pollen protein. Mucosal immune tolerance leading to a reduction of allergen-specific IgE, T-cell proliferative reactions, and histamine were induced, resulting in suppression of allergy-specific symptoms such as sneezing. Oral seed-based peptide immunotherapy offers a safe, simple, and cost-effective alternative to conventional allergen-specific immunotherapy using crude allergen extracts for treating allergic disease. A human version of rice seed-based edible vaccine containing seven T-cell epitopes from the Cry j 1 and Cry j 2 allergens was recently developed and is undergoing safety assessments.