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Antibody‐mediated immunity is highly protective against disease. The majority of current vaccines confer protection through humoral immunity, but there is high variability in responsiveness across populations. Identifying immune mechanisms that mediate low antibody responsiveness may provide potential strategies to boost vaccine efficacy. Here, we report diverse antibody responsiveness to unadjuvanted as well as adjuvanted immunization in substrains of BALB/c mice, resulting in high and low antibody response phenotypes. Furthermore, these antibody phenotypes were not affected by changes in environmental factors such as the gut microbiota composition. Antigen‐specific B cells following immunization had a marked difference in capability to class switch, resulting in perturbed IgG isotype antibody production. In vitro, a B‐cell intrinsic defect in the regulation of class‐switch recombination was identified in mice with low IgG antibody production. Whole genome sequencing identified polymorphisms associated with the magnitude of antibody produced, and we propose candidate genes that may regulate isotype class‐switching capability. This study highlights that mice sourced from different vendors can have significantly altered humoral immune response profiles, and provides a resource to interrogate genetic regulators of antibody responsiveness. Together these results further our understanding of immune heterogeneity and suggest additional research on the genetic influences of adjuvanted vaccine strategies is warranted for enhancing vaccine efficacy.

Background and objective Neutrophilic inflammation has been implicated in non-eosinophilic asthma (NEA) in adults, but little is known about NEA in children/adolescents. We assessed clinical and inflammatory characteristics of NEA in adolescent asthma. Methods Airway inflammation, sputum endotoxin, airway hyper-reactivity, atopy and lung function were assessed in 77 adolescents with asthma and 68 without asthma (12–17 years). Asthma was identified on the basis of wheeze and asthma history. Results The proportion of NEA (sputum eosinophils

A keenly sought therapeutic approach for the treatment of allergic disease is the identification and neutralization of the cytokine that regulates the differentiation of T helper 2 (Th2) cells. Th2 cells are exciting targets for asthma therapies. Recently, the cytokine IL-25 has been shown to enhance Th2-type immune activity and play important roles in mediating allergic inflammatory responses. To investigate this further, we crossed IL-25(-/-) C57BL/6 mice with G4 IL-4 C57BL/6 reporter mice and developed an assay for in vitro and in vivo IL-4-independent Th2-cell differentiation. These assays were used to determine whether IL-25 was critical for the formation of Th2 cells. We found there was no physiological role for IL-25 in either the differentiation of Th2 cells or their development to effector or memory Th2-cell subsets. Importantly, this data challenges the newly found and growing status of the cytokine IL-25 and its proposed role in promoting Th2-cell responses.

The immune mechanisms that orchestrate protection against tuberculosis as a result of BCG vaccination are not fully understood. We used the immunomodulatory properties of fingolimod (FTY720) treatment to test whether the lung-resident memory T lymphocytes generated by BCG vaccination were sufficient to maintain immunity against challenge infection with mycobacteria (BCG). Mice were given daily fingolimod treatment, starting either immediately before s.c. BCG vaccination or during subsequent BCG i.n. challenge, to prevent LN effector and memory lymphocytes from entering the periphery either during priming or challenge, respectively. Treatment with fingolimod during vaccination reduced vaccine-mediated protection against subsequent infection. By contrast, BCG-vaccinated mice were protected when fingolimod was given during the infectious challenge, suggesting that memory lymphocytes that migrate to the lung following vaccination are sufficient for protection. Notably, the antigen-reactive IFN-gamma or multicytokine-producing CD4(+) T cells present in the lung when fingolimod was given during BCG challenge did not correlate with protection; however, expression of MHC class II on macrophages isolated from the lungs post BCG challenge was increased in the protected mice. We conclude that protection conferred by BCG vaccination is dependent on memory lymphocytes retained in the lung, although IFN-gamma production by this population is not correlated with vaccine-mediated protection.

Improved analytical tools have revealed that the development and expression of a Th2 immune response can be broken down into distinct stages with respect to the cytokine microenvironment that is required. Although IL-4 and its STAT6-signalling pathway are critical for the expression of Th2 effector immune responses in peripheral tissues such as the skin, lung and gut, IL-4 and STAT6 signalling are not required for the initial generation of IL-4-producing Th2 cells in the lymph node. This finding reveals that we have yet to identify the key cytokine or microenvironment that stimulates the development of this most intriguing CD4(+) T-helper subset and emphasises the tissue specificity and timing of IL-4/STAT6-dependent Th2 effector responses.

Previous activation of effector Th2 cells is central to the development of allergic inflammatory responses. We have observed that priming of allergen-specific Th2 cells in C57BL/6 or B10.A mice with allergen delivered via the i.p. or s.c. routes results in very different outcomes following subsequent airway exposure to the same allergen. Systemic allergen immunization (via the i.p. route) resulted in the formation of a lung-resident population of allergen-specific T cells, and mice developed severe allergic airway inflammation in response to inhaled allergen. The localization of cells to the lung did not require the presence of antigen at this site, but reflected a large pool of circulating activated allergen-specific T cells. In contrast, localized immunization (via the s.c. route) resulted in a small T-cell response restricted to the draining lymph node, and mice were not responsive to inhaled allergen. These data indicate that prior sensitization to an allergen alone was not sufficient for the induction of allergic inflammation; rather, responsiveness was largely determined by precursor frequency and tissue localization of the allergen-specific effector Th2 cells.

Allergic asthma is responsible for widespread morbidity and mortality and its incidence has increased dramatically in industrialized countries over the past two decades. Here, we describe a new murine model of allergic asthma utilizing a novel allergen with intrinsic enzymatic activity similar to that reported for many environmental allergens. The allergen, NES, is excreted and secreted from the nematode Nippostrongylus brasiliensis, and can readily be isolated from in vitro parasite cultures. When NES is administered intranasally to presensitized mice, allergic airway disease develops, including airway hyper-responsiveness, airway eosinophilia, IgE antibody production and Th2 cytokine production. This disease is characteristic of atopic asthma and can be induced within 11 days, thus providing a platform for the rapid analysis of allergic disease and high throughput testing of immunomodulatory factors.

CTLA4 is currently viewed as a late-appearing T cell surface receptor which is able to inhibit the proliferation of activated T cells. We sought to identify how CTLA4 ligation exerts these anti-proliferative effects by studying its influence on the activities of the relevant nuclear transcription factors AP-1, NFAT and NF-kappaB. We found that cross-linking CTLA4 on activated T cells completely blocks AP-1 and NFAT transcription factor activity before any effects on T cell proliferation can be observed, with NF-kappaB activity affected to a lesser degree. The suppression of AP-1 and NFAT transcriptional activity correlates with reduced levels of AP-1 and NFAT DNA binding as early as 10 h after T cell activation, prior to detectable up-regulation of CTLA4 on the T cell surface. Additionally, inhibitory effects on T cell proliferation only occurred when CTLA4 molecules were ligated in proximity to signaling TCR complexes, and inhibition of transcription factor DNA binding and activity was observed in the absence of CD28 stimulation. CTLA4 can thus act early during T cell activation to reduce the activity of several key nuclear transcription factors important for continued T cell proliferation and differentiation.

Top of pageAbstract Mycobacterial infections in mice are normally characterized by a profound Th1 cell-mediated immune response, in which T cells secrete large amounts of IFN-. Recent evidence suggests that this response also includes a Th2 component. In order to investigate whether production of IL-4, IL-5, or IL-10 influenced the outcome of a Mycobacterium bovis-bacille Calmette-Guerin (BCG) infection, we intranasally infected IL-4, IL-5, and IL-10 gene-deficient and control mice and monitored the resulting immune response and bacterial clearance. IL-4, IL-5, and IL-10 deficient mice cleared the mycobacteria with the same kinetics as control mice. Furthermore, T cells of cytokine deficient and control mice produced similar levels of IFN- following in vitro stimulation with purified protein derivative (PPD) from M. bovis. We conclude that the cytokines IL-4, IL-5 and IL-10 are not essential for and do not negatively influence the protective immune response against M. bovis-BCG in the lung of mice. Keywords: IL-4, IL-5, IL-10, mycobacteria

We have studied the role of the CD28/B7 co-stimulatory pathway in the development of a Th2-type lung immune response. Mice injected two or three times intraperitoneally with ovalbumin in alum adjuvant and then re-exposed to the same antigen by intranasal (i.n.) inoculation show infiltration of the lung tissue and appearance in the broncho-alveolar lavage (BAL) fluid of significant numbers of eosinophils and lymphocytes, in a pattern which is reminiscent of asthmatic inflammation. The accumulation of eosinophils in the airways is completely dependent on interleukin (IL)-5 secretion by CD4+ T cells. We have used mice transgenic for a soluble form of murine CTLA-4 (mCTLA4-Hgamma1) which binds to B7 molecules on antigen-presenting cells, thereby preventing their interaction with T cell-expressed CD28. mCTLA4-Hgamma1-transgenic mice immunized intraperitoneally and challenged i.n. with ovalbumin failed to generate any eosinophil infiltration, suggesting that little or no IL-5 was secreted in the lungs of these mice. In contrast with the complete lack of eosinophils, the numbers and phenotypes of infiltrating lymphocytes were comparable in the lungs of mCTLA4-Hgamma1-transgenic and normal mice. Also, lung lymphocytes from immunized mCTLA4-Hgamma1-transgenic and normal mice could be shown to secrete comparable amounts of IL-4 and IL-5 when stimulated in culture in the absence of mCTLA4-Hgamma1. We conclude that mCTLA4-Hgamma1 can efficiently block the production of IL-5 during in vivo responses and inhibit eosinophil recruitment, but that it does not block the development of CD4+ T cells into Th2 cells with the potential to secrete IL-5.

The switch of activated B cells to IgE synthesis is an interleukin (IL)-3-dependent process. It is currently thought that specific T cells activated by antigen presented in the context of class II major histocompatibility complex are the major source of IL-4. Recently it has been demonstrated that a splenic non-T non-B cell population (termed NBNT) has the capacity to produce IL-4 following IgE and IgG receptor cross-linkage. In this study we demonstrate that IL-4 producing NBNT cells can induce the switch of lipopolysaccharide-activated B cells to the synthesis of IgG1 and IgE antibodies. Furthermore, it was found that not only IgE receptor cross-linkage but IL-3 was able to stimulate NBNT cells to produce IL-4 and induce the switch of B cells to IgE synthesis. NBNT cells derived from the spleen and bone marrow of SCID mice were able to produce IL-4 on exposure to IL-3. This suggested that the ability of IL-3 to stimulate IL-4 production was not dependent on prior exposure of the NBNT cells to antibody complexes in vivo. Taken together these findings represent the first observation that enough IL-4 is produced by NBNT cells to actually influence a B cell IgG/Ig response. The findings also clearly demonstrate that B cells do not need high concentrations of IL-4 to be directed to switch to IgG1 and IgE synthesis.

The cellular inflammatory responses which are seen in allergic and asthmatic diseases are viewed as being quite strongly dependent on the activities of T cells and their products. The major T cell subset involved appears to be the so-called CD4+ Th2 subset which produces interleukin-4 (IL-4) and interleukin-5 (IL-5). In vitro and in vivo experiments have indicated that IL-4 is a key regulator in these kinds of immune responses, not only switching B cells to IgE production, but acting on CD4+ T cells to drive their development towards a Th2 phenotype. Recent results have shown that the functional phenotype of CD8+ T cells can be switched from interferon gamma production to IL-4 and IL-5 production by the presence of IL-4. This could prove an especially important phenomenon since it is the production of interferon gamma by CD8 T cells which is seen as necessary for protection against virus infection. This short review updates our current knowledge of how IL-4 can act on CD4+ and CD8+ T cell subsets in in vivo models of asthma and allergic disease.

The uptake of alpha-ketoglutarate and malate by rat brain synaptosomal preparations was found to be affected by a variety of substances at physiologically relevant concentrations. Glutamine altered the uptake of alpha-ketoglutarate by causing an apparent reduction in the substrate-carrier affinity and an increase in Vmax. In contrast, glutamine did not appear to affect the Vmax of malate uptake, but it did increase markedly the uptake velocity at low concentrations of malate. L-Glutamate and L-aspartate were comparatively strong inhibitors of alpha-ketoglutarate and malate uptake. N-Acetylaspartate was a weak inhibitor of alpha-ketoglutarate uptake, a finding that contrasts with our previous observation that this compound potently inhibited alpha-ketoglutarate uptake by synaptosomes obtained from the cerebellum of 8- to 14-day-old mice. Ca2+ exhibited a variable effect but usually enhanced the uptake of alpha-ketoglutarate. The addition of small amounts of postmicrosomal supernatant to the incubation medium enhanced the uptake of alpha-ketoglutarate by low-density synaptosomes. By comparison, the uptake of glutamate, glutamine, gamma-aminobutyric acid, and several other amino acids was not affected. The enhancement of alpha-ketoglutarate uptake by the supernatant was due to a heat labile substance that was retained by dialysis tubing (MW cutoff = 8,000) and Amicon filter cones (CF 25), and was precipitated by ammonium sulfate at 60% saturation. In experiments in which the metabolic conversion of [U-14C] alpha-ketoglutarate to glutamate, aspartate, glutamine, and gamma-aminobutyric acid was determined, the presence of glutamine and glutamate in the incubation medium did not affect the pattern of labelling appreciably.

This study was undertaken to provide further evidence relevant to the hypothesis that astrocytes supply one or more citric acid cycle intermediates to synaptic terminals, thereby serving an anaplerotic function necessitated by the synthesis and release of amino acid neurotransmitters. In our experiments, two populations of synaptosomes obtained from the brain of rats were separated from myelin and mitochondria by using Percoll to generate continuous density gradients. Both synaptosomal populations readily accumulated 14C-labelled alpha-ketoglutarate and L-malate by high-affinity transport systems. Hofstee plots of uptake velocity as a function of substrate concentration were highly nonlinear, indicating that uptake was mediated by two or more carriers, or was subject to negative cooperativity. At least one carrier was selective for alpha-ketoglutarate and another for malate, whereas a third carrier appeared to be present which transported both substrates. At low concentrations (approximately 1 microM), alpha-ketoglutarate transport was almost totally Na+-dependent, whereas malate uptake exhibited little Na+-dependency. The transport of alpha-ketoglutarate was associated with a net influx, and therefore was not due to a homoexchange process. alpha-Ketoglutarate and malate were metabolized rapidly to glutamate and aspartate, respectively, by both synaptosomal preparations; however, in all cases, label accumulated in gamma-aminobutyric acid rather slowly. The incorporation of label into glutamine from alpha-ketoglutarate was much greater in the high-density synaptosomes that in low-density synaptosomes, an indication that the former contained a higher proportion of astrogliasomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibody-mediated immunity is highly protective against disease. The majority of current vaccines confer protection through humoral immunity, but there is high variability in responsiveness across populations. Identifying immune mechanisms that mediate low antibody responsiveness may provide potential strategies to boost vaccine efficacy. Here, we report diverse antibody responsiveness to unadjuvanted as well as adjuvanted immunization in substrains of BALB/c mice, resulting in high and low antibody response phenotypes. Furthermore, these antibody phenotypes were not affected by changes in environmental factors such as the gut microbiota composition. Antigen-specific B cells following immunization had a marked difference in capability to class switch, resulting in perturbed IgG isotype antibody production. In vitro, a B-cell intrinsic defect in the regulation of class-switch recombination was identified in mice with low IgG antibody production. Whole genome sequencing identified polymorphisms associated with the magnitude of antibody produced, and we propose candidate genes that may regulate isotype class-switching capability. This study highlights that mice sourced from different vendors can have significantly altered humoral immune response profiles, and provides a resource to interrogate genetic regulators of antibody responsiveness. Together these results further our understanding of immune heterogeneity and suggest additional research on the genetic influences of adjuvanted vaccine strategies is warranted for enhancing vaccine efficacy.