Alexander Faith

IL-10 directly acts on T cells by specifically altering the CD28 co-stimulation pathway

IL‐10 induces T cell anergy in numerous mouse models and specific immunotherapy of allergy in humans. Here, we demonstrate that IL‐10 directly acts on T cells which are stimulated via CD28 by efficiently blocking proliferation and cytokine production. T cells tolerized by IL‐10 showed high viability and the unresponsive state was reversed by anti‐CD3 monoclonal antibody (mAb) stimulation and IL‐2, but not by anti‐CD28 mAb stimulation. Signal transduction via CD28 requires CD28 tyrosine phosphorylation and binding of phosphatidylinositol 3‐kinase. IL‐10 inhibited tyrosine phosphorylation of CD28; thus, the phosphatidylinositol 3‐kinase binding to CD28 was blocked. Consequently, IL‐10 inhibited the antigen‐induced secretion of both Th1 and Th2 cytokines, including IL‐2, IFN‐γ, IL‐4, IL‐5 and IL‐13. Furthermore, neutralization of endogenously produced IL‐10 significantly increased T cell proliferation and both Th1 and Th2 cytokine production in vitro. Using superantigen stimuli, T cell suppression by IL‐10 was merely induced at low doses when co‐stimulation by CD28 was essential. Together, these data demonstrate that IL‐10 directly acts on the CD28 signaling pathway and this represents an important T cell suppression mechanism leading to anergy.

A molecular basis for T cell suppression by IL-10: CD28-associated IL-10 receptor inhibits CD28 tyrosine phosphorylation and phosphatidylinositol 3-kinase binding

Specific immune suppression and induction of anergy in T cells are essential processes in regulation and circumvention of immune defense. IL‐10, a suppressor cytokine of T cell proliferative and cytokine responses, plays a key regulatory role in tolerizing exogenous antigens during specific immunotherapy and natural exposure. The present study demonstrates that IL‐10 induces T cell suppression by blocking the CD28 costimulatory signal. T cell receptor counting and T cell proliferation studies by anti‐CD3 and anti‐CD28 stimulation in the presence or absence of IL‐10 revealed that IL‐10 only inhibits T cells stimulated by low numbers of triggered T cell receptors and that depend on CD28 costimulation. T cells receiving a strong signal by the T cell receptor alone and that do not require CD28 costimulation are therefore not affected by IL‐10. Coprecipitation experiments demonstrated that CD28 and the IL‐10 receptor are associated in activated T cells. IL‐10 inhibited CD28 tyrosine phosphorylation, the initial step of the CD28 signaling pathway. In consequence, phosphatidylinositol 3‐kinase p85 binding to CD28 was inhibited. Thus, IL‐10‐induced selective inhibition of the CD28 costimulatory pathway demonstrates a decisive mechanism in determining whether a T cell will contribute to an immune response or become anergic.

Determinants and Mechanisms of Human Immune Responses to Bee Venom Phospholipase A2

The elicitation of an immune response to protein antigens depends on the specific recognition of antigenic determinants (epitopes) by T and B lymphocytes. Bee venom phospholipase A2 (PLA) represents the major antigen/allergen of honey bee venom. It displays three dominant immunogenic peptide and one glycopeptide T cell recognition sites. These epitopes are equally recognized by both allergic and nonallergic individuals. A mixture of the three epitope containing peptides was successfully used in specific immunotherapy of bee venom–allergic patients. Both peptide and whole bee venom immunotherapy induced a state of specific anergy in T cells. The production of specific IgE and IgG4 antibodies directly correlated with the secreted interleukin–4:γ–interferon (IL–4:IFNγ) ratio, which itself depended on the concentration of available antigen and the strength of the T cell–activating signal. This signal comprises accumulated molecular interactions delivered by engagement of the antigenic peptide/MHC class II complex with the T cell receptor (TcR). Indeed the thermodynamic laws of chemical equilibrium reactions reveal that the antigen concentration, together with the equilibration constant Ki and the related Gibbs standard free energy ΔG° of the MHC–II/Ag/TcR complex reaction, may govern the secreted IL–4:IFNγ ratio, and in consequence, differential IgE and IgG4 antibody formation. Ki includes epitope and MHC–II haplotype variability and therefore represents a measure of immunological individuality. A major B cell epitope was determined by using point–mutated PLA. Specific antigen recognition by B cells can trigger distinct cytokine profiles in T cells and contribute to the differential regulation of specific IgE and IgG4 antibodies. Our results indicate that distinct cytokine profiles inducing allergic and nonallergic responses can be attributed to thresholds of T cell activation generated by the specific binding properties of individual MHC–II molecules to immunogenic T cell epitopes and their presentation to TcR.

Cloning, production, characterization and IgE cross-reactivity of different manganese superoxide dismutases in individuals sensitized to Aspergillus fumigatus.

BACKGROUND Manganese superoxide dismutase (MnSOD) from Aspergillus fumigatus has been demonstrated to be an allergen showing a high degree of homology with phylogenetically distant MnSODs. We describe cloning, production and characterization of MnSODs from different species. METHODS MnSODs were cloned by PCR, expressed as inclusion body protein in Escherichia coli, purified by Ni2+-chelate affinity chromatography and characterized. RESULTS The MnSODs from A. fumigatus, man, yeast, Drosophila melanogaster and E. coli show about 50% identity and 70% similarity on the primary structure. All proteins were produced at a high level in E. coli and refolded to achieve enzymatic activity. The proteins were able to bind IgE from sera of individuals sensitized to the A. fumigatus MnSOD. CONCLUSIONS MnSOD represents a novel pan-allergen restricted to individuals sensitized to A. fumigatus.

Impaired secretion of interleukin-4 and interleukin-13 by allergen-specific T cells correlates with defective nuclear expression of NF-AT2 and jun B: relevance to immunotherapy.

Background Allergen immunotherapy (IT) is a successful treatment associated with decreased Th2 cytokine production by allergen‐specific T cells. We have previously demonstrated (Faith et al., J Immunol 1997; 159:53–57) that inhibition of Th2 cytokine production in vitro correlates with impaired tyrosine kinase activity through the TCR. The transcription factor complex, nuclear factor of activated T cells (NF‐AT), which regulates Th2 cytokine production is controlled by the activity of tyrosine kinases.

T cells producing the anti-inflammatory cytokine IL-10 regulate allergen-specific Th2 responses in human airways.

Murine models suggest a critical functional role for the anti‐inflammatory cytokine IL‐10 in local regulation of allergic airways inflammation. There is little corresponding information on human airway cells. This study aimed to investigate whether local IL‐10 production regulates responses by respiratory mucosal leucocytes isolated from nasal polyps.

Counter regulation of the high affinity IgE receptor, FcεRI, on human airway dendritic cells by IL-4 and IL-10

Background:  Immunoglobulin E is a signalling molecule within the environment of the respiratory tract, the high affinity receptor for which, FcεRI, is expressed by dendritic cells (DC). Little is known, however, of the expression and function of FcεRI on DC in the human respiratory tract.

Role of cysteinyl leukotrienes in human allergen-specific Th2 responses induced by granulocyte macrophage-colony stimulating factor.

Background:  The pro‐inflammatory cytokine, granulocyte macrophage‐colony stimulating factor (GM‐CSF), which is elevated in the lungs of atopic asthmatic patients, has been shown to enhance major histocompatibility class II expression of alveolar macrophages (AM). We hypothesized that exposure of AM and monocytes from atopic asthmatic patients to GM‐CSF would enhance their antigen presenting function, and investigated putative mechanisms for this effect.

Urban Particulate Matter Suppresses Priming of T Helper Type 1 Cells by Granulocyte/Macrophage Colony–Stimulating Factor–Activated Human Dendritic Cells

Urban particulate matter (UPM) exacerbates asthmatic lung inflammation and depresses lung immunity. Lung dendritic cells (DCs) react to airway particulates, and have a critical role in linking innate and adaptive immunity, but the direct effects of UPM on DCs, that have been activated by granulocyte/macrophage colony-stimulating factor (GM-CSF), a product of stimulated normal human bronchial epithelial cells, has not been investigated. Human blood CD1c(+) DCs were purified and activated with UPM in the presence or absence of GM-CSF with and without LPS, and DC maturation was assessed by flow cytometry. DC stimulatory capacity and priming of 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester-labeled naive CD4 T cells was investigated using the allogeneic mixed lymphocyte reaction. T cell proliferation and effector function were assessed using flow cytometry and secreted cytokines were measured by combined bead array. UPM enhanced DC maturation in an LPS-independent manner. DCs activated by UPM plus GM-CSF (UPM + GM-CSF DCs) induced higher naive CD4 T cell proliferation in the allogeneic mixed lymphocyte reaction than DCs pretreated by GM-CSF alone (GM-CSF DCs), and elicited both substantially lower levels of IFN-γ, IL-13, and IL-5 secretion, and lower frequencies of alloantigen-specific T helper (Th) type 1 effector cells than naive CD4 T cells primed by GM-CSF DCs. UPM-stimulated DCs produced IL-6 and TNF-α. Neutralization of IL-6 decreased naive CD4 T cell proliferation stimulated by UPM + GM-CSF DCs, and significantly increased the frequency of alloantigen-specific Th1 effector cells, but did not reverse UPM-induced inhibition of IFN-γ secretion. We conclude that UPM enhances GM-CSF-induced DC maturation and stimulatory capacity, but inhibits the generation of Th1 cells. Thus, UPM exposure may impair Th1 responses to pulmonary pathogens.

Targeting the dendritic cell: the key to immunotherapy in cancer?

Immunotherapy denotes a strategy for manipulating a patients immune response [1]. In cancer or infectious disease the approach is designed to boost the patients response to tumour antigens or pathogens. Conversely, immunotherapeutic strategies in autoimmunity or allergy are designed to silence the patients response to autoantigens or allergens. Two principal approaches to immunotherapy, modulation of the immune system de novo by therapeutic vaccination or administration of exogenous reagents, such as cytokines and antibodies, in order to boost endogenous immune function, have been described [2].