Characterization of the human T cell response to antigen 5 from Vespula vulgaris (Ves v 5)

BACKGROUND: The T cell reactivity to the major allergen of bee venom, phospholipase A2, has been thoroughly characterized. In contrast, only little is known about the human cellular response to major allergens from wasp venom. OBJECTIVE: To characterize the human T cell response to antigen 5 from Vespula vulgaris, Ves v 5. METHODS: Recombinant Ves v 5 was used to establish allergen-specific T cell lines (TCL) and T cell clones (TCC) from the peripheral blood of vespid-allergic and non-allergic individuals. Ves v 5-specific TCL were mapped for T cell epitopes using overlapping synthetic peptides representing the complete amino acid sequence of Ves v 5. Ves v 5-specific TCC were analysed for antigen-induced secretion of IL-4, IFN-gamma and IL-10. RESULTS: Seventeen distinct T cell epitopes were recognized by allergic individuals among which Ves v 5(181-192) was identified as a dominant T cell epitope. Partially different epitopes were observed in TCL from non-allergic subjects and the dominant epitope Ves v 5(181-192) was not prevalent in these cultures. Ves v 5-specific TCC isolated from allergic individuals did not show the typical T helper type 2 (Th2)-like cytokine profile in response to specific stimulation, i.e. high amounts of IL-4 and low IFN-gamma. TCC from non-allergic individuals showed a Th1-like cytokine pattern. CONCLUSIONS: Our findings provide evidence that the allergic T cell response to Ves v 5 is not Th2-dominated and that different immunogenic sites on this major wasp venom allergen are recognized by allergic and non-allergic individuals.     

Impaired Toll‐like receptor 2 signalling in monocytes from 5‐year‐old allergic children

The relative composition of the two major monocytic subsets CD14⁺CD16⁻ and CD14⁺CD16⁺ is altered in some allergic diseases. These two subsets display different patterns of Toll-like receptor levels, which could have implications for activation of innate immunity leading to reduced immunoglobulin E-specific adaptive immune responses. This study aimed to investigate if allergic status at the age of 5 years is linked to differences in monocytic subset composition and their Toll-like receptor levels, and further, to determine if Toll-like receptor regulation and cytokine production upon microbial stimuli is influenced by the allergic phenotype. Peripheral blood mononuclear cells from 5-year-old allergic and non-allergic children were stimulated in vitro with lipopolysaccharide and peptidoglycan. Cells were analysed with flow cytometry for expression of CD14, Toll-like receptors 2 and 4 and p38-mitogen-activated protein kinase (MAPK). The release of cytokines and chemokines [tumour necrosis factor, interleukin (IL)-1β, IL-6, IL-8, IL-10, IL-12p70] into culture supernatants was measured with cytometric bead array. For unstimulated cells there were no differences in frequency of the monocytic subsets or their Toll-like receptor levels between allergic and non-allergic children. However, monocytes from allergic children had a significantly lower up-regulation of Toll-like receptor 2 upon peptidoglycan stimulation. Further, monocytes from allergic children had a higher spontaneous production of IL-6, but there were no differences between the two groups regarding p38-MAPK activity or cytokine and chemokine production upon stimulation. The allergic subjects in this study have a monocytic population that seems to display a hyporesponsive state as implicated by impaired regulation of Toll-like receptor 2 upon peptidoglycan stimulation.     

A subset of CD8+ T cells from allergic patients produce IL-4 and stimulate IgE production in vitro

Background and Objective A subset of IL-4 producing CD8⁺ T cells was recently identified in HIV patients. Based on these findings we examined whether IL-4 producing CD8⁺ T cells would also be present in allergic patients and what would be the functional relevance of this T-cell population. Methods We investigated the role of CD8⁺ T cells in IgE production of allergic diseases by analysing the cytokine profile of individual CD4⁺ and CD8⁺ T cells. Results In allergic patients about twice as many CD4⁺ T cells and six times as many CD8⁺ T cells produced IL-4 as in non-allergic controls. In contrast the frequency of IFNγ⁺ T-cell subsets did not significantly differ between the allergic and non-allergic individuals. The frequency of 1L4⁺CD8⁺ T cells correlated with the level of serum IgE. Coculture experiments with T cells or purified CD8⁺ T cells together with autologous B cells indicated that CD8⁺ T cells enhanced IgE in vitro, but not IgM production, even when they were physically separated from B cells. This effect could be partially blocked by addition of an IL-4 binding protein, a soluble IL-4 receptor indicating that lL-4 is involved in CD8⁺ T-cell mediated IgE production. Conclusions These data indicate a positive role of IL-4 secreting CD8⁺ T cells in IgE regulation in allergic patients.     

Decay-accelerating factor 1 (Daf1) deficiency exacerbates xenobiotic-induced autoimmunity

Absence of decay-accelerating factor 1 (Daf1) has been shown to enhance T-cell responses and autoimmunity via increased expression of specific cytokines, most notably interferon (IFN)-γ. To determine if Daf1 deficiency can exacerbate IFN-γ-dependent murine mercury-induced autoimmunity (mHgIA), C57/BL6 Daf1^+/+ and Daf1^−/− mice were exposed to mercuric chloride (HgCl₂) and examined for differences in cytokine expression, T-cell activation and features of humoral autoimmunity. In the absence of Daf1, mHgIA was exacerbated, with increased serum immunoglobulin G (IgG), anti-nuclear autoantibodies (ANAs) and anti-chromatin autoantibodies. This aggravated response could not be explained by increased T-cell activation but was associated with increased levels of IFN-γ, interleukin (IL)-2, IL-4 and IL-10 but not IL-17 in Daf1-deficient mice. Anti-CD3/anti-CD28 costimulation of Daf1^−/− CD4⁺ T cells in vitro was also found to increase cytokine expression, but the profile was different from that of mHgIA, suggesting that the cytokine changes observed in Daf1 deficiency reflect a response to mercury. The role of Daf1 in influencing cytokine expression was further examined by stimulation of CD4⁺ T cells in the presence of anti-CD3 and CD97, a molecular partner for Daf1. This resulted in increased IL-10, decreased IL-17 and IL-21 and decreased IFN-γ. These findings demonstrate that the absence of Daf1 exacerbates mHgIA, with changes in the profile of expressed cytokines. Interaction between Daf1 and its molecular partner CD97 was found to modify expression of mHgIA-promoting cytokines, suggesting a possible approach for the suppression of overaggressive cytokine production in autoimmunity.     

Importance of the inducible costimulator molecule for the induction of allergic immune responses and its decreased expression on T helper cells after venom immunotherapy

The inducible costimulator (ICOS), a newly identified member of the CD28 receptor family that is induced after T-cell activation, and its ligand (ICOSL), being expressed on activated monocytes and dendritic cells play a key role in T-cell-mediated immune responses. As ICOS costimulation also seems to regulate T helper 2 effector cells, the aim of this study was to analyse the function of this molecule in allergic immune responses and their specific therapy, mainly venom immunotherapy (VIT). CD4+ T cells from grass pollen-, or bee or wasp venom-allergic donors were stimulated in the presence of autologous mature dendritic cells, which were pulsed with different allergen doses. In this system, costimulation of ICOS strongly enhanced the production of the T helper 2 cytokines interleukin (IL)-4, IL-5 and IL-10 and, to a lesser extent, secretion of the T helper 1 cytokine, interferon-gamma. Expression of ICOS on CD4+ T cells was induced, in a dose-dependent manner, after a few days of stimulation with allergen-pulsed dendritic cells, reaching a peak on day 6. The upregulation of ICOS after stimulation with venom allergens was significantly reduced after VIT. Addition of exogenous IL-10 (which is induced during VIT) to the co-cultures before VIT also led to an inhibition of ICOS expression, while blocking of IL-10 in co-cultures after VIT partially restored the expression of ICOS. These data indicate that the inhibition of T cells after immunotherapy also involves decreased induction of the costimulatory molecule ICOS, which, in turn, seems to be dependent on the presence of IL-10, also associated with the inhibited status of T cells after VIT. This makes the ICOS-ICOSL pathway a potential target for therapeutic intervention in T helper 2-mediated diseases, such as allergic diseases.     

Defining the T cell antigen proteome of wasp venom

BACKGROUND: While modulation of T cell function is believed to be important in the successful acquisition of clinical tolerance during venom immunotherapy, little is known of the role of wasp venom specific T cell antigens. OBJECTIVE: We sought comprehensively to characterize the T cell proteome for wasp venom to facilitate the future development of T cell-based immunotherapeutic approaches. METHODS: Using peripheral blood mononuclear cells from wasp venom-allergic individuals and IL-4 ELISPOT analysis, we characterized T cell responses to whole venom and gel filtration/ion exchange-fractionated venom. Reactive fractions were purified and identified using highly sensitive electrospray ion-trap mass spectrometry. RESULTS: Wasp venom-allergic individuals have detectable whole wasp venom-specific T cells directly ex vivo, which show rapid IL-4 effector function. T cell responses to gel filtration/ion exchange fractionated venom were dominated by responses to phospholipase A(1), hyaluronidase and antigen 5. CONCLUSION: Although it is likely that there are many T cell antigens within wasp venom, the main responses are to proteins coincident with the known IgE-binding proteins.     

Venom immunotherapy modulates interleukin-4 and interferon-gamma messenger RNA expression of peripheral T lymphocytes.

The mechanism by which specific immunotherapy exerts its beneficial effect remains unclear. In order to evaluate the influence of venom immunotherapy on the T-cell cytokine pattern of allergic reactions, we studied interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) mRNA expression of peripheral T lymphocytes from 12 patients undergoing rush venom desensitization, before treatment at Day 0 (D0), at Day 15 (D15) and Day 90 (D90) after treatment, and from seven controls. Antigen-specific T-cell proliferation was also determined. Cytokine mRNA expression was evaluated using in situ hybridization, 24 hr after culture of peripheral T cells with medium, venom, or an unrelated allergen. Allergen-induced T-cell proliferation decreased at D15 and D90 of rush immunotherapy (P < or = 0.02). In venom-stimulated cultures of the patient group, there was a decrease in IL-4 mRNA-positive cells at D15 and D90 (P < or = 0.001). Before desensitization, IFN-gamma mRNA expression was lower in patients than in controls and did not increase after in vitro allergen stimulation. In contrast, after immunotherapy, spontaneous IFN-gamma mRNA expression increased, but only at D90 (P < or = 0.001). The cytokine pattern observed at D90 after immunotherapy was similar to that observed in control subjects. In conclusion, venom immunotherapy induced an altered cytokine mRNA pattern in allergen-stimulated T cells which was dissociated from the early changes of allergen-induced T-cell responsiveness.     

Insect venom immunotherapy induces interleukin-10 production and a Th2-to-Th1 shift,and changes surface marker expression in venom-allergic subjects

The current study was carried out to elucidate the immunoregulatory changes induced by venom immunotherapy (VIT) in bee or wasp allergic subjects. All subjects included in this study had a history of severe systemic allergic reactions to stings of the respective insect as well as positive skin tests with the respective venom or venom-specific IgE in the sera. Parameters assessed in peripheral blood mononuclear cells (PBMC) before and after initiation of VIT (rush therapy reaching a maintenance dose of 100 μg venom injected subcutaneously within 1 week) were expression of CD3, CD4, CD8, CD45RA, CD45R0, interleukin (IL)-2 receptor (R)α, IL-4R, IL-12R, FcσRII, CD40, and CD40 ligand (CD40L), cells producing interferon (IFN)-γ and IL-10 after stimulation with phorbol 12-myristate 13-acetate + ionomycin in the presence of monensin measured by flow cytometry; secretion of IFN-γ, IL-4, and IL-10 measured by ELISA (IFN-γ and IL-10 were additionally measured by PCR), and proliferation after stimulation with the respective venom. Significant decreases were observed after VIT for proliferative response to venom and venom + IL-4, IL-4 secretion, FcσRII, CD40, and CD40L expression. Significant increases were observed after VIT for IFN-γ concerning the amount secreted and the number of producing cells, and IL-10. IL-10 was mainly produced by CD4⁺ cells that were negative for IFN-γ, but some double-positive (IL-10 and IFN-γ) cells were always detected. Addition of blocking anti-IL-10 antibodies, but not isotype control antibodies, prevented down-regulation of proliferation (but not IL-4 secretion) and further enhanced IFN-γ secretion after VIT. These data indicate that in insect venom allergic subjects, VIT not only induces a rapid shift in cytokine expression from Th2 to Th1 cytokines, but also leads to induction of the immunosuppressive cytokine IL-10, which may be important for the limitation of potentially harmful allergen-specific Th1 responses. The described changes in cytokine expression may be responsible for subsequent increases in allergen-specific IgG and decreases in IgE production, as well as suppressive activity observed in earlier studies.     

Elevated basal serum tryptase and hymenoptera venom allergy: relation to severity of sting reactions and to safety and efficacy of venom immunotherapy

BACKGROUND: Mastocytosis and/or elevated basal serum tryptase may be associated with severe anaphylaxis. OBJECTIVE: To analyse Hymenoptera venom-allergic patients with regard to basal tryptase in relation to the severity of sting reactions and the safety and efficacy of venom immunotherapy. METHODS: Basal serum tryptase was measured in 259 Hymenoptera venom-allergic patients (158 honey bee, 101 Vespula). In 161 of these (104 honey bee, 57 Vespula), a sting challenge was performed during venom immunotherapy. RESULTS: Nineteen of the 259 patients had an elevated basal serum tryptase. Evidence of cutaneous mastocytosis as documented by skin biopsy was present in 3 of 16 patients (18.8%). There was a clear correlation of basal serum tryptase to the grade of the initial allergic reaction (P<0.0005). Forty-one of the 161 sting challenged patients reacted to the challenge, 34 to a bee sting and 7 to a Vespula sting. Thereof, 10 had an elevated basal serum tryptase, i.e. 1 (2.9%) of the reacting and 2 (2.9%) of the non-reacting bee venom (BV) allergic individuals, as compared to 3 (42.9%) of the reacting and 4 (8%) of the non-reacting Vespula venom-allergic patients. Thus, there was a significant association between a reaction to the sting challenge and an elevated basal serum tryptase in Vespula (chi2=6.926, P<0.01), but not in BV-allergic patients. Systemic allergic side-effects to venom immunotherapy were observed in 13.9% of patients with normal and in 10% of those with elevated basal serum tryptase. CONCLUSIONS: An elevated basal serum tryptase as well as mastocytosis are risk factors for severe or even fatal shock reactions to Hymenoptera stings. Although the efficacy of venom immunotherapy in these patients is slightly reduced, most of them can be treated successfully. Based on currently available data, lifelong treatment has to be discussed in this situation.     

A dual color ELISPOT method for the simultaneous detection of IL-2 and IFN-gamma HIV-specific immune responses

The single color IFN-γ ELISPOT assay has become a standard for assessing HIV-specific immune responses in HIV-infected subjects. However, recent data suggests that single cytokine detection for immune monitoring of HIV-infected individuals may not be sufficient to fully describe virus-specific immune responses. Here, we have designed and validated a dual color ELISPOT assay capable of detecting both IL-2 and IFN-γ secreting cells simultaneously in response to HIV antigens. We found that a cell input number of 200,000 cells/well provided a good balance between limited availability of cells due to blood volume restrictions and ability to detect all cytokine secretion patterns. The simultaneous detection of IL-2 and IFN-γ resulted in a decreased magnitude of IFN-γ but not IL-2 responses. Measures of intra- and inter-assay variability for the dual color ELISPOT assay were comparable to that seen for single cytokine ELISPOT assay with coefficients of variation below 20% for IL-2, IFN-γ and dual secretion. Although CD8+ T cells mediated most HIV-specific responses in infected subjects, CD4+ T cells mediated responses to HIV were also detected. Features of this assay such as high throughput, cell number requirement and cytokine choice should make this assay a valuable tool for screening for HIV-specific immune responses in several clinically relevant settings.     

In Vitro Induction of Allergen-Specific Interleukin-10-Producing Regulatory B Cell Responses by Interferon-γ in Non-Immunoglobulin E-Mediated Milk Allergy

Purpose

Specific oral immunotherapy (SOIT) using interferon-γ (IFN-γ) has been successful as a food allergy treatment. Interleukin-10 (IL-10)-producing regulatory B cells (Br1s) play a role in immune tolerance to food allergens. In addition, IFN-γ shows tolerogenic effects on allergen-induced Br1 responses.

Methods

Eleven patients that were allergic to cow''s milk and 12 milk-tolerant subjects were selected by double-blind placebo-controlled food challenge (DBPCFC) and clinical characteristics. The immunomodulatory effects of IFN-γ on allergen-specific Br1 responses were evaluated in 6 milk allergy patients and 8 milk-tolerant subjects. Peripheral blood mononuclear cells (PBMCs) from subjects were stimulated with casein and/or IFN-γ and analyzed by flow cytometry.

Results

IFN-γ had no effect on total cell counts or the proportion of Br1 cells in PBMCs. IFN-γ stimulation did not change total Br1 cell counts or the percentage of Br1s among CD5(+) B cells in the milk allergy or the milk-tolerant groups. In the milk allergy group, Br1 counts were not different between the control and the casein stimulation but significantly increased in the IFN-γ + casein stimulated cells, and the Br1 fractions were decreased after casein stimulation and recovered in the addition of IFN-γ for stimulation. In the milk-tolerant group, Br1 counts increased in the casein stimulated cells and in the IFN-γ + casein stimulated cells, but the increase was significantly less when IFN-γ was added, and the Br1 fractions were increased after casein stimulation and IFN-γ + casein stimulation, that was not significant when IFN-γ was added.

Conclusions

IFN-γ-induced allergen-specific Br1 responses in the PBMCs of milk allergy patients play a role in milk allergen-specific tolerance induction in vitro. Further investigations into the molecular immunological mechanisms underlying the induction of allergen-specific Br1 responses are needed.     

Systemic T-cell unresponsiveness during rush bee-venom immunotherapy

By rush bee-venom immunotherapy, subjects reacting allergically to the venom can be effectively anergized, although the mechanism of action is not known. Here we analyzed the systemic effects of rush desensitization on the T cells of allergic patients. In most patients, we found reduced frequencies of T cells recalled to express CD69 and the cytokines interleukin (IL)-4 and interferon-gamma (IFN-γ) after stimulation of peripheral blood mononuclear cells with phorbol 12-myristate 13-acetate (PMA) and ionomycin, as compared with normal donors. These frequencies are progressively reduced during immunotherapy. The frequency of cells expressing IL-2 does not change. A few patients show a different response to immunotherapy: frequencies of cells expressing CD69, IL-4, or IFN-γ do not change, and remain similar to those of normal donors. However, the frequency of cells able to express IL-2 is increased. The analysis of cytokine expression in CD45RO* vs CD45RO' T-cell populations revealed differences between normal and allergic donors. In allergic patients, higher frequencies of IL-4- and IFN-γ-expressing cells among the CD45RO subpopulation were found than in normal donors. This situation is not modified by immunotherapy. The results reveal a certain degree of heterogeneity in the response of allergic patients to bee-venom rush immunotherapy; however, all are clearly differentiated from normal controls as judged by cytokine expression of CD45RO T cells. In most allergic patients, a considerable percentage of TTi cells become unresponsive to mitogenic stimulation, and may be responsible for the desensitization itself     

Cytokine Gene Expression Occurs More Rapidly in Stimulated Peripheral Blood Mononuclear Cells from Human Immunodeficiency Virus-Infected Persons

Evaluation of cytokine gene expression following in vitro stimulation is one means of examining the dysregulation of the immune system in human immunodeficiency virus (HIV) infection. We have assessed differences in the immune status of non-HIV-infected (HIV−) and HIV-infected (HIV+) individuals by evaluating the kinetics of the expression of cytokine genes. We compared detailed time courses of cytokine mRNA expression in HIV− and HIV+ peripheral blood mononuclear cells (PBMC) and found that there is a significant shift (P < 0.01) for all cytokines examined (interleukin 2 [IL-2], IL-6, IL-10, gamma interferon, and tumor necrosis factor alpha [TNF-α]) to an earlier time of mean peak mRNA expression by HIV+ PBMC (between 4 and 8 h) compared to HIV− PBMC (8 h) in response to either phytohemagglutinin (PHA) or anti-CD3 stimulation. Additional studies showed that although PHA-stimulated HIV+ PBMC showed decreased median IL-2, IL-4, and TNF-α mRNA levels, they typically demonstrated more rapid kinetics (increased mean 4-h/24-h cytokine mRNA ratios), with significant differences for IL-4 (P < 0.05) and TNF-α (P < 0.005), compared to HIV− PBMC. The use of fresh or frozen cells gave comparable cytokine mRNA data; however, the secretion of some cytokine proteins (IL-2 receptor, IL-10, and TNF-α) appeared to be reduced in HIV+ PBMC that had been frozen and thawed. Our studies demonstrate that the kinetics of cytokine gene expression can reveal additional dysregulation of the immune system in HIV infection, suggesting that PBMC of HIV-infected persons exist in an activated state in vivo that permits them to express cytokine genes more rapidly than a normal PBMC.     

Defective suppression of Th2 cytokines by CD4+CD25+ regulatory T cells in birch allergics during birch pollen season

BACKGROUND: CD4(+)CD25+ regulatory T cells suppress proliferation and cytokine production by human T cells both to self-antigens and exogenous antigens. Absence of these cells in human newborns leads to multiple autoimmune and inflammatory disorders together with elevated IgE levels. However, their role in human allergic disease is still unclear. OBJECTIVE: This study aimed to evaluate the capacity of CD4(+)CD25+ regulatory T cells to suppress proliferation and cytokine production outside and during birch-pollen season in birch-allergic patients relative to non-allergic controls. METHODS: CD4+ cells were obtained from blood of 13 birch-allergic patients and six non-allergic controls outside pollen season and from 10 birch-allergic patients and 10 non-allergic controls during birch-pollen season. CD25+ and CD25- fractions were purified with magnetic beads and cell fractions, alone or together in various ratios, were cultured with antigen-presenting cells and birch-pollen extract or anti-CD3 antibody. Proliferation and levels of IFN-gamma, IL-13, IL-5 and IL-10 were measured by thymidin incorporation and ELISA, respectively. Numbers of CD25+ cells were analysed by flow cytometry. RESULTS: CD4(+)CD25+ regulatory T cells from both allergics and non-allergics potently suppressed T cell proliferation to birch allergen both outside and during birch-pollen season. However, during season CD4(+)CD25+ regulatory T cells from allergic patients but not from non-allergic controls were defective in down-regulating birch pollen induced IL-13 and IL-5 production, while their capacity to suppress IFN-gamma production was retained. In contrast, outside pollen season the regulatory cells of both allergics and non-allergic controls were able to inhibit T-helper 2 cytokine production. CONCLUSION: This is the first study to show differential suppression of Th1 and Th2 cytokines, with CD4(+)CD25+ regulatory T cells from birch-pollen-allergic patients being unable to down-regulate Th2, but not Th1 responses during birch-pollen season.