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Overproduction of immunoglobulin E (IgE) by a subset of B cells plays a key role in the pathogenesis of allergic asthma. Anti-IgE monoclonal antibodies have been successfully used to treat the disease, but long-term application is required.
For this study, cytotoxic T lymphocytes (CTLs) against IgE-producing B cells were generated ex vivo by stimulating naive CD8 T cells with IgE-derived peptides presented by Drosophila-derived artificial antigen-presenting cells. Based on the treatment of allergic asthma in mice, the inhibitive effect of this CTL on IgE responses and airway inflammation was determined with the enzyme-linked immunosorbent assay and histochemical method.
The IgE-specific CTLs effectively lysed target cells in vitro, while the adoptively transferred CTLs specifically inhibited IgE responses and airway inflammation in an asthmatic mouse model. The effect of IgE-specific CTLs is MHC (major histocompatibility complex) Class I-restricted and requires the expression of perforin.
IgE-specific CTLs generated ex vivo may provide a novel treatment for allergic asthma and lead to a new therapy for other immunological disorders.
Elevation of serum immunoglobulin E (IgE) level is correlated with atopic diseases such as allergic rhinitis and allergic asthma.(1) Circulated IgE in the blood binds to the high-affinity IgE receptor (FcεRI) on the surface of mast cells positioned along the mucosal lining and underneath the skin during an allergic response. IgE-mediated mast cell degranulation also results in the release of newly synthesised inflammatory cytokines and chemokines, including IL-4, IL-5, IL-6, TNF, MCP-1 and MIP-1, and thus plays a role in the late phase of allergic responses.(2) Therefore, IgE is usually used as a natural target for anti-allergic therapy. Indeed, anti-IgE monoclonal antibodies (mAbs) have successfully been developed for the treatment of IgE-mediated allergic disorders.(3-7) It has been demonstrated that anti-IgE mAbs attenuate both early- and late-phase asthmatic responses, improving both the symptom score and peak expiratory flow in patients with allergic asthma.(8-10) To benefit from this treatment, however, patients required the injection of a high dose of anti-IgE antibody (150–300 mg/injection) every 2–4 weeks.(11) Despite a near-complete suppression of free IgE, a prolonged and substantial rise in total IgE, in the form of IgE-anti-IgE complexes, was observed in patients who received anti-IgE antibody,(3) suggesting that IgE synthesis could not be stopped by the treatment.(9,12,13) Thus, the direct inhibition of IgE-producing cells by IgE-specific cytotoxic T lymphocytes (CTLs) may be an alternative approach to the long-term treatment of allergic disorders.
CTLs play an essential role in immunity against viral and intracellular pathogens by recognising antigenic peptides in the context of major histocompatibility complex (MHC) Class I molecules.(14) Previously, we showed that Drosophila-derived artificial antigen-presenting cells (aAPC) can be used to generate CTLs specific for tumour-associated antigenic peptides in vitro, and that the adoptive transfer of these CTLs inhibits tumour cell growth in vivo.(15) Therefore, we hypothesised that adoptive transfer of these IgE-specific CTLs should also be able to lyse IgE-producing B cells in vivo and consequently lead to inhibition of IgE-mediated pathology. This study aimed to evaluate whether IgE antigenic peptide-specific CTLs can be generated in vitro and to test if these CTLs could inhibit IgE responses in vivo. Using an established aAPC system,(16) we successfully generated IgE-specific CTLs in vitro, the adoptive transfer of which efficiently inhibited IgE responses and airway inflammation in an asthmatic mouse model.
C57BL/6J (B6), CB6F1/J (BALB/cJ F × C57BL/6J M), C57BL/6-Pfp
All IgE-derived peptides were synthesised by SynPep Corporation (San Diego, CA, USA) and purified with C18 reverse phase high-performance liquid chromatography. RMA-S cells and Ld-transfected RMA-S cells (RMA-S.Ld) were cultured and maintained as previously described.(16) Drosophila cells transfected with MHC Class I (Ld or Db), CD54 (also ICAM-1) and CD80 (also B7-1) were generated and maintained as previously described.(16) The MHC Class I stabilisation assay was applied, also as previously described.(17) During the assay, RMA-S or RMA-S.Ld cells were cultured overnight at room temperature with or without indicated peptides and then shifted to an additional two-hour culture at 37°C. The cells were stained with fluorescein isothiocyanate-labelled anti-H-2 Db or Ld mAb (BD Pharmingen, San Diego, CA, USA) and were analysed with a FACScan flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).
CD8 T cells were purified from lymph nodes of CB6F1/J, B6/J, IFN-g or PF-deficient mice (IFN-g-/- or PF-/-) using a positive selection magnetic-activated cell sorting system (Miltenyi Biotec, Somerville, MA, USA). Purified CD8 T cells were cultured with Drosophila cells transfected with Db, CD54 and CD80 with IgE 44 peptide (IgEp44) or IgE 366 peptide (IgEp366). IL-2 (20 hU/mL) was added into the culture medium at Days 3, 5 and 7, and cells were split on Day 5 and Day 7 based on cell density. CTLs were harvested at Day 9, and their specific activity was tested as previously described.(16) RMA-S or RMA-S.Ld cells were incubated with 51Cr for one hour with or without IgE peptides and were used as target cells. The CTL effector cells were cultured with the labelled target cells at indicated effector-to-target ratios in a U-bottom microplate at 37ºC for four hours. Specific 51Cr release was calculated as previously described.(16)
The mouse model of allergic asthma was generated as indicated in
Diagram shows the experimental design of a mouse model of allergic asthma. BAL: bronchoalveolar lavage; CTL: cytotoxic T cell; d: Day; IgE: immunoglobulin E; i.n: intranasal; i.p: intraperitoneal; OVA: ovalbumin
Total serum IgE and immunoglobulin G (IgG) isotypes (IgG1, IgG2a) and eotaxin from the mice were determined by sandwich ELISA. For this purpose, polystyrene 96-well plates (Costar, Corning, NY, USA) were coated overnight at 4°C with capture antibody of anti-mouse IgE (BD Pharmingen, San Diego, CA, USA) or anti-IgG (Southern Biotechnology, Birmingham, AL, USA) or anti-eotaxin (R&D, Minneapolis, MN, USA) in phosphate-buffered saline (PBS; 100 µL/well). Plates were then washed with PBS plus 0.02% Tween-20 and blocked with 2% bovine serum albumin (100 µL/well) for two hours at room temperature. After washing and blocking, diluted serum samples and standards were added to each well and incubated at room temperature for two hours. For measurement of total IgE, the plates were washed and incubated with biotinylated anti-mouse IgE for one hour, followed by incubation with horseradish peroxidase-conjugated streptavidin for an additional hour. The plates were washed and developed with 3,3’,5,5’-tetramethylbenzidine solution (BD Pharmingen, San Diego, CA, USA). For measurement of IgG1 and IgG2a, the plates were washed and incubated with alkaline phosphatase-conjugated rabbit anti-mouse IgG1 or IgG2a for one hour. After washing, the plates were developed with para-nitrophenyl phosphate substrate. For measurement of OVA-specific IgE or IgG, the determination procedure was carried out as described herein, but the plates were coated with 100 µL of OVA (10 µL/mL) instead of capture antibody. Absorbance was measured with a microplate reader at 450 nm wavelength.
GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA) was used for statistical analysis. Mean methacholine dose-response curves for each experimental group were compared using two-way analysis of variance. All data was presented as mean ± standard deviation. Student’s t-test was used for all comparisons. A p-value < 0.05 or < 0.01 was considered statistically significant or extremely significant, respectively.
The amino acid sequence of the constant region of IgE was analysed for 9-mer peptide sequences that contained binding motifs for H-2 Ld and Db MHC Class I molecules by using the BIMAS program of the Bioinformatics and Molecular Analysis Section (
Identification of antigenic peptides for mouse IgE.
We tested the capacity of these synthetic peptides to bind and stabilise Ld and Db Class I molecules expressed on Drosophila (Schneider 2) cells and RMA-S cells.(17,20,21) Two IgE-derived peptides, identified as IgEp11 and IgEp366, bound strongly to Ld, whereas IgEp114 bound only weakly to Ld. Of the five peptides predicted to bind to Db, only IgEp44 bound strongly, while IgEp16 and IgEp125 bound weakly to Db. Interestingly, IgEp366, which was originally predicted to bind Ld, bound Db as well. Therefore, a total of six nonapeptides were identified that bound to either Ld or Db MHC Class I molecules (
The ability of the IgE-derived peptides to elicit CTL responses was initially evaluated in vitro. As previously described,(16) Drosophila cells transfected with MHC Class I, CD54 and CD80 molecules are potent antigen-presenting cells (APC) for the activation of resting naive CD8 T cells in vitro. Resting naive CD8 T cells purified from mouse lymph nodes were cultured with transformed Drosophila cells expressing Ld or Db, CD54 and CD80 (Fly Ld.B7.ICAM or Fly Dd.B7.ICAM) in the presence of indicated peptides, and the CTL activity was measured on Day 9. The IgEp44-induced CTLs specifically lysed RMA-S cells loaded with the corresponding peptide. The target cells alone or those loaded with control IgE peptides were not recognised by IgEp44-specific CTLs (
Charts show (a) the generation and target specificity of anti-immunoglobulin E (IgE)-specific cytotoxic T lymphocytes (CTLs) in vitro; and (b) total serum IgE response and (c) antigen-specific IgE response before (Day 28) and after CTL treatment (Day 48), demonstrating the inhibition of IgE response by specific CTLs. CD8 T cells were purified from lymph nodes of CB6F1/J mice and stimulated by co-culturing with Drosophila-derived artificial antigen-presenting cells expressing H-2 Db, CD54 and CD80 molecules in the presence of indicated IgE peptide. CTL activity was measured with induced CD8 T cells against 51Cr labelled RMA-S cells in the presence of various IgE peptides. After the second immunisation, each mouse received 1 × 107 IgE-specific CTLs, except for the control mice, which received phosphate-buffered saline (PBS). There were five mice in each CTL or PBS group. E/T: effector-to-target ratio; OVA: ovalbumin
To determine if IgE-specific CTLs specifically inhibit IgE responses exclusively, both IgE and IgG responses were investigated (
Charts show the effect of anti-immunoglobulin E (IgE) cytotoxic T lymphocytes (CTLs) on the responses of (a) IgE, (b) IgG1 and (c) IgG2a, measured before CTL (Day 28) and after CTL treatment (Day 48). Anti-IgE CTLs were generated as described in
CTL cytotoxic activity primarily depends on the expression of PF alone. In addition, CTLs also produce some other cytokines, such as IFN-γ, to regulate Th1 response. It was therefore possible that the inhibition of IgE responses by CTLs was a consequence of regulation of Th1 responses by CTL-produced IFN-γ than a direct killing result of IgE-producing cells.(8) To distinguish the influence of the killing activity and IFN-γ release on IgE responses, we generated IgE-specific CTLs from either PF-/- or IFN-γ-/- mice.(23,24) CD8 T cells purified from PF-/- mice proliferated in response to IgE peptides. However, these PF-/- mice-derived CTLs were unable to kill target cells loaded with IgE peptides and therefore lost the ability to inhibit IgE responses in vivo (Figs.
Charts show the effect of perforin (PF) or interferon gamma (IFN-g) expression on (a) anti-immunoglobulin E (IgE) cytotoxic T lymphocyte (CTL) activity and (b) inhibition of IgE responses in vivo. IgE-specific CTLs were generated from either wild-type (WT) or knockout strains of mice (PF-/- or IFN-γ-/-). CTL activity was evaluated as described in
CTLs, as effector cells, recognise peptides presented by peptide-MHC complexes on target cells. Therefore, we investigated whether the activity of IgE-specific CTLs was dependent on MHC Class I molecule expression on target cells. IgE-specific peptide IgEp44 is restricted by MHC Class I molecule Db and induces specific CTLs (
We investigated the long-term in vivo effect of adoptively transferred IgE-specific CTLs in an asthmatic mouse model. Pre-sensitised mice were randomly divided into two groups: one was given IgE-specific CTLs (i.e. CTL group), while the other received PBS (i.e. PBS group) during the initial challenge. At the time intervals indicated, both groups of mice were re-challenged with OVA, and serum IgE was measured one week after each challenge. Before the IgE-specific CTL treatment, IgE levels were elevated in both groups (
Charts show long-term effect of specific cytotoxic T lymphocytes (CTLs) on immunoglobulin E (IgE) response in vivo, including: (a) the primary IgE-specific CTL treatment and (b) the second treatment with IgE-specific CTLs on mice that had recurrent IgE responses. The mice were re-challenged with antigen at the indicated times. IgE responses were measured one week after each challenge. Ten mice received phosphate-buffered saline (PBS) or IgE-specific CTLs.
Since IgE-specific CTLs effectively inhibited IgE responses in a mouse model of allergic asthma (Figs.
The effect of IgE-specific CTLs on airway inflammation was further confirmed with examination of lung histology (
Charts show (a) dose-dependent inhibition of immunoglobulin E (IgE) responses, measured two weeks after treatment with two different doses (5 × 106 and 10 × 106) of IgE-specific cytotoxic T lymphocytes (CTLs); (b) dose-dependent reduction of eosinophils and eotaxin in bronchoalveolar lavage five weeks post IgE-specific CTL treatment; and (c & d) reductions in airway hyperresponsiveness with increasing doses of methacholine, measured using whole body plethysmography, in two independent experiments. (e) Photomicrographs show reductions in airway inflammation following treatment with anti-IgE CTLs (5 × 106/mouse; haematoxylin & eosin). OVA: ovalbumin; PBS: phosphate-buffered saline
Effect of anti-IgE CTLs on airway inflammation for each mouse.
The hallmarks of asthma are airway hyperresponsiveness (AHR) and airway inflammation.(26) Since IgE-specific CTLs could inhibit IgE responses and reduce airway inflammation, we investigated whether CTL treatment could reduce AHR. Sensitised mice were treated with IgE-specific anti-IgE CTLs and re-challenged as described in
Immunotherapy has been developed to fight diseases such as cancer, autoimmune diseases and Alzheimer’s disease.(29-31) In this study, we demonstrated that one kind of T cell-based immunotherapy CTLs specific for IgE, a non-tumour self-antigen expressed on a subset of B cells, can be generated in vitro with aAPCs.(16) The in vitro generated IgE-specific CTLs effectively inhibited IgE responses to antigen in pre-sensitised mice, while the IgG responses were almost unaffected. Inhibition of IgE responses by CTLs required expression of both PF by CTLs and MHC Class I molecules on target cells. Enomoto et al has demonstrated that allergen-specific CTLs require PF expression to suppress allergic airway inflammation.(32) In contrast, IFN-γ released by IgE-specific CTLs was not essential for inhibition of the IgE responses. Thus, inhibition of IgE responses by IgE-specific CTLs required both recognition of peptide-MHC Class I complexes and subsequent PF-mediated killing. Apparently, alteration of the balance between Th1 and Th2 responses owing to IFN-γ release from the IgE-specific CTLs did not play a major role in the inhibition of IgE responses in vivo.
Eosinophilic inflammation in the lung is an important feature of allergic asthma.(33) Adoptively transferred IgE-specific CTLs could inhibit both IgE responses and airway eosinophilic inflammation in the animal model of asthma. The inhibition efficiency depended on the dose of IgE-specific CTLs transferred and was directly correlated with the reduction of airway inflammation. This data indicates that specific CTL-mediated inhibition of IgE responses leads to the reduction of airway eosinophilic inflammation.
Anti-IgE antibodies that compete in binding to IgE at the same site as its FcεRI have been developed for the treatment of asthma.(3,4,34) These antibodies inhibit IgE effector function by blocking IgE binding to its high affinity receptors expressed on mast cells and basophils, consequently blocking the inflammatory cascade induced by cross-linking of receptor-bound IgE. One of them, omalizumab (Xolair), has been developed and used successfully for the treatment of patients with moderate to severe asthma.(3,4,11,34,35) Xolair has been demonstrated to improve symptoms and reduce rescue medication and corticosteroid use in patients with allergic asthma.(34,36) However, despite the near-complete reduction of free IgE by the anti-IgE antibody, the total IgE level (IgE-anti-IgE complexes) continued to rise and was shown to be fivefold higher in patients who received anti-IgE for 16 weeks.(3,13) Within weeks after discontinuing the treatment, free IgE level returned to base level,(11) and most of the patients needed treatment with an anti-IgE antibody every 2–4 weeks for an extended period. In our study, IgE-specific CTLs recognised IgE-derived peptides presented by MHC Class I molecules. A single IgE-specific CTL treatment was sufficient to inhibit IgE responses to antigenic challenge for up to six months (
Additionally, IgE-specific CTLs enhanced IgG2a responses to the tested antigens in the sensitised mouse model (
Production of cytokines by T cells can be regulated by specific co-stimulatory molecules presented on the surface of APC.(37) Co-stimulation of CD8 T cells with APC expressing either CD80 or CD86 molecules induced both IL-2 and IL-4 expression. Interestingly, co-stimulation of CD8 T cells with APC expressing both CD80 and CD54 molecules enhanced gene expression of IL-2 as well as IL-10, whereas IL-4 expression was completely inhibited. Thus, activation of CD8 T cells with APC expressing different co-stimulatory molecules can be used to regulate cytokine production profile. In this study, IgE-specific CTLs were stimulated with APC expressing both CD54 and CD80. As expected, these CTLs express IL-10 rather than IL-4. It has been reported that IL-10 can be used to ameliorate allergen-induced asthma, airway hyper-reactivity and inflammation.(38-41) Thus, regulation of cytokine production by CTLs could be an additional strategy for regulation of immune response in vivo by IgE-specific CTLs.
These novel findings indicate that the central tolerance of T cells to IgE is incomplete and that IgE-specific T cells can be activated in vitro in the presence of optimal stimulation in the mouse model. We have also generated human IgE-specific CTLs ex vivo, and the function of these CTLs has been tested in vitro. Together with studies of CTLs targeting IgE in both mouse and human, these investigations are defining potential clinical applications to enhance CTL function to kill IgE-producing B cells in the airway in asthma cases. Although it is not known if IgE-specific CD8 T cells can be activated in vivo and play a role in the regulation of IgE responses, adoptive transfer of in vitro-generated IgE-specific CTLs can potentially provide a novel tool for the treatment of severe asthma and other IgE-mediated allergic disorders. In addition, generation of CTLs for other self-antigens that are transiently and specifically expressed on cells of the immune system may provide a new approach to immune regulation in vivo.
This study was funded by Zhejiang Provincial Top Key Discipline of Biology Open Foundation (11610331251507 to Chen J) and Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, Zhejiang, China.
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