Anna Russano

Human CD1-restricted T cell recognition of lipids from pollens

Plant pollens are an important source of environmental antigens that stimulate allergic responses. In addition to acting as vehicles for foreign protein antigens, they contain lipids that incorporate saturated and unsaturated fatty acids, which are necessary in the reproduction of higher plants. The CD1 family of nonpolymorphic major histocompatibility complex–related molecules is highly conserved in mammals, and has been shown to present microbial and self lipids to T cells. Here, we provide evidence that pollen lipids may be recognized as antigens by human T cells through a CD1-dependent pathway. Among phospholipids extracted from cypress grains, phosphatidyl-choline and phosphatidyl-ethanolamine were able to stimulate the proliferation of T cells from cypress-sensitive subjects. Recognition of phospholipids involved multiple cell types, mostly CD4+ T cell receptor for antigen (TCR)αβ+, some CD4−CD8− TCRγδ+, but rarely Vα24i + natural killer–T cells, and required CD1a+ and CD1d+ antigen presenting cell. The responding T cells secreted both interleukin (IL)-4 and interferon-γ, in some cases IL-10 and transforming growth factor-β, and could provide help for immunoglobulin E (IgE) production. Responses to pollen phospholipids were maximally evident in blood samples obtained from allergic subjects during pollinating season, uniformly absent in Mycobacterium tuberculosis–exposed health care workers, but occasionally seen in nonallergic subjects. Finally, allergic, but not normal subjects, displayed circulating specific IgE and cutaneous weal and flare reactions to phospholipids.

CD1-Restricted Recognition of Exogenous and Self-Lipid Antigens by Duodenal γδ+ T Lymphocytes

γδ T cells are present in the mucosal intestinal epithelia and secrete factors necessary to maintain tissue integrity. Ags recognized by these cells are poorly defined, although in mice non-classical MHC class I molecules have been implicated. Since MHC class I-like CD1 receptors are widely expressed at the surface of epithelial and dendritic intestinal cells and have the capacity to present lipid Ags to T cells, we hypothesized that these molecules might present autologous and/or exogenous phospholipids to intestinal γδ T lymphocytes. Intraepithelial T lymphocytes from normal human duodenal mucosal biopsies were cloned and exposed to natural and synthetic phospholipids using CD1a-, CD1b-, CD1c- or CD1d-transfected C1R lymphoblastoid or HeLa cell lines as APCs. Their cytolytic properties and regulatory cytokine secretion were also examined. Most clones obtained from duodenal mucosa (up to 70%) were TCRαβ+, and either CD4+ or CD8+, whereas 20% were CD4−CD8− (6 clones) or TCRγδ+ (12 clones). A relevant percentage (up to 66%) of TCRγδ+ but few (<5%) TCRαβ+ T cell clones responded to synthetic and/or natural phospholipids presented by CD1 molecules, as measured by both [3H]thymidine incorporation and IL-4 release assays. A Th1-like cytolytic and functional activity along with the ability to secrete regulatory cytokines was observed in most phospholipid-specific γδ T cell clones. Thus, a substantial percentage of TCRγδ+ but few TCRαβ+ from human duodenal mucosa recognize exogenous phospholipids in a CD1-restricted fashion. This adaptive response could contribute to mucosal homeostasis, but could also favor the emergence of inflammatory or allergic intestinal diseases.

Biological effects of montelukast, a cysteinyl-leukotriene receptor-antagonist, on T lymphocytes

Background Montelukast (MNT), a cysteinyl‐leukotriene receptor (Cys‐LTR) antagonist, has anti‐inflammatory activity in the treatment of allergic diseases. If this effect is due only to blocking leukotrienes or also owing to inhibiting proliferation and survival of inflammatory cells, is actually unknown.

Defective Expression of Fas Messenger RNA and Fas Receptor on Pulmonary T Cells from Patients with Asthma

During the past several years, considerable progress has been made toward a better understanding of the pathogenesis of allergic diseases [1]. Inflammation at the sites of target organs seems to be the pathologic hallmark of the disease process; among the various types of cells involved in tissue infiltration and damage, T lymphocytes are probably the main effector cells priming the local ongoing allergic immune response [2-5]. These cells are principally involved in the local recognition of aerodispersed allergens and act by secreting interleukin-4 and interleukin-5, the so-called T-helper type 2 (Th2) cytokines [6, 7], and thus determining local and systemic IgE synthesis and the mucosal recruitment of other inflammatory cells, such as neutrophils and eosinophils. The cell secreting interferon- or interleukin-2 (or both) is T-helper type 1 (Th1), and it is involved in the delayed hypersensitivity reactions [8]. Although our knowledge about the initiation of the allergic response has rapidly expanded in recent years [9], nothing is known about why the inflammatory response cannot be turned off in atopic persons. Because immune reactions in general are potentially dangerous in a physiologic setting [10], they must be carefully controlled (if the antigenic stimulus becomes too great) or extinguished (if the damaging agent is successfully eliminated). One important mechanism of lymphocyte control is programmed cell death (apoptosis), which occurs in every immune response [11, 12], is energy dependent, and is associated with endonuclease activation. The biochemical hallmark of apoptosis is the fragmentation of chromatin into oligonucleosomes with subsequent changes in nuclear structure (picnosis) and alterations in the surface membrane of the apoptotic cell; this allows the cell to be eliminated from the site of inflammation by phagocytosis without damage to the surrounding host tissue [12]. T cells have at least two apoptotic pathways: active death, which is antigen driven, and passive death, which occurs at the end of an immune response and is due to lymphokine withdrawal or other mechanisms [13]. These two forms of death are molecularly distinct because the cell surface molecule Fas/APO-1 (CD95), a 45-kD protein belonging to the tumor necrosis factor (TNF) receptor family [14], and TNF itself are major participants in active, but not passive, death. The cross-linking of Fas receptor with its recently identified ligand (Fas ligand) leads to the induction of apoptosis in lymphocytes [15] and provides a mechanism for the removal of antigen-activated T cells [16, 17]. This leads to resolution of the inflammatory response [18], protecting the host against the detrimental effects that would ensue if cell disintegration or necrosis were to occur. Spontaneous remission of respiratory allergic disease invariably following withdrawal of allergen [19, 20] underlies the mechanism of passive death. Because Fas and TNF are major participants in active antigen-driven death [13], we investigated whether T cells from the lungs of untreated persons with asthma express Fas receptor. We show that the persistence of allergen-specific T cells at the mucosal surfaces of atopic persons results from local defective surface expression of Fas receptor and subsequent impairment in active, allergen-driven cell death. Methods Study Participants Twelve patients (6 male and 6 female; age range, 8 to 54 years) with newly diagnosed, mildly symptomatic chronic asthma who underwent bronchoalveolar lavage were included in the study. At the time of recruitment, all patients had stable pulmonary function and an FEV1 at least 70% of that predicted for their age and height. None was receiving inhaled or oral corticosteroids, sodium chromoglycate, theophylline, or 2 agonists. All had increased airway responsiveness to methacholine (concentration producing a decrease of 20% from baseline in FEV1, <8 mg/mL); were atopic, as defined by positive skin prick tests done with purified Dermatophagoides pteronyssinus allergen extract (Neo Abello, Madrid, Spain); and had positive results on enzyme-linked immunosorbent assay (DPC Corp., Los Angeles, California) for circulating allergen-specific IgE. No patients smoked or had had an upper respiratory tract infection in the 8 weeks before the study. Ten age- and sex-matched normal nonsmoking persons served as controls. No control had a history of asthma, systemic illness, or recent respiratory illness or any evidence of airway hyperresponsiveness on methacholine challenge. All had normal results on pulmonary function tests; none had positive results on skin prick tests to a panel of allergens that included house dust mites and pollens. The study protocol was approved by the institutional review board for human studies of our university, and informed consent was given by each patient or by the patients relatives if the patient was a child. The study was conducted according to local ethical committee guidelines and the principles of the Declaration of Helsinki. Collection of Bronchoalveolar Lavage Fluid Fiberoptic bronchoscopy was performed as reported elsewhere [21]. Lavage of the right middle lobe was done with three consecutive 20-mL aliquots of prewarmed physiologic saline. Recovered bronchoalveolar lavage cells were spun immediately at 200 g for 10 minutes at 4C. After resuspension, cytospins were prepared for differential cell counts. Reagents and Flow Cytometry Immunophenotyping was done simultaneously on pulmonary and peripheral blood T cells from patients and controls by using the following monoclonal antibodies in various combinations: phycoerythrin-conjugated anti-CD3 (OKT3, Ortho, Raritan, New Jersey), which recognizes up to 90% of T cells bearing the or T-cell receptor heterodimer; anti-CD4 and anti-CD8 (OKT4 and OKT8, Ortho), which stain helper/inducer and cytotoxic T-cell subsets; anti-CD25 (anti-interleukin-2 receptor, Becton-Dickinson, Mountain View, California) and anti-HLA-DR, which recognize, respectively, the interleukin-2 receptor chain and the major histocompatibility complex class II molecule (both referring to activated T cells); anti-CD45R0 (Immunotech, Marseille, France), a common leukocyte antigen present on activated/memory T cells; fluorescein-conjugated anti-Fas receptor (Immunotech); and phycoerythrin-conjugated anti-TCR [T-cell receptor] delta1 (T-Cell Sciences, Cambridge, Massachusetts), a pan-reactive T-cell reagent. For staining, 5000 to 10 000 cells were resuspended in 50 L of saline, incubated at 4C for 30 minutes, washed, and analyzed by flow cytometry (FACScan, Becton-Dickinson). For analysis of two-color cytofluorometric data, an electronic gate was set on the lymphocyte population based on the forward-angle versus the right-angle light scatter histogram. Quadrant markers in fluorescence histograms were set by using matched isotype controls. The Lysis II program (Becton-Dickinson) was used to optimize gating of lymphocytes and to provide an objective way to exclude both debris (noncellular events due to particulate matter) and other cells from the lymphocyte gate. Separation of Enriched T-Cell Populations Bronchoalveolar lavage mononuclear cells from patients and controls were separated into sheep erythrocyte rosette-enriched (containing T cells) and rosette-depleted subsets [22]. On the basis of their reactivity with the anti-CD3 monoclonal antibody OKT3 (>98% by immunofluorescence), the rosetted mononuclear cells were considered to be highly purified T-cell subsets and were used for Fas messenger RNA (mRNA) polymerase chain reaction (PCR) analysis and apoptotic cell death experiments. Modulation of Surface Fas Receptor on Pulmonary T Cells T-cell-enriched populations from patients and controls were cultured in vitro on 24-well plastic plates (Kostar, Denmark) coated with anti-CD3 monoclonal antibody for 24 hours in the presence of recombinant human interleukin-2 (50 U/mL; Serotec, Oxford, United Kingdom) or interleukin-4 (400 IU/mL; Serotec). At the end of the culture time, cells were analyzed for surface expression of Fas molecule by double-color immunofluorescence. Evaluation of Fas-Induced Programmed Cell Death Enriched T lymphocytes derived from bronchoalveolar lavage were washed and resuspended in RPMI-1640 (Gibco, Grand Island, New York) supplemented with 10% fetal calf serum, 2 mmol of L-glutamine per L, 10 mmol of HEPES per L, 50 U of penicillin per L, and 50 g of streptomycin at 106 cells/mL. All samples were incubated for 18 hours with medium alone or with anti-Fas IgM (a specific antibody to the Fas receptor that can induce apoptosis of Fas+ target cells), washed, centrifuged at 200 g for 10 minutes, and dissolved in hypotonic lysing buffer (100 mmol of NaCl per L, 10 mmol of Tris per L, 1 mmol of EDTA per L, 1% sodium dodecyl sulfate, 200 g of proteinase K per mL, pH of 7.5). A standard DNA electrophoresis assay was used for qualitative evaluation of apoptotic cells, as described elsewhere [23]. Quantitative analysis of spontaneous and anti-Fas-induced programmed cell death was done by cytofluorometry with a fluorochrome solution containing propidium iodide [23]. Fas Messenger RNA Expression Expression of mRNA was detected by reverse transcription PCR. With the guanidinium isothiocyanate acid phenol method, RNA was isolated from bronchoalveolar lavage-enriched T cells from patients, controls, and the U937 cell line (as a positive control) [24]. First-strand complementary DNA synthesis was done by using total RNA, random hexadeoxynucleotide primers, and Moloney murine leukemia virus reverse transcriptase (Gibco BRL, Life Technologies, Milano, Italy), as described elsewhere [25]. Primers for Fas and -actin PCR amplification were designed according to previously published sequences [25]. After amplification, one tenth of the PCR product was run on 2% agarose gel and stained with ethidium bromide. Statistical Analysis Patients were stratified according to age. Pair

CD4+IL13+ T lymphocytes at birth and the development of wheezing and/or asthma during the 1st year of life.

Background: Despite our knowledge that maternal inheritance influences the development of asthma in childhood, attempts to identify a clear-cut Th2-oriented cytokine production by T lymphocytes at birth have given conflicting results. The prognostic significance of these cells for asthma development later in life remains to be determined. Methods: We evaluated at the single cell level Th1- and Th2-type cytokines in 208 randomly selected cord blood mononuclear cell (CBMC) samples obtained from pregnant women (group A, n = 68 with diagnosed respiratory allergic disease; group B, n = 140, with no evidence of atopy), and prospectively followed newborns for 1 year. Results: There was no difference in IFN-γ, IL-4 and IL-5 production at birth between both groups, whereas a correlation between CD4+IL13+ lymphocytes from CBMC samples derived from atopic mothers and the occurrence of wheezing and/or asthma during the 1st year of life was found. Conclusions: Our observations suggest that the intracellular cytokine profile of cord blood CD4+ cells, in terms of IL-13 production, could be considered a useful tool for a more accurate identification of newborns from atopic mothers who are at high risk of developing asthma.

Role of T-helper type 2 cytokines in down-modulation of Fas mRNA and receptor on the surface of activated CD4+ T cells: molecular basis for the persistence of the allergic immune response

The mechanisms responsible for persistence of T lymphocytes at the sites of allergic inflammation are not completely understood. Activated T cells, usually expressing Fas on their surface, undergo activation‐induced apoptotic death, thus limiting the dangerous consequences of a persistent immune reaction. We have previously shown that pulmonary T lymphocytes from untreated asthmatic subjects do not express surface Fas receptors nor do they contain Fas mRNA, yet they display normal levels of Fas ligand. This is not an inherited defect and is confined to mucosal T cells. To gain insights into the mechanism responsible for these findings, we performed a set of experiments with both purified Dermatophagoides pteronyssinus allergen and recombinant human cytokines: interleukin 2 (IL‐2), IL‐4, IL‐5, transforming growth factor β1, interferon γ, and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). In vitro exposure of purified CD4+ lymphocytes to allergen yielded only transient up‐regulation of surface Fas but did not influence susceptibility to Fas‐mediated cell death. T‐helper type 2 cytokines (IL‐4, IL‐5, and GM‐CSF) had a dose‐dependent and specific inhibitory effect on Fas mRNA, suggesting a new fundamental biological role in the survival of inflammatory cells during allergen exposure.—Spinozzi, F., Agea, E., Fizzotti, M., Bassotti, G., Russano, A., Droetto, S., Bistoni, O., Grignani, F., Bertotto, A. Role of T‐helper type 2 cytokines in down‐modulation of Fas mRNA and receptor on the surface of activated CD4+ T cells: molecular basis for the persistence of the allergic immune response. FASEB J. 12, 1747–1753 (1998)

Influenza viruses and cross-reactivity in healthy adults: Humoral and cellular immunity induced by seasonal 2007/2008 influenza vaccination against vaccine antigens and 2009 A(H1N1) pandemic influenza virus

We analyzed humoral and cellular immune responses against vaccine antigens and the new A(H1N1) virus in healthy adults before and after immunization with the 2007/2008 commercially available trivalent subunit MF59-adjuvanted influenza vaccine during the Fall 2007, prior to the emergence of the new virus. Antibody titers were significantly boosted only against the three vaccine antigens. Seasonal vaccination boosted pre-existing cellular responses upon stimulation of peripheral blood mononuclear cells not only with the homologous three vaccine antigens, but also with the heterologous new 2009 A(H1N1) and with a highly conserved peptide present in the stalk region of hemagglutinin (HA). These results show that cross-reactive cell responses against the new virus were present before the circulation of the virus and were boosted by seasonal vaccination. The cross-reactivity of cellular responses might, at least in part, explain the low pathogenicity of the new pandemic virus. The finding of cellular immunity, that can be increased by seasonal vaccination, against the conserved HA peptide, underline the potential use, in human vaccines, of conserved peptides of the stalk region of HA characterized by broad immunogenicity in experimental systems.

Complementary roles for lipid and protein allergens in triggering innate and adaptive immune systems.

Background:  Recent advances in allergy research mostly focussed on two major headings: improving protein allergen purification, which is aimed towards a better characterization of IgE‐ and T‐cell reactive epitopes, and the potential new role for unconventional innate and regulatory T cells in controlling airway inflammation. These advancements could appear to be in conflict each other, as innate T cells have a poorly‐defined antigen specificity that is often directed toward nonprotein substances, such as lipids.

Biochemical and Immunological Characterization of Pollen-Derived β-Galactosidase Reveals a New Cross-Reactive Class of Allergens among Mediterranean Trees

Background: The most potent allergens in the Spermatophytae family exhibit significant homology with enzymes. Some of these are though to be involved in pectin metabolism, recognition of compatible stigma and delivery of sperm cells to the ovule. Objective: To test if glycohydrolase activities from some Mediterranean tree pollens could act as allergens in sensitized hosts. Methods: Freshly collected Cupressus and Olea pollens were investigated for their glycohydrolase activities by means of synthetic fluorogenic substrates and isoenzymes characterized by DEAE-cellulose ion-exchange chromatography. Binding of specific IgE was investigated by immunoblotting in 30 tree-sensitive subjects, as well as in 20 atopic non-tree-sensitive and 15 healthy controls. The enzymes were also adopted to stimulate proliferation of allergen-specific T cell clones. Finally, they were tested in vivo in a cutaneous immediate wheal and flare reaction. Results: β-Galactosidase (β-GAL) is present with different isoenzymatic patterns on both pollen extracts, could be recognized by circulating IgE, as well as immunoprecipitated by sera from allergic subjects. The enzyme could stimulate the proliferation of T cells from allergic subjects, and favor the emergence of CD4+ T cell clones with specific in vitro reactivity to β-GAL. Finally, the enzyme induced in vivo a cutaneous wheal and flare reaction in clinically sensitive subjects. Conclusions: Despite different isoenzymatic patterns, Olea-derived β-GAL cross-reacted with that from cypress pollen, suggesting that these enzymatic glycoproteins may represent major native allergens among these Mediterranean trees.