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.

Loss of cardiolipin in palmitate-treated GL15 glioblastoma cells favors cytochrome c release from mitochondria leading to apoptosis.

Unlike oleate and linoleate, palmitate induced mitochondrial apoptosis in GL15 glioblastoma cells. Decrease in membrane potential in a subpopulation of mitochondria of palmitate-treated cells was revealed using the 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide probe. The diminished ability to reduce a tetrazolium salt indicated an impairment of mitochondrial function. Up to 50% cytochrome c (cyt c) was detached from the inner mitochondrial membrane and released outside mitochondria in palmitate-treated cells, whereas no release was detected after oleate and linoleate treatments. Cyt c release into the cytosol was followed by caspase 3 activation. Released cyt c and caspase 3 activity were not affected by neutral and acid sphingomyelinase inhibitors and by the inhibitor of serine palmitoyltransferase cycloserine, indicating that apoptosis was independent of the ceramide pathway, nor the mitochondrial pro-apoptotic AIF or Bcl-2/Bax factors appeared to be involved in the effect. Utilization of palmitate by GL15 cells altered phospholipid composition. Cardiolipin (CL), the lipid involved in cyt c interaction with the inner mitochondrial membrane, was decreased and highly saturated. This produced an imbalance in hydrophilic/hydrophobic interactions underlying the anchorage of cyt c, by weakening the hydrophobic component and facilitating detachment of the protein and activation of downstream processes. The primary role of CL was explored by supplying GL15 with exogenous CL through a fusion process of CL liposomes with cell plasma membrane. Fused CL moved to mitochondria, as detected by nonylacridine orange probe. Enrichment of mitochondrial membranes with CL prior to palmitate treatment of cells caused decreased cyt c release and caspase 3 activity.

Apoptosis, airway inflammation and anti-asthma therapy: from immunobiology to clinical application.

T lymphocyte apoptosis is essential for maintaining immune system homeostasis. Experimental evidence suggests apoptosis control mechanisms may be impaired in inflammatory conditions, particularly airway Th2-type allergic diseases. This review briefly examines the mucosal immune system homeostasis and common apoptotic pathways and discusses impaired apoptosis, allergy, airway inflammation, remodelling and fibrosis. Finally, the paper presents an update on pharmacological targeting of apoptosis to control airway inflammation in patients with allergic asthma.

CD1-restricted recognition of exogenous and self-lipid antigens by duodenal gammadelta+ T lymphocytes.

Gammadelta 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 gammadelta 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 TCRalphabeta+, and either CD4+ or CD8+, whereas 20% were CD4-CD8- (6 clones) or TCRgammadelta+ (12 clones). A relevant percentage (up to 66%) of TCRgammadelta+ but few (<5%) TCRalphabeta+ T cell clones responded to synthetic and/or natural phospholipids presented by CD1 molecules, as measured by both [(3)H]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 gammadelta T cell clones. Thus, a substantial percentage of TCRgammadelta+ but few TCRalphabeta+ 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.

Recognition of lipids from pollens by CD1-restricted T cells.

Allergic rhinitis and asthma should be considered as organ-specific inflammatory diseases in which the genetic background has determined a local overproduction of Th2-type cytokines and an over-expansion of particular APCs and T cells. Among the latter, a potential pathogenetic role could be assumed for natural killer T cells, expressing both invariant (Valpha24/Vbeta11) and classic alphabeta or gammadelta T-cell receptors. Recent studies support this notion and also suggest that surface pollen substances of nonprotein structure, such as lipid components recognized by CD1, could be viewed as one of the foreign materials against which the immune system of the allergic subject can mount a local inflammatory response.

Recognition of pollen-derived phosphatidyl-ethanolamine by human CD1d-restricted gamma delta T cells.

BACKGROUND: Evidences from mice and human beings indicate that gammadelta T cells could be relevant in recognition of stress-induced self and/or yet unidentified inhaled foreign antigens. Their specificity differs from classic MHC-restricted alphabeta T cells and involves the immunoglobulin-like structure of the gammadelta T-cell receptor with the recognition of small organic molecules, alkylamines, and self lipid compounds presented by CD1+ dendritic cells. OBJECTIVE: Because CD1 receptors are mainly devoted to lipid antigen presentation, we sought to determine whether exogenous pollen membrane lipids may act as allergens for CD1-restricted gammadelta T cells. METHODS: Peripheral blood and nasal mucosa-associated gammadelta T cells were cloned from normal controls and cypress-sensitive subjects and tested for their antigen specificity and CD1-restriction with phospholipids extracted from tree pollen grains, as well with other natural or synthetic compounds. Phospholipid reactivity of cloned gammadelta T cells was measured by mean of proliferative response and cytokine release as well as by testing their helper activity on IgE production in vitro and in vivo. RESULTS: Cloned gammadelta T lymphocytes from subjects with allergy, but not normal controls, were found to recognize pollen-derived phosphatidyl-ethanolamine (PE) in a CD1d-restricted fashion. Only 16:0/18:2 and 18:2/18:2 PE were stimulatory, whereas no response was recorded for disaturated PE, phosphatidylcholine, neutral lipids, or protein extract. Proliferating clones secreted both T(H)1-type and T(H)2-type cytokines and drove IgE production in vitro and in vivo. CONCLUSION: CD1d-restricted gammadelta T cells specific for phospholipids can represent a key mucosal regulatory subset for the control of early host reactivity against tree pollens. CLINICAL IMPLICATIONS: By knowing how lipid allergen constituents interact with mucosal immune system, we can expand our possibilities in diagnostic and therapeutic interventions.

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)alphabeta(+), some CD4(-)CD8(-) TCRgammadelta(+), but rarely Valpha24i(+) natural killer-T cells, and required CD1a(+) and CD1d(+) antigen presenting cell. The responding T cells secreted both interleukin (IL)-4 and interferon-gamma, in some cases IL-10 and transforming growth factor-beta, 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.

Interleukin 7-engineered stromal cells: a new approach for hastening naive T cell recruitment.

In this study we determined whether human stromal cells could be engineered with a retroviral vector carrying the interleukin 7 (IL-7) gene and investigated the effects on T cells in vitro and in vivo in a murine model. Transduced mesenchymal cells strongly express CD90 (98.15%), CD105 (87.6%), and STRO-1 (86.7%). IL-7 production was 16.37 (+/-2 SD) pg/ml, which remained stable for 60 days. In vitro-immunoselected naive T cells maintained the CD45RA+ CD45RO- naive phenotype (4.2 times more than controls) after 7 days of culture with IL-7-engineered stromal cells. The apoptosis rate (4.7%) of the naive T cells cultured with transduced stromal cells overlapped with that of freshly isolated cells. Immunohistological analysis detected stromal cells in bone marrow, spleen, and thymus. Cotransplantation of IL-7-engineered stromal cells with CD34+ cells improved engraftment in terms of CD45+ cells and significantly increased the CD3+ cell count in peripheral blood, bone marrow, and spleen. These data demonstrate the following: (1) human stromal cells can be transduced, generating a normal layer; (2) transduced stromal cells in vitro maintain the naive T cell phenotype; and (3) IL-7-transduced stromal cells in vivo home to lymphoid organs and produce sufficient IL-7 in loco, supporting T cell development in a cotransplantation model. Because of their efficient cytokine production and homing, IL-7-engineered stromal cells might be an ideal vehicle to hasten immunological reconstitution in T cell-depleted hosts.

Biochemical and immunological characterization of pollen-derived beta-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: beta-Galactosidase (beta-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 beta-GAL. Finally, the enzyme induced in vivo a cutaneous wheal and flare reaction in clinically sensitive subjects. CONCLUSIONS: Despite different isoenzymatic patterns, Olea-derived beta-GAL cross-reacted with that from cypress pollen, suggesting that these enzymatic glycoproteins may represent major native allergens among these Mediterranean trees.