Human TCR transgenic Bet v 1-specific Th1 cells suppress the effector function of Bet v 1-specific Th2 cells.

Pollinosis to birch pollen is a common type I allergy in the Northern Hemisphere. Moreover, birch pollen-allergic individuals sensitized to the major birch pollen allergen Bet v 1 frequently develop allergic reactions to stone fruits, hazelnuts, and certain vegetables due to immunological cross-reactivity. The major T cell epitope Bet v 1(142-153) plays an important role in cross-reactivity between the respiratory allergen Bet v 1 and its homologous food allergens. In this study, we cloned and functionally analyzed a human αß TCR specific for the immunodominant epitope Bet v 1(142-153). cDNAs encoding TCR α- and ß-chains were amplified from a Bet v 1(142-153)-specific T cell clone, introduced into Jurkat T cells and peripheral blood T lymphocytes of allergic and nonallergic individuals, and evaluated functionally. The resulting TCR transgenic (TCRtg) T cells responded in an allergen-specific and costimulation-dependent manner to APCs either pulsed with Bet v 1(142-153) peptide or coexpressing invariant chain::Bet v 1(142-153) fusion proteins. TCRtg T cells responded to Bet v 1-related food and tree pollen allergens that were processed and presented by monocyte-derived dendritic cells. Bet v 1(142-153)-presenting but not Bet v 1(4-15)-presenting artificial APCs coexpressing membrane-bound IL-12 polarized allergen-specific TCRtg T cells toward a Th1 phenotype, producing high levels of IFN-γ. Coculture of such Th1-polarized T cells with allergen-specific Th2-differentiated T cells significantly suppressed Th2 effector cytokine production. These data suggest that human allergen-specific TCR can transfer the fine specificity of the original T cell clone to heterologous T cells, which in turn can be instructed to modulate the effector function of the disease initiating/perpetuating allergen-specific Th2-differentiated T cells.

Lipid rafts, pseudotyping, and virus-like particles: relevance of a novel, configurable, and modular antigen-presenting platform.

Antigen presentation by professional antigen-presenting cells (APC) is the first step towards the initiation of an adaptive immune response carried out by naïve T lymphocytes. For this purpose, antigens are presented in the form of peptide/major histocompatibility complexes (pMHC) on APC to the antigen receptor of T cells. For sustained T cell activation to occur, numerous additional molecules specifically expressed on the surface of APC have to synergize with pMHC to stimulate a given T lymphocyte. Moreover, soluble factors such as cytokines critically contribute to the specific milieu during T cell activation. APC 'talk' to T cells only when they engage in intimate physical interaction. The cell biological correlate of this APC-T cell interaction is commonly referred to as the formation of an immunological synapse. In this review we aim to provide an overview of a novel cell-free antigen-presenting platform, i.e. virus-like particles (VLP) decorated with immune receptors of choice, which was devised to overcome the molecular and cell biological complexity of the APC side of the immunological synapse. In the past we have demonstrated that immune receptor-decorated VLP are able to activate, modulate, or abrogate antigen-specific T lymphocyte responses. Thus, antigen-specific VLP represent a valuable tool which might help to explore and understand the function of antigen-specific T lymphocytes in more detail and might thus open new avenues for the modulation of pathologic T lymphocyte responses, e.g. for the treatment of allergic diseases.

Fluorosomes: a convenient new reagent to detect and block multivalent and complex receptor-ligand interactions.

We describe for the first time fluorescent virus-like particles decorated with biologically active mono- and multisubunit immune receptors of choice and the basic application of such fluorosomes (FSs) to visualize and target immune receptor-ligand interactions. For that purpose, human embryonic kidney (HEK)-293 cells were stably transfected with Moloney murine leukemia virus (MoMLV) matrix protein (MA) GFP fusion constructs. To produce FSs, interleukins (ILs), IL-receptors (IL-Rs), and costimulatory molecules were fused to the glycosyl phosphatidyl inositol anchor acceptor sequence of CD16b and coexpressed along with MoMLV group-specific antigen-polymerase (gag-pol) in MA::GFP(+) HEK-293 cells. We show that IL-2 decorated but not control-decorated FSs specifically identify normal and malignant IL-2 receptor-positive (IL-2R(+)) lymphocytes by flow cytometry. In addition to cytokines and costimulatory molecules, FSs were also successfully decorated with the heterotrimeric IL-2Rs, allowing identification of IL-2(+) target cells. Specificity of binding was proven by complete inhibition with nonlabeled, soluble ligands. Moreover, IL-2R FSs efficiently neutralized soluble IL-2 and thus induced unresponsiveness of T cells receiving full activation stimuli via T-cell antigen receptor and CD28. FSs are technically simple, multivalent tools for assessing and blocking mono- and multisubunit immune receptor-ligand interactions with natural constituents in a plasma membrane context.

Modulation of allergen-specific T-lymphocyte function by virus-like particles decorated with HLA class II molecules.

BACKGROUND: T(H)2 lymphocytes play an important role in the induction and maintenance phase of type I allergy. Modulation of the responses of T(H)2 lymphocytes by novel forms of antigen-presenting platforms may help shape the immune response to allergen and palliate allergic diseases. OBJECTIVE: To present HLA class II/allergen-peptide complexes on virus-like particles (VLPs) and to evaluate their potential to modulate allergen-specific T-cell responses. METHODS: Virus-like particles that express the immunodominant T-cell epitope Art v 1(25-34) of the major mugwort pollen allergen in the context of HLA-DR1 and costimulatory molecules were produced by transfection of 293 cells. The effect of VLPs on IL-2 promoter activity, proliferation, and cytokine production of allergen-specific T cells derived from donors with and without mugwort pollen allergy was determined. RESULTS: Flow-cytometric analyses showed that HLA class II molecules, invariant chain::Art v 1 fusion proteins, and costimulatory molecules were expressed on 293 cells. Biochemical analyses confirmed that these molecules were efficiently targeted to VLPs. The engineered VLPs activated Art v 1-specific T cells in a costimulation-dependent manner. VLPs lacking costimulators induced T-cell unresponsiveness, which was overcome by addition of exogenous IL-2. Costimulation could be provided by CD80, CD86, or CD58 and induced distinct cytokine profiles in allergen-specific T cells. Unlike the other costimulatory molecules, CD58 induced IL-10/IFN-gamma-secreting T cells. CONCLUSION: Virus-like particles represent a novel, modular, acellular antigen-presenting system able to modulate the responses of allergen-specific T cells in a costimulator-dependent fashion. Allergen-specific VLPs show promise as tools for specific immunotherapy of allergic diseases.

Molecular and functional analysis of the antigen receptor of Art v 1-specific helper T lymphocytes.

BACKGROUND: Ninety-five percent of patients with mugwort allergy are sensitized to Art v 1, the sole major allergen in mugwort (Artemisia vulgaris) pollen. Sixty-nine percent of patients recognizing the single immunodominant T-cell epitope Art v 1(25-36) have an HLA-DRB1*01 phenotype. OBJECTIVE: We studied cloning and functional expression of a human alphabeta T-cell receptor (TCR) specific for Art v 1(25-36). METHODS: TCR chains were RT-PCR amplified from an Art v 1(25-36)-specific T-cell clone, retrovirally transferred, and functionally tested in Jurkat T cells or alternatively in peripheral blood T lymphocytes of nonallergic individuals. RESULTS: The alpha-chain of the TCR is composed of TRAV17 and TRAJ45 segments, and the beta-chain uses TRBV18, TRBD1, and TRBJ2-7. Analyses of 23 other Art v 1-specific T-cell clones did not reveal preferential usage of the TRAV17, TRBV18, or other TCR gene families. Efficient TCR transfer into Jurkat T cells was shown by binding of TCR Vbeta18-specific mAb and DRB1*0101/Art v 1 tetramers. Transgenic Jurkat T cells specifically recognized syngeneic EBV B cells pulsed with Art v 1(25-36) peptide and artificial antigen-presenting cells expressing invariant chain::Art v 1 fusion proteins. Moreover, transfer of the TCR into peripheral blood lymphocytes generated T cells that were Art v 1 reactive. Activation of transgenic T cells by artificial antigen-presenting cells was strictly dependent on costimulation. CONCLUSION: For the first time, a detailed molecular and functional analysis of a human allergen-specific TCR is presented.

General strategy for decoration of enveloped viruses with functionally active lipid-modified cytokines.

Viral particles preferentially incorporate extra- and intracellular constituents of host cell lipid rafts, a phenomenon central to pseudotyping. Based on this mechanism, we have developed a system for the predictable decoration of enveloped viruses with functionally active cytokines that circumvents the need to modify viral proteins themselves. Human interleukin-2 (hIL-2), hIL-4, human granulocyte-macrophage colony-stimulating factor (hGM-CSF), and murine IL-2 (mIL-2) were used as model cytokines and fused at their C terminus to the glycosylphosphatidylinositol (GPI) acceptor sequence of human Fcgamma receptor III (CD16b). We show here that genetically modified cytokines are all well expressed on 293 producer cells. However, only molecules equipped with GPI anchors but not those linked to transmembrane/intracellular regions of type I membrane proteins are efficiently targeted to lipid rafts and consequently to virus-like particles (VLP) induced by Moloney murine leukemia virus Gag-Pol. hIL-4::GPI and hGM-CSF::GPI coexpressed on VLP were found to differentiate monocytes towards dendritic cells. Apart from myeloid-committed cell types, VLP-bound cytokines also act efficiently on lymphocytes. hIL-2::GPI strongly costimulated T-cell receptor (TCR)/CD3 dependent T-cell activation in vitro and mIL-2::GPI-coactivated antigen-specific T cells in vivo. On a molar basis, the functional activity of VLP-bound hIL-2::GPI was found to be comparable to that of soluble hIL-2. VLP decorated with hIL-2::GPI and coexpressing a TCR/CD3 ligand have an IL-2-specific activity of 5 x 10(4) units/mg protein. Virus particles decorated with lipid-modified cytokines might help to improve viral strains for vaccination purposes, the propagation of factor-dependent cell types, as well as gene transfer by viral systems in the future.

Direct stimulation of T lymphocytes by immunosomes: virus-like particles decorated with T cell receptor/CD3 ligands plus costimulatory molecules.

Many infectious viruses coevolved with the vertebrate immune system. During the assembly of enveloped viruses, lipid ordered domains of the host cell plasma membrane, called lipid rafts, frequently function as a natural meeting point for viral proteins. The role of lipid rafts in the organization of complex combinations of immune receptors during antigen presentation and T cell signaling is widely recognized. In our studies, we determined whether lipid rafts, virus budding, and molecular interactions during T cell activation could be brought into a novel context to create artificial antigen-presenting particles. We show here that cell-free virus-like particles (VLP) expressing a surrogate TCR/CD3 ligand (OKT3scFv) and the costimulator CD80 polyclonally activate human T cells independently of accessory cells. VLP expressing the glycoprotein epitope 33-41 of the lymphocytic choriomeningitis virus in the context of H-2D(b) activate and expand naïve, antigen-specific CD8(+) T lymphocytes and differentiate them into cytotoxic effector cells. Efficient targeting of T cell ligands to lipid rafts and ultimately to VLP is achieved by C-terminal introduction of glycosyl phosphatidyl inositol acceptor sequences, replacing transmembrane and intracellular domains. In this work, basic functions of immunostimulatory molecules meet virus biology and translate into a reductionist antigen-specific T lymphocyte-stimulating vehicle, which we refer to as immunosomes. A large variety of agonistic and antagonistic accessory molecules on genuine antigen-presenting cells may complicate the predictable manipulation of T cells as well as the analysis of selected receptor combinations, making immunosomes potentially useful reagents for such purposes in the future.