Alina Neunkirchner

Naturally occurring regulatory T cells: markers, mechanisms, and manipulation

Naturally occurring CD4+CD25high forkhead box protein 3 (FOXP3) + regulatory T cells (nTregs) are key mediators of immunity, which orchestrate and maintain tolerance to self and foreign antigens. In the recent 1.5 decades, a multitude of studies have aimed to define the phenotype and function of nTregs and to assess their therapeutic potential for modulating immune mediated disorders such as autoimmunity, allergy, and episodes of transplant rejection. In this review, we summarize the current knowledge on the biology of nTregs. We address the exact definition of nTregs by specific markers and combinations thereof, which is a prerequisite for the state‐of‐the‐art isolation of defined nTreg populations. Furthermore, we discuss the mechanism by which nTregs mediate immunosuppression and how this knowledge might translate into novel therapeutic modalities. With first clinical studies of nTreg‐based therapies being finished, questions concerning the reliable sources of nTregs are becoming more and more eminent. Consequently, approaches allowing conversion of CD4+ T cells into nTregs by coculture with antigen‐presenting cells, cytokines, and/or pharmacological agents are discussed. In addition, genetic engineering approaches for the generation of antigen‐specific nTregs are described.—Schmetterer, K. G., Neunkirchner, A., Pickl, W. F. Naturally occurring regulatory T cells: markers, mechanisms, and manipulation. FASEB J. 26, 2253‐2276 (2012). www.fasebj.org

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-2Db 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.

General Strategy for Decoration of Enveloped Viruses with Functionally Active Lipid-Modified Cytokines

ABSTRACT 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 Fcγ 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 × 104 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.

Bet v 1–specific T-cell receptor/forkhead box protein 3 transgenic T cells suppress Bet v 1–specific T-cell effector function in an activation-dependent manner

BACKGROUND Regulatory T (Treg) cells establish and maintain tolerance to self-antigens and many foreign antigens, such as allergens, by suppressing effector T-cell proliferation and function. We have previously shown that human T-cell receptor (TCR) αβ-chains specific for allergen-derived epitopes confer allergen specificity on peripheral blood T cells of individuals with and without allergy. OBJECTIVE To study the feasibility of generating allergen-specific human Treg cells by retroviral transduction of a transcription unit encoding forkhead box protein 3 (FOXP3) and allergen-specific TCR αβ-chains. METHODS cDNAs encoding the α and β-chains of a Bet v 1(142-153)-specific TCR (TCR alpha variable region 6/TCR beta variable region 20) and human FOXP3 were linked via picornaviral 2A sequences and expressed as single translational unit from an internal ribosomal entry site-green fluorescence protein-containing retroviral vector. Retrovirally transduced peripheral blood T cells were tested for expression of transgenes, Treg phenotype, and regulatory capacity toward allergen-specific effector T cells. RESULTS Transduced T cells displayed a Treg phenotype with clear-cut upregulation of CD25, CD39, and cytotoxic T-lymphocyte antigen 4. The transduced cells were hyporesponsive in cytokine production and secretion and, like naturally occurring Treg cells, did not proliferate after antigen-specific or antigen-mimetic stimulation. However, proliferation was inducible upon exposure to exogenous IL-2. In coculture experiments, TRAV6(+)TRBV20(+)FOXP3(+) transgenic T cells, unlike FOXP3(+) single transgenic T cells or naturally occurring Treg cells, highly significantly suppressed T cell cytokine production and proliferation of corresponding allergen-specific effector T cells in an allergen-specific, dose-dependent manner. CONCLUSION We demonstrate a transgenic approach to engineer human allergen-specific Treg cells that exert their regulatory function in an activation-dependent manner. Customized Treg cells might become useful for tolerance induction therapies in individuals with allergic and other immune-mediated diseases.

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.

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 1142–153 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 αβ TCR specific for the immunodominant epitope Bet v 1142–153. cDNAs encoding TCR α- and β-chains were amplified from a Bet v 1142–153-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 1142–153 peptide or coexpressing invariant chain::Bet v 1142–153 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 1142–153-presenting but not Bet v 14–15-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.

CD23 surface density on B cells is associated with IgE levels and determines IgE-facilitated allergen uptake, as well as activation of allergen-specific T cells

Background: Increasing evidence suggests that the low‐affinity receptor for IgE, CD23, plays an important role in controlling the activity of allergen‐specific T cells through IgE‐facilitated allergen presentation. Objective: We sought to determine the number of CD23 molecules on immune cells in allergic patients and to investigate whether the number of CD23 molecules on antigen‐presenting cells is associated with IgE levels and influences allergen uptake and allergen‐specific T‐cell activation. Methods: Numbers of CD23 molecules on immune cells of allergic patients were quantified by using flow cytometry with QuantiBRITE beads and compared with total and allergen‐specific IgE levels, as well as with allergen‐induced immediate skin reactivity. Allergen uptake and allergen‐specific T‐cell activation in relation to CD23 surface density were determined by using flow cytometry in combination with confocal microscopy and T cells transfected with the T‐cell receptor specific for the birch pollen allergen Bet v 1, respectively. Defined IgE‐allergen immune complexes were formed with human monoclonal allergen‐specific IgE and Bet v 1. Results: In allergic patients the vast majority of CD23 molecules were expressed on naive IgD+ B cells. The density of CD23 molecules on B cells but not the number of CD23+ cells correlated with total IgE levels (RS = 0.53, P = .03) and allergen‐induced skin reactions (RS = 0.63, P = .008). Uptake of allergen‐IgE complexes into B cells and activation of allergen‐specific T cells depended on IgE binding to CD23 and were associated with CD23 surface density. Addition of monoclonal IgE to cultured PBMCs significantly (P = .04) increased CD23 expression on B cells. Conclusion: CD23 surface density on B cells of allergic patients is correlated with allergen‐specific IgE levels and determines allergen uptake and subsequent activation of T cells.

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 (ILRs), 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.—Kueng, H. J., Manta, C., Haiderer, D., Leb, V. M., Schmetterer, K. G., Neunkirchner, A., Byrne, R. A., Scheinecker, C., Steinberger, P., Seed, B., Pickl, W. F. Fluorosomes: a convenient new reagent to detect and block multivalent and complex receptor‐ligand interactions. FASEB J. 24, 1572–1582 (2010). www.fasebj.org

STAT3 governs hyporesponsiveness and granzyme B-dependent suppressive capacity in human CD4+ T cells

Signal transducer and activator of transcription 3 (STAT3) integrates key signals of cell surface immune receptors, yet its precise role in cluster of differentiation (CD)4+ T cells is not well‐established. Current research has indicated T‐helper cell 17–inducing roles but also tolerogenic roles. To address this issue, human T cells were transduced with the constitutively active STAT3 mutant STAT3C. Following stimulation, STAT3C+ T cells up‐regulated IL‐10 (4.1 ± 0.5‐fold; P < 0.001) and granzyme B (2.5 ± 1.2, P < 0.05) secretion, combined with significantly reduced IFN‐γ (35 ± 5%), IL‐2 (57 ± 4%), TNF‐α (64 ± 8%), and IL‐13 (89 ± 3%) secretion (P < 0.001). CD3/CD2‐or CD3/CD28‐activated STAT3C+ T cells revealed reduced proliferation (53.4 ± 23.5% and 70.5 ± 10.4%, respectively), which was independent of IL‐10 production and significantly suppressed effector T cell proliferation by 68.7 ± 10.6% and 65.9 ± 2.6%, respectively (P < 0.001). Phenotypically, STAT3C‐transgenic CD4+ T cells resembled effector T cells regarding expression of T regulatory cell markers, but up‐regulated granzyme B expression levels by 2.4‐fold (P < 0.05). Suppression was cell contact dependent and mediated by granzyme B‐induced cell death, but was independent of IL‐10 and TGF‐β. Notably, peripheral blood CD4+CD45RA– lymphocyte activation gene‐3+CD49+ type 1 regulatory T cells revealed activation‐induced hyperphosphorylation of STAT3. In agreement, pharmacological inhibition of STAT3 activation partially reverted hyporesponsiveness of peripheral type 1 regulatory T cells (increasing their division index from 0.46 ± 0.11 to 0.89 ± 0.04; P < 0.01). These observations indicate a clear‐cut relation between activation of STAT3 and the acquisition of a tolerogenic program, which is also used by peripheral blood type 1 regulatory T cells.—Schmetterer, K. G., Neunkirchner, A., Wojta‐Stremayr, D., Leitner, J., Steinberger, P., Pickl, W. F., STAT3 governs hyporesponsiveness and granzyme dependent suppressive capacity in human CD4+ T cells. FASEB J. 29, 759–771 (2015). www.fasebj.org

Blocking antibodies induced by allergen-specific immunotherapy ameliorate allergic airway disease in a human/mouse chimeric model

Allergen‐specific immunotherapy (AIT) induces specific blocking antibodies (Ab), which are claimed to prevent IgE‐mediated reactions to allergens. Additionally, AIT modulates cellular responses to allergens, for example, by desensitizing effector cells, inducing regulatory T and B lymphocytes and immune deviation. It is still enigmatic which of these mechanisms mediate(s) clinical tolerance. We sought to address the role of AIT‐induced blocking Ab separately from cellular responses in a chimeric human/mouse model of respiratory allergy.