Cor a 1-reactive T cells and IgE are predominantly cross-reactive to Bet v 1 in patients with birch pollen-associated food allergy to hazelnut.

BACKGROUND: IgE- and T-cell cross-reactivity contribute to the birch pollen-food syndrome. OBJECTIVES: We performed a comprehensive analysis of T-cell cross-reactivity in primary cell cultures, facilitating the identification of allergen-specific T-cell subpopulations from individual patients. METHODS: Patients with birch pollen allergy and associated food allergy to hazelnuts, carrots, or both were analyzed for IgE cross-reactivity, T-cell responses, and T-cell cross-reactivity to recombinant Bet v 1.0101 (Bet v 1; birch), Cor a 1.0401 (Cor a 1; hazelnut), and Dau c 1.0104 (Dau c 1; carrot). A novel flow cytometry-based method using a 2-step staining process with fluorescent dyes was established to identify subpopulations of cross-reactive T cells. RESULTS: IgE-binding inhibition tests of individual sera revealed that the vast majority of Cor a 1-reactive IgE was cross-reactive to Bet v 1, whereas Bet v 1-reactive IgE was only partially inhibited by preincubation with Cor a 1. Primary stimulation of T cells with Bet v 1 or Cor a 1 resulted in a significant increase in specific responses to Cor a 1 or Bet v 1 after secondary stimulation, respectively, indicating T-cell cross-reactivity between birch and hazelnut allergens in all patients of the study cohort. Preactivation with Dau c 1 induced less pronounced effects. A novel flow cytometry-based proliferation assay identified a predominant Cor a 1/Bet v 1-cross-reactive T-cell subpopulation within highly Bet v 1/Cor a 1-responsive T cells. CONCLUSION: Analysis of primary allergen-specific T cells combined with flow cytometry-based proliferation assays facilitates investigation of allergen-specific T-cell subpopulations in subjects and might be helpful to evaluate the effect of birch-specific immunotherapy on pollen-associated food allergies.

Neuronal nitric oxide synthase modulates maturation of human dendritic cells.

Dendritic cells (DCs) are the most potent APCs of the immune system. Understanding the intercellular and intracellular signaling processes that lead to DC maturation is critical for determining how these cells initiate T cell-mediated immune processes. NO synthesized by the inducible NO synthase (iNOS) is important for the function of murine DCs. In our study, we investigated the regulation of the arginine/NO-system in human monocyte-derived DCs. Maturation of DCs induced by inflammatory cytokines (IL-1beta, TNF, IL-6, and PGE(2)) resulted in a pronounced expression of neuronal NOS (nNOS) but only minimal levels of iNOS and endothelial NOS were detected in human mature DCs. In addition, reporter cell assays revealed the production of NO by mature DCs. Specific inhibitors of NOS (N-nitro-L-arginine methyl ester) or of the NO target guanylyl cyclase (H-(1,2,4)-oxadiazolo [4,3-a] quinoxalin-1-one) prevented DC maturation (shown by decreased expression of MHC class II, costimulatory and CD83 molecules and reduced IL-12 production) and preserved an immature phenotype, indicating an autocrine effect of nNOS-derived NO on human DC maturation. Notably, inhibitor-treated DCs were incapable of inducing efficient T cell responses after primary culture and generated an anergic T cell phenotype. In conclusion, our results suggest that, in the human system, nNOS-, but not iNOS-derived NO, plays an important regulatory role for the maturation of DCs and, thus, the induction of pronounced T cell responses.

MAP kinase p38 and its relation to T cell anergy and suppressor function of regulatory T cells.

Diverse regulatory T cell populations (Treg) are important for the control of self tolerance and immune homeostasis. These include naturally occurring CD4+CD25+ Treg (nTreg) and induced Treg (iTreg). Tolerogenic dendritic cells, modulated by IL-10, are able to convert peripheral T cells into iTreg. These are anergic and characterized by a G(1) cell cycle arrest, dependent on elevated levels of the cdk inhibitor p27(Kip1). Novel data revealed a distinct pattern of MAP kinase activation in iTreg different from clonal T cell anergy, with enhanced activation of the p38-MAPKAP-K2/3 pathway. p38 is involved in cell cycle control and its activity is a prerequisite for the induction and maintenance of the anergic state in iTreg. Inhibition of p38 leads to down regulation of p27(Kip1), cell cycle progress and loss of regulatory T cell function. Here, we discuss these data in light of the role of p38 and p27(Kip1) in T cell activation, anergy induction and cell cycle control.

Tolerogenic dendritic cells in health and disease: friend and foe!

Dendritic cells (DC) are professional antigen presenting cells and crucial mediators of immune defence and tolerance. Experimental evidence in vitro and in vivo has shown that tolerogenic DCs (tDCs) contribute to prevention of autoimmunity and allergic reactions but might also worsen the course of cancer and certain infectious diseases. The development of in vitro protocols for the generation of human tDCs has greatly improved the knowledge of basic principles for tolerance induction by tDCs and fosters approaches for the therapeutic manipulation of tDCs in conditions such as severe autoimmunity, allergy or transplantation.

Activation of MAP kinase p38 is critical for the cell-cycle-controlled suppressor function of regulatory T cells.

Regulatory T cells play an essential role in the control of self-tolerance and processes of adaptive immunity. Tolerogenic IL-10-modulated human dendritic cells (IL-10DCs) induce anergic T cells with strong suppressive properties (iTregs) that inhibit the activation of effector T cells. In this study, we evaluated the interaction between cell-cycle regulation and intracellular signaling in these iTregs. Analysis of signal transduction events revealed a down-regulation of the mitogen-activated protein kinases (MAPKs) Jun N-terminal kinase (JNK) and a nonactivation of extracellular-signal-regulated kinase (ERK) in contrast to a marked activation of p38 MAPK and the p38 effector MAPK-activated protein kinases 2/3 (MAPKAP2/3). The elevated activation of p38 is critical for the induction and maintenance of anergy controlled by an increased expression of the cell-cycle inhibitor p27(Kip1). Moreover, blocking experiments with the specific inhibitor SB203580 demonstrated that the regulatory function of iTregs is associated with an enhanced p38 MAPK activity. In contrast to other Treg populations, the suppressor function of iTregs is independent of IL-10. In conclusion, our data indicate that a cross-talk of cell-cycle regulation and p38-dependent signal transduction is required for the suppressor function of iTregs.

Simian immunodeficiency viruses with defective nef genes show increased susceptibility to the noncytotoxic antiviral activity of CD8+ lymphocytes.

The noncytotoxic soluble factor produced by CD8+ T cells inhibits replication of HIV and SIV in vitro and is thought to play a crucial role in combatting infection in vivo. We determined the effect of human CD8+ lymphocytes on the in vitro replication potential of both wild-type and nef-defective mutants of the simian immunodeficiency virus SIVmac251. Although replication of wild-type SIVmac251 in unstimulated human PBMC supplemented with IL-2 was unaffected by the presence of CD8+ T cells, the nef mutants were susceptible to the inhibitory effects. The effect of exogenous IL-2 depended upon the culture conditions: (i) in nonstimulated human PBMC depleted of CD8+ T cells, addition of IL-2 had a positive effect on the growth of the nef-defective viruses; (ii) in total human PBMC, IL-2 appeared to reinforce the CD8+ T-cell-dependent inhibition of the same mutant viruses. This strongly suggests that IL-2 stimulates the noncytotoxic anti-HIV/SIV response of CD8+ cells present in PBMC cultures. PHA stimulation of unfractionated human PBMC overrode the suppression of viral replication by CD8+ T cells. Depletion of activated T cells expressing the IL-2 receptor alpha-chain (CD25+ T cells), present in small amounts in these primary T cell cultures, dramatically reduced viral replication, indicating that the depleted cell population harbors the target cells permissive for viral replication. Furthermore, using neutralizing antibodies we could show that inhibition by the beta-chemokines MIP-1alpha, MIP-1beta, and RANTES and the inhibitory effect of CD8+ lymphocytes on nef mutant SIVmac viruses are harbored on different levels.