Human B cells secrete migration inhibition factor (MIF) and present a naturally processed MIF peptide on HLA-DRB1*0405 by a FXXL motif.

A better knowledge of peptide structures interacting with major histocompatibility complex (MHC) molecules is of great interest for better understanding of the molecular basis of immune recognition. We have isolated naturally processed peptides from a continuously growing antigen-presenting Epstein-Barr virus-transformed human B-cell line. HLA-DR complexes were purified by specific affinity chromatography and complexed peptides were released by acid treatment. The isolated peptides were separated by reversed phase chromatography and fractions were analysed by Edman degradation at picomolar ranges. From 30 fractions that were examined seven peptides bound to the HLA-DRB1*0405 and two peptides from the human leucocyte antigen (HLA) class II associated invariant chain bound to HLA-DRB1*1302. In addition, a N-terminal beta-chain peptide of the 0405 allele was identified. Evaluation of amino acid sequences revealed a refined FXXL motif for the 0405 allele, in which F (phenylalanine) stands for any aromatic amino acid and L (leucine) can be exchanged by either I (isoleucine) or V (valine). In total, three fractions contained a peptide derived from the human migration inhibition factor (MIF), a pro-inflammatory cytokine that is normally produced by activated T lymphocytes and monocytes/macrophages. Indeed, cytokine analysis revealed high amounts of MIF secreted by the B-cell line, confirming that MHC class II expressing cells can present any intrinsic peptide that contains the distinct motif for HLA-binding. For MIF, the amino acid sequence Y36IAV39 represents the required binding motif for HLA-DRB1*0405. Nevertheless, it is the first time that cytokine fragments were found to bind to HLA molecules on human B cells.

An altered peptide ligand specifically inhibits Th2 cytokine synthesis by abrogating TCR signaling.

Altered peptide ligands (APL) can modify T cell effector function by their diversity in binding to the TCR or MHC class II-presenting molecules. The capacity to inhibit Th2 cytokine production by allergen-specific T cells would contribute to combating allergic inflammation. The presence of APL generated by Ala-substitutions in a synthetic dodeca-peptide spanning an immunodominant epitope of bee venom phospholipase A2 (PLA) was investigated in human T cells. Four of five substituted peptides reduced proliferation, IL-4, and IFN-gamma production by cloned PLA-specific Th0 cells proportionately. However, one APL, PLA-F82A, inhibited IL-4 but had no effect on IFN-gamma production. This uncoupling of IL-4 from IFN-gamma production was also observed on immunogenic restimulation of the cloned T cells pre-exposed to the APL/APCs. It appeared to result from lower affinity of binding to MHC class II by the APL compared with the native peptide. The APL also inhibited IL-4 production by polyclonal T cells. In consequence of the change in cytokine secretion, the production of IgG4 in vitro increased by PLA-F82A stimulation, compared with the native peptide. Exposure of the cloned T cells to either the APL or the native peptide, in the absence of professional APC, induced anergy such that proliferation and production of IL-4, IL-5, and IL-13 was abrogated on immunogenic rechallenge. Defective T cell activation appeared to result from alterations in transmembrane signaling through the TCR, specifically to lack of tyrosine phosphorylation of the tyrosine kinase, ZAP-70.

Enzymatic activity of soluble phospholipase A2 does not affect the specific IgE, IgG4 and cytokine responses in bee sting allergy.

BACKGROUND: The soluble bee venom phospholipase A2 (PLA) represents the major allergen/antigen for allergic and hyperimmune individuals following bee sting. A number of studies implicate enzymes, and PLA in particular, as potent allergens. We have studied specific activation of T cells by enzymatically active and inactive mutants of PLA, and secretion of cytokines regulating IgE and IgG4 antibody formation. METHODS: Recombinant (r) wild type PLA (rPLA-WT) and an enzymatically inactive rPLA (rPLA-H34Q) were produced in Escherichia coli. Eleven bee venom allergic patients and three hyperimmune, healthy individuals were included in the study. After specific stimulation of PBMC with the rPLA variants, proliferative response, IFNgamma, IL-2, IL-4, IL-5, and IL-13 production, as well as total and PLA-specific IgE and IgG4 production, were analysed. RESULTS: Similar levels of specific B cell recognition, proliferative and cytokine responses were observed after stimulation with either enzymatically active or inactive rPLA. In addition, equal amounts of antigen-specific and total IgE and IgG4 antibodies were produced by stimulation with both forms of rPLA. CONCLUSIONS: The enzymatic activity of PLA does not influence the specific activation and cytokine production by T cells from bee venom-sensitized or hyperimmune individuals, or the IgE/IgG4 antibodies synthesis by B cells in vitro.

Differential regulation of human T cell cytokine patterns and IgE and IgG4 responses by conformational antigen variants.

Bee venom phospholipase A2 (PLA) represents the major allergen and antigen in allergic and non-allergic individuals sensitized to bee sting. We have studied specific activation of peripheral T cells by different structural and conformational variants of PLA and secretion of cytokines regulating IgE and IgG4 antibody (Ab) formation. PLA molecules expressing the correctly folded tertiary structure, which show high affinity to membrane phospholipids and were recognized by Ab from bee sting allergic patients, induced high IL-4, IL-5 and IL-13 production in peripheral blood mononuclear cell cultures. In contrast, non-refolded recombinant PLA (rPLA) and reduced and alkylated native PLA (nPLA) induced more IFN-gamma and IL-2 and higher proliferative responses. Differences in proliferation and cytokine patterns among correctly folded and non-refolded PLA resulted from conformation-dependent involvement of different antigen-presenting cell (APC) types. Antigen (Ag)-presenting B cells recognized PLA only in its natural conformation, stimulated Th2 type cytokines and induced IgE Ab. Non-refolded PLA was recognized, processed and presented exclusively by monocytes and induced a Th1 dominant cytokine profile leading to IgG4 production by B cells. The possibility that production of particular cytokine patterns and Ig isotype was influenced by the enzymatic activity of PLA was excluded by using enzymatically inactive H34Q point-mutated, refolded rPLA. These findings demonstrate the decisive role of specific Ag recognition by different APC, depending on structural features, membrane phospholipid binding property and the existence of conformational B cell epitopes, in the differential regulation of memory IgE and IgG4 Ab. Furthermore, they show that a change from IgE-mediated allergy to normal immunity against a major allergen can be induced by rPLA variants that are not recognized by specific Ab and B cells but still carry the T cell epitopes. These features may enable new applications for safer immunotherapy.

Increased specificity of diagnostic tests with recombinant major bee venom allergen phospholipase A2.

BACKGROUND: In diagnosis of type I allergy recombinant allergens have potential advantages over conventional allergenic extracts, both regarding specificity and reproducibility. OBJECTIVES: We therefore decided to study honey bee venom (BV) and its major allergen phospholipase A2 (PLA) in native and recombinant form for diagnosis of bee sting allergy. METHOD: We investigated 85 patients with a history of a recent systemic allergic bee sting reaction and positive intracutaneous skin test to BV, and 21 controls with no history of allergic bee sting reaction and negative skin test to BV. Intracutaneous skin tests and determination of specific IgE by ImmunoCAP(R) to BV, native PLA (nPLA) and recombinant PLA (rPLA) were done in all patients and controls. RESULTS: In skin testing 84 (99%) of the 85 patients reacted to nPLA and 81 (95%) to rPLA, while none of the 21 controls was positive with nPLA or rPLA. Specific serum IgE to BV could be detected in 82 of the patients (96%), to nPLA in 73 (86%) and to rPLA in 66 (78%). Four (19%) of the controls had a positive CAP to BV, one (4.8%) to nPLA and none to rPLA. Analysis of discordant results in CAP showed, that most patients with specific IgE to BV, but not to nPLA and rPLA, had positive skin tests to both PLA preparations and low levels of BV specific IgE. Patients with specific IgE to nPLA but not to rPLA were usually sensitized to minor allergens of BV which contaminated the commercial nPLA. CONCLUSIONS: PLA is the major allergen in BV. While diagnostic tests with BV are more sensitive, the specificity of tests with PLA, especially rPLA is clearly increased as compared with BV.

Epitope-specific T cell tolerance to phospholipase A2 in bee venom immunotherapy and recovery by IL-2 and IL-15 in vitro.

Bee venom phospholipase A2 (PLA) is the major allergen in bee sting allergy. It displays three peptide and a glycopeptide T cell epitopes, which are recognized by both allergic and non-allergic bee venom sensitized subjects. In this study PLA- and PLA epitope-specific T cell and cytokine responses in PBMC of bee sting allergic patients were investigated before and after 2 mo of rush immunotherapy with whole bee venom. After successful immunotherapy, PLA and T cell epitope peptide-specific T cell proliferation was suppressed. In addition the PLA- and peptide-induced secretion of type 2 (IL-4, IL-5, and IL-13), as well as type 1 (IL-2 and IFN-gamma) cytokines were abolished, whereas tetanus toxoid-induced cytokine production and proliferation remained unchanged. By culturing PBMC with Ag in the presence of IL-2 or IL-15 the specifically tolerized T cell response could be restored with respect to specific proliferation and secretion of the type 1 T cell cytokines, IL-2 and IFN-gamma. In contrast, IL-4, IL-5, and IL-13 remained suppressed. Treatment of tolerized T cells with IL-4 only partially restored proliferation and induced formation of distinct type 2 cytokine pattern. In spite of the allergen-specific tolerance in T cells, in vitro produced anti-PLA IgE and IgG4 Ab and their corresponding serum levels slightly increased during immunotherapy, while the PLA-specific IgE/IgG4 ratio changed in favor of IgG4. These findings indicate that bee venom immunotherapy induces a state of peripheral tolerance in allergen-specific T cells, but not in specific B cells. The state of T cell tolerance and cytokine pattern can be in vitro modulated by the cytokines IL-2, IL-4, and IL-15, suggesting the importance of microenvironmental cytokines leading to success or failure in immunotherapy.