Characterization of the human T cell response to antigen 5 from Vespula vulgaris (Ves v 5)

BACKGROUND: The T cell reactivity to the major allergen of bee venom, phospholipase A2, has been thoroughly characterized. In contrast, only little is known about the human cellular response to major allergens from wasp venom. OBJECTIVE: To characterize the human T cell response to antigen 5 from Vespula vulgaris, Ves v 5. METHODS: Recombinant Ves v 5 was used to establish allergen-specific T cell lines (TCL) and T cell clones (TCC) from the peripheral blood of vespid-allergic and non-allergic individuals. Ves v 5-specific TCL were mapped for T cell epitopes using overlapping synthetic peptides representing the complete amino acid sequence of Ves v 5. Ves v 5-specific TCC were analysed for antigen-induced secretion of IL-4, IFN-gamma and IL-10. RESULTS: Seventeen distinct T cell epitopes were recognized by allergic individuals among which Ves v 5(181-192) was identified as a dominant T cell epitope. Partially different epitopes were observed in TCL from non-allergic subjects and the dominant epitope Ves v 5(181-192) was not prevalent in these cultures. Ves v 5-specific TCC isolated from allergic individuals did not show the typical T helper type 2 (Th2)-like cytokine profile in response to specific stimulation, i.e. high amounts of IL-4 and low IFN-gamma. TCC from non-allergic individuals showed a Th1-like cytokine pattern. CONCLUSIONS: Our findings provide evidence that the allergic T cell response to Ves v 5 is not Th2-dominated and that different immunogenic sites on this major wasp venom allergen are recognized by allergic and non-allergic individuals.     

Characterization of T cell epitopes in αs1-casein in cow''s milk allergic, atopic and non-atopic children

BACKGROUND: One to two percent of infants suffer from IgE-mediated allergic reactions against cow's milk proteins. Most children develop clinical tolerance, but approximately 15% are still allergic by the age of 10 years. Little is known about the T cell epitopes in individual cow's milk protein in relation to allergy and tolerance. OBJECTIVE: To identify T cell epitopes in alphas1-casein, the most abundant milk protein, and to investigate T cell responses toward these epitopes in allergic, atopic and non-atopic children. METHODS: Allergen-specific T cell lines (TCLs) were derived from peripheral blood mononuclear cells of 11 cow's milk allergic, nine atopic and nine non-atopic children. T cell responses were measured to alphas1-casein and to overlapping peptides (18-mers), spanning the alphas1-casein molecule. Proliferation was determined by incorporation of (3)H-thymidine, and cytokine production (IL-10, IL-13 and IFN-gamma) was measured by ELISA. RESULTS: Four main regions (amino acid (AA) residues 43-66, 73-96, 91-114 and 127-180) in the alphas1-casein molecule were immunogenic to T cells, among which the AA residues 133-156 spanned the immunodominant part. Only subtle differences were found in peptide recognition between the subject groups. Some of the peptides induced slightly Th1- or Th2-skewed cytokine responses. The increased levels of IL-10 in response to alphas1-casein observed in TCLs from atopic children appeared not to be linked to recognition of specific IL-10-inducing epitopes. CONCLUSIONS: The immunodominant sequence in alphas1-casein is spanned by AA residues 133-156. Tolerance towards alphas1-casein in atopic children may be mediated by an overall induction of IL-10 and not by recognition of certain T cell epitopes. The identified T cell epitopes in children with cow's milk allergy may be useful targets in developing peptide immunotherapy.     

The correlation between ovomucoid-derived peptides, human leucocyte antigen class II molecules and T cell receptor-complementarity determining region 3 compositions in patients with egg-white allergy

BACKGROUND: Food allergies are more prevalent in children, due to the immature gastrointestinal epithelial membrane barrier allowing more proteins through the barrier and into circulation. Ovomucoid (OM) is one of the major allergens that is found in egg white. OBJECTIVE: The aim of this study was to determine T cell epitopes, antigen-presenting human leucocyte antigen (HLA) class II molecules of the T cell lines (TCLs) and T cell clones (TCCs), and complementarity determining region (CDR) 3 loops of the T cell receptor (TCR) alpha and beta chains of the TCCs specific to OM. METHODS: We established TCLs and TCCs specific to OM from peripheral blood mononuclear cells (PBMCs) of four atopic patients with egg-white allergy using a mixture of a panel of overlapping synthetic peptides corresponding to the amino acid sequence of the entire OM. We identified the T cell epitopes by antigen-induced proliferative responses, antigen-presenting molecules using allogeneic PBMCs and CDR3 loops of the TCR alpha and beta chains by cloning and sequence analysis. RESULTS: The TCLs and TCCs responded to seven different peptides, and their antigen-presenting molecules were different from each other. Sequence analysis of the TCR alpha and beta gene usage of the TCCs showed marked heterogeneity, and the usage of the CDR3 loop of the TCCs involved heterogenous amino acid residues. Interestingly, TCCs 'IH3.3' and 'YT6.1' recognized the same OM peptides, and had the same TCR Vbeta-Jbeta gene usage. Considering that peptide motifs bind to HLA class II molecules, the electrically charged residue (positive or negative) on the CDR3alpha and the CDR3beta loops of TCR of TCC may form ionic bonds with a charged residue on the HLA class II molecules-peptide complex. CONCLUSIONS: TCCs that have the same TCR gene usage were established from patients who had shown similar hypersensitivity-type, indicating that antigen recognition by a specific TCR is closely associated with the characteristics of each patient's symptoms.     

Characterization of the human T cell response to rye grass pollen allergens Lol p 1 and Lol p 5

BACKGROUND: Knowledge of dominant T cell epitopes of major allergens recognized by allergic individuals is required to improve efficacy and safety of allergen immunotherapy. Rye grass pollen (RGP) is the most important source of seasonal aeroallergens in temperate climates and Lol p 1 and Lol p 5 are the two major IgE-reactive allergens. This study aimed to characterize the T cell response to these allergens using a large panel of RGP-sensitive individuals. METHODS: Short-term RGP-specific T cell lines (TCL) were generated from 38 RGP-sensitive subjects and stimulated with Lol p 1 and/or Lol p 5 allergens and synthetic 20-mer peptides. Proliferative responses were determined by 3H-thymidine uptake and IL-5 and IFN-gamma in culture supernatants analysed by ELISA. RESULTS: Of 17 subjects tested for reactivity to both allergens 16 (94%) responded to Lol p 1 and/or Lol p 5, establishing these as major T cell-reactive allergens. Sites of T cell reactivity were spread throughout the allergen molecules but regions of high reactivity were found. For Lol p 1 these spanned residues 19-38, 109-128, 154-173, 190-209, and for Lol p 5 37-56, 100-119, 145-164, 154-173, 190-209, 217-236 and 226-245. IL-5 and IFN-gamma were produced by T cells cultured with proliferation-inducing peptides. CONCLUSIONS: T cell responses to RGP major allergens have been extensively characterized, providing fundamental information for developing T cell-targeted immunotherapy for RGP allergy.     

The hevein domain of the major latex-glove allergen Hev b 6.01 contains dominant T cell reactive sites

BACKGROUND: Sensitization to natural rubber latex (Hevea brasiliensis) is a major cause of occupational asthma and rhinitis affecting frequent latex-glove users. Hev b 6.01, a known major latex allergen, is cleaved naturally into hevein (4.7 kDa) and a C-terminal fragment (14 kDa). Hevein is an abundant protein in latex-glove extracts. As the immune response to allergens is initiated by activation of allergen-specific CD4(+) T cells, identification of dominant T cell epitopes is crucial for the development of specific immunotherapy. OBJECTIVE: To identify dominant T cell epitopes of Hev b 6.01 in latex-allergic glove users. METHODS: Ten latex-allergic frequent glove users and six non-latex-allergic atopic control subjects were selected, based on clinical symptoms and positive latex-specific serum IgE. Serum IgE reactivity to glove extract and recombinant Hev b 6.01 (rHev b 6.01) were analysed by ELISA. Latex-specific short-term oligoclonal T cell lines were generated from peripheral blood of latex-allergic subjects. These lines were tested for proliferative responses to overlapping 20-mer peptides of the Hev b 6.01 molecule. CD4(+) T cell intracellular cytokines, IL-4 and IFN-gamma were assessed following stimulation with immobilized anti-CD3 in the presence of IL-2. RESULTS: All ten of the latex-allergic patients showed serum IgE binding to glove extract while eight of these also showed IgE binding to rHev b 6.01 by ELISA. Western blotting confirmed reactivity with rHev b 6.01 at around 20 kDa. T cell proliferation assays showed that latex-specific T cell lines from all subjects responded to one or more peptides, with greatest frequency of reactivity to peptides Hev b 6.01 p(10-29) and Hev b 6.01 p(19-38) in the hevein domain. An allergic-type cytokine profile with considerable IL-4 in addition to IFN-gamma was evident from intracellular cytokine staining. CONCLUSION: Hevein is an important T cell as well as B cell immunogen and contains dominant T cell reactive sites.     

Specific T cell lines for ovalbumin, ovomucoid, lysozyme and two OA synthetic epitopes, generated from egg allergic patients' PBMC

Background Proteitis of hen egg white are common ingredients of food and difficult to eliminate. Allergens of egg while induce allergic symptoms among relatively high numbers of palients suffering from food allergy. B cell epitopes to hen egg white tnajor allergens have been reported. Considering that IgE antibody formation is mostly T cell dependent, the study of T cell epitopes is essential for both T cell dependent and independent IgE response. Objectives Little information on T cell epitopes recognizing food allergens has been reported. T cell responses to hen egg white allergens and two synthetic OA peptides located at amino acid residues No. 105–122 and 323–339 were investigated. Methods Peripheral blood mononuclear cells from hen egg allergic patients were investigated. Various allergens of hen egg white were used for stimulation. Primary proliferation responses were detected followed by the generation of long-term cultures which were examined for their specificity, phenotype, cytokine profile and IgE production. The allergen specific T cell lines were mapped using a panel of 13 synthetic peptides of ovalbumin. Results Human T cells recognizing ovomucoid, lysozyme and ovalbumin epitope 105–122 are reported for the first time. The cell lines were enriched CD4⁺/CD8⁺ T cells (CD2⁺ 95%). Ovomucoid and ovalbumin induced IgE synthesis by a small fraction of B cells (1%) present in the ovalbumin and ovomucoid specific T cell lines. Conclusions Human T cells recognized several egg white allergens and epitopes within the ovalbumin molecule. Specific IgE was produced in cultures stimulated with ovalbumin and ovomucoid. OA peptides 105–122 and 323–339 have no affinity to the specific IgE of the two patients; an observation which could be of particular interest regarding the mechanisms of peptide-based immunotherapy.     

Immunogenic peptides: B & T Cell Epitopes of Per a 10 Allergen of Periplaneta americana

Mapping of B and T cell epitopes of an allergen can be utilised in the development of alternative therapeutic modalities and diagnostics. The present study was aimed to identify B and T cell epitopes of Per a 10, a major cockroach allergen, by computational tools and subsequent validation by in vitro experiments. Per a 10 three-dimensional structure was homology modelled using structure of anionic trypsin from pacific chum salmon as a template. Seven B cell epitopes (B-P1 to B-P7) were predicted by sequence and structure based methods. Three T cell epitopes (T-P8 to T-P10) were predicted by binding score and inhibitory concentration dependent prediction tools. Predicted epitopes were synthesized and biological activity was assessed by ELISA, ELISA inhibition and PBMC proliferation assays. B cell peptides B-P5, B-P6 and B-P7 showed significantly high IgE binding with pooled and individual cockroach hypersensitive patients’ sera while the T cell peptides did not show IgE binding. ELISA inhibition was performed to determine the potency of the predicted peptides. Fifty nanogram of peptide B-P7 was required for 50% IgE binding inhibition of surface bound Per a 10 whereas seventy five nanogram and ninety nanogram of B-P5 and B-P6 were required for the same respectively. Upon stimulation with T-P8 and T-P10 peptides, PBMCs from cockroach allergic patients’ (n = 15) showed significant lymphocyte proliferation and induced IL-4 and IL-5 cytokine release in the culture supernatant demonstrating Th2 dominant cell mediated response of predicted T cell peptides. In conclusion, Per a 10 3-D structure obtained by homology modelling was used to identify B and T cell epitopes, followed by in vitro validation. The identified peptides can be potentially used in designing diagnostics and therapies for cockroach allergy.     

Identification of human T cell epitopes in Japanese cypress pollen allergen, Cha o 1, elucidates the intrinsic mechanism of cross-allergenicity between Cha o 1 and Cry j 1, the major allergen of Japanese cedar pollen, at the T cell level

BACKGROUND: Pollens from species of Cupressaceae family are one of the most important causes of respiratory allergies worldwide. In Japan, many patients with pollinosis have specific IgE to both pollens of Japanese cypress (Chamaecyparis obtusa) and Japanese cedar (Cryptomeria japonica). The sequences between Cha o 1 and Cry j 1, the major allergens of Japanese cypress and Japanese cedar pollens, respectively, are 80% identical. OBJECTIVE: This study was undertaken to identify T cell epitopes in Cha o 1, and to elucidate the mechanism of cross-allergenicity between Cha o 1 and Cry j 1, at the T cell level. METHODS: T cell epitopes in Cha o 1 were identified by the reactivity of T cell lines, generated from 19 patients, to stimulation with overlapping peptides. The subsets of T cell clones specific to rCha o 1 were determined according to their ability to produce IL-4 and IFN-gamma. Peptide specificities of two T cell clones were determined by stimulation with the peptides from Cha o 1 and Cry j 1. RESULTS: Four dominant and six subdominant T cell epitopes were identified in Cha o 1. While four T cell epitopes, p11-30, p211-230, p251-270 and p331-350, were common to Cha o 1 and Cry j 1, 4 T cell epitopes, p61-80, p71-90, p311-330 and p321-340, were considered to be unique to Cha o 1. The subsets of T cell clones were predominantly of T helper2-type. One T cell clone recognized p16-30, which is common to Cha o 1 and Cry j 1, but another recognized p321-330, which is unique to Cha o 1. CONCLUSION: Presence of both T cells reactive to T cell epitopes common to Cha o 1 and Cry j 1 and T cells specific to T cell epitopes unique to Cha o 1 in patients with pollinosis contributes to prolonged symptoms after the cedar pollen season in March and the following cypress pollen season in April.     

Definition of three minimal T helper cell epitopes of rubella virus E1 glycoprotein

To characterize T cell-recognized epitopes on rubella virus (RV) E1 glycoprotein, IL-2-dependent RV-specific T cell lines were established from 14 rubella-seropositive healthy donors. The responses of these lines were studied by using a panel of 94 partially overlapping synthetic peptides of 15 amino acids (aa) length covering the known nucleotide sequence of RVE1 glycoprotein. Two to seven peptide-defined epitopes were recognized by the T cell lines, but a large interindividual variation was found. T cell reactivity was most often localized to the regions between aa 276 and 290, aa 381 and 395 and aa 410 and 420. Analysis of overlapping, truncated peptides revealed three minimal T helper cell epitopes VIGSQARK, KFVTAALLN and RVIDPAAQ in aa positions 280–287, 385–393 and 412–419, respectively.     

Aberrant interleukin (IL)-4 and IL-5 production in vitro by CD4+ helper T cells from atopic subjects.

The cytokine secretion profiles of T cell lines (TCL) specific for purified protein derivative (PPD) or streptokinase (SK), contemporarily derived from nine atopic and nine nonatopic individuals, were compared. Upon stimulation with phorbol myristate acetate (PMA) plus anti-CD3 monoclonal antibody (mAb), all TCL from both atopics and nonatopics produced interleukin (IL)-2 and interferon (IFN)-gamma. The mean IL-2 production by PPD- or SK-specific TCL from both atopics and nonatopics was similar, whereas the mean IFN-gamma production by TCL derived from atopics was significantly lower. In addition, both PPD- and SK-specific TCL from atopics produced detectable amounts of IL-4 and IL-5, whereas the corresponding TCL derived from nonatopics did not. A total number of 107 and 99 PPD-specific CD4+ T cell clones (TCC) were then derived from TCL of 4 atopic and 4 nonatopic donors and assessed for their profile of cytokine production in response to stimulation with either PMA plus anti-CD3 mAb or the specific antigen. Under both these experimental conditions, virtually all PPD-specific TCC from both atopic and nonatopic individuals produced IL-2 and IFN-gamma. In contrast, the great majority of PPD-specific TCC derived from nonatopic individuals did not produce IL-4 and IL-5, whereas high proportions of PPD-specific TCC derived from atopic donors displayed the ability to produce noticeable amounts of IL-4 and IL-5 besides IL-2 and IFN-gamma. These data indicate that CD4+ T cells from atopic individuals are able to produce IL-4 and IL-5 in response to bacterial antigens, such as PPD and SK, that usually evoke responses with a restricted type-1 T helper (Th1)-like cytokine profile in nonatopic individuals. Aberrant IL-4 production by Th cells may represent one of the immune alterations responsible for enhanced IgE antibody production in atopic people.     

T cell recognition pattern of bovine milk alphaS1-casein and its peptides

T cell recognition patterns of CAS1_Bovin, its limited hydrolysis, oxidized, reduced/alkylated, cyanogen bromide cleavage fractions and synthetic peptides were examined. Thirteen overlapping peptides covering the intact molecule, with chain lengths varied between 17 and 20 AA, were prepared by f-moc SPPS. In addition, six CNBr-cleavage fragments were obtained and extensively purified using RP/HPLC. Likewise, chemically modified derivatives and limited pepsin hydrolysate, were performed and the specificities were confirmed. Stimulation of PBMC and TCL cultures by the intact CAS1_Bovin molecule, synthetic peptides and modified derivatives were screened by [methyl-3H] thymidine incorporations. PBMC phenotype was performed by flow cytometry and the mean CD4+/CD8+ ratio of freshly prepared PBMC was compared with the ratio following specific CAS1_Bovin stimulation. CD4+ phenotypes (TH1/, TH2 and TH0) were assigned by assay of four marker cytokines IL-4, IL-6, IL-10 and IFN-gamma. Five CNBr fragments and seven of the thirteen tested peptides were recognized by specific TCL. The most reactive epitopes of CAS1_Bovin comprised seven motifs namely: peptides Cas 1-18, Cas 16-35, Cas 67-85, Cas 91-110, Cas 136-155, Cas 152-169 and Cas 166-183. The stimulation range for the seven peptides was 1058-2383 cpm. Stimulation for the CNBr fragments were, respectively, 8670, 5808, 3324, 5465, 2255 and 321 cpm. Cytokine assay showed that CD4+ TH2 phenotype was dominant for half the number of patients, while TH1 solely or combined TH0 were represented in the other four cell culture filtrates. The T cell reactive epitopes described and their antibodies will be useful tools for methods in progress for the detection of masked casein epitopes encompassed in processed food. In conclusion, T cell recognition pattern of CAS1_Bovin was examined using extensively purified synthetic peptides and CNBr fragments. Five large and seven small peptides were clearly recognized. Peptides of chain length less than six AA were left unrecognised. CD4+ TH2 phenotype was the most dominant TCL subpopulations found in atopic patients while CD4+ TH1 was representative in the non-IgE mediated type IV hypersensitivity.     

日本血吸虫重组28GST T细胞表位谱的预测及鉴定

目的 :用软件预测日本血吸虫重组 2 8GST抗原分子T细胞表位谱 ,并鉴定其Th1型细胞表位。方法 :用软件预测重组2 8GSTT细胞表位谱 ,并筛选几种较好的T细胞表位 ,人工合成表位肽或用基因工程制备重组表位肽融合蛋白。体外刺激经照射的尾蚴感染并用 2 8GST加强免疫的C5 7BL/ 6小鼠 (H 2 b)脾细胞 ,通过淋巴细胞增殖试验、ELISA及流式细胞术等 ,分析各种表位的免疫刺激作用 ,鉴定Th1型细胞表位。结果 :在 9个候选的表位中 ,P6 (73～ 86aa)是刺激作用最强的Th1型细胞表位。结论 :重组 2 8GST含有功能性Th1型细胞表位     

T cell proliferation and cytokine responses to ovalbumin and ovomucoid detected in children with and without egg allergy

BACKGROUND: The specific T cell responses in egg allergy and resolution have not been fully elucidated. OBJECTIVE: To characterize egg allergen-specific T cells of children with active and resolved egg allergy, in comparison with non-allergic controls. METHOD: We studied children with active (n=35) or resolved (n=20) egg allergy determined by oral challenge, and non-allergic controls (n=15). Peripheral blood mononuclear cells were labelled with carboxyfluorescein succinimidyl ester (CFSE) and stimulated with ovalbumin (OVA), ovomucoid (OM) or tetanus toxoid. Flow cytometry was used to detect divided CD3+ CFSE(lo) cells that expressed intra-cytoplasmic IL-4 or IFN-gamma. The cell division index (CDI) was calculated as a measure of allergen-specific proliferation. Peanut-specific T cells of a subgroup of children who also had peanut allergy were also studied. RESULTS: OVA-specific T cells were found in subjects with active (87%) or resolved (75%) egg allergy and in controls (67%), with a trend towards increased T cell proliferation in allergy. OM-induced weaker T cell responses than OVA, stimulating fewer responders (46% allergic, 50% resolved, 60% controls) and 10-fold less proliferation [CDI(OVA) 2.0 (median), 25.6 (maximum) vs. CDI(OM) 0.2 (median), 15.1 (maximum); P<0.01]. Both egg allergens induced significant IL-4+ (median 10%, range 1.4-58%) and IFN-gamma+ (median 28%, range 4.5-63%) cells in responders, including non-allergics. There were no significant differences in IFN-gamma+ or IL-4+ cells or in IFN-gamma/IL-4 ratios between groups. Peanut-specific T cell proliferation was significantly higher in peanut allergy [CDI(CPE) 16.5 (median), 24.8 (maximum)] compared with controls [CDI(CPE) 2.1 (median), 16.1 (maximum)] but cytokine profiles were not different. Tetanus-specific T cells were seen in 90% of the subjects, with no significant inter-group differences in responses. CONCLUSION: Egg allergen-specific T cells are readily detected in all groups and not restricted to egg allergy. In contrast, peanut-specific proliferation was significantly higher in peanut allergy. This suggests that T cell responses in peanut and egg allergy may differ. We did not find T helper type 2-deviated cytokine responses in egg or peanut allergy.     

IL-12-Dependent enhancement of CTL response to weak class I-restricted peptide immunogens requires coimmunization with T helper cell immunogens

The effect of in vivo administration of rmIL-12 on the CTL response to immunization with a weakly immunogenic class I-restricted peptide emulsified in incomplete Freund's adjuvant was investigated. In the absence of IL-12, peptide-specific CTL responses were significantly greater following coimmunization with class I-restricted peptide and T helper cell antigens than following immunization with the class I-restricted peptide alone. IL-12-dependent enhancement of the CTL response to peptide immunization was demonstrated in the presence of, but not in the absence of, coimmunization with T helper cell antigen. These findings indicate that IL-12 enhancement of the CTL response to weak class I-restricted immunogens is T helper cell dependent. Treatment with rmIL-12 also enhanced the CTL response to immunization with cDNA encoding both CTL and T helper cell epitopes. These findings are relevant to the design of vaccines containing tumor-associated class I-restricted peptides currently being tested as an immunotherapy for cancer patients.     

Functional inhibition related to structure of a highly potent insulin-specific CD8 T cell clone using altered peptide ligands

Insulin-reactive CD8 T cells are amongst the earliest islet-infiltrating CD8 T cells in NOD mice. Cloned insulin B15-23-reactive cells (designated G9C8), restricted by H-2K(d), are highly diabetogenic. We used altered peptide ligands (APL) substituted at TCR contact sites, positions (p)6 and 8, to investigate G9C8 T cell function and correlated this with structure. Cytotoxicity and IFN-gamma production assays revealed that p6G and p8R could not be replaced by any naturally occurring amino acid without abrogating recognition and functional response by the G9C8 clone. When tested for antagonist activity with APL differing from the native peptide at either of these positions, the peptide variants, G6H and R8L showed the capacity to reduce the agonist response to the native peptide. The antagonist activity in cytotoxicity and IFN-gamma production assays can be correlated with conformational changes induced by different structures of the MHC-peptide complexes, shown by molecular modeling. We conclude that p6 and p8 of the insulin B15-23 peptide are very important for TCR stimulation of this clone and no substitutions are tolerated at these positions in the peptide. This is important in considering the therapeutic use of peptides as APL that encompass both CD4 and CD8 epitopes of insulin.     

Characterization of the T cell response to the major hazelnut allergen, Cor a 1.04: evidence for a relevant T cell epitope not cross-reactive with homologous pollen allergens

BACKGROUND: IgE antibodies specific for the major birch-pollen allergen, Bet v 1, cross-react with homologous allergens in particular foods, e.g. apples, carrots and hazelnuts. In a high number of tree pollen-allergic individuals, this cross-reactivity causes clinical symptoms, commonly known as the 'birch-fruit-syndrome'. OBJECTIVE: To characterize the T cell response to the Bet v 1-related major allergen in hazelnuts, Cor a 1.04, and its cellular cross-reactivity with Bet v 1 and the homologous hazel pollen allergen, Cor a 1. METHODS: Using recombinant Cor a 1.04, T cell lines (TCL) and T cell clones (TCC) were established from peripheral blood mononuclear cells of tree pollen-allergic patients with associated food allergy. T cell epitopes were determined using overlapping synthetic peptides in Cor a 1.04-reactive TCL and TCC. In parallel, reactivity to Bet v 1 and Cor a 1 was tested. RESULTS: In total, 20 distinct T cell epitopes on the hazelnut allergen were identified. Several Cor a 1.04-specific TCL and TCC reacted with pollen allergens albeit less pronounced than with the hazelnut allergen. Several Cor a 1.04-specific TCC did not react with pollen allergens. Interestingly, these clones were found to react with the Bet v 1-related major allergen in carrots, Dau c 1. The cellular cross-reactivity between both food allergens could be associated with the most frequently recognized T cell epitope of Cor a 1.04, Cor a 1.04(142-153). CONCLUSIONS: The major hazelnut allergen cross-reacts with the major allergens of birch and hazel pollen but apparently contains a relevant T cell epitope not shared with pollen allergens. Our finding may have important implications for the specific immunotherapy of tree pollen-allergic patients suffering from concomitant hazelnut allergy.     

Inhibition of mucosal and systemic T(h)2-type immune responses by intranasal peptides containing a dominant T cell epitope of the allergen Der p 1

Although the intranasal administration of peptides containing T cell epitopes has been shown to be a potent method of inhibiting responses to the allergen Der p 1, the experiments to date have concentrated on their ability to regulate immune responses to the injection of antigen in a T(h)1-type adjuvant. Their ability to regulate responses to a T(h)2-type immunization and to sensitization via the respiratory mucosa has not been examined. Here it is shown that peptide used in doses required to block delayed-type hypersensitivity can also readily inhibit IgE responses to Der p 1 injected in alum. To examine responses induced in the respiratory mucosa, mice pretreated with intranasal peptide were sensitized with an intranasal dose of Der p 1 in conjunction with a mutated enterotoxin adjuvant. Intranasal peptide even in very high doses did not reduce IgE titers, but the ability of cells from the draining lymph nodes to release IL-4 and IL-13 but not IL-2, IL-5, IL-10 or IFN-gamma was reduced. These are the first reports on the effect of intranasal peptides containing T cell epitopes on IgE in T(h)2 immunization and on responses to respiratory immunization. Thus the effect of the peptide-induced mucosal tolerance differs depending on the type of immunization used for sensitization, but the potential to inhibit T(h)2 responses and responses to respiratory sensitization as well as T(h)1 responses has been demonstrated.     

Cross-reactive epitopes and HLA-restriction elements in human T cell recognition of the Mycobacterium leprae 18-kD heat shock protein

We have previously demonstrated that the Mycobacterium leprae 18-kD heat shock protein (HSP18) is represented among the antigenic targets of human T cell responses induced by M. leprae immunization and that the peptide 38-50 serves as an immunodominant epitope recognized by CD4+ T cell clones. By using peripheral blood mononuclear cells and T cell lines from the same donor group, we have in this study shown that the M. leprae HSP18 and peptide 38-50 were recognized by memory T cells 8 years after immunization with M. leprae. The finding that M. bovis BCG-induced T cell lines responded to M. leprae HSP18, but not to the peptide 38-50, suggested the existence of additional T cell epitopes of a cross-reactive nature. Consistent with this, testing of the T cell lines for proliferative responses to the complete HSP18 molecule, truncated HSP18 (amino acid (aa) residues 38-148) and overlapping synthetic peptides, made it possible to identify two cross-reactive epitope regions defined by aa residues 1-38 and 41-55. While peptide 38-50-reactive T cell clones showed limited cross-reactivity by responding to M. leprae, M. avium and M. scrofulaceum, the T cell lines specific to the epitopes 1-38 and 41-55 were broadly cross-reactive, as demonstrated by their response to M. leprae, M. tuberculosis complex, M. avium and other mycobacteria. MHC restriction analysis of the HSP18-responding T cell lines showed that the epitopes 1-38 and 38-50 were presented by one of the two HLA-DR molecules expressed from self HLA-DRB1 genes, whereas the epitope 41-55 was recognized in the presence of autologous as well as HLA-DR and HLA-DQ mismatched allogeneic antigen-presenting cells. The results obtained in this study made it possible to identify cross-reactive T cell epitopes of the M. leprae HSP18, and provide an explanation for T cell recognition of this antigen in individuals infected with species of the M. tuberculosis complex or environmental mycobacteria.     

Study of antibody and T cell responses in rabbits immunized with synthetic human B cell epitope analogues of La (SSB) autoantigen

The aim of this study was to investigate the immunogenicity of four synthetic peptides, representing linear B cell epitopes of the human La/SSB autoantigen: 145-164 aa (p1), 289-308 aa (p2), 301-318 aa (p3) and 349-364 aa (p4), in rabbits. New Zealand White rabbits were immunized with each of the above peptides attached in four copies on tetrameric sequential oligopeptide carriers (SOC) in duplicate. Control immunizations were also performed (one rabbit each, immunized with Freud's complete adjuvant alone or with the SOC carrier alone). Animals were bled at regular intervals and sera were analysed for anti-La/SSB activity by ELISA assays using as antigen the various synthetic peptides, as well as the whole La/SSB protein. Four months after the last immunization, the animals were killed and peripheral blood mononuclear and spleen cells were co-cultured with either the peptides, the SOC carrier, or 27 peptides, covering the entire length of the human La/SSB molecule (23 amino acids long, overlapping by eight residues to each other). A specific, IgG, anti-peptide antibody response was detected, initially directed against the priming peptide, and subsequently expanded to the other La/SSB synthetic peptides. The antibody titres remained high, even 4 months after the last immunization. Sera from rabbits immunized with either p2 or p3 reacted also with the whole La/SSB protein, as was demonstrated by ELISA and immunoblot assays. No reactivities against either Ro60 or Ro52 autoantigen were found. Rabbit spleen cell reacted not only with the epitope used for the immunization but also with other La/SSB peptides. Immunization of rabbits with the major human La/SSB B cell antigenic determinants, linked to SOC carrier, induces strong and sustained antibody and T cell responses against multiple epitopes of the human La/SSB protein. Thus, La/SSB B cell linear epitopes are probably capable also of functioning as T cell epitopes, in this experimental animal.     

一种探找超抗原T细胞识别表位的新方法

目的建立一种有效的探找超抗原Ｔ细胞识别表位的新方法。方法以检测超抗原合成肽或其在ＣＤ２８抗体辅助下诱导外周血单个核细胞（ＰＢＭＣ）的增殖与否为指标，确定超抗原的合成肽是否含有超抗原Ｔ细胞识别表位。结果一个超抗原合成肽单独并不能活化ＰＢＭＣ，但在ＣＤ２８抗体的辅助下却可激活ＰＢＭＣ。结论超抗原合成肽结合ＣＤ２８抗体的方法对寻找超抗原Ｔ细胞识别表位是切实可行的。