Toshiyuki Katsuki

Determinant analysis of IgE and IgG4 antibodies and T cells specific for bovine αs1-casein from the same patients allergic to cow's milk: Existence of αs1-casein–specific B cells and T cells characteristic in cow's-milk allergy

In an effort to clarify the etiology of milk allergy from the standpoint of allergen-specific immune reactions, we investigated the determinants of IgE, IgG4, and T cells specific for bovine alpha(s)1-casein from the same individual patients by using its synthetic peptides and cyanogen bromide-digested fragments. Alpha(s)1-casein is a major allergen in cows milk, and its unique conformation enabled us to investigate the determinants of antibodies without consideration about missing the reactivities because of conformational changes. Nine patients were selected as subjects from among 129 milk-sensitive infants screened by ELISA to assess the anti-alpha(s)1-casein IgE levels in their sera. By using ELISA for epitope mapping, a C-terminal region of alpha(s)1-casein was identified as a common binding site for IgE from all of these patients, whereas those for anti-alpha(s)1-casein IgG4 were located in multiple regions of alpha(s)1-casein. We determined the specificities of seven alpha(s)1-casein-specific T-cell lines established from peripheral blood mononuclear cells of two of the patients. These T cells have been shown to secrete IL-4. All of the T-cell lines had different specificities to alpha(s)1-casein. However, a common amino acid residue use was found among the determinants of various T-cell lines from each patient. The results suggest that patients allergic to cows milk have characteristic B cells recognizing a limited region of alpha(s)1-casein and secreting alpha(s)1-casein-specific IgE. These B cells may interact particularly with T cells recognizing determinants with a common structure.

A common T-cell epitope between human thyroglobulin and human thyroid peroxidase is related to murine experimental autoimmune thyroiditis

We have investigated functional common T-cell epitopes between human thyroglobulin (hTg) and human thyroid peroxidase (hTPO) in mice. Four hTg peptides, Tg-P1, Tg-P2, Tg-P3 and Tg-P4, in which 5 amino acid residues are identical to those of hTPO, and 1 hTPO peptide, TPO-P4 relevant to Tg-P4, were prepared. Among these peptides, only Tg-P4 (residues 2730-2743) and TPO-P4 (residues 118-131) were highly antigenic and both peptides shared the common T-cell epitope. In addition, when the spleen cells from mice immunized with mouse Tg (mTg) were restimulated in vitro by Tg-P4 or TPO-P4 as well as by mTg, these cells transferred thyroiditis to naive recipient mice. These findings indicate that this common T-cell epitope between hTg and hTPO is immunogenic and related to the development of murine experimental autoimmune thyroiditis.

Establishment and Characterization of Ovalbumin-Specific T Cell Lines from Patients with Egg Allergy

In order to investigate T cell recognition of allergens in hen egg allergy, we have established 30 ovalbumin (OVA)-specific T cell lines (TCLs) from peripheral blood mononuclear cells of 6 patients with atopic dermatitis, who are positive for IgE antibodies to OVA and clinically allergic to hen egg, and characterized them for their cytokine production pattern. All TCLs we could study were mainly composed of CD4+ T cells. Most TCLs produced significant amounts of interleukin 4 (IL-4) and IL-5 but no or very little interferon gamma on antigen stimulation, suggesting that these TCLs belong to TH2-type T cells. Restriction elements and epitope specificities were further studied on some TCLs. Antibody blocking of the proliferative responses of the TCLs to OVA indicated that HLA-DR are acting as the dominant restriction elements for these TCLs with minor contribution of HLA-DQ. By use of 187 overlapping synthetic peptides covering the whole sequence of OVA, at least 3 different T cell epitopes were identified.

Establishment and characterization of αs1-casein–specific T-cell lines from patients allergic to cow’s milk: Unexpected higher frequency of CD8+ T-cell lines

Abstract To study cows milk allergy at the cellular level, we assessed the reactivity of peripheral blood mononuclear cells from patients allergic to cows milk to α s1 -casein, which is one of the major allergens in cows milk. Proliferation of the cells to α s1 -casein activation showed a rather weak response. Therefore to understand T-cell reactivity to α s1 -casein in more detail, we prepared α s1 -casein–specific T-cell lines from patients allergic to cows milk and established 26 T-cell lines. These T-cell lines could be classified into three groups by analyzing their surface marker expression: those containing predominantly CD4 + CD8 - T cells, those containing both CD4 + CD8 - and CD4 - CD8 + T cells, and those containing predominantly CD4 - CD8 + T cells. The CD8 + T cells were obtained at an unexpectedly higher frequency from the patients. These T-cell lines produced interferon-γ and IL-4. These results suggest that CD8 + T cells specific for α s1 -casein and CD4 + T cells were primed by the stimulation with α s1 -casein in patients allergic to milk and that both T cells may play a key role in the onset, progression of, or recovery from cows milk allergy. (J ALLERGY CLIN IMMUNOL 1996;97:1342-9.)

Diminished interferon‐gamma (IFN‐γ) production by bacterial antigen‐specific T cells in atopic patients

In this study, we established and studied cytokine production of T cell lines (TCL) specific to either a purified protein derivative of Mycobacterium tuberculosis (PPD) or Dermatophagoides farinae (Df) from atopic patients and non‐atopic healthy subjects. IFN‐γ was detected in the culture supernatants of all of 36 PPD‐specific TCL established from healthy controls, whereas only 24 of 38 PPD‐specific TCL from patients produced IFN‐γ. Furthermore, the amounts of IFN‐γ produced by PPD‐specific TCL from patients were significantly lower than those from healthy controls. No IL‐4 was detected in any PPD‐specific TCL from either healthy controls or atopic patients. The amounts of IL‐4 production from Df‐specific TCL from atopic patients were much higher than from healthy controls, while few TCL produced IFN‐γ. These results suggest that the skewing to the Th2‐type T cell response in atopic patients is a response not only to allergens, but also to bacterial antigens, compared with non‐atopic subjects. Activation of PPD‐specific TCL from patients with calcium ionophore A23187 plus phorbol myristate acetate resulted in much higher IFN‐γ production than in TCL established from healthy controls, indicating that the low production of IFN‐γ by PPD‐specific T cells from atopic patients is not due to an intrinsic T cell defect but to some regulatory mechanisms.

Homing Receptor Expression on Cord Blood T Lymphocytes and the Development of Atopic Eczema in Infants

Expression of the gut-homing receptor integrin αEβ7, but not cutaneous lymphocyte-associated antigen (CLA), on milk allergen-stimulated cord blood T lymphocytes precedes the development of milk-induced eczema in early infancy. The data indicate the involvement of integrin αEβ7 in the development of infantile allergic eczema and provide a clue to the avoidance of specific allergens and novel therapy targeting homing receptors in food allergy.

Thyroglobulin-specific t cell line from a healthy individual does not produce proinflammatory cytokines on antigenic stimulation: an implication for possible fail-safe mechanism to avoid autoimmunity

In order to investigate the regulation of autoimmune response to thyroglobulin (Tg), one of the thyroid autoantigens, we established a Tg-specific T cell line by stimulation of peripheral blood mononuclear cells from a healthy volunteer with Tg and characterized its cytokine production pattern. The Tg-specific T cell line, designated DH5D1, obtained from a limiting dilution culture bore alpha beta T cell receptor and was CD4 and CD45RO positive. Upon stimulation with Tg, DH5D1 secreted little or no titers of IL-2, TNF-alpha, and IFN-gamma, whereas activation with combination of phorbol myristate acetate and calcium ionophore produced measurable levels of these cytokines. These results indicate that the Tg-specific T cell line is not defective in its capacity to produce proinflammatory cytokines and suggest that the inability of cytokine production by autoreactive T cells of healthy individuals is one fail-safe mechanism for preventing aggression of harmful autoimmune response.

Comparison of specificity between IgG, IgE and T cells to three casein components: Implication for the role of circulating allergenspecific T cells in food allergy

In order to investigate the role of food antigen‐specific T cells circulating in the blood of patients with food allergy, we compared T cell response to three casein components (αs‐, β‐ and, K‐casein) with specificities of IgG and IgE binding to the casein components in four milk‐allergic patients (P1‐4) with atopic dermatitis. In all patients, the binding activities of IgG antibodies to αs‐casein were most dominant, followed by those to β‐ and to K‐casein. The major component of casein bound by IgE antibodies was αs‐casein in P1 and P3, K‐casein in P2, and αs‐casein as well as K‐casein in P4; the order of casein components bound by IgE antibodies was different from that by IgG antibodies. Proliferative responses of peripheral blood mononuclear cells (PBMC) to casein components were so low that the dominance of casein recognition could not be clearly demonstrated. However, short‐term T cell lines that specifically respond to casein were successfully established from PBMC of the four patients and the proliferative responses of the T cell lines to the three components of casein were in accord with the IgE antibody specificity to casein components but not with that of IgG antibody specificity. When taken together, these results indicate that casein‐specific T cells circulating in the blood are involved in or reflect an allergic reaction against casein.

T Helper Lymphocyte Response to Respiratory Syncytial Virus and Its Components in Patients with Respiratory Allergy and Nonatopic Controls

Background: Respiratory syncytial virus (RSV) G protein is involved in Th2-shifted immune response, while F protein has a reverse effect on RSV infection in Th2-prone BALB/c mice. Studies on the human T cell response to F or G protein are few, and the relationship between the immune response to G protein and atopy is not known. Methods: We established CD4+ RSV-specific T cell lines (TCLs) from adult patients with respiratory allergic diseases (allergics) or nonatopic controls (controls), and examined proliferative responses and γ-interferon (IFN-γ) and interleukin 4 (IL-4) production in these TCLs upon stimulation with RSV, F or G proteins. Results: 32 and 29 RSV-specific oligoclonal TCLs were established from allergics and controls, respectively. IL-4/IFN-γ in the culture supernatant of antigen-stimulated TCLs was significantly higher in allergics than in controls (p = 0.042). IL-4/IFN-γ ratios in the culture supernatants of G-protein-reactive TCLs were significantly higher in allergics than in controls (p = 0.016), while no differences in IL-4/IFN-γ in culture supernatants of F-protein-reactive TCLs were found between allergics and controls (p = 0.787). IL-4/IFN-γ in the culture supernatants of G-protein-reactive TCLs was significantly higher than those of F-protein-reactive TCLs in allergics (p = 0.023) but not in controls (p = 0.768). Conclusion: The results suggest that the T cell response to RSV is influenced by the atopic diathesis as well as by individual RSV antigens, and that G protein may be an important antigen involved in allergy in humans.

Low Interleukin 10 Production at Birth Is a Risk Factor for Atopic Dermatitis in Neonates with Bifidobacterium Colonization

Background: Altered regulatory immune responses to microbial stimuli and intestinal colonization of beneficial bacteria early in life may contribute to the development of allergic diseases (e.g., atopic dermatitis [AD]). However, few reports have investigated these factors simultaneously. The purpose of this study was to analyze neonatal immune responses to microbial stimuli as well as intestinal colonization of beneficial bacteria, in relation to the development of AD in a birth cohort. Methods: Pregnant women were recruited, and their infants were followed up until 7 months of age. Levels of interleukin (IL)-10 released from cord-blood mononuclear cells (CBMCs) stimulated with heat-killed gram-positive bacteria (Bifidobacterium bifidum and Lactobacillus rhamnosus GG) and Lactobacillus-derived peptidoglycan were measured. Fecal Bifidobacterium counts at 4 days and 1 month were quantified using real-time polymerase chain reaction. The development of AD was determined by means of a questionnaire at 7 months of age. Results: The levels of released IL-10 were significantly lower in infants with AD (n = 17) than in infants without AD (n = 53) for all stimuli. In infants with fecal Bifidobacterium, the incidence of AD was inversely associated with the release of IL-10 from cord blood mononuclear cells. Conclusion: Our findings suggest that impaired IL-10 production in response to microbial stimuli at birth may be associated with an increased risk of developing infantile AD, even in infants with early colonization of intestinal bifidobacteria.