Shu-Chen Lyu

Peanut oral immunotherapy results in increased antigen-induced regulatory T-cell function and hypomethylation of forkhead box protein 3 (FOXP3)

BACKGROUND The mechanisms contributing to clinical immune tolerance remain incompletely understood. This study provides evidence for specific immune mechanisms that are associated with a model of operationally defined clinical tolerance. OBJECTIVE Our overall objective was to study laboratory changes associated with clinical immune tolerance in antigen-induced T cells, basophils, and antibodies in subjects undergoing oral immunotherapy (OIT) for peanut allergy. METHODS In a phase 1 single-site study, we studied participants (n = 23) undergoing peanut OIT and compared them with age-matched allergic control subjects (n = 20) undergoing standard of care (abstaining from peanut) for 24 months. Participants were operationally defined as clinically immune tolerant (IT) if they had no detectable allergic reactions to a peanut oral food challenge after 3 months of therapy withdrawal (IT, n = 7), whereas those who had an allergic reaction were categorized as nontolerant (NT; n = 13). RESULTS Antibody and basophil activation measurements did not statistically differentiate between NT versus IT participants. However, T-cell function and demethylation of forkhead box protein 3 (FOXP3) CpG sites in antigen-induced regulatory T cells were significantly different between IT versus NT participants. When IT participants were withdrawn from peanut therapy for an additional 3 months (total of 6 months), only 3 participants remained classified as IT participants, and 4 participants regained sensitivity along with increased methylation of FOXP3 CpG sites in antigen-induced regulatory T cells. CONCLUSION In summary, modifications at the DNA level of antigen-induced T-cell subsets might be predictive of a state of operationally defined clinical immune tolerance during peanut OIT.

Successful immunotherapy induces previously unidentified allergen-specific CD4+ T-cell subsets

Significance The mechanisms through which successful immunotherapy induces possible deletion, replacement, or reprogramming of T cells are unknown. By evaluating the expression of T-cell–related genes, and using appropriate multivariate statistical approaches, our data show that successful immunotherapy can induce previously unidentified CD4+ T-cell subtypes during treatment that could help to predict an “immune-tolerant” clinical phenotype identified after cessation of treatment. The ability to use “anergic” transcriptional phenotypes in single T cells to predict successful “immune tolerance” induction in the clinic setting, as suggested by our findings, could lead to transformative impacts in the field of immunotherapy. Allergen immunotherapy can desensitize even subjects with potentially lethal allergies, but the changes induced in T cells that underpin successful immunotherapy remain poorly understood. In a cohort of peanut-allergic participants, we used allergen-specific T-cell sorting and single-cell gene expression to trace the transcriptional “roadmap” of individual CD4+ T cells throughout immunotherapy. We found that successful immunotherapy induces allergen-specific CD4+ T cells to expand and shift toward an “anergic” Th2 T-cell phenotype largely absent in both pretreatment participants and healthy controls. These findings show that sustained success, even after immunotherapy is withdrawn, is associated with the induction, expansion, and maintenance of immunotherapy-specific memory and naive T-cell phenotypes as early as 3 mo into immunotherapy. These results suggest an approach for immune monitoring participants undergoing immunotherapy to predict the success of future treatment and could have implications for immunotherapy targets in other diseases like cancer, autoimmune disease, and transplantation.

Krüppel-Like Transcription Factor 13 Regulates T Lymphocyte Survival In Vivo

Krüppel-like transcription factor (KLF)13, previously shown to regulate RANTES expression in vitro, is a member of the Krüppel- like family of transcription factors that controls many growth and developmental processes. To ascertain the function of KLF13 in vivo, Klf13-deficient mice were generated by gene targeting. As expected, activated T lymphocytes from Klf13−/− mice show decreased RANTES expression. However, these mice also exhibit enlarged thymi and spleens. TUNEL, as well as spontaneous and activation-induced death assays, demonstrated that prolonged survival of Klf13−/− thymocytes was due to decreased apoptosis. Microarray analysis suggests that protection from apoptosis-inducing stimuli in Klf13−/− thymocytes is due in part to increased expression of BCL-XL, a potent antiapoptotic factor. This finding was confirmed in splenocytes and total thymocytes by real-time quantitative PCR and Western blot as well as in CD4+CD8− single-positive thymocytes by real-time quantitative PCR. Furthermore, EMSA and luciferase reporter assays demonstrated that KLF13 binds to multiple sites within the Bcl-XL promoter and results in decreased Bcl-XL promoter activity, making KLF13 a negative regulator of BCL-XL.

Tissue-specific differences in the establishment of tolerance. Tolerogenic effects of lung allografts in rats.

With the increasing frequency of transplantation of two or more organs into a single recipient, it has become evident that different organs are rejected with different kinetics. In this study the kinetics of skin, lung, and heart allograft rejection were compared in a rodent model. To study the influence of different allografts on the recipients immune system, simultaneous or sequential skin, lung, or heart transplants were performed in various combinations, using DA rats as recipients for PVG allografts. Recipients receiving primary allografts were treated postoperatively with ten doses of cyclosporine (CsA) or preoperatively with 4 doses of rabbit antirat thymocyte globulin (ATG). Subsequent transplants were performed a minimum of 40 days later without additional immunosuppression. All primary skin allografts and 60% of primary lung allografts were rejected, while 100% of the heart allografts were accepted indefinitely. Recipients of primary skin allografts rejected subsequent skin, lung, or heart allografts with accelerated kinetics. Recipients of primary heart allografts accepted subsequent skin, lung, and heart allografts indefinitely without further immunosuppression. Surprisingly, animals that had rejected a primary lung allograft accepted subsequent skin or heart allografts indefinitely. Simultaneously transplanted skin and lung allografts were concordantly rejected. However, these animals accepted a subsequent heart allograft indefinitely, suggesting a strong tolerizing effect of lung allografts. Our results indicate that tissue-specific differences are critical, not only in determining acceptance or rejection of a primary allograft but also in determining the fate of subsequent allografts.

TISSUE-SPECIFIC DIFFERENCES IN THE ESTABLISHMENT OF TOLERANCE

With the increasing frequency of transplantation of two or more organs into a single recipient, it has become evident that different organs are rejected with different kinetics. In this study the kinetics of skin, lung, and heart allograft rejection were compared in a rodent model. To study the influence of different allografts on the recipients immune system, simultaneous or sequential skin, lung, or heart transplants were performed in various combinations, using DA rats as recipients for PVG allografts. Recipients receiving primary allografts were treated postoperatively with ten doses of cyclosporine (CsA) or preoperatively with 4 doses of rabbit antirat thymocyte globulin (ATG). Subsequent transplants were performed a minimum of 40 days later without additional immunosuppression. All primary skin allografts and 60% of primary lung allografts were rejected, while 100% of the heart allografts were accepted indefinitely. Recipients of primary skin allografts rejected subsequent skin, lung, or heart allografts with accelerated kinetics. Recipients of primary heart allografts accepted subsequent skin, lung, and heart allografts indefinitely without further immunosuppression. Surprisingly, animals that had rejected a primary lung allograft accepted subsequent skin or heart allografts indefinitely. Simultaneously transplanted skin and lung allografts were concordantly rejected. However, these animals accepted a subsequent heart allograft indefinitely, suggesting a strong tolerizing effect of lung allografts. Our results indicate that tissue-specific differences are critical, not only in determining acceptance or rejection of a primary allograft but also in determining the fate of subsequent allografts.

Purification and Characterization of a Black Walnut (Juglans nigra) Allergen, Jug n 4

Tree nuts as a group cause a significant number of fatal anaphylactic reactions to foods. Walnuts (Juglans spp.) are one of the leading causes of allergic reactions to tree nuts in the U.S. and Japan. The purpose of this study was to purify and characterize potential food allergens from black walnut. Here, we report the isolation of the black walnuts allergen Jug n 4 (an 11S globulin) by ammonium sulfate precipitation, hydrophobic interaction, and size exclusion chromatography. Reducing SDS-PAGE analysis indicated that purified Jug n 4 consists of three major bands. N-Terminal sequencing data of these bands indicated that they were the results of a post-transcriptional protease cleavage of the mature protein at a site that consists of a known conserved protease recognition motif, NGXEET. Western blot experiments revealed that 32% of the sera from 25 patients with double-blind, placebo-controlled clinical walnut allergy contained IgE antibodies that recognized Jug n 4, indicating that it is a walnut allergen. Identifying this and additional allergens may facilitate the understanding of the allergenicity of seed storage proteins in tree nuts and their cross-reactivity.

Identification, characterization, and initial epitope mapping of pine nut allergen Pin k 2

The aims of this study were to predict, identify and characterize pine nut allergens. Korean pine (Pinus koraiensis) vicilin was predicted to be a pine nut allergen. Recombinant Korean pine vicilin was expressed in E. coli and purified. Natural Korean pine vicilin isolated from pine nuts (which displayed multiple bands in SDS-gels due to posttranslational digestion) and its full length recombinant counterpart were used to test whether it is a food allergen. The recognition of the protein (and its fragments) by patient serum IgE was analyzed by Western blot. The study included fourteen patients diagnosed with clinical pine nut allergy. Twenty nine percent of the patient sera recognized both the natural and recombinant pine nut vicilin, indicating that Korean pine vicilin is a bona fide food allergen. The serum recognition patterns of the naturally occurring protein fragments suggested that some of linear IgE epitopes may be mapped to the fragment boundaries. The chemical and thermal stability of the recombinant protein was investigated. It underwent a thermal transition with a Tm=76.6°C. The transition was accompanied by an increase in the amplitude of the circular dichroism signal at 220nm. Urea induced unfolding of the recombinant protein had a Cm of 4.6M.

Isotype-specific agglutination-PCR (ISAP): A sensitive and multiplex method for measuring allergen-specific IgE

To the Editor: Component-resolved diagnostics (CRD) is a rapidly growing class of in vitro tests for evaluating individuals thought to have allergic diseases. In contrast to using whole-allergen extracts, CRD uses individual, molecularly pure allergen components to measure allergen-specific IgE (sIgE), thereby helping to assess the risk of allergy to such allergens with improved accuracy. For example, the presence of sIgE against certain peanut components (eg, Ara h 1, Ara h 2, and Ara h 3) indicates a higher risk for anaphylaxis than does anti–Ara h 8 sIgE (which can reflect cross-reactivity with birch pollen). Although ELISAbased CRD tests may lack analytical sensitivity, the alternative more sensitive assays currently used clinically (eg, ImmunoCAP) are not multiplexible and may omit certain critical allergens. These problems can be mitigated by collecting more blood for additional tests. However, this approach can be problematic when studying small animals or testing young children. Here, we report isotype-specific agglutination-PCR (ISAP), a highly sensitive and multiplexible approach for measuring