Identification of Novel Th2-Associated Genes in T Memory Responses to Allergens Anthony Bosco, Kathy L. McKenna, Catherine J. Devitt, Martin J. Firth, Peter D. Sly and Patrick G. Holt This information is current as of October 1, 2021. J Immunol 2006; 176:4766-4777; ; doi: 10.4049/jimmunol.176.8.4766 http://www.jimmunol.org/content/176/8/4766 Downloaded from References This article cites 119 articles, 43 of which you can access for free at: http://www.jimmunol.org/content/176/8/4766.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on October 1, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Identification of Novel Th2-Associated Genes in T Memory Responses to Allergens1 Anthony Bosco, Kathy L. McKenna, Catherine J. Devitt, Martin J. Firth, Peter D. Sly, and Patrick G. Holt2 Atopic diseases are associated with hyperexpression of Th2 cytokines by allergen-specific T memory cells. However, clinical trials with recently developed Th2 inhibitors in atopics have proven disappointing, suggesting underlying complexities in atopy patho- genesis which are not satisfactorily explained via the classical Th1/Th2 paradigm. One likely possibility is that additional Th2- associated genes which are central to disease pathogenesis remain unidentified. The aim of the present study was to identify such novel Th2-associated genes in recall responses to the inhalant allergen house dust mite. In contrast to earlier human microarray studies in atopy which focused on mitogen-activated T cell lines and clones, we concentrated on PBMC-derived primary T cells stimulated under more physiological conditions of low dose allergen exposure. We screened initially for allergen-induced gene Downloaded from activation by microarray, and validated novel genes in independent panels of subjects by quantitative RT-PCR. Kinetic analysis of allergen responses in PBMC revealed an early wave of novel atopy-associated genes involved in signaling which were coex- pressed with IL-4 and IL-4R, followed by a later wave of genes encoding the classical Th2 effector cytokines. We further dem- onstrate that these novel activation-associated Th2 genes up-regulate in response to another atopy-associated physiological stim- ulus bacterial superantigen, but remain quiescent in nonphysiological responses in primary T cells or cell lines driven by potent mitogens, which may account for their failure to be detected in earlier microarray studies. The Journal of Immunology, 2006, 176: http://www.jimmunol.org/ 4766–4777. t is well-established from studies in humans and animal mod- number of differentially expressed genes identified, however, the els that Th2 lymphocytes secreting signature cytokines such lack of consistency in the gene lists reported by each study is I as IL-4, IL-5, IL-9, and IL-13 are central to the development striking. of atopy (1, 2). Accordingly, these cytokines and their downstream In the current study, a different approach was undertaken. We products have become major foci for drug development for control reasoned that while the pattern of T cell gene expression induced of diseases such as atopic asthma. However, the level of clinical in vivo at sites such as the airway mucosa is ultimately controlled efficacy achieved in recent trials with newly developed Th2 an- by local tissue microenvironmental factors, significant elements of by guest on October 1, 2021 tagonists including anti-IgE (3), rIL-12 (4), anti-IL-4 (5), and anti- the potential gene response “program” of allergen-specific T mem- IL-5 (6) have been disappointing, suggesting that additional (as yet ory cells (exemplified by the IL-4/IL-5-dominant cytokine profile unrecognized) components of the Th2 cascade which escape reg- of T cell clones from atopics) can be accurately revealed by low- ulation via these approaches play key roles in atopy pathogenesis. intensity in vitro stimulation of recirculating memory cells har- The likelihood that additional atopy-associated genes remain to vested from peripheral blood. In adopting this approach, we have be identified can be inferred from recent experiences reported in minimized in vitro manipulations and avoided the use of strong the immunological literature following the introduction of mi- activation stimuli, which have the potential to distort patterns of croarray technology. Collectively, these studies indicate that indi- gene expression in T cells (22, 23), and report for the first time the vidual immune responses typically involve up-regulation of mul- results of microarray analysis of PBMC-derived T cell responses tiple hundreds of genes (7–9). This technology has also been to the house dust mite (HDM)3 allergen in short-term primary applied in the allergy field to study Th2 responses and related culture. The fidelity of the experimental system was evident by the signaling pathways in freshly isolated T cells (10), polyclonally parallel detection of Th2 cytokine hyperexpression in atopics as stimulated T cells (11, 12), allergen-specific T cell clones and lines pooled samples by microarray, and individually by ELISA. In ad- (13, 14), polarized Th1/Th2 cell lines (15–20), and in animal mod- dition, several novel atopy-associated genes were identified, and els (21). A consistent finding in the studies was the substantial the preferential expression of these genes in atopics was validated in HDM-stimulated CD4ϩ T cells from an additional panel of atopic patients and nonatopic controls, both as pooled samples by Telethon Institute for Child Health Research, and Centre for Child Health Research, Faculty of Medicine and Dentistry, University of Western Australia, Perth, Western microarray and individually by quantitative RT-PCR. We addi- Australia tionally demonstrate differential expression of these novel genes in Received for publication November 1, 2005. Accepted for publication January an unrelated T cell response system, notably activation which is 30, 2006. driven by the bacterial superantigen staphylococcal enterotoxin B The costs of publication of this article were defrayed in part by the payment of page (SEB). charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by the National Health and Medical Research Council of Australia and Pfizer Pharmaceuticals. 3 Abbreviations used in this paper: HDM, house dust mite; SEB, staphylococcal en- 2 Address correspondence and reprint requests to Prof. Patrick G. Holt, Division of terotoxin B; PPD, purified protein derivative; qRT-PCR, quantitative RT-PCR; QT, Cell Biology, Telethon Institute for Child Health Research, P.O. Box 855, West Perth, quality threshold; MB, multivariate empirical Bayes; SR, stimulation ratio; DTH, WA 6872, Australia. E-mail address: [email protected] delayed-type hypersensitivity. Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 The Journal of Immunology 4767 Materials and Methods the novel Th2-associated genes detected by microarray screening, using Subjects more precise qRT-PCR methodology. Microarray data were analyzed in the open-source statistical software R Subjects were volunteers aged 11–58 years. Atopic status to HDM was (͗www.r-project.org/͘), using several additional add-on packages from the determined by skin prick test (wheal Ն5 mm) and/or positive serum HDM- Bioconductor Project (͗www.bioconductor.org/͘) (35) including affy, specific IgE (Ն0.35 kU/L). The study was approved by our institutional affyQCReport, affyPLM, annotate, hgu133a, hgu133plus2, limma, q human ethics committee. value, and time course. The probe-level model algorithm (Refs. 34 and 36, ͗http://stat-www.berkeley.edu/users/bolstad/Dissertation/ Cell preparation and culture methodologies Bolstad_2004_Disseration.pdf͘), which is based on the robust multi- array average algorithm (37), was used for background subtraction, nor- PBMC were cultured as detailed (24) with medium alone or containing ␮ malization, and summarization of probe set intensities. All microarrays optimal stimulatory concentrations of 10 g/ml HDM (Dermatophagoides within each experiment were of comparable quality as assessed using the pteronyssinus; CSL) or 10 ␮g/ml purified protein derivative (PPD; Myco- ␮ affyQCReport package. Microarray scan images were checked for spatial bacterium tuberculosis; CSL), 200 ng/ml SEB (Sigma-Aldrich), 1 g/ml defects/artifacts using diagnostic plots from the affyPLM package. RNA PHA (Murex Biotech), or soluble anti-CD3 and 20 U/ml rIL-2 (Cetus). integrity was confirmed by the Affymetrix RNA degradation controls Optimal concentrations of in vitro stimuli were established in forerunner (GAPDH and actin, 3Ј–5Ј ratios Ͻ3). dose response experiments using