T H 17 Cell Frequency in Peripheral Blood from Children with Allergic Asthma Correlates with the Level of Asthma Control Sebastian Kerzel, MD 1 , Jana Dehne, MS 1 , Tobias Rogosch, PhD 1 , Bianca Schaub, MD 2 , Rolf F. Maier, MD 1 , and Michael Zemlin, MD 1 Here we show that the frequency of T H 17 cells (CD3 + CD4 + CD161 + CCR6 + lymphocytes) is increased in peripheral blood of children with allergic asthma. Moreover, we found a significant relationship between the frequency of T H 17 cells and level of asthma control, with reduced asthma control correlated with a higher proportion of T H 17 cells. (J Pediatr 2012;161:1172-4). I n Western countries, asthma is the most frequent chronic disease in childhood. 1 For many years, the paradigm of un- derlying immune dysregulation dominated mainly by T helper (T H ) 2 cells was widely accepted. With the recent de- scription of T H 17 cells and further T H cell subtypes, new aspects have been added to the understanding of immune regulation during the allergic response. 2 Although much of the data on the role of T H 17 immunity in the allergic response are derived from animal models, 3 it has been shown that interleukin (IL)-17 is increased in spu- tum and bronchoalveolar lavage fluid from patients with asthma. 4 Subsequent studies implied a role for IL-17 in per- petuating allergic inflammation in asthma by enhancing the local accumulation of neutrophils. 5 A recent study reported an increased T H 17 response in adults with allergic asthma. 6 The only previous study addressing the role of T H 17 in pedi- atric allergy found that enhanced allergen-induced IL-17 re- sponses during immunotherapy were associated with high clinical symptom scores in children with allergic rhinitis. 7 However, although childhood represents the most critical pe- riod for the development of asthma, T H 17 immunity has not yet been studied in this context. Thus, the aim of the present study was to evaluate whether the frequency of T H 17 cells (CD3 + CD4 + CD161 + CCR6 + lymphocytes) is increased in children with allergic asthma and is correlated with disease activity. Methods Twenty-five children with allergic asthma were diagnosed ac- cording to standardized criteria. 8 Children with nonallergic asthma were excluded. Asthma control was classified as con- trolled, partly controlled, or uncontrolled, according to Global Initiative Against Asthma guidelines. 9 The use of in- haled steroids did not differ significantly among the 3 asthma groups (c 2 test; P = .46). Patient characteristics are summa- rized in the Table. A control group of 15 healthy children was recruited. Ex- clusion criteria for the control group were symptoms or a doctor’s diagnosis of pulmonary or allergic disease, chronic immune disorder, acute infection (assessed clinically and by laboratory measurements) during the previous 2 weeks, and 1 or more positive items on the modified asthma predictive index. 10 Children with sensitization detected by a radioaller- gosorbent test or skin prick test were not included in the con- trol group. After informed consent, 1.2 mL of venous blood was col- lected during a routinely performed blood withdrawal. The study was approved by the local Ethics Committee. Erythro- cytes were lysed, and peripheral blood mononuclear cells were recovered by density centrifugation using a Ficoll- Hypaque gradient (PAA, Linz, Austria). After washing, cells were stained with the following fluorescence-labeled antibodies: anti-CD3 (FITC), anti- CD4 (PerCP), anti-CD161 (PE) (all from BD Biosciences, Heidelberg, Germany), and anti-CCR6 (AlexaFluor 647; Biozol, Eching, Germany). Isotype controls were purchased from BD Biosciences. Analyses were performed with a 4- color FacsCalibur flow cytometer (BD Biosciences). Cells were gated into a physical lymphocyte gate in the forward scatter/side scatter plot. T H 17 cells were defined as CD3 + CD4 + CD161 + CCR6 + lymphocytes. 11 At least 30 000 lymphocytes were analyzed in each sample. Statistical analyses were performed using Prism 5.0 (GraphPad Software, La Jolla, California) and SPSS version 17.0 (SPSS Inc, Chicago, Illinois). Normal distribution was assessed using the Kolmogorov-Smirnov test. Differences were assessed with the 2-tailed Student t test for normally dis- tributed data and the Mann-Whitney U test for nonnormally distributed data. The level of statistical correlation was deter- mined by Pearson product-moment correlation. Adjustment From the 1 Department of Pediatrics, Philipps University, Marburg and 2 Department of Pulmonary and Allergy, University Children’s Hospital Munich, Ludwig Maximillian University, Munich, Germany Funded by Deutsche Forschungsgemeinschaft SFB/TR22 (TP A17 and A22), a re- search grant from the University Medical Center Giessen and Marburg, and Behr- ing-Roentgen-Stiftung. The authors declare no conflicts of interest. 0022-3476/$ - see front matter. Copyright ª 2012 Mosby Inc. All rights reserved. http://dx.doi.org/10.1016/j.jpeds.2012.07.051 T H T helper IL Interleukin 1172