and luminol (50 mg/ml), D-luciferin (461025M) (Roche Diagnos- tics) and H2O2 were injected. EDN and cytokine release Eosinophils (26106/ml) were activated with the same stimuli as for ROS production. After 18 h culture, supernatants were collected. EDN levels were measured by specific ELISA (MBL). The lower detection limit was 0.62 ng/ml. IFNc and TNFa cytokines were measured by specific ELISA (Diaclone). Detection limit was 5 pg/ml for IFNc and 8 pg/ml for TNFa. Activation with Mycobacteria Mycobacterium bovis-BCG Pasteur strain was obtained from Dr C. Locht (Inserm U629, Institut Pasteur de Lille, France) and was grown in Sauton medium at 37uC. Eosinophils were incubated with various BCG numbers (Eosinophil/BCG: 20/1, 10/1 and 5/ 1). ROS and EPO release were determined as described above. For inhibition experiments, eosinophils (20/1 ratio) were incubat- ed with a blocking anti-cdTCR mAb (10 mg/ml) or isotype control for 30 min at 37uC, before BCG addition. Tumor cell apoptosis Colo 205 cells were labelled with 10 mM PKH26 (Sigma) according to manufacturer’s instructions. Then, eosinophil- mediated cytotoxicity against PKH-26-labelled Colo-205 (10 mM) was measured in complete medium at 1.66104 targets/ well into U-bottom plates containing eosinophils (25:1 Effector:- Target ratio) [61]. After different time points, apoptosis was measured by flow cytometry after annexinV-FITC staining (Pharmingen) for 15 min at RT. For inhibition experiments, blocking antibodies against cdTCR or isotype-matched control (10 mg/ml) were added to eosinophils immediately prior to the incubation in the presence of targets. In some experiments, eosinophils were preincubated for 30 min with 50 mM SBA or 20 mM TUBAg 1 before addition to the target cells. Statistical analysis Individual data or mean6S.E.M values were presented. All statistical analyses were performed using SPSS software. Normal- ity of data samples was assessed with the Normality test of Shapiro and Wilk. Then parametric paired-samples Student’s t-test was used to compare variables and the one-tailed statistical significance level was as represented on Figures. Supporting Information Figure S1 Gating strategy and specific identification of purified peripheral blood eosinophils in flow cytometry analysis. (A–B) Gating of purified peripheral blood eosinophils on forward and side scatter parameters immediately after purification. (A) Ellipsoids circle the analyzed population. (B). Specificity of eosinophil identification by intracellular detection of EPO, MBP References 1. Giembycz MA, Lindsay MA (1999) Pharmacology of the eosinophil. Pharmacol Rev 51: 213–340. 2. Rothenberg ME, Hogan SP (2006) The eosinophil. Annu Rev Immunol 24: 147–174. 3. Jacobsen EA, Ochkur SI, Pero RS, Taranova AG, Protheroe CA, et al. (2008) Allergic pulmonary inflammation in mice is dependent on eosinophil-induced recruitment of effector T cells. J Exp Med 205: 699–710. 4. Walsh ER, Sahu N, Kearley J, Benjamin E, Kang BH, et al. (2008) Strain- specific requirement for eosinophils in the recruitment of T cells to the lung during the development of allergic asthma. J Exp Med 205: 1285–1292. 5. Young JD, Peterson CG, Venge P, Cohn ZA (1986) Mechanism of membrane damage mediated by human eosinophil cationic protein. Nature 321: 613–616. Eosinophil cdTCR Expression or MPO using PE- or biotin-conjugated specific antibodies (grey shaded histograms). Staining on purified neutrophils as well as gated lymphocytes and monocytes from purified PBMC is shown as control. Staining with control isotype matched antibodies is represented in white histograms. Found at: doi:10.1371/journal.pone.0005926.s001 (6.41 MB TIF) Figure S2 Surface expression of CD3, cdTCR, Vgamma9, Vdelta2,Vdelta1, abTCR, CD8 and NKG2D detected by flow cytometry in double staining on immune cell populations. (A) In vitro-generated BrHPP-induced gamma9 delta2 TCR+ lympho- cytes. (B) Scatter-gated lymphocytes from PBMC. (C) Scatter-gated monocytes from PBMC. (D) CD16+-purified blood neutrophils. Staining with control isotype matched antibodies is represented. Found at: doi:10.1371/journal.pone.0005926.s002 (4.23 MB TIF) Figure S3 Surface expression of cdTCR/CD3 complex by human eosinophils following induction by a mycobacterial cdTCR ligand. (A–B) CD3, cdTCR, abTCR and CD8 surface expression on EPO+-purified peripheral blood eosinophils incubated with culture medium (A) or with 40 nM TubAg for 2 h (B). Staining with control isotype matched antibodies is represented. Found at: doi:10.1371/journal.pone.0005926.s003 (3.02 MB TIF) Figure S4 Surface expression of CD3, cdTCR and CD8 on cord blood-derived eosinophils. Eosinophils derived from CD34+ cord blood cells (day 21) were analysed for CD3, cdTCR and CD8 cell surface expression after gating on EPO+ cells. Staining with control isotype antibodies is represented. Found at: doi:10.1371/journal.pone.0005926.s004 (1.28 MB TIF) Table S1 List, and characteristics of antibody used in this manuscript. Mb: Membrane staining. IC: Intracellular staining Found at: doi:10.1371/journal.pone.0005926.s005 (0.06 MB DOC) Table S2 Gene-specific primer sequences Found at: doi:10.1371/journal.pone.0005926.s006 (0.04 DOC) MB Acknowledgments The authors wish to thank M. Bonneville (Inserm, Nantes, France), J. Khalife (Inserm, Lille) for helpful advices. They are grateful to L. Prin (CHRU, Lille France) and the National Network on Hypereosinophilia for access to eosinophilic patients, C. Rouanet and C. Locht (Inserm, Lille, France) for providing BCG and technical suggestions and Dr D. Plager (Mayo Clinic, Rochester) for anti-MBP2 antibody. The authors also with to thank M. Delbecke and J. Bertout for their technical assistance. Author Contributions Conceived and designed the experiments: FL VD GW FS DDD MC. Performed the experiments: FL VD GW SL EH LH VM FS. Analyzed the data: FL VD GW SL JJF MLG DDD MC. Contributed reagents/ materials/analysis tools: JJF. Wrote the paper: DDD MC. 6. Gounni AS, Lamkhioued B, Ochiai K, Tanaka Y, Delaporte E, et al. (1994) High-affinity IgE receptor on eosinophils is involved in defence against parasites. Nature 367: 183–186. 7. Stenfeldt AL, Wenneras C (2004) Danger signals derived from stressed and necrotic epithelial cells activate human eosinophils. Immunology 112: 605–614. 8. Jacobsen EA, Taranova AG, Lee NA, Lee JJ (2007) Eosinophils: singularly destructive effector cells or purveyors of immunoregulation? J Allergy Clin Immunol 119: 1313–1320. 9. Kayaba H, Dombrowicz D, Woerly G, Papin JP, Loiseau S, et al. (2001) Human eosinophils and human high affinity IgE receptor transgenic mouse eosinophils express low levels of high affinity IgE receptor, but release IL-10 upon receptor activation. J Immunol 167: 995–1003. PLoS ONE | www.plosone.org 15 June 2009 | Volume 4 | Issue 6 | e5926