software (BD). Multiparameter data analysis was performed with FCS Express Version 3 (Denovo Software). Immunofluorescence microscopy Fetal tissues and post-natal thymus were fixed in formalin and embedded in paraffin by the UCLA Tissue Procurement Core Laboratory. Four mm sections were cut and placed on slides. The slides were heated at 60uC for 30 minutes and washed in xylenes, followed by rehydration in 100%, 95%, and 70% ethanol and incubation in target retrieval solution pH 9.0 for 20 minutes at 95–99uC. Samples were stained with monoclonal antibodies to MxA (1:500), IRF-7 (1:50), or LL-37 (1:50) and CD1a (1:50) or CD27 (neat) overnight at 4uC. Samples were washed and stained with secondary antibodies (1:250) for 4 hours at room tempera- ture. Slides were mounted in fluorescence mounting medium and coverslips were sealed with clear nail polish. For staining with CD123 and MxA slides were stained first with CD123 (1:40) overnight and washed. Slides were then stained with rabbit anti- mouse IgG1 (1:500) for 2 hours followed by Goat anti Rabbit AlexaFluor-594 for 2 hours. MxA was conjugated with Zenon anti IgG2a AlexaFluor-488 following the manufacturer’s protocol. Slides were stained for 2 hours and fixed with 2% paraformalde- hyde for 10 minutes before samples were mounted in fluorescence mounting medium and coverslips were sealed with nail polish. Images were taken with a Zeiss compound microsope and analyzed with Axiovision LE version 4.6 (Carl Zeiss Microima- ging). Immunohistochemistry Postnatal thymus and fetal tissues were formalin fixed and paraffin embedded. 4 um sections were mounted on slides and stained with a monoclonal antibody to LL-37 (1:50) or CD19 (1:50) and control IgG2a antibody followed by biotinylated rabbit anti-mouse antibody and streptavidin-biotin-peroxidase complex using the DAKO Catalyzed Signal Amplification System. Isolation and stimulation of thymic pDC pDC were isolated from human thymus tissue by MACS-based negative selection (Miltenyi). The purity of pDC was checked by flow cytometry using CD45RA FITC, BDCA4-APC, CD123PE, CD3 PerCP antibodies. Cells were stained with 7AAD to determine the percentage of apoptotic cells. pDC were stimulated with CpG-A(2216) oligonucleotides (6 mg/ml) or LL-37 (10 mg) in the presence or absence of DNAse I (Sigma; final conc.5 mg/ml). LL37 premixed with 2 mg autologous DNA (DNAsy Kit, Qiagen) (peptide: DNA mass ratio of 5:1) and incubated 30 min. at room temperature (50 mg/ml of LL-37 and 10 mg/ml of DNA final concentration) was added to pDC in some experiments. After overnight culture (18–20 hours) at 37uC, 5%CO2 IFN-a level was measured by enzyme-linked immunosorbent assay (ELISA; PBL Biomedical Laboratories). Remaining cells were frozen in Trizol and used to isolate RNA for quantitative real-time PCR for MxA expression. To obtain a purified population of pDC the cells were first enriched with BDCA4 selection kit (Miltenyi) followed by cell sorting of CD123+CD45RA+ cells using a FACSAria II cell sorter (Becton Dickinson). The purity of sorted pDC exceeded 95%. pDC were stimulated with CpG-A(2216) oligonucleofo- tides (10 mg/ml) or LL-37 peptide (Netherlands Cancer Insti- tute) (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) (50 mg/ml) in the presence or absence of DNAse I (Roche) (2000 U/ml) and RNAse (Roche) (50 ug/ml). LL-37 (50 mg/ml) was premixed with eukaryotic DNA (10 mg/ ml) and RNA from cell line supernatant for 30 min. at room Constitutive IFN-a Expression in the Human Thymus temperature and added to pDC in some experiments. After 4 hours of culture at 37uC, 5% CO2 pDC were resuspended in Trizol. RNA was isolated and cDNA synthesis was performed by using the transcription high fidelity cDNA synthesis kit (Roche). RNA isolation and real-time PCR 106107 cells were frozen in TriReagent (Invitrogen) and stored at 280uC until RNA was isolated, when samples were incubated with poly-acryl carrier for 5 minutes at room temperature, 100 ul 1-Bromo-3-chloropropane per mL of TriReagent was added for 10 minutes, spun at 12,000 g for 10 minutes at 4uC and the aqueous phase was transferred to a fresh tube. 500 uL of isopropanol was added for 10 minutes and samples were spun at 12,000 g for 8 minutes at room temperature. Pellets were washed with 75% ethanol and allowed to air dry before resuspension in 100 ml water. Taq-man gene expression assays were used to measure gene expression (Applied Biosystems). Real-time PCR for analysis of MxA (Mx1 HS_00182073_m1) IFNA1 (Hs00256882_s1), IFNA2 (Hs00265051_s1) was performed following manufacturer’s instruc- tions. 96 well plates were run on an ABI 7300 and analyzed with the 7300 system software. Q-PCR on sorted pDC and thymocytes for relative expression of IFN-a and MxA mRNA was performed by Sybr Green PCR (iCycler Bio-Rad CFX96). Primers: IFN-a: 59-GACTC- CATCTTGGCTGTGA-39, 59- TGATTTCTGCTCTGACAA- CCT-39, MxA: 59-GGTGGCTGAGAACAACCTGT-39, 59-GG- TCCTGCTCCACACCTAGA-39, Beta-Actin and GAPDH were used as controls. (Beta-Actin: 59-CTCTTCCAGCCTTCCT- TCCT-39, 59-AGCACTGTGTTGGCGTACAG-39) (GAPDH: 59- GAGTCAACGGATTTGGTCGT-39, 59-GACAAGCTTC- CCGTTCTCAG-39). Statistics Non-parametric Spearman rank correlation was used to compare percent MxA expression versus age of thymus tissue; two sided Wilcoxon-Mann-Whitney test to compare mean total MxA expression between males and females; two sided Wilcoxon signed rank sum test and sign test to compare mean fluorescence intensity of MxA in thymocyte subsets. Results Expression of MxA in thymus, but not peripheral lymphoid organs Previously we observed that IFN-a was detected in normal human fetal thymus/liver implants from SCID-hu mice [17]. The current study was designed to obtain further insight in the localization of endogenous constitutively produced IFN-a, the cell type and the mechanism of induction in the thymus. The inability to reliably detect all IFN-a subtypes and differences in the kinetics of IFN-a secretion by different cell types makes the study of IFN-a secretion technically challenging. However, studies have shown that the ISG MxA is only synthesized in the presence of type I IFNs, and is absent when type I IFNs are not secreted [9] and requires signaling though STAT1 [33]. Hence, we first set out to analyze the expression of MxA as a surrogate marker to detect the presence of secreted type I interferons. We confirmed that type I IFNs are constitutively secreted in the thymus by analysis of intracellular MxA expression using flow cytometry (Fig.1). We extended these findings by showing that postnatal (18/18 donors, age 1 day-30 years) and fetal thymus (3/3 donors) expressed MxA (Fig.1A). While MxA was detected in all thymus tissues (range 0.9–45%), there was no correlation between PLoS ONE | www.plosone.org 3 August 2011 | Volume 6 | Issue 8 | e24252