we have shown they retain the ability to secrete IFN-a in response to CpG [17]. Peripheral blood pDC may also secrete IFN-a in response to tissue damage as observed in the skin lesions in psoriasis [29]. In this case, the TLR-7/9 stimulus is eukaryotic nucleic acids, RNA or DNA, in complex with an anti-microbial peptide (LL-37) derived from the CAMP gene [29,30]. LL-37 mRNA is expressed in the thymus [31], but it has not been investigated whether the resultant peptide can stimulate thymic pDC in the presence of autologous DNA or RNA. Given that thymocytes undergoing negative selection die through induction of apoptosis [32] it is attractive to speculate that this process provides an ample source of self-RNA/DNA, which in complex with LL-37 induces the secretion of IFN-a from pDC. In this report we not only examine IFN-a expression in thymocytes, but also expression of surrogate markers that are indicative of type I interferon receptor signaling activity, including MxA, IRF-7, and phosphorylated STAT1 (pSTAT1). We report that IFN-a and MxA are constitutively expressed in normal postnatal and fetal thymus tissues ex vivo, corroborating our previous results that IFN-a and MxA are expressed in normal thymus/liver implants in the SCID-hu mouse [17]. Furthermore, MxA, IRF-7, and pSTAT1 proteins are preferentially expressed in mature thymocytes that are located in the thymic medulla. Not only does this area coincide with the known localization pattern of pDC, but in addition we observed that pDC constitutively expressed the highest levels of IFN-a in the normal thymus. Furthermore low, but reproducible, expression of LL-37 was detected within the thymic medulla. This, together with our observation that LL-37 complexed with eukaryotic RNA or DNA can stimulate pDC to express IFN-a, suggests that thymic pDCs are involved in LL-37/DNA-RNA induced secretion of IFN-a in the absence of infection. We speculate that thymic pDC may play a role in regulating the rate of normal T cell development or alter the requirements for negative selection. Materials and Methods Ethics statement The use of anonymous thymus, peripheral lymphoid and fetal liver tissues and peripheral blood mononuclear cells was reviewed by the UCLA Institutional Review Board (IRB), which concluded that these activities did not involve human subjects, and therefore did not require IRB review or certification. Reagents and mAbs mAbs CD1a, CD3, CD4, CD8, CD27, CD45RA, CD123, and STAT1 (pY701); isotype controls IgG1 and IgG2 conjugated with FITC, PE, PerCP, PE-Cy7 or APC; and goat anti-mouse FITC were obtained from Becton Dickinson (BD); CD1a conjugated with PE from Beckman Coulter Immunotech; BDCA4-APC from Miltenyi Biotec; CD8-APC-Alexa750, CD45RA-PerCP-Cy5.5, CD1a-Pacific Blue and CD3-650 eFluor (Nano-crystals) from eBioscience; IFN-a -biotin (Endogen); isotype control IgG2a pure, Goat anti-Rabbit PE from Caltag; mAb to CD27 from Lab Vision; Goat anti-mouse IgG1 AlexaFluor-488, Goat anti-mouse IgG2a AlexaFluor-594, Goat anti-Rabbit AlexaFluor-594, and Zenon anti IgG2a AlexaFluor-488 from Molecular Probes. mAb to MxA was generously provided by Drs. Haller and Kochs (University of Freiburg, Germany). mAb CD1a pure, TBST, Antibody Diluent, and Target Retrieval Solution were obtained from DAKO (Carpinteria, CA); rabbit anti-mouse IgG1 pure from Zymed; mAb IRF-7 from Santa Cruz Biotechnology; mAb LL-37 from Cell Sciences. Mounting Medium for fluorescence was Constitutive IFN-a Expression in the Human Thymus obtained from Vector Laboratories. Polyoxyethylenesorbitanmo- nolaurate (Tween-20) was purchased from Sigma-Aldrich, para- formaldehyde from Polysciences. Recombinant human IFN-a was obtained from BioSource. Cell cultures Fetal tissues were obtained from Advanced Biosciences Resources; adult PBMC from the UCLA CFAR Virology Core Laboratory; normal human postnatal thymus specimens from children undergoing corrective cardiac surgery. Thymocytes were prepared and cultured, as previously described, in serum-free medium consisting of IMDM (Omega Scientific) supplemented with delipidated BSA (Sigma-Aldrich) at 1100 mg/ml, transferrin (Sigma-Aldrich) at 85 mg/ml, 2 mM glutamine, and penicillin/ streptomycin at 25 U/25 mg/ml[17]. Thymocytes were cultured at 1–26107 cells/ml in serum-free medium as pellet cultures at 37uC in 5% CO2 in round-bottom tissue culture tubes. Immunofluorescent intracellular staining and flow cytometry For combined surface and intracellular staining using the unconjugated mAb to MxA, 16106 cells were fixed in 1% paraformaldehyde, permeabilized with 0.2% Tween-20, and stained intracellularly with a 1/500 dilution of mAb to MxA or isotype control Ab for 20 min at 4uC as previously described [17]. The cells were washed with 0.2% Tween-20, and goat anti-mouse FITC mAb was added at 10 ml/tube and incubated for 20 min at 4uC. Cells were washed with 0.2% Tween-20 and incubated for 10 min with 50 ml/tube of a 1/15 dilution of normal mouse IgG (Caltag Laboratories). PE, PerCP, and APC-conjugated mAb were added for 20 min at 4uC and washed with 0.2% Tween-20. Cells were finally resuspended in FACS buffer before acquisition on a FACSCalibur flow cytometer (BD Immunocytometry Systems). For combined surface and intracellular staining using uncon- jugated IRF-7, 16106 cells were first surface-immunophenotyped with FITC, TC, or APC antibodies and fixed with 1% paraformaldehyde. Cells were permeabilized with 0.2% Tween- 20 and incubated with 400 ng IRF-7 antibody for 40 minutes. Cells were washed and incubated with 10 ml Goat anti-Rabbit PE for 20 minutes. Cells were resuspended in FACS buffer before acquisition on a FACSCalibur or LSRII flow cytometer. For combined surface and intracellular staining using IFN-a- biotin, 16106 cells were first surface-immunophenotyped with FITC, PerCP-Cy5.5, PE-Cy7, APC, APC-Ax750, Pacific Blue and eFluor650 nanocristal antibodies, then fixed with BD FACS Lysing Solution. Cells were then permeabilized with BD FACS Permeabilizing Solution 2 and incubated with 2 mg of IFN-a- biotin (Thermo Scientific) or mouse IgG1 (BD Biosciences) antibody for 20 minutes. Cells were then washed and incubated with 5 ml of SAV-PE (BD Biosciences) for 10 minutes. Cells then were washed and resuspended in FACS buffer before acquisition on a LSRII (BD). For combined surface and intracellular staining using PE conjugated pSTAT1, 16106 cells were washed with FACS buffer and surface-immunophenotyped with FITC and APC labeled antibodies. The cells were fixed in 1.5% paraformaldehyde for 10 minutes at room temperature and permeabilized with 1 ml cold (4uC) methanol for 10 minutes at 4uC. After washing the cells twice with FACS buffer, they were stained with 10 ml pSTAT1-PE or IgG2-PE control antibody for 30 minutes at room temperature. The cells were resuspended in 250 ml FACS buffer before acquisition on a FACSCalibur or LSRII flow cytometer. Multiparameter data acquisition was performed with CellQuest PLoS ONE | www.plosone.org 2 August 2011 | Volume 6 | Issue 8 | e24252