preferentially expressed in mature thymocytes (Fig. S1B) confirm- ing the expression pattern observed with MxA. Taken together, the presence of MxA, pSTAT1 and IRF-7 support the notion that type I IFNs are constitutively secreted in the thymus. Furthermore, the phenotype of the cells that have responded to type I IFNs and as a result phosphorylate STAT1 and express MxA and IRF-7 ex vivo suggest that type I IFNs are secreted locally within the medulla where mature thymocytes are located. Stimulation of thymocytes with exogenous IFN-a upregulates MxA and pSTAT1 While all thymocyte subsets express CD118, we cannot exclude the possibility that immature thymocytes may not respond to stimulation by IFN-a. To test this notion, thymocytes were stimulated with 1000 U/mL recombinant IFN-a overnight and stained for surface expression of CD4, CD8, CD1a, CD3, CD27, CD45RA and CD123 combined with intracellular MxA expres- sion. Stimulation with exogenous IFN-a showed a robust increase in total MxA expression in CD272CD1a+ thymocytes, confirming the ability of immature thymocytes to respond to IFN-a. Notably, no further increase in MxA expression in CD27+CD1a2 mature thymocytes was observed (Fig. 3C). Similarly, pSTAT1 levels increased in immature, but not in mature thymocytes after stimulation with 1000 U/mL exogenous IFN-a (data not shown). These results indicate that all thymocytes have the potential to respond to IFN-a ex vivo. However in vivo, immature thymocytes are less stimulated and mature thymocytes may have already been stimulated to maximal levels by constitutively produced IFN-a. pDC express IFN-a and higher levels of MxA in the thymus compared to other thymocyte subsets We have previously shown that thymic pDC are necessary for the expression of MxA in response to HIV-1 infection of the thymus [17]. Their role as the natural interferon producing cells suggests that thymic pDC are likely involved in the constitutive secretion of IFN-a and subsequent expression of MxA by thymocytes. Using real-time PCR analysis, we observed that IFN-a mRNA is transcribed in the thymus and that IFN-a transcripts are preferentially transcribed in MACS enriched or sorted pDC compared to total thymocytes or non-pDC (Fig. S2). To confirm IFN-a protein expression in pDC, we electronically gated on CD123+CD45RA+ thymocytes which lacked expression of CD1a and CD3, the phenotype of pDC. As can be seen in Figure 4A, .88% of pDC expressed IFN-a, while non-pDC did not. Since it is known that pDC themselves are able to respond to type I IFNs, it is expected that pDC express MxA. By flow cytometry we measured MxA and pSTAT1 expression ex vivo in pDC (identified here as CD45RA + CD123+/hi cells), isolated from post-natal thymus tissues and found that (8969%) of pDC are MxA positive (n = 15) (Fig. 4B). In addition,MxA mRNA was also measured and observed to be expressed at high levels in pDC (Fig. S2). Post-natal thymic pDC also expressed pSTAT1 (Fig. 4B) and IRF-7 (data not shown). Furthermore, thymic pDC showed a significantly higher MxA mean fluorescence intensity (3–13 fold, p,0.00001, (n = 15)) than other MxA positive thymocyte subsets (Fig. 4C). To confirm our observation that pDC express high levels of MxA in the thymus, but not in other lymphoid tissues, we stained single cell suspensions from fetal tissues or adult PBMC for surface expression of CD123, CD45RA, CD3 and intracellular expression of MxA. MxA expression in pDC was determined by gating on CD45RA + CD123+/hi cells that lacked CD3. We observed that in 3/3 experiments pDC from the thymus were MxA positive, while Constitutive IFN-a Expression in the Human Thymus pDC from autologous fetal spleen and lymph node lacked MxA expression (Fig. S3). Adult peripheral blood pDC also lacked MxA expression (Fig. S3) and a lack of MxA expression was also observed in Cord blood (data not shown). To verify MxA expression in thymic pDC and to analyze co- localization with MxA positive thymocytes, post-natal thymus tissue was stained with CD123 and anti-MxA antibodies. In line with previous findings, pDC (CD123+) are located within the medulla and at the cortico-medullary junction, but not found within the cortex (Fig. 4D) [18,19,35]. As expected from the flow cytometric data, the majority of cells that express high levels of CD123 also expressed MxA (Fig. 4D, Arrows). Moreover, pDC were located in close proximity to thymocytes that expressed MxA (Fig. 4D, Merge). The co-localization of pDC with MxA positive cells in the medulla suggests that pDC are directly responsible for the secretion of IFN-a in the thymus. Furthermore, the finding that MxA is highly expressed in thymic pDC adds value to the notion that type I IFN can act in an autocrine or paracrine manner as a pDC survival factor and is in agreement with a previous report [36]. The anti-microbial peptide LL-37 co-localizes with pDC in the medulla Our data suggest that there is a thymus-specific trigger for pDC to induce IFN-a/b secretion. Recent publications demonstrated that the anti-microbial peptide LL-37 isolated from psoriasis skin lesions can bind eukaryotic DNA and RNA and trigger peripheral blood pDC to secrete IFN-a in a TLR-9 dependent manner [29,30]. We hypothesized that expression of LL-37 in the thymus could result in interferon secretion, since DNA/RNA would be readily available from medullary thymocytes undergoing apoptosis as a result of negative selection. To assess whether and where LL- 37 protein is expressed, thymic tissue sections were stained with an anti- LL-37 monoclonal antibody. We found that LL-37 was mainly expressed in the medulla in the fetal thymus, although some expression was also observed in the cortex by immunoflu- orescence (data not shown) and immunohistochemistry (Fig. 5, brown staining). Thus the combined presence of pDC, LL-37, and autologous DNA/RNA derived from negatively selected thymo- cytes in the medulla can explain the presence of MxA in normal thymus tissue. While LL-37 was not detected in the fetal lymph node (same donor as fetal thymus), it was found in several (n = 3) non-B (CD19+) cell zones of the fetal spleen. We speculate that there is likely no/little apoptosis occurring in the fetal spleen resulting in no/limited amounts of autologous DNA/RNA. This could account for the observed lack of MxA expression in lymphocytes and pDC in the fetal spleen (Fig. 1 and Fig. S3). Exogenous LL-37 complexed with DNA/RNA upregulates IFN-a Based on findings that IFN-a production by peripheral pDC can be induced by LL-37 complexed with eukaryotic DNA or RNA [29,30], we examined whether exogenous LL-37 complexed with eukaryotic DNA/RNA increases IFN-a secretion by thymic pDC. PDC were sorted from thymus and stimulated with LL-37 complexed with DNA/RNA obtained from supernatants of apoptotic cells. After 6 hours of culture, we detected 9.1-fold more IFN-a transcripts compared to either medium or eukaryotic DNA/RNA alone (Fig. 6). Notably, pretreatment of the supernatant containing eukaryotic DNA/RNA with DNAse and RNAse partially inhibited IFN-a mRNA expression. Stimulation with the TLR9 agonist CpG-A (2216), which was used as a positive control, induced 3-fold higher levels of IFN-a expression PLoS ONE | www.plosone.org 6 August 2011 | Volume 6 | Issue 8 | e24252