Constitutive IFN-a Expression in the Human Thymus Figure 1. Constitutive expression of MxA in thymocytes. A,B, Total thymocytes were isolated from fetal and postnatal thymus tissues. Cells were stained for surface expression of CD3, CD4, CD8 and intracellular expression of MxA ex vivo. Three fetal and 18 postnatal thymus tissues were examined to determine the effect of age (A) and sex (B) on MxA expression. C, Cells from postnatal thymus, fetal thymus spleen and lymph node tissues and adult PBMC were stained for intracellular expression of MxA. Fetal tissues were obtained from the same donor. doi:10.1371/journal.pone.0024252.g001 total MxA expression and age (R2 = 0.0301, p = 0.46) (Fig. 1A) or mean total MxA expression and sex (p = 0.80) (Fig. 1B). The presence of IFN-a secretion and resulting MxA expression in normal tissues may not be unique to the thymus. To investigate this, we compared the MxA expression in different lymphoid tissues by staining postnatal thymus and fetal thymus, spleen, and lymph node from the same fetal donor. In 3/3 experiments, only the thymus, but not spleen or lymph node from the same fetal donor expressed MxA (Fig. 1C). Also adult PBMC (n = 3) (Fig. 1C) lacked MxA expression. These findings were confirmed at the RNA level by real time PCR (data not shown). Thus, MxA expression in the thymus is not a result of localization of cells within lymphoid tissue as both fetal spleen and lymph nodes lack MxA expression. This finding is of note since fetal tissue by definition is devoid of bacterial or viral infections, yet the presence of MxA in the thymus suggests that IFN-a/b is secreted constitutively. MxA expression is located in the thymic medulla To determine whether type I IFNs are secreted in specific regions in the thymus in vivo, we performed immunofluorescence microscopy using an anti-MxA monoclonal antibody. Fetal, postnatal, and adult thymus tissues were stained with CD1a to distinguish the cortex and CD27 to distinguish the medulla. In 3/3 fetal and 9/9 postnatal thymus samples (age 3 months-30 years) MxA co-localized with the CD27+ thymocytes in the medulla (Fig.2A), but not with the CD1a+ thymocytes in the cortex (Fig. 2B). Within the medulla, both CD27+ and CD272 cells expressed MxA (Fig. 2A). To confirm these results, post-natal thymus tissue sections were also stained for expression of IRF-7, a factor required for the transcription of downstream interferon stimulated genes including MxA [2]. Like MxA, IRF-7 was preferentially expressed in the medulla, but not in the cortex (data not shown). Localization of MxA and IRF-7 in areas of the thymus that stain with CD27 confirms that IFN-a/b is secreted within the thymic medulla. MxA, IRF-7, and phosphorylated STAT1 are preferentially expressed in mature thymocytes We and others have shown that most thymocyte subsets express the receptor for IFN-a (CD118) and can respond to stimulation with type I IFNs [17],[34]. If IFN-a is secreted throughout the thymic cortex and medulla then the phenotype of MxA expressing cells should match the phenotypic distribution of all developing thymocytes. To determine the immunophenotypic profile of MxA expressing cells, total thymocytes were stained for cell surface expression of CD4, CD8, CD1a, CD3, CD27, CD45RA and CD123 in combination with intracellular MxA expression. We observed that MxA is preferentially expressed in cells that exhibit a more mature phenotype (ie. CD45RA+, CD4+ or CD8+, CD3+/hi, CD1a- and CD27+) (Fig. 3A). Furthermore, when focusing on the mature, medullary, thymocyte subsets (ie. CD27+ cells), MxA expression was enriched in the mature CD45RA + CD4+ and CD45RA + CD8+ cells that are ready to emigrate from the thymus to the periphery (Fig. 3B). These data are consistent with the notion that type I IFNs are secreted locally in the medulla. We also assessed expression of phosphorylated STAT1 (pSTAT1) and IRF-7, which are upstream of MxA, and dependent on signals received from the type I IFN receptor [2]. Phosphorylated STAT1 (Fig. S1A) and IRF-7 were also PLoS ONE | www.plosone.org 4 August 2011 | Volume 6 | Issue 8 | e24252