stained for surface expression of CD4, CD8, CD1a, CD3 and CD27 and intracellular expression of pSTAT1 (A) or IRF-7 (B). Differences of thymocyte subset expression patterns in all thymocytes and pSTAT1 and IRF-7 negative and positive cells are shown. (TIF) Figure S2 Thymic pDC constitutively express high levels of IFN-a mRNA. mRNA was prepared from postnatal total thymocytes, BCDA4-MACS enriched pDC and the BDCA4 negative fraction, and from sorted pDC. Q-PCR was performed for IFN-a mRNA. (TIF) Figure S3 pDC from post-natal and fetal thymus express MxA, but not pDC from fetal spleen or fetal lymph node or adult PBMC. Cells from fetal tissues or adult PBMC were stained for surface expression of CD123, CD3 and CD45RA and intracellular expression of MxA. MxA expression in pDC was determined by gating on CD45RA + CD123+/hi cells. (TIF) References 1. Pestka S, Krause CD, Walter MR (2004) Interferons, interferon-like cytokines, and their receptors. Immunol Rev 202: 8–32. 2. Grandvaux N, tenOever BR, Servant MJ, Hiscott J (2002) The interferon antiviral response: from viral invasion to evasion. CurrOpinInfectDis 15: 259–267. 3. Horisberger MA, Staeheli P, Haller O (1983) Interferon induces a unique protein in mouse cells bearing a gene for resistance to influenza virus. Proc Natl Acad Sci U S A 80: 1910–1914. 4. Lindenmann J (1964) Inheritance of Resistance to Influenza Virus in Mice. Proc Soc Exp Biol Med 116: 506–509. 5. Staeheli P, Haller O, Boll W, Lindenmann J, Weissmann C (1986) Mx protein: constitutive expression in 3T3 cells transformed with cloned Mx cDNA confers selective resistance to influenza virus. Cell 44: 147–158. 6. Kochs G, Trost M, Janzen C, Haller O (1998) MxA GTPase: oligomerization and GTP-dependent interaction with viral RNP target structures. Methods 15: 255–263. 7. Turan K, Mibayashi M, Sugiyama K, Saito S, Numajiri A, et al. (2004) Nuclear MxA proteins form a complex with influenza virus NP and inhibit the transcription of the engineered influenza virus genome. Nucleic Acids Res 32: 643–652. 8. Kochs G, Janzen C, Hohenberg H, Haller O (2002) Antivirally active MxA protein sequesters La Crosse virus nucleocapsid protein into perinuclear complexes. Proc Natl Acad Sci U S A 99: 3153–3158. 9. Holzinger D, Jorns C, Stertz S, Boisson-Dupuis S, Thimme R, et al. (2007) Induction of MxA gene expression by influenza A virus requires type I or type III interferon signaling. J Virol 81: 7776–7785. 10. Chieux V, Hober D, Harvey J, Lion G, Lucidarme D, et al. (1998) The MxA protein levels in whole blood lysates of patients with various viral infections. J VirolMethods 70: 183–191. 11. von Wussow P, Jakschies D, Block B, Tschechne B, Schedel I, et al. (1990) The interferon-induced Mx-homologous protein in people with symptomatic HIV-1 infection. AIDS 4: 119–124. 12. von Wussow P, Jakschies D, Hochkeppel HK, Fibich C, Penner L, et al. (1990) The human intracellular Mx-homologous protein is specifically induced by type I interferons. EurJ Immunol 20: 2015–2019. 13. Lin Q, Dong C, Cooper MD (1998) Impairment of T and B cell development by treatment with a type I interferon. JExpMed 187: 79–87. 14. Binder D, Fehr J, Hengartner H, Zinkernagel RM (1997) Virus-induced transient bone marrow aplasia: major role of interferon-alpha/beta during acute infection with the noncytopathic lymphocytic choriomeningitis virus. J ExpMed 185: 517–530. 15. Baron ML, Gauchat D, La Motte-Mohs R, Kettaf N, Abdallah A, et al. (2008) TLR Ligand-Induced Type I IFNs Affect Thymopoiesis. J Immunol 180: 7134–7146. 16. Schmidlin H, Dontje W, Groot F, Ligthart SJ, Colantonio AD, et al. (2006) Stimulated plasmacytoid dendritic cells impair human T-cell development. Blood 108: 3792–3800. 17. Gurney KB, Colantonio AD, Blom B, Spits H, Uittenbogaart CH (2004) Endogenous IFN-alpha production by plasmacytoid dendritic cells exerts an antiviral effect on thymic HIV-1 infection. J Immunol 173: 7269–7276. 18. Bendriss-Vermare N, Barthelemy C, Durand I, Bruand C, Dezutter- Dambuyant C, et al. (2001) Human thymus contains IFN-alpha-producing CD11c(2), myeloid CD11c(+), and mature interdigitating dendritic cells. JClinInvest 107: 835–844. Constitutive IFN-a Expression in the Human Thymus Acknowledgments We thank the cardiac surgery team and staff at UCLA and Dr. M. Hazekamp and staff at the Leiden University Medical Center and the Amsterdam Medical Center for providing us with the thymus specimens. The UCLA Virology Core, supported by the UCLA Center for AIDS Research (CFAR) for providing us with peripheral blood specimens, and the staff of the UCLA CFAR Flow Cytometry Core for their assistance with the multicolor flow cytometry. We are grateful for the help of Berend Hooijbrink with FACS sorting and maintenance of the FACS facility at the AMC; Hergen Spits and Heike Schmidlin for their critical review of the manuscript, Otto Haller and George Kochs for the antibody to MxA, Deborah Anisman-Posner, Beverly Redsar, Vanda Farahmand and Fabienne Ropers for their excellent technical assistance. Author Contributions Conceived and designed the experiments: ADC CHU. Performed the experiments: ADC ME MJ LJ. Analyzed the data: ADC ME MJ LJ BB CHU. Wrote the paper: ADC BB CHU. 19. Res PC, Couwenberg F, Vyth-Dreese FA, Spits H (1999) Expression of pTalpha mRNA in a committed dendritic cell precursor in the human thymus. Blood 94: 2647–2657. 20. Martin-Gayo E, Sierra-Filardi E, Corbi AL, Toribio ML (2010) Plasmacytoid dendritic cells resident in human thymus drive natural Treg cell development. Blood 115(26): 5366–5375. 21. Hanabuchi S, Ito T, Park WR, Watanabe N, Shaw JL, et al. (2010) Thymic stromal lymphopoietin-activated plasmacytoid dendritic cells induce the generation of FOXP3+ regulatory T cells in human thymus. J Immunol 184: 2999–3007. 22. Cella M, Jarrossay D, Facchetti F, Alebardi O, Nakajima H, et al. (1999) Plasmacytoid monocytes migrate to inflamed lymph nodes and produce large amounts of type I interferon. NatMed 5: 919–923. 23. Ferbas JJ, Toso JF, Logar AJ, Navratil JS, Rinaldo CR (1994) CD4+ blood dendritic cells are potent producers of IFN-a in response to in vitro HIV-1 infection. JImmunol 152: 4649. 24. Fong L, Mengozzi M, Abbey NW, Herndier BG, Engleman EG (2002) Productive infection of plasmacytoid dendritic cells with human immuno- deficiency virus type 1 is triggered by CD40 ligation. J Virol 76: 11033–11041. 25. Foster GR, Germain C, Jones M, Lechler RI, Lombardi G (2000) Human T cells elicit IFN-alpha secretion from dendritic cells following cell to cell interactions. EurJImmunol 30: 3228–3235. 26. Kadowaki N, Antonenko S, Lau JY, Liu YJ (2000) Natural interferon alpha/ beta-producing cells link innate and adaptive immunity. JExpMed 192: 219–226. 27. Siegal FP, Kadowaki N, Shodell M, Fitzgerald-Bocarsly PA, Shah K, et al. (1999) The nature of the principal type 1 interferon-producing cells in human blood. Science 284: 1835–1837. 28. Yonezawa A, Morita R, Takaori-Kondo A, Kadowaki N, Kitawaki T, et al. (2003) Natural alpha interferon-producing cells respond to human immunode- ficiency virus type 1 with alpha interferon production and maturation into dendritic cells. J Virol 77: 3777–3784. 29. Lande R, Gregorio J, Facchinetti V, Chatterjee B, Wang YH, et al. (2007) Plasmacytoid dendritic cells sense self-DNA coupled with antimicrobial peptide. Nature 449: 564–569. 30. Ganguly D, Chamilos G, Lande R, Gregorio J, Meller S, et al. (2009) Self-RNA- antimicrobial peptide complexes activate human dendritic cells through TLR7 and TLR8. J Exp Med 206: 1983–1994. 31. Bals R, Wang X, Zasloff M, Wilson JM (1998) The peptide antibiotic LL-37/ hCAP-18 is expressed in epithelia of the human lung where it has broad antimicrobial activity at the airway surface. Proc Natl Acad Sci U S A 95: 9541–9546. 32. Surh CD, Sprent J (1994) T-cell apoptosis detected in situ during positive and negative selection in the thymus. Nature 372: 100–103. 33. Haller O, Kochs G (2011) Human MxA protein: an interferon-induced dynamin-like GTPase with broad antiviral activity. J Interferon Cytokine Res 31: 79–87. 34. Keir ME, Stoddart CA, Linquist-Stepps V, Moreno ME, McCune JM (2002) IFN-alpha secretion by type 2 predendritic cells up-regulates MHC class I in the HIV-1-infected thymus. J Immunol 168: 325–331. 35. Vandenabeele S, Hochrein H, Mavaddat N, Winkel K, Shortman K (2001) Human thymus contains 2 distinct dendritic cell populations. Blood 97: 1733–1741. PLoS ONE | www.plosone.org 10 August 2011 | Volume 6 | Issue 8 | e24252