An IFN-alpha heteroduplex-tracking assay (IFN-HTA) was developed to quantify the frequency of expression of the 16 genes coding for related interferon-alpha (IFN-alpha) subtypes in mice. In mLN of mice treated with Poly (I:C), we observed the induction of three sequential waves of type I IFN production, instead of two as is commonly described: early IFNs after 1 h (IFN-beta), late IFNs after 3 h (mostly IFN-alpha1, -alpha2, -alpha 4 and -alpha 5) and "secondary late IFNs" after 6 h (IFN-alpha 6T and -alpha 8/6). The late IFN wave was associated with the upregulation of the interferon regulatory factor (IRF)-7 mRNA and proteins, whereas the secondary late IFN wave was associated with a slight upregulation of IRF-8 mRNA. Type I IFNs produced in the thymus were associated with a distinct IRF mRNA expression pattern. This IFN-HTA strategy can serve as a useful tool to qualify and quantify the expression of various IFN-alpha subtypes under distinct immune responses and thus provides a first step in evaluating their function.

The interactions between TLRs and their ligands have profound immune modulation properties. Attention has focused mostly on the impact of TLR ligands on peripheral innate and adaptive immunity during viral infections, whereas little impact of TLR activation has been shown on thymic development. Here we show that treatment of murine fetal thymic organ cultures (FTOCs) with TLR3 or TLR7 ligands induced rapid expression of IFN-α and -β mRNA, hallmarks of acute and chronic viral infections. This resulted in an early developmental blockade, increased frequencies of apoptotic cells, and decreased proliferation of thymocytes, which led to an immediate decrease in cellularity. FTOCs infected with vesicular stomatitis virus, known to act through TLR7, were similarly affected. Down-regulation of IL-7R α-chain expression, together with an increased expression of suppressor of cytokine signaling-1 and a concomitant decreased expression of the transcriptional regulator growth factor independence 1 were observed in TLR ligands or IFN-treated FTOCs. This indicates a role for these pathways in the observed changes in thymocyte development. Taken together, our data demonstrate that TLR activation and ensuing type I IFN production exert a deleterious effect on T cell development. Because TLR ligands are widely used as vaccine adjuvants, their immunomodulatory actions mediated mainly by IFN-α suggested by our results should be taken in consideration.