Antioxidant treatment of thymic organ cultures decreases NF-κB and TCF1(α) transcription factor activities and inhibits αβ T cell development

Using electrophoretic mobility shift assays (EMSA), we have recently shown that nuclear extracts of 14-day mouse fetal thymocytes contain abundant NF-κB transcription factor activity. To determine the functional role of NF-κB in early thymocyte development, we have exposed fetal thymus organ cultures to inhibitors of NF-κB activation, namely the antioxidants N-acetyl-L-cysteine and butylated hydroxyanisole. Both compounds caused a dose-dependent arrest of thymocyte differentiation toward αβ, but not γδ T cells. This was associated with a profound decrease in nuclear content of NF-κB and TCF1 (α) transcription factor activity, as determined by EMSA. In contrast, NF-Y was affected less strongly, and cyclic AMP-response-element-binding protein levels remained essentially unchanged by antioxidants. To test the idea that αβ T cell development is correlated with NF-κB and TCF1(α) activity, we conducted additional experiments in a submersion culture system in which the generation of αβ T cells can be manipulated. Standard submersion culture supports γδ but not αβ T cell development. Under these conditions, EMSA showed that transcription factor activities were similar to those seen in the presence of antioxidants. Importantly, when the generation of αβ T cells in submersion culture was restored by elevating oxygen concentrations, there was a dramatic increase in TCF1(α) activity, and both NF-κB and NF-Y returned to control levels. Taken together, these results strongly suggest that NF-κB and TCF1(α), presumably in concert with other transcription factors, play an important role in the development of αβ T cells.