Human T-cell leukemia virus type 1 (HTLV-1) establishes a lifelong persistent infection in humans. Approximately 3% of the infected individuals will develop adult T-cell leukemia/lymphoma (ATLL), an aggressive malignancy of mature CD4+ T-cells. The viral protein Tax plays a major role in HTLV-1 pathogenicity by activating the NF-κB pathway. Tax activates both the canonical and non-canonical NF-κB pathways, promoting NF-κB translocation to the nucleus and transcription of genes that favour T-cell proliferation and survival. Our previous studies showed that the p13 protein of HTLV-1 enhances mitochondrial ROS production, resulting in activation of normal T-cells. ROS constitute a homeostatic rheostat that controls the activity of several key pathways, including the NF-κB pathway.Thus, we hypothesized that the effects of p13 on ROS production could affect the activation of the NF-κB pathway by Tax in primary T-cells.
The work described in the present thesis was aimed at testing the hypothesis that Tax and p13 might act in concert to activate the NF-κB signal transduction pathway in primary T-cells. To this end, we optimized a transfection protocol for primary T-cells using an innovative approach based on the electroporation of in vitro-transcribed RNA. Activation of the NF-κB pathway was then analysed by measuring expression of the NF-κB target genes CD25 and 4-1BB.
Results showed that the co-transfection of Tax and p13 resulted in a synergistic activation of the NF-κB pathway in primary T-cells measured as an increase in the expression levels of both CD25 and 4-1BB. In addition to being a transcriptional target of NF-κB, CD25 is also an early marker of T-cell activation. To further test the effects of Tax and p13 on cell activation, we measured CD38 expression by flow cytometry. Jurkat T-cells, which exhibit a constitutively activated CD38 positive phenotype, were used as a control. Results of this analysis confirmed the synergy of Tax and p13, although the effect was not so prominent as that observed for the CD25 marker, suggesting that, within the time frame of our experiments, Tax and p13 drove T-cells to an early-intermediate stage of activation.
Taken together, these findings suggest that, in contrast to the well-established role of Tax as an activator of the NF-κB pathway in tumor cell lines, in the context of normal T-cells, the induction of NF-κB target genes requires the concerted action of Tax and p13.
Current studies are aimed at verifying the ROS-dependence of this effect and testing the functional interaction of Tax and p13 in the context of the complete HTLV-1 genome using wild type HTLV-1 and a p13-knock-out HTLV-1 molecular clone. These experiments will be carried out in primary T-cells as well as in dendritic cells, which have recently emerged as an important target of the virus in vivo
