Abstract
Non-classical HLA-G and HLA-E molecules have well-recognized immunomodulatory effects on peripheral blood immune-competent cells; however, little is known regarding the effect of these molecules in central tolerance. Aire is responsible for promiscuous gene expression of tissue-related antigens by thymic cells during induction of central tolerance. We characterized the differential transcript profiles of murine MHC class Ib (Qa-1/HLA-E, Qa-2/HLA-G) and Aire genes during ontogeny of thymus, and identified that the transcriptional profile of the functional homolog of HLA-G, (H2-Q7/Qa2) was positively correlated with Aire during thymus fetal development, indicating that non-classical MHC class I genes could be under the transcriptional control of Aire. This study aimed to evaluate the transcriptional and post-transcriptional relation between Aire and HLA-G. Using previously validated Aire plasmid-based constructs, we transfected different cell-lines: HLA-G + Cells (JEG3), HLA-G- Cells (U251) and thymic cells (4D6 and LT-TEC2), and assessed HLA-G gene and protein expression. Using an in silico approach, we found 4 putative binding sites for Aire-complex at the 5’-promoter region of the HLA-G gene. We cloned, selected (enzyme digestion: EcoRI/HindIII) and validated (sequencing/alignment) the bacterial-vectors containing Aire. Initially, we observed that only JEG-3 cells were HLA-G + with transcript levels being 10.000-fold greater than the other cell lines and 50% of cell population presenting HLA-G in cell surface. After transient transfection, HLA-G transcript levels were increased only in 4D6 and LT-TEC2 cells (P = 0.03 and P = 0.007, respectively) compared to control (empty-vector). We did not observe any modification in the HLA-G cell surface expression in all cell lines. Concluding, Aire may play a role in the transcriptional regulation of HLA-G, which appears to be cell type-dependent, i. e., primarily inducted in thymic cell lines.
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