"Our studies demonstrated that ID proteins are critical for NKT cell development and differentiation. This project aimed at determining how ID/E proteins and their targets regulate NKT cell development. We focused on two transcription factors, Lef1 and Bcl6, because there was no previous evidence regarding their function in innate-like T lymphocyte development.
Our results revealed that BCL6 is expressed in the earliest NKT cell progenitors and controls a subset of NKT differentiation-associated genes. In the absence of BCL6, NKT cells fail to develop in the thymus and their differentiation is blocked at the most immature stage. BCL6 regulates expression of the NKT-signature transcription factor PLZF and a subset of its target genes, especially tissue homing receptors and transcription factors. Therefore, BCL6 is crucial for the implementation of the differentiation program of the NKT cells.
Our results showed that Id3-/- mice had increased expression of LEF1 accompanied by an expansion of NKT2 and a loss of NKT1 and NKT17, while mice deficient for the antagonistic E protein E2A, have decreased LEF1 expression in NKT cells and decreased NKT2 cell numbers. We generated mice deficient for both ID3 and LEF to examine whether LEF1 was responsible for the expansion of NKT2 cells in Id3-/- mice. We showed that NKT2 cell development was restored in Id3-/-;Lef1-/- mice, whereas NKT1 cell development was not. Surprisingly, we found that NKT17 were expanded in Id3-/-;Lef1-/- mice and are accumulating in the lungs. Therefore, LEF1 and ID/E proteins regulate the bifurcation point of NKT2/NKT17 cells, while NKT1 cell differentiation relies only on ID/E proteins and is independent of LEF1.
Our results also revealed the accessibility landscape of the chromatin in the earliest NKT cell progenitors and how this changes during differentiation. Studies in our genetically modified mice, in correlation with global gene expression profiling, will reveal potential new regulatory regions that are LEF1-, BCL6- and/or ID-dependent.
Our preliminary results showed that NKT cells are depleted in the course of chemically-induced hepatocellular carcinoma in mice, a cancer model that recapitulates the human condition. Further studies are warrant to determine the role of NKT cell in liver cancer and the molecular mechanisms involved.
Through EpiNKT, Dr. Verykokakis received training to diversify his competences and develop skills that helped him secure a position as Group Leader at Fleming and independent funding for his laboratory. He received training to enhance his research potential and his lab and personnel management skills and also generate a network of collaborations. Dr. Verykokakis is also teaching courses on innate lymphocyte biology in the M.Sc. program “Molecular Biomedicine”.
Part of this work has been presented in international meetings, including the CD1-MR1 2015 international Conference, the EEBMB2015 international conference and the EEBE2017 national meeting. Upon completion, these studies are scheduled to be presented in additional meetings, including the upcoming Next Gen Immunology international meeting, in Rehovot, Israel in February 2018. Moreover, part of this study was also presented in the internal seminar series of the host institute. Furthermore, several concepts emerged from this work are currently in press in an invited, peer-reviewed, review article in Current Opinion in Immunology. Two manuscripts regarding the results of the original research, intended for peer-review publication are currently in preparation. These findings have been discussed in class with graduate students of the M.Sc. program ""Molecular Biomedicine"". Dr. Verykokakis has also accepted and trained undergraduate students in his laboratory from various departments of the University of Athens and other national Universities.
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