Periodic Reporting for period 1 - EpiNKT (Transcriptional and epigenetic control of innate-like T lymphocyte development) Reporting period: 2015-08-31 to 2017-08-30 Summary of the context and overall objectives of the project Natural Killer T (NKT) cells constitute a prototypical population of innate-like T lymphocytes. Their effector programs are acquired during thymic development, prior to microbial exposure, and are polarized into three distinct populations. While the functional heterogeneity of NKT cells is starting to be appreciated, the molecular mechanisms involved in NKT lineage specification and subset polarization are currently not well understood. We recently found that the balance between the E protein family of transcription factors (TF) and their inhibitors, the ID proteins, which is pivotal in the bifurcation of adaptive and innate lymphoid lineages and is associated with human lymphomas, regulates NKT cell development. The overall objective of this grant proposal was to identify genes regulated by the E/ID pathway that determine NKT cell development.NKT cells produce a vast amount of various cytokines rapidly after activation, thereby influencing the functions of innate and adaptive immune cells and orchestrating the early phases of an immune response. Although rare, NKT cells modulate responses to a wide range of diseases, including microbial infection, hematopoietic malignancies, cancer, and inflammation, thus rendering them attractive targets in immune therapies and vaccination strategies. Our studies contribute to the understanding of the developmental and molecular pathways that determine NKT cell effector fate choices and help harness their immunotherapeutic potential. Our studies are therefore relevant to public health because they will provide alternatives for interception of the immune response and thereby alter the course of disease. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far "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." Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) In this project, we have identified at least three transcriptional target genes of the E/ID pathway in early stages of NKT cell development. We have validated the function of two of these genes, which are known oncogenes, while that of the third candidate is currently under investigation. We are currently characterizing the chromatin accessibility landscape of the earliest NKT cell progenitors and how changes in these patterns may influence NKT cell development and function. Our study provided significant insight into the transcriptional networks that regulate innate T cell development at its inception, while identifying novel functions for two proteins with known roles in normal lymphopoiesis and human lymphomagenesis. Therefore, our research will inform further studies on putative cancer immunotherapeutic approaches. Furthermore, we initiated a new line of research regarding the function of NKT cells in hepatocellular carcinoma, which is not currently understood, with the aim to exploit the unique properties of NKT cells in cancer therapy. EpiNKT had a significant impact on Dr. Verykokakis’ integration at BSRC “Alexander Fleming” and the Greek scientific community. Dr. Verykokakis is currently an Associate Researcher (Researcher C level) and he runs an independent laboratory at Fleming, investigating the molecular mechanisms of innate lymphocyte development and function. He is a Stavros Niarchos Foundation fellow and he has secured start-up funding for his laboratory until 2020. Dr. Verykokakis has also been invited in high schools and other outreach events with the aim to help students explore their career options in science. Therefore, during the implementation of this project, Dr. Verykokakis fully developed his potential as a researcher and educator to smoothly integrate in the Greek scientific community and also educate the public on issues related to Immunology.