4D C4D C4D C2TRE2TRETAL2TRETALTALWe identified THEMIS as an indispensable molecule for T cell develop-⃝THEMIS-ERT2: THEMIS is retained in the cytoplasm by ERT2. With tamox-ifen treatment, cytoplasmic THEMIS-ERT2 is able to translocate into the nucleus.All these THEMIS mutant mice showed a deficiency in T cell development similar to Themis germline knock-out mice, indicating that THEMIS in the nucleus or cytoplasm is not sufficient for its function. Rather THEMIS needs to be able to translocate between compartments to carry out its normal function. To provide proof-of-concept for this, THEMIS-ERT2 mutant mice were given several doses of Tamoxifen, which caused recovery of conventional T cell devel-opment and seeding of the periphery. This result indicated that dynamic move-ment of THEMIS from cytoplasm to nucleus is indispensable for its function. A follow-up study aims to elucidate the mechanism of THEMIS translocation and to examine its nuclear function and identify nuclear binding partners.GRB2SHP1GRB2SHP183%3%93%3%CD8α3%2%CD8αGRB2SHP111%83%3%4%CD8α35tamoxifenCyt(-)tamoxifen(+)Themis ERT2NucCytNucTHEMISATM10%CDC374% abThemis wt tamoxifen(-)(-)THEMISTHEMISTHEMIStamoxifenThemis ERT2tamoxifen(+)THEMISTHEMISTCRTCRnucleusnucleusnucleusTCRtranslocate into the other compartment.ment. GWAS linked the THEMIS locus with autoimmune diseases such as Celiac Disease, Multiple Sclerosis, Rheumatoid Arthritis, and Atopic Der-matitis. THEMIS functions as an adapter that modulates T cell receptor (TCR) signal strength and THEMIS-deficiency causes impaired conventional TCR αβ T cell development in mice. The mechanisms used by THEMIS to control TCR signaling remain unclear and controversial. Besides the cytoplasm, THEMIS also localizes in the nucleus. Although the functions of cytoplasmic THEMIS have been studied intensively, its nuclear function is poorly elucidated. To ad-dress this gap, we generated various THEMIS mouse mutants to manipulate the subcellular localization of THEMIS and examined the consequences of this for T cell development and function.⃝nTHEMIS: THEMIS exists exclusively in the nucleus.⃝cTHEMIS: THEMIS exists exclusively in the cytoplasm.⃝n/cTHEMIS: THEMIS exists both in cytoplasm and nucleus but cannot Our overall goal is to dissect the mechanisms employed by THEMIS to modulate TCR signals and control gene expression and ultimately to elucidate the correlation between THEMIS and protective immunity, as well as its role in the T cell-specific inflammatory diseases mentioned above.Invited presentationsCheroutre H. “Dietary Antigens build ‘De-Fence’ at the Mucosal Border of the Intestine” Asociacion Chilena de Immunologia (Santiago, Chile/Online) November 2020Cheroutre H. “The Cd8 Intergenic Region Controls the Generation of Cytotoxic CD4 Effector Cells” Meeting at La Jolla Institute (La Jolla, USA/Online) June 2020Cheroutre H. “The Cd8 Intergenic Region Controls the Generation of Cytotoxic CD4 Effector Cells” Seminar at National Institute of Health (Washington, USA/Online) May 2020Figure: Dynamic subcellular translocation of THEMIS is necessary for its function in T cells(a) The THEMIS-ERT2 fusion protein is retained in the cytoplasm at steady state. Administration of Tamoxifen induces translocation of THEMIS from the cytoplasm to the nucleus. Lysate from total thymocytes was separated into the cytoplasmic (Cyt) and nuclear (Nuc) fraction and blotted with anti-THEMIS and anti-ATM (nuclear protein) and anti-CDC37 (cytoplasmic protein) as controls.(b) Subcellular localization of THEMIS and T cell develop-ment in the thymus. THEMIS is present in the cytoplasm and nucleus of normal thymocytes and TCRαβ+ T cells (left panel). Artificial retention of THEMIS-ERT2 in the cytoplasm abrogates T cell development (middle panel), resulting in reduced frequencies of CD4+CD8– and CD4–CD8+ mature thymocytes. Addition of Tamoxifen restores translocation of THEMIS from the cytoplasm to the nucleus and results in normal T cell development (right). Dot profiles show the CD4 and CD8 expression pattern by thymocytes.Laboratory for Immune CrosstalkTeam Leader: Hilde Cheroutre
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