ecnacifingS?nucleusWe discovered THEMIS as an indispensable gene for T cell development. 36TurboIDTurboIDcytoplasmABiFold change(unstimulated/stimulated)TCRstimulationbiotinTHEMIS??THEMIS?To investigate the importance of nuclear THEMIS function, we established ERT2-Themis knock-in mice. In these mice, without tamoxifen, THEMIS remains exclusively in the cytoplasm, which results in a severe deficiency in conventional T cell development similar to germline Themis knock-out mice. Tamoxifen administration in vivo, induced import of THEMIS protein into the nucleus and resulted in total recovery of normal T cell development and matu-ration. These results clearly indicate that THEMIS in T cells plays essential and non-redundant roles in the cytoplasm and nucleus and that dynamic subcellu-lar localization is important for its function.We next aimed to identify THEMIS interacting proteins in the cytoplasm and nucleus with/without TCR stimulation by biotinylating enzyme and mass spectrometry in collaboration with Dr. Yibo (IMS, YCI Laboratory for Next-Generation Proteomics). We introduced a TurboID-Themis fusion construct into the mouse T cell hybridoma, 2B4, and compared the array of biotinylated proteins after TCR stimulation with those of unstimulated 2B4 and found that, besides cytoplasmic components, several important nuclear proteins were biotinylated. These include histone modification enzymes, components of the nuclear pore complex and chromatin-remodeling proteins. These findings sug-gest roles of nuclear THEMIS in epigenetic modulation and gene transcription in response to TCR stimulation.Many GWAS studies revealed that the THEMIS locus associates with T cell-driven autoimmune diseases such as Celiac Disease, Multiple Sclerosis, Rheumatoid Arthritis, and Atopic Dermatitis. THEMIS is involved in T cell re-ceptor (TCR) signaling as an adapter molecule able to modulate signal strength. THEMIS-deficiency results in a reduction of conventional mature T cells in mice. However, the mechanisms used by THEMIS to control TCR signaling remain unclear and controversial. Although functions of cytoplasmic THEMIS have been studied intensively, THEMIS is also present in the nucleus but its nuclear function is poorly understood.Figure: Identification of proteins proximal to THEMIS by proteomicsA. We introduced cDNA encoding a TurboID-Themis fu-sion protein. TurboID is a biotinylation enzyme that adds biotin to proximal proteins.B. Volcano plot showing fold change and significance of biotinylated proteins. We compared biotinylated proteins in the lysates from an unstimulated and TCR-stimulated T cell line.Recent Major PublicationsLiu W, Chou TF, Garrett-Thomson SC, Seo GY, Fedorov E, Ramagopal UA, Bonanno JB, Wang Q, Kim K, Garforth SJ, Kakugawa K, Cheroutre H, Kronenberg M, Almo SC. HVEM structures and mutants reveal distinct functions of binding to LIGHT and BTLA/CD160. J Exp Med 218, e20211112 (2021)Invited presentationsCheroutre H. “New Members in an Old Club: TCR signalo-some Revised” Distinguished Lecture at the Research Forum of the Oklahoma Medical Research Foundation (OMRF), University of Oklahoma Medical Center (Okla-homa City, USA) November 2021Cheroutre H. “How and Why: do CD4 Th Cells Convert to CTL in the Intestine?” Kenneth Rainin Foundation 2021 Innovation Symposium (Oakland, USA/Online) July 2021Cheroutre H. “Oral Non-Pathogen Antigens Induce Protective Tolerance in the Intestine” MIST NIH/NIAID (Rockville, USA) February 2021Laboratory for Immune CrosstalkTeam Leader: Hilde Cheroutre
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