ARTICLES https://doi.org/10.1038/s41590-019-0570-3 1 Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore. 2 Laboratory of Immunobiology, Rega Institute for Medical Research, KU Leuven, Leuven, Belgium. 3 State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, China. 4 Immunology Programme, Life Sciences Institute, National University of Singapore, Singapore, Singapore. *e-mail: micnrjg@nus.edu.sg T hemis1 (henceforth referred to as Themis) is a T cell-specific molecule whose function has been contentious. Germline Themis deficiency leads to a striking defect in thymocyte development, with a block in double-positive to single-positive progression and maturation 1–5 . Themis constitutively binds to the adapter Grb2 (refs. 2–4,6,7 ) and the phosphatase Shp1 (refs. 8–12 ), whereas T cell receptor (TCR) stimulation leads to interaction with the adapter molecule linker for activation of T cells (LAT) 6,7 . Currently, there are two conflicting models of Themis function during thymocyte development. It was previously observed that pre-selection Themis-deficient thymocytes show enhanced TCR signaling in response to low- or intermediate-affinity ligands 8 , lead- ing to the proposal that Themis is a negative regulator of TCR signal though positive regulation of Shp1 (refs. 13,14 ). The defective thymic selection in Themis-deficient mice is then a result of negative selec- tion in response to low- or intermediate-affinity self-peptide–major histocompatibility complex (self-pMHC). However, this model has been challenged by subsequent work proposing that Themis acts as a negative regulator of Shp1 to positively modulate TCR signals in response to low-affinity pMHC to allow positive selection 9,11,15 . The role of Themis in peripheral T cells has not yet been estab- lished. Themis is expressed most strongly in double-positive thy- mocytes 1–4 , but is readily detectable in single-positive thymocytes and mature T cells. Germline Themis deficiency reduces peripheral CD4 + and CD8 + T cell numbers, but increases the percentage of CD44 hi memory phenotype T cells, and of Foxp3 + regulatory T cells (T reg cells) 1–4 . It is not known whether this phenotype is due to impaired thymic development, or to a role of Themis in peripheral T cells independent of its functions during thymic development. Peripheral T cell responses and homeostasis depend on TCR and cytokine receptor signals. Tonic TCR signaling from recognition of self-pMHC correlates with CD8 + T cell responses to antigenic stimulation 16–18 , and with the capacity to undergo lymphopenia- induced proliferation (LIP) 19,20 . Signaling pathways induced by self- pMHC recognition in mature T cells are poorly understood, and the signaling pathways that allow integration of low-affinity TCR and cytokine signals are unknown. Identification of molecules regulat- ing mature T cell responses to low-affinity ligands and cytokines is of clinical importance, as these can be targeted to modulate T cell activation during anti-tumor responses or in autoimmunity. Here, we describe a new function of Themis in CD8 + T cell homeostasis through regulation of signal synergy between low-affinity TCR and cytokine signals. Results Themis is required for maintenance of peripheral CD8 + T cells. To investigate the role of Themis in mature T cells, we generated Themis conditional knockout (cKO) mice, where Themis exon 4 is flanked by LoxP sites (‘floxed’), using distal LckCre (dlLck- Cre) 21 . To validate the kinetics of dlLck-Cre recombinase activity, we crossed wild-type dlLck-Cre + mice with the Ai14 Cre activ- ity reporter strain 22 , in which Cre activity-dependent removal of a floxed STOP cassette permits tdTomato expression. dlLck-Cre resulted in tdTomato signal in a small fraction of double-positive thymocytes, a significant proportion of single-positive thymocytes and more than 90% of peripheral T lymphocytes (Fig. 1a). This pat- tern of Cre activity allows Themis-sufficient thymocyte develop- ment and Themis deletion in mature T cells. We crossed dlLck-Cre + mice with the floxed Themis-bearing mice. We confirmed that the expression of Themis protein in double-positive thymocytes from Themis-floxed dlLck-Cre + mice (hereafter referred to as cKO) was comparable to that from Themis-floxed Cre – controls (henceforth referred to as wild type) (Fig. 1b), but we observed some reduction in Themis protein in single-positive thymocytes. Peripheral CD4 + T cell receptor and cytokine signal integration in CD8 + T cells is mediated by the protein Themis Joanna Brzostek  1 , Namrata Gautam 1 , Xiang Zhao 1 , Elijah W. Chen 1 , Monika Mehta 1 , Desmond W. H. Tung 1 , Yen Leong Chua 1 , Jiawei Yap 1 , Su H. Cho 1 , Shvetha Sankaran 1 , Vasily Rybakin 1,2 , Guo Fu  3 and Nicholas R. J. Gascoigne  1,4 * T cell homeostasis and functional responsiveness require signals from self-peptide–major histocompatibility complex (self- pMHC) and cytokines, but the mechanisms controlling this signal integration are unknown. Using a conditional deletion of the T cell lineage-specific protein Themis, we show that Themis is required for the maintenance of peripheral CD8 + T cells and for proliferative CD8 + T cell responses to low-affinity pMHC aided by cytokines. Themis-deficient peripheral T cells show a phenotype indicative of reduced tonic signaling from self-pMHC, strongly suggesting that Themis is a positive regulator of T cell receptor signal strength in response to low-affinity self-pMHC in peripheral T cells. Signals from low-affinity pMHC and cytokines synergistically induce phosphorylation of the kinase Akt, metabolic changes and c-Myc transcription factor induction in CD8 + T cells only in the presence of Themis. This function of Themis is mediated through Shp1 phosphatase, as peripheral Themis and Shp1 double deletion rescues the peripheral CD8 + T cell maintenance. NATURE IMMUNOLOGY | www.nature.com/natureimmunology