Development of a self-tolerant T-cell receptor (TCR) repertoire with the potential to recognize the universe of infectious agents depends on proper regulation of TCR signalling. The repertoire is whittled down during T-cell development in the thymus by the ability of quasi-randomly generated TCRs to interact with self-peptides presented by major histocompatibility complex (MHC) proteins. Low-affinity TCR interactions with self-MHC proteins generate weak signals that initiate ‘positive selection’, causing maturation of CD4- or CD8αβ-expressing ‘single-positive’ thymocytes from CD4+CD8αβ+ ‘double-positive’ precursors1. These develop into mature naive T cells of the secondary lymphoid organs. TCR interaction with high-affinity agonist self-ligands results in ‘negative selection’ by activation-induced apoptosis or ‘agonist selection’ of functionally differentiated self-antigen-experienced T cells2,3. Here we show that positive selection is enabled by the ability of the T-cell-specific protein Themis4,5,6,7,8,9 to specifically attenuate TCR signal strength via SHP1 recruitment and activation in response to low- but not high-affinity TCR engagement. Themis acts as an analog-to-digital converter translating graded TCR affinity into clear-cut selection outcome. By dampening mild TCR signals Themis increases the affinity threshold for activation, enabling positive selection of T cells with a naive phenotype in response to low-affinity self-antigens.
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Morris, G. P. & Allen, P. M. How the TCR balances sensitivity and specificity for the recognition of self and pathogens. Nature Immunol. 13, 121–128 (2012)
Cheroutre, H. & Lambolez, F. The thymus chapter in the life of gut-specific intra epithelial lymphocytes. Curr. Opin. Immunol. 20, 185–191 (2008)
Stritesky, G. L., Jameson, S. C. & Hogquist, K. A. Selection of self-reactive T cells in the thymus. Annu. Rev. Immunol. 30, 95–114 (2012)
Fu, G. et al. Themis controls thymocyte selection through regulation of T cell antigen receptor-mediated signaling. Nature Immunol. 10, 848–856 (2009)
Johnson, A. L. et al. Themis is a member of a new metazoan gene family and is required for the completion of thymocyte positive selection. Nature Immunol. 10, 831–839 (2009)
Lesourne, R. et al. Themis, a T cell-specific protein important for late thymocyte development. Nature Immunol. 10, 840–847 (2009)
Patrick, M. S. et al. Gasp, a Grb2-associating protein, is critical for positive selection of thymocytes. Proc. Natl Acad. Sci. USA 106, 16345–16350 (2009)
Kakugawa, K. et al. A novel gene essential for the development of single positive thymocytes. Mol. Cell. Biol. 29, 5128–5135 (2009)
Brockmeyer, C. et al. T cell receptor (TCR)-induced tyrosine phosphorylation dynamics identifies THEMIS as a new TCR signalosome component. J. Biol. Chem. 286, 7535–7547 (2011)
Lesourne, R. et al. Interchangeability of Themis1 and Themis2 in thymocyte development reveals two related proteins with conserved molecular function. J. Immunol. 189, 1154–1161 (2012)
Paster, W. et al. GRB2-mediated recruitment of THEMIS to LAT is essential for thymocyte development. J. Immunol. 190, 3749–3756 (2013)
Daniels, M. A. et al. Thymic selection threshold defined by compartmentalization of Ras/MAPK signalling. Nature 444, 724–729 (2006)
Hogquist, K. A. et al. T cell receptor antagonist peptides induce positive selection. Cell 76, 17–27 (1994)
Oh-Hora, M. et al. Dual functions for the endoplasmic reticulum calcium sensors STIM1 and STIM2 in T cell activation and tolerance. Nature Immunol. 9, 432–443 (2008)
Mariathasan, S. et al. Duration and strength of extracellular signal-regulated kinase signals are altered during positive versus negative thymocyte selection. J. Immunol. 167, 4966–4973 (2001)
Fischer, A. M., Katayama, C. D., Pages, G., Pouyssegur, J. & Hedrick, S. M. The role of erk1 and erk2 in multiple stages of T cell development. Immunity 23, 431–443 (2005)
McNeil, L. K., Starr, T. K. & Hogquist, K. A. A requirement for sustained ERK signaling during thymocyte positive selection in vivo . Proc. Natl Acad. Sci. USA 102, 13574–13579 (2005)
Lorenz, U. SHP-1 and SHP-2 in T cells: two phosphatases functioning at many levels. Immunol. Rev. 228, 342–359 (2009)
Zhang, J. et al. Involvement of the SHP-1 tyrosine phosphatase in regulation of T cell selection. J. Immunol. 163, 3012–3021 (1999)
Plas, D. R. et al. Cutting edge: the tyrosine phosphatase SHP-1 regulates thymocyte positive selection. J. Immunol. 162, 5680–5684 (1999)
Carter, J. D., Neel, B. G. & Lorenz, U. The tyrosine phosphatase SHP-1 influences thymocyte selection by setting TCR signaling thresholds. Int. Immunol. 11, 1999–2014 (1999)
Štefanová, I. et al. TCR ligand discrimination is enforced by competing ERK positive and SHP-1 negative feedback pathways. Nature Immunol. 4, 248–254 (2003)
Rybakin, V. & Gascoigne, N. R. J. Negative selection assay based on stimulation of T cell receptor transgenic thymocytes with peptide-MHC tetramers. PLoS ONE 7, e43191 (2012)
Bouillet, P. et al. BH3-only Bcl-2 family member Bim is required for apoptosis of autoreactive thymocytes. Nature 415, 922–926 (2002)
Moran, A. E. et al. T cell receptor signal strength in Treg and iNKT cell development demonstrated by a novel fluorescent reporter mouse. J. Exp. Med. 208, 1279–1289 (2011)
Johnson, D. J. et al. Shp1 regulates T cell homeostasis by limiting IL-4 signals. J. Exp. Med. 210, 1419–1431 (2013)
Dubois, P. C. et al. Multiple common variants for celiac disease influencing immune gene expression. Nature Genet. 42, 295–302 (2010)
Trynka, G. et al. Dense genotyping identifies and localizes multiple common and rare variant association signals in celiac disease. Nature Genet. 43, 1193–1201 (2011)
Sawcer, S. et al. Genetic risk and a primary role for cell-mediated immune mechanisms in multiple sclerosis. Nature 476, 214–219 (2011)
Fu, G. & Gascoigne, N. R. J. Multiplexed labeling of samples with cell tracking dyes facilitates rapid and accurate internally controlled calcium flux measurement by flow cytometry. J. Immunol. Methods 350, 194–199 (2009)
Fu, G. et al. Protein kinase C η is required for T cell activation and homeostatic proliferation. Sci. Signal. 4, ra84 (2011)
Huang, Y. H. et al. Positive regulation of Itk PH domain function by soluble IP4. Science 316, 886–889 (2007)
Minoo, P., Zadeh, M. M., Rottapel, R., Lebrun, J. J. & Ali, S. A novel SHP-1/Grb2-dependent mechanism of negative regulation of cytokine-receptor signaling: contribution of SHP-1 C-terminal tyrosines in cytokine signaling. Blood 103, 1398–1407 (2004)
Simoneau, M. et al. Activation of Cdk2 stimulates proteasome-dependent truncation of tyrosine phosphatase SHP-1 in human proliferating intestinal epithelial cells. J. Biol. Chem. 283, 25544–25556 (2008)
Hammond, K. J. et al. CD1d-restricted NKT cells: an interstrain comparison. J. Immunol. 167, 1164–1173 (2001)
Gangadharan, D. et al. Identification of pre- and postselection TCRαβ+ intraepithelial lymphocyte precursors in the thymus. Immunity 25, 631–641 (2006)
Hogquist, K. A., Jameson, S. C. & Bevan, M. J. Strong agonist ligands for the T cell receptor do not mediate positive selection of functional CD8+ T cells. Immunity 3, 79–86 (1995)
Hogquist, K. A. et al. Identification of a naturally occurring ligand for positive selection. Immunity 6, 389–399 (1997)
Santori, F. R. et al. Rare, structurally homologous self-peptides promote thymocyte positive selection. Immunity 17, 131–142 (2002)
Rosette, C. et al. The impact of duration versus extent of TCR occupancy on T cell activation: a revision of the kinetic proofreading model. Immunity 15, 59–70 (2001)
Huang, J. et al. The kinetics of two-dimensional TCR and pMHC interactions determine T-cell responsiveness. Nature 464, 932–936 (2010)
Alam, S. M. et al. T cell receptor affinity and thymocyte positive selection. Nature 381, 616–620 (1996)
Alam, S. M. et al. Qualitative and quantitative differences in T cell receptor binding of agonist and antagonist ligands. Immunity 10, 227–237 (1999)
Juang, J. et al. Peptide-MHC heterodimers show that thymic positive selection requires a more restricted set of self-peptides than negative selection. J. Exp. Med. 207, 1223–1234 (2010)
We thank X. L. Chen and Y. Xing for technical advice. We thank J. Ampudia, S. Vallée, J. Hu and S. Feldstein for help, and the National Institutes of Health (NIH) Tetramer Core Facility for production of MHC-I tetramers. Supported by NIH grants AI073870, DK094173 and GM065230 to N.R.J.G., DP1OD006433 to H.C., GM100785 and AI070845 to K.S.; Wellcome Trust Grant GR076558MA to O.A., and by the National University of Singapore. J.C. was supported by a fellowship from the Spanish Ministerio de Ciencia e Innovacion (MICIIN), J.A.H.H. by the Irving S. Sigal Fellowship of the American Chemical Society and NIH training grant T32AI07244, and K.S. by The Leukemia & Lymphoma Society Scholar Award 1440-11. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Allergy and Infectious Diseases, the NIH, or other funding agencies. This is manuscript number 21592 from The Scripps Research Institute.
The authors declare no competing financial interests.
Extended data figures and tables
Thymocytes from wild-type or Themis-deficient mice were first stained with saturated amount of anti-CD3/CD4 antibodies and subsequently cross-linked with titrated amount of streptavidin (S.Av). Two independent experiments are shown here.
Extended Data Figure 2 OT-I TCR system and comparison of Ca2+ flux between Themis-sufficient and Themis-deficient pre-selection thymocytes.
a, Summary of responses of OT-I T cells and thymocytes to different peptides. Data from references 12, 13 and 37, 38, 39, 40, 41, 42, 43, 44. b, Representative FACS plots from Fig. 1a are shown here again for illustration (top panel), statistical analysis of Ca2+ flux between Themis-sufficient (+/+) and Themis-deficient (−/−) thymocytes were calculated using Wilcoxon signed rank test (P value listed), each line links cells from a pair of mice being compared in the same tube as described30 (bottom panel). Results are pooled from multiple experiments as described in Fig. 1 legend.
Thymocytes from indicated mice were either stimulated with peptide presented on thymocytes themselves (left) or with Kb-tetramers (right). As shown, similar results were obtained by both stimulation methods.
Thymocytes were stimulated with ionomycin/PMA (phorbol-12-myristate-13-acetate) (Iono+PMA) to obtain maximal extent of NFATC2 nuclear translocation as a positive control for image analysis. NFATC2 translocation in non-stimulated cells (CONTROL) is used as negative control for image analysis. DAPI and NFATC2 staining are colour-coded as indicated.
ERK1 and 2 phosphorylation of indicated thymocytes in response to different stimuli, normalized to VAV. Representative of 4 experiments.
Thymocytes were prepared and stimulated with Kb-tetramers. Representative FACS plots are shown in a. Data compiled from several experiments. PMA and ionomycin (P+I) treatment was used as a positive control for stimulation to obtain maximal ERK phosphorylation. b, Same p-ERK data as in Fig. 2b, presented as responses to the different ligands overlaid within the same mouse genotype.
Extended Data Figure 7 Three-dimensional reconstruction images of negative-selection-like ERK signalling in
Themis−/−thymocytes in response to positive-selecting ligands. a–c, Themis+/+ or Themis−/− OT-I Tap1−/− pre-selection thymocytes were stimulated with Kb–OVA (a), Kb–G4 (b) or Kb–VSV (c). Localization of p-ERK was determined by specific staining with anti-p-ERK antibody. Nuclei were counterstained with Hoechst 33342, and plasma membrane was labelled with Cy3.5. Top panels represent each separate channel of a single centred plane. Bottom panels represent two different 3D reconstructions of 30 planes (step = 0.2 μm), surface rendering (left) and volume rendering (right). d, Fluorescence line profile analysis of representative cells in a, b and c, green line (p-ERK) blue line (Hoechst) and red line (Cy3.5).
Phosphorylation of SLP-76 was determined in cell lysates. Representative of 2 experiments.
Themis−/−double-positive cells. a, Phosphorylation of SHP1 was determined in cell lysates. In this experiment, cell lysates from the same time point after stimulation (0.5, 2 and 5 min, respectively) were grouped together and directly compared on the same gel. b, Quantitation: the intensity ratio of p-SHP1 to total ERK was determined by LiCor Odyssey software.
HEK293 cells were transiently transfected with the indicated expression vectors. a, b, Pull-down assays using Streptactin beads were performed two days after transfection and the precipitate subjected to SDS–PAGE and immunoblotting with anti-haemagglutinin (HA) tag (a) and anti-SHP2 (b) antibodies, respectively. Representative of 3 (a) and 2 (b) similar experiments, respectively. Note that HEK293 cells express SHP2 but little SHP1. Cells originally from ATCC, tested negative for mycoplasma within previous 3 months, not short tandem repeat profiled. Constitutive binding of GRB2 to THEMIS has been reported previously.
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Fu, G., Casas, J., Rigaud, S. et al. Themis sets the signal threshold for positive and negative selection in T-cell development. Nature 504, 441–445 (2013). https://doi.org/10.1038/nature12718
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