Themis sets the signal threshold for positive and negative selection in T-cell development

Abstract

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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Figure 1: Differentially regulated Ca2+ flux in Themis−/− thymocytes.
Figure 2: Themis-deficiency allows low-affinity ligands to elicit negative selection-like characteristics of ERK activation.
Figure 3: Proximal TCR signalling in Themis-deficient thymocytes responding to positive selecting ligands.
Figure 4: Themis-deficient mice show enhanced negative selection that can be rescued by Bim deficiency, but normal agonist selection.

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Acknowledgements

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.

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G.F., J.C., S.R., V.R., F.L., J.B., K.S. and J.A.H.H. performed experiments and analysed data; W.P. and O.A. performed initial experiments on Themis–SHP1/2 interaction, G.F. and N.R.J.G. designed the project with help and insight from W.P., O.A., H.C. and K.S.; G.F. and N.R.J.G. wrote the manuscript with help from the other authors.

Corresponding author

Correspondence to Nicholas R. J. Gascoigne.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Ca2+ flux in Themis-deficient thymocytes stimulated by TCR crosslinking.

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.

Extended Data Figure 3 Comparison of Ca2+ flux induced by different methods.

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.

Extended Data Figure 4 Quantification of NFATC2 nuclear translocation.

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.

Extended Data Figure 5 Biochemical analyses of ERK phosphorylation in Themis-deficient thymocytes.

ERK1 and 2 phosphorylation of indicated thymocytes in response to different stimuli, normalized to VAV. Representative of 4 experiments.

Extended Data Figure 6 Flow-cytometric analysis of ERK phosphorylation.

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. ac, 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).

Extended Data Figure 8 SLP-76 phosphorylation is not affected in Themis-deficient thymocytes.

Phosphorylation of SLP-76 was determined in cell lysates. Representative of 2 experiments.

Extended Data Figure 9 Decreased SHP1 phosphorylation in

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.

Extended Data Figure 10 THEMIS forms complexes with SHP1 and SHP2.

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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