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Quantitative proteomics analysis of signalosome dynamics in primary T cells identifies the surface receptor CD6 as a Lat adaptor–independent TCR signaling hub

Nature Immunology volume 15, pages 384–392 (2014)Cite this article

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Abstract

T cell antigen receptor (TCR)-mediated activation of T cells requires the interaction of dozens of proteins. Here we used quantitative mass spectrometry and activated primary CD4+ T cells from mice in which a tag for affinity purification was knocked into several genes to determine the composition and dynamics of multiprotein complexes that formed around the kinase Zap70 and the adaptors Lat and SLP-76. Most of the 112 high-confidence time-resolved protein interactions we observed were previously unknown. The surface receptor CD6 was able to initiate its own signaling pathway by recruiting SLP-76 and the guanine nucleotide–exchange factor Vav1 regardless of the presence of Lat. Our findings provide a more complete model of TCR signaling in which CD6 constitutes a signaling hub that contributes to the diversification of TCR signaling.

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Figure 1: Normal development and function of T cells isolated from Slp76OST mice.
Figure 2: Purification of SLP-76-associated protein complexes from Slp76OST CD4+ T cells.
Figure 3: Protein-protein interactions in the Zap70–Lat–SLP-76 interaction network of mouse CD4+ T cells.
Figure 4: Kinetics of the binding of proteins to Zap70, Lat and SLP-76.
Figure 5: CD6 associates with SLP-76 after engagement of the TCR.
Figure 6: SLP-76 associates with CD6 in a Lat-independent manner.
Figure 7: Limited transcriptional changes occur when the TCR is engaged in the absence of Lat.

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References

  1. Balagopalan, L., Coussens, N.P., Sherman, E., Samelson, L.E. & Sommers, C.L. The LAT story: a tale of cooperativity, coordination, and choreography. Cold Spring Harb. Perspect. Biol. 2, a005512 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  2. Roncagalli, R., Mingueneau, M., Gregoire, C., Malissen, M. & Malissen, B. LAT signaling pathology: an “autoimmune” condition without T cell self-reactivity. Trends Immunol. 31, 253–259 (2010).

    Article  CAS  PubMed  Google Scholar 

  3. Sommers, C.L. et al. A LAT mutation that inhibits T cell development yet induces lymphoproliferation. Science 296, 2040–2043 (2002).

    Article  CAS  PubMed  Google Scholar 

  4. Mingueneau, M. et al. Loss of the LAT adaptor converts antigen-responsive T cells into pathogenic effectors that function independently of the T cell receptor. Immunity 31, 197–208 (2009).

    Article  CAS  PubMed  Google Scholar 

  5. Chuck, M.I., Zhu, M., Shen, S. & Zhang, W. The role of the LAT-PLC-γ1 interaction in T regulatory cell function. J. Immunol. 184, 2476–2486 (2010).

    Article  CAS  PubMed  Google Scholar 

  6. Ou-Yang, C.W. et al. Role of LAT in the granule-mediated cytotoxicity of CD8 T cells. Mol. Cell. Biol. 32, 2674–2684 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Zheng, Y. et al. Temporal regulation of EGF signalling networks by the scaffold protein Shc1. Nature 499, 166–171 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Glatter, T., Wepf, A., Aebersold, R. & Gstaiger, M. An integrated workflow for charting the human interaction proteome: insights into the PP2A system. Mol. Syst. Biol. 5, 237 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. Varjosalo, M. et al. Interlaboratory reproducibility of large-scale human protein-complex analysis by standardized AP-MS. Nat. Methods 10, 307–314 (2013).

    Article  CAS  PubMed  Google Scholar 

  10. Mayya, V. et al. Quantitative phosphoproteomic analysis of T cell receptor signaling reveals system-wide modulation of protein-protein interactions. Sci. Signal. 2, ra46 (2009).

    Article  PubMed  Google Scholar 

  11. Mbonye, U.R. et al. Phosphorylation of CDK9 at Ser175 enhances HIV transcription and is a marker of activated P-TEFb in CD4+ T lymphocytes. PLoS Pathog. 9, e1003338 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Astoul, E., Edmunds, C., Cantrell, D.A. & Ward, S.G. PI 3-K and T-cell activation: limitations of T-leukemic cell lines as signaling models. Trends Immunol. 22, 490–496 (2001).

    Article  CAS  PubMed  Google Scholar 

  13. Junttila, M.R., Saarinen, S., Schmidt, T., Kast, J. & Westermarck, J. Single-step Strep-tag purification for the isolation and identification of protein complexes from mammalian cells. Proteomics 5, 1199–1203 (2005).

    Article  CAS  PubMed  Google Scholar 

  14. Secrist, J.P., Burns, L.A., Karnitz, L., Koretzky, G.A. & Abraham, R.T. Stimulatory effects of the protein tyrosine phosphatase inhibitor, pervanadate, on T-cell activation events. J. Biol. Chem. 268, 5886–5893 (1993).

    Article  CAS  PubMed  Google Scholar 

  15. Carvalho, P.C., Hewel, J., Barbosa, V.C. & Yates, J.R. III. Identifying differences in protein expression levels by spectral counting and feature selection. Genet. Mol. Res. 7, 342–356 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Harkiolaki, M. et al. Structural basis for SH3 domain-mediated high-affinity binding between Mona/Gads and SLP-76. EMBO J. 22, 2571–2582 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Di Bartolo, V. et al. A novel pathway down-modulating T cell activation involves HPK-1-dependent recruitment of 14–3-3 proteins on SLP-76. J. Exp. Med. 204, 681–691 (2007).

    Article  CAS  PubMed  Google Scholar 

  18. Lasserre, R. et al. Release of serine/threonine-phosphorylated adaptors from signaling microclusters down-regulates T cell activation. J. Cell Biol. 195, 839–853 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Evans, R., Lellouch, A.C., Svensson, L., McDowall, A. & Hogg, N. The integrin LFA-1 signals through ZAP-70 to regulate expression of high-affinity LFA-1 on T lymphocytes. Blood 117, 3331–3342 (2011).

    Article  CAS  PubMed  Google Scholar 

  20. Salmerón, A. et al. Transferrin receptor induces tyrosine phosphorylation in T cells and is physically associated with the TCR ζ-chain. J. Immunol. 154, 1675–1683 (1995).

    PubMed  Google Scholar 

  21. Kobarg, J., Whitney, G.S., Palmer, D., Aruffo, A. & Bowen, M.A. Analysis of the tyrosine phosphorylation and calcium fluxing of human CD6 isoforms with different cytoplasmatic domains. Eur. J. Immunol. 27, 2971–2980 (1997).

    Article  CAS  PubMed  Google Scholar 

  22. Wee, S. et al. Tyrosine phosphorylation of CD6 by stimulation of CD3: augmentation by the CD4 and CD2 coreceptors. J. Exp. Med. 177, 219–223 (1993).

    Article  CAS  PubMed  Google Scholar 

  23. Hassan, N.J. et al. CD6 regulates T-cell responses through activation-dependent recruitment of the positive regulator SLP-76. Mol. Cell. Biol. 26, 6727–6738 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Williams, B.L. et al. Genetic evidence for differential coupling of Syk family kinases to the T-cell receptor: reconstitution studies in a ZAP-70-deficient Jurkat T-cell line. Mol. Cell. Biol. 18, 1388–1399 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Finco, T.S., Kadlecek, T., Zhang, W., Samelson, L.E. & Weiss, A. LAT is required for TCR-mediated activation of PLCγ1 and the Ras pathway. Immunity 9, 617–626 (1998).

    Article  CAS  PubMed  Google Scholar 

  26. Letourneur, F. & Klausner, R.D. Activation of T cells by a tyrosine kinase activation domain in the cytoplasmic tail of CD3ɛ. Science 255, 79–82 (1992).

    Article  CAS  PubMed  Google Scholar 

  27. Wegener, A.M. et al. The T cell receptor/CD3 complex is composed of at least two autonomous transduction modules. Cell 68, 83–95 (1992).

    Article  CAS  PubMed  Google Scholar 

  28. Irving, B.A., Chan, A.C. & Weiss, A. Functional characterization of a signal transducing motif present in the T cell antigen receptor ζ chain. J. Exp. Med. 177, 1093–1103 (1993).

    Article  CAS  PubMed  Google Scholar 

  29. Gimferrer, I. et al. Relevance of CD6-mediated interactions in T cell activation and proliferation. J. Immunol. 173, 2262–2270 (2004).

    Article  CAS  PubMed  Google Scholar 

  30. Zimmerman, A.W. et al. Long-term engagement of CD6 and ALCAM is essential for T-cell proliferation induced by dendritic cells. Blood 107, 3212–3220 (2006).

    Article  CAS  PubMed  Google Scholar 

  31. Oliveira, M.I. et al. CD6 attenuates early and late signaling events, setting thresholds for T-cell activation. Eur. J. Immunol. 42, 195–205 (2012).

    Article  CAS  PubMed  Google Scholar 

  32. Wakamatsu, E., Mathis, D. & Benoist, C. Convergent and divergent effects of costimulatory molecules in conventional and regulatory CD4+ T cells. Proc. Natl. Acad. Sci. USA 110, 1023–1028 (2013).

    Article  CAS  PubMed  Google Scholar 

  33. Diehn, M. et al. Genomic expression programs and the integration of the CD28 costimulatory signal in T cell activation. Proc. Natl. Acad. Sci. USA 99, 11796–11801 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Ibáñez, A. et al. Mitogen-activated protein kinase pathway activation by the CD6 lymphocyte surface receptor. J. Immunol. 177, 1152–1159 (2006).

    Article  PubMed  Google Scholar 

  35. Ménasché, G. et al. RIAM links the ADAP/SKAP-55 signaling module to Rap1, facilitating T-cell-receptor-mediated integrin activation. Mol. Cell. Biol. 27, 4070–4081 (2007).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  36. Burdette, D.L. & Vance, R.E. STING and the innate immune response to nucleic acids in the cytosol. Nat. Immunol. 14, 19–26 (2013).

    Article  CAS  PubMed  Google Scholar 

  37. Hassan, N.J., Barclay, A.N. & Brown, M.H. Frontline: Optimal T cell activation requires the engagement of CD6 and CD166. Eur. J. Immunol. 34, 930–940 (2004).

    Article  CAS  PubMed  Google Scholar 

  38. De Jager, P.L. et al. Meta-analysis of genome scans and replication identify CD6, IRF8 and TNFRSF1A as new multiple sclerosis susceptibility loci. Nat. Genet. 41, 776–782 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Kofler, D.M., Severson, C.A., Mousissian, N., De Jager, P.L. & Hafler, D.A. The CD6 multiple sclerosis susceptibility allele is associated with alterations in CD4+ T cell proliferation. J. Immunol. 187, 3286–3291 (2011).

    Article  CAS  PubMed  Google Scholar 

  40. Grégoire, C. et al. Deletion of the LIME adaptor protein minimally affects T and B cell development and function. Eur. J. Immunol. 37, 3259–3269 (2007).

    Article  PubMed  CAS  Google Scholar 

  41. May, R.M. et al. Murine natural killer immunoreceptors use distinct proximal signaling complexes to direct cell function. Blood 121, 3135–3146 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Mingueneau, M. et al. The proline-rich sequence of CD3ɛ controls T cell antigen receptor expression on and signaling potency in preselection CD4+CD8+ thymocytes. Nat. Immunol. 9, 522–532 (2008).

    Article  CAS  PubMed  Google Scholar 

  43. Zhang, Y., Muyrers, J.P., Testa, G. & Stewart, A.F. DNA cloning by homologous recombination in Escherichia coli. Nat. Biotechnol. 18, 1314–1317 (2000).

    Article  CAS  PubMed  Google Scholar 

  44. Köntgen, F., Suss, G., Stewart, C., Steinmetz, M. & Bluethmann, H. Targeted disruption of the MHC class II Aa gene in C57BL/6 mice. Int. Immunol. 5, 957–964 (1993).

    Article  PubMed  Google Scholar 

  45. Pettitt, S.J. et al. Agouti C57BL/6N embryonic stem cells for mouse genetic resources. Nat. Methods 6, 493–495 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Malissen, M. et al. Altered T cell development in mice with a targeted mutation of the CD3-ɛ gene. EMBO J. 14, 4641–4653 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Craig, R. & Beavis, R.C. TANDEM: matching proteins with tandem mass spectra. Bioinformatics 20, 1466–1467 (2004).

    Article  CAS  PubMed  Google Scholar 

  48. Deutsch, E.W. et al. A guided tour of the Trans-Proteomic Pipeline. Proteomics 10, 1150–1159 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Fermin, D., Basrur, V., Yocum, A.K. & Nesvizhskii, A.I. Abacus: a computational tool for extracting and pre-processing spectral count data for label-free quantitative proteomic analysis. Proteomics 11, 1340–1345 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Donnadieu, E. et al. Reconstitution of CD3ζ coupling to calcium mobilization via genetic complementation. J. Biol. Chem. 269, 32828–32834 (1994).

    Article  CAS  PubMed  Google Scholar 

  51. Irizarry, R.A. et al. Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4, 249–264 (2003).

    Article  PubMed  Google Scholar 

  52. Lee, H.J. et al. Direct transfer of α-synuclein from neuron to astroglia causes inflammatory responses in synucleinopathies. J. Biol. Chem. 285, 9262–9272 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Smyth, G.K. Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat. Appl. Genet. Mol. Biol. 3, 1–29 (2004).

    Article  Google Scholar 

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Acknowledgements

We thank A. Weiss (University of California, San Francisco) for the Jurkat T cell lines E6.1, P116 and JCaM2.5; members of the SYBILLA consortium; O. Acuto, M. Brown, K. Reginald and A.-M. Schmitt-Verhulst for discussions; and E. Caccomo, A.-M. Mura, F. Danjan and M. Richelme and the Finnish Microarray and Sequencing Centre at Turku for technical assistance. Supported by Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, GIS-IBISA, PHENOMIN, ANR (ADAPT project to M.M. and iSa project to B.M.), the European Communities (SYBILLA project to B.M., M.G., H.L. and R.L., and MASTERSWITCH project to B.M.), the Academy of Finland Centre for Excellence in Molecular Systems Immunology and Physiology Research, the Sigrid Jusélius Foundation (R.L. and H.L.), the European Union 7th Framework project PROSPECTS, the European Research Council ('Proteomics v3.0' to R.A., and 'Integrate' to B.M.) and the China Scholarship Council (Y.L.).

Author information

Author notes

  1. Romain Roncagalli and Simon Hauri: These authors contributed equally to this work.

Authors and Affiliations

  1. Centre d'Immunologie de Marseille-Luminy, UM2 Aix-Marseille Université, Marseille, France

    Romain Roncagalli, Yinming Liang, Rachel Joly, Aurélie Malzac, Marie Malissen & Bernard Malissen

  2. INSERM U1104, Marseille, France

    Romain Roncagalli, Yinming Liang, Rachel Joly, Aurélie Malzac, Marie Malissen & Bernard Malissen

  3. Centre National de la Recherche Scientifique UMR7280, Marseille, France

    Romain Roncagalli, Yinming Liang, Rachel Joly, Aurélie Malzac, Marie Malissen & Bernard Malissen

  4. Department of Biology, Institute of Molecular Systems Biology, Eidgenossische Technische Hochschule Zurich, Zurich, Switzerland

    Simon Hauri, Ruedi Aebersold & Matthias Gstaiger

  5. Competence Center for Systems Physiology and Metabolic Diseases, Eidgenossische Technische Hochschule Zurich, Zurich, Switzerland

    Simon Hauri, Ruedi Aebersold & Matthias Gstaiger

  6. Centre d'Immunophénomique, UM2 Aix-Marseille Université, Marseille, France

    Fréderic Fiore, Amandine Sansoni & Bernard Malissen

  7. INSERM US012, Marseille, France

    Fréderic Fiore, Amandine Sansoni & Bernard Malissen

  8. CNRS UMS3367, Marseille, France

    Fréderic Fiore, Amandine Sansoni & Bernard Malissen

  9. Turku Centre for Biotechnology, University of Turku and Abo Akademi University, Turku, Finland

    Zhi Chen, Kartiek Kanduri, Harri Lähdesmäki & Riitta Lahesmaa

  10. Department of Information and Computer Science, Aalto University, Aalto, Finland

    Harri Lähdesmäki

  11. Division of Molecular Immunology, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan

    Sho Yamasaki

  12. RIKEN Center for Integrative Medical Sciences, Yokohama, Japan

    Takashi Saito

  13. Faculty of Science, University of Zurich, Zurich, Switzerland

    Ruedi Aebersold

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  1. Romain Roncagalli
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Contributions

R.A., M.G. and B.M. conceived of the project; R.R. and S.H. designed and did the experiments for Figures 3 and 4 and Supplementary Figures 5 and 8; R.R., A.S. and R.J. did the experiments for Figures 1, 2, 5 and 6 and Supplementary Figures 2, 3 and 6; F.F. and B.M. designed the experiments for Supplementary Figure 1; S.Y. and T.S. developed the maT-Cre mice; Y.L., Z.C., K.K., A.M., H.L., R.L. and M.M. designed and did the experiments for Figure 7; and R.R., S.H., M.G. and B.M. wrote the manuscript.

Corresponding authors

Correspondence to Matthias Gstaiger or Bernard Malissen.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Generation and validation of knock-in mice expressing endogenous SLP-76, Zap70 and Lat proteins tagged with a One-STrEP-tag (OST) sequence.

(a) Strategy used to produce Slp76OST knock-in mice. Left panel: (1) Partial restriction map of the Slp76 (Lcp2) gene. Exons are shown as filled black boxes and numbered. The stop codon (STOP) is indicated. The 5′- and 3′-single copy probes used to verify proper homologous recombination events by Southern blot analysis are shown in red. B: BamHI, H: HindIII. (2) Targeting vector used for the introduction of the OST sequence in the Slp76 gene. A OST-(Stop)2-loxP-tACE-CRE-PGK-gb2-neor-loxP cassette (OST-NEO) was introduced at the 3′ end of the Slp76 coding sequence (see Online Methods). The tACE-CRE-PGK-gb2-neor sequence is bracketed by loxP sites (triangles) and directs its own excision as it passes through the male germline. TK: thymidine kinase expression cassette. (3) Structure of the targeted Slp76OST allele following homologous recombination. (4) Structure of the Slp76OST allele following homologous recombination and Cre-mediated self-excision of the tACE-CRE-PGK-gb2-neor cassette. Right panel: Southern blot analysis of two appropriately recombined ES clones. ES cell DNA was digested with HindIII (5′ probe) or BamHI (3′ probe) and hybridized to the 5′ or 3′ single-copy probe shown in line 1 of the left panel. (b) Expression of the SLP-76 and SLP7-6-OST proteins in thymocytes and splenocytes from wild-type (WT) and Slp76OST mice was detected using an anti-SLP-76. Blotting with an antibody against Lat served as a loading control. (c) Strategy used to produce Zap70OST knock-in mice. Left panel: (1) Partial restriction map of the Zap70 gene. Exons are shown as filled black boxes and numbered. The stop codon (STOP) is indicated. The 5′- and 3′-single copy probes used to verify proper homologous recombination events by Southern blot analysis are shown in red. B: BamHI, X: XbaI. (2) Targeting vector used for the introduction of the OST in the Zap70 gene as described in (a). B: BamHI, X: XbaI. (3) Structure of the targeted Zap70 OST allele following homologous recombination. (4) Structure of the Zap70OST allele following homologous recombination and Cre-mediated self-excision of the tACE-CRE-PGK-gb2-neor cassette. Right panel: Southern blot analysis of four appropriately recombined ES clones. ES cell DNA was digested by XbaI (5′ probe) or BamHI (3′ probe) and hybridized to the 5′ or 3′single-copy probe shown in line 1 of the left panel. (d) Expression of Zap70 and Zap70-OST proteins in thymocytes and splenocytes from wild-type (WT) and Zap70OST mice was detected using an anti-Zap70. Blotting with an antibody against Lat served as a loading control. (e) Strategy used to produce LatOST knock-in mice. Left panel: (1) Partial restriction map of the Lat gene. Exons are shown as filled black boxes and numbered. The initiation (ATG) and stop (STOP) codons are indicated. The 3′-single copy probe used to verify proper homologous recombination events by Southern blot analysis is shown in red. X: XbaI. (2) Targeting vector used for the introduction of the OST in the Lat gene as described in (a). X: XbaI. (3) Structure of the targeted Lat OST allele following homologous recombination. The pair of PCR primers (red arrows) used to verify proper homologous recombination events at the 5′ end is shown (4) Structure of the Lat OST allele following homologous recombination and Cre-mediated self-excision of the tACE-CRE-PGK-gb2-neor cassette. Right panel: Southern blot analysis of three appropriately recombined ES clones. ES cell DNA was digested by XbaI and hybridized to the 5′ single-copy probe shown in line 1 of left panel. PCR analysis of tail DNA of the three ES clones using the pair of primers shown in line 4 of left panel. (f) Expression of Lat and Lat-OST proteins in thymocytes from wild-type (WT) and LatOST mice was detected using an anti-Lat. To estimate the levels reduction in Lat-OST as compared to Lat, increasing amounts (1X, 2X and 4X) of LatOST lysates were analyzed. Blotting with an antibody against Zap70 served as a loading control.

Supplementary Figure 2 Normal development and function of T cells isolated from knock-in mice homozygous for the Zap70OST allele.

(a) Flow cytometry analysis of thymus and spleen. Wild-type (WT) and Zap70OST thymocytes were analyzed for expression of CD4 and CD8 (left) and TCRαβ and TCRγδ (right). Expression of CD4 and CD8 (left) and TCRαβ and TCRγδ (right) by wild-type (WT) and Zap70OST thymocytes. Numbers adjacent to outlined areas indicate percent double-positive cells (top right), CD4+ single-positive cells (top left), CD8+ single-positive cells (bottom right) and double negative CD4−CD8− cells (bottom left). Wild-type and Zap70OST splenocytes were analyzed for expression of CD5 and CD19. Numbers adjacent to outlined areas indicate percent B cells (top left) and T cells (bottom right). Expression of CD4 and CD8 and of TCRαβ and TCRγδ by T cells (identified as CD19−CD5+ cells). Numbers adjacent to outlined areas indicate percent CD4+ and CD8+ cells (middle) and TCRαβ and TCRγδd cells (right). Data are representative of at least three experiments with two mice per genotype. (b) Cellularity of thymus, pooled axillary, brachial, inguinal and mesenteric lymph nodes (LN) and spleen of wild-type and Zap70OST mice. Data are expressed as mean value ±SEM (n=6). (c) CFSE dilution by wild-type, and Zap70OST CD4+ T cells activated for 72 h with plate-bound anti-CD3 (3 μg/ml) in the presence or absence (key) of soluble anti-CD28 (1 μg/ml). Black lines, CFSE dilution at initiation of culture. (d) IL-2 in supernatants of wild-type, and Zap70OST CD4+ T cells activated for 72 h as in (c). (e) Changes in intracellular calcium in wild-type, and Zap70OST CD4+ T cells stimulated with biotinylated anti-CD3 and avidin. Arrow corresponds to the time of addition of avidin. Data are representative of at least two experiments. Wild-type and Zap70OST CD8+ T cells were analyzed in parallel and showed similar changes in intracellular calcium (data not shown).

Supplementary Figure 3 Normal development and function of T cells isolated from knock-in mice homozygous for the LATOST allele.

(a) Flow cytometry analysis of thymus and spleen. Wild-type (WT) and LatOST thymocytes were analyzed for expression of CD4 and CD8 (left) and TCRαβ and TCRγδ (right). Numbers adjacent to outlined areas indicate percent double-positive cells (top right), CD4+ single-positive cells (top left), CD8+ single-positive cells (bottom right) and double negative CD4−CD8− cells (bottom left). Wild-type and LatOST splenocytes were analyzed for expression of CD5. Numbers adjacent to outlined areas indicate percent B cells (top left) and T cells (bottom right). Expression of CD4 and CD8 and of TCRαβ and TCRγδ by T cells (identified as B220−CD5+ cells). Numbers adjacent to outlined areas indicate percent CD4+ and CD8+ cells (middle) and TCRαβ and TCRγδ cells (right). Data are representative of at least three experiments with two mice per genotype. (b) Cellularity of thymus, pooled axillary, brachial, inguinal and mesenteric lymph nodes (LN) and spleen of wild-type and LatOST mice. Data are expressed as mean value ±SEM (n=6). (c) CFSE dilution by wild-type, and LatOST CD4+ T cells activated for 72 h with plate-bound anti-CD3 (3 μg/ml) in the presence or absence (key) of soluble anti-CD28 (1 μg/ml). Black lines, CFSE dilution at initiation of culture. (d) IL-2 in supernatants of wild-type, and LatOST CD4+ T cells activated for 72 h as in (c). (e) Changes in intracellular calcium in wild-type, and LatOST CD4+ T cells stimulated with biotinylated anti-CD3 and avidin. Arrow corresponds to the time of addition of avidin. Data are representative of at least two experiments. Wild-type and LatOST CD8+ T cells were analyzed in parallel and showed similar changes in intracellular calcium (data not shown).

Supplementary Figure 4 Flow chart of quantitative mass spectrometry of primary CD4+ T cells.

(a) Schematic overview of affinity purification and mass-spectrometry (AP-MS) analysis of primary mouse CD4+ T cells isolated from knock-in mice expressing endogenous proteins belonging to the TCR signaling pathway and tagged with a One-STrEP-tag (OST). (b) Representation of dynamic changes in the indicated THMS1 peptide LTLLSLAEER during pervanadate stimulation by label-free quantification of the precursor ion intensity.

Supplementary Figure 5 The Zap70−Lat−SLP-76 network shows enrichment for proteins with domains involved in protein-protein and in protein-phospholipid interactions.

(a) Protein domain classification1 is showed in the right margin. The graph shows the distribution of the specified molecular domains among the proteins of the Zap70-Lat-SLP-76 network. Statistical validation of the enrichment for Src-homology 2 (SH2) and 3 (SH3) domains and Pleckstrin Homology (PH) domains is shown below the graph. (b) Representation of Protein-Protein Interactions (PPIs) identified in this study and in public PPI databases2. The numbers of interactions corresponding to each of the specified category (see key) are shown. The new interactions identified in this study are displayed in blue. Previously described interactions not identified in this study are categorized on the basis of their occurrence (green: one occurrence; purple: more than one occurrence). Shared interactions between the PINA database and this study are shown in red.

Supplementary Figure 6 CD25 and CD6 expression in Jurkat E6.1 T cells and their Zap70-deficient (P116) and Lat-deficient (JCaM2.5) variants before or after transfection with CD25ζ and human CD6 constructs.

(a) Expression of the CD3ɛ chains on the specified cells (see key). An isotype control staining is also shown (control). (b) CD25 and CD6 expression on the specified cells. An isotype control staining is also shown (control). (c) E6.1 and E6.1 E6.1-CD25ζ-CD6 cells were left untreated (-) of stimulated (+) with anti-CD3 or anti-CD25 for 2 min at 37°C. Equivalent amounts of lysates were analyzed by immunoblot with antibody specific for phosphotyrosine (Anti-P-Tyr). An anti-SLP-76 was used as a loading control.

Supplementary Figure 7 Early TCR signals proceed via two distinct signal nucleation platforms involving Lat and the surface receptor CD6.

(a) Right part. Once bound to their antigenic ligand, TCR expressed on the surface of T cells recruit the protein tyrosine kinase Lck and Zap70 that in turn phosphorylates the tyrosine residues (red dots) found in Lat molecules. As a result, phosphorylated Lat molecules recruit the adaptors SLP-76-GADS and the assembly of a Lat-SLP-76 signalosome ensues. Note that SLP-76 constitutively associates with the GADS adaptor and uses the SH2 domain of GADS to dock onto phosphorylated Lat molecules. Left part. The CD6 surface receptor is recruited to the immunological synapse via interaction with CD166 on antigen-presenting cells. The docking of the SLP-76-GADS complex to phosphorylated CD6 occurred through the SLP-76 SH2 domain3. In contrast to CD6, Lat is palmitoylated and associated with lipid rafts. Accordingly, the Lat- and CD6-based signalosomes may be located within distinct plasma membrane lipidic environments and contribute non-redundant signals. (b) In the absence of Lat, the TCR initiates the formation of an incomplete CD6-SLP-76 signalosome that is capable of inducing a number of post-translational events that have limited transcriptional consequences but result for instance in the formation of TCR-triggered T cell-APC conjugates.

Supplementary Figure 8 Combined Cytoscape map of the Zap70−Lat−SLP-76 network based on public human and mouse protein-protein interaction (PPI) data and on PPIs observed in this study.

The overall organization of the map is as shown in Fig. 3 and protein-protein interactions were visualized using Cystoscape 2.8.3. Colors and shapes of nodes indicate the type of identified proteins according to their domains and putative functions (key shown in the top right hand corner). Edge line colors define the PPI that are proper to the present study, the PPI that are found in public database, and the PPI that are found in both the present study and public database (shared interactions). Pink boxes highlight the subsets of proteins that interact with Zap70, Lat or SLP-76 and have not been previously identified in public databases. Each of the proteins is denoted by its Uniprot symbol (see Supplementary Table 1) except LCP2 that is called SLP-76.

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Supplementary Figures 1–8 (PDF 2432 kb)

Supplementary Table 1

List of the proteins associated with Zap70, Lat and SLP-76 in resting and pervanadate-activated CD4+ T cells (Figure 3) and with SLP-76 in anti-CD3 and anti-CD4 activated CD4+ T cells (Figure 2d) and identified in the present study by affinity purification and mass-spectrometry. Each of the proteins is denoted by its Uniprot symbol (http://www.uniprot.org) except LCP2 that is called SLP-76. (XLSX 633 kb)

Supplementary Table 2

List of the protein-protein interactions identified in the present study and the PINA public PPI database. Direct interactions with the bait and inter-prey interactions are listed. When available, the mouse or human interacting proteins are characterized by their Uniprot AC number. References (PubMed; http://www.ncbi.nlm.nih.gov/pubmed) and methods of analysis are provided for each specified interaction. (XLSX 35 kb)

Supplementary Table 3

List of genes differentially expressed in Lat+CD4+ T cells activated for 4 hours with anti-CD3 and anti-CD28 antibodies versus Lat+CD4+ T cells kept for 4 hours without stimuli and in Lat–CD4+ T cells activated for 4 hours with anti-CD3 and anti-CD28 antibodies versus Lat–CD4+ T cells kept for 4 hours without stimuli. Data were normalized and differentially expressed genes were identified based on adj.p > 0.05 and logFC >1 or <-1. (XLSX 391 kb)

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Roncagalli, R., Hauri, S., Fiore, F. et al. Quantitative proteomics analysis of signalosome dynamics in primary T cells identifies the surface receptor CD6 as a Lat adaptor–independent TCR signaling hub. Nat Immunol 15, 384–392 (2014). https://doi.org/10.1038/ni.2843

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