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Newly generated T cell receptor microclusters initiate and sustain T cell activation by recruitment of Zap70 and SLP-76

Nature Immunology volume 6, pages 1253–1262 (2005)Cite this article

Abstract

T cell receptor (TCR) activation and signaling precede immunological synapse formation and are sustained for hours after initiation. However, the precise physical sites of the initial and sustained TCR signaling are not definitively known. We report here that T cell activation was initiated and sustained in TCR-containing microclusters generated at the initial contact sites and the periphery of the mature immunological synapse. Microclusters containing TCRs, the tyrosine kinase Zap70 and the adaptor molecule SLP-76 were continuously generated at the periphery. TCR microclusters migrated toward the central supramolecular cluster, whereas Zap70 and SLP-76 dissociated from these microclusters before the microclusters coalesced with the TCR-rich central supramolecular cluster. Tyrosine phosphorylation and calcium influx were induced as microclusters formed at the initial contact sites. Inhibition of signaling prevented recruitment of Zap70 into the microclusters. These results indicated that TCR-rich microclusters initiate and sustain TCR signaling.

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Figure 1: Generation of microclusters of CD3ζ, Zap70 and SLP-76 throughout the cell-bilayer contact area in a spatio-temporal way.
Figure 2: Recruitment of Zap70 and SLP-76 to CD3 microclusters immediately after cell-bilayer contact.
Figure 3: Phosphorylated Zap70 and tyrosine-phosphorylated proteins are localized only in CD3ζ microclusters at the periphery of immunological synapse.
Figure 4: Induction of calcium influx accompanies microcluster formation at the initial contact sites.
Figure 5: Src family kinases regulate Zap70 recruitment to microclusters but not the formation of CD3ζ microclusters.
Figure 6: CD3ζ, Zap70 and SLP-76 microclusters are continuously generated at the periphery after mature immunological synapse formation.
Figure 7: Localization of TCR and Zap70 together in newly generated microclusters at the periphery after mature immunological synapse formation.

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Acknowledgements

We thank R. Varma, G. Campi and T. Starr for instructions on the preparation of planar bilayers and for sharing data before publication, and H. Yamaguchi for secretarial assistance. Supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan (T.Y., M.T. and T.S.), New Energy Development Organization (M.T.), and the National Institutes of Health (AI043542 and AI044931 to M.L.D.).

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Authors and Affiliations

  1. Laboratory for Cell Signaling, Tsurumi-ku, Yokohama, 230-0045, Kanagawa, Japan

    Tadashi Yokosuka, Wakana Kobayashi, Akiko Hashimoto-Tane & Takashi Saito

  2. Single-Molecule Immunoimaging, RIKEN Center for Allergy and Immunology, Tsurumi-ku, Yokohama, 230-0045, Kanagawa, Japan

    Kumiko Sakata-Sogawa, Michio Hiroshima & Makio Tokunaga

  3. Structural Biology Center, National Institute of Genetics, Shizuoka, 411-8540, Japan

    Makio Tokunaga

  4. Department of Genetics, The Graduate University for Advanced Studies, Mishima, Shizuoka, 411-8540, Japan

    Makio Tokunaga

  5. Skirball Institute of Biomolecular Medicine and Department of Pathology, Program in Molecular Pathogenesis, New York University School of Medicine, New York, 10021, New York, USA

    Michael L Dustin

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Supplementary information

Supplementary Fig. 1

Statistical analysis of the movement of CD3ζ-MCs at the initiation of T cell-bilayer contact. (PDF 952 kb)

Supplementary Fig. 2

The area and relative fluorescence intensity of CD3ζ-MCs. (PDF 1076 kb)

Supplementary Fig. 3

Generation of CD3ζ-, ZAP-70- and SLP-76-MCs under suboptimal concentrations of the agonistic peptide. (PDF 4662 kb)

Supplementary Fig. 4

CD3ζ-, ZAP-70- and SLP-76-MCs are generated at the initiation of the T cell-APC interaction. (PDF 1926 kb)

Supplementary Fig. 5

Microclusters are formed in the initial interface of the normal T cell – APC interaction. (PDF 708 kb)

Supplementary Fig. 6

ZAP-70 is continuously phosphorylated throughout IS formation. (PDF 1093 kb)

Supplementary Fig. 7

Three-dimensional views of the CD3ζ-expressing cells on bilayers, in the presence or absence of PP2. (PDF 1336 kb)

Supplementary Fig. 8

Quantification of CD3ζ-EGFP proteins within individual MC before and after IS formation. (PDF 841 kb)

Supplementary Fig. 9

Statistical analysis of the movement of CD3ζ-, ZAP-70- and SLP-76-MCs after mature IS formation. (PDF 700 kb)

Supplementary Table 1

AND-Tg T cells expressing ZAP-70-EGFP or SLP-76-EGFP, or both ZAP-70-mRFP and SLP-76-EGFP were plated on a PCC88-104-prepulsed planar bilayer containing I-Ek and ICAM-1. (PDF 276 kb)

Supplementary Video 1

AND-Tg T cells expressing CD3ζ-EGFP were plated on PCC88-104-prepulsed planar bilayers containing non-labeled I-Ek and ICAM-1. Images were collected at video rate (30 frames/sec) by TIRFM, 8-frame averaged, and rebuilt at 1 frame/sec, from time zero to 300 sec. (MOV 2932 kb)

Supplementary Video 2

AND-Tg T cells expressing ZAP-70-EGFP were plated on PCC88-104-prepulsed planar bilayers containing non-labeled I-Ek and ICAM-1. Images were collected at video rate (30 frames/sec) by TIRFM, 8-frame averaged, and rebuilt at 1 frame/sec, from time zero to 300 sec. (MOV 2932 kb)

Supplementary Video 3

AND-Tg T cells expressing SLP-76-EGFP were plated on PCC88-104-prepulsed planar bilayers containing non-labeled I-Ek and ICAM-1. Images were collected at video rate (30 frames/sec) by TIRFM, 8-frame averaged, and rebuilt at 1 frame/sec, from time zero to 300 sec. (MOV 2903 kb)

Supplementary Video 4

AND-Tg T cells expressing CD3ζ-EGFP were plated on PCC88-104-prepulsed planar bilayers containing non-labeled I-Ek and ICAM-1. Images were collected at video rate (30 frames/sec) from 15 min after the cell-bilayer contacts. They were 8-frame averaged and rebuilt at 1 frame/sec for the duration of 90 sec. (MOV 1956 kb)

Supplementary Video 5

AND-Tg T cells expressing ZAP-70-EGFP were plated on PCC88-104-prepulsed planar bilayers containing non-labeled I-Ek and ICAM-1. Images were collected at video rate (30 frames/sec) from 15 min after the cell-bilayer contacts. They were 8-frame averaged and rebuilt at 1 frame/sec for the duration of 90 sec. (MOV 1956 kb)

Supplementary Video 6

AND-Tg T cells expressing SLP-76-EGFP were plated on PCC88-104-prepulsed planar bilayers containing non-labeled I-Ek and ICAM-1. Images were collected at video rate (30 frames/sec) from 15 min after the cell-bilayer contacts. They were 8-frame averaged and rebuilt at 1 frame/sec for the duration of 90 sec. (MOV 1956 kb)

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Yokosuka, T., Sakata-Sogawa, K., Kobayashi, W. et al. Newly generated T cell receptor microclusters initiate and sustain T cell activation by recruitment of Zap70 and SLP-76. Nat Immunol 6, 1253–1262 (2005). https://doi.org/10.1038/ni1272

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