DOI: 10.1126/science.1067710. T Cell Receptor Signaling Precedes Immunological Synapse Formation

T Cell Receptor Signaling Precedes Immunological Synapse Formation

10.1126/science.1067710

Crossref journal-article

American Association for the Advancement of Science (AAAS)

Science (221)

Abstract

The area of contact between a T cell and an antigen-presenting cell (APC) is known as the immunological synapse. Although its exact function is unknown, one model suggests that it allows for T cell receptor (TCR) clustering and for sustained signaling in T cells for many hours. Here we demonstrate that TCR-mediated tyrosine kinase signaling in naı̈ve T cells occurred primarily at the periphery of the synapse and was largely abated before mature immunological synapses had formed. These data suggest that many hours of TCR signaling are not required for T cell activation. These observations challenge current ideas about the role of immunological synapses in T cell activation.

Bibliography

Lee, K.-H., Holdorf, A. D., Dustin, M. L., Chan, A. C., Allen, P. M., & Shaw, A. S. (2002). T Cell Receptor Signaling Precedes Immunological Synapse Formation. Science, 295(5559), 1539â1542.

Authors 6

Kyeong-Hee Lee (first)
Amy D. Holdorf (additional)
Michael L. Dustin (additional)
Andrew C. Chan (additional)
Paul M. Allen (additional)
Andrey S. Shaw (additional)

References 32 Referenced 566

S. K. Bromley et al. Annu. Rev. Immunol. 19 375 (2001). (10.1146/annurev.immunol.19.1.375)
10.1016/S0960-9822(00)00870-8
A. Grakoui et al. Science 285 221 (1999). (10.1126/science.285.5425.221)
10.1038/25764
J. Kaye et al. Nature 341 746 (1989). (10.1038/341746a0)
K.-H. Lee A. Shaw unpublished data.
Naı̈ve splenic T cells were prepared from AND TCR transgenic mice (5). After depletion of plastic-adherent splenocytes for 2 hours at 37°C T cells were purified with antibody to MHC II MACS macro beads (Miltenyi Biotec Auburn CA). Splenic APCs were prepared from B10.BR mice. APCs were enriched for macrophages and dendritic cells by depleting splenocytes of T and B cells with Thy 1.2- and B220-conjugated microbeads. Splenic APCs were loaded with 1 μM moth cytochrome c (residues 88 to 103) at 37°C overnight. T cells and APCs were mixed in a 1:5 ratio conjugated by briefly spinning and subsequently incubated at 37°C in serum-free medium. Costaining with antibodies to macrophage or dendritic markers demonstrated that about 90% of the conjugates formed were with macrophages. At the indicated time points cells were fixed with 4% paraformaldehyde and mounted on poly- l -lysine–coated glass slides. For TCR staining antibody to TCR Vβ 3 (KJ25) was used. For LFA-1 monoclonal antibody to LFA-1 (H155) (25) was used. For the staining of intracellular signaling molecules cell conjugates were permeabilized with 0.05% TX-100 at room temperature for 5 min after fixation. Cells were labeled with rabbit antisera against pY394 Lck and pY319 ZAP70 and biotinylated antibodies against Lck ZAP70 and phosphotyrosine (4G10; Upstate Biotechnology). Donkey antibody to rabbit rat or hamster secondary antibodies conjugated to Cy3 and Cy5 were used (Jackson Immuno Research West Grove PA). For biotinylated antibodies Alexa-488–conjugated streptavidin was used (Molecular Probes Eugene OR). Cells were mounted with ProLong Mounting medium (Molecular Probes). Two- and three-color confocal images and differential interference contrast (DIC) images were acquired with a Zeiss LSM 510. The three-dimensional reconstruction of the T cell–APC contact area was generated with 0.2 μM serial sections of x - y images along the z axis and subsequent analysis with LSM 510 software.
10.1126/science.7509083
A. D. Holdorf K.-H. Lee W. R. Burack P. M. Allen A. S. Shaw Nature Immunol. in press.
L. K. Gauen et al. Mol. Cell. Biol. 14 3729 (1994). (10.1128/MCB.14.6.3729)
B. L. Williams et al. EMBO J. 18 1832 (1999). (10.1093/emboj/18.7.1832)
Supplementary figures are available on Science Online at www.sciencemag.org/cgi/content/full/295/5559/1539/DC1.
10.1073/pnas.88.20.9166
10.1016/0092-8674(94)90080-9
Itoh Y., Hemmer B., Martin R., Germain R. N., J. Immunol. 162, 2073 (1999). (10.4049/jimmunol.162.4.2073) / J. Immunol. by Itoh Y. (1999)
10.1084/jem.185.10.1859
; C. Cenciarelli et al. Science 257 795 (1992); (10.1126/science.1323144)
10.1038/375148a0
10.1016/S1074-7613(00)80461-6
10.1046/j.1440-1711.1999.00877.x
Purified splenic T cells from AND TCR transgenic mice were labeled with 500 nM CFSE (Molecular Probes). The CFSE-labeled T cells were conjugated with splenic APCs from B10.BR mice as described above. At the indicated time points cell conjugates were disrupted by two rounds of complement lysis with antibody to I-E k (14.4.4) and rabbit complement (Cedarlane Lab: 1:50 dilution). T cells were reisolated and recultured with unloaded APCs. After 24 48 and 72 hours T cell division was determined by flow cytometric measurement of CFSE intensity of the T cells.
10.1038/87730
Weiss A., Shields R., Newton M., Manger B., Imboden J., J. Immunol. 138, 2169 (1987); (10.4049/jimmunol.138.7.2169) / J. Immunol. by Weiss A. (1987)
10.1126/science.3259335
10.1016/S0960-9822(01)00165-8
10.1126/science.274.5295.2086
10.1016/S0167-5699(97)80027-8
S. Miyamoto et al. J. Cell Biol. 131 791 (1995). (10.1083/jcb.131.3.791)
A. Hutloff et al. Nature 397 263 (1999) (10.1038/16717)
P. S. Linsley et al. J. Exp. Med. 176 1595 (1992). (10.1084/jem.176.6.1595)
S. Pont et al. J. Immunol. 136 3750 (1986). (10.4049/jimmunol.136.10.3750)
We wish to thank E. Unanue for reading the manuscript and R. Burack and E. Hailman for intellectual support. This work was supported by grants from the NIH.

Dates

Type	When
Created	23 years, 1 month ago (July 27, 2002, 5:35 a.m.)
Deposited	1 year, 7 months ago (Jan. 9, 2024, 4:28 p.m.)
Indexed	1 month, 3 weeks ago (July 11, 2025, 9:19 p.m.)
Issued	23 years, 6 months ago (Feb. 22, 2002)
Published	23 years, 6 months ago (Feb. 22, 2002)
Published Print	23 years, 6 months ago (Feb. 22, 2002)

Funders 0

None

BibTeX

@article{Lee_2002, title={T Cell Receptor Signaling Precedes Immunological Synapse Formation}, volume={295}, ISSN={1095-9203}, url={http://dx.doi.org/10.1126/science.1067710}, DOI={10.1126/science.1067710}, number={5559}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Lee, Kyeong-Hee and Holdorf, Amy D. and Dustin, Michael L. and Chan, Andrew C. and Allen, Paul M. and Shaw, Andrey S.}, year={2002}, month=feb, pages={1539–1542} }

JSON

{
  "indexed": {
    "date-parts": [
      [
        2025,
        7,
        12
      ]
    ],
    "date-time": "2025-07-12T01:19:56Z",
    "timestamp": 1752283196147
  },
  "reference-count": 32,
  "publisher": "American Association for the Advancement of Science (AAAS)",
  "issue": "5559",
  "content-domain": {
    "domain": [],
    "crossmark-restriction": false
  },
  "published-print": {
    "date-parts": [
      [
        2002,
        2,
        22
      ]
    ]
  },
  "abstract": "<jats:p>The area of contact between a T cell and an antigen-presenting cell (APC) is known as the immunological synapse. Although its exact function is unknown, one model suggests that it allows for T cell receptor (TCR) clustering and for sustained signaling in T cells for many hours. Here we demonstrate that TCR-mediated tyrosine kinase signaling in na\u0131\u0308ve T cells occurred primarily at the periphery of the synapse and was largely abated before mature immunological synapses had formed. These data suggest that many hours of TCR signaling are not required for T cell activation. These observations challenge current ideas about the role of immunological synapses in T cell activation.</jats:p>",
  "DOI": "10.1126/science.1067710",
  "type": "journal-article",
  "created": {
    "date-parts": [
      [
        2002,
        7,
        27
      ]
    ],
    "date-time": "2002-07-27T09:35:18Z",
    "timestamp": 1027762518000
  },
  "page": "1539-1542",
  "source": "Crossref",
  "is-referenced-by-count": 566,
  "title": "T Cell Receptor Signaling Precedes Immunological Synapse Formation",
  "prefix": "10.1126",
  "volume": "295",
  "author": [
    {
      "given": "Kyeong-Hee",
      "family": "Lee",
      "sequence": "first",
      "affiliation": [
        {
          "name": "Department of Pathology and Immunology, Washington University School of Medicine, 660 South Euclid, Box 8118, Saint Louis, MO 63110, USA."
        }
      ]
    },
    {
      "given": "Amy D.",
      "family": "Holdorf",
      "sequence": "additional",
      "affiliation": [
        {
          "name": "Department of Pathology and Immunology, Washington University School of Medicine, 660 South Euclid, Box 8118, Saint Louis, MO 63110, USA."
        }
      ]
    },
    {
      "given": "Michael L.",
      "family": "Dustin",
      "sequence": "additional",
      "affiliation": [
        {
          "name": "Program in Molecular Pathogenesis, Skirball Institute of Biomolecular Medicine, New York University, 540 First Avenue, New York, NY 10016, USA."
        }
      ]
    },
    {
      "given": "Andrew C.",
      "family": "Chan",
      "sequence": "additional",
      "affiliation": [
        {
          "name": "Genentech, Department of Immunology, 530 Forbes Boulevard, One DNA Way, South San Francisco, CA 94080, USA."
        }
      ]
    },
    {
      "given": "Paul M.",
      "family": "Allen",
      "sequence": "additional",
      "affiliation": [
        {
          "name": "Department of Pathology and Immunology, Washington University School of Medicine, 660 South Euclid, Box 8118, Saint Louis, MO 63110, USA."
        }
      ]
    },
    {
      "given": "Andrey S.",
      "family": "Shaw",
      "sequence": "additional",
      "affiliation": [
        {
          "name": "Department of Pathology and Immunology, Washington University School of Medicine, 660 South Euclid, Box 8118, Saint Louis, MO 63110, USA."
        }
      ]
    }
  ],
  "member": "221",
  "reference": [
    {
      "key": "e_1_3_1_2_2",
      "doi-asserted-by": "crossref",
      "unstructured": "S. K. Bromley et al. Annu. Rev. Immunol. 19 375 (2001).",
      "DOI": "10.1146/annurev.immunol.19.1.375"
    },
    {
      "key": "e_1_3_1_3_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0960-9822(00)00870-8"
    },
    {
      "key": "e_1_3_1_4_2",
      "doi-asserted-by": "crossref",
      "unstructured": "A. Grakoui et al. Science 285 221 (1999).",
      "DOI": "10.1126/science.285.5425.221"
    },
    {
      "key": "e_1_3_1_5_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/25764"
    },
    {
      "key": "e_1_3_1_6_2",
      "doi-asserted-by": "crossref",
      "unstructured": "J. Kaye et al. Nature 341 746 (1989).",
      "DOI": "10.1038/341746a0"
    },
    {
      "key": "e_1_3_1_7_2",
      "unstructured": "K.-H. Lee A. Shaw unpublished data."
    },
    {
      "key": "e_1_3_1_8_2",
      "unstructured": "Na\u0131\u0308ve splenic T cells were prepared from AND TCR transgenic mice (5). After depletion of plastic-adherent splenocytes for 2 hours at 37\u00b0C T cells were purified with antibody to MHC II MACS macro beads (Miltenyi Biotec Auburn CA). Splenic APCs were prepared from B10.BR mice. APCs were enriched for macrophages and dendritic cells by depleting splenocytes of T and B cells with Thy 1.2- and B220-conjugated microbeads. Splenic APCs were loaded with 1 \u03bcM moth cytochrome c (residues 88 to 103) at 37\u00b0C overnight. T cells and APCs were mixed in a 1:5 ratio conjugated by briefly spinning and subsequently incubated at 37\u00b0C in serum-free medium. Costaining with antibodies to macrophage or dendritic markers demonstrated that about 90% of the conjugates formed were with macrophages. At the indicated time points cells were fixed with 4% paraformaldehyde and mounted on poly- l -lysine\u2013coated glass slides. For TCR staining antibody to TCR V\u03b2 3 (KJ25) was used. For LFA-1 monoclonal antibody to LFA-1 (H155) (25) was used. For the staining of intracellular signaling molecules cell conjugates were permeabilized with 0.05% TX-100 at room temperature for 5 min after fixation. Cells were labeled with rabbit antisera against pY394 Lck and pY319 ZAP70 and biotinylated antibodies against Lck ZAP70 and phosphotyrosine (4G10; Upstate Biotechnology). Donkey antibody to rabbit rat or hamster secondary antibodies conjugated to Cy3 and Cy5 were used (Jackson Immuno Research West Grove PA). For biotinylated antibodies Alexa-488\u2013conjugated streptavidin was used (Molecular Probes Eugene OR). Cells were mounted with ProLong Mounting medium (Molecular Probes). Two- and three-color confocal images and differential interference contrast (DIC) images were acquired with a Zeiss LSM 510. The three-dimensional reconstruction of the T cell\u2013APC contact area was generated with 0.2 \u03bcM serial sections of x - y images along the z axis and subsequent analysis with LSM 510 software."
    },
    {
      "key": "e_1_3_1_9_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1126/science.7509083"
    },
    {
      "key": "e_1_3_1_10_2",
      "unstructured": "A. D. Holdorf K.-H. Lee W. R. Burack P. M. Allen A. S. Shaw Nature Immunol. in press."
    },
    {
      "key": "e_1_3_1_11_2",
      "doi-asserted-by": "crossref",
      "unstructured": "L. K. Gauen et al. Mol. Cell. Biol. 14 3729 (1994).",
      "DOI": "10.1128/MCB.14.6.3729"
    },
    {
      "key": "e_1_3_1_12_2",
      "doi-asserted-by": "crossref",
      "unstructured": "B. L. Williams et al. EMBO J. 18 1832 (1999).",
      "DOI": "10.1093/emboj/18.7.1832"
    },
    {
      "key": "e_1_3_1_13_2",
      "unstructured": "Supplementary figures are available on Science Online at www.sciencemag.org/cgi/content/full/295/5559/1539/DC1."
    },
    {
      "key": "e_1_3_1_14_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.88.20.9166"
    },
    {
      "key": "e_1_3_1_14_3",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0092-8674(94)90080-9"
    },
    {
      "key": "e_1_3_1_14_4",
      "doi-asserted-by": "crossref",
      "first-page": "2073",
      "DOI": "10.4049/jimmunol.162.4.2073",
      "volume": "162",
      "author": "Itoh Y.",
      "year": "1999",
      "unstructured": "Itoh Y., Hemmer B., Martin R., Germain R. N., J. Immunol. 162, 2073 (1999).",
      "journal-title": "J. Immunol."
    },
    {
      "key": "e_1_3_1_15_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1084/jem.185.10.1859"
    },
    {
      "key": "e_1_3_1_15_3",
      "doi-asserted-by": "crossref",
      "unstructured": "; C. Cenciarelli et al. Science 257 795 (1992);",
      "DOI": "10.1126/science.1323144"
    },
    {
      "key": "e_1_3_1_15_4",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/375148a0"
    },
    {
      "key": "e_1_3_1_16_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S1074-7613(00)80461-6"
    },
    {
      "key": "e_1_3_1_17_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1046/j.1440-1711.1999.00877.x"
    },
    {
      "key": "e_1_3_1_18_2",
      "unstructured": "Purified splenic T cells from AND TCR transgenic mice were labeled with 500 nM CFSE (Molecular Probes). The CFSE-labeled T cells were conjugated with splenic APCs from B10.BR mice as described above. At the indicated time points cell conjugates were disrupted by two rounds of complement lysis with antibody to I-E k (14.4.4) and rabbit complement (Cedarlane Lab: 1:50 dilution). T cells were reisolated and recultured with unloaded APCs. After 24 48 and 72 hours T cell division was determined by flow cytometric measurement of CFSE intensity of the T cells."
    },
    {
      "key": "e_1_3_1_19_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/87730"
    },
    {
      "key": "e_1_3_1_20_2",
      "doi-asserted-by": "crossref",
      "first-page": "2169",
      "DOI": "10.4049/jimmunol.138.7.2169",
      "volume": "138",
      "author": "Weiss A.",
      "year": "1987",
      "unstructured": "Weiss A., Shields R., Newton M., Manger B., Imboden J., J. Immunol. 138, 2169 (1987);",
      "journal-title": "J. Immunol."
    },
    {
      "key": "e_1_3_1_20_3",
      "doi-asserted-by": "publisher",
      "DOI": "10.1126/science.3259335"
    },
    {
      "key": "e_1_3_1_21_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0960-9822(01)00165-8"
    },
    {
      "key": "e_1_3_1_22_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1126/science.274.5295.2086"
    },
    {
      "key": "e_1_3_1_23_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0167-5699(97)80027-8"
    },
    {
      "key": "e_1_3_1_24_2",
      "doi-asserted-by": "crossref",
      "unstructured": "S. Miyamoto et al. J. Cell Biol. 131 791 (1995).",
      "DOI": "10.1083/jcb.131.3.791"
    },
    {
      "key": "#cr-split#-e_1_3_1_25_2.1",
      "doi-asserted-by": "crossref",
      "unstructured": "A. Hutloff et al. Nature 397 263 (1999)",
      "DOI": "10.1038/16717"
    },
    {
      "key": "#cr-split#-e_1_3_1_25_2.2",
      "doi-asserted-by": "crossref",
      "unstructured": "P. S. Linsley et al. J. Exp. Med. 176 1595 (1992).",
      "DOI": "10.1084/jem.176.6.1595"
    },
    {
      "key": "e_1_3_1_26_2",
      "doi-asserted-by": "crossref",
      "unstructured": "S. Pont et al. J. Immunol. 136 3750 (1986).",
      "DOI": "10.4049/jimmunol.136.10.3750"
    },
    {
      "key": "e_1_3_1_27_2",
      "unstructured": "We wish to thank E. Unanue for reading the manuscript and R. Burack and E. Hailman for intellectual support. This work was supported by grants from the NIH."
    }
  ],
  "container-title": "Science",
  "original-title": [],
  "language": "en",
  "link": [
    {
      "URL": "https://www.science.org/doi/pdf/10.1126/science.1067710",
      "content-type": "unspecified",
      "content-version": "vor",
      "intended-application": "similarity-checking"
    }
  ],
  "deposited": {
    "date-parts": [
      [
        2024,
        1,
        9
      ]
    ],
    "date-time": "2024-01-09T21:28:44Z",
    "timestamp": 1704835724000
  },
  "score": 1,
  "resource": {
    "primary": {
      "URL": "https://www.science.org/doi/10.1126/science.1067710"
    }
  },
  "subtitle": [],
  "short-title": [],
  "issued": {
    "date-parts": [
      [
        2002,
        2,
        22
      ]
    ]
  },
  "references-count": 32,
  "journal-issue": {
    "issue": "5559",
    "published-print": {
      "date-parts": [
        [
          2002,
          2,
          22
        ]
      ]
    }
  },
  "alternative-id": [
    "10.1126/science.1067710"
  ],
  "URL": "http://dx.doi.org/10.1126/science.1067710",
  "relation": {
    "has-review": [
      {
        "id-type": "doi",
        "id": "10.3410/f.1004974.58104",
        "asserted-by": "object"
      }
    ]
  },
  "ISSN": [
    "0036-8075",
    "1095-9203"
  ],
  "subject": [],
  "container-title-short": "Science",
  "published": {
    "date-parts": [
      [
        2002,
        2,
        22
      ]
    ]
  }
}