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Molecular anatomy of antigen-specific CD8+ T cell engagement and synapse formation in vivo

Abstract

Antigen-specific CD8+ T cells are required for the clearance of most viral infections and several cancers. However, it is not clear in vivo whether CD8+ T cells can engage multiple targets simultaneously, engagement results in the formation of an immunologic synapse or molecules involved in CD8 function are redistributed to the synapse. We used here high-resolution microscopy to visualize interactions between virus-specific effectors and target cells in vivo. Using either in situ tetramer staining or green fluorescent protein–labeled virus-specific T cells, we have shown that a single CD8+ T cell can engage two or three targets, a synapse occurs at the site of engagement and molecules involved in attachment (lymphocyte function–associated antigen 1), signaling (Lck) and lytic activity (perforin) are differentially positioned on the T cell. In addition, we have established an in vivo approach for assessing the intricacies of antigen-specific T cell activation, migration, engagement, memory and other defining elements of adaptive immunity.

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Figure 1: Tetramer staining of virus-specific CD8+ T cells in the spleens and CNS of GP33 TCR–Tg mice.
Figure 2: Visualization of antiviral immunity in the CNS of B6 mice during the induction of lethal meningitis.
Figure 3: Expansion and migration of GFP+ Db-GP(33–41)-specific T cells after an i.c. infection.
Figure 4: Cellular reorganization of Db-GP(33–41)-specific T cells juxtaposed to LCMV-infected targets.
Figure 5: Patterns of interfacial LFA-1, Lck and perforin staining on Db-GP(33–41)-specific T cells in the CNS.
Figure 6: LFA-1 and Lck 3D localization on Db-GP(33–41)-specific T cells in the CNS.

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References

  1. Zinkernagel, R.M. & Doherty, P.C. Restriction of in vitro T cell–mediated cytotoxicity in lymphocytic choriomeningitis within a syngeneic or semiallogeneic system. Nature 248, 701–702 (1974).

    Article  CAS  Google Scholar 

  2. Kagi, D., Ledermann, B., Burki, K., Zinkernagel, R.M. & Hengartner, H. Molecular mechanisms of lymphocyte-mediated cytotoxicity and their role in immunological protection and pathogenesis in vivo. Annu. Rev. Immunol. 14, 207–232 (1996).

    Article  CAS  Google Scholar 

  3. von Andrian, U.H. & Mackay, C.R. T-cell function and migration. Two sides of the same coin. N. Engl. J. Med. 343, 1020–1034 (2000).

    Article  CAS  Google Scholar 

  4. Skinner, P.J., Daniels, M.A., Schmidt, C.S., Jameson, S.C. & Haase, A.T. Cutting edge: In situ tetramer staining of antigen-specific T cells in tissues. J. Immunol. 165, 613–617 (2000).

    Article  CAS  Google Scholar 

  5. Haanen, J.B. et al. In situ detection of virus- and tumor-specific T-cell immunity. Nature Med. 6, 1056–1060 (2000).

    Article  CAS  Google Scholar 

  6. Flugel, A. et al. Migratory activity and functional changes of green fluorescent effector cells before and during experimental autoimmune encephalomyelitis. Immunity 14, 547–560 (2001).

    Article  CAS  Google Scholar 

  7. Reinhardt, R.L., Khoruts, A., Merica, R., Zell, T. & Jenkins, M.K. Visualizing the generation of memory CD4 T cells in the whole body. Nature 410, 101–105 (2001).

    Article  CAS  Google Scholar 

  8. Allan, J.E., Dixon, J.E. & Doherty, P.C. Nature of the inflammatory process in the central nervous system of mice infected with lymphocytic choriomeningitis virus. Curr. Top. Microbiol. Immunol. 134, 131–143 (1987).

    CAS  PubMed  Google Scholar 

  9. Fung-Leung, W.P., Kundig, T.M., Zinkernagel, R.M. & Mak, T.W. Immune response against lymphocytic choriomeningitis virus infection in mice without CD8 expression. J. Exp. Med. 174, 1425–1429 (1991).

    Article  CAS  Google Scholar 

  10. Kagi, D. et al. Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin–deficient mice. Nature 369, 31–37 (1994).

    Article  CAS  Google Scholar 

  11. Joly, E., Mucke, L. & Oldstone, M.B. Viral persistence in neurons explained by lack of major histocompatibility class I expression. Science 253, 1283–1285 (1991).

    Article  CAS  Google Scholar 

  12. Gallimore, A. et al. A protective cytotoxic T cell response to a subdominant epitope is influenced by the stability of the MHC class I/peptide complex and the overall spectrum of viral peptides generated within infected cells. Eur. J. Immunol. 28, 3301–3311 (1998).

    Article  CAS  Google Scholar 

  13. van der Most, R.G. et al. Identification of Db- and Kb-restricted subdominant cytotoxic T-cell responses in lymphocytic choriomeningitis virus-infected mice. Virology 240, 158–167 (1998).

    Article  CAS  Google Scholar 

  14. Gairin, J.E., Mazarguil, H., Hudrisier, D. & Oldstone, M.B. Optimal lymphocytic choriomeningitis virus sequences restricted by H-2Db major histocompatibility complex class I molecules and presented to cytotoxic T lymphocytes. J. Virol. 69, 2297–2305 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Pircher, H., Burki, K., Lang, R., Hengartner, H. & Zinkernagel, R.M. Tolerance induction in double specific T-cell receptor transgenic mice varies with antigen. Nature 342, 559–561 (1989).

    Article  CAS  Google Scholar 

  16. Janes, P.W., Ley, S.C. & Magee, A.I. Aggregation of lipid rafts accompanies signaling via the T cell antigen receptor. J. Cell. Biol. 147, 447–461 (1999).

    Article  CAS  Google Scholar 

  17. Monks, C.R., Freiberg, B.A., Kupfer, H., Sciaky, N. & Kupfer, A. Three-dimensional segregation of supramolecular activation clusters in T cells. Nature 395, 82–86 (1998).

    Article  CAS  Google Scholar 

  18. Sanderson, C.J. The mechanism of T cell mediated cytotoxicity. II. Morphological studies of cell death by time-lapse microcinematography. Proc. R. Soc. Lond. B 192, 241–255 (1976).

    Article  CAS  Google Scholar 

  19. Rothstein, T.L., Mage, M., Jones, G. & McHugh, L.L. Cytotoxic T lymphocyte sequential killing of immobilized allogeneic tumor target cells measured by time-lapse microcinematography. J. Immunol. 121, 1652–1656 (1978).

    CAS  PubMed  Google Scholar 

  20. Hahn, K. et al. Antigen presentation and cytotoxic T lymphocyte killing studied in individual, living cells. Virology 201, 330–340 (1994).

    Article  CAS  Google Scholar 

  21. Okabe, M., Ikawa, M., Kominami, K., Nakanishi, T. & Nishimune, Y. 'Green mice' as a source of ubiquitous green cells. FEBS Lett. 407, 313–319 (1997).

    Article  CAS  Google Scholar 

  22. Kucik, D.F., Dustin, M.L., Miller, J.M. & Brown, E.J. Adhesion-activating phorbol ester increases the mobility of leukocyte integrin LFA-1 in cultured lymphocytes. J. Clin. Invest. 97, 2139–2144 (1996).

    Article  CAS  Google Scholar 

  23. Krummel, M.F. & Davis, M.M. Dynamics of the immunological synapse: finding, establishing and solidifying a connection. Curr. Opin. Immunol. 14, 66–74 (2002).

    Article  CAS  Google Scholar 

  24. Stinchcombe, J.C., Bossi, G., Booth, S. & Griffiths, G.M. The immunological synapse of CTL contains a secretory domain and membrane bridges. Immunity 15, 751–761 (2001).

    Article  CAS  Google Scholar 

  25. Kane, L.P., Lin, J. & Weiss, A. Signal transduction by the TCR for antigen. Curr. Opin. Immunol. 12, 242–249 (2000).

    Article  CAS  Google Scholar 

  26. Veillette, A., Bookman, M.A., Horak, E.M. & Bolen, J.B. The CD4 and CD8 T cell surface antigens are associated with the internal membrane tyrosine-protein kinase p56lck. Cell 55, 301–308 (1988).

    Article  CAS  Google Scholar 

  27. Bachmann, M.F. et al. Developmental regulation of Lck targeting to the CD8 coreceptor controls signaling in naive and memory T cells. J. Exp. Med. 189, 1521–1530 (1999).

    Article  CAS  Google Scholar 

  28. Yannelli, J.R., Sullivan, J.A., Mandell, G.L. & Engelhard, V.H. Reorientation and fusion of cytotoxic T lymphocyte granules after interaction with target cells as determined by high resolution cinemicrography. J. Immunol. 136, 377–382 (1986).

    CAS  PubMed  Google Scholar 

  29. Kuhn, J.R. & Poenie, M. Dynamic polarization of the microtubule cytoskeleton during CTL-mediated killing. Immunity 16, 111–121 (2002).

    Article  CAS  Google Scholar 

  30. Grakoui, A. et al. The immunological synapse: a molecular machine controlling T cell activation. Science 285, 221–227 (1999).

    Article  CAS  Google Scholar 

  31. Bromley, S.K. et al. The immunological synapse. Annu. Rev. Immunol. 19, 375–396 (1902).

    Article  Google Scholar 

  32. Miller, M.J., Wei, S.H., Parker, I. & Cahalan, M.D. Two-photon imaging of lymphocyte motility and antigen response in intact lymph node. Science 296, 1869–1873 (2002).

    Article  CAS  Google Scholar 

  33. Stoll, S., Delon, J., Brotz, T.M. & Germain, R.N. Dynamic imaging of T cell-dendritic cell interactions in lymph nodes. Science 296, 1873–1876 (2002).

    Article  Google Scholar 

  34. Bousso, P., Bhakta, N.R., Lewis, R.S. & Robey, E. Dynamics of thymocyte-stromal cell interactions visualized by two-photon microscopy. Science 296, 1876–1880 (2002).

    Article  CAS  Google Scholar 

  35. Busch, D.H., Pilip, I.M., Vijh, S. & Pamer, E.G. Coordinate regulation of complex T cell populations responding to bacterial infection. Immunity 8, 353–362 (1998).

    Article  CAS  Google Scholar 

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Acknowledgements

Supported by NIH grant AI09484, training grant AG00080 (to D. B. M.) and Juvenile Diabetes Research Foundation Award 3-2000-510 (to U. C.)

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Correspondence to Dorian B. McGavern.

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Supplementary information

Web Movie 1.

A 3D rotation of a GFP+ Db-GP(33-41)-specific T cell (green) in juxtaposition with three nucleated (blue) LCMV-infected targets (red) in the CNS. (MOV 6527 kb)

Web Movie 2.

A 3D rotation illustrating the distribution of LFA-1 (red) on the same GFP+ Db-GP(33-41)-specific T cell (green) shown in Web Movie 1. Nuclei are shown in blue. Note the polarization of LFA-1 toward the virus-infected targets and the LFA-1 coated process that the CTL has extended. (MOV 6150 kb)

Web Movie 3.

A 3D rotation illustrating the distribution of LFA-1 (red) on GFP+ Db-GP(33-41)-specific T cell (green) that has trafficked to the CNS but is not engaged with a LCMV-infected target. Note the homogenous clusters of LFA-1 around the cell membrane. Nuclei are shown in blue. (MOV 2824 kb)

Web Movie 4.

A 3D rotation of a GFP+ Db-GP(33-41)-specific T cell (green) in juxtaposition with a single LCMV-infected target (blue) in the CNS. Note the interfacial aggregation of Lck (red) on the CTL. (MOV 3029 kb)

Web Movie 5.

A 3D rotation showing the distribution of Lck (red) on a GFP+ Db-GP(33-41)-specific T cell (green) not engaged with an LCMV-infected target. Note the homogenous distribution of Lck on the CTL membrane. Nuclei are shown in blue. (MOV 2817 kb)

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McGavern, D., Christen, U. & Oldstone, M. Molecular anatomy of antigen-specific CD8+ T cell engagement and synapse formation in vivo. Nat Immunol 3, 918–925 (2002). https://doi.org/10.1038/ni843

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