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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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).
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).
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).
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).
Haanen, J.B. et al. In situ detection of virus- and tumor-specific T-cell immunity. Nature Med. 6, 1056–1060 (2000).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
Hahn, K. et al. Antigen presentation and cytotoxic T lymphocyte killing studied in individual, living cells. Virology 201, 330–340 (1994).
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).
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).
Krummel, M.F. & Davis, M.M. Dynamics of the immunological synapse: finding, establishing and solidifying a connection. Curr. Opin. Immunol. 14, 66–74 (2002).
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).
Kane, L.P., Lin, J. & Weiss, A. Signal transduction by the TCR for antigen. Curr. Opin. Immunol. 12, 242–249 (2000).
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).
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).
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).
Kuhn, J.R. & Poenie, M. Dynamic polarization of the microtubule cytoskeleton during CTL-mediated killing. Immunity 16, 111–121 (2002).
Grakoui, A. et al. The immunological synapse: a molecular machine controlling T cell activation. Science 285, 221–227 (1999).
Bromley, S.K. et al. The immunological synapse. Annu. Rev. Immunol. 19, 375–396 (1902).
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).
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).
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).
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).
Supported by NIH grant AI09484, training grant AG00080 (to D. B. M.) and Juvenile Diabetes Research Foundation Award 3-2000-510 (to U. C.)
The authors declare no competing financial interests.
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)
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)
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)
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)
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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