Direct observation of ligand recognition by T cells

The activation of T cells through interaction of their T-cell receptors with antigenic peptide bound to major histocompatibility complex (MHC) on the surface of antigen presenting cells (APCs) is a crucial step in adaptive immunity. Here we use three-dimensional fluorescence microscopy to visualize individual peptide–I-Ek class II MHC complexes labelled with the phycobiliprotein phycoerythrin in an effort to characterize T-cell sensitivity and the requirements for forming an immunological synapse in single cells. We show that T cells expressing the CD4 antigen respond with transient calcium signalling to even a single agonist peptide–MHC ligand, and that the organization of molecules in the contact zone of the T cell and APC takes on the characteristics of an immunological synapse when only about ten agonists are present. This sensitivity is highly dependant on CD4, because blocking this molecule with antibodies renders T cells unable to detect less than about 30 ligands.

[1]  H. Eisen,et al.  Evidence that a single peptide-MHC complex on a target cell can elicit a cytolytic T cell response. , 1996, Immunity.

[2]  Ellis L. Reinherz,et al.  Crystal structure of the human CD4 N-terminal two-domain fragment complexed to a class II MHC molecule , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[3]  K. Triantafilou,et al.  Phycobiliprotein-Fab conjugates as probes for single particle fluorescence imaging. , 2000, Cytometry.

[4]  P. Marrack,et al.  THE MAJOR HISTOCOMPATIBILITY COMPLEX RESTRICTED ANTIGEN RECEPTOR ON T CELLS , 2003 .

[5]  A. Williams,et al.  Molecular associations between the T-lymphocyte antigen receptor complex and the surface antigens CD2, CD4, or CD8 and CD5. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[6]  H. Grey,et al.  The minimal number of antigen‐major histocompatibility complex class II complexes required for activation of naive and primed T cells , 1997, European journal of immunology.

[7]  H. Grey,et al.  The minimal number of class II MHC-antigen complexes needed for T cell activation. , 1990, Science.

[8]  M. Davis,et al.  Visualizing the dynamics of T cell activation: intracellular adhesion molecule 1 migrates rapidly to the T cell/B cell interface and acts to sustain calcium levels. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[9]  D. Baylor,et al.  Responses of retinal rods to single photons. , 1979, The Journal of physiology.

[10]  Emil R. Unanue,et al.  Quantitation of antigen-presenting cell MHC class II/peptide complexes necessary for T-cell stimulation , 1990, Nature.

[11]  S. Murray,et al.  The prolyl isomerase Pin1 is a regulator of p53 in genotoxic response , 2002, Nature.

[12]  Ellis L. Reinherz,et al.  T Cell Receptor Binding to a pMHCII Ligand Is Kinetically Distinct from and Independent of CD4* , 2001, The Journal of Biological Chemistry.

[13]  Colin R. F. Monks,et al.  Three-dimensional segregation of supramolecular activation clusters in T cells , 1998, Nature.

[14]  P. Negulescu,et al.  Polarity of T cell shape, motility, and sensitivity to antigen. , 1996, Immunity.

[15]  C. Heldin,et al.  Dimerization of cell surface receptors in signal transduction , 1995, Cell.

[16]  P. Anderson,et al.  Comodulation of CD3 and CD4. Evidence for a specific association between CD4 and approximately 5% of the CD3:T cell receptor complexes on helper T lymphocytes. , 1988, Journal of immunology.

[17]  J. Altman,et al.  Initiation of signal transduction through the T cell receptor requires the multivalent engagement of peptide/MHC ligands [corrected]. , 1998, Immunity.

[18]  R. Tsien.  Fluorescent probes of cell signaling. , 1989, Annual review of neuroscience.

[19]  G. Nolan,et al.  Production of high-titer helper-free retroviruses by transient transfection. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[20]  P. Marrack,et al.  The major histocompatibility complex-restricted antigen receptor on T cells. , 1984, Annual review of immunology.

[21]  Y. Chien,et al.  CD4 augments the response of a T cell to agonist but not to antagonist ligands. , 1997, Immunity.

[22]  M. Davis,et al.  A receptor/cytoskeletal movement triggered by costimulation during T cell activation. , 1998, Science.

[23]  M. Luscher,et al.  Peptide binding to class I MHC on living cells and quantitation of complexes required for CTL lysis , 1991, Nature.

[24]  R C Brower,et al.  Minimal requirements for peptide mediated activation of CD8+ CTL. , 1994, Molecular immunology.

[25]  P. Marrack,et al.  Detection of antigen-specific T cells with multivalent soluble class II MHC covalent peptide complexes. , 1998, Immunity.

[26]  P. Allen,et al.  Differential requirements for CD4 in TCR-ligand interactions. , 1999, Journal of immunology.

[27]  J. Cochran,et al.  The relationship of MHC-peptide binding and T cell activation probed using chemically defined MHC class II oligomers. , 2000, Immunity.

[28]  Mark M. Davis,et al.  Determination of the Relationship Between T Cell Responsiveness and the Number of MHC-Peptide Complexes Using Specific Monoclonal Antibodies , 2000 .

[29]  M. Davis,et al.  Differential clustering of CD4 and CD3zeta during T cell recognition. , 2000, Science.

[30]  Toshio Yanagida,et al.  Single-molecule imaging of EGFR signalling on the surface of living cells , 2000, Nature Cell Biology.

[31]  Christoph Wülfing,et al.  Costimulation and endogenous MHC ligands contribute to T cell recognition , 2002, Nature Immunology.

[32]  S. Bromley,et al.  The immunological synapse: a molecular machine controlling T cell activation. , 1999, Science.