Imaging synapse formation during thymocyte selection: inability of CD3zeta to form a stable central accumulation during negative selection.
TCR signaling can result in cell fates ranging from activation to tolerance to apoptosis. Organization of molecules in an "immunological synapse" between mature T cells and APCs correlates with the strength of TCR signaling. To investigate synapse formation during thymic selection, we have established a reaggregate system in which molecular recruitment of GFP fusion proteins to thymocyte:stromal cell interfaces can be visualized in real time. We demonstrate that negative selection is associated with efficient conjugate formation and rapid recruitment of p56(lck) and CD3zeta to an immunological synapse. Interestingly, CD3zeta-GFP does not accumulate at the center of the synapse, as in mature T cells, but at the periphery across a wide range of ligand densities. This implicates differences in synapse geometry in initiation of alternate signals downstream of the TCR.
Richie
Lauren I
LI
Program in Immunology, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305, USA.
Ebert
Peter J R
PJ
Wu
Lawren C
LC
Krummel
Matthew F
MF
Owen
John J T
JJ
Davis
Mark M
MM
D000818:Animals; D017252:CD3 Complex / Q000235:genetics / Q000276:immunology*; D002454:Cell Differentiation; D049452:Green Fluorescent Proteins; D007091:Image Processing, Computer-Assisted; D008164:Luminescent Proteins / Q000235:genetics; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000235:genetics / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D013569:Synapses; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology*; D013950:Thymus Gland / Q000166:cytology / Q000276:immunology
D017252:CD3 Complex; C121704:CD3 antigen, zeta chain; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)