title: NSOM/QD-based direct visualization of CD3-induced and CD28-enhanced nanospatial coclustering of TCR and coreceptor in nanodomains in T cell activation.
abstract: Direct molecular imaging of nano-spatial relationship between T cell receptor (TCR)/CD3 and CD4 or CD8 co-receptor before and after activation of a primary T cell has not been reported. We have recently innovated application of near-field scanning optical microscopy (NSOM) and immune-labeling quantum dots (QD) to image Ag-specific TCR response during in vivo clonal expansion, and now up-graded the NSOM/QD-based nanotechnology through dipole-polarization and dual-color imaging. Using this imaging system scanning cell-membrane molecules at a best-optical lateral resolution, we demonstrated that CD3, CD4 or CD8 molecules were distinctly distributed as single QD-bound molecules or nano-clusters equivalent to 2-4 QD fluorescence-intensity/size on cell-membrane of un-stimulated primary T cells, and approximately 6-10% of CD3 were co-clustering with CD4 or CD8 as 70-110 nm nano-clusters without forming nano-domains. The ligation of TCR/CD3 on CD4 or CD8 T cells led to CD3 nanoscale co-clustering or interaction with CD4 or CD8 co-receptors forming 200-500 nm nano-domains or >500 nm micro-domains. Such nano-spatial co-clustering of CD3 and CD4 or CD3 and CD8 appeared to be an intrinsic event of TCR/CD3 ligation, not purely limited to MHC engagement, and be driven by Lck phosphorylation. Importantly, CD28 co-stimulation remarkably enhanced TCR/CD3 nanoscale co-clustering or interaction with CD4 co-receptor within nano- or micro-domains on the membrane. In contrast, CD28 co-stimulation did not enhance CD8 clustering or CD3-CD8 co-clustering in nano-domains although it increased molecular number and density of CD3 clustering in the enlarged nano-domains. These nanoscale findings provide new insights into TCR/CD3 interaction with CD4 or CD8 co-receptor in T-cell activation.
authors.affiliation: Department of Microbiology and Immunology, Center for Primate Biomedical Research, University of Illinois College of Medicine, Chicago, Illinois, United States of America.
authors.forename: Liyun
authors.initials: L
authors.lastname: Zhong
authors.forename: Gucheng
authors.initials: G
authors.lastname: Zeng
authors.forename: Xiaoxu
authors.initials: X
authors.lastname: Lu
authors.forename: Richard C
authors.initials: RC
authors.lastname: Wang
authors.forename: Guangming
authors.initials: G
authors.lastname: Gong
authors.forename: Lin
authors.initials: L
authors.lastname: Yan
authors.forename: Dan
authors.initials: D
authors.lastname: Huang
authors.forename: Zheng W
authors.initials: ZW
authors.lastname: Chen
mesh_terms: D000818:Animals; D018106:CD28 Antigens / Q000378:metabolism*; D017252:CD3 Complex / Q000378:metabolism*; D015704:CD4 Antigens / Q000096:biosynthesis; D016827:CD8 Antigens / Q000096:biosynthesis; D016000:Cluster Analysis; D007091:Image Processing, Computer-Assisted; D008213:Lymphocyte Activation; D008253:Macaca mulatta; D053758:Nanoparticles / Q000737:chemistry; D036103:Nanotechnology / Q000379:methods; D045663:Quantum Dots; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D012984:Software
chemical_list: D018106:CD28 Antigens; D017252:CD3 Complex; D015704:CD4 Antigens; D016827:CD8 Antigens; D011948:Receptors, Antigen, T-Cell