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 ∼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.

The coreceptor CD4 is expressed in distinct nanoclusters and does not colocalize with T-cell receptor and active protein tyrosine kinase p56lck

“…The spatial distribution of TCR-CD3 and CD4 was previously examined using another nondiffraction-limited optical microscopy technique, namely near-field scanning optical microscopy and immune-labeling quantum dots (46). In their best resolution of around 50 nm, the authors showed that the TCR-CD3 and CD4 reside in their separate nanoclusters with minimal association in a nonactivated state, which agrees well with our findings.…”

Quantum Dots in Bioanalysis: A Review of Applications across Various Platforms for Fluorescence Spectroscopy and Imaging

“…Building on this approach, these authors subsequently used two-color NSOM to visualize the clustering and interactions of membrane receptors during TCR/CD3-mediated signaling in T cells. 293 Images of QD 605 and QD 650 PL were combined with topographical information (Fig. 19) and indicated co-clustering of CD3 with CD4 or CD8 co-receptors during T-cell activation.…”

Fluorescence Imaging on the Nanoscale: Bioimaging Using Near-field Scanning Optical Microscopy

“…42,43 In one case they demonstrated that only 10% of the  T-cell receptor (TCR) clusters were >90 nm in diameter, whereas 40% of -TCR clusters were larger than 90 nm. 42 Significant changes in cluster size for V2V2 T cells were observed following antigen-induced 325 in vivo clonal expansion of the cells.…”

“…A second study showed that TCRs (CD3, CD4 and CD8) were found as either isolated receptors or small nanoclusters labeled with 2-4 quantum dots in unstimulated T 330 cells with relatively low levels of colocalization (<10% of CD3 with either CD4 or CD8). 43 Activation of the cells led to significant changes in the distribution of the receptors with co-clustering of CD3 with CD4 or CD8 to give 200-500 nm nanodomains and >500 nm microdomains. In these studies the brightness and resistance to photobleaching of quantum dots provided an advantage over standard 335 immunofluorescence with dye-labeled antibodies.…”