NSOM/QD-based nanoscale immunofluorescence imaging of antigen-specific T-cell receptor responses during an in vivo clonal Vγ2Vδ2 T-cell expansion

Abstract

Nanoscale imaging of an in vivo antigenspecific T-cell immune response has not been reported. Here, the combined nearfield scanning optical microscopy-and fluorescent quantum dot-based nanotechnology was used to perform immunofluorescence imaging of antigen-specific T-cell receptor (TCR) response in an in vivo model of clonal T-cell expansion. The near-field scanning optical microscopy/quantum dot system provided a best-optical-resolution (Ͻ50 nm) nanoscale imaging of V␥2V␦2 TCR on the membrane of nonstimulated V␥2V␦2 T cells. Before Ag-induced clonal expansion, these nonstimulating V␥2V␦2 TCRs appeared to be distributed differently from their ␣␤ TCR counterparts on the cell surface. Surprisingly, V␥2V␦2 TCR nanoclusters not only were formed but also sustained on the membrane during an in vivo clonal expansion of V␥2V␦2 T cells after phosphoantigen treatment or phosphoantigen plus mycobacterial infection. IntroductionT-cell receptors (TCRs) play a crucial role in recognition of antigens and development of immune responses. Whereas immune events for TCR-mediated recognition, signaling, and activation are well described, 1-4 nanoscale imaging of immunobiology of antigenspecific TCR during the in vivo clonal T-cell expansion has not been studied. Because TCRs trigger downstream signaling and activation after antigen recognition, some unique TCR nanostructures may develop after TCR contact on Ag/antigen-presenting cell (APC) and thus contribute to selected functions, such as clonal expansion, effector function, contracting (clonal exhaustion), or differentiation. Whereas this presumption can be tested by imaging or visualization of antigen-specific TCR during the in vivo T-cell response, conventional imaging techniques using fluorescent or confocal microscopy do not have nanoscale optical resolution power to reveal individual TCRs and their dynamics during clonal expansion-maturation. [1][2][3][4] Nanotechology-based imaging may make it possible to reveal TCR nanostructures in the context of T-cell recognition of antigens and therefore provide new insight into T-cell response or ultimately immunity. 5 Nanotechnology is emerging as a multidisciplinary tool to advance life science and medicine. 6,7 However, nanoscale imaging or dissecting of functional biologic molecules in cells remain challenging. Near-field scanning optical microscopy (NSOM) has proved to be a useful nanotechnology tool for studying hard and flat materials, but its application in biomedical research is still limited. [8][9][10][11] Complicated natures of cell membranes or biologic molecules make it difficult for NSOM to generate high spatial resolution images. Whereas home-made NSOM operating in liquid can yield images of biologic molecules, the current commercial NSOM instruments are all designed for in-air imaging, 12-15 posing a challenge for nanoscale imaging of cell membrane proteins. Although NSOM combined with some common fluorescent materials were used for imaging, 16 the absence of highly photostable fluorophores for use in NSOM is perhap...

Human γδ T Cells: A Lymphoid Lineage Cell Capable of Professional Phagocytosis

“…1B) anti-TCR␥␦ staining both on the surface and inside the cell. This punctate staining was consistent with TCR-CD3 clustering on activated ␣␤ T cells (20) and reports of TCR␥␦ clustering on ␥␦ T cells after TCR ligation (21), and was likely due to some activation during selection. ␥␦ T cells were also delineated from the few contaminating monocytes by their distinct morphology.…”

Fluorescence-topographic NSOM directly visualizes peak-valley polarities of GM1/GM3 rafts in cell membrane fluctuations

“…Shown are confocal images of untreated QD stock solution (left), QD supernatant after the first centrifugation at 5,000 g for 5 min (middle) and final QD supernatant treated by all the procedures: the first centrifugation, dilution in PBS, filtration (?80-100 nm-pore filter) and the second centrifugation at 12,000 g for 5 min. The nearfield scanning optical microscopy (NSOM) (or transmission electron microscopy) images of the single-QD particles from the final supernatant were shown in our previous study (4). B and C show the confocal images of GM1 (B stained first with biotinylated CTB and then streptavidin-conjugated QD655) or GM3 (C stained sequentially with anti-GM3 IgM, biotinylated anti-IgM IgG, and streptavidin-conjugated QD655) microdomains on cell membrane of MDCK cells.…”

“…All negative control experiments showed negative results (data now shown). To remove QD aggregates in commercial QD solution, the following procedures were followed to prepare QD dyes for use in each experiment, as mentioned previously (4). Briefly, the QD solution was spin down at 5,000 g for 5 min, a small amount (in the order of ml) of the supernatant was diluted in PBS, and then filtered/centrifuged through the Ultrafree-MC centrifugal filter devices (Millipore Corp., Bedford, MA) with ?80-100 nm pores at 12,000 g for 5 min.…”

“…The high-resolution electron microscopy (EM) is often limited by uncertain fidelity or reliability due to the complicated sample-preparing procedures and demanding imaging conditions. Near-field scanning optical microscopy (NSOM) should be the best candidate for nanoscale imaging of topographic distribution of membrane gangliosides or lipid rafts, since NSOM can simultaneously provide topographic and fluorescence visualization of cell-surface molecules in nanoscale resolution (4,5). In this study, the simultaneous fluorescence-topographic NSOM imaging, in combination with fluorescent quantum dot (QD) (4), was utilized to directly visualize nanoscale topographic-distributions of gangliosides or lipid rafts in the apical plasma membranes of Madin-Darby canine kidney cells (MDCK) cells.…”

What do diffusion measurements tell us about membrane compartmentalisation? Emergence of the role of interprotein interactions

“…This methodology has been successfully used to demonstrate that free heavy chains and class I HLA heterodimers co-cluster in B cells [81]. It has also been used to observe nanometre domains of β-adrenergic receptor complexes on the surface of cardiac myocytes [71,82], clustering of the pathogen receptor DC-SIGN on dendritic cells [83] and T cell receptor reorganisation induced by antigenic stimulation [84].These high resolution methodologies share a common drawback of a low time resolution as they rely on scanning the surface of interest. In a perpetually moving landscape, this would not provide a reliable image at a given instant.…”