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. 2015 Aug 1;195(3):1320-30.
doi: 10.4049/jimmunol.1403143. Epub 2015 Jun 29.

Imaging of Cell-Cell Communication in a Vertical Orientation Reveals High-Resolution Structure of Immunological Synapse and Novel PD-1 Dynamics

Joon Hee Jang  1 , Yu Huang  2 , Peilin Zheng  3 , Myeong Chan Jo  4 , Grant Bertolet  5 , Michael Xi Zhu  6 , Lidong Qin  7 , Dongfang Liu  8
Affiliations

Imaging of Cell-Cell Communication in a Vertical Orientation Reveals High-Resolution Structure of Immunological Synapse and Novel PD-1 Dynamics

Joon Hee Jang et al. J Immunol. .

Abstract

The immunological synapse (IS) is one of the most pivotal communication strategies in immune cells. Understanding the molecular basis of the IS provides critical information regarding how immune cells mount an effective immune response. Fluorescence microscopy provides a fundamental tool to study the IS. However, current imaging techniques for studying the IS cannot sufficiently achieve high resolution in real cell-cell conjugates. In this study, we present a new device that allows for high-resolution imaging of the IS with conventional confocal microscopy in a high-throughput manner. Combining micropits and single-cell trap arrays, we have developed a new microfluidic platform that allows visualization of the IS in vertically "stacked" cells. Using this vertical cell pairing (VCP) system, we investigated the dynamics of the inhibitory synapse mediated by an inhibitory receptor, programed death protein-1, and the cytotoxic synapse at the single-cell level. In addition to the technique innovation, we have demonstrated novel biological findings by this VCP device, including novel distribution of F-actin and cytolytic granules at the IS, programed death protein-1 microclusters at the NK IS, and kinetics of cytotoxicity. We propose that this high-throughput, cost-effective, easy-to-use VCP system, along with conventional imaging techniques, can be used to address a number of significant biological questions in a variety of disciplines.

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Figures

Figure 1
Figure 1
Optical geometry and device design. (A) The optical geometry (left) for imaging the IS by conventional methods and corresponding simulated point-spread-function (PSF, right) of the excitation beam. (B) The optical geometry (left) for imaging the IS by the VCP system and corresponding simulated PSF (right) of the excitation beam. (C) Overall design of the microfluidic platform and flow pathways during cell loading. VCP ver.3 is shown. (D) Wide field fluorescent microscopic image merged with bright field image of the microfluidic device after cell loading. Red and green channels correspond to K562 and KHYG-1 cells, respectively. Scale bar indicates 100 µm (left) and 20 µm (right), respectively. (E) Percentage of trap-captured cells in each step during the cell loading procedure. The graph shows average and standard deviation (SD) of cell capture efficiency over four different area of the device. The results are from three independent experiments.
Figure 2
Figure 2
Simulated flow velocity distribution on the top layer. (A) Overview of the flow velocity in VCP ver.3. (B) Flow velocity distribution around a single microstructure without cell. Red lines show bottom layer and white blocks indicate top PDMS structure. (C) Flow velocity distribution changes around a single microstructure with a trapped cell. The flow velocity is pseudo-colored with cool and warm colors indicating low and high flow velocity, respectively.
Figure 3
Figure 3
Comparison of IS structure by conventional (Conv.) and VCP systems imaged by confocal fluorescence microscopy. CD16-KHYG-1 (NK) cell conjugated with K562 (target) cell on cover glass coated with poly-L-lysine (A) and vertical stack of NK-target cell pair by VCP ver.2 (B) are fixed, permeabilized, and stained for F-actin (red), perforin (green), and α-tubulin (cyan). Scale bars indicate 5 µm. The three-dimensional (3D) fluorescent images centered at the IS are z-projected. Magnified areas (8×8 µm, white boxes) from the fluorescent microscope image obtained by using coverslips coated with poly-L-lysine (C) and by using the VCP ver.2 system (D). Perforin (green) and F-actin (red) are visualized. Scale bars indicate 2 µm. (E) Fluorescence intensity profile of F-actin and perforin was measured across the white line. Fluorescence intensity of the perforin and F-actin is shown in green and red line, respectively. (F) Full width at half maximum (FWHM) and standard error of fluorescence intensity for individual perforin granule along the vertical line (blue line in C and D). (G) Pearson’s correlation coefficient (r) was calculated for perforin and F-actin from 3D colocalization analysis. Each dot represents each pair (n = 46 for conventional, n = 45 for VCP imaging). (H) Pearson’s correlation coefficient (r) was calculated from 2D colocalization analysis. The same set of data as (G) was used. (I) Costes P-value (indicating the reliability of Pearson’s correlation coefficient analysis) obtained during 2D colocalization analysis. VCP ver.3 was used for (G-I).
Figure 4
Figure 4
Time-lapse images of ‘Dispersed→Centralized→Dispersed’ (D→C→D) PD-1/PD-L1 clusters by 3D confocal fluorescence microscope using VCP ver.3. (A) shows two examples of a subpopulation of NK-target cell pairs (68.2%, 15 out of 22 observations) whose PD-1/PD-L1 microclusters coalesced, then dispersed. Scale bars indicate 5 µm. (B) Proposed model of PD-1/PD-L1 cluster movement during cell-cell communication within the D→C→D subpopulation.
Figure 5
Figure 5
Time-lapse images of ‘stay-Dispersed’ (sD) PD-1/PD-L1 clusters by 3D confocal fluorescence microscope. (A) and (B) represent two examples of a subpopulation of PD-1/PD-L1 cluster movement on two cell pairs imaged using VCP ver.3. During imaging, the clusters did not coalesced in this sub-population (22.7%, 5 out of 22 observations). Scale bars indicate 5 µm.
Figure 6
Figure 6
Time-lapse images of ‘Dispersed→stay-Centralized’ (D→sC) PD-1/PD-L1 clusters (9.1%, 2 out of 22 observations) by 3D confocal fluorescence microscope. (A) Schematic model (left) and time series taken from Supplemental Movie 3 (right). Fluorescent images of selected time points from live cell imaging by using the VCP ver.2 system for IS formation between PD-L1-mCherry+ K562 (bottom, red) and PD-1-GFP+ CD16-KHYG-1 (top, green) cells. PD-1-GFP, PD-L1-mCherry, bright field, merged, and colocalization of PD-1 and PD-L1 are presented. Scale bar, 10 µm. (B) Track path of the clusters in the colocalized fluorescence image. Black dashed line indicates central cluster region. (C) Fluorescent image merged with bright field image after disconnecting negative pressure.
Figure 7
Figure 7
Kinetics of cytotoxicity mediated by NK cells imaged under wide-field fluorescence microscope. (A) Live cell time-lapse images from the VCP ver.3 system used to establish kinetics of CD16-KHYG-1 (red)-mediated cytotoxicity against K562 (green) target cell. Scale bars indicate 100 µm (left) and 10 µm (right), respectively. (B) Mean fluorescent intensity (MFI) of K562 cells paired with CD16-KHYG-1 cells (gray), K562 cells alone (green), and CD16-KHYG-1 cells alone (red) during the 6-hour acquisition. (C–F) Classification of killing kinetics of NK cells when paired with K562 target cells. (C) ‘Slow decay’ of normalized MFI from K562 cells conjugated with NK cells for 6 hrs. (D) ‘Single-drop’ of normalized MFI from K562 cells conjugated with NK cells (left) and quantification of ‘single-drop’ occurrence in K562 cells after conjugation with NK cells (right). (E) ‘Fast decay’ of normalized MFI from K562 cells conjugated with NK cells for 6 hrs. (F) ‘Multiple-drop’ of normalized MFI from K562 cells conjugated with NK cells (left) and quantification of ‘multiple-drop’ occurrence in K562 cells after conjugation with NK cells (right). Black lines indicate average of the each population.
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