Patent Application
20180156777
This invention relates to cell imaging in general, and more particularly to high resolution imaging of cell-cell communication.
The ability of cells to communicate with each other is one of the most essential and fundamental activities in eukaryotic organisms. The cells of the immune system communicate with each other through a variety of strategies. One of the most important of these is the formation of an interface structure known as the immunological synapse (IS). Since its original description, an enormous body of evidence has been accumulated showing that the IS plays a pivotal role in the immune response, mediating such vital functions as immune recognition, adhesion, activation and inhibition.
Given the critical role of the IS in immune responses against cancer and infection, it is essential to understand the molecular mechanisms underlying IS formation, signaling and function in real cell-cell conjugates. Conventional confocal microscopy of both fixed and live cells represents the most common imaging technique available to study the IS. Traditionally, immune cells are mixed with target cells or antigen-presenting cells (APCs). After fixation, cell-cell conjugates are imaged under a confocal microscope. However, current methods of imaging the synapse face several limitations. One of the most obvious of these is the low resolution of IS images in lateral cell-cell conjugates (i.e., where the two cells are disposed in side-by-side relation). In this situation the cell pairs extend parallel to the plane of focus of the confocal microscope, and the synaptic interface lies perpendicular to the plane of focus, along the Z axis (
Furthermore, successful operation of optical tweezers requires extensive training. In addition, even when cell stacking is successfully achieved using optical tweezers, lateral movement of the cells (particularly live cells) can occur during cell imaging, which can disrupt imaging of the synapse interface.
Recently, a micropit system has been developed to achieve high-resolution IS imaging between effector and target cells at low cost. However, the loading efficiency of cells in the micropit system alone is low—about 10-15%. In addition, the frequency of vertical orientation stacking between effector and target cells is also relatively low, which makes imaging time-consuming.
Therefore, development of a novel system for vertically stacking cells would fill a significant unmet need to facilitate the study of IS formation and cell-cell communication.
The present invention comprises the provision and use of a new system that allows for high-resolution imaging of the IS with conventional confocal microscopy in a high-throughput manner. By combining micropits and single cell trap arrays, a new microfluidic platform has been developed that allows visualization of the IS in vertically “stacked” cells. This vertical cell pairing (VCP) system has been used to investigate the dynamics of the inhibitory synapse mediated by an inhibitory receptor, programmed death protein-1 (PD-1) and the cytotoxic synapse at the single cell level. In addition to the technique innovation, novel biological findings were demonstrated using this VCP system, including novel distribution of F-actin and cytolytic granules at the IS, PD-1 microclusters in the human natural killer (NK) IS, and kinetics of cytotoxicity. The 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.
In one preferred form of the invention, there is provided a vertical cell pairing (VCP) system comprising:
a substrate having a top surface;
at least one micropit formed in the top surface of the substrate, the at least one micropit being sized to seat a cell; and
at least one microtrap positioned adjacent the top surface of the substrate, the at least one microtrap comprising a body having a vertical slot, wherein the vertical slot has an opening and an exit, the vertical slot being sized to pass fluid therethrough but to prevent a cell from passing therethrough;
wherein the at least one microtrap is disposed relative to the at least one micropit so that a cell seated in the at least one micropit is in cell-cell communication with a cell disposed at the opening of the vertical slot.
In another preferred form of the invention, there is provided a method for vertically pairing cells, the method comprising:
providing a vertical cell pairing (VCP) system comprising:
flowing a first slurry of cells over the top surface of the substrate and seating a first cell in the at least one micropit; and
flowing a second slurry of cells over the top surface of the substrate so that a second cell is disposed at the opening of the vertical slot in the at least one microtrap.
In another preferred form of the invention, there is provided a vertical cell pairing (VCP) system comprising:
a substrate having a top surface; and
at least one microtrap positioned adjacent the top surface of the substrate, the at least one microtrap comprising a body having a vertical groove and a vertical slot, wherein the vertical groove has an opening and an exit, the vertical groove being sized to seat a pair of cells therein when the pair of cells are vertically aligned, wherein the vertical slot has an opening and an exit, the vertical slot being sized to pass fluid therethrough but to prevent a cell from passing therethrough, wherein the exit of the vertical groove is in fluid communication with the opening of the vertical slot.
In another preferred form of the invention, there is provided a method for vertically pairing cells, the method comprising:
providing a vertical cell pairing (VCP) system comprising:
flowing a first slurry of cells over the top surface of the substrate and seating a first cell in the vertical groove of the at least one microtrap; and
flowing a second slurry of cells over the top surface of the substrate and seating a second cell in the vertical groove of the at least one microtrap.
These and other objects and features of the present invention will be more fully disclosed or rendered obvious by the following detailed description of the preferred embodiments of the invention, which is to be considered together with the accompanying drawings.
The present invention comprises the provision and use of a novel system to study the IS horizontally in a high-resolution and high-throughput manner. Combining micropits with single cell trap arrays, this newly-developed microfluidic platform provides various technical novelties that answer the previously-discussed difficulties in IS imaging. The vertical cell orientation of the paired cells enables the IS to be imaged in a horizontal plane and enhances resolution on both fixed and live cell imaging. This vertical cell pairing (VCP) system can minimize the lateral cell drift at the focal plane and constrain the conjugated cells in the vertical position. This system can also capture more than 3000 conjugates at a time with high loading efficiency. Using this VCP system, a “face-to-face” look at the structure of the human natural killer (NK) cell IS is presented. Compared to conventional cell-cell conjugates, the organization of F-actin at the IS can be clearly observed at the VCP system with high-resolution using conventional confocal microscopy. The VCP system of the present invention was able to detect the positioning of perforin-positive lytic granules over regions of low F-actin density at the IS, a detail which has been previously reported under super-resolution microscopy, but one that has proved difficult to image using conventional approaches. In addition, bright F-actin puncta that were segregated from the cytolytic granules were observed at the center of NK synapse, which is usually indiscernible with conventional confocal microscopy. Furthermore, the novel dynamics of PD-1 microclusters at NK synapse and target cell lysis mediated by NK cells at a real cell-cell interface were also observed in the VCP system. Thus, the VCP system provides a high-throughput, high-efficiency, and user-friendly approach to IS imaging that not only successfully addresses many of the problems of previous techniques, but also possesses broad potential applications in a variety of other biological disciplines.
Looking first at
Prior art micropit system 5 suffers from a number of disadvantages, including (i) low yield (due to the difficulty of inducing a cell to enter micropit 20), and (ii) poor cell seating (the cell of the second cell type may move out of vertical alignment with the cell of the first cell type even when both cells are seated in micropit 20).
Microtrap system 25 suffers from a number of disadvantages, including (i) low yield (due to the difficulty of inducing a cell to enter vertical groove 50), and (ii) low pairing efficiency for two different types of cells (i.e., the same types of cells are paired in majority of the microtraps).
In accordance with the present invention, and looking now at
On account of the foregoing, when a first slurry of cells of a first cell type is flowed over top planar surface 110, a cell of the first cell type will be captured by vertical slot 130 of microtrap 120 directly over micropit 115. Thereafter, when cell pairing system 100 is subjected to a centrifugal force (e.g., using a centrifuge), the cell of the first cell type captured by microtrap 120 will be caused to enter, and seat in, micropit 115. A washing step can then be effected (e.g., flowing a buffer solution over top planar surface 110) so as to wash away any cells which have not entered micropit 115. Then a second slurry of cells of a second cell type is flowed over top planar surface 110 so that a cell of the second cell type is captured by microtrap 120, i.e., so that the cell of the second cell type is disposed atop, and in communication with, the cell of the first cell type which is captured in micropit 115. If desired, cell pairing system 100 may be subjected to a further application of centrifugal force (e.g., using a centrifuge) so as to ensure that the cell of the second cell type (captured by microtrap 120) is disposed in secure contact with the cell of the first cell type (captured in micropit 115). In a preferred form of the invention, a cover (not shown in
Significantly, by pairing microtrap 120 with micropit 115, vertical cell pairing system 100 provides (i) high yield (due to the ease with which a cell of the first cell type can be captured by microtrap 120 and then loaded into micropit 115, and due to the ease with which a cell of the second cell type can be captured by microtrap 120 adjacent to the cell of the first cell type), and (ii) the ensured alignment of the cell of the second type with the cell of the first cell type (due to the alignment of the vertical slot 130 of microtrap 120 with micropit 115).
1. Microfluidic VCP System Fabrication
The schematic flow of the fabrication procedure is summarized in
Polydimethylsiloxane (PDMS, Sylgard 184, Dow Corning Corp.) was mixed with curing agent at a ratio of 10:1. The micropit array mold (bottom mold) was dipped into the PDMS and pressed on a number 1.5 cover glass (170 μm thickness) with 40 Newtons by a heated mechanical press (CH4386, Carver press). The PDMS was cured at 80° C. for 2 hours, and the mold was removed to obtain the bottom layer. The PDMS was poured onto the trap array mold (top mold) and cured at 77° C. for 1 hr. The PDMS was lifted from the mold to obtain the top layer. The inlet hole (4 mm diameter) and negative pressure port (0.5 mm diameter) were punched (Accu-Punch, Syneo) into the top PDMS layer. A drop of methanol was applied for lubrication and the top layer was snapped on the bottom layer to assemble the two layers.
2. Cells Culture
The K562 myelogenous leukemia cell line (American Type Culture Collection, ATCC) was cultured in RPMI medium (Gibco, USA). The KHYG-1 human NK cell line (provided by Dr. David T. Evans, Harvard Medical School) was maintained in R10 medium consisting of RPMI 1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 10 mM HEPES (Gibco, USA), 1 mM sodium pyruvate (Cellgro, USA), 2 mM L-Glutamin (Gibco, USA), and 1% MEM non-essential amino acids solution (Gibco, USA). Additionally, 1 μg/ml cyclosporine A (CsA, Sigma-Aldrich, USA), 50 μg/ml Primocin, and 10 U/ml interleukin-2 (IL-2) were freshly added into the R10 medium for each passage. The culture medium for CD16-KHYG-1 cells was replaced with CsA-free medium one day before use. The Human Embryonic Kidney 293T (HEK293T) cells were cultured in DMEM (Gibco, USA) with 10% FBS, 10 mM HEPES, and 2 mM L-Glutamine.
3. Plasmids and Transduction of CD16-KHYG-1 and K562 Cell Lines
To generate the PD-1-GFP construct, a full length of PD-1-GFP fusion protein sequence (OriGene, MD) was amplified by the primers 5′-AATCCGGAATTCGCCGCCGCGATCGCCATGC-3′ (Forward) and 5′-AATCGCGGATCCTTAAACTCTTTCTTCACC-3′ (Reverse). The PCR product digested with EcoRI and BamHI (Thermo Scientific) was ligated with EcoRI and BamHI digested lentivector pCDH (23). Similarly, to generate the C-terminal mCherry-tagged PD-L1 construct, the PD-L1 cDNA (OriGene, MD) was amplified by the primers 5′-AATCCGGAATTCATGAGGATATTTGCTGTCT-3′ (Forward) and 5′-AATCGCGGATCCCGTCTCCTCCAAATGTGTA-3′ (Reverse). The PCR product was digested with EcoRI and BamHI and ligated with an mCherry-N1 vector (Clontech) to generate C-terminal mCherry-tagged PD-L1. The sequence of PD-L1-mCherry was then amplified by the primers 5′-TAGAGCTAGCGAATTATGAGGATATTTGCTGTCTTTA-3′ (Forward) and 5′-ATTTAAATTCGAATTTCACGCCTTGTACAGCTCGTCC-3′ (Reverse). The PCR product was inserted into EcoRI digested pCDH lentivector by the In-fusion cloning system (Clontech). All plasmids were verified by sequencing.
To generate the PD-1-GFP+ CD16-KHYG-1 and PD-L1-mCherry+ K652 cell lines, both CD16-KHYG-1 and K562 cell lines were transduced with the pCDH cDNA cloning and expression lentivirus system (SBI, System Biosciences), respectively. The lentivirus was generated by co-transfecting Human Embryonic Kidney 293T (HEK293T) cells with a lentiviral vector containing PD-1-GFP or PD-L1-mCherry and three packaging plasmids (pMLg/pRRE, pRSV-Rev, pMD2.g) by Lipofectamine reagent (Invitrogen). Briefly, 0.8 μg total DNA (0.128 μg pCDH, 0.32 μg pMLg/pRRE, 0.16 μg pRSV-Rev, 0.192 μg pMD2.g) was mixed with 4 μl Lipofectamine and added into one well of 293T cells cultured in a 6-well plate with approximately 90% confluence. Viral particles were harvested and filtered by a 0.45 μm filter (GE) after 48 hours transfection. Then, 2×105 CD16-KHYG-1 or K562 cells suspended in 4 ml R10 medium were infected with 4 ml viral supernatant. Transduced cells were cultured for 24 hours in the presence of 8 μg/ml polybrene and subsequently sorted by an Aria II cell sorter (BD). The expression of PD-1-GFP and PD-L1-mCherry were verified by flow cytometry (BD LSR Fortessa).
4. Cell Loading
The fabricated VCP microfluidic system was placed on a vacuum desiccator for 15 min. A 1 ml syringe was connected to the port through a tube to generate negative pressure and a drop of 2% bovine serum albumin (BSA) in phosphate buffered saline (PBS) was introduced into the inlet. The VCP system was incubated for 30 min at 37° C. Then R10 medium was subsequently used to replace the BSA solution in the VCP system. 10 μl of the first cell suspension with a concentration range of 106-107 cells/ml was added into the inlet. The flow rate of 15 μl/min was used to seed the cells using a syringe pump. After 30 s of seeding, the rest of the cell suspension was washed with PBS three times. The microfluidic VCP system was disconnected from the syringe and centrifuged (Sorvall Legend X1R, Thermo scientific) at 2000 rpm (700×g) for 10 min to spin down the cells into micropit array. After centrifugation, the syringe was re-connected and the second cell suspension was added into the inlet. The parameters for seeding the second cell suspension were the same as those for the first cell suspension.
For live cell imaging, the K562 cells stably expressing PD-L1-mCherry were first injected into the VCP system. The VCP system was mounted on the inverted fluorescence microscope stage. A single cell was focused upon with a 63x oil immersion objective lens. Then the CD16-KHYG-1 cells expressing PD-1-GFP were injected. After the effector cells were anchored on top of the target cells, the cell suspension in the inlet was replaced with fresh cell medium containing 25 mM HEPES buffer. Live cell imaging was performed at the synapse plane at every 30 s for 10 min. During the live cell imaging, the flow rate is maintained at 0.5 μl/min. For fixed cell imaging, the CD16-KHYG-1 cells were injected first. The VCP system was incubated for 1 hour at 37° C. The cell-cell conjugates were fixed by 4% formaldehyde solution in PBS for 15 min, then washed with PBS for 5 min. Permeabilization buffer containing 5% of normal donkey serum (NDS) and 0.5% Triton X-100 in PBS was added into the VCP system. The VCP system was incubated in 4° C. overnight. The permeabilization buffer was washed with PBS for 5 min. Then, primary antibody against α-tubulin (Abcam) in antibody buffer containing 3% NDS and 0.5% Triton X-100 in PBS was pumped into the VCP system. The VCP system was incubated at 4° C. overnight. The antibody buffer was washed with PBS for 5 min. The cells were stained by fluorescently labeled secondary antibody (Life Technologies). The antibody buffer was washed with PBS for 5 min, and finally, a drop of ProLong Gold antifade reagent mounting medium (Life Technologies) was added into the VCP system.
5. Confocal Microscope
High-resolution images were captured with a confocal fluorescence microscope (Leica TCS SP8, Leica Microsystem) equipped with a 63x oil immersion objective lens (NA 1.47, Leica Microsystem). In the fixed cell images, z-stacks of tubulin, perforin and actin were sequentially captured. The high-resolution live cell images were carried out at 37° C. 4.5 μm z-stacks centered at the IS were acquired to account for movement within the live cell. The fluorescence from PD-1-GFP, PD-L1-mCherry, and the bright field image was detected simultaneously. The images were acquired by LAS AF software (Leica) and analyzed with ImageJ and Imaris (Bitplane) software.
1. Design Optimization of Vertical Cell Pairing Microfluidic System
The development and optimization process of the VCP system design is summarized in
The fabrication method is described in greater detail in the Materials And Methods section above. Briefly, a polydimethylsiloxane (PDMS) pre-polymer-coated mold of the bottom layer, which contains 4000 individual micropits, was directly stamped onto a No. 1.5, 170 μm thick cover glass. The top layer, which includes the microtrap array, was prepared by standard photolithography procedures.
The microfluidic VCP system features four areas including inlet, negative pressure port, zone 1, and zone 2 to control the flow rate and direction (
The gravitational force (red arrow in
To test the loading efficiency of the VCP system, the fraction of the captured cells in each step was measured as shown in
2. High-Resolution Imaging Intracellular Structure of IS on Fixed Cells by VCP System
After successfully developing this VCP system, further testing was done to determine whether it would permit high-resolution imaging with a conventional confocal microscope, a common instrument in most research institutes. CD16-KHYG-1 cells were used as effector cells and the K562 cell line as susceptible target cells. The CD16-KHYG-1 effector cells were loaded into the VCP system, followed by the target cells. After fixation, cell-cell conjugates were stained for F-actin, perforin, and tubulin. As a control, conventional microscopy slides were prepared simultaneously, as previously described. Three-dimensional (3D) images were acquired in each fluorescence channel. After reconstruction of the 3D-stack, a z-projection of the interface between two cells was presented (
The representative image obtained by the VCP system demonstrates significant improvements over that of the image obtained by the conventional method in two respects. First, the present system prevents deformation of the IS. In the traditional method, cells spread out due to the interaction between the plasma membrane and the polylysine-coated coverslip (
To further quantify the unique features of F-actin puncta that appear well segregated from the perforin positive cytolytic granules in the IS, colocalization analysis was carried out and Pearson's correlation coefficient (Pearson's r) was calculated for the images obtained using the VCP system and the conventional method (
3. High-Resolution Imaging of Dynamics of PD-1 Microclusters on Live Cells by VCP System
Live imaging provides unprecedented information on the dynamics of the inhibitory IS, which cannot be obtained from images of fixed cells. After successfully demonstrating the high-resolution imaging of the IS made possible by the VCP system, the VCP system was further tested to determine whether the VCP system allows live-cell imaging of the IS. Here, a fluorescently tagged programmed cell death-1 (PD-1) was used, an important inhibitory receptor expressed on lymphocytes, as a mode to study the dynamics of the inhibitory synapse. To test whether the VCP system could be used to visualize the dynamics of PD-1 at the IS, CD16-KHYG-1 cells expressing PD-1-GFP were used, together with susceptible K562 target cells expressing PD-1 ligand (PD-L1)-mCherry (
There was also observed a second pattern of PD-1/PD-L1 microcluster movement within the synapse, in which PD-1/PD-L1 does not form a centralized cluster during the 10 min time-lapsed imaging acquisition (
In addition to the “D→C→D” and “sD” patterns, there was also observed that a small minority of PD-1/PD-L1 microclusters (9.1%, 2 out of 22 conjugates) form at the periphery of the IS and coalesce at the center of the synapse, where they remain clustered (
From these observations it was concluded that the dynamics of inhibitory synapse microclusters in live cells can be readily observed in the VCP system. It is believed that this is the first observation of single microcluster dynamics at IS in a real cell-cell conjugate with high resolution. Meanwhile, the distinct patterns of PD-1/PD-L1 microclusters in the IS is a striking feature of inhibitory synapses formed between PD-1 positive NK cells and PD-L1 positive target cells.
4. Kinetics of Live NK Cell Cytotoxicity Detected by the VCP System
Next, the VCP system was tested to see whether cytolytic killing could be monitored by the VCP system in live cells. To test this, K562 cells were loaded with calcein AM green viability dye. To distinguish the effectors from the target cells, CD16-KHYG-1 cells were labeled with CellTracker red. Cell-cell conjugates were imaged by wide-field fluorescence microscopy (
In addition to the “slow decay” fluorescence profile from calcein AM green, there was also observed three different kinetics of disappearance of calcein AM green in K562 cells. The second pattern of calcein AM (33.8%, 69 out of 204 conjugates) displayed a “single-drop” profile, indicating lysis of K562 cells by NK cells (
The third fluorescence profile of calcein AM (11.3%, 23 out of 204 conjugates) was termed “fast-decay” (
Notably, multiple stepwise decreases over time (“multiple-drop” profile) in calcein AM fluorescence (9.3%, 19 out of 204 conjugates) were also observed (
Altogether, these data show that the kinetics of cytotoxicity over time can be readily detected by the VCP system in both a high-throughput manner and at the single cell level, refinements which are all lost by conventional 4-hour 51Cr release assay, which only provides a blanket measure of killing activity over the entire culture after a certain time period, and is incapable of distinguishing between individual cytolytic and non-cytolytic cell pairings.
The immunological synapse is a critical platform for mediating an effective immune response in both the adaptive and innate immune systems. Current research on synaptic geometry is restricted in artificial systems such as antibody-coated coverslips or glass-supported planar lipid bilayer systems. The present invention provides a novel high-throughput VCP microfluidic system to study the IS in an actual cell-cell communication setting. A detailed protocol for generating this system is provided here. Using this system, a high-resolution image of the IS formed between and effector and target cell has been achieved under conventional confocal microscopy. Additionally, potential applications of this system have been demonstrated by investigating the dynamics of PD-1/PD-L1 microcluster formation at the IS, providing the first observation of microclusters at the IS in vertically oriented real cell-cell conjugates, as well as the kinetics of killing at the single cell level. The present invention provides a user-friendly VCP system and demonstrates the feasibility and application of this novel technique for studying cell-cell communications in a variety of disciplines. In addition, by using this VCP system, it is possible to provide insights on several significant biological questions.
Compared to the micropit system developed by Biggs and colleagues, the VCP system possesses two distinct advantages. The rate of successful vertical conjugates is significantly higher in the VCP system (˜73% vs. 10-15%) due to the novel combination of the micropit system with a single cell trap array, which efficiently guides the cells into the pits. Secondly, the VCP system addresses one of the major difficulties of the original micropit system, wherein there was an unavoidable tradeoff between loading efficiency (i.e., success rate of two-cell pairs entering single pits) and vertical orientation. In the micropit system, a larger pit diameter allows for higher loading efficiency, but because of the large pericellular area of the micropit, the top cell will often not be perfectly aligned on top of the bottom one.
Conversely, a narrower pit diameter significantly decreases the loading efficiency. Guiding the cells into narrow micropits by the single cell trap array, the VCP system is able to maintain a vertical orientation without compromising loading efficiency.
As a result of its innovative design, the VCP system has addressed a number of significant questions. The organization of F-actin at cytotoxic immunological synapse is still highly controversial. In the T cell IS, it has been shown that F-actin is totally cleared from the center of the synapse, which facilities degranulation events at the center of IS. However, this phenomenon is different in the NK cell synapse. Using super-resolution stimulated emission depletion (STED) and structured illumination microscopy (SIM), two independent research groups have shown that F-actin is not totally cleared at the center of IS. Instead, a punctuated, low density F-actin meshwork has been observed at the center of the NK cell synapse, where perforin-positive cytolytic granules are well segregated by the F-actin puncta. In agreement with these observations, perforin-positive cytolytic granules are seen positioned at low-density F-actin regions in the VCP system, a crucial detail that is usually indiscernible with conventional confocal microscopy. Using this VCP system, for the first time, it was cleanly observed that perforin-positive cytolytic granules are located among low density F-actin regions in the center of the IS formed between NK and target cells, without the need for super-resolution imaging techniques. Therefore, the results obtained with the VCP system support the phenomenon that F-actin “hypodensities” are present at the center of the IS in human NK cells and that cytolytic granules converge at these hypodensities.
In addition to the information gained from fixed-cell imaging, live imaging with the VCP system provides additional, critical information on the dynamics of inhibitory immunological synapses not available from previous studies of fixed NK cells. Programmed cell death protein 1 (PD-1) is an emerging immune checkpoint protein highly up-regulated in T, B, and NK cells in the setting of chronic viral infection and tumorigenesis. Similar to other inhibitory receptors such as KIR and CD94/NKG2A, the intracellular domain of PD-1 contains an immunoreceptor tyrosine inhibitory motif (ITIM), which plays a critical role in NK cell inhibition.
Engagement of PD-1 with PD-L1 inhibits cytotoxic killing of K562 target cells by CD16-KHYG1 cells, which indicates the occurrence of an inhibitory synapse. However, whether NK cells can form a stable inhibitory synapse is still controversial. Previous studies have shown that NK cells do not form stable synapses in the presence of inhibitory receptors. Using the VCP platform, it was observed that the microclusters of PD-1 and PD-L1 at the IS coalesce into a central cluster at the synapse. This pattern is reminiscent of previous studies that show that human primary NK cells form stable inhibitory synapse on lipid bilayers carrying HLA-E, a ligand for inhibitory receptor CD94/NKG2A. In addition, the results obtained by this novel VCP system demonstrate three distinct PD-1/PD-L1 microcluster patterns at the NK IS, which is the first to describe the dynamics of PD-1 microclusters in a real cell-cell conjugate at high resolution without the need of an expensive super-resolution imaging system.
An understanding of the forces that govern the stability of the IS is essential to identify its function. One of the most important functions of the IS is to mediate directed cytotoxicity towards target cells. Typical methods of monitoring NK cell cytotoxicity, such as 51Cr release assays with obvious radiation safety implications, are expensive and time-consuming. The VCP platform discussed herein allows rapid, quantitative, high-throughput functional measurements to monitor NK cell cytotoxicity in real time. The disappearance of the dye in the target cell within a single pit provides a clear readout for cell death at the single cell level, unobscured by the presence of other effector-target pairs, which can be an issue in a large open-well system. Therefore, in addition to providing a platform for enhanced study of the IS, the VCP system provides an alternate method for studying IS-mediated cellular cytotoxicity.
Thus it will be seen that there has been developed a novel, high-throughput VCP system to study cell-cell communications for both fixed and live cell imaging. The feasibility and potential application of this VCP system has been successfully demonstrated. This high-throughput and user-friendly VCP system offers a powerful new imaging platform that can be used to address a number of significant questions in immunology and cell biology, such as single cell analysis, cell fusion, cell-cell communication, and cell surface ligand mobility.
It should be understood that many additional changes in the details, materials, steps and arrangements of parts, which have been herein described and illustrated in order to explain the nature of the present invention, may be made by those skilled in the art while still remaining within the principles and scope of the invention.
This patent application claims benefit of: (i) pending prior U.S. Provisional Patent Application Ser. No. 62/155,762, filed May 1, 2015 by The Methodist Hospital and Joon Hee Jang et al. for HIGH-RESOLUTION IMAGING OF CELL-CELL COMMUNICATION (Attorney's Docket No. METHODIST-21 PROV); and (ii) pending prior U.S. Provisional Patent Application Ser. No. 62/243,257, filed Oct. 19, 2015 by The Methodist Hospital and Lidong Qin et al. for HIGH-RESOLUTION IMAGING OF CELL-CELL COMMUNICATION (Attorney's Docket No. METHODIST-21R PROV). The two (2) above-identified patent applications are hereby incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2016/030408 | 5/2/2016 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62243257 | Oct 2015 | US | |
| 62155762 | May 2015 | US |
