Name: an endothelial planar cell model for imaging immunological synapse dynamics
Uploaded: 2015-12-24T13:00:00.000+00:00
Duration: 9 min 25 s
Description: Watch this Scientific Journal Video about An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics at JoVE.com

We thank Dr. Peter T. Sage for his assistance in generating some of the representative images. This work was supported by an NIH R01 grant to C.V.C. (HL104006).

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The authors have nothing to disclose.

Overall, this protocol describes methods for investigating endothelial cells as i) understudied physiologic APCs and ii) as a novel type of &#39;planar cellular APC model&#39;. With respect to the former, it has become increasingly appreciated that non-hematopoietic peripheral (or &#39;stromal&#39;) APCs play critical, non-redundant roles (i.e., compared to hematopoietic APCs) in shaping adaptive immune responses 16-18. Among such &#39;semi-professional&#39; APCs, vascular and lymphatic endothelial cells (which vastly outnumber professional APCs) are among the best appreciated 16-18. However, the details of signaling events and their coupled immunological synapses remain largely uninvestigated. The methods described herein provide a basis for advancing understanding in this emerging area. For example, applying these approaches to the study of ISs formed on endothelial from different tissue that are known to exhibit distinct adaptive immunological function (e.g., liver and lymphatic endothelia) may be particularly instructive.
Significantly, this method helps to bridge the gap between professional APCs and artificial APC substrate models in order to enhance the ability to interrogate basic mechanisms of adaptive immune responses. Whereas planar APC substrates (i.e., lipid bilayers, antibody-coated glass) provide optimal imaging (which has lead to many critical insights 7,11-14), such models are inherently limited. Alternatively, details of physiologic cell-cell scanning dynamics have thus far been profoundly obscured by orientation-related imaging issues 8-10. The imaging capabilities provided in the current approach overcome this obstacle to uniquely reveal otherwise undetectable three-dimensional subcellular architecture and rapid remodeling of a physiologic APC-T cell interface. These offer potential for new understanding of antigenic signaling.
For example, the appreciation that T cells form ILPs enriched with TCR, actin and signaling molecules (e.g., Figure 2D, Figure 3), suggest that these likely represent the physiologic 3-dimensional counter parts to the planar TCR signaling micro-clusters observed on artificial APC substrates 6,7,12-14,29-31. This implies that under physiologic settings, lymphocytes may employ ILPs as discrete subcellular &#39;3D-reaction volumes&#39; with &#39;signalosome&#39;-like properties that amplify and sustain signaling by concentrating important molecules/activities 32,33. Additionally, the presence of significant biomechanical inputs (i.e., cell-cell force application) at sights if ILP protrusion against the APC can be inferred from the imaging. This is nontrivial as mechanical forces are increasingly viewed as critical facilitators of antigenic signaling, while precisely how such forces are manifested remains unclear 14,34-37.
The overall relevance of this model as a reasonable surrogate for professional APCs is supported by our direct demonstration that similar ILP arrays could be seen in synapses formed on ECs and DCs (and to a lesser extent B cells) when analogous imaging approaches were employed (e.g., use of the same fluorescence makers and control of the IS plane orientation plane 19). Nonetheless, it needs to be considered that the relative biomechanical properties of EC versus professional APC membrane surfaces are unknown and that differences in this parameter could affect details of IS topology and antigenic responses.
The key to the approach described herein is strongly related to its resolution benefits. This, in turn is highly related to the strength of the fluorescence signal. Thus, it is critical to pay special attention in optimizing transfection and staining of fluorescent reporters and imaging parameters/technique (e.g., exposure time, focus, etc&#8230;). With respect to the former, the protocol for live-cell studies that here described are limited to transfection of generic membrane and cytoplasmic markers that report topological changes at the T cell-APC interface in real-time (e.g., Figure 2, Movies 1, 2). Additionally, this protocol can be readily modified to for more sophisticated analysis by concomitantly introducing/imaging further reporters in the endothelium (e.g., fluorescent protein-tagged MHCI/II and adhesion, co-stimulatory and co-inhibitory molecules and biosensors for signaling and biomechanical dynamics). Unfortunately, however, a general limitation is that lymphocytes are notoriously difficult to transfect. This precludes convenient use of fluorescent proteins (e.g., tagged to TCR, PKC-Q, etc.) in T cells for live cell imaging.
While the current protocol involves activation of human effector/memory CD4+ Th1 type lymphocytes through SAg (a widely used model), much broader possibilities exist. As previously demonstrated 19,20 this approach is readily adaptable to a broad range of other settings such as responses of other CD4+ subsets and activation of CD8+ lymphocyte killing responses. Through isolation of lymphocytes and endothelial cells from existing TCR-transgenic strains this method is also readily adaptable to study responses to specific peptide antigens 19,20. Finally, the current approach is limited to examination of effector/memory T cells. However, transfection of endothelial cells with the essential co-stimulatory molecules (CD80/86, not normally expressed on endothelium) might allow for study of na&#239;ve cell priming dynamics in this model.

T lymphocytes are a branch of the adaptive immune system characterized by the ability to efficiently recognize peptide antigen (Ag) bound to major histocompatibility complex (MHC) molecules through their T cell receptors (TCRs) 1. Na&#239;ve lymphocytes constitutively migrate and scan &#39;professional Ag presenting cells&#39; (APCs; e.g., dendritic cells) within lymph nodes, while memory/effector T cells need to effectively survey an extremely wide range of APCs and potential target cells within peripheral tissues.
In the min following initial recognition of cognate Ag on an APC, lymphocytes arrest their migration and begin to form a specialized intimate cell-cell interface termed &#39;immunological synapse&#39; (IS). Sustained (i.e., 30-60 min) IS contacts are required to amplify and sustain signaling 2-7. Emerging studies identify that within the IS, it is the continuous formation and rapid remodeling of discrete sub-cellular signaling micro-clusters (i.e., containing MHC/Ag-TCR, F-actin, adhesion and signaling molecules) that determine the strength and quality of resulting immune responses 2-7. However, dynamic details and regulatory mechanism of this process are incompletely understood 8,9. This stems largely from technical challenges associated with irregular topologies of APC surfaces and poorly controlled orientation of the cell-cell interaction planes, issues that profoundly limit the requisite spatiotemporal imaging approaches 8-10 (Figure1A).
<img alt="Figure 1" src="/files/ftp_upload/53288/53288fig1.jpg" />
Figure 1.&#160;A Physiologic Planar Cell APC Model for Imaging Immunological Synapse Dynamics. The schematic illustrates traditional imaging of immunological synapse between a T cell and a professional APC (A) and T cell and a traditional planar lipid bilayer APC model (B) in comparison to this novel endothelial planar APC model (C). Professional APCs provide physiologic immunological synapses but offer poorly oriented cell-cell interface (i.e., with respect to the optimal x-y imaging plane; resolution ~0.2 &#956;m), which dramatically compromises spatial (z imaging plane resolution ~1 &#956;m) and temporal (i.e., due to the need to repeatedly scan through all z imaging planes) resolution of imaging. Bilayer models have a planar topology that provides optimal spatiotemporal imaging resolution, but are also highly simplified, non-physiological and rigid. This endothelial cell model combines the planar topology of lipid bilayers with the physiologic substrate of a classic APC to deliver optimal spatial and temporal imaging resolution in a physiologic setting. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53288/53288fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Previous work has partially circumvented these obstacles by developing planar substrate models (i.e., lipid bilayers and antibody-coated surfaces) that provide optimal spatiotemporal resolution (i.e., via fixing the T cell activation surface into a single plan that is parallel to the optimal x-y imaging plane) 11-15 (Figure 1B). These models have facilitated important insights into the subcellular/molecular dynamics that control antigenic signaling in T cells, including the discovery of dynamic actin/TCR signaling micro-clusters 7,11-14. However, such models are inherently oversimplified, as well as rigid (precluding the development/study of 3-dimensional topological features) (Figure 1B). Therefore, it remains uncertain how to relate such findings to physiologic cell-cell immune surveillance.
Though still understudied, vascular and lymphatic endothelial cells are emerging as a large (i.e., greater in numbers than all professional APCs, by ~1,000-fold) peripheral compartment of &#39;semi-professional&#39; APCs 16-18. These cells express MHC-I-, MHC-II- and a multitude of co-stimulator molecules (e.g., CD40, LFA3, ICOSL, 4-1BB, OX40L, TL1A, PD-L1; but not CD80 and CD86) and are strategically positioned at the blood-tissue interface where they serve specialized sentinel functions 16-18. Previous studies demonstrated that endothelial cells can effectively re-stimulate effector/memory, but not na&#239;ve, T cells 19-25. Thus, endothelial cells are likely to play unique APC roles in effector phase of adaptive immune responses within the peripheral tissues, such as local influence on T cell activation, differentiation, memory and tolerance 16,17,26. Critically, when grown in vitro, endothelial cells form virtually planar cell surfaces and are readily transfectable (e.g., with fluorescent protein reporters). These features are ideal for high spatiotemporal resolution imaging of topological dynamics during cell-cell interactions 19,27. Thus endothelial cells might serve as a physiologic &#39;planar cellular APC&#39; model distinctly suited for the study of the subcellular/molecular remodeling mechanisms that drive antigen recognition and regulate responses (Figure 1C) 19,20.
Previously established complementary imaging techniques (including transfection of endothelia cells with fluorescent protein makers of the plasma membrane and cytosol) for studying the details of leukocyte-endothelial interaction during adhesion and transendothelial migration 27, showed that leukocytes actively probe the surface of the endothelium by dynamic insertion and retraction of sub-micron-scale, actin-rich cylindrical protrusions (~200-1,000 nm in diameter and depth) termed invadosome-like protrusions (i.e., &#39;ILPs&#39;) 27,28. These imaging approaches have been further expanded along with the creation of protocols to take advantage of endothelial APC function to develop the first methods for high spatiotemporal resolution imaging of the T cell-endothelial immunological synapse as reported 19,20 and further describe herein. A central finding derived from this novel planar cellular APC model is that T cell ILPs function both in promoting initial Ag detection and in sustaining subsequent signaling. Indeed, arrays of multiple ILPs (that were stabilized and accrued in response to initial calcium flux) show enrichment in TCR and molecules suggestive of active signaling such PKC-Q, ZAP-70, phosphotyrosine and HS1. Therefore, ILPs seem to represent a three-dimensional physiologic equivalent to the TCR-signaling microclusters seen in planar bilayer models. This approach, thus, sensitively reveals/reports molecular and architectural (and implied biomechanical) dynamics not otherwise detectable.
The method described herein should be useful to bridging the gap between professional APC and artificial APC substrate models in order to enhance our ability to interrogate basic mechanisms of adaptive immune responses. While here the focus is on the activation of CD4+ Th1-type effector/memory cell, this basic approach can be readily modified to study a wide range of T cell types and Ags, as discussed below.

<table><tbody><tr><td>BD Vacutainer stretch latex free tourniquet</td><td>BD Biosciences</td><td>367203</td><td></td></tr><tr><td>BD alcohol swabs</td><td>BD Biosciences</td><td>326895</td><td></td></tr><tr><td>BD Vacutainer Safety-Lok</td><td>BD Biosciences</td><td>367861</td><td>K2 EDTA</td></tr><tr><td>BD Vacutainer Push Button Blood Collection Set</td><td>BD Biosciences</td><td>367335</td><td></td></tr><tr><td>RPMI-1640</td><td>Sigma-Aldrich</td><td>R8758-1L</td><td></td></tr><tr><td>Ficoll-Paque</td><td>Sigma-Aldrich</td><td>GE17-1440-02</td><td>Bring to RT before use</td></tr><tr><td>FCS-Optima</td><td>Atlanta Biologics</td><td>s12450</td><td>Heat inactivated</td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma-Aldrich</td><td>P4458-100ML</td><td></td></tr><tr><td>Trypan blue</td><td>Sigma-Aldrich</td><td>T8154-20ML</td><td></td></tr><tr><td>staphylococcal enterotoxin B</td><td>Toxin Technology</td><td>BT202RED</td><td>Stock solution 1mg/ml in PBS</td></tr><tr><td>toxic shock syndrome toxin 1</td><td>Toxin Technology</td><td>TT606RED</td><td>Stock solution 1mg/ml in PBS</td></tr><tr><td>human IL-15</td><td>R&amp;amp;D Systems</td><td>247-IL-025</td><td>Stock solution 50ug/ml in PBS</td></tr><tr><td>PBS</td><td>Life Technologies</td><td>10010-049</td><td></td></tr><tr><td>Fibronectin</td><td>Life Technologies</td><td>33016-015</td><td>Stock solution 1mg/ml in H20</td></tr><tr><td>HMVEC-d Ad-Dermal MV Endo Cells</td><td>Lonza</td><td>CC-2543</td><td>Other Human Microvascular ECs can be used, i.e. HLMVECs</td></tr><tr><td>EGM-2 MV bullet kit</td><td>Lonza</td><td>CC-3202</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Sigma-Aldrich</td><td>T-4174</td><td>Stock solution 10x, dilute in PBS</td></tr><tr><td>amaxa-HMVEC-L Nucleofector Kit</td><td>Lonza</td><td>vpb1003</td><td>Required Kit for step 4</td></tr><tr><td>IFN-g</td><td>Sigma-Aldrich</td><td>I3265</td><td>Stock solution 1mg/ml in H20</td></tr><tr><td>TNF-alpha 10ug, human</td><td>Life Technologies</td><td>PHC3015</td><td>Stock solution 1mg/ml in H20</td></tr><tr><td>phenol Red-free HBSS</td><td>Life Technologies</td><td>14175-103</td><td></td></tr><tr><td>Hepes</td><td>Fisher Scientific</td><td>BP299-100</td><td></td></tr><tr><td>Calcium Chloride</td><td>Sigma-Aldrich</td><td>C1016-100G</td><td>Stock solution 1M in H20</td></tr><tr><td>Magnesium chloride</td><td>Sigma-Aldrich</td><td>208337</td><td>Stock solution 1M in H20</td></tr><tr><td>Human Serum albumin</td><td>Sigma-Aldrich</td><td>A6909-10ml</td><td></td></tr><tr><td>Immersol 518 F fluorescence free Immersion oil</td><td>Fisher Scientific</td><td>12-624-66A</td><td></td></tr><tr><td>Fura-2 AM 20x50ug</td><td>Life Technologies</td><td>F1221</td><td>Stock solution 1mM in DMSO</td></tr><tr><td>pEYFP-Mem (Mem-YFP)</td><td>Clontech</td><td>6917-1</td><td></td></tr><tr><td>pDsRed-Monomer (Soluble Cytoplasmic DsRed)</td><td>Clontech</td><td>632466</td><td></td></tr><tr><td>pDsRed-Monomer Membrane (Mem-DsRed)</td><td>Clontech</td><td>632512</td><td></td></tr><tr><td>pEGFP-Actin</td><td>Clontech</td><td>6116-1</td><td></td></tr><tr><td>Alexa Fluor 488 Phalloidin</td><td>Life Technologies</td><td>A12379</td><td></td></tr><tr><td>Formaldehyde solution 37%</td><td>Fisher Scientific</td><td>BP531-500</td><td>Toxic, use fumehood</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100-5ML</td><td></td></tr><tr><td>&amp;nbsp;Falcon 15mL Conical Centrifuge Tubes</td><td>Fisher Scientific</td><td>14-959-70C</td><td></td></tr><tr><td>Falcon 50mL Conical Centrifuge Tubes</td><td>Fisher Scientific</td><td>14-959-49A&amp;nbsp;</td><td></td></tr><tr><td>Falcon Tissue Culture Treated Flasks T25</td><td>Fisher Scientific</td><td>10-126-9</td><td></td></tr><tr><td>&amp;nbsp;Falcon Tissue Culture Treated Flasks T75</td><td>Fisher Scientific</td><td>&amp;nbsp; 13-680-65</td><td></td></tr><tr><td>&amp;nbsp;Corning Cell Culture Treated T175</td><td>Fisher Scientific</td><td>10-126-61&amp;nbsp;</td><td></td></tr><tr><td>Glass coverslips&amp;nbsp;</td><td>Fisher Scientific</td><td>12-545-85&amp;nbsp;</td><td>12 mm diameter</td></tr><tr><td>&amp;nbsp;Falcon Tissue Culture Plates 24-well</td><td>Fisher Scientific</td><td>08-772-1</td><td></td></tr><tr><td>Delta-T plates</td><td>Bioptechs</td><td>04200415B</td><td></td></tr><tr><td>Wheaton Disposable Pasteur Pipets</td><td>Fisher Scientific</td><td>13-678-8D</td><td></td></tr><tr><td>1.5 ml Eppendorf tube&amp;nbsp;</td><td>Fisher Scientific</td><td>05-402-25</td><td></td></tr><tr><td>&amp;nbsp;ICAM1 mouse anti-human</td><td>BD Biosciences</td><td>555509</td><td></td></tr><tr><td>HS1 mouse anti-human</td><td>BD Biosciences</td><td>610541</td><td></td></tr><tr><td>Anti-Human CD11a (LFA-1alpha) Purified</td><td>ebioscience</td><td>BMS102</td><td></td></tr><tr><td>Anti-Human CD3 Alexa Fluor&amp;reg; 488</td><td>ebioscience</td><td>53-0037-41</td><td></td></tr><tr><td>Anti-MHC Class II antibody&amp;nbsp;</td><td>Abcam</td><td>ab55152</td><td></td></tr><tr><td>Anti-Talin 1 antibody</td><td>Abcam</td><td>ab71333</td><td></td></tr><tr><td>Anti-PKC theta antibody&amp;nbsp;</td><td>Abcam</td><td>ab109481</td><td></td></tr><tr><td>phosphotyrosine (4G10 Platinum)</td><td>Millipore</td><td>50-171-463</td><td></td></tr><tr><td>Nucleofector II</td><td>Amaxa Biosystems</td><td></td><td>Required electroporator for step 4</td></tr><tr><td>Zeiss Axiovert</td><td>Carl Zeiss MicroImaging</td><td></td><td></td></tr><tr><td>Zeiss LSM510&amp;nbsp;</td><td>Carl Zeiss MicroImaging</td><td></td><td></td></tr><tr><td>Zeiss Axiovison Software</td><td>Carl Zeiss MicroImaging</td><td></td><td></td></tr><tr><td>NU-425 (Series 60) Biological Safety Cabinet</td><td>NuAIRE</td><td>Nu-425-600</td><td></td></tr><tr><td>&amp;nbsp;Forma STRCYCLE 37 &amp;deg;C, 5% CO2 Cell culture Incubator</td><td>Fisher Scientific</td><td>202370</td><td></td></tr><tr><td>Centrifuge 5810</td><td>Eppendorf</td><td>EW-02570-02</td><td></td></tr><tr><td>Hemocytometer</td><td>Sigma-Aldrich</td><td>&amp;nbsp;Z359629</td><td>Bright-Line Hemocytometer</td></tr><tr><td>Isotemp Waterbath model 202</td><td>Fisher Scientific</td><td>15-462-2</td><td></td></tr></tbody></table>

an endothelial planar cell model for imaging immunological synapse dynamics

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological synapses&#39;. Within these intimate cell-cell interfaces discrete sub-cellular clusters of MHC/Ag-TCR, F-actin, adhesion and signaling molecules form and remodel rapidly. These dynamics are thought to be critical determinants of both the efficiency and quality of the immune responses that develop and therefore of protective versus pathologic immunity. Current understanding of immunological synapses with physiologic APCs is limited by the inadequacy of the obtainable imaging resolution. Though artificial substrate models (e.g., planar lipid bilayers) offer excellent resolution and have been extremely valuable tools, they are inherently non-physiologic and oversimplified. Vascular and lymphatic endothelial cells have emerged as an important peripheral tissue (or stromal) compartment of &#39;semi-professional APCs&#39;. These APCs (which express most of the molecular machinery of professional APCs) have the unique feature of forming virtually planar cell surface and are readily transfectable (e.g., with fluorescent protein reporters). Herein a basic approach to implement endothelial cells as a novel and physiologic &#39;planar cellular APC model&#39; for improved imaging and interrogation of fundamental antigenic signaling processes will be described.

A novel imaging approach using endothelial cells and combining the resolution advantages of the planar lipid bilayers model with the physiologic complexity and deformability of professional APCs was developed (Figure 1). Figure 2 provides examples of typical migration, calcium flux and topological dynamics observed with this approach. In the absence of SAg on endothelium, SAg-specific CD4+ Th1 lymphocytes rapidly spread, polarize and laterally migrate over the endothelial surface (Figure 2A) without fluxing calcium (not shown). Within ~5-10 min these lymphocyte initiate transmigration across the endothelium. In the presence of SAg loading onto endothelium, lymphocytes spread symmetrically with a &#39;fried egg&#39; topology, initiate sustained (i.e., for 30-60 min) intracellular calcium flux (Figure 2B; Movie 1) during which time they exhibit little or no migration (Figure 2C).
<img alt="Figure 2" src="/files/ftp_upload/53288/53288fig2.jpg" /> 
Figure 2.&#160;Live-Cell Analysis of T cell Activation and Migration and Immunological Synapse Topology of Endothelial APCs. (A) Panels show an example of time-series imaging of T cell migration on endothelium in the absence of SAg. Upper Panel shows DIC images. The initial position of the migrating T cell is indicated by the red dotted line. Lower Panel shows invaginations formed on the endothelium by of the migration T cell. Arrows indicate formation of transient rings (~0.5-1 &#956;m diameter) of membrane-YFP fluorescence formed as a result of T cell generation of &#39;invadosome-like protrusions&#39; (ILPs) against the endothelial APC surface (see D). (B) Shows an example of a similar series in the presence of SAg-loaded endothelium (left; See also corresponding Movie 1) and quantitative trace of the dynamic changes in calcium levels (right). Arrows indicates initial ILPs formation that correlates with the initiation of calcium flux (i) and subsequent stabilization of multiple ILPs that correlates with high calcium flux (ii). (C) Experiments as in A and B were subjected to cell tracking analysis. DIC images on left show representative T cell migration paths over the duration of 10 min (red traces). Graph on right shows the calculated migration velocities in the absence and presence of SAg. (D) Dynamic analysis of immunological synapse topology (modified from 19). Schematic (i) illustrates a sensitive endothelial cell/APC &#39;topology reporter system&#39; consisting of membrane-targeted-YFP and soluble cytosolic RFP. Actin-mediated ILP that protrude into the endothelial cell surface generate cylinder-shaped invaginations (a type of cellular foot-print, termed a &#39;podo-print&#39;) giving rise to sharply bent membrane that appear as rings of membrane-YFP fluorescence (i.e., the walls of the cylinder viewed en face). Cytosol displacement and exclusion at these loci appear as dark circles of cytosolic RFP. DIC and fluorescence images in (ii) show an example of such analysis in the presence of SAg (See also corresponding Movie 2). A single initial podo-print/ILP develops into an array of &#62;20 stabilized ILPs over 15 min. Blue box at 15 min (iii) illustrates a high-resolution view of a single podo-print/ILP whereby a ring of membrane-YFP overlaps with a region of excluded cytosolic RFP. Dashed line (iv) shows a quantitative line scan analysis of fluorescence intensities. Scale bars = 5 &#956;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53288/53288fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
As previously established, lymphocytes actively probe the surface of the endothelium by extending actin and HS1 (a cortactin homologue)-enriched and WASp- and src-dependent protrusions termed &#39;invadosome-like protrusions; (ILPs; each ~0.5-1 &#956;m in depth in diameter) 27,28. Transfection of membrane-targeted fluorescence protein (FP) has been demonstrated to provide highly sensitive reporters for subcellular topological dynamics at cell-cell surfaces. In this way, it has been shown that bright rings of fluorescent membrane-FP are indicative of membrane bending during T cell-endothelial interaction as a result of T cell ILPs protrusions. It is shown here, and in previous studies 19,20, that whereas small clusters of ILPs form and turnover rapidly (i.e., lifetimes of ~10-30 sec) during lateral migration of T cells on endothelium in the absence of SAg (Figure 2A), they become highly stabilized (i.e., lifetimes of ~30 min) and accumulate into dense arrays in the presence of SAg (Figure 2B). Moreover, analysis of the ILPs and calcium initiation kinetics (&#39;Offset time&#39;) show that ILPs precede calcium signaling, suggesting they aid in the MHC-II/Ag recognition process (Figure 2Bii). Additionally, stabilized ILPs arrays form commensurate with maximal calcium flux (Figure 2Bii). The idea that the rings of membrane fluorescence correspond to T cell protrusions driving discrete invagination spots in endothelial APCs, is confirmed by the demonstration that these rings in all cases correlate spatially and kinetically with zones of displaced/excluded cytoplasm (as reported by soluble DsRed; Figure 2D and Movie 2).
Figure 3 provides examples of fixed endpoint confocal imaging studies. After 10 min of co-incubation in the presence of SAg, ISs formed between T cells and endothelial APCs were imaged by fixation, immuno-fluorescence staining and confocal microscopy. These studies provide analysis of the subcellular distribution dynamics of critical molecules and signaling activities within ISs in particular relation to ILPs. Specifically, cytoskeletal/cytoskeletal regulator (actin, HS1; Figure 3A), antigen presentation/recognition (CD3/MHC-II; Figure 3B) adhesion (ICAM-1, LFA-1, talin; Figure 3C), and antigenic signaling (PKC-Q; Figure 3D) are seen to be co-enriched in podo-prints/ILP. Digital reconstruction of confocal serial-section z-stacks provides complementary evidence to those shown in Figure 2D that the T cell-endothelial endothelial IS has a discrete 3-dimension architecture punctuated by T cell ILP protruding in the APC surface (Figure 3Aii, Ci, iii, D). (Figure 3Civ).
<img alt="Figure 3" src="/files/ftp_upload/53288/53288fig3.jpg" /> 
Figure 3.&#160;Fixed-Sample Analysis of Molecular Distribution Dynamics in T cell-Endothelial Cell Immunological Synapses. Lymphocytes were incubated on SAg-loaded endothelium for 30 min and fixed and stained. In indicated panels endothelial cell were pre-transfected to express membrane-YFP or -DsRed. (A) Representative imaging show actin (i) and HS1 (a cortactin homologue; ii) co-enriched at the periphery of the immunological synapse in micro-clusters that correlate with the center of endothelial fluorescent rings of membrane-YFP (i.e., &#39;podo-prints&#39;; See Figure 2D) confirming that T cell ILPs are responsible for appearance of podo-prints. A side view (ii. 90&#176; Projection) illustrates that ILPs represent 3-dimensional protrusion in the z imaging plane (modified from 19). (B) Representative enrichment of T cell TCR (i) and endothelial MHC-II (ii) within the immunological synapse, show discrete subcellular partitioning to the ILP/podo-prints. (C) (i) Representative co-enrichment of ICAM-1 and LFA-1 in podo-prints and ILP, respectively of the IS. (i) Shows a 3-D digital reconstruction of confocal sections of the IS rotated 60&#176; (upper panels) and 90&#176; (orthogonal view; lower panels). (ii) Shows a zoomed view of the IS en face. (iii) Shows an orthogonal cross-sectional view of the boxed region in (ii) (modified from 19). (iv) Representative co-enrichment of talin (an integtin/actin linker protein) and actin in IS ILPs. (D) Representative co-enrichment of the TCR signaling molecule PKC-Q with actin in IS ILPs. Each panel shows an en face view (lower left portion) and two distinct orthogonal views (90&#176; Projection). The far left and right panels use are replicates of their, respective neighboring panels, projected using a rainbow fluorescence intensity scale (modified from 19). Scale bars = 5 &#956;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53288/53288fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Movie 1" src="/files/ftp_upload/53288/53288movie1.jpg" />
<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53288/53288movie1.mov">Movie 1: Live-Cell Dynamic Imaging of T cell Calcium Signaling and Immunological Synapse Topology. </a>Time-lapse live-cell imaging of Fura2-loaded CD4+ Th1 lymphocyte interacting with endothelium expressing membrane-YFP and loaded with SAg, corresponding to Figure 2B. Left panel shows intracellular calcium levels (rainbow: cool colors, low calcium; warm colors high calcium) in T cell. Right panel shown membrane-YFP fluorescence on the surface of the endothelial APC. Lymphocytes are seen to initiate spreading and ILP formation (as seen via fluorescent rings in mem-YFP channel; i.e., &#39;podo-prints&#39;; See Figure 2Bi), followed by induction of a sustained (30-60 min) intracellular calcium flux, which occurs commensurate with formation of peripheral arrays of ILPs/podo-prints analogous to TCR/actin &#39;signaling micro-clustered&#39; that are seen in planar bilayer APC models (See Figure 2Bii). The interval between frames is 20 sec. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53288/Movie1_finale_02_18_15.mov" target="_blank">Please click here to view this video.</a>
<img alt="Movie 2" src="/files/ftp_upload/53288/53288movie2.jpg" />
<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53288/53288movie2.mov">Movie 2: Live-Cell Dynamic Imaging of Immunological Synapse Topology via Fluorescent Reporters in Endothelial APCs. </a>Time-lapse live-cell imaging of CD4+ Th1 lymphocyte (DIC, left) interacting with endothelium co-expressing membrane-YFP (green) and soluble cytosolic DsRed (red) and loaded with SAg, corresponding to Figure 2D. Right panel shows merge of DIC, membrane and cytoplasm signaling. The combined signals of membrane and cytoplasmic fluorescence sensitively report formation and remodeling of multiple ~0.2-1 &#956;m-scale surface invaginations (podo-prints) that result from arrays T cell probing with ILP protrusions. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53288/Movie2_finale_02_18_15.mov" target="_blank">Please click here to view this video.</a>

Watch this Scientific Journal Video about An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics at JoVE.com

An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

Adaptive immunity is controlled by dynamic 'immunological synapses' formed between T cells and antigen presenting cells. This protocol describes methods for investigating endothelial cells both as understudied physiologic APCs and as a novel type of 'planar cellular APC model'.

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological ...

an-endothelial-planar-cell-model-for-imaging-immunological-synapse

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

8.9K Views.  Harvard Medical School - BIDMC. Adaptive immunity is controlled by dynamic 'immunological synapses' formed between T cells and antigen presenting cells. This protocol describes methods for investigating endothelial cells both as understudied physiologic APCs and as a novel type of 'planar cellular APC model'.

Video: An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

Supported Planar Bilayers for the Formation of Study of Immunological Synapses and Kinapse

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse.   To mimic the interactions between lymphocytes and antigen presenting cells, we use Ni2+-chelating lipids to anchor recombinant cell adhesion and MHC proteins to the upper leaflet of a bilayer with poly-histidine tags. Planar bilayers are generated by preparing lipid, treating the glass surfaces where the bilayer will form, and then forming the bilayer in a specialized chamber containing a flow-cell where the lymphocytes will be added.  Then, bilayers are charged with Ni and his-tagged recombinant proteins are added.  Finally,  lymphocytes are injected into the flow cell and TIRF microscopy can be used to image synapse formation and the mechanisms that control T cell locomotion, sites of receptor sorting,  and sites of receptor degradation.

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse. Here, we show methods for anchoring cell adhesion proteins known to modulate synapse formation to the upper leaflet of the lipid bilyer and visualize synapse formation using TIRF microscopy.

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse.   To mimic the interactions ...

Biology

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion (STED) Nanoscopy

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic actin reorganization is critical to the function of NK cells and the formation of the IS. Imaging of F-actin at the synapse has traditionally utilized confocal microscopy, however the diffraction limit of light restricts resolution of fluorescence microscopy, including confocal, to approximately 200 nm. Recent advances in imaging technology have enabled the development of subdiffraction limited super-resolution imaging. In order to visualize F-actin architecture at the IS we recapitulate the NK cell cytotoxic synapse by adhering NK cells to activating receptor on glass. We then image proteins of interest using two-color stimulated emission depletion microscopy (STED). This results in &#60;80 nm resolution at the synapse. Herein we describe the steps of sample preparation and the acquisition of images using dual color STED nanoscopy to visualize F-actin at the NK IS. We also illustrate optimization of sample acquisition using Leica SP8 software and time-gated STED. Finally, we utilize Huygens software for post-processing deconvolution of images.

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion STED Nanoscopy

Here we illustrate the protocol for imaging by two-color STED nanoscopy the cytotoxic immune synapse of NK cells recapitulated on glass. Using this method we obtain sub-100 nm resolution of synapse proteins and the cytoskeleton.

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic ...

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.

Leukocytes cross the endothelial monolayer using the paracellular or the transcellular route. We developed a simple assay to follow the distribution of endogenous junctional VE-cadherin and PECAM-1 during leukocyte transendothelial migration under physiological flow to discriminate between the two transmigration routes.

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins towards the immune synapse to assure a stable binding and signal exchange. Classical confocal, TIRF, or super-resolution microscopy have been used to study the immune synapse. Since these methods require manual image acquisition and time-consuming quantification, the imaging of rare events is challenging. Here, we describe a workflow that enables the morphological analysis of tens of thousands of cells. Immune synapses are induced between primary human T cells in pan-leukocyte preparations and Staphylococcus aureus enterotoxin B (SEB)-loaded Raji cells as APCs. Image acquisition is performed with imaging flow cytometry, also called In-Flow microscopy, which combines features of a flow cytometer and a fluorescence microscope. A complete gating strategy for identifying T cell/APC couples and analyzing the immune synapses is provided. As this workflow allows the analysis of immune synapses in unpurified pan-leukocyte preparations and hence requires only a small volume of blood (i.e., 1 mL), it can be applied to samples from patients. Importantly, several samples can be prepared, measured, and analyzed in parallel.

Here, we describe a complete workflow for the qualitative and quantitative analysis of immune synapses between primary human T cells and antigen-presenting cells. The method is based on imaging flow cytometry, which allows the acquisition and evaluation of several thousand cell images within a relatively short period of time.

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. Transcytosis across the BBB regulates the delivery of hormones, metabolites, and therapeutic antibodies to the brain parenchyma. Here, we present a protocol that combines immunofluorescence of free-floating sections with laser scanning confocal microscopy and image analysis to visualize subcellular organelles within endothelial cells at the BBB. Combining this data-set with 3D image analysis software allows for the semi-automated segmentation and quantification of capillary volume and surface area, as well as the number and intensity of intracellular organelles at the BBB. The detection of mouse endogenous immunoglobulin (IgG) within intracellular vesicles and their quantification at the BBB is used to illustrate the method. This protocol can potentially be applied to the investigation of the mechanisms controlling BBB transcytosis of different molecules in vivo.

Here we present a microscopy-based protocol for high-resolution imaging and a three-dimensional reconstruction of the mouse neurovascular unit and blood-brain barrier using brain free-floating sections. This method allows for the visualization, analysis, and quantification of intracellular organelles at the BBB.

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. ...

Neuroscience

Imaging the Human Immunological Synapse

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) and an effector helper T lymphocyte (Th) cell, and to record the images corresponding to the first stages of the IS formation and the subsequent trafficking events (occurring both in the APC and in the Th cell). These events will eventually lead to polarized secretion at the IS. In this protocol, Jurkat cells challenged with Staphylococcus enterotoxin E (SEE)-pulsed Raji cells as a cell synapse model was used, because of the closeness of this experimental system to the biological reality (Th cell-APC synaptic conjugates). The approach presented here involves cell-to-cell conjugation, time-lapse acquisition, wide-field fluorescence microscopy (WFFM) followed by image processing (post-acquisition deconvolution). This improves the signal-to-noise ratio (SNR) of the images, enhances the temporal resolution, allows the synchronized acquisition of several fluorochromes in emerging synaptic conjugates and decreases fluorescence bleaching. In addition, the protocol is well matched with the end point cell fixation protocols (paraformaldehyde, acetone or methanol), which would allow further immunofluorescence staining and analyses. This protocol is also compatible with laser scanning confocal microscopy (LSCM) and other state-of-the-art microscopy techniques. As a main caveat, only those T cell-APC boundaries (called IS interfaces) that were at the right 90&#176; angle to the focus plane along the Z-axis could be properly imaged and analyzed. Other experimental models exist that simplify imaging in the Z dimension and the following image analyses, but these approaches do not emulate the complex, irregular surface of an APC, and may promote non-physiological interactions in the IS. Thus, the experimental approach used here is suitable to reproduce and to confront some biological complexities occurring at the IS.

This protocol images both the immunological synapse formation and the subsequent polarized secretory traffic towards the immunological synapse. Cellular conjugates were formed between a superantigen-pulsed Raji cell (acting as an antigen-presenting cell) and a Jurkat clone (acting as an effector helper T lymphocyte).

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) ...

Precise Brain Mapping to Perform Repetitive In Vivo Imaging of Neuro-Immune Dynamics in Mice

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. Although studying these cells in isolation in vitro or together ex vivo provides useful physiological information; salient features of neural cell physiology will be missed in such contexts. Therefore, there is a need for studying neural cells in their native in vivo environment. The protocol detailed here describes repetitive in vivo two-photon imaging of neural cells in the rodent cortex as a tool to visualize and study specific cells over extended periods of time from hours to months. We describe in detail the use of the grossly stable brain vasculature as a coarse map or fluorescently labeled dendrites as a fine map of select brain regions of interest. Using these maps as a visual key, we show how neural cells can be precisely relocated for subsequent repetitive in vivo imaging. Using examples of in vivo imaging of fluorescently-labeled microglia, neurons, and NG2+ cells, this protocol demonstrates the ability of this technique to allow repetitive visualization of cellular dynamics in the same brain location over extended time periods, that can further aid in understanding the structural and functional responses of these cells in normal physiology or following pathological insults. Where necessary, this approach can be coupled to functional imaging of neural cells, e.g., with calcium imaging. This approach is especially a powerful technique to visualize the physical interaction between different cell types of the CNS in vivo when genetic mouse models or specific dyes with distinct fluorescent tags to label the cells of interest are available.

This protocol describes a chronic cranial window implantation technique that can be used for longitudinal imaging of neuro-glio-vascular structures, interactions, and function in both healthy and diseased conditions. It serves as a complementary alternative to the transcranial imaging approach that, while often preferred, possesses some critical limitations.

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. ...