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).

<ol>
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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 ce...

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/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 border="0" cellpadding="0" cellspacing="0" width="914">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">BD Vacutainer stretch latex free tourniquet</td>
			<td style="width:149px;">BD Biosciences</td>
			<td style="width:152px;">367203</td>
			<td style="width:221px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD alcohol swabs</td>
			<td style="width:149px;">BD Biosciences</td>
			<td>326895</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD Vacutainer Safety-Lok</td>
			<td style="width:149px;">BD Biosciences</td>
			<td style="width:152px;">367861</td>
			<td>K2 EDTA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">BD Vacutainer Push Button Blood Collection Set</td>
			<td style="width:149px;">BD Biosciences</td>
			<td style="width:152px;">367335</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">RPMI-1640</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td>R8758-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ficoll-Paque&#160;</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td style="width:152px;">GE17-1440-02</td>
			<td>Bring to RT before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">FCS-Optima</td>
			<td style="width:149px;">Atlanta Biologics</td>
			<td>s12450</td>
			<td>Heat inactivated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin&#160;</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td>&#160;P4458-100ML&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan blue</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td>T8154-20ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">staphylococcal enterotoxin B&#160;</td>
			<td>Toxin Technology</td>
			<td>BT202RED</td>
			<td>Stock solution 1mg/ml in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">toxic shock syndrome toxin 1&#160;</td>
			<td>Toxin Technology</td>
			<td>TT606RED</td>
			<td>Stock solution 1mg/ml in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">human IL-15</td>
			<td style="width:149px;">R&#38;D Systems</td>
			<td>247-IL-025</td>
			<td>Stock solution 50ug/ml in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">PBS</td>
			<td>Life Technologies</td>
			<td>10010-049</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:392px;">Fibronectin</td>
			<td style="width:149px;">Life Technologies</td>
			<td>33016-015</td>
			<td>Stock solution 1mg/ml in H20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HMVEC-d Ad-Dermal MV Endo Cells</td>
			<td style="width:149px;">Lonza</td>
			<td>CC-2543</td>
			<td>Other Human Microvascular ECs can be used, i.e. HLMVECs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGM-2 MV bullet kit</td>
			<td style="width:149px;">Lonza</td>
			<td>CC-3202</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">Trypsin-EDTA</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td>T-4174</td>
			<td>Stock solution 10x, dilute in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">amaxa-HMVEC-L Nucleofector Kit</td>
			<td style="width:149px;">Lonza</td>
			<td>vpb1003</td>
			<td>Required Kit for step 4</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:392px;">IFN-g</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td>I3265</td>
			<td>Stock solution 1mg/ml in H20</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">TNF-alpha 10ug, human</td>
			<td style="width:149px;">Life Technologies</td>
			<td>PHC3015</td>
			<td>Stock solution 1mg/ml in H20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">phenol Red-free HBSS&#160;</td>
			<td style="width:149px;">Life Technologies</td>
			<td>14175-103</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hepes</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td>BP299-100</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:392px;">Calcium Chloride</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td style="width:152px;">C1016-100G</td>
			<td>Stock solution 1M in H20</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:392px;">Magnesium chloride</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td style="width:152px;">208337</td>
			<td>Stock solution 1M in H20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">Human Serum albumin</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td>A6909-10ml</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immersol 518 F fluorescence free Immersion oil</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td>12-624-66A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fura-2 AM 20x50ug</td>
			<td style="width:149px;">Life Technologies</td>
			<td>F1221</td>
			<td>Stock solution 1mM in DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">pEYFP-Mem (Mem-YFP)</td>
			<td style="width:149px;">Clontech</td>
			<td style="width:152px;">6917-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">pDsRed-Monomer (Soluble Cytoplasmic DsRed)</td>
			<td style="width:149px;">Clontech</td>
			<td style="width:152px;">632466</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">pDsRed-Monomer Membrane (Mem-DsRed)</td>
			<td style="width:149px;">Clontech</td>
			<td style="width:152px;">632512</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">pEGFP-Actin</td>
			<td style="width:149px;">Clontech</td>
			<td style="width:152px;">6116-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">Alexa Fluor 488 Phalloidin</td>
			<td style="width:149px;">Life Technologies</td>
			<td>A12379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">Formaldehyde solution 37%</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td>BP531-500</td>
			<td>Toxic, use fumehood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">Triton X-100</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td style="width:152px;">X100-5ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">&#160;Falcon 15mL Conical Centrifuge Tubes</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td>14-959-70C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">Falcon 50mL Conical Centrifuge Tubes</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td>14-959-49A&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">Falcon Tissue Culture Treated Flasks T25</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td>10-126-9</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">&#160;Falcon Tissue Culture Treated Flasks T75</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td>&#160; 13-680-65</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">&#160;Corning Cell Culture Treated T175</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:152px;">10-126-61&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">Glass coverslips&#160;</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:152px;">12-545-85&#160;</td>
			<td>12 mm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">&#160;Falcon Tissue Culture Plates 24-well</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td>08-772-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">Delta-T plates</td>
			<td style="width:149px;">Bioptechs</td>
			<td>04200415B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wheaton Disposable Pasteur Pipets</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:152px;">13-678-8D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5 ml Eppendorf tube&#160;</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td>05-402-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">&#160;ICAM1 mouse anti-human</td>
			<td style="width:149px;">BD Biosciences</td>
			<td>555509</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HS1 mouse anti-human</td>
			<td>BD Biosciences</td>
			<td>610541</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Human CD11a (LFA-1alpha) Purified</td>
			<td>ebioscience</td>
			<td>BMS102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Human CD3 Alexa Fluor&#174; 488</td>
			<td>ebioscience</td>
			<td>53-0037-41</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-MHC Class II antibody&#160;</td>
			<td style="width:149px;">Abcam</td>
			<td>ab55152</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Talin 1 antibody</td>
			<td style="width:149px;">Abcam</td>
			<td>ab71333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">Anti-PKC theta antibody&#160;</td>
			<td style="width:149px;">Abcam</td>
			<td>ab109481</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">phosphotyrosine (4G10 Platinum)</td>
			<td style="width:149px;">Millipore</td>
			<td>50-171-463</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">Nucleofector II</td>
			<td style="width:149px;">Amaxa Biosystems</td>
			<td style="width:152px;"></td>
			<td>Required electroporator for step 4</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:392px;">Zeiss Axiovert</td>
			<td style="width:149px;">Carl Zeiss MicroImaging</td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:392px;">Zeiss LSM510&#160;</td>
			<td style="width:149px;">Carl Zeiss MicroImaging</td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:392px;">Zeiss Axiovison Software</td>
			<td style="width:149px;">Carl Zeiss MicroImaging</td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">NU-425 (Series 60) Biological Safety Cabinet</td>
			<td style="width:149px;">NuAIRE</td>
			<td style="width:152px;">Nu-425-600</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:392px;">&#160;Forma STRCYCLE 37 &#176;C, 5% CO2 Cell culture Incubator</td>
			<td>Fisher Scientific</td>
			<td>202370</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">Centrifuge 5810</td>
			<td style="width:149px;">Eppendorf</td>
			<td style="width:152px;">EW-02570-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">Hemocytometer</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td style="width:152px;">&#160;Z359629</td>
			<td>Bright-Line Hemocytometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">Isotemp Waterbath model 202</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:152px;">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 ...

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

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 ...

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 ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

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 ...

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not na&#239;ve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

Studying Organelle Dynamics in B Cells During Immune Synapse Formation

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both signaling&#160;and antigen uptake are coordinated. IS formation involves dynamic actin remodeling accompanied by the polarized recruitment to the synaptic membrane of the centrosome and associated intracellular organelles such as lysosomes and the Golgi apparatus. Initial stages of actin remodeling allow B cells to increase their cell surface and maximize the quantity of antigen-BCR complexes gathered at the synapse. Under certain conditions, when B cells recognize antigens associated to rigid surfaces, this process is coupled to the local recruitment and secretion of lysosomes, which can facilitate antigen extraction. Uptaken antigens&#160;are internalized into specialized endo-lysosome compartments for processing into peptides, which are loaded onto major histocompatibility complex II (MHC-II) molecules for further presentation to T helper cells. Therefore, studying organelle dynamics associated with the formation of an IS is crucial to understanding how B cells are activated. In the present article we will discuss both imaging and a biochemical technique used to study changes in intracellular organelle positioning and cytoskeleton rearrangements that are associated with the formation of an IS in B cells.

Herein we describe two approaches to characterize cell polarization events in B lymphocytes during the formation of an IS. The first, involves quantification of organelle recruitment and cytoskeleton rearrangements at the synaptic membrane. The second is a biochemical approach, to characterize changes in composition of the centrosome, which undergoes polarization to the immune synapse.

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both ...

Implantation and Monitoring by PET/CT of an Orthotopic Model of Human Pleural Mesothelioma in Athymic Mice

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. MPM development is mainly associated with asbestos. Treatments provide only modest survival since the median survival average is 9&#8211;18 months from the time of diagnosis. Therefore, more effective treatments must be identified. Most data describing new therapeutic targets were obtained from in vitro experiments and need to be validated in reliable in vivo preclinical models. This article describes one such reliable MPM orthotopic model obtained after injection of a human MPM cell line H2052/484 into the pleural cavity of immunodeficient athymic mice. Transplantation in the orthotopic site allows studying the progression of tumor in the natural in vivo environment. Positron emission tomography/computed tomography (PET/CT) molecular imaging using the clinical [18F]-2-fluoro-2-deoxy-D-glucose ([18F]FDG) radiotracer is the diagnosis method of choice for examining patients with MPM. Accordingly, [18F]FDG-PET/CT was used to longitudinally monitor the disease progression of the H2052/484 orthotopic model. This technique has a high 3R potential (Reduce the number of animals, Refine to lessen pain and discomfort, and Replace animal experimentation with alternatives) since the tumor development can be monitored non-invasively and the number of animals required could be significantly reduced.
This model displays a high development rate, a rapid tumor growth, is cost-efficient and allows for rapid clinical translation. By using this orthotopic xenograft MPM model, researchers can assess biological responses of a reliable MPM model following therapeutic interventions.

This article describes the generation of an orthotopic mouse model of human pleural mesothelioma by implantation of H2052/484 mesothelioma cells into the pleural cavity of immunocompromised athymic mice. The longitudinal monitoring of the development of intrapleural tumors was assessed by non-invasive multimodal [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography imaging.

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. ...

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) ...

An Immunological Model for Heterotopic Heart and Cardiac Muscle Cell Transplantation in Rats

Heterotopic heart transplantation in rats has been a commonly used model for diverse immunological studies for more than 50 years. Several modifications have been reported since the first description in 1964. After 30 years of performing heterotopic heart transplantation in rats, we have developed a simplified surgical approach, which can be easily taught and performed without further surgical training or background.
After dissection of the ascending aorta and the pulmonary artery and ligation of superior and inferior caval and pulmonary veins, the donor heart is harvested and subsequently perfused with ice-cold saline solution supplemented with heparin. After clamping and incising the recipient abdominal vessels, the donor ascending aorta and pulmonary artery are anastomosed to the recipient abdominal aorta and inferior vena cava, respectively, using continuous running sutures.
Depending on different donor-recipient combinations, this model allows analyses of either acute or chronic rejection of allografts. The immunological significance of this model is further enhanced by a novel approach of in-ear injection of vital cardiac muscle cells and subsequent analysis of draining cervical lymphatic tissue.

We describe a model of heterotopic abdominal heart transplantation in rats, implying modifications of current strategies, which lead to a simplified surgical approach. Additionally, we describe a novel rejection model by in-ear injection of vital cardiac muscle cells, allowing further transplant immunological analyses in rats.

Heterotopic heart transplantation in rats has been a commonly used model for diverse immunological studies for more than 50 years. Several modifications ...