Name: quantitative 3d imaging of trypanosoma cruzi-infected cells, dormant amastigotes, and t cells in intact clarified organs
Uploaded: 2022-06-23T13:30:00
Description: Watch this Scientific Journal Video about Quantitative 3D Imaging of Trypanosoma cruzi-Infected Cells, Dormant Amastigotes, and T Cells in Intact Clarified Organs at JoVE.com

We thank Dr. Etsuo Susaki for their valuable help and recommendations regarding tissue-clearing and immunostaining protocols. Also, we are grateful to M. Kandasamy from the CTEGD Biomedical Microscopy Core for technical support using LSFM and confocal imaging. We also thank all the members of Tarleton Research Group for helpful suggestions throughout this study.

This study was carried out in strict accordance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals and Association for Assessment and Accreditation of Laboratory Animal Care accreditation guidelines. The Animal Use Protocol (control of T. cruzi infection in mice-A2021 04-011-Y1-A0) was approved by the University of Georgia Institutional Animal Care and Use Committee. B6.C+A2:A44g-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J, B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J...

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The absence of extensive, whole-organ imaging of parasites and the immune response limits the understanding of the complexity of the host-parasite interactions and impedes the evaluation of therapies for Chagas disease. The present study adopted the CUBIC pipeline to clarify and stain intact organs and tissues of T. cruzi-infected mice.
Multiple tissue clearing protocols were tested in this study (PACT32, ECi33, FLASH34...

Chagas disease, caused by the protozoan parasite T. cruzi, is among the world&#39;s most neglected tropical diseases, causing approximately 13,000 annual deaths. The infection often progresses from an acute to a chronic stage producing cardiac pathology in 30% of the patients, including arrhythmias, heart failure, and sudden death1,2. Despite the strong host immune response elicited against the parasite during the acute phase, low numbers of parasites chronically persist throughout the host&#39;s life in tissues such as the heart and skeletal muscle. Several factors, including the delayed onset of adaptive immune responses and the presence of non-replicating forms of the parasite, may contribute to the capacity of T. cruzi to avoid a complete elimination by the immune system3,4,5,6. Furthermore, non-replicating dormant forms of the parasite display a low susceptibility to trypanocidal drugs and may in part be responsible for the treatment failure observed in many cases7,8.
The development of new imaging techniques provides an opportunity to gain insight into the spatial distribution of the parasites in the infected tissues and their relationship with the immune cells involved in their control. These characteristics are crucial for a better understanding of the processes of parasite control by the immune system and tracking the rare dormant parasites present in chronic tissues.
Light-sheet fluorescence microscopy (LSFM) is one of the most comprehensive and unbiased methods for 3D imaging of large tissues or organs without thin-sectioning. Light-sheet microscopes utilize a thin sheet of light to only excite the fluorophores in the focal plane, reduce photobleaching and phototoxicity of samples, and record images of thousands of tissue layers using ultra-fast cameras. The high level of tissue transparency necessary for the proper penetration of the laser light in tissues is obtained by homogenizing the refractive index (RI) following tissue delipidation and decolorization, which reduces the scattering of light and renders high-quality images9,10,11.
Tissue clearing approaches have been developed for the imaging of whole mice12,13,14, organoids15,16,17, organs expressing reporter fluorescent markers18,19,20,21,22,23, and recently a limited number of human tissues24. The current methods for tissue clearing are classified into three families: (1) organic solvent-based methods such as DISCO protocols25,26, (2) hydrogel-based methods, such as CLARITY27, and aqueous methods, such as CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis)18,19,28,29. CUBIC protocols maintain organ shape and tissue integrity, preserving the fluorescence of endogenously expressed reporter proteins. The most recent update of this technique, CUBIC-HistoVision (CUBIC-HV), also permits the detection of epitopes using fluorescently-tagged antibodies and DNA labeling28.
In the present protocol, the CUBIC pipeline for detecting T. cruzi expressing fluorescent proteins in clarified intact mouse tissues was used. Optically transparent tissues were LSFM imaged, 3D reconstructed, and the precise total number of T. cruzi infected cells, dormant amastigotes, and T cells per organ were automatically quantified. Also, this protocol was successfully adopted to obtain uniform labeling of cleared organs with antibodies and nuclear stains. These approaches are essential for understanding the expansion and control of T. cruzi in infected hosts and are useful for fully evaluating chemo- and immuno-therapeutics for Chagas disease.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1-methylimidazole</td>
			<td>Millipore Sigma</td>
			<td>616-47-7</td>
			<td></td>
		</tr>
		<tr>
			<td>2,3-Dimethyl-1-phenyl-5-pyrazolone (Antipyrine</td>
			<td>TCI</td>
			<td>D1876</td>
			<td></td>
		</tr>
		<tr>
			<td>6-wells cell culture plates</td>
			<td>ThermoFisher Scientific</td>
			<td>140675</td>
			<td></td>
		</tr>
		<tr>
			<td>AlexaFluor 647 anti-mouse Fab fragment</td>
			<td>Jackson Immuno Research Laboratories</td>
			<td>315-607-003</td>
			<td></td>
		</tr>
		<tr>
			<td>AlexaFluor 647 anti-rabbit Fab fragment</td>
			<td>Jackson Immuno Research Laboratories</td>
			<td>111-607-003</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-GFP nanobody Alexa Fluor 647</td>
			<td>Chromotek</td>
			<td>gb2AF647-50</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-RFP</td>
			<td>Rockland</td>
			<td>600-401-379</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-&#945;-SMA</td>
			<td>Sigma</td>
			<td>A5228</td>
			<td></td>
		</tr>
		<tr>
			<td>B6.C+A2:A44g-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J mouse</td>
			<td>The Jackson Laboratory</td>
			<td>Strain #007914</td>
			<td>Common Name: Ai14 , Ai14D or Ai14(RCL-tdT)-D</td>
		</tr>
		<tr>
			<td>B6.Cg-Gt(ROSA)26Sor tm14(CAG-tdTomato)Hze/J mouse</td>
			<td>The Jackson Laboratory</td>
			<td>Strain #007914</td>
			<td>Common Name: Ai14 , Ai14D or Ai14(RCL-tdT)-D</td>
		</tr>
		<tr>
			<td>BOBO-1 Iodide</td>
			<td>ThermoFisher Scientific</td>
			<td>B3582</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>#A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J-Tg(Cd8a*-cre)B8Asin/J mouse</td>
			<td>The Jackson Laboratory</td>
			<td>Strain #032080</td>
			<td>Common Name: Cd8a-Cre (E8III-Cre)</td>
		</tr>
		<tr>
			<td>CAPSO</td>
			<td>Sigma</td>
			<td>#C2278</td>
			<td></td>
		</tr>
		<tr>
			<td>Cleaning wipes Kimwipes</td>
			<td>&#160;Kimberly-Clark</td>
			<td>T8788</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Laser Scanning Microscope</td>
			<td>Zeiss</td>
			<td>LSM 790</td>
			<td></td>
		</tr>
		<tr>
			<td>CUBIC-HV 1 3D immunostaining kit</td>
			<td>TCI</td>
			<td>C3699</td>
			<td></td>
		</tr>
		<tr>
			<td>CUBIC-HV 1 3D nuclear staining kit</td>
			<td>TCI</td>
			<td>C3698</td>
			<td></td>
		</tr>
		<tr>
			<td>CUBIC-L</td>
			<td>TCI</td>
			<td>T3740</td>
			<td></td>
		</tr>
		<tr>
			<td>CUBIC-P</td>
			<td>TCI</td>
			<td>T3782</td>
			<td></td>
		</tr>
		<tr>
			<td>CUBIC-R+</td>
			<td>TCI</td>
			<td>T3741</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanoacrylate-based gel superglue</td>
			<td>Scotch</td>
			<td>571605</td>
			<td></td>
		</tr>
		<tr>
			<td>DiR (DiIC18(7); 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide) Company: Biotium</td>
			<td>Biotium</td>
			<td>#60017</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene diamine tetra acetic acid (EDTA)</td>
			<td>Millipore Sigma</td>
			<td>60-00-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Centrifuge tubes 15 mL</td>
			<td>Corning</td>
			<td>CLS430791</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Centrifuge tubes 50&#160; mL</td>
			<td>Corning</td>
			<td>CLS430290</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin</td>
			<td>Sigma-Aldrich</td>
			<td>HT501128</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>ThermoFisher Scientific</td>
			<td>J16920.BBR</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>Sigma</td>
			<td>#H3884 or #H4272</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris File Converter x64</td>
			<td>BitPlane</td>
			<td>v9.2.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris software</td>
			<td>BitPlane</td>
			<td>v9.3</td>
			<td></td>
		</tr>
		<tr>
			<td>ImSpector software</td>
			<td>LaVision BioTec, Miltenyi Biotec</td>
			<td>v6.7</td>
			<td></td>
		</tr>
		<tr>
			<td>Intravenous injection needle 23-G</td>
			<td>Sartori, Minisart Syringe filter</td>
			<td>16534</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td></td>
			<td></td>
			<td>lint free wipes</td>
		</tr>
		<tr>
			<td>Light-sheet fluorescent microscope</td>
			<td>Miltenyi Biotec</td>
			<td>ULtramicroscope II imaging system</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>ThermoFisher Scientific</td>
			<td>041838.K2</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette tips, 10 &#181;L, 200 &#181;L and 1,000 &#181;L</td>
			<td>Axygen</td>
			<td>T-300, T-200-Y and T-1000-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Motorized pipet dispenser</td>
			<td>Fisher Scientific, Fisherbrand</td>
			<td>03-692-172</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting Solution</td>
			<td>TCI</td>
			<td>M3294</td>
			<td></td>
		</tr>
		<tr>
			<td>N-butyldiethanolamine</td>
			<td>TCI</td>
			<td>B0725</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>TCI</td>
			<td>N0078</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Methylnicotinamide</td>
			<td>TCI</td>
			<td>M0374</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Sigma-Aldrich</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr>
			<td>RedDot 2 Far-Red Nuclear Stain</td>
			<td>Biotium</td>
			<td>#40061</td>
			<td></td>
		</tr>
		<tr>
			<td>Sacrifice Perfusion System</td>
			<td>Leica</td>
			<td>10030-380</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools</td>
			<td>91460-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipettes</td>
			<td>Costar Sterile</td>
			<td>4488</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaking incubator</td>
			<td>TAITEC</td>
			<td>BR-43FM MR</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide (NaN3)</td>
			<td>ThermoFisher Scientific</td>
			<td>447815000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium carbonate (Na2CO3)</td>
			<td>ThermoFisher Scientific</td>
			<td>L13098.36</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>ThermoFisher Scientific</td>
			<td>447302500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydrogen carbonate (NaHCO3)</td>
			<td>ThermoFisher Scientific</td>
			<td>014707.A9</td>
			<td></td>
		</tr>
		<tr>
			<td>SYTOX-G Green Nucleic Acid Stain</td>
			<td>ThermoFisher Scientific</td>
			<td>S7020</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitative 3d imaging of trypanosoma cruzi-infected cells, dormant amastigotes, and t cells in intact clarified organs

Chagas disease is a neglected pathology that affects millions of people worldwide, mainly in Latin America. The Chagas disease agent, Trypanosoma cruzi (T. cruzi), is an obligate intracellular parasite with a diverse biology that infects several mammalian species, including humans, causing cardiac and digestive pathologies. Reliable detection of T. cruzi in vivo infections has long been needed to understand Chagas disease&#39;s complex biology and accurately evaluate the outcome of treatment regimens. The current protocol demonstrates an integrated pipeline for automated quantification of T. cruzi-infected cells in 3D-reconstructed, cleared organs. Light-sheet fluorescent microscopy allows for accurately visualizing and quantifying of actively proliferating and dormant T. cruzi parasites and immune effector cells in whole organs or tissues. Also, the CUBIC-HistoVision pipeline to obtain uniform labeling of cleared organs with antibodies and nuclear stains was successfully adopted. Tissue clearing coupled with 3D immunostaining provides an unbiased approach to comprehensively evaluate drug treatment protocols, improve the understanding of the cellular organization of T. cruzi-infected tissues, and is expected to advance discoveries related to anti-T. cruzi immune responses, tissue damage, and repair in Chagas disease.

CUBIC fixed tissues were washed with PBS to remove fixatives and then incubated with CUBIC-L cocktails, a basic buffered solution of amino alcohols that extract pigments and lipids from the tissue resulting in decolorization of tissue while maintaining tissue architecture. Grid lines in the paper can be seen through the tissues indicating an appropriate clearing of the organs (Figure 2A). After delipidation, tissues were washed and immersed in CUBIC-R+ and mounting solution ...

Watch this Scientific Journal Video about Quantitative 3D Imaging of Trypanosoma cruzi-Infected Cells, Dormant Amastigotes, and T Cells in Intact Clarified Organs at JoVE.com

Quantitative 3D Imaging of Trypanosoma cruzi-Infected Cells, Dormant Amastigotes, and T Cells in Intact Clarified Organs

The present protocol describes light-sheet fluorescent microscopy and automated software-assisted methods to visualize and precisely quantify proliferating and dormant Trypanosoma cruzi parasites and T cells in intact, cleared organs and tissues. These techniques provide a reliable way to evaluate treatment outcomes and offer new insights into parasite-host interactions.

Quantitative 3D Imaging of Trypanosoma cruzi-Infected Cells, Dormant Amastigotes, and T Cells in Intact Clarified Organs

Chagas disease is a neglected pathology that affects millions of people worldwide, mainly in Latin America. The Chagas disease agent, Trypanosoma cruzi ...

This technique provided a more complete and 3D image of entire transparent organs and was useful for quantification, not only of replicating, but also dormant parasite and immune factor cells. This technique allows us to determine the special association of parasites, immune cells, and the tissue damage resulting from the infection. Epitope immunostaining and DNA labeling were also comparable with this technique.This protocol was effectively used for assessing treatment outcomes in mouse models of Chagas disease. A treatment of higher individual doses, given less frequently over an extended treatment period, eliminates both replicating and dormant parasites. Demonstrating the procedure will be Caleb Hawkins, a research technician from Rick Tarleton's lab.After euthanizing, dissecting, and fixing the organs of an infected mouse as described in the manuscript, immerse individual organs in 10 milliliters of 50%water-diluted CUBIC-L, with gentle shaking at room temperature in 50-milliliter conical tubes overnight protected from light. Next day, replace 50%CUBIC-L with 10 milliliters of 100%CUBIC-L, and incubate for six days at 37 degrees Celsius, while keeping the tube flat on the shaker incubator. At the end of this incubation period, the tissues should be almost completely transparent.Wash the transparent organs twice with PBS for two hours at 37 degrees Celsius, with gentle shaking. With each wash, transfer the tissues to a new 50-milliliter conical tube to remove Triton X-100. For DNA staining, dilute the commercially available nucleic acid dye in five milliliters of staining buffer at 1:2, 500.Then, immerse the tissue in a 15-milliliter conical tube containing nuclear dye solution, and incubate at 37 degrees Celsius, with gentle rotation for five days in a standing position. Wash the tissue three times with 15 milliliters of 3D nuclear staining wash buffer for two hours at room temperature, with gentle shaking. Calculate the required amount of primary and secondary antibodies.Mix primary and secondary antibodies in an amber two-milliliter tube, and incubate at 37 degrees Celsius for 1.5 hours. For buffer exchange, mix 7.5 milliliters of 2x HV1 3D immunostaining buffer with 7.5 milliliters of double-distilled water. Immerse the tissue sample in the buffer, and incubate for 1.5 hours at 32 degrees Celsius, with gentle shaking in a 15-milliliters conical tube in a horizontal position.Collect the tissue sample from the buffer exchange media, and immerse it in the antibody staining solution. Incubate tissues individually for one week at 32 degrees Celsius, gently shaking the tubes in a standing position away from the light. Move the tissue samples to four degrees Celsius, and incubate in a standing position.After cooling the HV1 3D immunostaining wash buffer to four degrees Celsius, wash the sample with 15 milliliters of buffer twice at four degree Celsius for 30 minutes, each with gentle shaking in a horizontal position. Dilute formalin to 1%in HV1 3D immunostaining wash buffer, and immerse the sample in eight milliliters of the solution for 24 hours at four degrees Celsius, with gentle shaking. Now, incubate the sample in fresh 1%formalin solution for one hour at 37 degrees Celsius, and wash in 15 milliliters of PBS for two hours at 25 degrees Celsius.Immerse transparent organs in a 50-milliliter conical tube containing five milliliters of 50%water diluted CUBIC-R+solution at room temperature, with gentle shaking. Replace it with five milliliters 100%CUBIC-R+and incubate with gentle shaking for two days. Dry the tissues using Kimwipes, and then, transfer them to five milliliters of mounting solution in a six-well culture plate, and incubate them at room temperature.Adhere the tissues to the microscope sample holder using gel super glue. Immerse the samples in the microscope quartz cuvette filled with 120 milliliters of mounting solution. Image them transversely to their longitudinal axis, and adhere the heart, with the apex and the aorta horizontally aligned.3D reconstructed Trypanosoma cruzi-infected heart displayed a higher proportion of infected cells in the atrium, compared with ventricles. Dormant parasites can be identified in the heart, as depicted in the 3D-enlarged insets. ZsGreen1-expressing T cells, as well as Trypanosoma cruzi-infected cells, were visualized in the skeletal muscle from a CD8 reporter mouse infected with Trypanosoma cruzi.3D zoom-ins of 3D-reconstructed image identified T cells in the region of an infected cell. The interface of T cells in host-infected cells was visualized by confocal microscopy. In the heart of an immunodeficient mouse, host cells infected with two different strains of Trypanosoma cruzi were detected, and slicing through the tissues showed abundant parasite-infected cells in the heart atria at various tissue depths.Immunodetection of vasculature in Trypanosoma cruzi-infected and cleared heart reveals the intricate vasculature of the heart. Parasite-infected cells could be observed in the heart atria. Nuclear staining of skeletal muscle showed an accumulation of nuclei in areas with few or undetectable tdTomato parasites.Boosting tdTomato and GFP parasite reporter markers with antibodies against RFP, and GFP, and whole cleared skeletal muscle resulted in strong fluorescence. So the critical points are the incubation time in the CUBIC buffer, the dilution and incubation time in the DNA dye, the incubation time in the antibodies, and the incubation time in the refracted index-matching solution. The use of additional staining, with specific antibodies dye and quick reaction, could significantly increase the potential of this technique, allowing for the detection of multiple cells, processes, and structure in intact organs.We now have a better and more complete way to explore the immune cells responsible for killing the parasites, the tissues where the parasites persist chronically, and the distribution of the specific drugs into the tissues.

quantitative-3d-imaging-trypanosoma-cruzi-infected-cells-dormant

Research

JoVE Journal

Immunology and Infection

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 
1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&amp;#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell interaction known as the immunological synapse. F-actin is also speculated to play a role in regulating molecular distributions at the membrane of cells including sub-membranous vesicle dynamics and protein clustering. While standard light microscope techniques allow generalized and diffraction-limited observations to be made, many cellular and molecular events including clustering and molecular flow occur in populations at length-scales far below the resolving power of standard light microscopy. By combining total internal reflection fluorescence with the super resolution imaging method structured illumination microscopy, the two-dimensional molecular flow of F-actin at the immune synapse of T cells was recorded. Spatio-temporal image correlation spectroscopy (STICS) was then applied, which generates quantifiable results in the form of velocity histograms and vector maps representing flow directionality and magnitude. This protocol describes the combination of super-resolution imaging and STICS techniques to generate flow vectors at sub-diffraction levels of detail. This technique was used to confirm an actin flow that is symmetrically retrograde and centripetal throughout the periphery of T cells upon synapse formation.

To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy.

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell ...

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and multiplexing ability. Using fluorescent nano-diamond fiducial markers, we describe the drift correction and alignment procedures required to obtain high precision in single molecule localization microscopy. In addition, a new multiplexing strategy, madSTORM, is described in which multiple molecules are targeted in the same cell using sequential binding and elution of fluorescent antibodies. madSTORM is demonstrated on an activated T cell to visualize the locations of different components within a membrane-bound, multi-protein structure called the T cell receptor microcluster. In addition, application of madSTORM as a general tool for visualization of multi-protein structures is discussed.

We demonstrate a method to image multiple molecules within heterogeneous nano-structures at single molecule accuracy using sequential binding and elution of fluorescently labeled antibodies.