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 and C57BL/6J-Tg(Cd8a*-cre)B8Asin/J mice (female, 1-2 months old) were used for the present study. The mice were obtained from commercial sources (see Table of Materials).
1. Infection, perfusion, and dissection
<ol>
	<li>Intraperitoneally infect mice with tissue culture-derived trypomastigotes of Colombiana (DTU TcI) or Brazil (DTU TcV) T. cruzi strain expressing tdTomato or GFP fluorescent proteins, respectively. The infection dose could range from 50,000 to 200,000 trypomastigotes diluted in 100 &#181;L of 1x Phosphate-Buffered Saline (PBS). 
	NOTE: Specific details about the generation of reporter parasites and mouse models of infection are available in Canavaci et al.29 and Bustamante et al.30.</li>
	<li>Euthanize the mice by carbon dioxide inhalation at a flow rate of 3-7 L/min. As soon as the animals stop showing any pedal reflex, make a longitudinal incision through the skin from the abdomen towards the sternum. Then cut the body wall from the abdomen and continue through the ribs on each side of the thorax until the sternum can be lifted away, exposing the heart.</li>
	<li>As described in Figure 1, make a 2.5 mm incision in the heart&#39;s right auricle and collect the draining blood using a 1 mL micropipette tip.</li>
	<li>Insert a butterfly needle (connected to one end of the perfusion system) into the apex of the left ventricle until it reaches the ascending aorta. Use gel-based glue (see Table of Materials) to seal the inlet hole around the needle and maintain the needle in position during perfusions.</li>
	<li>Perfuse the mice30 with 50 mL of cold Heparin-PBS (pH 7.4, 10 U/mL of Heparin) or until the fluid that comes out of the mouse toward the collection tray is clear of blood.</li>
	<li>Perfuse with 50 mL of cold 4% (w/v) paraformaldehyde (PFA) (pH 7.4) in PBS. 
	NOTE: PFA tissue fixation is likely one of the critical steps of the protocol, especially for maintaining epitope structures and subsequent immunodetection. PFA degrades over time, so it must be freshly prepared. The pH of the solution is also important to avoid over-fixation, which could lead to poor clearing of tissues. 
	CAUTION: PFA is moderately toxic by skin contact. Acute exposure is also highly irritating to the nose, eyes, and throat. Long-term exposure to low levels in the air or skin may cause skin irritation such as dermatitis and itching, and asthma-like respiratory problems. Wear face and eye protection and do not breathe dust, gas, mist, fumes, or vapors.
	<ol>
		<li>Following the PFA perfusion, perfuse the mouse with 100 mL of CUBIC-P buffer to reach the clarification levels mentioned in step 1.5.</li>
		<li>To prepare CUBIC-P buffer, dissolve 10% N-butyldiethanolamine, 5% Triton X-100, and 5% 1-N-Methylimidazole in double-distilled water or use the commercial cocktails (see Table of Materials). 
		NOTE: Steps 1.6.1.-1.6.2. are recommended only for highly pigmented organs such as the heart and kidney since they require a pre-clearing step to obtain increased levels of transparency. After using CUBIC-P, dissect the organs and proceed directly to step 2: Tissue clearing.</li>
	</ol>
	</li>
	<li>Dissect the tissue samples/organs30 to be imaged and post-fix them in 4% (w/v) PFA in PBS (~10 mL/whole organ) overnight (ON) at 4 &#176;C with gentle shaking (no more than 5 x g) in 50 mL conical tubes. 
	NOTE: All the incubations from hereafter must be performed on tubes laying horizontally at 20-30 &#176;C protected from light.</li>
	<li>Wash the sample in 10 mL of PBS (supplemented with 0.05% sodium azide (NaN3) for 3 h (three times) at room temperature (RT) with gentle shaking (5 x g). 
	&#8203;NOTE: Tissues can be frozen by incubating in 10 mL of 30% sucrose in PBS with gentle shaking (5 x g) at 4 &#176;C ON in 50 mL conical tubes. After tissues sink to the bottom of the tube, embed them in the OCT compound and keep them at -80 &#176;C. Thaw at RT until the OCT compound completely melts, then wash in PBS (~10 mL/whole organ) ON at 4 &#176;C with gentle shaking (5 x g) in 50 mL conical tubes. Proceed to step 2.</li>
</ol>
2. Tissue clearing
NOTE: All the tissue clearings performed in this work were done using CUBIC protocol I22. Three different cocktails were used: CUBIC-P for delipidation and rapid decolorization during perfusions, CUBIC-L for delipidation and decolorization, and CUBIC-R for RI matching. DNA staining and immunostainings were performed using CUBIC-HV 1 3D nuclear staining kit and CUBIC-HV 1 3D immunostaining kit, respectively (see Table of Materials).
<ol>
	<li>Immerse individual organs in 10 mL of 50% water-diluted CUBIC-L (see Table of Materials) with gentle shaking (5 x g) at RT (ON) in 50 mL conical tubes. Keep tubes flat on the shaking plate. 
	NOTE: To avoid tissue damage, organs are maintained in the same tube, and solutions are collected using a vacuum system. To prepare CUBIC-L, dissolve 10% N-butyldiethanolamine and 10% Triton X-100 in double-distilled water using the commercial cocktails (see Table of Materials). 
	CAUTION: CUBIC-L causes serious eye damage.Wear eye and face protection. Dispose to an approved waste disposal plant.</li>
	<li>Immerse sample in 10 mL of 100% CUBIC-L for 6 days (refreshing the solution on day 3). 
	NOTE: At the end of this incubation period, the tissues must be almost completely transparent.</li>
	<li>Wash the transparent organs with PBS (supplemented with 0.05% NaN3) for 2 h (three times) at 37 &#176;C with gentle shaking (5 x g). Transfer tissues to a new 50 mL conical tube with each wash to remove Triton X-100. 
	NOTE: As described in Figure 2B, if the goal of the experiment is to visualize endogenously-expressed reporter proteins from transgenic T. cruzi parasites or mice (Figure 2C-F), skip steps 3, 4, and 5 and continue directly to step 6.</li>
</ol>
3. DNA staining
<ol>
	<li>Dilute the commercially available nucleic acid dye (see Table of Materials) in 5 mL of staining buffer (included in the kit) at 1/2,500 dilution.</li>
	<li>Immerse tissue in the nuclear dye solution and incubate at 37 &#176;C with gentle rotation for 5 days using 15 mL conical tubes in a standing position.</li>
	<li>Wash with 15 mL of 3D nuclear staining wash buffer (included in the kit) for 2 h (three times) at RT with gentle shaking (5 x g). 
	&#8203;NOTE: Other DNA dyes can be used in these concentrations and incubation times: DAPI (included in the kit): 1/200, 5 days incubation; BOBO-1: 1/400, 5 days incubation; Propidium Iodide (PI) (included in the kit): 1/100, 3 days incubation; RedDot2: 1/250, 3 days incubation. If the specific aim of the experiment is to visualize both endogenously-expressed reporter proteins and use nuclear stains, skip steps 4 and 5 and continue directly to step 6.</li>
</ol>
4. Extracellular matrix (ECM) digestion
NOTE: Hyaluronidase digestion of the ECM must be performed to facilitate the proper penetration of the antibodies into deep regions of the tissues28.
<ol>
	<li>Immerse individual organs in 15 mL of hyaluronidase reaction buffer for 2 h at 37 &#176;C in a 50 mL conical tube in flat position protected from light. 
	NOTE: To prepare hyaluronidase reaction buffer, dissolve 10 mM of CAPSO; 150 mM of Sodium Chloride (NaCl), and 0.05% of NaN3 (see Table of Materials) in double-distilled water and adjust pH to 10.</li>
	<li>Prepare Enzyme Solution by mixing 75 &#181;L of 20 mg/mL of hyaluronidase stock into 425 &#181;L of hyaluronidase reaction buffer. To prepare 20 mg/mL of hyaluronidase stock, dissolve hyaluronidase in 50 mM of Carbonate buffer, 150 mM of NaCl, 0.01% of BSA, and 0.05% of NaN3 (see Table of Materials). Adjust pH to 10 and aliquot in volumes of 77 &#181;L at -30 &#176;C.</li>
	<li>Discard hyaluronidase reaction buffer using a pipette and immerse the organ in the Enzyme Solution (500 &#181;L in a 15 mL conical tube) in a standing position protected from light for 24 h at 37 &#176;C with gentle shaking (5 x g).</li>
	<li>Wash the sample in 15 mL of hyaluronidase wash buffer in a 50 mL conical tube in a horizontal position protected from light for 2 h (three times) at 37 &#176;C with gentle shaking (5 x g).
	<ol>
		<li>To prepare hyaluronidase wash buffer, dissolve 50 mM of Carbonate buffer, 150 mM of NaCl, 0.1% (v/v) of Triton X-100, 5% (v/v) of Methanol, and 0.05% NaN3. Adjust pH to 10. To prepare 10x Carbonate buffer-NaCl stock, dissolve 2.96 g of Sodium Carbonate, 1.86 g of Sodium Hydrogen Carbonate, and 8.77 g of NaCl in 100 mL of double-distilled water with 0.05% NaN3 (see Table of Materials) and adjust pH to 10.</li>
	</ol>
	</li>
</ol>
5. Immunostaining
<ol>
	<li>Label vasculature using anti-&#945;-SMA (alpha-small muscle actin, see Table of Materials) antibodies following the steps below.
	<ol>
		<li>Generate primary plus conjugated Fab fragment secondary antibody complex. Start this reaction 1.5 h prior to the staining procedure.
		<ol>
			<li>Calculate the required amount of primary and secondary antibodies (mix at a 1:0.5 ratio by weight). 
			NOTE: For the primary antibody anti-&#945;-SMA, 3.5 &#181;g is needed to label the entire heart or a fragment of skeletal muscle of similar dimensions. For 2.5 mg/mL product, 3.5/2.5 = 1.4 &#181;L of antibody solution is needed. For secondary antibody AlexaFluor 647 anti-mouse Fab fragment, 1.75 &#181;g is needed to label the entire heart or a fragment of skeletal muscle of similar dimensions. For 1.7 mg/mL product, 1.75/1.7 = 1 &#181;L of antibody solution is needed.</li>
			<li>Mix primary and secondary antibodies in an amber 2 mL tube and incubate for 1.5 h at 37 &#176;C.</li>
		</ol>
		</li>
		<li>For buffer exchange, mix 7.5 mL of 2x HV1 3D immunostaining buffer (included in the kit, see Table of Materials) with 7.5 mL of double-distilled water and immerse the tissue sample for 1.5 h at 32 &#176;C with gentle shaking (5 x g) in a 15 mL conical tube in a horizontal position. Start this reaction simultaneously as the generation of antibody complex (1.5 h prior to the immunostaining procedure).</li>
	</ol>
	</li>
	<li>Perform 3D immunostaining following the steps below.
	<ol>
		<li>In a 15 mL conical tube, prepare the antibody staining solution following Supplementary File 1.</li>
		<li>Collect the tissue sample from the buffer exchange media (step 5.1.2) and immerse it in the antibody staining solution. Incubate tissues individually for 1 week at 32 &#176;C with gentle shaking (5 x g) of the tubes in a standing position protected from light. Seal the tube with paraffin film to avoid evaporation.</li>
		<li>Move to 4 &#176;C and incubate ON in a standing position.</li>
		<li>Cool 1x HV1 3D immunostaining wash buffer (included in the kit, see Table of Materials) to 4 &#176;C and wash the sample with 15 mL buffer (two times) for 30 min each at 4 &#176;C with gentle shaking (5 x g). Keep 15 mL conical tubes in a horizontal position until step 5.2.7.</li>
		<li>Dilute formalin to 1% in 1x HV1 3D immunostaining wash buffer and immerse the sample in 8 mL of the solution for 24 h at 4 &#176;C with gentle shaking (5 x g).</li>
		<li>Incubate in fresh 1% formalin solution for 1 h at 37 &#176;C with gentle shaking (5 x g).</li>
		<li>Wash in 15 mL of PBS for 2 h at 25 &#176;C with gentle shaking (5 x g).</li>
	</ol>
	</li>
	<li>Boost tdTomato signal using anti-Red Fluorescent Protein (RFP) antibodies following the steps below.
	<ol>
		<li>Follow the same incubation times and temperatures as in step 5.2.2. Calculate the amount of primary and secondary antibodies (see Table of Materials) and mix them at a 1:0.5 ratio by weight. 
		NOTE: For primary antibody anti-RFP, 5 &#181;g is needed to label the entire heart or a fragment of skeletal muscle of similar dimensions. For 1.25 mg/mL product, 5/1.25 = 4 &#181;L of the solution is required. For secondary antibody Alexa Fluor 647 anti-rabbit Fab fragment, 2.5 &#181;g is needed to label the entire heart or a fragment of skeletal muscle of similar dimensions. For 1.5 mg/mL product, 2.5/1.5 = 1.7 &#181;L of the solution is needed.</li>
		<li>Prepare the antibody staining solution as mentioned in Supplementary File 1.</li>
	</ol>
	</li>
	<li>Boost GFP signals using anti-GFP nanobodies following the steps below.
	<ol>
		<li>Follow the same incubation times and temperatures as mentioned in step 5.2.2. 
		NOTE: Anti-GFP nanobodies (see Table of Materials) are conjugated with Alexa Fluor 647, so the antibody complex generation is unnecessary.</li>
		<li>Prepare the antibody staining solution as mentioned in Supplementary File 1.</li>
	</ol>
	</li>
</ol>
6. RI matching
<ol>
	<li>Immerse transparent organs in 5 mL of 50% water-diluted CUBIC-R+ solution (see Table of Materials) at RT (ON) with gentle shaking (5 x g) in a 50 mL conical tube. Keep tubes in a standing position during the entire RI matching step. 
	NOTE: Recycled CUBIC R+ solutions from previous experiments could be reused (up to four times) in this step. To prepare CUBIC-R+ solution, dissolve 45% of 2,3-Dimethyl-1-phenyl-5-pyrazolone (Antipyrine), 30% of Nicotinamide or N-Methylnicotinamide, and 0.5% of N-butyldiethanolamine in double-distilled water or use the commercial CUBIC-R+ buffer (see Table of Materials). 
	CAUTION: CUBIC-R+ causes skin irritation, serious eye irritation, and damage to organs. Wear protective gloves, and ensure eye and face protection. Do not breathe dust, fumes, gas, mist, or vapors. Dispose of to an approved waste disposal plant.</li>
	<li>Replace 50% CUBIC-R+ with 5 mL of 100% CUBIC-R+ and incubate with gentle shaking (5 x g) at RT for 2 days. Then, transfer the tissues onto a stack of lint-free wipes and carefully turn the tissues to remove CUBIC-R+ solution from the organ surfaces for 5 min.</li>
</ol>
7. Mounting
<ol>
	<li>After drying the tissues, transfer them to 5 mL of Mounting Solution (RI = 1.520) (see Table of Materials) in a six-well cell culture plate and incubate them (ON) at RT. Frequently turn the tissues to eliminate air bubbles, especially on the surfaces of the organs. 
	NOTE: Organs can be stored for more than 6 months in Mounting Solution or CUBIC-R+.</li>
	<li>Adhere the tissues to the microscope sample holder by using cyanoacrylate-based gel glue.</li>
	<li>Immerse the samples in the microscope quartz cuvette filled with 120 mL of Mounting Solution and image them transversally to their longitudinal axis. Adhere the heart with the apex and the aorta horizontally aligned. 
	&#8203;NOTE: Cleared and mounted organs can be sectioned with a vibratome and imaged at high magnification by confocal microscopy. Embed the organs in 2% agarose and cut sections of 100-500 &#181;m. Organs can also be manually sectioned with a sharp blade to produce thick-tissue sections (&#62;1000 &#181;m). After sectioning, place the slices into glass-bottom 35 mm Petri dishes, mount using the same mounting solution, seal with nail polish, and image with a confocal microscope.</li>
</ol>
8. Image acquisition
<ol>
	<li>Image the mounted samples with a light-sheet microscope (see Table of Materials). Set magnification and step size between individual slices to 3 &#181;m, and use right and left light sheet lasers with 5 &#181;m thicknesses and 100% width. Set the exposure time constant at 50-100 ms, and adjust the laser power from 10% to 80% depending on the fluorescence signal intensity.
	<ol>
		<li>For the co-detection of tdTomato-expressing parasites and DiR-stained dormant amastigotes (Figure 2C), use red (Ex/Em 561/620-660 nm) and infra-red (Ex/Em 785/845-55 nm) channels, respectively. For the co-detection of T cells and tdTomato-expressing parasites in CD8 reporter mouse (Figure 2D), use green (Ex/Em 488/525-50 nm) and red channel, respectively.</li>
		<li>For the coinfections assays (Figure 2E), as well as for the co-detection of parasites expressing GFP in nuclei reporter mice (Figure 2F), use the green and red channels, respectively. For the detection of tdTomato-expressing parasites, nuclear dye, and heart vasculature (Figure 3B,C), use the red, green, and far-red (Ex/Em 640/680-730 nm) channels, respectively.</li>
		<li>Detect anti-RFP antibodies with the far-red channel and anti-GFP nanobodies using the green channel (Figure 3D).</li>
	</ol>
	</li>
	<li>Convert the acquired TIFF image stacks and 3D reconstruct the organs using Imaris v9.7.2 software (see Table of Materials).</li>
</ol>
9. Surface reconstruction and quantification with Imaris software
<ol>
	<li>Select surfaces detection algorithm tool and initiate the wizard in the image analysis software.</li>
	<li>Perform an initial analysis in a 3D region of interest (randomly selected) and then apply it to the entire 3D organ reconstruction. After choosing a region of interest (ROI), select the channel to be analyzed, uncheck the smooth button, and select background subtraction. 
	NOTE: Background subtraction calculates a unique local background value for every voxel and subtracts this from the original pixel value. The diameter of the largest sphere which fits into the object must be up to 200 &#181;m for T. cruzi-infected cells, 10 &#181;m for T cells, and 5 &#181;m for individual parasites.</li>
	<li>Use the histogram to adjust the classification and the filter drop-down list to select the measurements for classification. 
	NOTE: Use the histogram to adjust the classification: ellipticity (oblate) and sphericity measurements are recommended.</li>
	<li>Finalize the wizard, press statistics button, and retrieve the total number of parasite-infected cells or T cells as the number of disconnected components per time point.</li>
</ol>

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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+three-dimensional+single-cell-resolution+whole-brain+atlas+using+CUBIC-X+expansion+microscopy+and+tissue+clearing.">A three-dimensional single-cell-resolution whole-brain atlas using CUBIC-X expansion microscopy and tissue clearing.</a> Nature Neuroscience. 21, 625-637 (2018).</li><li>Zhao, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+molecular+probing+of+intact+human+organs.">Cellular and molecular probing of intact human organs.</a> Cell. 180 (4), 796-812 (2020).</li><li>Erturk, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+imaging+of+solvent-cleared+organs+using+3DISCO.">Three-dimensional imaging of solvent-cleared organs using 3DISCO.</a> Nature Protocols. 7, 1983-1995 (2012).</li><li>Renier, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iDISCO:+a+simple,+rapid+method+to+immunolabel+large+tissue+samples+for+volume+imaging.">iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging.</a> Cell. 159 (4), 896-910 (2014).</li><li>Chung, K., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLARITY+for+mapping+the+nervous+system.">CLARITY for mapping the nervous system.</a> Nature Methods. 10, 508-513 (2013).</li><li>Susaki, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile+whole-organ/body+staining+and+imaging+based+on+electrolyte-gel+properties+of+biological+tissues.">Versatile whole-organ/body staining and imaging based on electrolyte-gel properties of biological tissues.</a> Nature Communications. 11, 1982 (2020).</li><li>Kubota, S. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-body+profiling+of+cancer+metastasis+with+single-cell+resolution.">Whole-body profiling of cancer metastasis with single-cell resolution.</a> Cell Reports. 20 (1), 236-250 (2017).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> Journal of Visualized Experiments. (65), e3564 (2012).</li><li>Bustamante, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+drug+regimen+clears+active+and+dormant+trypanosomes+in+mouse+models+of+Chagas+disease.">A modified drug regimen clears active and dormant trypanosomes in mouse models of Chagas disease.</a> Science Translational Medicine. 12 (567), (2020).</li><li>Wang, H., Khoradmehr, A., Tamadon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FACT+or+PACT:+A+comparison+between+free-acrylamide+and+acrylamide-based+passive+sodium+dodecyl+sulfate+tissue+clearing+for+whole+tissue+imaging.">FACT or PACT: A comparison between free-acrylamide and acrylamide-based passive sodium dodecyl sulfate tissue clearing for whole tissue imaging.</a> Cell Journal. 21 (2), 103-114 (2019).</li><li>Hofmann, J., Gadjalova, I., Mishra, R., Ruland, J., Keppler, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+tissue+clearing+and+multi-organ+volumetric+imaging+enable+quantitative+visualization+of+sparse+immune+cell+populations+during+inflammation.">Efficient tissue clearing and multi-organ volumetric imaging enable quantitative visualization of sparse immune cell populations during inflammation.</a> Frontiers in Immunology. 11, 599495 (2020).</li><li>Messal, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen+retrieval+and+clearing+for+whole-organ+immunofluorescence+by+FLASH.">Antigen retrieval and clearing for whole-organ immunofluorescence by FLASH.</a> Nature Protocols. 16, 239-262 (2021).</li><li>Kolesova, H., Capek, M., Radochova, B., Janacek, J., Sedmera, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+different+tissue+clearing+methods+and+3D+imaging+techniques+for+visualization+of+GFP-expressing+mouse+embryos+and+embryonic+hearts.">Comparison of different tissue clearing methods and 3D imaging techniques for visualization of GFP-expressing mouse embryos and embryonic hearts.</a> Histochemistry and Cell Biology. 146 (2), 141-152 (2016).</li><li>Chen, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+versatile+tiling+light+sheet+microscope+for+imaging+of+cleared+tissues.">A versatile tiling light sheet microscope for imaging of cleared tissues.</a> Cell Reports. 33, 108349 (2020).</li></ol>

The authors declare that they have no competing interests.

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 ...

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

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Immunology and Infection

3.0K Views.  University of Georgia. 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.

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

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 ...

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.

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...