Name: induction and scoring of graft-versus-host disease in a xenogeneic murine model and quantification of human t cells in mouse tissues using digital pcr
Uploaded: 2019-05-23T12:30:00
Description: Watch this Scientific Journal Video about Induction and Scoring of Graft-Versus-Host Disease in a Xenogeneic Murine Model and Quantification of Human T Cells in Mouse Tissues using Digital PCR at JoVE

We would like to acknowledge the laboratory of Lane Christenson for providing the digital PCR machine used in these experiments and for the technical support provided. We would also like to thank Dr. Thomas Yankee for his guidance and mentorship. These studies were supported by the Tripp Family Foundation.

All mouse experiments were performed in compliance, and with approval from, the University of Kansas Medical Center Institutional Animal Care and Use Committee. All healthy human blood samples were obtained under informed consent and with approval from the Institutional Review Board at the University of Kansas Medical Center.
1. Irradiation of NSG Mice
<ol>
	<li>One day prior to PBMC injection, irradiate 8&#8211;12-week old NSG mice (either sex can be used). In a sterile biosafety cabinet, p...

<ol>
	<li>Jagasia, 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=Risk+factors+for+acute+GVHD+and+survival+after+hematopoietic+cell+transplantation.">Risk factors for acute GVHD and survival after hematopoietic cell transplantation.</a> Blood. 119 (1), 296-307 (2012).</li><li>Bolanos-Meade, J., 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=Phase+3+clinical+trial+of+steroids/mycophenolate+mofetil+vs+steroids/placebo+as+therapy+for+acute+GVHD:+BMT+CTN+0802.">Phase 3 clinical trial of steroids/mycophenolate mofetil vs steroids/placebo as therapy for acute GVHD: BMT CTN 0802.</a> Blood. 124 (22), 3221-3227 (2014).</li><li>Gooley, T. 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=Reduced+mortality+after+allogeneic+hematopoietic-cell+transplantation.">Reduced mortality after allogeneic hematopoietic-cell transplantation.</a> New England Journal of Medicine. 363 (22), 2091-2101 (2010).</li><li>Hahn, T., 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=Significant+improvement+in+survival+after+allogeneic+hematopoietic+cell+transplantation+during+a+period+of+significantly+increased+use,+older+recipient+age,+and+use+of+unrelated+donors.">Significant improvement in survival after allogeneic hematopoietic cell transplantation during a period of significantly increased use, older recipient age, and use of unrelated donors.</a> Journal of Clinical Oncology. 31 (19), 2437-2449 (2013).</li><li>Khoury, H. J., 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=Improved+survival+after+acute+graft-versus-host+disease+diagnosis+in+the+modern+era.">Improved survival after acute graft-versus-host disease diagnosis in the modern era.</a> Haematologica. 102 (5), 958-966 (2017).</li><li>King, M. 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=Human+peripheral+blood+leucocyte+non-obese+diabetic-severe+combined+immunodeficiency+interleukin-2+receptor+gamma+chain+gene+mouse+model+of+xenogeneic+graft-versus-host-like+disease+and+the+role+of+host+major+histocompatibility+complex.">Human peripheral blood leucocyte non-obese diabetic-severe combined immunodeficiency interleukin-2 receptor gamma chain gene mouse model of xenogeneic graft-versus-host-like disease and the role of host major histocompatibility complex.</a> Clinical &amp; Experimental Immunology. 157 (1), 104-118 (2009).</li><li>Lucas, P. J., Shearer, G. M., Neudorf, S., Gress, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+antimurine+xenogeneic+cytotoxic+response.+I.+Dependence+on+responder+antigen-presenting+cells.">The human antimurine xenogeneic cytotoxic response. I. Dependence on responder antigen-presenting cells.</a> Journal of Immunology. 144 (12), 4548-4554 (1990).</li><li>Ito, R., 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=Highly+sensitive+model+for+xenogenic+GVHD+using+severe+immunodeficient+NOG+mice.">Highly sensitive model for xenogenic GVHD using severe immunodeficient NOG mice.</a> Transplantation. 87 (11), 1654-1658 (2009).</li><li>Kawasaki, 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=Comprehensive+Analysis+of+the+Activation+and+Proliferation+Kinetics+and+Effector+Functions+of+Human+Lymphocytes,+and+Antigen+Presentation+Capacity+of+Antigen-Presenting+Cells+in+Xenogeneic+Graft-Versus-Host+Disease.">Comprehensive Analysis of the Activation and Proliferation Kinetics and Effector Functions of Human Lymphocytes, and Antigen Presentation Capacity of Antigen-Presenting Cells in Xenogeneic Graft-Versus-Host Disease.</a> Biology of Blood and Marrow Transplantation. 24 (8), 1563-1574 (2018).</li><li>Ito, R., 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+Novel+Xenogeneic+Graft-Versus-Host+Disease+Model+for+Investigating+the+Pathological+Role+of+Human+CD4(+)+or+CD8.">A Novel Xenogeneic Graft-Versus-Host Disease Model for Investigating the Pathological Role of Human CD4(+) or CD8.</a> T Cells Using Immunodeficient NOG Mice. American Journal of Transplantation. 17 (5), 1216-1228 (2017).</li><li>Trotta, E., 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+human+anti-IL-2+antibody+that+potentiates+regulatory+T+cells+by+a+structure-based+mechanism.">A human anti-IL-2 antibody that potentiates regulatory T cells by a structure-based mechanism.</a> Nature Medicine. 24 (7), 1005-1014 (2018).</li><li>Dijke, I. E., 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=Discarded+Human+Thymus+Is+a+Novel+Source+of+Stable+and+Long-Lived+Therapeutic+Regulatory+T+Cells.">Discarded Human Thymus Is a Novel Source of Stable and Long-Lived Therapeutic Regulatory T Cells.</a> American Journal of Transplantation. 16 (1), 58-71 (2016).</li><li>Huang, F., 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=Human+Gingiva-Derived+Mesenchymal+Stem+Cells+Inhibit+Xeno-Graft-versus-Host+Disease+via+CD39-CD73-Adenosine+and+IDO+Signals.">Human Gingiva-Derived Mesenchymal Stem Cells Inhibit Xeno-Graft-versus-Host Disease via CD39-CD73-Adenosine and IDO Signals.</a> Frontiers in Immunology. 8, 68 (2017).</li><li>Stockis, J., 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=Blocking+immunosuppression+by+human+Tregs+in+vivo+with+antibodies+targeting+integrin+alphaVbeta8.">Blocking immunosuppression by human Tregs in vivo with antibodies targeting integrin alphaVbeta8.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (47), E10161-E10168 (2017).</li><li>Vogelstein, B., Kinzler, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+PCR.">Digital PCR.</a> Proceedings of the National Academy of Sciences of the United States of America. 96 (16), 9236-9241 (1999).</li><li>Quan, P. L., Sauzade, M., Brouzes, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dPCR:+A+Technology+Review.">dPCR: A Technology Review.</a> Sensors (Basel). 18 (4), (2018).</li><li>Sykes, P. J., 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=Quantitation+of+targets+for+PCR+by+use+of+limiting+dilution.">Quantitation of targets for PCR by use of limiting dilution.</a> Biotechniques. 13 (3), 444-449 (1992).</li><li>Ma, H., 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=Evaluation+of+Bacterial+Contamination+in+Goat+Milk+Powder+Using+PacBio+Single+Molecule+Real-Time+Sequencing+and+Droplet+Digital+PCR.">Evaluation of Bacterial Contamination in Goat Milk Powder Using PacBio Single Molecule Real-Time Sequencing and Droplet Digital PCR.</a> Journal of Food Protection. , 1791-1799 (2018).</li><li>Vitale, S. R., 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=An+optimized+workflow+to+evaluate+estrogen+receptor+gene+mutations+in+small+amounts+of+cell-free+DNA.">An optimized workflow to evaluate estrogen receptor gene mutations in small amounts of cell-free DNA.</a> Journal of Molecular Diagnostics. , (2018).</li><li>Gorgannezhad, L., Umer, M., Islam, M. N., Nguyen, N. T., Shiddiky, M. J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+DNA+and+liquid+biopsy:+opportunities,+challenges,+and+recent+advances+in+detection+technologies.">Circulating tumor DNA and liquid biopsy: opportunities, challenges, and recent advances in detection technologies.</a> Lab Chip. 18 (8), 1174-1196 (2018).</li><li>Yardeni, T., Eckhaus, M., Morris, H. D., Huizing, M., Hoogstraten-Miller, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retro-orbital+injections+in+mice.">Retro-orbital injections in mice.</a> LabAnimal. 40 (5), 155-160 (2011).</li><li>Machholz, E., Mulder, G., Ruiz, C., Corning, B. F., Pritchett-Corning, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manual+restraint+and+common+compound+administration+routes+in+mice+and+rats.">Manual restraint and common compound administration routes in mice and rats.</a> Journal of Visualized Experiments. (67), (2012).</li><li>Cooke, K. R., 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=An+experimental+model+of+idiopathic+pneumonia+syndrome+after+bone+marrow+transplantation:+I.+The+roles+of+minor+H+antigens+and+endotoxin.">An experimental model of idiopathic pneumonia syndrome after bone marrow transplantation: I. The roles of minor H antigens and endotoxin.</a> Blood. 88 (8), 3230-3239 (1996).</li><li>Weisdorf, D. J., 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=Prospective+grading+of+graft-versus-host+disease+after+unrelated+donor+marrow+transplantation:+a+grading+algorithm+versus+blinded+expert+panel+review.">Prospective grading of graft-versus-host disease after unrelated donor marrow transplantation: a grading algorithm versus blinded expert panel review.</a> Biology of Blood and Marrow Transplantation. 9 (8), 512-518 (2003).</li><li>Nervi, B., 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=Factors+affecting+human+T+cell+engraftment,+trafficking,+and+associated+xenogeneic+graft-vs-host+disease+in+NOD/SCID+beta2mnull+mice.">Factors affecting human T cell engraftment, trafficking, and associated xenogeneic graft-vs-host disease in NOD/SCID beta2mnull mice.</a> Experimental Hematology. 35 (12), 1823-1838 (2007).</li><li>Leon-Rico, D., 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=Comparison+of+haematopoietic+stem+cell+engraftment+through+the+retro-orbital+venous+sinus+and+the+lateral+vein:+alternative+routes+for+bone+marrow+transplantation+in+mice.">Comparison of haematopoietic stem cell engraftment through the retro-orbital venous sinus and the lateral vein: alternative routes for bone marrow transplantation in mice.</a> LabAnimal. 49 (2), 132-141 (2015).</li><li>Ali, 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=Xenogeneic+graft-versus-host-disease+in+NOD-scid+IL-2Rgammanull+mice+display+a+T-effector+memory+phenotype.">Xenogeneic graft-versus-host-disease in NOD-scid IL-2Rgammanull mice display a T-effector memory phenotype.</a> PLoS One. 7 (8), e44219 (2012).</li><li>van Rijn, R. 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=A+new+xenograft+model+for+graft-versus-host+disease+by+intravenous+transfer+of+human+peripheral+blood+mononuclear+cells+in+RAG2-/-+gammac-/-+double-mutant+mice.">A new xenograft model for graft-versus-host disease by intravenous transfer of human peripheral blood mononuclear cells in RAG2-/- gammac-/- double-mutant mice.</a> Blood. 102 (7), 2522-2531 (2003).</li><li>Wunderlich, 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=OKT3+prevents+xenogeneic+GVHD+and+allows+reliable+xenograft+initiation+from+unfractionated+human+hematopoietic+tissues.">OKT3 prevents xenogeneic GVHD and allows reliable xenograft initiation from unfractionated human hematopoietic tissues.</a> Blood. 123 (24), e134-e144 (2014).</li><li>Schroeder, M. A., DiPersio, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+graft-versus-host+disease:+advances+and+limitations.">Mouse models of graft-versus-host disease: advances and limitations.</a> Disease Models &amp; Mechanisms. 4 (3), 318-333 (2011).</li><li>Zeiser, R., Blazar, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+models+of+acute+and+chronic+graft-versus-host+disease:+how+predictive+are+they+for+a+successful+clinical+translation?.">Preclinical models of acute and chronic graft-versus-host disease: how predictive are they for a successful clinical translation?.</a> Blood. 127 (25), 3117-3126 (2016).</li></ol>

Disease progression is generally consistent in the xenoGVHD model, even with injection of PBMC from different donors, so multiple experiments can be combined. The key steps required to maintain this consistency are proper i.v. injection technique, blinding and consistent scoring. A study by Nervi et al.25 demonstrated that compared to intravenous tail vein injection, retro-orbital injections of PBMC resulted in more consistent engraftment and more severe GVHD. Leon-Rico et al.26<...

Allogeneic hematopoietic stem cell transplant (HSCT) has become routine treatment for patients suffering from hematological malignancies such as leukemia with poor prognosis. A significant complication of HSCT is acute graft-versus-host disease (GVHD). A 2012 study reported that acute GVHD developed in 39% of HSCT patients receiving transplants from sibling donors and 59% of patients receiving transplants from unrelated donors1. Acute GVHD occurs when donor-derived T cells attack recipient&#8217;s organs. The only successful therapy for GVHD is treatment with highly immunosuppressive drugs2, which are highly toxic and increase the risk of infection and tumor recurrence. Thus, despite improvements that have been made in acute GVHD survival in recent years3,4,5, there is still a critical need for improved GVHD therapies with minimal toxicity that promote long-term remission.
The overall goal of the following methods is to induce and score xenogeneic GVHD (xenoGVHD). The xenoGVHD model was developed as a tool to induce acute GVHD with human cells rather than murine cells allowing for more direct translation of pre-clinical GVHD research to clinical trials6. This model involves intravenously injecting human peripheral blood mononuclear cells (PBMC) into NOD-SCID IL-2R&#947;null (NSG) mice that are sublethally irradiated. Injected human T cells are activated by human antigen presenting cells (APCs) presenting murine antigen and the activated T cells migrate to distant tissues resulting in systemic inflammation and ultimately death6,7,8,9,10. Disease pathology and progression in the xenoGVHD model closely mimic human acute GVHD. Specifically, the pathogenic human T cells are reactive to murine major histocompatibility complex (MHC) proteins, which is similar to the T cell alloreactivity in human GVHD6,9. The primary advantage of the xenoGVHD model over the mouse MHC-mismatch model, the other widely used GVHD model, is it allows for testing of therapies on human cells rather than murine cells. This allows for testing of products that can directly be translated to the clinic without any modifications because they are made to target human cells. Recently, this model has been used to test a human anti-IL-2 antibody11, human thymic regulatory T cells (Tregs)12 and human mesenchymal stem cells13 as potential treatments for acute GVHD. In a wider context, this model can be used as an in vivo suppression assay for any drug or cell type that can suppress human T cell activity. For example, Stockis et al.14 used the xenoGVHD model to study the effect of blocking integrin &#945;V&#946;8 on Treg suppressive activity in vivo. Thus, the xenoGVHD model can provide insight into the mechanism of any therapy targeting T cells in an in vivo setting.
An additional method described in this protocol is how to detect human T cells in mouse tissues using digital polymerase chain reaction (dPCR). The goal of this method is to offer a tool to quantify migration and proliferation of T cells in target tissues, which measure efficacy of immunosuppressive therapies being tested in this model. dPCR is a relatively novel method for quantification of nucleic acids15. Briefly, the PCR reaction mixture is divided into partitions that contain small numbers of the target sequence or no target at all. The target sequence is then amplified and detected using DNA intercalating dyes or fluorescent target-specific probes. dPCR quantifies the number of copies of target sequence based on the fraction of positive partitions and Poisson&#8217;s statistics15,16. Detecting T cells with dPCR requires much less tissue compared with other alternative methods, including flow cytometry and histology, and can be performed on frozen or fixed tissue. dPCR does not require a standard curve to determine copy numbers, nor are technical replicates required. This reduces the amount of reagent and template DNA needed for dPCR compared to traditional quantitative PCR (qPCR)16. Partitioning the PCR reaction into sub-reactions in dPCR effectively concentrates targets17. Thus, dPCR is primarily a tool for detection of rare targets in a large amount of non-target DNA. For example, dPCR is being used to detect bacterial contamination in milk18, identify rare mutations in the estrogen receptor gene19, and detect circulating tumor DNA in the blood of patients20. In this protocol, dPCR serves as an efficient tool for detecting and quantifying human T cells in tissues of mice with xenoGVHD.

<table>
	<tbody>
		<tr>
			<td>1.5 mL eppendorf tubes</td>
			<td>Fisher</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL serological pipet</td>
			<td>VWR International</td>
			<td>89130-898</td>
			<td></td>
		</tr>
		<tr>
			<td>10mL BD Vacutainers - Green capped with Sodium Heparin</td>
			<td>Becton Dickinson</td>
			<td>366480</td>
			<td></td>
		</tr>
		<tr>
			<td>250 &#181;L Ranin pipette tips</td>
			<td>Rainin</td>
			<td>17001118</td>
			<td>Do not use other pipettes or pipet tips for droplet generation</td>
		</tr>
		<tr>
			<td>50 mL conical tube</td>
			<td>VWR International</td>
			<td>89039-656</td>
			<td></td>
		</tr>
		<tr>
			<td>96-Well ddPCR plate</td>
			<td>Bio-Rad</td>
			<td>12001925</td>
			<td></td>
		</tr>
		<tr>
			<td>ACK (Ammonium-Chloride-Potassium) Lysing Buffer</td>
			<td>Lonza</td>
			<td>10-548E</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Alcohol Wipes</td>
			<td>Fisher Scientific</td>
			<td>6818</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia Chamber</td>
			<td>World Precision Instruments</td>
			<td>EZ-178</td>
			<td>Provided by animal facility</td>
		</tr>
		<tr>
			<td>Anesthesia Machine</td>
			<td>Parkland Scientific</td>
			<td>PM1002</td>
			<td>Provided by animal facility</td>
		</tr>
		<tr>
			<td>BD Vacutainer Safety-Lok Blood Collection Set</td>
			<td>Becton Dickinson</td>
			<td>367281</td>
			<td></td>
		</tr>
		<tr>
			<td>DG8 Cartridges and Gaskets for QX100/QX200 Droplet Generator</td>
			<td>Bio-Rad</td>
			<td>1864007</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse and RNAse free Molecular Grade H2O</td>
			<td>Life Technologies</td>
			<td>1811318</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl alcohol, Pure,200 proof, for molecular biology</td>
			<td>Sigma-Aldrich</td>
			<td>E7023-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll</td>
			<td>Fisher Scientific</td>
			<td>45001750</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin Syringe</td>
			<td>Fisher Scientific</td>
			<td>329424</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Sigma-Aldrich</td>
			<td>CDS019936</td>
			<td>Provided by animal facility</td>
		</tr>
		<tr>
			<td>Liquid nitrogen</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Irradiator Pie Cage</td>
			<td>Braintree Scientific, Inc.</td>
			<td>MPC 1</td>
			<td>Holds up to 11 mice</td>
		</tr>
		<tr>
			<td>Nexcare Gentle Paper Tape (a.k.a. 3M Micropore Surgical Tape / 3/4&#34;)</td>
			<td>Fisher Scientific</td>
			<td>19-027-761</td>
			<td></td>
		</tr>
		<tr>
			<td>P1000 pipetman</td>
			<td>MidSci</td>
			<td>A-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>P200 pipetman</td>
			<td>MidSci</td>
			<td>A-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierceable Foil Heat Seal</td>
			<td>Bio-Rad</td>
			<td>1814040</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetaid Gilson Macroman</td>
			<td>Fisher Scientific</td>
			<td>F110756</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet-Lite Multi Pipette L8-200XLS+</td>
			<td>Rainin</td>
			<td>17013805</td>
			<td>Do not use other pipettes or pipet tips for droplet generation</td>
		</tr>
		<tr>
			<td>Qiagen DNeasy Blood and Tissue Kit</td>
			<td>Qiagen</td>
			<td>69506</td>
			<td></td>
		</tr>
		<tr>
			<td>qPCR plates</td>
			<td>VWR International</td>
			<td>89218-292</td>
			<td></td>
		</tr>
		<tr>
			<td>QX200 Droplet Digital PCR System</td>
			<td>Bio-Rad</td>
			<td>12001925</td>
			<td>Includes droplet generator, droplet reader, laptop computer, software, associated component consumables, for EvaGreen or probe-based digital PCR applications</td>
		</tr>
		<tr>
			<td>QX200 Droplet Generation Oil for EvaGreen</td>
			<td>Bio-Rad</td>
			<td>1864006</td>
			<td></td>
		</tr>
		<tr>
			<td>QX200 ddPCR EvaGreen Supermix</td>
			<td>Bio-Rad</td>
			<td>1864033</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase and DNase-free plate seal</td>
			<td>Thermo Scientific</td>
			<td>12565491</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI Advanced 1640</td>
			<td>Life Technologies</td>
			<td>12633012</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Gauze Pads (2&#34; x 2&#34;, 12-Ply)</td>
			<td>Fisher Scientific</td>
			<td>67522</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Phosphate Buffered Saline</td>
			<td>Fisher Scientific</td>
			<td>21040CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile reservoir</td>
			<td>VWR International</td>
			<td>89094-662</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgial Scissors</td>
			<td>Kent Scientific</td>
			<td>INS600393-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Forceps</td>
			<td>Kent Scientific</td>
			<td>INS650914-4</td>
			<td></td>
		</tr>
	</tbody>
</table>

induction and scoring of graft-versus-host disease in a xenogeneic murine model and quantification of human t cells in mouse tissues using digital pcr

Acute graft-versus-host disease (GVHD) is a significant limitation for patients receiving hematopoietic stem cell transplant as therapy for hematological deficiencies and malignancies. Acute GVHD occurs when donor T cells recognize host tissues as a foreign antigen and mount an immune response to the host. Current treatments involve toxic immunosuppressive drugs that render patients susceptible to infection and recurrence. Thus, there is ongoing research to provide an acute GVHD therapy that can effectively target donor T cells and reduce side effects. Much of this pre-clinical work uses the xenogenic GVHD (xenoGVHD) murine model that allows for testing of immunosuppressive therapies on human cells rather than murine cells in an in vivo system. This protocol outlines how to induce xenoGVHD and how to blind and standardize clinical scoring to ensure consistent results. Additionally, this protocol describes how to use digital PCR to detect human T cells in mouse tissues, which can subsequently be used to quantify efficacy of tested therapies. The xenoGVHD model not only provides a model to test GVHD therapies but any therapy that can suppress human T cells, which could then be applied to many inflammatory diseases.

Sublethally irradiated 8-12-week old NSG mice of both sexes that received human PBMC started displaying clinical signs of GVHD around day 10 post injection compared to negative control mice that received PBS only (Figure 1A). XenoGVHD mice had a median survival of 23.5 days (Figure 1B). With digital PCR, CD3 epsilon positive human T cells could be detected in the lung and liver samples of mice that received human PBMC. Tissue sam...

Watch this Scientific Journal Video about Induction and Scoring of Graft-Versus-Host Disease in a Xenogeneic Murine Model and Quantification of Human T Cells in Mouse Tissues using Digital PCR at JoVE

Induction and Scoring of Graft-Versus-Host Disease in a Xenogeneic Murine Model and Quantification of Human T Cells in Mouse Tissues using Digital PCR

Here, we present a protocol to induce and score disease in a xenogeneic graft-versus-host disease (xenoGVHD) model. xenoGVHD provides an in vivo model to study immunosuppression of human T cells. Additionally, we describe how to detect human T cells in tissues with digital PCR as a tool to quantify immunosuppression.

Acute graft-versus-host disease (GVHD) is a significant limitation for patients receiving hematopoietic stem cell transplant as therapy for hematological ...

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 ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

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piggyBac Transposon System Modification of Primary Human T Cells

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Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

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There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

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Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

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Induction of Intestinal Graft-versus-host Disease and Its Mini-endoscopic Assessment in Live Mice

Acute graft-versus-host disease (GvHD) represents the most severe complication that patients previously undergoing allogeneic hematopoietic stem cell ...

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Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear ...

Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to ...