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, place mice in a sterilized pie cage or microisolator. Irradiate mice in a Cs137 source or small animal irradiator (e.g., RS 2000) with a total dose of 150 cGy with slow rotation to ensure even irradiation.</li>
	<li>Place mice into clean cages in a sterile biosafety cabinet.</li>
</ol>
2. Preparation of human PBMC for injection
<ol>
	<li>Collect enough healthy human blood to isolate 1.1 x 107 PBMC per mouse. Dilute the heparinized blood in an equal volume of 2% fetal bovine serum (FBS) in phosphate buffered saline (PBS). 
	NOTE: With this protocol, the PMBC yield is generally 0.5&#8211;1 x 107 per 10 mL of whole blood. Each mouse will receive 107 PBMC in 100 &#181;L of PBS. The extra 1 x 106 in 10 &#181;L per mouse ensures that each mouse receives the full dosage of PBMC, should there be any issues with filling syringes.</li>
	<li>Add 15 mL lymphocyte separation density gradient medium (e.g., Ficoll) to a 50 mL conical tube and then carefully overlay up to 25 mL of diluted blood on top of the density gradient. Centrifuge the tube containing density gradient and diluted blood at 400 x g for 40 min at room temperature without brake.</li>
	<li>Harvest the PBMC interface into 10 mL of PBS in a 50 mL conical tube. Centrifuge the cells at 400 x g for 10 min. Remove the supernatant.</li>
	<li>Loosen pellet by flicking tube and resuspend in 10 mL PBS to wash the PBMC. Centrifuge the cells at 400 x g for 5 min.</li>
	<li>(Optional) Discard the supernatant and lyse red blood cells (RBCs) in the cell pellet by adding a volume of Ammonium-Chloride-Potassium (ACK) lysis buffer that is equal to the pellet volume. Gently resuspend the pellet and swirl the tube for 30&#8211;60 s. Fill tube with serum-free RPMI media and centrifuge the tube at 400 x g for 5 min.</li>
	<li>Remove the supernatant and re-suspend the cell pellet in 5 mL of PBS. Count the cells using trypan blue exclusion with a hemocytometer and a microscope.</li>
	<li>Centrifuge the cells at 400 x g for 5 min. Remove the supernatant and resuspend cells at 1 x 108 cells/mL in PBS.</li>
</ol>
3. Retro-orbital injection of human PBMC into mice21
<ol>
	<li>Place an anesthesia chamber in a laminar flow hood to maintain sterility. Pre-charge the anesthesia chamber with 5% isoflurane and 1 L/min oxygen flow rate for 5 min.</li>
	<li>Reduce isoflurane to 2% and put mice from the same cage into the anesthesia chamber. Once mice lose righting, anesthetize mice for 5 min in chamber. During this time, pre-fill syringe with 100 &#181;L (1 x 107 cells) of cell suspension.</li>
	<li>Place mouse on a heating pad with its nose in a nose cone to maintain anesthesia. Check for loss of consciousness by pinching a foot pad and checking for lack of reflex.</li>
	<li>Restrain mouse with the thumb and middle finger. Insert the 28 G 1/2 in needle with the bevel down lateral to the medial canthus, through the conjunctival membrane until the back of the eye is reached. Slightly retract the needle and slowly inject 100 &#956;L of cells (1 x 107 cells).</li>
	<li>Fully retract the needle and properly dispose of the syringe and needle. Close the eyelid and apply mild pressure to the injection site with a gauze sponge.</li>
	<li>Examine the injection site for swelling or other visible trauma. Allow the mouse to regain consciousness in a sterile cage lined with paper towels to prevent aspiration of bedding before moving to home cage.&#160;After any bleeding has stopped, apply a single drop of proparacaine to the eye for analgesia. 
	NOTE: Tail vein injection is an alternative method of intravenous injection of PBMC for this protocol as described by Macholz et al.22.</li>
</ol>
4. Clinical Scoring of acute GVHD in Mice (Figure 1)23
<ol>
	<li>Measure GVHD score every other day until mice reach a score of a 2 and then every day until day of sacrifice.
	<ol>
		<li>Place the cage in a laminar flow hood, remove the food and water and put the lid back on. Score activity by observing mice for 5 min and assign scores as follows: 0 = mouse starts walking within a couple minutes and keeps walking around cage, 1 = mouse takes a more than a couple minutes to get up and walks slowly around cage, 2 = mouse doesn&#8217;t get up in 5 min and only walks when touched.</li>
	</ol>
	</li>
	<li>Weigh each mouse in a glass beaker and give a weight loss score: 0 = &#60;10% change, 1 = 10&#8211;25% change and 2 = &#62;25% change.</li>
	<li>While the mouse is still in the beaker, inspect for posture: 0 = normal, 1 = hunching at rest, 2 = hunching impairs movement; fur texture: 0 = normal, 1 = mild to moderate ruffling, 2 = severe ruffling, and skin integrity : 0 = normal, 1 = scaling of paws/tail, 2 = obvious areas of denuded skin (look at ears, tail and paws for scaling).</li>
	<li>With five categories and a score of 0&#8211;2 for each category, a mouse can reach a maximum score of 10. When a mouse reaches a score of 7 or greater or reaches 42 days post-injection, euthanize mouse via CO2 euthanasia or other method approved by the local Institutional Animal Care and Use Committee. 
	NOTE: To ensure accuracy of results, the scoring is performed by a researcher who is blinded to the treatment groups24. Additionally, although &#62;20% of body weight loss is the recommended humane endpoint for many IACUC protocols, with additional justification IACUC approval of this protocol can be obtained.</li>
</ol>
5. Harvesting tissues for genomic DNA from euthanized mice and isolating genomic DNA
<ol>
	<li>Dissect mice using sterile surgical tools. Cut a small piece of tissue about 3 mm x 0.5 mm in size from an organ of interest, e.g., lung, liver, or spleen. Weigh the tissue sample and place the tissue piece in a sterile 1.5 mL tube. If collecting multiple tissues, wash tools with ethanol between cutting each organ.</li>
	<li>Freeze tissues by submerging the tubes containing the tissue in liquid nitrogen until it stops bubbling and store in -80 &#176;C overnight. Thaw the tissue samples and lyse them according to a genomic DNA (gDNA) isolation kit.</li>
	<li>Isolate genomic DNA according to manufacturer&#8217;s instructions as described in the kit. Be sure to note the amount of tissue that was processed per microliter of eluted gDNA (mg/&#956;L). 
	NOTE: Frozen tissues can be stored at -80 &#176;C for processing at a later time.</li>
</ol>
6. Quantification of human T cells using digital PCR (Figure 2)
<ol>
	<li>Prepare digital PCR reactions according to the protocol for DNA binding dyes for the digital PCR machine being used. Use the following primers specific for human CD3 epsilon genomic DNA (NCBI Reference Sequence: NG_007383.1); Forward Primer: AGGCTGCCTTAACTCCCAAG, Reverse Primer: GCCCTACCAGCTGTGGAAAC. These primers will yield a single band of 105 bp. 
	NOTE: Add 0.5 &#956;L of a restriction enzyme, such as HindIII, to each reaction to digest the genomic DNA. Be sure to test out different gDNA dilutions to optimize separation of positive and negative droplets. Additionally, a subset of infiltrating T cells may be non-pathogenic regulatory T cells. These cells can be quantified using additional primers for regulatory T cell markers.</li>
	<li>Carry out the digital PCR reaction under the following conditions: Lid at 105 &#176;C, 95 &#176;C for 10 min (1 cycle); 95 &#176;C for 30 s, ramp 2 &#176;C/s and 55 &#176;C for 1 min, ramp 2 &#176;C/s (40 cycles); 72 &#176;C for 10 min, 12 &#176;C hold. 
	NOTE: These parameters may need to be adjusted depending on digital PCR equipment.</li>
	<li>Acquire digital PCR data with analysis software. Report data as numbers of copies per mg of tissue using the following equation: (number of copies/&#956;L) x (&#956;L of gDNA in reaction) x (dilution factor)/(mg of tissue/&#956;L of total gDNA )</li>
</ol>

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No conflicts of interest declared.

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 also demonstrated that retro-orbital injections resulted in more consistent hematopoietic stem cell engraftment in mice compared to tail vein injection. However, if necessary, tail vein injections can be used as an alternative method in the xenoGVHD model27,28,29.
The problem of variability of outcomes associated with tail vein injections can be reduced by increasing the number of subjects. Additionally, it is important that the person scoring the mice is blinded to mouse treatment to avoid bias in scoring of more subjective criteria such as activity or fur texture. The importance of blinded GVHD scoring has also been demonstrated in the clinic24. The person injecting the mice should not be the same person scoring the mice. If this is not possible, control and treatment tubes can be randomized and labeled with new ID&#8217;s by another person (i.e., control is A, treatment is B) so injections can be blinded. Mouse scoring should be performed around the same time every day and on the same days post-injection between different experiments.
One potential obstacle in this model is the lack of GVHD development. This could be due to reduced viability of PBMC which can be addressed by checking viability of PBMC by counting cells with trypan blue. If trouble with cell viability is encountered, the cells can be put in PBS supplemented with 2% FBS to improve survival. Also, fewer mice can be injected at a time to reduce the time cells sit at room temperature. The efficacy of the retro-orbital injection can be the problem. Mice that are injected with PBMC but do not develop GVHD can be euthanized and immune cells isolated from their spleens can be analyzed for presence of human cells via dPCR or flow cytometry. If there is poor engraftment, then there is likely a problem with injection technique. As a test of injection technique, mice can be injected retro-orbitally with 200 &#956;L of Evans blue dye. If the injection is successful, the ears, paws and tail of the mouse will turn blue.
The xenoGVHD model closely mimics human acute GVHD disease pathogenesis and progression30. Unlike the murine MHC mismatch GVHD model, the xenoGVHD model allows testing of the effect of immunosuppressive therapies, including human cell therapies, on human cells rather than murine cells. This reduces the variation due to species differences when applying research results to the clinic. The xenoGVHD model can also serve as an in vivo suppression assay in other fields of T cell research. Thus, results from experiments using the xenoGVHD model can be applied to any human T cell-mediated inflammatory disease in addition to GVHD.
There are limitations of the xenoGVHD model. These include experimental variability and possible differences in GVHD treatment compared to the clinic. Experimental inconsistencies can stem from differences in mouse strain, sites of PBMC injection, radiation dose and microbial environment30,31. Thus, laboratories using this model should attempt to standardize these parameters to ensure consistent outcomes. In this protocol, we describe scoring methods that help reduce variability in scoring. Factors that may limit comparability of xenoGVHD data to clinical outcomes include the lack of control groups treated with GVHD prophylactic drugs and the use of irradiation as the only source of conditioning in the xenoGVHD model31. Additionally, the mechanism of xenoGVHD does not completely recapitulate the underlying pathogenesis in human GVHD. For example, it is donor APCs rather than host APCs that activate human T cells in the xenoGVHD model, whereas host APCs play a significant role in human GVHD7. Thus, as with most pre-clinical models, there are inconsistencies and incompatibilities between xenoGVHD and human GVHD that may limit the application of data generated from the xenoGVHD to the clinic.

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 samples from mice injected with PBS were used as controls (Figure 2).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59107/59107fig1.jpg" /> 
Figure 1: GVHD disease progression. Sublethally irradiated 8-12-week-old male and female NSG mice were retro-orbitally injected with 1 x 107 human PBMC (n = 6) or PBS (n = 4) as a negative control. Data shown are the combined results of three independent experiments. (A) GVHD score was measured every other day until mice reached a score of a 2 and then every day until day of sacrifice. Data reported at each time point for GVHD score is the mean &#177; SEM score of the live mice combined with the last scores of any deceased mice in each group. * p &#60; 0.05 as determined by Mann Whitney U test. (B) Kaplan-Meier curve of survival. Death was marked when GVHD score was &#8805;7. * p &#60; 0.05 as determined by log-rank test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59107/59107fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59107/59107fig2.jpg" /> 
Figure 2: Detection of human T cells using digital PCR. Lung and liver samples were collected from mice that were retro-orbitally injected with PBS (n = 3) or 1 x 107 human PBMC (n = 3) when mice reached a GVHD score of &#8805;7 or 42 days post injection. Data were collected from 3 independent experiments. gDNA was isolated and digital PCR was used to determine the copies of human CD3 epsilon per milligram of tissue. (A) Representative digital PCR plot of lung and liver samples from a mouse injected with PBS or PBMC. (B) Quantification of copies of human CD3 epsilon per millgram of tissue from mice injected with PBS or PBMC. * p &#8804; 0.05 as determined by Mann Whitney U test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59107/59107fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

induction-scoring-graft-versus-host-disease-xenogeneic-murine-model

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

7.0K Views.  University of Kansas Medical Center. 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.

Video: 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

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 ...

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

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

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

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

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

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 ...

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

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

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

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

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

Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) resulting in cognitive and motor impairment. Experimental autoimmune encephalomyelitis (EAE) is a useful animal model of MS, because it is also characterized by lesion formation in the CNS, motor impairment and is also driven by autoimmune and inflammatory reactions. One of the EAE models is induced with a peptide derived from the myelin oligodendrocyte protein (MOG)35-55 in mice. The EAE mice develop a progressive disease course. This course is divided into three phases: the preclinical phase (day 0 - 9), the disease onset (day 10 - 11) and the acute phase (day 12 - 14). MS and EAE are induced by autoreactive T cells that infiltrate the CNS. These T cells secrete chemokines and cytokines which lead to the recruitment of further immune cells. Therefore, the immune cell distribution in the spinal cord during the three disease phases was investigated. To highlight the time point of the disease at which the activation/proliferation/accumulation of T cells, B cells and monocytes starts, the immune cell distribution in lymph nodes, spleen and blood was also assessed. Furthermore, the levels of several cytokines (IL-1&#946;, IL-6, IL-23, TNF&#945;, IFN&#947;) in the three disease phases were determined, to gain insight into the inflammatory processes of the disease. In conclusion, the data provide an overview of the functional profile of immune cells during EAE pathology.

This manuscript describes the methods for induction and scoring of the experimental autoimmune encephalomyelitis (EAE) model, together with the assessment of immune cell distribution and mRNA cytokine levels in lymph nodes, spleen, blood and spinal cord using flow cytometry and quantitative PCR, respectively, at various disease phases.

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) ...

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

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

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 transplantation (allo-HCT) face and is frequently associated with a poor clinical outcome. While, for instance, GvHD manifestations of the skin are usually responsive to established immune-suppressive therapies and are, hence, not taking a fatal course, the presence and the intensity of intestinal GvHD, especially of the mid-to-lower parts of the gut, strongly influence the outcome and overall survival of patients with acute GvHD. Therapeutic options are essentially limited to the classic immune-suppressive agents yielding only moderate disease-mitigating effects. Hence, detailed knowledge about the tissue-resident immune cascade, changes in the intestinal microbiota, and the stromal response prior, upon, and after intestinal GvHD onset are urgently needed to understand the events and mechanisms underlying its pathogenesis and to develop innovative therapeutic options. Murine models of GvHD are frequently employed to identify and functionally assess molecules and pathways putatively driving intestinal GvHD. However, means to specifically monitor and evaluate intestinal inflammation over time are essentially lacking since established scores to assess and grade acute GvHD are routinely comprised of various parameters which rather reflect&#160;systemic GvHD manifestations. The detailed evaluation of intestinal GvHD has been restricted to studies using euthanized mice, thereby essentially excluding longitudinal (i.e., kinetic) analyses of the colonic compartment under a given experimental condition (e.g., antibody-mediated blockade of a proinflammatory cytokine) in live mice (i.e., in vivo). The mini-endoscopic in situ assessment of the distal colon of allo-HCT-treated mice described here allows a) a detailed macroscopic evaluation of different aspects of intestinal inflammation and b) the option to collect tissue samples for downstream analyses at various time points over the course of the observation period. Overall, the mini-endoscopic approach provides a major advance in preclinical noninvasive monitoring and assessment of intestinal GvHD.

Here, we present a protocol that describes allogeneic hematopoietic stem cell transplantation and allows repetitive mini-endoscopic evaluations of the distal colon in situ for the presence, characteristics, and severity of colonic inflammation within live mice suffering from intestinal graft-versus-host disease.

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

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear cells (PBMCs). The development of stable, non-toxic, fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE) allows for rare, antigen-specific T cells to be distinguished from bystanders by diminution in fluorescent staining, as detected by flow cytometry. This method has the following advantages over alternative approaches: (i) it is very sensitive to low-frequency T cells, (ii) no knowledge of the antigen or epitope is required, (iii) the phenotype of the responding cells can be analyzed, and (iv) viable, responding cells can be sorted and used for further analysis, such as T cell cloning.

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Presented here is a protocol for measuring proliferating CD4+ T cells in response to antigenic proteins or peptides using dye dilution. This assay is particularly sensitive to rare antigen-specific T cells and can be modified to facilitate cloning of antigen-specific cells.

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

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

Summary

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Chapters in this video

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

Seven Steps to Stellate Cells

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

piggyBac Transposon System Modification of Primary Human T Cells

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4⁺CD45RB^high T Cells into Immunodeficient Mice

Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

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

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

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

Quantification of Proliferating Human Antigen-specific CD4⁺ T Cells using Carboxyfluorescein Succinimidyl Ester