This study is supported by University of Central Florida College of Medicine start-up grant (to HN), the University of Pittsburgh Medical Center Hillman Cancer Center start-up grant (to HL), the United States NIH Grant #1P20CA210300-01 and Vietnamese Ministry of Health Grant #4694/QD-BYT (to PTH). We thank Dr. Xue-zhong Yu at Medical University of South Carolina for providing materials for the study.

The experimental procedures were approved by the Institutional Animal Care and Use Committee of University of Central Florida.
1. GVHD Induction
NOTE: Allogeneic bone marrow (BM) cell transplantation (step 1.2) is performed within 24 h after irradiation. All procedures described below are performed in a sterile environment. Perform the procedure in a tissue culture hood and use filtered reagents.
<ol>
	<li>Day 0: Prepare the recipient mice.
	<ol>
		<li>Use female wild type (WT) mice on a BALB/c background (CD45.2+ H2kd+), 10&#8722;12 weeks old as recipients.</li>
		<li>Ear-tag and weigh the recipients before TBI. Then, place up to 11 mice in the irradiation chamber and keep it in the irradiator. Irradiate at a single dose of 700 cGy for 10&#8722;15 min.</li>
		<li>Return irradiated animals to the cages and house them in pathogen-free facilities before the transplant. 
		NOTE: The minimal body weight of the recipients should be around 20 g. Use this weight as the reference to calculate the body weight loss during the experimental period.</li>
	</ol>
	</li>
	<li>Day 1: Prepare T-cell depleted bone marrow (TCD-BM) from the donor mice.
	<ol>
		<li>Euthanize CD45.1/Ly5.1 C57BL/6 mice by CO2 asphyxiation and wait for 2&#8722;3 min until they are unconscious. Perform cervical dislocation as a secondary euthanasia if necessary.</li>
		<li>Put each mouse on a clean working board. Sanitize the fur and the skin with 70% isopropanol.</li>
		<li>Collect the tibia and femur from both legs using forceps and scissors. Put them in ice-cold RPMI containing 1% FCS and 100 U/mL penicillin/streptomycin and 2 mM L-glutamine (1% RPMI) in 50 mL conical tubes. Clean the femur and the tibia bones thoroughly by removing all the muscle tissues using forceps and scissors. Transfer the femur and the tibia from the 50 mL conical tubes to a 92 mm diameter Petri dish containing 1% RPMI.</li>
		<li>Using scissors, cut the ends of the femur or tibia. Fill a 50 mL tube with 25 mL of 1% RPMI 1640 medium. Use this to fill a 3 mL syringe attached to a 26 G needle with 3 mL of 1% RPMI. Insert this syringe into the bone and push the plunger to flush the bone marrow (BM) out of the cavity into the collection tube with 1% RPMI (~3 mL/bone).</li>
		<li>Repeat step 1.2.4 for all the remaining tibia and femur bones from other donor mice. Keep all the collection tubes on ice.</li>
		<li>Make the single cell suspension by flushing the bone marrow pieces through a 75 &#181;m mesh cell strainer. Collect the single cell suspension in a 50 mL tube.</li>
		<li>Centrifuge tubes at 800 x g for 5 min at 4 &#176;C. Aspirate and discard the supernatant. Resuspend the pellet in PBS buffer containing 0.5% bovine serum albumin (BSA) at 20 x 106 cells/mL. Save an aliquot of 2 x 106 cells for purity staining.</li>
		<li>Add Thy 1 antibody at 0.05 &#181;g/106 cells14 and incubate for 30 min at 4 &#176;C. Wash once with 25 mL ice-cold PBS. Resuspend at 20 x 106/mL in 0.5% BSA/10% young rabbit complement/2% DNase (10,000 U/mL in sterile H2O). Incubate for 45 min at 37 &#176;C and wash 2x as before. 
		NOTE: T-cells are depleted using a mAb specific for Thy1, a protein expressed by all T-cells, but not other leukocytes.</li>
		<li>Resuspend the cells in 20 mL of PBS buffer containing 0.5% BSA. Count the bone marrow cells in 1% acetic acid using a hemocytometer. Keep an aliquot of 2 x 106 cells for a purity check by flow cytometry after cell purification. 
		NOTE: One donor mouse normally generates about 25 x 106 TCD-BM.</li>
		<li>Use flow cytometry to confirm a successful T-cell depletion. Stain 1 x 106 cells, preserved from steps 1.2.7 (before T-cell depletion) and 1.2.9 (TCD-BM after T-cell depletion) with the following antibodies: &#945;-CD3 (17A2), &#945;-CD4 (GK1.5), and &#945;-CD8&#945; (53-6.7).</li>
	</ol>
	</li>
	<li>Day 1: Prepare T-cells from the donor mice
	<ol>
		<li>Use CD45.2+ H2kb+ C57BL/6 wild type (WT) mice as donors for T-cells.</li>
		<li>Euthanize C57BL/6 mice by carbon dioxide (CO2) asphyxiation as described in 1.2.1.</li>
		<li>Clean the fur and skin of the mouse thoroughly with 70% ethanol. Excise spleens and lymph nodes and separate them into a single cell suspension of splenocytes using a syringe plunger and 40 &#181;m mesh strainers. Wash the strainer and syringe plunger with 1% RPMI (1% FBS containing RPMI media) to collect all splenocytes.</li>
		<li>Centrifuge the cell suspension at 800 x g for 5 min at 4 &#176;C. Add 5 mL of ACK lysing buffer (1 mM Na2EDTA, 10 mM KHCO3, 144 mM NH4Cl, pH 7.2) after discarding the supernatant. Incubate the cell suspension for 5 min at room temperature. 
		NOTE: ACK lysis buffer is used for lysing red blood cells.</li>
		<li>Add 5 mL of 1% RPMI to stop lysis. Centrifuge at 800 x g for 5 min at 4 &#176;C. Discard the supernatant.</li>
		<li>Prepare ice-cold magnetic-activated cell sorting (MACS) buffer (0.5% BSA, 2 mM EDTA in PBS, pH 7.2). Degas the buffer before use. Resuspend the cell pellets in 5 mL of MACS buffer.</li>
		<li>Count the splenocytes and check for the live and dead cells using a hemocytometer and 1% trypan blue. Save an aliquot of 2 x 106 cells to evaluate the purification yield with flow cytometry analysis.</li>
		<li>Resuspend the splenocytes at the concentration of 200 x 106/mL in MACS buffer. Add 0.03 &#181;L of biotin anti-mouse-Ter-119, 0.03 &#181;L of biotin anti-mouse-CD11b, 0.03 &#181;L of biotin anti-mouse-CD45R, and 0.03 &#181;L of biotin anti-mouse-DX5 per 106 cells. Incubate for 15 min at 4 &#176;C. 
		NOTE: Biotin anti-mouse-Ter-119, biotin anti-mouse-CD11b, biotin anti-mouse-CD45R, and biotin anti-mouse-DX5 were used to react with erythroid, granulocytes, B-cells, and NK cells respectively. Therefore, these cell subsets are depleted in following step15.</li>
		<li>Add 10 mL of ice-cold MACS buffer to the cell suspension. Centrifuge at 800 x g for 5 min at 4 &#176;C. Discard the supernatant.</li>
		<li>Resuspend the cell pellets in the MACS buffer at a concentration of 100 x 106/mL. Add anti-biotin microbeads (0.22 &#181;L/106 cells) to the splenocyte suspension. Mix well and incubate for an additional 15 min at 4 &#176;C. Wash the cell suspension once with 10 mL of ice-cold MACS buffer. Centrifuge at 800 x g for 5 min at 4 &#176;C and discard the supernatant.</li>
		<li>Put a magnetic separating column in the magnetic field. Rinse the column with 3 mL of MACS buffer. Drop the cell suspension onto the column. Collect the flow-through consisting of unbound, enriched T-cells, in a new 15 mL conical tube. Wash the MS column with 3 mL of ice-cold MACS buffer. 
		NOTE: Ensure that the column is empty prior to performing the washing steps.</li>
		<li>Centrifuge the cell suspension at 800 x g for 5 min at 4 &#176;C. Resuspend the cell pellet in 5 mL of MACS buffer.</li>
		<li>Count the cells in 1% trypan blue using a hemocytometer. Save an aliquot of 2 x 106 cells for a purity check by flow cytometry. 
		NOTE: The average yield of splenic T-cells isolated by this method is ~20&#8722;25 x 106 cells per mouse.</li>
		<li>Confirm the yield of T-cell enrichment by flow cytometry. Stain 1 x 106 cells, preserved from steps 1.3.7 (before T-cell depletion) and 1.3.13 (TCD-BM after T-cell depletion), with the following antibodies: &#945;-CD3 (17A2), &#945;-CD4 (GK1.5), and &#945;-CD8&#945; (53-6.7).</li>
	</ol>
	</li>
	<li>Day 1: Inject irradiated mice with donor T-cells and TCD-BM.
	<ol>
		<li>Wash the TCD-BM and T-cells 2x with PBS (800 x g for 5 min at 4 &#176;C). Resuspend the cells in ice-cold PBS for injection. Adjust the cell concentration to 20 x 106/mL for TCD BM and 4 x 106/mL for T-cells.</li>
		<li>Heat the animal using a heating lamp to increase the visibility of the bilateral tail veins. If necessary, place the mouse in a restrainer.</li>
		<li>Clean the surface of the tail using 70% isopropanol. Inject TCD-BM (5 x 106 cells/mouse) with or without T-cells (0.75 x 106 cells/mouse). Be careful not to introduce any air into the syringe.</li>
		<li>Remove the needle and apply an antiseptic swab directly to the injection site 5&#8722;10 s to stop any bleeding.</li>
	</ol>
	</li>
	<li>Assess GVHD on days 2&#8722;80.
	<ol>
		<li>Keep track of the animal survival. Monitor the clinical signs of GVHD adapted from the scoring system established previously by Cook et al.16 and the body weight of the recipient mice 2x per week. Use the body weight determined prior to the TBI to calculate the body weight loss.</li>
		<li>Weigh each mouse individually. Score the weight loss as follows: grade 0 = less than 10%; grade 1 = 10%&#8722;20%; grade 2 = more than 20%.</li>
		<li>Score the posture sign of the recipients: grade 0 = no hunch; grade 0.5 = slight hunch but straightens when walking; grade 1 = animal stays hunched when walking; grade 1.5 = animal does not straighten out; grade 2.0 = animal stand on rear toes.</li>
		<li>Score the mobility sign of the recipients: grade 0 = very active; grade 0.5 = slower than naive mice; grade 1.0 = moves only when poked; grade 1.5 = moves slightly when poked; grade 2.0 = does not move when poked.</li>
		<li>Score the skin of the recipients: grade 0 = no abrasions, lesions, or scaling; grade 0.5 = redness in one specific area; grade 1 = abrasion in one area or mild abrasion in two areas; grade 1.5 = serious abrasions in two or more areas; grade 2.0 = severe abrasions, cracking skin, dried blood.</li>
		<li>Score the fur of the recipients: grade 0 = no abnormal signs; grade 0.5 = ridging on the side of belly or nape of neck, grade 1.0 = ridging across or the side of belly and neck; grade 1.5 = unkempt matted and ruffed fur; grade 2.0 = badly matted fur on belly and back.</li>
		<li>Score the diarrhea of the recipients: grade 0 = no diarrhea; grade 0.5 = slight and soft stool; grade 1.0 = mild (yellow stool); grade 1.5 = moderate (yellow stool with a little blood); grade 2 = severe (light yellow and bloody stool, &#34;dried cake stools&#34; appear at the anal area).</li>
	</ol>
	</li>
</ol>
2. Cotransplantation Model
<ol>
	<li>Generate bone marrow-derived dendritic cells (BM-DCs).
	<ol>
		<li>Isolate bone marrow from the femurs and tibias of WT or factor B (fB)-/- mice on the B6 background as described in steps 1.2.3&#8722;1.2.4. Spin the cells at 800 x g for 5 min.</li>
		<li>Resuspend the pellet in 5 mL of ACK lysing buffer for red blood cell lysis. Incubate the cell suspension for 5 min on ice. Add 10 mL of 1% RPMI to the cell suspension to stop the lysis and centrifuge at 800 x g for 5 min at 4 &#176;C. Discard the supernatant.</li>
		<li>Resuspend the cell pellets in 10 mL of culture media (RPMI 1460 containing 10% FBS, 100 U/mL of penicillin/streptomycin, 2 mM l-glutamine, and 50 mM &#946;-mercaptoethanol) and adjust the volume to meet the final concentration of 2 x 106 cells/mL.</li>
		<li>Add 20 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF) to the cell suspension. Culture the bone marrow cells in 100 x 15 mm Petri dishes at 37 &#176;C in 5% CO2 for 6 days.</li>
		<li>Replace half of the ongoing culture media (about 5 mL) with fresh media containing 40 ng/mL GM-CSF on day 3.</li>
		<li>Prewarm the culture media in a water bath at 37 &#176;C. Collect about 5 mL of the media from the bone marrow culture dishes. Centrifuge at 800 x g for 5 min at 4 &#176;C. Discard the supernatant. Resuspend the cell pellet in 5 mL culture media containing 40 ng/mL GM-CSF.</li>
		<li>Add 25 &#181;g/mL lipopolysaccharide (LPS) to the media on day 6 of the culture to mature the BM-DCs. Save an aliquot of 2 x 106 cells to determine the DC differentiation efficacy by flow cytometry.</li>
		<li>Collect matured BM-DCs: Use cell lifters to softly scrape DCs from the Petri dishes. Collect all the cell suspensions in 50 mL conical tubes. Centrifuge at 800 x g, for 5 min at 4 &#176;C. Discard the supernatant. Wash the cell pellets 3x with 50 mL of ice-cold PBS. Save about 2 x 106 BM-DCs for immunological phenotype analysis by flow cytometry.</li>
	</ol>
	</li>
	<li>Perform dendritic cell co-transplantation of BMT (DC-cotransplanting BMT). 
	NOTE: Use FVB (H2kq) mice as donors for T-cells and BM cells. Irradiate B6 Ly5.1 recipient mice at a dose of 1,100 cGy (2 doses, 3 h interval). See details in step 1.1.
	<ol>
		<li>Isolate BM from femurs and tibias of the FVB donor mice. See details in steps 1.2.3&#8722;1.2.10. 
		NOTE: Use total isolated bone marrow instead of TCD-BM in this model.</li>
		<li>Purify T-cells from the spleens and lymph nodes of the FVB donor mice. See details in steps 1.3.1-1.3.13.</li>
		<li>Inject BM (5 x 106/mouse), T-cells (0.5 x 106/mouse) with BM-DCs (2 x 106/mouse) on day 0 of the experimental course.</li>
		<li>On day 3 after the transplantation, examine the donor DC reconstitution by flow cytometry.
		<ol>
			<li>Collect blood from the eyes of the recipients.</li>
			<li>Anesthetize the CD45.1/Ly5.1 B6 mice by 3% isoflurane inhalation. Check for the depth of anesthesia by the lack of response to a toe pinch.</li>
			<li>Place a sterile glass pipette tube in the medial canthus of the eye directed caudally at a 30&#8722;45&#176; angle from the plane of the nose. Apply pressure while gently rotating the tube.</li>
			<li>Drop the blood into a 10 &#181;L heparin-containing sterile 1.5 mL microcentrifuge tubes. Transfer 50 &#181;L of blood into 5 mL glass flow tubes.</li>
			<li>Add 2 mL of ACK lysing buffer. Incubate at 37 &#176;C in water bath for 45 min. Add 2 mL of FACS staining buffer. Centrifuge at 800 x g for 5 min at 4 &#730;C. Discard the supernatant.</li>
			<li>Resuspend the cell pellets with 200 &#181;L of FACS buffer containing appropriate flow staining antibodies (live/death yellow, &#945;-H-2Kb, &#945;-CD45.1, &#945;-CD45.2, &#945;-CD11c, and &#945;-MHCII). Incubate for 15 min at 4 &#176;C in the dark. Wash 2x with 1 mL FACS buffer. Resuspend the cell pellets in 200 &#181;L of FACS buffer and perform the analysis by flow cytometry.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. GVHD/GVL Models of BMT
<ol>
	<li>Perform GVHD/GVL induction.
	<ol>
		<li>Culture the luciferase transduced A20 B cell lymphoma in RPMI culture media.</li>
		<li>Irradiate BALB/c background WT or Ly5.1 recipient at 700 cGy (single dose). See details in step 1.1.</li>
		<li>Isolate TCD-BM from the femurs and tibias of B6. Ly5.1 donor mice. See details in steps 1.2.1&#8722;1.2.10.</li>
		<li>Purify T-cells from spleens and lymph nodes of the C57BL/6 donor mice. See details in steps 1.3.1&#8722;1.3.15.</li>
		<li>Wash the A20 lymphoma 2x with 25 mL of PBS. Resuspend the cell pellets in 10 mL of ice-cold PBS. Take a cell suspension aliquot (10 &#181;L) and count the cells using 1% trypan blue and a hemocytometer. Adjust the cell concentration to 20,000 cells/mL.</li>
		<li>Inject TCD-BM (5 x 106/mouse) with or without T-cells (0.75 x 106/mouse) and A20 lymphoma (5,000 cells/mouse).</li>
		<li>Follow up on the recipient survival, GVHD clinical signs, and body weight loss during the experimental course. See details in step 1.5.</li>
	</ol>
	</li>
	<li>Perform bioluminescent imaging.
	<ol>
		<li>Monitor the tumor growth in the transplanted recipient by injecting the recipient mice with 4 mg of D-luciferin. Incubate for 5 min to ensure luciferin reacts with luciferase.</li>
		<li>Anesthetize the mouse using 3% isoflurane in the chamber of a bioluminescence imager and image the recipients for 5 min in field D and the exposure time of 1 min.</li>
		<li>Analyze data using image-analyzing software. Change the scale of the pseudo color images for best results. 
		NOTE: All the pictures must be at the same scale across experiments.</li>
		<li>Use the image-analyzing software to determine the regions of interest and quantify the signal density by calculating the flux (photons/s) being emitted from each region of interest.</li>
	</ol>
	</li>
</ol>

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C., Perreault, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Major+vs+minor+histocompatibility+antigens.">Major vs minor histocompatibility antigens.</a> Blood. 129, 664-666 (2017).</li><li>Gendelman, 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=Host+conditioning+is+a+primary+determinant+in+modulating+the+effect+of+IL-7+on+murine+graft-versus-host+disease.">Host conditioning is a primary determinant in modulating the effect of IL-7 on murine graft-versus-host disease.</a> Journal of Immunology. 172, 3328-3336 (2004).</li><li>Li, 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=HY-Specific+Induced+Regulatory+T+Cells+Display+High+Specificity+and+Efficacy+in+the+Prevention+of+Acute+Graft-versus-Host+Disease.">HY-Specific Induced Regulatory T Cells Display High Specificity and Efficacy in the Prevention of Acute Graft-versus-Host Disease.</a> Journal of Immunology. 195, 717-725 (2015).</li><li>Zeiser, 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=Early+CD30+signaling+is+critical+for+adoptively+transferred+CD4+CD25++regulatory+T+cells+in+prevention+of+acute+graft-versus-host+disease.">Early CD30 signaling is critical for adoptively transferred CD4+CD25+ regulatory T cells in prevention of acute graft-versus-host disease.</a> Blood. 109, 2225-2233 (2007).</li><li>Sadeghi, 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=GVHD+after+chemotherapy+conditioning+in+allogeneic+transplanted+mice.">GVHD after chemotherapy conditioning in allogeneic transplanted mice.</a> Bone Marrow Transplant. 42, 807-818 (2008).</li></ol>

The authors have no conflicts of interest.

The use of stem cells to suit a particular individual is an effective approach to treat advanced and resistant cancers18. Small molecule pharmaceuticals, however, have long remained a primary focus of personalized cancer therapy. On the other hand, in cellular therapy a multitude of interactions between donor and host can decisively influence the treatment outcomes, such as the development of GVHD after BMT1.
Major MHC-mismatched mouse models of ...

Allogeneic hematopoietic stem cell transplantation (BMT) is an effective therapy to treat hematological malignancies1,2 through the graft-versus-leukemia (GVL) effect3. However, donor lymphocytes always mount unwanted immunological attacks against recipient tissues, a process called graft-versus-host disease (GVHD)4.
Murine models of GVHD are an effective tool to study the biology of GVHD and the GVL response5. Mice are a cost-effective research animal model. They are small and efficiently dosed with molecules and biologics at early phases of development6. Mice are ideal research animals for genetic manipulation studies because they are genetically well defined, which is ideal for studying biological pathways and mechanisms6. Several mouse major histocompatibility complex (MHC) MHC-mismatched models of GVHD have been well established, such as C57BL/6 (H2b) to BALB/c (H2d) and FVB (H2q)&#8594;C57BL/6 (H2b)5,7. These are particularly valuable models to determine the role of individual cell types, genes, and factors that affect GVHD. Transplantation from C57/BL/6 (H2b) parental donors to recipients with mutations in MHC I (B6.C-H2bm1) and/or MHC II (B6.C-H2bm12) revealed that a mismatch in both MHC class I and class II is an important requirement for the development of acute GVHD. This suggests that both CD4+ and CD8+ T-cells are required for disease development7,8. GVHD is also involved in an inflammatory cascade known as the &#39;pro-inflammatory cytokine storm&#39;9. The most common conditioning method in murine models is total body irradiation (TBI) by X-ray or 137Cs. This leads to the recipient&#39;s bone marrow ablation, thereby allowing donor stem cell engraftment and preventing rejection of the graft. This is done by limiting the proliferation of recipient T-cells in response to donor cells. Additionally, genetic disparities play an important role in disease induction, which also depends on minor MHC-mismatch10. Therefore, myeloablative irradiation dose varies in different mouse strains (e.g., BALB/c&#8594;C57BL/6).
Activation of donor T-cells by host antigen presenting cells (APCs) is essential for GVHD development. Among the APCs, dendritic cells (DCs) are the most potent. They are inheritably capable of inducing GVHD due to their superior antigen uptake, expression of T-cell co-stimulatory molecules, and production of pro-inflammatory cytokines that polarize T-cells into pathogenic subsets. Recipient DCs are critical for facilitating T-cell priming and GVHD induction after transplantation11,12. Accordingly, DCs have become interesting targets in the treatment of GVHD12.
TBI is required to enhance the donor cell engraftment. Due to the TBI effect, recipient DCs are activated and survive for a short time after the transplantation12. Despite major advancements in the usage of bioluminescence or fluorescence, establishing an effective model to study the role of recipient DCs in GVHD is still challenging.
Because donor T-cells are the driving force for GVL activity, treatment strategies using immunosuppressive drugs such as steroids to suppress T-cell alloreactivity often cause tumor relapse or infection13. Therefore, targeting recipient DCs may provide an alternative approach to treat GVHD while preserving the GVL effect and avoiding infection.
In brief, the current study provides a platform to understand how different types of signaling in recipient DCs regulates GVHD development and the GVL effect after BMT.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5 M EDTA pH 8.0 100ML</td>
			<td>Fisher Scientific</td>
			<td>BP2482100</td>
			<td>MACS buffer</td>
		</tr>
		<tr>
			<td>10X PBS</td>
			<td>Fisher Scientific</td>
			<td>BP3994</td>
			<td>MACS buffer</td>
		</tr>
		<tr>
			<td>A20 B-cell lymphoma</td>
			<td>University of Central Florida</td>
			<td>In house</td>
			<td>GVL experiment</td>
		</tr>
		<tr>
			<td>ACC1 fl/fl</td>
			<td>Jackson Lab</td>
			<td>30954</td>
			<td>GVL experiment</td>
		</tr>
		<tr>
			<td>ACC1 fl/fl CD4cre</td>
			<td>University of Central Florida</td>
			<td></td>
			<td>GVL experiment</td>
		</tr>
		<tr>
			<td>Anti-Biotin MicroBeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-485</td>
			<td>T-cell enrichment</td>
		</tr>
		<tr>
			<td>Anti-Human/Mouse CD45R (B220)</td>
			<td>Thermo Fisher Scientific</td>
			<td>13-0452-85</td>
			<td>T-cell enrichment</td>
		</tr>
		<tr>
			<td>Anti-mouse B220 FITC</td>
			<td>Thermo Fisher Scientific</td>
			<td>10452-85</td>
			<td>Flow cytometry analysis</td>
		</tr>
		<tr>
			<td>Anti-mouse CD11c- AF700</td>
			<td>Thermo Fisher Scientific</td>
			<td>117319</td>
			<td>Flow cytometry analysis</td>
		</tr>
		<tr>
			<td>Anti-Mouse CD25 PE</td>
			<td>Thermo Fisher Scientific</td>
			<td>12-0251-82</td>
			<td>Flow staining</td>
		</tr>
		<tr>
			<td>Anti-Mouse CD4 Biotin</td>
			<td>Thermo Fisher Scientific</td>
			<td>13-0041-86</td>
			<td>T-cell enrichment</td>
		</tr>
		<tr>
			<td>Anti-Mouse CD4 eFluor&#174; 450 (Pacific Blue&#174; replacement)</td>
			<td>Thermo Fisher Scientific</td>
			<td>48-0042-82</td>
			<td>Flow staining</td>
		</tr>
		<tr>
			<td>Anti-mouse CD45.1 PE</td>
			<td>Thermo Fisher Scientific</td>
			<td>12-0900-83</td>
			<td>Flow cytometry analysis</td>
		</tr>
		<tr>
			<td>Anti-Mouse CD8a APC</td>
			<td>Thermo Fisher Scientific</td>
			<td>17-0081-83</td>
			<td>Flow cytometry analysis</td>
		</tr>
		<tr>
			<td>Anti-mouse H-2Kb PerCP-Fluor 710</td>
			<td>Thermo Fisher Scientific</td>
			<td>46-5958-82</td>
			<td>Flow cytometry analysis</td>
		</tr>
		<tr>
			<td>Anti-mouse MHC Class II-antibody APC</td>
			<td>Thermo Fisher Scientific</td>
			<td>17-5320-82</td>
			<td>Flow cytometry analysis</td>
		</tr>
		<tr>
			<td>Anti-Mouse TER-119 Biotin</td>
			<td>Thermo Fisher Scientific</td>
			<td>13-5921-85</td>
			<td>T-cell enrichment</td>
		</tr>
		<tr>
			<td>Anti-Thy1.2</td>
			<td>Bio Excel</td>
			<td>BE0066</td>
			<td>BM generation</td>
		</tr>
		<tr>
			<td>B6 fB-/- mice</td>
			<td>University of Central Florida</td>
			<td>In house</td>
			<td>Recipients</td>
		</tr>
		<tr>
			<td>B6.Ly5.1 (CD45.1+) mice</td>
			<td>Charles River</td>
			<td>564</td>
			<td>Donors</td>
		</tr>
		<tr>
			<td>BALB/c mice</td>
			<td>Charles River</td>
			<td>028</td>
			<td>Transplant recipients</td>
		</tr>
		<tr>
			<td>C57BL/6 mice</td>
			<td>Charles River</td>
			<td>027</td>
			<td>Donors/Recipients</td>
		</tr>
		<tr>
			<td>CD11b</td>
			<td>Thermo Fisher Scientific</td>
			<td>13-0112-85</td>
			<td>T-cell enrichment</td>
		</tr>
		<tr>
			<td>CD25-biotin</td>
			<td>Thermo Fisher Scientific</td>
			<td>13-0251-82</td>
			<td>T-cell enrichment</td>
		</tr>
		<tr>
			<td>CD45R</td>
			<td>Thermo Fisher Scientific</td>
			<td>13-0452-82</td>
			<td>T-cell enrichment</td>
		</tr>
		<tr>
			<td>CD49b Monoclonal Antibody (DX5)-biotin</td>
			<td>Thermo Fisher Scientific</td>
			<td>13-5971-82</td>
			<td>T-cell enrichment</td>
		</tr>
		<tr>
			<td>Cell strainer 40 uM</td>
			<td>Thermo Fisher Scientific</td>
			<td>22363547</td>
			<td>Cell preparation</td>
		</tr>
		<tr>
			<td>Cell strainer 70 uM</td>
			<td>Thermo Fisher Scientific</td>
			<td>22363548</td>
			<td>Cell preparation</td>
		</tr>
		<tr>
			<td>D-Luciferin</td>
			<td>Goldbio</td>
			<td>LUCK-1G</td>
			<td>Live animal imaging</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Atlanta Bilogicals R&#38;D system</td>
			<td>D17051</td>
			<td>Cell Culture</td>
		</tr>
		<tr>
			<td>Flow cytometry tubes</td>
			<td>Fisher Scientific</td>
			<td>352008</td>
			<td>Flow cytometry analysis</td>
		</tr>
		<tr>
			<td>FVB/NCrl</td>
			<td>Charles River</td>
			<td>207</td>
			<td>Donors</td>
		</tr>
		<tr>
			<td>Lipopolysacharide (LPS)</td>
			<td>Millipore Sigma</td>
			<td>L4391-1MG</td>
			<td>DC mature</td>
		</tr>
		<tr>
			<td>LS column</td>
			<td>Mitenyi Biotec</td>
			<td>130-042-401</td>
			<td>Cell preparation</td>
		</tr>
		<tr>
			<td>MidiMACS</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-302</td>
			<td>T-cell enrichment</td>
		</tr>
		<tr>
			<td>New Brunswick Galaxy 170R incubator</td>
			<td>Eppendorf</td>
			<td>Galaxy 170 R</td>
			<td>Cell Culture</td>
		</tr>
		<tr>
			<td>Penicilin+streptomycinPenicillin/Streptomycin (10,000 units penicillin / 10,000 mg/ml strep)</td>
			<td>GIBCO</td>
			<td>15140</td>
			<td>Media</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Thermo Fisher Scienctific</td>
			<td>11875-093</td>
			<td>Media</td>
		</tr>
		<tr>
			<td>TER119</td>
			<td>Thermo Fisher Scientific</td>
			<td>13-5921-82</td>
			<td>T-cell enrichment</td>
		</tr>
		<tr>
			<td>Xenogen IVIS-200</td>
			<td>Perkin Elmer</td>
			<td>Xenogen IVIS-200</td>
			<td>Live animal imaging</td>
		</tr>
		<tr>
			<td>X-RAD 320 Biological Irradiator</td>
			<td>Precision X-RAY</td>
			<td>X-RAD 320</td>
			<td>Total Body Irradiation</td>
		</tr>
	</tbody>
</table>

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 eradicate tumors. However, its application is limited by the development of graft-versus-host disease (GVHD), a major complication of BMT. GVHD is evoked when T-cells in the donor grafts recognizealloantigen expressed by recipient cells and mount unwanted immunological attacks against recipient healthy tissues. Thus, traditional therapies are designed to suppress donor T-cell alloreactivity. However, these approaches substantially impair the GVL effect so that the recipient&#39;s survival is not improved. Understanding the effects of therapeutic approaches on BMT, GVL, and GVHD, is thus essential. Due to the antigen-presenting and cytokine-secreting capacities to stimulate donor T-cells, recipient dendritic cells (DCs) play a significant role in the induction of GVHD. Therefore, targeting recipient DCs becomes a potential approach for controlling GVHD. This work provides a description of a novel BMT platform to investigate how host DCs regulate GVH and GVL responses after transplantation. Also presented is an effective BMT model to study the biology of GVHD and GVL after transplantation.

The major MHC-mismatched B6 (H2kb)-BALB/C (H2kd) model closely corresponded to GVHD development after the transplantation (Figure 2). All six GVHD clinical signs established previously by Cooke et al.16 occurred in the recipients transplanted with WT-B6 T-cells but not in the recipients transplanted with BM alone (step 1.5), which represented the GVHD-negative group. There are two phases in GVHD development in thi...

Watch this Scientific Journal Video about Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease at JoVE.com

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

Graft-versus-host disease is a major complication after allogeneic bone marrow transplantation. Dendritic cells play a critical role in the pathogenesis of graft-versus-host disease. The current article describes a novel bone marrow transplantation platform to investigate the role of dendritic cells in the development of graft-versus-host disease and the graft-versus-leukemia effect.

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

bone-marrow-transplantation-platform-to-investigate-role-dendritic

Research

JoVE Journal

Immunology and Infection

7.7K Views.  University of Central Florida. Graft-versus-host disease is a major complication after allogeneic bone marrow transplantation. Dendritic cells play a critical role in the pathogenesis of graft-versus-host disease. The current article describes a novel bone marrow transplantation platform to investigate the role of dendritic cells in the development of graft-versus-host disease and the graft-versus-leukemia effect.

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

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

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 ...

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played by T cells in an immune response. This protocol describes the in vitro differentiation of bone marrow (BM) progenitors to obtain granulocyte macrophage colony-stimulating factor (GM-CSF) derived-dendritic cells (DCs). The protocol also describes the adoptive transfer of ovalbumin peptide (OVAp)-loaded GM-CSF-derived DCs and na&#239;ve CD4 T cells from OTII transgenic mice in order to analyze the in vivo activation, proliferation, and Th1 differentiation of the transferred CD4 T cells. This protocol circumvents the limitation of purely in vivo methods imposed by the inability to specifically manipulate or select the studied cell population. Moreover, this protocol allows studies in an in vivo environment, thus avoiding alterations to functional factors that may occur in vitro and including the influence of cell types and other factors only found in intact organs. The protocol is a useful tool for generating changes in DCs and T cells that modify adaptive immune responses, potentially providing important results to understand the origin or development of numerous immune associated diseases.

An &lt;em&gt;In Vivo&lt;/em&gt; Mouse Model to Measure Na&amp;#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Here, we present a protocol for the in vivo determination of na&#239;ve CD4 T cell (T cell) activation, proliferation, and Th1 differentiation induced by GM-CSF bone marrow (BM)-derived dendritic cells (DCs). In addition, this protocol describes BM and T-cell isolation, DC generation, and DC and T-cell adoptive transfer.

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...

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 ...

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.

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 ...