Name: bone marrow transplantation platform to investigate the role of dendritic cells in graft-versus-host disease
Uploaded: 2020-03-17T12:00:00
Description: 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

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 ...

<ol>
	<li>Shlomchik, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Graft-versus-host+disease.">Graft-versus-host disease.</a> Nature Reviews Immunology. 7, 340-352 (2007).</li><li>Appelbaum, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Haematopoietic+cell+transplantation+as+immunotherapy.">Haematopoietic cell transplantation as immunotherapy.</a> Nature. 411, 385-389 (2001).</li><li>Blazar, B. R., Murphy, W. J., Abedi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+graft-versus-host+disease+biology+and+therapy.">Advances in graft-versus-host disease biology and therapy.</a> Nature Reviews Immunology. 12, 443-458 (2012).</li><li>Pasquini, M. C., Wang, Z., Horowitz, M. M., Gale, R. 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G., Radaszkiewicz, T., Pals, S. T., van der Meer, W. G., Gleichmann, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allosuppressor+and+allohelper+T+cells+in+acute+and+chronic+graft-vs-host+disease.+I.+Alloreactive+suppressor+cells+rather+than+killer+T+cells+appear+to+be+the+decisive+effector+cells+in+lethal+graft-vs.-host+disease.">Allosuppressor and allohelper T cells in acute and chronic graft-vs-host disease. I. Alloreactive suppressor cells rather than killer T cells appear to be the decisive effector cells in lethal graft-vs.-host disease.</a> The Journal of Experimental Medicine. 155, 1501-1522 (1982).</li><li>Lu, Y., Waller, E. 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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=Posaconazole+or+fluconazole+for+prophylaxis+in+severe+graft-versus-host+disease.">Posaconazole or fluconazole for prophylaxis in severe graft-versus-host disease.</a> New England Journal of Medicine. 356, 335-347 (2007).</li><li>Dittel, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+of+specific+cell+populations+by+complement+depletion.">Depletion of specific cell populations by complement depletion.</a> Journal of Visualized Experiments. , (2010).</li><li>Nguyen, H. 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The roles of minor H antigens and endotoxin.</a> Blood. 88, 3230-3239 (1996).</li><li>Nguyen, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complement+C3a+and+C5a+receptors+promote+GVHD+by+suppressing+mitophagy+in+recipient+dendritic+cells.">Complement C3a and C5a receptors promote GVHD by suppressing mitophagy in recipient dendritic cells.</a> Journal of Clinical Investigation Insight. 3, (2018).</li><li>McNutt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+immunotherapy.">Cancer immunotherapy.</a> Science. 342, 1417 (2013).</li><li>Negrin, R. S., Contag, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+using+bioluminescence:+a+tool+for+probing+graft-versus-host+disease.">In vivo imaging using bioluminescence: a tool for probing graft-versus-host disease.</a> Nature Reviews in Immunology. 6, 484-490 (2006).</li><li>Roy, D. 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 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.

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	<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 ...

This novel bone marrow transplant protocol can be used to investigate how host dendritic cells regulate graft-versus-host and graft-versus-leukemia responses after transplantation. Ex vivo cell regenerations allows this protocol to be adapted to test the role of other immune cell population such as macrophage and neutrophils simply by modifying the culture condition. Demonstrating the procedure with me will be lab manager Krystal Hossack and undergraduate Sanjeev Gurshaney.For donor T-cell-depleted C57BL/6 bone marrow preparation, harvest the femur and tibia according to standard protocols from euthanized donor mice and transfer the bones into a 92 millimeter diameter Petri dish containing 1%RPMI medium. Using scissors, remove the ends from each bone and fill a three milliliter syringe equipped with a 26 gauge needle with fresh 1%RPMI medium. Insert the needle into one end of one bone and depress the plunger to flush the bone marrow out of the cavity and into a collection cell strainer.When all of the bone marrow has been collected, strain the bone marrow pieces through a 75 micrometer mesh filter and transfer the single-cell suspension into a new 50 milliliter tube. Sediment the cells by centrifugation and resuspend the pellet at a 20 times 10 to the six cells per milliliter concentration in PBS supplemented with 0.5%BSA. Next, add 0.05 micrograms of THI1 antibody per one times 10 to the six cells for a 30-minute incubation at four degrees Celsius.At the end of the incubation, wash the cells twice with 10 milliliters of ice cold PBS and resuspend the pellet at a two times 10 to the seven cells per milliliter concentration in 0.5%BSA supplemented with 10%young rabbit complement and 2%DNAse in sterile water for a 45-minute incubation at 37 degrees Celsius. At the end of the incubation, wash the cells twice with 15 milliliters of fresh PBS per wash and resuspend the pellet in 20 milliliters of PBS containing 0.5%BSA. Pool the lymph nodes and the spleen in a 40 micrometer pore strainer and use a syringe plunger to macerate the tissues through the filters to release the cells.Wash the strainer and plunger with RPMI medium supplemented with 1%FBS to collect all of the cells and sediment the cells by centrifugation. Add five milliliters of ACK lysis buffer to the cell pellet. After five minutes at room temperature, stop the reaction with five milliliters of medium supplemented with 1%FBS and pellet the white blood cells by centrifugation.Resuspend the pellet in five milliliters of MACS buffer for counting and dilute the cells to a concentration of two times 10 to the eight cells per milliliter in fresh MACS buffer. Add the appropriate concentration of anti-erythroid, anti-granulocyte, anti-B-cell, and anti-NK-cell depletion antibodies per one times 10 to the six cells for a 15-minute incubation at four degrees Celsius. At the end of the incubation, wash the cells with 10 milliliters of ice cold MACS buffer and resuspend the pellet at a one times 10 to the eight cells per milliliter concentration in fresh MACS buffer.Next, add 0.22 microliters of Anti-Biotin MicroBeads per one times 10 to the six cells with mixing for a 15-minute incubation at four degrees Celsius. At the end of the incubation, wash the cells with 10 milliliters of ice cold MACS buffer and collect the unbound enriched T-cells by magnetic bead separation using MACSxpress separator according to standard bead isolation protocols. At the end of the separation, sediment the T-cells by centrifugation and resuspend the pellet in five milliliters of fresh MACS buffer for counting.To generate bone marrow-derived dendritic cells, isolate the bone marrow from the femurs of wild type or factor B knockout mice as demonstrated and lyse the red blood cells in five milliliters of ACK lysis buffer, stopping the reaction with 10 milliliters of RPMI supplemented with 1%FBS after five minutes. After collecting the whole blood cells by centrifugation, resuspend the pellet in 10 milliliters of culture medium to a two times 10 to the sixth cells per milliliter concentration and add 20 nanograms per milliliter of GM-CSF to the cells before seeding 10 milliliters of cells onto individual 100 by 15 millimeter Petri dishes at 37 degrees Celsius and 5%carbon dioxide for six days. On day six, activate the bone marrow-derived dendritic cell cultures with 25 micrograms per milliliter of LPS per plate.At least 85%of the cultured bone marrow cells differentiate into dendritic cells. The T-cell purity after magnetic bead enrichment is 90%The major MHC mismatched C57BL/6 BALB/c model closely corresponds to GVHD development after transplantation. The peak of severity occurs approximately 11 days after cell transfer followed by a reduction in the clinical scores and body weight recovery up to day 16.The recipients uniformly succumbed to GVHD by 30 to 40 days posttransplant. Interestingly, transplantation with factor B knockout dendritic cells improves recipient survival and GVHD clinical scores. Luciferase-transduced A20 B-cell lymphoma allows the monitoring of tumor growth in live animals.In this representative experiment, all of the wild type BALB/c recipients that received bone marrow alone plus A20 died of a tumor relapse. Wild type BALB/c recipients transplanted with bone marrow-derived ACC1-depleted donor T-cells died of GVHD. In addition, animals that received donor bone marrow and ACC1-depleted T-cells died of both GVHD and tumor relapse.To generate enough bone marrow cell, the collected tibia and femur bones must be completely free of muscle tissue before the bone marrow correction. Negative purification of the bone marrow cells with biotinylase anti-CD3 and Anti-Biotin MicroBeads can also per performed to deplete the lymphocyte from the bone marrow.

Research

JoVE Journal

Immunology and Infection

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 ...

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

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

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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

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