This work was supported by grants from NIH (5K22A1119151-01 and 1R56DK104903-01) to M.L.B, Pilot/Feasibility Program of the Diabetes Research Center (P30-DK079638) at BCM, JDRF 1-FAC-2014-243-A-N APF, ADA 1-15-JF-07, AAI Careers in Immunology Fellowship to M.B., and The Robert and Janice McNair Foundation.

Ethics Statement: Every effort is made to keep animal discomfort or stress to a minimum during irradiation and tail vein injections. Mice are used as a source of cells in these experiments; as such there are no procedures or manipulations apart from euthanasia. Mice will be euthanized by CO2 inhalation followed by cervical dislocation to confirm death. This procedure is consistent with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association.
1. Prepare Retroviral Construct
<ol>
	<li>Sub-clone the T-cell receptor (TCR) alpha and beta chains, separated by a 2A linker, into a MSCV-based retroviral11 vector (example, pMIAII or pMIGII)11. 
	NOTE: The 2A linker allows for stoichiometric and concordant expression of the alpha and beta TCR chains from a single open reading frame. The vector includes an IRES fluorescent protein cassette (ametrine or GFP) that is used to track transduced cells and transduction efficiency. For a detailed protocol please see Holst et al.11</li>
	<li>Isolate Plasmid DNA using commercially obtained plasma prep kit or a similar product. Use a working concentration of 1 - 2 &#956;g &#956;l-1.</li>
</ol>
2. Generation of Retroviral Producer Cell Lines
<ol>
	<li>Utilize a two-step process to produce the retroviral producer cell lines. 
	NOTE: For a detailed protocol, please view Holst et al.11.
	<ol>
		<li>Generate retrovirus by transient transfection of 293T cells with three plasmids (pVSVG, pEQ-Pam3(-E) and vector of interest11) and take the retroviral supernatant from the 293T cells to transduce the GP+E86 ecotropic retroviral producer cell line.</li>
		<li>Isolate the top 40% of fluorescent expressing GP+E86 producer cells by FACS and propagate the transdcuced-GP+E86 producer cells as a source of virus. 
		NOTE: The generation of high-titer producers is critical for efficient transduction of bone marrow cells. For a complete protocol please view Holst et al.11</li>
	</ol>
	</li>
</ol>
3. Retrovirus-mediated Stem Cell Gene Transfer (Day -5)&#160;
<ol>
	<li>Thaw out and culture 0.5 -1.0 x 106 of each GP+E86 producer cell line in complete DMEM 10% (vol/vol) FBS to allow sufficient growth for two confluent 150-mm plates by Day 0 of this protocol. 
	NOTE: Thawing 0.5 - 1.0 x 106 of each GP+E86 producer cell line in a 10 cm plate will allow for 30 - 50% confluency at Day 5. This also allows for 5 days of culture in order to expand the GP+E86 producer cell lines to two confluent 150-mm plates by Day 0.
	<ol>
		<li>Culture retroviral producer cell line in complete tissue culture media containing 5% feta calf serum, and ciprofloxacin (10 &#956;g/ml) to avoid mycoplasma contamination. 
		NOTE: Discontinue use of ciprofloxacin when generating the viral supernatant for bone marrow transduction. Avoid over-growth of the producer cell line, as this will negatively impact bone marrow transduction efficiency.</li>
	</ol>
	</li>
</ol>
4. Inject Donor Mice with 5-Fluorouracil (5-FU) (Day -3)
<ol>
	<li>Weigh the donor mice (5 - 12 weeks old) to determine the amount of 5-FU required. Using mice younger than 5 weeks of age results in low bone marrow yield. To ensure expression of a single TCR, use a T cell deficient mouse strain such as SCID, RAG or TCR alpha deficient mice as bone marrow donors.</li>
	<li>Working in the tissue culture hood, dilute the 5-FU stock with sterile PBS in order to make a sufficient amount of 10 mg ml-1 working solution of 5-FU to deliver 0.15 mg 5-FU per gram of body weight.</li>
	<li>Using a 1 ml syringe with a 37 G needle, intraperitoneal inject 0.15 mg 5-FU per gram of body weight per donor mouse. 
	NOTE: Use 1 donor mouse per one recipient to start and adjust the number of donor mice according to the cell yield. An alternative method to the 5-FU treatment for the enrichment and isolation of bone marrow progenitors is negative selection of lineage positive cells as described in Viret et al.</li>
</ol>
5. Bone Marrow Extraction Procedure (Day 0)
<ol>
	<li>Obtain dissection scissors and forceps for bone isolation. Prepare media (HBSS supplemented with 5% FCS (vol/vol)) to collect bones in a 50 ml conical tube. Keep 50 ml conical containing media on ice. Euthanize mice by CO2 inhalation followed by cervical dislocation to confirm death. Pinch paw to ensure no reflexes are detected.</li>
	<li>Sterilize the surgical site and surgical tools with ethanol or methanol. Remove the femur, tibia, humerus, and pelvic girdle carefully by first cutting up and under the pelvic girdle. Cut back down along the femur to the tibia. Remove all surrounding tissue using scissors and forceps to obtain clean bones. Keep bones hydrated in HBSS + 5% (vol/vol) FBS.</li>
	<li>Separate the bones by cutting at the joints, making sure to cut both ends off each bone. Insert a 25-gauge needle attached to a 10-ml syringe containing HBSS + 5% (vol/vol) FBS. Apply pressure and flush all bone marrow through a 70-&#956;m strainer resting in a 50 ml conical.</li>
	<li>Periodically, push the bone marrow through the 70-&#956;m filter using the plunger from a 1 ml or 3 ml syringe. Rinse the strainer with HBSS + 5% (vol/vol) FBS. This will ensure a single cell suspension that is free of bone fragments and tissue. Keep cells sterile by performing the procedure in a sterile hood.</li>
	<li>Centrifuge bone marrow cells at 300 x g for 10 min at 4 &#176;C.</li>
</ol>
6. Bone Marrow Culture (Day 0)
<ol>
	<li>Decant or aspirate media and resuspend cell pellet in 1 ml ammonium chloride based red blood lysis buffer (or RBC lysis buffer equivalent) per 50 ml conical tube. After 1 min incubation at room temperature, quench the red cell lysis buffer by adding 10 ml of complete DMEM 20% (vol/vol) FBS.</li>
	<li>Centrifuge cells at 300 x g for 10 min at 4 &#176;C.</li>
	<li>Decant or aspirate supernatant and resuspend bone marrow in 5 ml complete DMEM 20% (vol/vol) FBS. Count the cells with a Neubauer counting chamber. 
	NOTE: General yield should be approximately 5 - 10 x 106 cells per mouse; however, the yield can vary among different strains of mice.</li>
	<li>Plate bone marrow cells at a density of 4 x 107 cells per 150-mm plate in 30 ml of complete DMEM 20% (vol/vol) FBS supplemented with IL-3 (20 g ml-1), IL-6 (50 g ml-1) and SCF (50 g ml-1). 
	NOTE: Use freshly thawed-aliquoted cytokines. Do not use cytokines that have been frozen and thawed more than once or kept at 4 &#176;C for more than 2 weeks.</li>
	<li>Incubate bone marrow overnight (approximately 24 hr) at 37 &#176;C with 5% CO2.</li>
</ol>
7. Retroviral Producer Cell Culture (Day 0)
<ol>
	<li>Plate 3 x 106 retroviral producer cells per 150-mm plate in 18 ml complete DMEM 20% (vol/vol) FBS.</li>
	<li>Incubate retroviral producer cells at 37 &#176;C overnight. Anticipate one 150-mm plate for 15 million bone marrow cells (approximately 2 - 3 donor mice).</li>
</ol>
8. Retroviral Supernatant (Day 1)
<ol>
	<li>The next day (approximately 24 hr later), harvest the retroviral supernatant from the producer plates (set up in step 7) by drawing up the retroviral supernatant directly off of the 150-mm plate with a 10-ml syringe and filter using a 0.45-&#956;m syringe filter.</li>
	<li>Carefully replace the removed retroviral supernatant with 18 ml of fresh complete DMEM 20% (vol/vol) FBS.</li>
	<li>Supplement the filtered retroviral supernatant with IL-3 (20 g ml-1), IL-6 (50 g ml-1), MSCF (50 g ml-1) and Hexadimethrine bromide (Polybrene) (6 &#956;g ml-1). 
	NOTE: Hexadimethrine bromide should be made fresh every 2 months to avoid a drop in efficacy of retroviral transduction.</li>
</ol>
9. Bone Marrow Harvest (Day 1)
<ol>
	<li>After completing step 8.3, harvest the bone marrow cells from Step 6.4 and transfer the media containing non-adherent cells into sterile 50-ml tubes.</li>
	<li>Wash the plates vigorously with 10 ml PBS and collect in the 50 ml conical from step 9.1. If necessary, repeat the wash step.</li>
	<li>Centrifuge the cells at 300 x g for 10 min at 4 &#176;C, decant the supernatant, resuspend cells in a small volume (1 - 3 ml) complete DMEM 20% (vol/vol) FBS, and count. Anticipate 50 - 75% recovery from day 0. Resuspend bone marrow at 1 x 106 cells per 1 ml of the retroviral supernatant containing cytokines and Hexadimethrine bromide.</li>
</ol>
10. Bone Marrow Transduction (Day 1)
<ol>
	<li>Plate the retroviral supernatant/bone marrow mixture at a volume of 3 ml per well in a six-well tissue culture plate. Wrap the plates in plastic wrap to minimize gas exchange during the centrifugation in step 10.2.</li>
	<li>Spin the wrapped six-well plates at 1,000 x g for 60 min a 37 &#176;C.</li>
	<li>After the spin is complete, remove the plastic wrap and place the plates in a 37 &#176;C CO2 incubator for 24 hr.</li>
</ol>
11. Retroviral Supernatant and Transduction (Day 2)
<ol>
	<li>After 24 hr, collect fresh viral supernatant from the viral producer cell line. Filter the retroviral supernatant using a 10-ml syringe and a 0.45-&#956;m syringe filter. Supplement the filtered retroviral supernatant with IL-3 (20 g ml-1), IL-6 (50 g ml-1) and SCF (50 g ml-1) and Hexadimethrine bromide (6 g ml-1).</li>
	<li>Next, carefully remove with pipette or aspirate the top 2 ml of media from each well of the six-well plate containing the bone marrow cells. 
	NOTE: Work carefully not to aspirate the bone marrow, which is usually concentrated in the middle of the well. Alternatively, the removed supernatant can be spun down to recover any lost bone marrow cells.</li>
	<li>To each well in the bone marrow 6-well plate add 3 ml of fresh viral supernatant containing cytokines and Hexadimethrine bromide. Wrap the plates in plastic, and repeat the spin transduction at 1,000 x g for 60 min a 37 &#176;C. Unwrap the plates, and incubate cells at 37 &#176;C and 5% CO2 overnight (about 24 hr).</li>
</ol>
12. Addition of Fresh Supplemented DMEM (Day 3)
<ol>
	<li>After 24 hr, remove the top 2 ml of media from each well of the six-well plate containing the bone marrow cells, as in step 11.2. Replace the removed media with fresh DMEM 20% (vol/vol) FBS supplemented with final concentrations of IL-3 (20 g ml-1), IL-6 (50 g ml-1) and SCF (50 g ml-1).</li>
	<li>Incubate bone marrow plates at 37 &#176;C CO2 incubator for another 24 hr.</li>
</ol>
13. Harvest Transduced Bone Marrow (Day 4)
<ol>
	<li>After 24 hr, collect the bone marrow cells from the six-well plates. Wash the plates vigorously with PBS to remove all non-adherent cells.
	<ol>
		<li>Centrifuge the cells at 300 x g for 10 min at 4 &#176;C and decant the supernatant.</li>
		<li>Resuspend the bone marrow in a small volume (1 ml) of PBS + 0.5% (vol/vol) heat-inactivated FBS. Keep cells on ice.</li>
	</ol>
	</li>
	<li>Take a 10-&#956;l sample of each bone marrow condition and count the cells using a Neubauer counting chamber. Assuming 1 - 2 donor per recipient, the yield will be sufficient to transfer at least 4 x 106 cells per recipient mouse. Resuspend the bone marrow in a sufficient volume of PBS + 0.5% (vol/vol) heat-inactivated FBS to inject 200 - 300 &#956;l per mouse.
	<ol>
		<li>Inject at least 2 x 106 cells with a transduction efficiency of 25% (can inject up to 8 x106 cells) per mouse intravenously. 
		NOTE: We routinely inject five recipient mice per two 6-well plates of bone marrow cells. If the yield is substantially lower, more donor mice should be used until the sufficient bone marrow number can be obtained. In order to achieve optimal reconstitution, at least 5 x 105 transduced (fluorescent positive) should be injected into each recipient.</li>
	</ol>
	</li>
	<li>With a micro pipettor, take a 10-&#956;l sample of each bone marrow condition and analyze transduction efficiency by flow cytometry based on expression of the fluorescent marker (Figure 1A)11. 
	NOTE: Generally, the percentage of fluorescent positive cells is between 25% and 70%, depending on the construct and retroviral titer. The number of transduced bone marrow cells necessary to reconstitute a sub-lethally irradiate mouse can vary depending on the transduction efficiency. The lower the transduction efficiency, the more bone marrow cells required and conversely, the higher the bone marrow transduction efficiency, the lower number of cells required for reconstitution. Transduction efficiency below 25% is suboptimal and may result in insufficient reconstitution and survival. In order to ensure optimal bone marrow recovery and transduction efficiency, use freshly prepared tissue culture media and cytokines. When expressing a new TCR utilizing the retrogenic system, the selection of that TCR in vivo is unknown. Depending on each individual TCR affinity, TCR selection in the thymus and peripheral T cell expression will vary17.</li>
</ol>
14. Mouse Irradiation, Bone Marrow Injection and Care
<ol>
	<li>Irradiate mice the day before bone marrow injection (Same day as Step 13). Use the following doses: C57BL/6 mice (and other wild type strains), 900 rads; Rag1-/- mice, 500 rads; scid mice, 300 rads).
	<ol>
		<li>Place recipient mice on Amoxicillin (50 mg/kg/day) or another antibiotic water treatment before irradiation, and keep on antibiotics for the first two weeks after bone marrow injection. 
		NOTE: Optimal irradiation dose may have to be determined empirically.</li>
	</ol>
	</li>
	<li>For injection, load the cells from Step 13.1 into a 1-ml syringe using blunt needles immediately before injection, then change the needle to 27-gauge for injection, expel any air to avoid air embolism. Heat mice under a heating lamp and inject 0.2 ml of bone marrow per mouse intravenously. 
	NOTE: Do not let cells sit in the syringe for any length of time or the cells will settle.</li>
	<li>Monitor the mice weekly to ensure that they remain healthy.</li>
	<li>After 5 - 6 weeks, bleed the mice to check for reconstitution on the basis of GFP+/ametrine+ and TCR co-expression (Figure 1B)11.</li>
</ol>

<ol>
	<li>Qi, Q., 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=Diversity+and+clonal+selection+in+the+human+T-cell+repertoire.">Diversity and clonal selection in the human T-cell repertoire.</a> Proceedings of the National Academy of Sciences of the United States of America. 111, 13139-13144 (2014).</li><li>Zarnitsyna, V. I., Evavold, B. D., Schoettle, L. N., Blattman, J. N., Antia, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimating+the+diversity,+completeness,+and+cross-reactivity+of+the+T+cell+repertoire.">Estimating the diversity, completeness, and cross-reactivity of the T cell repertoire.</a> Frontiers in immunology. 4, 485 (2013).</li><li>Kisielow, P., Bluthmann, H., Staerz, U. D., Steinmetz, M., von Boehmer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tolerance+in+T-cell-receptor+transgenic+mice+involves+deletion+of+nonmature+CD4+8++thymocytes.">Tolerance in T-cell-receptor transgenic mice involves deletion of nonmature CD4+8+ thymocytes.</a> Nature. 333, 742-746 (1988).</li><li>Hogquist, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+receptor+antagonist+peptides+induce+positive+selection.">T cell receptor antagonist peptides induce positive selection.</a> Cell. 76, 17-27 (1994).</li><li>Verdaguer, 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=Spontaneous+autoimmune+diabetes+in+monoclonal+T+cell+nonobese+diabetic+mice.">Spontaneous autoimmune diabetes in monoclonal T cell nonobese diabetic mice.</a> J Exp Med. 186, 1663-1676 (1997).</li><li>Katz, J. D., Wang, B., Haskins, K., Benoist, C., Mathis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Following+a+diabetogenic+T+cell+from+genesis+through+pathogenesis.">Following a diabetogenic T cell from genesis through pathogenesis.</a> Cell. 74, 1089-1100 (1993).</li><li>Pauza, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-cell+receptor+transgenic+response+to+an+endogenous+polymorphic+autoantigen+determines+susceptibility+to+diabetes.">T-cell receptor transgenic response to an endogenous polymorphic autoantigen determines susceptibility to diabetes.</a> Diabetes. 53, 978-988 (2004).</li><li>Jasinski, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+insulin+(B:9-23)+T-cell+receptor+mice+develop+autoimmune+diabetes+dependent+upon+RAG+genotype,+H-2g7+homozygosity,+and+insulin+2+gene+knockout.">Transgenic insulin (B:9-23) T-cell receptor mice develop autoimmune diabetes dependent upon RAG genotype, H-2g7 homozygosity, and insulin 2 gene knockout.</a> Diabetes. 55, 1978-1984 (2006).</li><li>Kersh, G. 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=TCR+transgenic+mice+in+which+usage+of+transgenic+alpha-+and+beta-chains+is+highly+dependent+on+the+level+of+selecting+ligand.">TCR transgenic mice in which usage of transgenic alpha- and beta-chains is highly dependent on the level of selecting ligand.</a> Journal of immunology. 161, 585-593 (1998).</li><li>Bettini, M. L., Bettini, M., Vignali, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TCR+retrogenic+mice:+A+rapid,+flexible+alternative+to+TCR+transgenic+mice.">TCR retrogenic mice: A rapid, flexible alternative to TCR transgenic mice.</a> Immunology. 136 (3), 265-272 (2012).</li><li>Holst, 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=Generation+of+T-cell+receptor+retrogenic+mice.">Generation of T-cell receptor retrogenic mice.</a> Nat Protoc. 1, 406-417 (2006).</li><li>Holst, J., Vignali, K. M., Burton, A. R., Vignali, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+analysis+of+T-cell+selection+in+vivo+using+T+cell-receptor+retrogenic+mice.">Rapid analysis of T-cell selection in vivo using T cell-receptor retrogenic mice.</a> Nat Methods. 3, 191-197 (2006).</li><li>Bettini, M. L., Bettini, M., Nakayama, M., Guy, C. S., Vignali, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+T+cell+receptor-retrogenic+mice:+improved+retroviral-mediated+stem+cell+gene+transfer.">Generation of T cell receptor-retrogenic mice: improved retroviral-mediated stem cell gene transfer.</a> Nat Protoc. 8, 1837-1840 (2013).</li><li>Donnelly, M. L., 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=Analysis+of+the+aphthovirus+2A/2B+polyprotein+'cleavage'+mechanism+indicates+not+a+proteolytic+reaction,+but+a+novel+translational+effect:+a+putative+ribosomal+'skip'.">Analysis of the aphthovirus 2A/2B polyprotein 'cleavage' mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal 'skip'.</a> J Gen Virol. 82, 1013-1025 (2001).</li><li>Atkins, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+case+for+"StopGo":+reprogramming+translation+to+augment+codon+meaning+of+GGN+by+promoting+unconventional+termination+(Stop)+after+addition+of+glycine+and+then+allowing+continued+translation+(Go).">A case for "StopGo": reprogramming translation to augment codon meaning of GGN by promoting unconventional termination (Stop) after addition of glycine and then allowing continued translation (Go).</a> RNA. 13, 803-810 (2007).</li><li>Doronina, V. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+release+of+nascent+chains+from+ribosomes+at+a+sense+codon.">Site-specific release of nascent chains from ribosomes at a sense codon.</a> Mol Cell Biol. 28, 4227-4239 (2008).</li><li>Bettini, 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=TCR+affinity+and+tolerance+mechanisms+converge+to+shape+T+cell+diabetogenic+potential.">TCR affinity and tolerance mechanisms converge to shape T cell diabetogenic potential.</a> Journal of immunology. 193, 571-579 (2014).</li><li>Brehm, M. A., Wiles, M. V., Greiner, D. L., Shultz, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+improved+humanized+mouse+models+for+human+infectious+diseases.">Generation of improved humanized mouse models for human infectious diseases.</a> J Immunol Methods. 410, 3-17 (2014).</li><li>Brehm, M. A., Shultz, L. D., Greiner, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+mouse+models+to+study+human+diseases.">Humanized mouse models to study human diseases.</a> Curr Opin Endocrinol Diabetes Obes. 17, 120-125 (2010).</li><li>Chaplin, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+interleukin-12+as+a+self-processing+2A+polypeptide.">Production of interleukin-12 as a self-processing 2A polypeptide.</a> J Interferon Cytokine Res. 19, 235-241 (1999).</li><li>Collison, L. W., 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=The+inhibitory+cytokine+IL-35+contributes+to+regulatory+T-cell+function.">The inhibitory cytokine IL-35 contributes to regulatory T-cell function.</a> Nature. 450, 566-569 (2007).</li><li>Holst, 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=Scalable+signaling+mediated+by+T+cell+antigen+receptor-CD3+ITAMs+ensures+effective+negative+selection+and+prevents+autoimmunity.">Scalable signaling mediated by T cell antigen receptor-CD3 ITAMs ensures effective negative selection and prevents autoimmunity.</a> Nature immunology. 9, 658-666 (2008).</li><li>Kalos, 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=T+cells+with+chimeric+antigen+receptors+have+potent+antitumor+effects+and+can+establish+memory+in+patients+with+advanced+leukemia.">T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia.</a> Sci Transl Med. 3, 95ra73 (2011).</li><li>VanSeggelen, 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=T+Cells+Engineered+With+Chimeric+Antigen+Receptors+Targeting+NKG2D+Ligands+Display+Lethal+Toxicity+in+Mice.">T Cells Engineered With Chimeric Antigen Receptors Targeting NKG2D Ligands Display Lethal Toxicity in Mice.</a> Mol Ther. 23, 1600-1610 (2015).</li></ol>

No conflicts of interest declared.

In the protocol, we detail several critical steps to ensure optimal bone marrow health, transduction efficiency and reconstitution. First critical step is the generation and proper maintenance of the GP+E86 viral producer cells. Use early passage producer cell lines and maintain at 80% confluency or lower prior to use. When making fresh GP+E86 viral producer cells, ensure the 293T cells are early passage and growing in culture for 24 - 48 hr. Plating too many GP+E86 cells during the transduction step will lower viral tit...

T cell receptor (TCR) repertoire of humans and mice has been estimated at 1 x 108 and 2 x 106 unique TCRs respectively1,2. This large diversity allows T cells to recognize a vast array of antigen epitopes derived from self-peptides as well as from pathogens presented by the major histocompatibility complex (MHC) on antigen presenting cells (APCs). The subtle differences in the interactions of the TCRs with unique peptide-MHC complexes dictate whether a T cell will undergo apoptosis, anergy, activation, differentiation, cytokine production or cytotoxicity. However, due to the large TCR repertoire, analysis of how a specific TCR will respond to a particular antigen requires the use of single TCR systems.
Various TCR transgenic mice have been generated in order to study the function of a single TCR in an in vivo model 3-9. However, there are caveats to TCR transgenic mice including the cost, the length of time to generate a single transgenic mouse and the so called founder effect of random transgene insertion into germline DNA10. Therefore, relatively few TCR transgenic mice have been generated for any given antigen and the functional implications of high and low TCR affinity for the same epitope are rarely addressed. To address the need for a rapid approach to screen and study multiple TCRs individually or in combination, retrogenic (&#39;retro&#39; from retrovirus and &#39;genic&#39; from transgenic) mice have been utilized as an alternative to TCR transgenic mice11-13.
The 2A peptide consensus motif found within several viruses consist of an 2A-Asp-Val/Ile-Glut-X-Asn-Pro-Gly-2B-Pro, in which cleavage occurs between the glycine of the 2A and the proline of the 2B from cis-acting hydrolase activity, resulting in ribosomal skipping during translation10,14-16. For a detailed diagram depicting the cleavage of the various 2A peptides (F2A, E2A, T2A and P2A) please refer to references 10 - 12. In this manner, 2 cistrons (TCR alpha and TCR beta) can be linked resulting in stoichiometric translation in a single vector. Utilizing this approach, we are able to express and directly compare multiple antigen specific TCRs in vivo.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DMEM, high glucose + glutamine</td>
			<td>Corning Cellgro</td>
			<td>10-013-CV</td>
			<td>Dulbecco&#39;s Modification of Eagle&#39;s Medium with 4.5 g/L glucose, L-glutamine &#38; sodium pyruvate</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Atlanta Biological</td>
			<td>S11550</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-Versene</td>
			<td>Lonza</td>
			<td>17-161F</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 um syringe filter</td>
			<td>Thermo Scientific</td>
			<td>194-2545</td>
			<td></td>
		</tr>
		<tr>
			<td>polybrene</td>
			<td>Sigma</td>
			<td>H9268-10G</td>
			<td>Sterile Filtered in dH2O</td>
		</tr>
		<tr>
			<td>Ciprofloxacin&#160;</td>
			<td>VWR</td>
			<td>AAJ61970-06</td>
			<td></td>
		</tr>
		<tr>
			<td>5-fluorouracil (5-FU)</td>
			<td>VWR</td>
			<td>AAA13456-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Corning Cellgro</td>
			<td>25-000-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM nonessential Amino Acids</td>
			<td>Corning Cellgro</td>
			<td>25-025-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES 1M solution</td>
			<td>Corning Cellgro</td>
			<td>25-060-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Gibco by Life Technologies</td>
			<td>21985-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen/Strept</td>
			<td>Corning Cellgro</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Corning Cellgro</td>
			<td>25-005-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>150 mm tissue culture dishes</td>
			<td>Greiner Bio-one</td>
			<td>639160</td>
			<td></td>
		</tr>
		<tr>
			<td>Tisue culture-treated 6-well flat plate</td>
			<td>Greiner Bio-one</td>
			<td>657160</td>
			<td></td>
		</tr>
		<tr>
			<td>70 um nylon cell strainers</td>
			<td>Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IL-3</td>
			<td>Invitrogen</td>
			<td>PMC0033</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IL-6</td>
			<td>Invitrogen</td>
			<td>&#160;PHC0063</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Stem Cell Factor</td>
			<td>Invitrogen</td>
			<td>PMC2113L</td>
			<td></td>
		</tr>
		<tr>
			<td>10x PBS</td>
			<td>Corning Cellgro</td>
			<td>46-D13-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>HANKS Buffer</td>
			<td>Corning Cellgro</td>
			<td>21020147</td>
			<td></td>
		</tr>
		<tr>
			<td>BD 10 mL Syringe</td>
			<td>BD</td>
			<td>300912</td>
			<td></td>
		</tr>
		<tr>
			<td>BD 1 mL Syringe</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>27G x 1/2 BD Precision Glide Needle</td>
			<td>BD</td>
			<td>305109</td>
			<td></td>
		</tr>
		<tr>
			<td>30G x 1/2 BD Precision Glide Needle</td>
			<td>BD</td>
			<td>305106</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Bone marrow transduction efficiency is checked at step 13.3 of the protocol before the bone marrow is injected into tail vein i.v. In the representative bone marrow transduction figure (Figure 1A), approximately 10-&#956;l of the harvested bone marrow was added to 100-&#956;l of PBS and analyzed for ametrine expression. Generally the percentage of fluorescent positive cells is between 25% and 70%, depending on the construct and retroviral titer. After 6 weeks bone marrow ...

Watch this Scientific Journal Video about Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice at JoVE.com

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

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 ...

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Research

JoVE Journal

Immunology and Infection

9.5K Views.  Baylor College of Medicine. 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.

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

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged antigen-specific T cell receptor (TCR) have been an indispensable tool to study T cell development and function. However, such TCRs are usually isolated from the relevant T cells after long-term culture often following repeated antigen stimulation, which unavoidably selects for T cells with high affinity. Random genomic integration of the TCR &alpha;- and &beta;-chain and expression from non-endogenous promoters can lead to variations in expression level and kinetics.
Epigenetic reprogramming via somatic cell nuclear transfer provides a tool to generate embryonic stem cells and mice from any cell of interest. Consequently, when SCNT is applied to T cells of known specificity, these genetic V(D)J rearrangements are transferred to the SCNT-embryonic stem cells (ESCs) and the mice derived from them, while epigenetic marks are reset. We have demonstrated that T cells with pre-defined specificities against Toxoplasma gondii can be used to generate mouse models that express the specific TCR from their endogenous loci, without experimentally introduced genetic modification. The relative ease and speed with which such transnuclear models can be obtained holds promise for the construction of other disease models.

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

We demonstrate here that epigenetic reprogramming via Somatic Cell Nuclear Transfer (SCNT) can be used as a tool to generate mouse models with pre-defined T cell receptor (TCR) specificities. These transnuclear mice express the corresponding TCR from their endogenous locus under the control of the endogenous promoter.

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged ...

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 ...

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, biomechanics, and stem cell biology. Despite the importance of the bone in healthy and pathologic states, however, it is a largely under-researched organ due to lack of specialized knowledge of bone dissection and bone marrow isolation. Mice are a common model organism to study effects on bone and bone marrow, necessitating a standardized and efficient method for long bone dissection and bone marrow isolation for processing of large experimental cohorts. We describe a straightforward dissection procedure for the removal of the femur and tibia that is suitable for downstream applications, including but not limited to histomorphologic analysis and strength testing. In addition, we outline a rapid procedure for isolation of bone marrow from the long bones via centrifugation with limited handling time, ideal for cell sorting, primary cell culture, or DNA, RNA, and protein extraction. The protocol is streamlined for rapid processing of samples to limit experimental error, and is standardized to minimize user-to-user variability.

Here we present a protocol for the dissection of hind limb long bones (femurs and tibiae) from the laboratory mouse. We further describe a rapid technique for bone marrow isolation from these bones that utilizes centrifugation for removal of bone marrow from the bone marrow space.

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, ...

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCR&#945; or TCR&#946; hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCR&#945; or TCR&#946; of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

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.

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 ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...

Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous antigens and modulate responses to potent microbially derived immune stimuli. For this reason, the intestine is a major target site of immune dysregulation and inflammation in many diseases including but, not limited to inflammatory bowel diseases such as Crohn&#8217;s disease and ulcerative colitis, graft-versus-host disease (GVHD) after bone marrow transplantation (BMT), and many allergic and infectious conditions. Murine models of gastrointestinal inflammation and colitis are heavily used to study GI complications and to pre-clinically optimize strategies for prevention and treatment. Data gleaned from these models via isolation and phenotypic analysis of immune cells from the intestine is critical to further immune understanding that can be applied to ameliorate gastrointestinal and systemic inflammatory disorders. This report describes a highly effective protocol for the isolation of mononuclear cells (MNC) from the colon using a mixed silica-based density gradient interface. This method reproducibly isolates a significant number of viable leukocytes while minimizing contaminating debris, allowing subsequent immune phenotyping by flow cytometry or other methods.

The goal of this protocol is to isolate mononuclear cells that reside in the lamina propria of the colon by enzymatic digestion of the tissue using collagenase. This protocol allows for the efficient isolation of mononuclear cells resulting in a single cell suspension which in turn can be used for robust immunophenotyping.

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous ...

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.

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 ...