Name: in vivo augmentation of gut-homing regulatory t cell induction
Uploaded: 2020-01-22T13:00:00.000+00:00
Duration: 8 min 2 s
Description: Watch this Scientific Journal Video about In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction at JoVE.com

This work was supported by the Office of the Assistant Secretary of Defense for Health Affairs through the Peer Reviewed Medical Research Program under Award No. W81XWH-15-1-0240 (XT). Opinions, interpretations, conclusions, and recommendations are those of the author and are not necessarily endorsed by the Department of Defense. This work was also partially supported by Research Innovation Grants from the Department of Medicine at Loma Linda University (681207-2967 [XT and GG], 681205-2967 [XT], and 325491 [DJB]).

All in vivo animal study protocols were reviewed and approved by the Loma Linda University Institutional Animal Care and Use Committee (IACUC) as well as the Animal Care and Use Review Office (ACURO) of the US Army Medical Research and Materiel Command (USAMRMC) of the Department of Defense.
1. Preparation of the Lentivirus that Expresses both 1&#945;-hydroxylase and RALDH2 (lenti-CYP-ALDH Virus)
<ol>
	<li>Day 0: In the early morning, prepare 5 x 105 cells/mL of 293T cells in the CM-10-D cell culture medium.</li>
	<li>Seed 20 mL/plate in 150 mm x 25 mm culture dishes. Culture the cells at 37 &#176;C and 5% CO2 for 24 h. Confluency will reach ~80&#8211;90% after 24 h.</li>
	<li>Day 1: Make 2x HBS (50 mM HEPES, 280 mM NaCl, and 1.5 mM Na2HPO4, pH 7.1). Aliquot and store at -20 &#176;C.</li>
	<li>Make 2 M CaCl2 and store at room temperature.</li>
	<li>For each 150 mm x 25 mm culture dish, prepare a DNA precipitation mixture in a sterile 50 mL culture tube. First, add 1,620 &#181;L of 2x HBS, 9.5 &#181;g of PMD2G (VSVG), 17.5 &#181;g of pCMVR8.74 (Capsid), 27 &#181;g of Lenti-CYP-ALDH plasmid (a lentiviral vector that carries both CYP27B1 and ALDH1a2). Second, add H2O to a final volume of 3,037.5 &#181;L.</li>
	<li>In the last, add 202.5 &#181;L of 2 M CaCl2 dropwise while mixing the solution to final concentrations of 1x HBS and 125 mM CaCl2. CaCl2 must be the last to be added. Leave the transfection mixtures at room temperature for 20 min with occasional mixing. For multiple 150 mm x 25 mm culture dishes, scale up accordingly.</li>
	<li>Add the 3,240 &#181;L of DNA precipitation mixture dropwise to one 150 mm x 25 mm culture dish containing the 293T cells. Gently swirl the culture dish side to side while adding the DNA precipitation mixture. Incubate the dish at 37 &#176;C and 5% CO2 for 24 h.</li>
	<li>Day 2: Remove the calcium phosphate transfection solution, wash gently with 1x PBS, and refeed the cell culture with CM-4-D cell culture medium. Incubate the cells at 37 &#176;C and 5% CO2 for 24 h.</li>
	<li>Day 3: Harvest the supernatants in sterile storage bottles and store at 4&#8211;8 &#176;C. Replenish the cell culture with fresh CM-4-D cell culture medium. Culture the cells at 37 &#176;C and 5% CO2 for another 24 h.</li>
	<li>Day 4: Harvest the supernatants into sterile storage bottles. Filter the supernatants from Day 3 and Day 4 through a 0.45 &#181;m filter. To concentrate the VSVG pseudotype virus, transfer the supernatants into centrifuge tubes and spin at 4,780 x g for 24 h at 4 &#176;C.</li>
	<li>Day 5: Pour the supernatants off the pellets (the pellet is often visible) and allow the tubes to drain on a paper towel in a sterile biosafety cabinet for several minutes.</li>
	<li>Resuspend the pellets by pipetting with sterile PBS containing 5% glycerol. The volume for resuspension will be 33.3 &#181;L per 150 mm x 25 mm culture dish.</li>
	<li>Aliquot 200 &#181;L/tube and store at -80 &#176;C. Make a separate aliquot of 50 &#181;L/vial for titration purposes. Titers should be within the range of 108&#8211;109 transducing units (TUs)/mL.</li>
	<li>Add bleach into the culture dishes and discard them. 
	NOTE: These transfected cells are the biggest safety concern in the protocol since all lentiviral elements are expressed in the cells.</li>
</ol>
2. Generation of Bone Marrow Derived DCs (BMDCs)
<ol>
	<li>Harvest tibias and femurs from 4&#8211;5 Balb/c mice into a 50 mL sterile polypropylene tube containing the culture medium6.</li>
	<li>Transfer the tibias and femurs into a 100 mm x 20 mm culture dish containing the culture medium. Remove tissues such as muscles from the tibias and femurs using dissecting scissors and forceps.</li>
	<li>Cut both ends of the tibias and femurs to expose the bone marrow cavity.</li>
	<li>Draw 10 mL culture medium in a 10 mL syringe attached to a 30 G needle.</li>
	<li>Carefully insert the needle into the bone marrow cavity and flush the bone marrow into a clean 50 mL sterile polypropylene tube. Repeat this procedure if necessary, to ensure that the bone marrow has been completely flushed out.</li>
	<li>Pellet the bone marrow cells by centrifugation (400 x g) at 2&#8211;8 &#176;C for 5 min. Aspirate the supernatant.</li>
	<li>Resuspend the pellet with 5 mL of 1x red blood cell lysis buffer.</li>
	<li>Incubate the cells at room temperature for 4&#8211;5 min with occasional shaking.</li>
	<li>Stop the reaction by adding 30 mL of culture medium.</li>
	<li>Pellet the cells by centrifugation (400 x g) at 2&#8211;8 &#176;C for 5 min. Resuspend the cells in 10 mL of CM-10-R cell culture medium. If needed, the cells can be passed through a 40 &#181;m cell strainer to remove debris.</li>
	<li>Perform a cell count and adjust the cells to 1 x 106 cells/mL using CM-10-R cell culture medium.</li>
	<li>Add recombinant murine granulocyte/macrophage colony-stimulating factor (GM-CSF, final concentration = 100 U/mL) and murine interleukin-4 (IL-4, final concentration = 10 U/mL). 
	NOTE: The stock solutions of GM-CSF and IL-4 are stored at -80 &#176;C at 100,000 U/mL (1,000x) and 10,000 U/mL (1,000x), respectively.</li>
	<li>Distribute the cells into 6 well culture plates at 4 mL/well and culture the cells at 37 &#176;C and 5% CO2 for 48 h.</li>
	<li>Remove nonadherent cells with gentle pipetting.</li>
	<li>Add fresh CM-10-R cell culture medium containing the GM-CSF (100 U/mL) and IL-4 (10 U/mL). Culture the cells for another 48 h.</li>
	<li>Harvest nonadherent cells (BMDCs) for transduction by the lenti-CYP-ALDH virus.</li>
</ol>
3. Transduction of DCs with lenti-CYP-ALDH Virus to Generate DC-CYP-ALDH Cells
<ol>
	<li>Prepare 1 x 106 DCs/well in a total volume of 0.5 mL of CM-10-R cell culture medium containing 50 &#181;L of virus and 8 &#181;g/mL protamine in a 6 well culture plate.</li>
	<li>Culture the cells at 37 &#176;C and 5% CO2 for 24 h.</li>
	<li>Replace the medium with fresh CM-10-R cell culture medium and culture the cells at 37 &#176;C and 5% CO2 for another 24 h. At this time point, the enzymatic activities of 1&#945;-hydroxylase and RALDH2 can be evaluated (see step 4). If necessary, repeat steps 3.1&#8211;3.3 for a second transduction.</li>
	<li>Add lipopolysaccharide (100 ng/mL) and culture the cells for 24 h.</li>
	<li>Harvest the cells (DC-CYP-ALDH cells) for experiments.</li>
</ol>
4. Evaluation of the Overexpressed 1&#945;-hydroxylase and RALDH2 in DC-CYP-ALDH Cells
<ol>
	<li>Evaluation of the overexpressed 1&#945;-hydroxylase in DC-CYP-ALDH cells
	<ol>
		<li>Seed 1.0 x 106 DC-CYP-ALDH cells/well in 2 mL of CM-10-R cell culture medium into 12 well culture plates.</li>
		<li>Add 25(OH)D to the final concentration of 2.5 &#956;M. Incubate the DC-CYP-ALDH cells for 24 h.</li>
		<li>Harvest the supernatants and store at -20 &#176;C.</li>
		<li>Assay for 1,25(OH)2D concentrations in the supernatants using a commercially available radioimmunoassay (RIA) (see Table of Materials).</li>
		<li>Determine the expression of 1&#945;-hydroxylase in DC-CYP-ALDH cells by fluorescence activated cell sorting (FACS) using an anti-CYP27B1 antibody.</li>
	</ol>
	</li>
	<li>Evaluation of the overexpressed RALDH2 in DC-CYP-ALDH cells 
	NOTE: The expression and activity of the overexpressed RALDH2 are determined using a commercial aldehyde dehydrogenase (ALDH) detection kit (see Table of Materials).
	<ol>
		<li>Seed 1 mL of DC-CYP-ALDH or control cells (1 x 105 cells/mL in assay buffer) in each of two 5 mL tubes (one labeled as &#34;Control&#34; and the other labeled as &#34;Test&#34;).</li>
		<li>Add 10 &#181;L of diethylaminobenzaldehyde (DEAB) solution (1.5 mM in 95% ethanol) to each of the control tubes and mix immediately. DEAB is a RALDH2 inhibitor.</li>
		<li>Add 5 &#181;L of activated RALDH fluorescence substrate (300 &#181;M) to the Control and Test tubes and mix immediately.</li>
		<li>Incubate the tubes for 45 min.</li>
		<li>Pellet the cells by centrifugation at 400 x g at 2&#8211;8 &#176;C for 5 min. Aspirate the supernatants.</li>
		<li>Reconstitute the cells in 200 &#181;L of assay buffer.</li>
		<li>Keep the cells on ice and proceed for analysis by FACS.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Abraham, C., Cho, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+bowel+disease.">Inflammatory bowel disease.</a> New England Journal of Medicine. 361 (21), 2066-2078 (2009).</li><li>Kaser, A., Zeissig, S., Blumberg, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+bowel+disease.">Inflammatory bowel disease.</a> Annual Reviews in Immunology. 28, 573-621 (2010).</li><li>Clifford, D. 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=Natalizumab-associated+progressive+multifocal+leukoencephalopathy+in+patients+with+multiple+sclerosis:+lessons+from+28+cases.">Natalizumab-associated progressive multifocal leukoencephalopathy in patients with multiple sclerosis: lessons from 28 cases.</a> Lancet Neurology. 9 (4), 438-446 (2010).</li><li>Linda, 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=Progressive+multifocal+leukoencephalopathy+after+natalizumab+monotherapy.">Progressive multifocal leukoencephalopathy after natalizumab monotherapy.</a> New England Journal of Medicine. 361 (11), 1081-1087 (2009).</li><li>Fischer, 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=Differential+effects+of+alpha4beta7+and+GPR15+on+homing+of+effector+and+regulatory+T+cells+from+patients+with+UC+to+the+inflamed+gut+in+vivo.">Differential effects of alpha4beta7 and GPR15 on homing of effector and regulatory T cells from patients with UC to the inflamed gut in vivo.</a> Gut. 65 (10), 1642-1664 (2016).</li><li>Xu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vivo+Generation+of+Gut-Homing+Regulatory+T+Cells+for+the+Suppression+of+Colitis.">In Vivo Generation of Gut-Homing Regulatory T Cells for the Suppression of Colitis.</a> Journal of Immunology. 202 (12), 3447-3457 (2019).</li><li>Rosenblum, M. D., Way, S. S., Abbas, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+T+cell+memory.">Regulatory T cell memory.</a> Nature Reviews Immunology. 16 (2), 90-101 (2016).</li><li>Grimm, A. J., Kontos, S., Diaceri, G., Quaglia-Thermes, X., Hubbell, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memory+of+tolerance+and+induction+of+regulatory+T+cells+by+erythrocyte-targeted+antigens.">Memory of tolerance and induction of regulatory T cells by erythrocyte-targeted antigens.</a> Science Report. 5, 15907 (2015).</li><li>Kim, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+inhibitory+activity+of+regulatory+T+cells+requires+the+transcription+factor+Helios.">Stable inhibitory activity of regulatory T cells requires the transcription factor Helios.</a> Science. 350 (6258), 334-339 (2015).</li><li>Bhela, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Plasticity+and+Stability+of+Regulatory+T+Cells+during+Viral-Induced+Inflammatory+Lesions.">The Plasticity and Stability of Regulatory T Cells during Viral-Induced Inflammatory Lesions.</a> Journal of Immunology. 199 (4), 1342-1352 (2017).</li><li>Bluestone, J. 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=Type+1+diabetes+immunotherapy+using+polyclonal+regulatory+T+cells.">Type 1 diabetes immunotherapy using polyclonal regulatory T cells.</a> Science Translational Medicine. 7 (315), (2015).</li><li>Marek-Trzonkowska, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapy+of+type+1+diabetes+with+CD4(+)CD25(high)CD127-regulatory+T+cells+prolongs+survival+of+pancreatic+islets+-+results+of+one+year+follow-up.">Therapy of type 1 diabetes with CD4(+)CD25(high)CD127-regulatory T cells prolongs survival of pancreatic islets - results of one year follow-up.</a> Clinical Immunology. 153 (1), 23-30 (2014).</li><li>Desreumaux, P., 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=Safety+and+efficacy+of+antigen-specific+regulatory+T-cell+therapy+for+patients+with+refractory+Crohn's+disease.">Safety and efficacy of antigen-specific regulatory T-cell therapy for patients with refractory Crohn's disease.</a> Gastroenterology. 143 (5), 1201-1202 (2012).</li><li>Di Ianni, 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=Tregs+prevent+GVHD+and+promote+immune+reconstitution+in+HLA-haploidentical+transplantation.">Tregs prevent GVHD and promote immune reconstitution in HLA-haploidentical transplantation.</a> Blood. 117 (14), 3921-3928 (2011).</li><li>Brunstein, C. G., 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=Infusion+of+ex+vivo+expanded+T+regulatory+cells+in+adults+transplanted+with+umbilical+cord+blood:+safety+profile+and+detection+kinetics.">Infusion of ex vivo expanded T regulatory cells in adults transplanted with umbilical cord blood: safety profile and detection kinetics.</a> Blood. 117 (3), 1061-1070 (2011).</li><li>Kang, S. 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=1,25-Dihyroxyvitamin+D3+promotes+FOXP3+expression+via+binding+to+vitamin+D+response+elements+in+its+conserved+noncoding+sequence+region.">1,25-Dihyroxyvitamin D3 promotes FOXP3 expression via binding to vitamin D response elements in its conserved noncoding sequence region.</a> Journal of Immunology. 188 (11), 5276-5282 (2012).</li><li>Correale, J., Ysrraelit, M. C., Gaitan, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunomodulatory+effects+of+Vitamin+D+in+multiple+sclerosis.">Immunomodulatory effects of Vitamin D in multiple sclerosis.</a> Brain. 132, 1146-1160 (2009).</li><li>Iwata, 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=Retinoic+acid+imprints+gut-homing+specificity+on+T+cells.">Retinoic acid imprints gut-homing specificity on T cells.</a> Immunity. 21 (4), 527-538 (2004).</li><li>Steinman, R. M., Banchereau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taking+dendritic+cells+into+medicine.">Taking dendritic cells into medicine.</a> Nature. 449 (7161), 419-426 (2007).</li><li>Vicente-Suarez, I., Brayer, J., Villagra, A., Cheng, F., Sotomayor, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TLR5+ligation+by+flagellin+converts+tolerogenic+dendritic+cells+into+activating+antigen-presenting+cells+that+preferentially+induce+T-helper+1+responses.">TLR5 ligation by flagellin converts tolerogenic dendritic cells into activating antigen-presenting cells that preferentially induce T-helper 1 responses.</a> Immunology Letters. 125 (2), 114-118 (2009).</li><li>Danova, K., 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=NF-kappaB,+p38+MAPK,+ERK1/2,+mTOR,+STAT3+and+increased+glycolysis+regulate+stability+of+paricalcitol/dexamethasone-generated+tolerogenic+dendritic+cells+in+the+inflammatory+environment.">NF-kappaB, p38 MAPK, ERK1/2, mTOR, STAT3 and increased glycolysis regulate stability of paricalcitol/dexamethasone-generated tolerogenic dendritic cells in the inflammatory environment.</a> Oncotarget. 6 (16), 14123-14138 (2015).</li><li>Liu, P. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptor+triggering+of+a+vitamin+D-mediated+human+antimicrobial+response.">Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response.</a> Science. 311 (5768), 1770-1773 (2006).</li><li>Cao, 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=Application+of+vitamin+D+and+vitamin+D+analogs+in+acute+myelogenous+leukemia.">Application of vitamin D and vitamin D analogs in acute myelogenous leukemia.</a> Experimental Hematology. 50, 1-12 (2017).</li><li>Anderson, 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=Lasting+Impact+of+Clostridium+difficile+Infection+in+Inflammatory+Bowel+Disease:+A+Propensity+Score+Matched+Analysis.">Lasting Impact of Clostridium difficile Infection in Inflammatory Bowel Disease: A Propensity Score Matched Analysis.</a> Inflammatory Bowel Disease. 23 (12), 2180-2188 (2017).</li><li>Tsai, J. 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=Association+of+Aneuploidy+and+Flat+Dysplasia+With+Development+of+High-Grade+Dysplasia+or+Colorectal+Cancer+in+Patients+With+Inflammatory+Bowel+Disease.">Association of Aneuploidy and Flat Dysplasia With Development of High-Grade Dysplasia or Colorectal Cancer in Patients With Inflammatory Bowel Disease.</a> Gastroenterology. 153 (6), 1492-1495 (2017).</li><li>Lee, H. 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=Tracking+of+dendritic+cell+migration+into+lymph+nodes+using+molecular+imaging+with+sodium+iodide+symporter+and+enhanced+firefly+luciferase+genes.">Tracking of dendritic cell migration into lymph nodes using molecular imaging with sodium iodide symporter and enhanced firefly luciferase genes.</a> Science Reports. 5, 9865 (2015).</li><li>Shen, Z., Reznikoff, G., Dranoff, G., Rock, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloned+dendritic+cells+can+present+exogenous+antigens+on+both+MHC+class+I+and+class+II+molecules.">Cloned dendritic cells can present exogenous antigens on both MHC class I and class II molecules.</a> Journal of Immunology. 158 (6), 2723-2730 (1997).</li><li>Okada, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Administration+route-dependent+vaccine+efficiency+of+murine+dendritic+cells+pulsed+with+antigens.">Administration route-dependent vaccine efficiency of murine dendritic cells pulsed with antigens.</a> British Journal of Cancer. 84 (11), 1564-1570 (2001).</li><li>Li, C. 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=Dendritic+cells,+engineered+to+overexpress+25-hydroxyvitamin+D+1alpha-hydroxylase+and+pulsed+with+a+myelin+antigen,+provide+myelin-specific+suppression+of+ongoing+experimental+allergic+encephalomyelitis.">Dendritic cells, engineered to overexpress 25-hydroxyvitamin D 1alpha-hydroxylase and pulsed with a myelin antigen, provide myelin-specific suppression of ongoing experimental allergic encephalomyelitis.</a> FASEB J. , (2017).</li><li>Narula, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+High-Dose+Vitamin+D3+Supplementation+in+Patients+with+Crohn's+Disease+in+Remission:+A+Pilot+Randomized+Double-Blind+Controlled+Study.">Impact of High-Dose Vitamin D3 Supplementation in Patients with Crohn's Disease in Remission: A Pilot Randomized Double-Blind Controlled Study.</a> Digestive Disease Science. 62 (2), 448-455 (2017).</li><li>Ahmad, S. 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=Vitamin+A+Supplementation+during+Pregnancy+Enhances+Pandemic+H1N1+Vaccine+Response+in+Mothers,+but+Enhancement+of+Transplacental+Antibody+Transfer+May+Depend+on+When+Mothers+Are+Vaccinated+during+Pregnancy.">Vitamin A Supplementation during Pregnancy Enhances Pandemic H1N1 Vaccine Response in Mothers, but Enhancement of Transplacental Antibody Transfer May Depend on When Mothers Are Vaccinated during Pregnancy.</a> Journal of Nutrition. 148 (12), 1968-1975 (2018).</li><li>Noronha, S. M. 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=Aldefluor+protocol+to+sort+keratinocytes+stem+cells+from+skin.">Aldefluor protocol to sort keratinocytes stem cells from skin.</a> Acta Cirurgica Brasileira. 32 (11), 984-994 (2017).</li><li>Ferreira, G. 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=Vitamin+D3+Induces+Tolerance+in+Human+Dendritic+Cells+by+Activation+of+Intracellular+Metabolic+Pathways.">Vitamin D3 Induces Tolerance in Human Dendritic Cells by Activation of Intracellular Metabolic Pathways.</a> Cell Reports. 10 (5), 711-725 (2015).</li><li>Bakdash, G., Vogelpoel, L. T., van Capel, T. M., Kapsenberg, M. L., de Jong, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+primes+human+dendritic+cells+to+induce+gut-homing,+IL-10-producing+regulatory+T+cells.">Retinoic acid primes human dendritic cells to induce gut-homing, IL-10-producing regulatory T cells.</a> Mucosal Immunology. 8 (2), 265-278 (2015).</li></ol>

Drs. Xiaolei Tang and David J. Baylink are inventors of a pending patent related to this study.

In this article we describe the use of DC-CYP-ALDH cells, for augmenting the induction of gut-homing Treg cells in peripheral lymphoid tissues. Our data have shown that the DC-CYP-ALDH cells can synthesize locally high concentrations de novo of both 1,25(OH)2D and RA in vitro in the presence of corresponding substrates (i.e., 25[OH]D and retinol, respectively). Because sufficient blood concentrations of 25(OH)D and retinol can be easily achieved through vitamin D and A supplementations respectively in patients who have deficiencies30,31, we reason that the DC-CYP-ALDH cells can augment the induction of gut-homing Treg cells in peripheral lymphoid tissues when normal blood concentrations of 25(OH)D and retinol are present. To support this reasoning, our data demonstrates that in normal healthy animals that do not have vitamin D and vitamin A deficiencies, the DC-CYP-ALDH cells augment the induction of Treg cells that express both regulatory molecules (i.e., foxp3 and IL-10) and a gut-homing receptor (i.e., CCR9). Therefore, this technology can be used for further investigation of gut-homing Treg cells for the treatment of IBD.
One critical step of this protocol is the production of lenti-CYP-ALDH virus with high titers. The preferred virus titers should be 108&#8211;109 TUs/mL. A high titer of the lenti-CYP-ALDH virus is necessary for a high transduction efficiency in DCs.
Another critical step of this protocol is the transduction efficiency in DCs. Because DC-CYP-ALDH cells are not tolerized in vitro in this technology, it is essential that the transduction rate be more than 90% to ensure that the DC-CYP-ALDH cells can efficiently augment the induction of gut-homing Treg cells in vivo. In addition, the DC-CYP-ALDH cells can be further purified by FACS before in vivo administration32.
A unique advantage of this protocol is that the DC-CYP-ALDH cells do not need to be tolerized in vitro before in vivo administration. It is expected that the DC-CYP-ALDH cells, as a result of the combined actions of 1,25(OH)2D and RA, will be maintained in a tolerogenic status in vivo because both 1,25(OH)2D and RA have been shown to tolerize DCs33,34. Therefore, we anticipate that the DC-CYP-ALDH cells will not have instability concerns in an in vivo proinflammatory environment.
Currently, we have only demonstrated that DC-CYP-ALDH cells can increase the frequency (number) of gut-homing Treg cells in peripheral lymphoid tissues and intestines6. Consequently, regulatory function in the intestines as a whole is enhanced because the percent of Treg cells in the intestines is increased. Figure 4 shows that when compared to those with control treatments, intraperitoneal treatment with DC-CYP-ALDH cells significantly increased the percentage of CCR9+foxp3+ Treg cells in the mesenteric lymph nodes, meaning that more Treg cells in the mesenteric lymph nodes are able to specifically home into the intestinal tissues. Figure 4 further shows that most foxp3+ T cells in mesenteric lymph nodes are negative for CCR9 and therefore do not have gut-homing capacity. However, whether DC-CYP-ALDH cells can also enhance the regulatory function of each Treg cell per se (such as enhanced expression levels of foxp3 and/or IL-10) requires further investigation.
The reagents and materials described here are for animal studies only. However, the protocol is applicable for human studies by using corresponding human reagents and materials, except that DCs will be generated from peripheral blood monocytes in humans. The ultimate goal of this protocol is the generation of clinical grade DC-CYP-ALDH cells for the treatment of IBD.

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 million IBD patients. It is generally accepted that a dysregulated immune response to gut bacteria initiates the disease and disrupts the mucosal epithelial barrier1,2. For this reason, currently available U.S. Food and Drug Administration (FDA)-approved drugs inhibit the functions of inflammatory mediators or block the homing of immune cells into the gut. However, the inflammatory mediators and immune cells that are targeted are also necessary for immune defenses. As a result, the inflammatory mediator inhibitors compromise systemic immune defense and the immune cell homing blockers weaken gut immune defense, both of which can lead to severe consequences3,4. In addition, the immune cell homing blockers can also block the homing of regulatory T (Treg) cells into the gut and hence can worsen the already compromised gut immune tolerance in IBD patients. Furthermore, blocking of Treg cell homing into the gut may also lead to systemic immune suppression due to the accumulation of Treg cells in the blood5. Finally, inhibitors and blockers function transiently and, thereby, require frequent administrations. Frequent administration of these inhibitors and blockers may further exacerbate the untoward side effects.
Recently, we proposed a novel strategy that can potentially mitigate or even eliminate the side effects associated with current drugs for IBD treatment6. This strategy augments the induction of gut-homing Treg cells in peripheral lymphoid tissues6. The rationale of this strategy is that gut-homing Treg cells specifically home to the gut and hence will not compromise systemic immune defenses. In addition, since Treg cells can potentially form memory7,8, gut-homing Treg cells can potentially provide a stable control of the chronic gut inflammation in IBD patients and, thereby, treatment should not need to be administered as frequently. Furthermore, since this strategy augments the induction of gut-homing Treg cells in vivo, it does not have the concern of in vivo instability in a highly proinflammatory environment that is associated with adoptive transfer of in vitro generated Treg cells9,10. In this regard, in vitro generated Treg cells are one of the proposed strategies for the treatment of autoimmune diseases11,12,13 and transplant rejection14,15. Finally, in this strategy, dendritic cells (DCs) are engineered to produce locally high concentrations of two molecules de novo: active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and active vitamin A (retinoic acid or RA). We chose 1,25(OH)2D and RA because 1,25(OH)2D can induce the expression of regulatory molecules (e.g., forkhead box P3 [foxp3] and interleukin-10 [IL-10])16,17 and that RA can stimulate the expression of gut-homing receptors in T cells18. Because both 1,25(OH)2D and RA can also tolerize DCs28,29, we reason that the engineered DCs will be stably maintained in a tolerogenic status in vivo and hence circumvent the in vivo instability concerns that are associated with in vitro generated tolerogenic DCs (TolDCs)19,20,21. In this respect, TolDCs are also one of the proposed strategies for in vivo augmentation of Treg cell functions19,20,21. To support our reasoning, we have shown that the engineered DCs, upon in vivo delivery, can augment the induction of gut-homing Treg cells in peripheral lymphoid tissues6.
An additional advantage of our proposed strategy is that 1,25(OH)2D also has other functions that can potentially benefit IBD patients. These other functions include the ability of 1,25(OH)2D to stimulate the secretion of antimicrobials22 and to suppresses carcinogenesis23. Infections and cancers are frequently associated with IBD24,25.
To generate the DCs that can produce locally high concentrations of both 1,25(OH)2D and RA de novo, we use a lentiviral vector to engineer DCs to overexpress two genes. One gene is the cytochrome P450 family 27 subfamily B member 1 (CYP27B1) that encodes 25-hydroxyvitamin D 1&#945;-hydroxylase (1&#945;-hydroxylase), which physiologically catalyzes the synthesis of 1,25(OH)2D. The other gene is the aldehyde dehydrogenase 1 family member A2 (ALDH1a2) that encodes retinaldehyde dehydrogenase 2 (RALDH2), which physiologically catalyzes the synthesis of RA6.
Because in vivo augmentation of gut-homing Treg cell induction is potentially important in the treatment of IBD, in the following protocol we will detail the procedures for the generation of the 1&#945;-hydroxylase-RALDH2-overexpressing DCs (DC-CYP-ALDH cells) that can be used for the future investigation of gut-homing Treg cells in vivo.

<table><tbody><tr><td>10 mL syringes</td><td>ThermoFisher Scientific</td><td>Cat# 03-377-23</td><td></td></tr><tr><td>100 mm x 20 mm culture dishes</td><td>Sigma-Aldrich</td><td>Cat# CLS430167</td><td></td></tr><tr><td>12-well culture plates</td><td>ThermoFisher Scientific</td><td>Cat# 07-200-82</td><td></td></tr><tr><td>150 mm x 25 mm culture dishes</td><td>Sigma-Aldrich</td><td>Cat# CLS430559</td><td></td></tr><tr><td>25-hydroxycholecalciferol (25[OH]D)</td><td>Sigma-Aldrich</td><td>Cat# H4014</td><td></td></tr><tr><td>293T cells</td><td>ATCC</td><td>CRL-3216</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>ThermoFisher Scientific</td><td>Cat#: 21985023</td><td></td></tr><tr><td>6-well culture plates</td><td>ThermoFisher Scientific</td><td>Cat# 07-200-83</td><td></td></tr><tr><td>ALDEFLUOR kit</td><td>Stemcell Technologies</td><td>Cat# 01700</td><td></td></tr><tr><td>Anti-CYP27B1</td><td>Abcam</td><td>Cat# ab95047</td><td></td></tr><tr><td>BD FACSAria II</td><td>BD Biosciences</td><td>N/A</td><td></td></tr><tr><td>CaCl2</td><td>Sigma-Aldrich</td><td>Cat# C1016</td><td></td></tr><tr><td>CM-10-D cell culture medium</td><td></td><td></td><td>DMEM medium containing 10% fetal bovine serum (FBS), 100 U/ml penicillin/streptomycin, 0.055 mM 2-mercaptoethanol (2-ME), 1 mM sodium pyruvate, 0.1 mM nonessential amino acid, and 2 mM L-glutamine.</td></tr><tr><td>CM-10-R cell culture medium</td><td></td><td></td><td>RPMI 1640 medium (no glutamine) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin/streptomycin, 0.055 mM 2-mercaptoethanol (2-ME), 1 mM sodium pyruvate, 0.1 mM nonessential amino acid, and 2 mM L-glutamine.</td></tr><tr><td>CM-4-D cell culture medium</td><td></td><td></td><td>DMEM medium containing 4% fetal bovine serum (FBS), 100 U/ml penicillin/streptomycin, 0.055 mM 2-mercaptoethanol (2-ME), 1 mM sodium pyruvate, 0.1 mM nonessential amino acid, and 2 mM L-glutamine.</td></tr><tr><td>Corning bottle-top vacuum filters, 0.22 mM, 500 mL</td><td>Sigma-Aldrich</td><td>Cat# CLS430513</td><td></td></tr><tr><td>Corning bottle-top vacuum filters, 0.45 mM, 500 mL</td><td>Sigma-Aldrich</td><td>Cat# CLS430514</td><td></td></tr><tr><td>Dissecting scissor</td><td>ThermoFisher Scientific</td><td>Cat# 08-940</td><td></td></tr><tr><td>DMEM medium</td><td>ThermoFisher Scientific</td><td>Cat# 11960044</td><td></td></tr><tr><td>Fetal bovine serum</td><td>ThermoFisher Scientific</td><td>Cat# 16000044</td><td></td></tr><tr><td>Forceps</td><td>ThermoFisher Scientific</td><td>Cat# 22-327379</td><td></td></tr><tr><td>Gibco ACK lysing buffer</td><td>ThermoFisher Scientific</td><td>Cat# A1049201</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>Cat# G5516</td><td></td></tr><tr><td>Goat anti-rabbit IgG</td><td>Abcam</td><td>Cat# ab205718</td><td></td></tr><tr><td>HEPES</td><td>Millipore</td><td>Cat# 391340</td><td></td></tr><tr><td>Lenti-CYP-ALDH</td><td>Custom-made</td><td>1.6-kb mouse CYP27B1 and ALDH1a2 cDNAs were amplified by PCR using a plasmid containing the CYP27B1 cDNA and a plasmid containing the ALDH1a2 cDNA respectively (GeneCopoeia). The amplified CYP27B1 cDNA fragment with a 5&#039; KOZAK ribosome entry sequence was cloned into the pRRL-SIN.cPPt.PGKGFP.WPRE lentiviral vector (Addgene). The resulting construct was designated as lenti-CYP-GFP. The amplified ALDH1a2 cDNA fragment was cloned into the lenti-CYP-GFP to replace the GFP and was designated as lenti-CYP-ALDH. This bicistronic plasmid expresses CYP27B1 controlled by SFFV promoter and ALDH1a2 controlled by PGK promoter.</td><td></td></tr><tr><td>L-glutamine</td><td>ThermoFisher Scientific</td><td>Cat#25030081</td><td></td></tr><tr><td>Lipopolysaccharide</td><td>Sigma-Aldrich</td><td>Cat# L3755</td><td></td></tr><tr><td>Murine GM-CSF</td><td>Peprotech</td><td>Cat# 315-03</td><td></td></tr><tr><td>Murine IL-4</td><td>Peprotech</td><td>Cat# 214-14</td><td></td></tr><tr><td>Na2HPO4</td><td>Sigma-Aldrich</td><td>Cat# NIST2186II</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>Cat# S9888</td><td></td></tr><tr><td>Needles</td><td>ThermoFisher Scientific</td><td>Cat# 14-841-02</td><td></td></tr><tr><td>Nonessential Amino Acids</td><td>ThermoFisher Scientific</td><td>Cat#: 11140076</td><td></td></tr><tr><td>pCMVR8.74</td><td>Addgene</td><td>Plasmid# 22036</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>ThermoFisher Scientific</td><td>Cat#15140148</td><td></td></tr><tr><td>Phoshate Balanced Solution (PBS)</td><td>ThermoFisher Scientific</td><td>Cat#: 20012027</td><td></td></tr><tr><td>PMD2G</td><td>Addgene</td><td>Plasmid# 12259</td><td></td></tr><tr><td>Polypropylene tube, 15 mL</td><td>ThermoFisher Scientific</td><td>Cat# AM12500</td><td></td></tr><tr><td>Polypropylene tube, 50 mL</td><td>ThermoFisher Scientific</td><td>Cat# AM12502</td><td></td></tr><tr><td>Protamine sulfate</td><td>Sigma-Aldrich</td><td>Cat# P3369</td><td></td></tr><tr><td>Rabbit polycloncal IgG isotype control</td><td>Abcam</td><td>Cat# ab171870</td><td></td></tr><tr><td>Radioimmunoassay for 1,25(OH)&lt;sub&gt;2&lt;/sub&gt;D measurement</td><td>Heartland Assays</td><td></td><td></td></tr><tr><td>RPMI 1640 medium, no glutamine</td><td>ThermoFisher Scientific</td><td>Cat# 21870076</td><td></td></tr><tr><td>Sodium pyruvat</td><td>ThermoFisher Scientific</td><td>Cat#: 11360070</td><td></td></tr><tr><td>Sorvall Legend XTR Centrifuge</td><td>ThermoFisher Scientific</td><td>Cat# 75004521</td><td></td></tr><tr><td>Sterile Cell strainers, 40 mm</td><td>ThermoFisher Scientific</td><td>Cat# 07-201-430</td><td></td></tr><tr><td>Sterile storage bottles, 500 mL</td><td>ThermoFisher Scientific</td><td>Cat# CLS431432</td><td></td></tr></tbody></table>

in vivo augmentation of gut-homing regulatory t cell induction

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 million IBD patients. It is generally accepted that a dysregulated immune response to gut bacteria initiates the disease and disrupts the mucosal epithelial barrier. We recently show that gut-homing regulatory T (Treg) cells are a promising therapy for IBD. Accordingly, this article presents a protocol for in vivo augmentation of gut-homing Treg cell induction. In this protocol, dendritic cells are engineered to produce locally high concentrations of two molecules de novo, active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and active vitamin A (retinoic acid or RA). We chose 1,25(OH)2D and RA based on previous findings showing that 1,25(OH)2D can induce the expression of regulatory molecules (e.g., forkhead box P3 and interleukin-10) and that RA can stimulate the expression of gut-homing receptors in T cells. To generate such engineered dendritic cells, we use a lentiviral vector to transduce dendritic cells to overexpress two genes. One gene is the cytochrome P450 family 27 subfamily B member 1 that encodes 25-hydroxyvitamin D 1&#945;-hydroxylase, which physiologically catalyzes the synthesis of 1,25(OH)2D. The other gene is the aldehyde dehydrogenase 1 family member A2 that encodes retinaldehyde dehydrogenase 2, which physiologically catalyzes the synthesis of RA. This protocol can be used for future investigation of gut-homing Treg cells in vivo.

DC-CYP-ALDH cells expressed significantly increased amount of 1&#945;-hydroxylase. To determine whether DC-CYP-ALDH cells generated from BMDCs expressed a significantly increased amount of 1&#945;-hydroxylase, BMDCs were transduced with the lenti-CYP-ALDH virus to produce bone-marrow-derived DC-CYP-ALDH cells (BMDC-CYP-ALDH cells). Subsequently, the BMDC-CYP-ALDH cells were examined for the expression of 1&#945;-hydroxylase by FACS. Our data showed that the BMDC-CYP-ALDH cells, when compared to the parental BMDCs, displayed enhanced expression of the 1&#945;-hydroxylase (Figure 1A). We also determined the enzymatic activity of the 1&#945;-hydroxylase in the BMDC-CYP-ALDH cells. To do so, 1.0 x 106 BMDC-CYP-ALDH cells in 2 mL of CM-10-R cell culture medium were added into 12 well culture plates. 25(OH)D was then added to the cell culture at the final concentration of 2.5 &#956;M. The cells were cultured at 37 &#730;C and 5% CO2 for 24 h and the supernatants were harvested for the measurement of 1,25(OH)2D using a radioimmunoassay (RIA). Our data showed that 1,25(OH)2D concentrations in the culture supernatants of the BMDC-CYP cells (BMDCs transduced with lenti-CYP-GFP virus) and the BMDC-CYP-ALDH cells were each approximately 20x higher than those of the parental BMDCs and the BMDC-ALDH cells (BMDCs transduced with lenti-ALDH virus) (Figure 1B).
DC-CYP-ALDH cells expressed significantly increased amount of RALDH2. To determine whether BMDC-CYP-ALDH cells expressed a significantly increased amount of RALDH2, a RALDH2 substrate was added into the cell cultures in the presence or absence of the RALDH2 inhibitor diethylaminobenzaldehyde (DEAB) (15 &#181;M). The fluorescent product retained inside the cells was analyzed by FACS. Our data showed that mean fluorescence intensities (MFIs) of the BMDC-CYP-ALDH cells were approximately 6x higher than those of the parental BMDCs, suggesting that the BMDC-CYP-ALDH cells, when compared to the parental BMDCs, expressed significantly enhanced RALDH2 enzymatic activity (Figure 2A,B).
DC-CYP-ALDH cells augmented the induction of foxp3+CCR9+ gut-homing Treg cells in vitro. To investigate whether DC-CYP-ALDH cells were able to augment the induction of gut-homing Treg cells in vitro, we transduced DC2.4 cells (a bone marrow-derived DC line26,27,28,29), with lenti-CYP-ALDH virus and generated DC2.4-CYP-ALDH cells. Subsequently, we determined whether the DC2.4-CYP-ALDH cells were able to augment the induction of gut-homing Treg cells in vitro. Accordingly, naive CD4+ T cells were purified from C57BL/6 mice. Purified naive CD4+ T cells at 5 x 105 cells/well were then cocultured with either the parental DC2.4 cells (1 x 105 cells/well) or the DC2.4-CYP-ALDH cells (1 x 105 cells/well) in 24 well culture plates in a serum-free medium in the presence of an anti-CD3 monoclonal antibody (5 &#181;g/mL) and recombinant human IL-2 (50 U/mL). In addition, 25(OH)D and retinol at various concentrations were also added into the cultures. The cells were incubated at 37 &#730;C and 5% CO2. Five days later, the cells were analyzed by FACS for the expressions of foxp3 and c-c chemokine receptor type 9 (CCR9). Our data showed that in the presence of the substrates, the DC2.4 cells did not significantly change the abundance of foxp3+CCR9+ cells in the CD4+ T cell populations (Figure 3A,B). In contrast, the DC2.4-CYP-ALDH cells significantly enhanced the abundance of foxp3+CCR9+ cells among CD4+ T cells. In addition, the more 25(OH)D added, the greater the ability of the DC2.4-CYP-ALDH cells to increase the abundance of foxp3+CCR9+ cells among CD4+ T cells. Therefore, our data support that the DC-CYP-ALDH cells can augment the induction of gut-homing Treg cells in vitro.
DC-CYP-ALDH cells augmented the induction of foxp3+CCR9+ gut-homing Treg cells in vivo. To determine whether DC-CYP-ALDH cells could augment the induction of gut-homing Treg cells in vivo, we intraperitoneally administered one of the following cells into Balb/c mice: the parental DC2.4 cells, the DC2.4-CYP cells (DC2.4 cells transduced with lenti-CYP-GFP virus), and the DC-2.4-CYP-ALDH cells. Four days after the cell administration, mesenteric lymph nodes were examined by FACS (Figure 4A). Our data showed that the DC2.4-CYP-ALDH cells, when compared to the controls, significantly increased the abundance of foxp3+CCR9+ cells among CD3+ T cells (Figure 4B,C). Based on these results, we conclude that the DC-CYP-ALDH cell administration significantly augments the induction of foxp3+CCR9+ T cells in peripheral lymphoid tissues.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60585/60585fig1a.jpg" /> 
Figure 1: DC-CYP-ALDH cells expressed significantly increased amount of 1&#945;-hydroxylase. (A) BMDC-CYP-ALDH cells were generated and analyzed by FACS. A representative FACS plot shows the expression of 1&#945;-hydroxylase in the parental BMDCs and the BMDC-CYP-ALDH cells (gated on live cells). (B) 1&#945;-hydroxylase substrate (i.e., 25(OH)D) was added into the DC cultures. 24 h later, the supernatants were collected and 1,25(OH)2D concentrations were measured. The data show concentrations of 1,25(OH)2D in the cultures of the parental BMDCs, the BMDC-CYP cells, the BMDC-ALDH cells, and the BMDC-CYP-ALDH cells. **p &#60; 0.01. ANOVA test. n = 4. This figure is adapted from Xu et al.6. Copyright 2019. The American Association of Immunologist, Inc. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60585/60585fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60585/60585fig2a.jpg" /> 
Figure 2: DC-CYP-ALDH cells expressed significantly increased amount of RALDH2. (A) BMDC-CYP-ALDH cells were generated and analyzed as described in the protocol. Representative overlaid FACS plots show the BODIPY aminoacetate fluorescence in the BMDCs and the BMDC-CYP-ALDH cells in the presence (+DEAB) or absence (-DEAB) of the RALHD2 inhibitor diethylaminobenzaldehyde (DEAB). (B) Mean fluorescence intensities (MFIs) of BODIPY aminoacetate in the BMDCs and the BMDC-CYP-ALDH cells in the absence of DEAB. *p &#60; 0.05; t-test; n = 4. This figure is adapted from Xu et al.6. Copyright 2019. The American Association of Immunologist, Inc. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60585/60585fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60585/60585fig3.jpg" /> 
Figure 3: DC-CYP-ALDH cells augmented the induction of foxp3+CCR9+ gut-homing Treg cells in vitro. (A) CD4+ naive T cells were isolated from C57BL/6 mouse spleens. The CD4+ T cells were then activated in cultures by an anti-CD3 mAb (5 &#181;g/mL) in the presence of either the parental DC2.4 cells or the DC2.4-CYP-ALDH cells. Additionally, the cultures were added with the indicated concentrations of 25(OH)D and retinol. Five days later, the cells were collected and analyzed by FACS for the expressions of foxp3 and CCR9 in CD3+CD4+ T cell population. Representative FACS plots show the expressions of foxp3 and CCR9 in the CD3+CD4+ T cell populations. (B) Cumulative data from (A). *p &#60; 0.05; ANOVA test; n = 4. This figure is adapted from Xu et al.6. Copyright 2019. The American Association of Immunologist, Inc. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60585/60585fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60585/60585fig4.jpg" /> 
Figure 4: DC-CYP-ALDH cells augmented the induction of foxp3+CCR9+ gut-homing Treg cells in vivo. (A) Balb/c mice intraperitoneally (i.p.) received one of the following DC transfers (Transfer): no DC transfer (No transfer), parental DC2.4 cells, DC2.4-CYP cells, and DC2.4-CYP-ALDH cells. Four days later, mesenteric lymph nodes (MLNs) were analyzed by FACS. (B) Representative FACS plots show the expressions of foxp3 and CCR9 in CD3+ T cell population. (C) Cumulative data from (B) show the percentage of foxp3+CCR9+ cells in the CD3+ T cell population. Cells were gated on CD3+ T cells for all the analyses. Where applicable, the data presented are means &#177; SEM. *p &#60; 0.05; ANOVA test; n = 4&#8211;6. This figure is adapted from Xu et al.6. Copyright 2019. The American Association of Immunologist, Inc. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60585/60585fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction at JoVE.com

In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction

Here we present a protocol for in vivo augmentation of gut-homing regulatory T cell induction. In this protocol, dendritic cells are engineered to locally produce high concentrations of the active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and the active vitamin A (retinoic acid or RA) de novo.

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 ...

in-vivo-augmentation-of-gut-homing-regulatory-t-cell-induction

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

5.3K Views.  Loma Linda University. Dit protocol beschrijft een potentieel nieuwe therapeutische strategie die gemakkelijk kan worden gewijzigd voor menselijke toepassingen. Deze techniek verbetert direct de inductie van darm-homing regulerende T-cellen in perifere lymfeweefsels en kan zelfs in zeer pro-inflammatoire omgevingen stabiel worden geïmplementeerd. Deze technieken kunnen ook worden gebruikt om de rol van actieve vitamine D en vitamine A binnen het perifere immuunsysteem te onderzoeken.Deze visuele demonstrati...

Video: In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction

Competitive Homing Assays to Study Gut-tropic T Cell Migration

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial cells and tissue extravasation is a multistep process controlled by different adhesion molecules (homing receptors) expressed on lymphocytes and their respective ligands (addressins) displayed on endothelial cells 1 2. Even though the function of these adhesion receptors can be partially studied ex vivo, the ultimate test for their physiological relevance is to assess their role during in vivo lymphocyte adhesion and migration. Two complementary strategies have been used for this purpose: intravital microscopy (IVM) and homing experiments. Although IVM has been essential to define the precise contribution of specific adhesion receptors during the adhesion cascade in real time and in different tissues, IVM is time consuming and labor intensive, it often requires the development of sophisticated surgical techniques, it needs prior isolation of homogeneous cell populations and it permits the analysis of only one tissue/organ at any given time. By contrast, competitive homing experiments allow the direct and simultaneous comparison in the migration of two (or even more) cell subsets in the same mouse and they also permit the analysis of many tissues and of a high number of cells in the same experiment.
Here we describe the classical competitive homing protocol used to determine the advantage/disadvantage of a given cell type to home to specific tissues as compared to a control cell population. We chose to illustrate the migratory properties of gut-tropic versus non gut-tropic T cells, because the intestinal mucosa is the largest body surface in contact with the external environment and it is also the extra-lymphoid tissue with the best-defined migratory requirements. Moreover, recent work has determined that the vitamin A metabolite all-trans retinoic acid (RA) is the main molecular mechanism responsible for inducing gut-specific adhesion receptors (integrin a4b7and chemokine receptor CCR9) on lymphocytes. Thus, we can readily generate large numbers of gut-tropic and non gut-tropic lymphocytes ex vivoby activating T cells in the presence or absence of RA, respectively, which can be finally used in the competitive homing experiments described here.

Competitive homing experiments allow to directly assessing the migratory properties of two different cell populations in a single mouse. Here we illustrate this procedure by comparing the migration of ex vivo-generated gut-tropic versus non-gut tropic T cells.

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial ...

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a promising source for adoptive T cell therapy (ACT). However, classical in vitro methods for producing regenerated T cells from iPSC result in either innate-like or terminally differentiated T cells, which are phenotypically and functionally distinct from na&#239;ve T cells. Recently, a novel three-dimensional (3D) thymic culture system was developed to generate a homogenous subset of CD8&#945;&#946;+ antigen-specific T cells with a na&#239;ve T cell-like functional phenotype, including the capacity for proliferation, memory formation, and tumor suppression in vivo. This protocol avoids aberrant developmental fates, allowing for the generation of clinically relevant iPSC-derived T cells, designated as iPSC-derived thymic emigrants (iTE), while also providing a potent tool to elucidate the subsequent functions necessary for T cell maturation after thymic selection.

This article describes a novel method to generate tumor antigen-specific induced pluripotent stem cell-derived thymic emigrants (iTE) by a three-dimensional (3D) thymic culture system. iTE are a homogenous subset of T cells closely related to na&#239;ve T cells with the capacity for proliferation, memory formation, and tumor suppression.

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

Cancer Research

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 ...

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

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

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

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

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

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

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. 
Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human na&#239;ve T cells using a range of Treg-inducing factors (TGF-&#946; plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR. 
These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained. 
A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-&#946; plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.

This protocol describes the reproducible generation and phenotyping of human induced regulatory T cells (iTregs) from na&#239;ve CD4+ T cells in vitro. Different protocols for FOXP3 induction allow for the study of specific iTreg phenotypes obtained with respective protocols.

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing ...

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for TCD8 &#8220;immunodominance&#8221; in this powerful system. Accordingly, the bulk of the T Ag-specific TCD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor TCD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape TCD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

We describe here a flow cytometry-based in vivo killing assay that enables examination of immunodominance in cytotoxic T lymphocyte (CTL) responses to a model tumor antigen. We provide examples of how this elegant assay may be employed for mechanistic studies and for drug efficacy testing.

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...

Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or microbial lipid antigens presented by the non-polymorphic MHC class I-related molecule CD1d. Preclinical and clinical studies support a role for iNKT cells in cancer, autoimmunity and infectious diseases. iNKT cells are very conserved throughout species and their investigation has been facilitated by mouse models, including CD1d-deficient or iNKT-deficient mice, and the possibility to unequivocally detect them in mice and men with CD1d tetramers or mAbs specific for the semi-invariant TCR. However, iNKT cells are rare and they need to be expanded to reach manageable numbers for any study. Because the generation of primary mouse iNKT cell line in vitro has proven difficult, we have set up a robust protocol to purify and expand splenic iNKT cells from the iV&#945;14-J&#945;18 transgenic mice (iV&#945;14Tg), in which iNKT cells are 30 times more frequent. We show here that primary splenic iV&#945;14Tg iNKT cells can be enriched through an immunomagnetic separation process, yielding about 95-98% pure iNKT cells. The purified iNKT cells are stimulated by anti-CD3/CD28 beads plus IL-2 and IL-7, resulting in 30-fold expansion by day +14 of the culture with 85-99% purity. The expanded iNKT cells can be easily genetically manipulated, providing an invaluable tool to dissect mechanisms of activation and function in vitro and, more importantly, also upon adoptive transfer in vivo.

We describe a rapid and robust protocol to enrich invariant natural killer T (iNKT) cells from mouse spleen and expand them in vitro to suitable numbers for in vitro and in vivo studies.

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or ...

Retroviral Transduction of Guide RNA for Gene Editing in Th17 Differentiation

Retroviral CRISPR/Cas9-Mediated Gene Targeting for the Study of Th17 Differentiation in Vitro

T helper cells that produce IL-17A, known as Th17 cells, play a critical role in immune defense and are implicated in autoimmune disorders. CD4 T cells can be stimulated with antigens and well-defined cytokine cocktails in vitro to mimic Th17 cell differentiation in vivo. Research has been conducted extensively on the Th17 differentiation regulation mechanisms using the in vitro Th17 polarization assay.
Conventional Th17 polarization methods typically involve obtaining na&#239;ve CD4 T cells from genetically modified mice to study the effects of specific genes on Th17 differentiation and function. These methods can be time-consuming and costly and may be influenced by cell-extrinsic factors from the knockout animals. Thus, a protocol using retroviral transduction of guide RNA to introduce gene knockout in CRISPR/Cas9 knockin primary mouse T cells serves as a very useful alternative approach. This paper presents a protocol to differentiate na&#239;ve primary T cells into Th17 cells following retroviral-mediated gene targeting, as well as the subsequent flow cytometry analysis methods for assaying infection and differentiation efficiency.

We present a protocol for retroviral transduction of guide RNA into primary T cells from Cas9 transgenic mice, providing an efficient alternative for gene editing in studying Th17 differentiation.

T helper cells that produce IL-17A, known as Th17 cells, play a critical role in immune defense and are implicated in autoimmune disorders. CD4 T cells ...

In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video