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>

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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...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#39; 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)2D 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 paren...

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 ...

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5.3K Views.  Loma Linda University. 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.

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

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 ...

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&amp;#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 ...

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3+CD4+CD25+CD127low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.

CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of immunotherapies. Here we describe the use of fluorescent target array (FTA) technology, which utilizes vital dyes such as carboxyfluorescein succinimidyl ester (CFSE), violet laser excitable dyes (CellTrace Violet: CTV) and red laser excitable dyes (Cell Proliferation Dye eFluor 670: CPD) to combinatorially label mouse lymphocytes into &#62;250 discernable fluorescent cell clusters. Cell clusters within these FTAs can be pulsed with major histocompatibility (MHC) class-I and MHC class-II binding peptides and thereby act as target cells for CD8+ and CD4+ T cells, respectively. These FTA cells remain viable and fully functional, and can therefore be administered into mice to allow assessment of CD8+ T cell-mediated killing of FTA target cells and CD4+ T cell-meditated help of FTA B cell target cells in real time in vivo by flow cytometry. Since &#62;250 target cells can be assessed at once, the technique allows the monitoring of T cell responses against several antigen epitopes at several concentrations and in multiple replicates. As such, the technique can measure T cell responses at both a quantitative (e.g.&#160;the cumulative magnitude of the response) and a qualitative (e.g.&#160;functional avidity and epitope-cross reactivity of the response) level. Herein, we describe how these FTAs are constructed and give an example of how they can be applied to assess T cell responses induced by a recombinant pox virus vaccine.

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in detail in vivo is important for the development of our understanding of the immune response. Here we describe the use of fluorescent target arrays (FTAs) in an in vivo T cell assay that assesses &#62;250 parameters simultaneously by flow cytometry.

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of ...

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.

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

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 ...

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

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

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

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

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model offers accessibility to rare populations of antigen-specific T-cells representative of the cellular memory response as well as the cytokine microenvironment in situ.
This report describes the practical procedure of a cutaneous recall, an SB induction, and a harvest of antigen-specific T-cells. To exemplify the method, the tuberculin skin test is used for antigenic recall in individuals who, prior to this study, underwent a Bacillus Calmette-Gu&#233;rin vaccination against an infection with Mycobacterium tuberculosis. Finally, examples of multiplex and flow cytometric analyses of SB specimens are provided, illustrating high fractions of antigen-specific polyfunctional CD4+ T-cells available by this sampling method compared with cells isolated from the blood.
The method described here is safe and minimally invasive, provides a unique opportunity to study both innate and adaptive immune responses in vivo, and may be beneficial to a broad community of researchers working with cell-mediated immunity and human memory responses, in the context of vaccine development.

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Here, we provide a demonstration of the suction blister cutaneous recall model. The model allows a simple access to study human in vivo adaptive immune responses, for instance in the context of vaccine development.

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model ...