eoe sub articles

EoE Sub Articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1.&#160;In Vivo&#160;Maturation and Suppression of Autoimmune Arthritis
<ol>
	<li>Generate the constructs to be used in retroviral transduction by sub-cloning the OT-II T cell receptor (TCR) genes and forkhead box protein 3 (FoxP3) into the MiDR plasmid&#160;linked with self-cleaving peptide 2A to make the MiDR-TCR&#945;-2A-TCR&#946;-2A-FoxP3 construct.</li>
	<li>Perform retroviral transduction&#160;of induced pluripotent stem cells&#160;(iPSCs) using Plat E cells as the packaging cell line.</li>
	<li>In vivo&#160;differentiation and arthritis induction in mice.
	<ol>
		<li>Differentiate OT-II TCR/FoxP3-transduced iPSCs (OT-II-FoxP3/iPSCs), FoxP3-transduced iPSCs, and DsRed transduced iPSCs on the OP9-DL1-DL4-I-Ab&#160;stromal cells in the presence of cytokines mFlt-3L and mIL-7 for 8 days.</li>
		<li>On day 0, seed OP9-DL1-DL4-I-Ab cells at a minimum density of 104 cells/cm2 by using OP-9 media (&#945;-MEM media containing 20% fetal calf serum (FCS) and 2.2 g/L sodium bicarbonate. Plate 1 x 106 cells per 10 cm dish.</li>
		<li>On day 3, when OP9-DL1-DL4-I-Ab cells become 80-90% confluent, remove the media and seed 0.5 - 1 x 105 iPSCs over OP9-DL1-DL4--I-Ab cells in OP-9 media. 
		&#8203;NOTE: This establishes the co-culture of iPSCs on OP9-DL1-DL4-I-Ab cells and is considered day 0 of differentiation.</li>
		<li>On day 5, remove the media from the 10 cm dish by aspiration, wash the cells with 10 ml of 1x phosphate-buffered saline (PBS), and aspirate the PBS. Add 4 ml of 0.25% trypsin and incubate at 37 &#176;C for 10 min. Add another 8 ml of iPSC media to the cells, re-suspend them, and centrifuge at 400 x g for 5 min at room temperature.
		<ol>
			<li>Aspirate the supernatant and re-suspend the cells in 10 ml of iPSC media. Incubate these re-suspended cells on a fresh 10 cm dish and return to the incubator for 30 min. 
			&#8203;NOTE: Remove the OP9-DL1-DL4-I-Ab feeder cells and keep the differentiating iPSCs in the media. Maintain OP9-DL1-DL4-I-Ab cells continuously to achieve 80 - 90% confluency for further co-culture.</li>
			<li>After 30 minutes, collect the floating cells, filter them through a 70 &#181;m cell strainer, and count them with a hemocytometer.</li>
		</ol>
		</li>
		<li>Seed 5 x 105 of iPSCs to a fresh 80 - 90% confluent of OP9-DL1-DL4-I-Ab cells in OP9 media. Supplement the media with mFlt-3L at a final 5 ng/ml concentration.</li>
		<li>Trypsinize all three kinds of cells from the 10 cm plate and re-suspend cells from each 10 cm plate in 10 ml of fresh media. Add the cells to a fresh 10 cm plate and return to the incubator for 30 min. After 30 min, collect floating cells.</li>
		<li>Pass cells through a 70 &#181;m cell strainer to remove cell clusters and count using a hemocytometer. Adjust the cells to a concentration of 1.5 x 107&#160;cells/ml in cold PBS and filter again if needed. Keep cells on ice until adoptive cell transfer into mice.</li>
		<li>Inject 200 &#181;l of the cell suspension (3 x 106&#160;cells) into three different groups of 4 - 6 week-old female C57BL/6 mice through the tail vein.</li>
		<li>On day 10, after the cell transfer, inject mice with 100 &#181;g of methylated bovine serum albumin (mBSA) emulsified in Freund&#39;s complete adjuvant at the base of the tail using a 1 ml syringe.</li>
		<li>On day 17, anesthetize mice using an isoflurane vaporizer. Utilize 4 - 5% isoflurane for induction and 1 - 2% for maintenance. Induce arthritis by intra-articular injection of 20 &#181;g mBSA in 10 &#181;l PBS in the left knee joint and of 20 &#181;g mBSA and 100 &#181;g whole ovalbumin (OVA) in 10 &#181;l PBS in the right knee joint. Food and a gel pack can be placed on the bedding floor after arthritis has developed.&#160;Mice will be euthanized: Body Condition Score (BCS) of 2/5 or moribund, severe cachexia and/or persistent recumbency (a 24-hr&#160;period), and severity score of 4.&#160; In score 4, erythema and severe swelling encompass the ankle, foot, and digits, or ankyloses of the limb.&#160;</li>
	</ol>
	</li>
	<li>Characterization of the OT-II TCR/FoxP3-transduced iPSCs.
	<ol>
		<li>Use a fluorescent microscope (20X) to visualize unfixed live DsRed+&#160;GFP+&#160;cells.</li>
		<li>Examine gene integration and expression by both Western blot and flow cytometry.</li>
	</ol>
	</li>
	<li>Arthritis suppression assay
	<ol>
		<li>Measure the swelling of both knees of the mice before arthritis induction by using a dial-gauge caliper to establish a baseline.</li>
		<li>Follow steps (1.3.1 - 1.3.10) for arthritis induction and Treg transfer. Measure mouse knees with a dial-gauge caliper post-Treg transfer.</li>
		<li>Calculate the percent increase in the knee diameter: Percent Increase = (Knee diameter on day 1 - Knee diameter on day 0) / Knee diameter on day 0.</li>
	</ol>
	</li>
	<li>Measurement of joint destruction and inflammatory cell infiltration in the knees by histology (Figure 1).
	<ol>
		<li>On day 7 post-arthritis induction, euthanize the mice by carbon dioxide (CO2)&#160;inhalation followed by cervical dislocation. Remove hair from the knees with an electric clipper and excise the knees by surgical procedure.</li>
		<li>Remove the skin from the legs by making an incision in the hind leg with blunt-end scissors and cut the skin across the thigh and down to the ankle. Gently peel the skin over the leg and foot to expose the muscle. Remove the muscle from the leg carefully without damaging the knee joint.
		<ol>
			<li>Cut the hind leg just above the pelvic/hip joint and cut the tibia using sharp dissecting scissors.</li>
		</ol>
		</li>
		<li>Fix the knees in 4% formalin for 48 hr. Decalcify the knees by using 2.5 M formic acid; rinse thrice in xylene for 3 min, rinse twice in 100% ethanol, rinse twice in 95% ethanol, rinse twice in deionized water for 2 min, and decalcify in 1 mM ethylenediaminetetraacetic acid (EDTA). Treat at a sub-boiling temperature (90 &#176;C) for 20 min.</li>
		<li>Remove both knees, fix in 10% formalin, and calcify in Formical-4. Embed the tissues in Paraffin, section at 4 &#181;m, and stain with hematoxylin and eosin (H &#38; E) or Safranin O staining and observe under a microscope.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>C57BL/6j mice</td>
			<td>Jackson Laboratory</td>
			<td>664</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-MEM</td>
			<td>Invitrogen</td>
			<td>A10490-01</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Hyclone</td>
			<td>SH3007.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>Sigma</td>
			<td>107689</td>
			<td></td>
		</tr>
		<tr>
			<td>Genejammer</td>
			<td>Integrated science</td>
			<td>204130</td>
			<td></td>
		</tr>
		<tr>
			<td>mFlt-3L</td>
			<td>peprotech</td>
			<td>250-31L</td>
			<td></td>
		</tr>
		<tr>
			<td>mIL-7</td>
			<td>peprotech</td>
			<td>217-17</td>
			<td></td>
		</tr>
		<tr>
			<td>mBSA</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Ova albumin</td>
			<td>Avantor</td>
			<td>0440-01</td>
			<td></td>
		</tr>
		<tr>
			<td>CFA</td>
			<td>Difco</td>
			<td>2017014</td>
			<td></td>
		</tr>
		<tr>
			<td>Tailveiner restrainer</td>
			<td>Braintree scientific</td>
			<td>RTV 150-STD</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148-500G</td>
			<td>Caution: Allergenic, Carcenogenic, 
			Toxic</td>
		</tr>
	</tbody>
</table>

a cell-based therapy for autoimmune arthritis in a murine model

Source: Haque, M. et al., <a href="https://app.jove.com/v/54720">Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity.</a>&#160;J. Vis. Exp. (2016)
This video showcases a cell-based therapy for autoimmune arthritis in mice. iPSC-derived T regulatory cells expressing ovalbumin-specific receptors are injected with methylated bovine serum albumin (mBSA) emulsified in an adjuvant. Murine antigen-presenting cells (APCs) process and present the mBSA to T cells, prompting T cell activation and migration to the knee joint&#39;s synovial membrane. Subsequent injections involve mBSA alone in the left knee and a combination of mBSA and ovalbumin in the right knee. In the left knee, the immune response to mBSA triggers arthritis, while in the right knee, T-regulatory cells interacting with ovalbumin mitigate inflammation, improving arthritis symptoms.

<img alt="Figure 1" src="/files/ftp_upload/22237/22237_Figure1.jpg" /> 
Figure 1: Adoptive Transfer of Ag-specific iPSC-Tregs Ameliorates AIA in Mice. Murine iPSCs were transduced with the retroviral construct MiDR, MiDR-FoxP3, or MiDR-TCR-FoxP3 and were co-cultured on the OP9-DL1/DL4/I-Ab cells. On day 7, the gene-transduced cells (3 &#215; 106/mouse) were adoptively transferred into female C57BL/6 mice induced with AIA two wee...

A Cell-Based Therapy for Autoimmune Arthritis in a Murine Model

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
...

Take iPSC-derived regulatory T cells, or Tregs, expressing ovalbumin-specific receptors. Inject them into the tail vein of a restrained mouse.
The cells reach the synovial membrane lining the inner surface of the knee joint capsule.
Next, administer methylated bovine serum albumin or mBSA, emulsified with an adjuvant.
Adjuvants enhance the presentation of mBSA to antigen-presenting cells, or APCs, which process and present the fragments via MHC molecules.
The APCs migrate to the lymph nodes, inducing T cell activation. Activated T cells migrate to the knee joint.
Inject mBSA alone and mBSA with ovalbumin in the left and right knee joints, respectively.
In the left knee, APCs present the mBSA fragments to activated T cells, triggering pro-inflammatory cytokine release and leading to tissue damage and joint swelling, termed arthritis.
In the right knee, Tregs interact with ovalbumin-presenting APCs and release anti-inflammatory cytokines, reducing tissue damage and joint swelling, thereby ameliorating arthritis.

a-cell-based-therapy-for-autoimmune-arthritis-in-a-murine-model

Research

JoVE Encyclopedia of Experiments

Immunology

Immunotherapy

Model Development and Disease Study

70 次观看. 来源:Haque， M. et al.， 干细胞衍生的抗原特异性调节性 T 细胞对抗自身免疫的开发。J. Vis. Exp. （2016 年） 该视频展示了一种基于细胞的小鼠自身免疫性关节炎疗法。将表达卵清蛋白特异性受体的 iPSC 衍生的 T 调节细胞注射在佐剂中乳化的甲基化牛血清白蛋白 （mBSA）。小鼠抗原呈递细胞 （APC） 加工 mBSA 并将其呈递给 T 细胞，促进 T 细胞活化并迁移到膝关节滑膜。随后的注射包...

视频: 小鼠模型中基于细胞的自身免疫性关节炎疗法 - 概念

Enrich and Expand Rare Antigen-specific T Cells with Magnetic Nanoparticles

We have developed a tool to both enrich and expand antigen-specific T cells. This can be helpful in cases such as to A) detect the existence of antigen-specific T cells, B) probe the dynamics of antigen-specific responses, C) understand how antigen-specific responses affect disease state such as autoimmunity, D) demystify heterogeneous responses for antigen-specific T cells, or E) utilize antigen-specific cells for therapy. The tool is based on a magnetic particle that we conjugate antigen-specific and T cell co-stimulatory signals, and that we term as artificial antigen presenting cells (aAPCs). Consequently, since the technology is simple to produce, it can easily be adopted by other laboratories; thus, our purpose here is to describe in detail the fabrication and subsequent use of the aAPCs. We explain how to attach antigen-specific and co-stimulatory signals to the aAPCs, how to utilize them to enrich for antigen-specific T cells, and how to expand antigen-specific T cells. Furthermore, we will highlight engineering design considerations based on experimental and biological information of our experience with characterizing antigen-specific T cells.

Antigen-specific T cells are difficult to characterize or utilize in therapies due to their extremely low frequency. Herein, we provide a protocol to develop a magnetic particle which can bind to antigen-specific T cells to enrich these cells and then to expand them several hundred-fold for both characterization and therapy.

利用磁性纳米粒子丰富和扩大罕见抗原特异性 t 细胞

We have developed a tool to both enrich and expand antigen-specific T cells. This can be helpful in cases such as to A) detect the existence of ...

科研

JoVE Journal

工程学

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.

在从幼稚的CD4 + T细胞的人CD4 + FOXP3 +诱导调节性T细胞（iTregs的）使用含有TGF-β-协议的体外分化

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

免疫与感染

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.

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.

在 Vivo 增强的古特-霍姆调节 T 细胞诱导

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

A Cell-Based Therapy for Autoimmune Arthritis in a Murine Model

成績單

A Cell-Based Therapy for Autoimmune Arthritis in a Murine Model