Name: induction of intestinal inflammation by adoptive transfer of cbir1 tcr transgenic cd4+ t cells to immunodeficient mice
Uploaded: 2021-12-16T14:30:00
Description: Watch this Scientific Journal Video about Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice at JoVE.com

This work was supported in part by the National Institutes of Health grants DK125011, AI150210, and DK124132, the University of Texas System STARs award (Y.C.), and the James W. McLaughlin Fellowship Fund from The University of Texas Medical Branch at Galveston (W.Y.). Figure 1 was created with BioRender.com.

All animal procedures were performed according to the University of Texas Medical Branch&#39;s Committee on the Use and Care of the animals. CBir1 TCR Tg mice were provided by Dr. Charles Elson of the University of Alabama at Birmingham. CBir1 TCR Tg mice can be female or male but should be at 8-12 weeks. Rag1-/- mice on the C57BL/6 background were obtained from the Jackson Laboratory10. Rag1-/- mice must be gender and age-matched, and either male or female ...

<ol>
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Although every step is essential for the reproducibility of this colitis model, there are several critical steps. The recipient Rag-/- mice should receive adequate viable na&#970;ve CD4+ T cells to induce intestinal inflammation. We used spleens for the isolation of na&#239;ve CD4+ T cells instead of MLNs. Because the yield of na&#239;ve CD4+ T cells in MLNs is much lower than in spleens. CD62L is highly expressed in na&#239;ve T cells, and CD44 and CD25 are the activa...

Inflammatory bowel diseases (IBD), mainly including Crohn&#39;s disease (CD) and ulcerative colitis (UC), are characterized by chronic, relapsing-remitting inflammation of the gastrointestinal tract, affecting millions worldwide1. Several factors have been implicated in the development and pathogenesis of IBD, including genetic susceptibility, gut microbiota, immune responses, diet, and lifestyle2. However, the exact mechanism of IBD is still not completely understood.
One of the particular interests is the interaction between gut microbiota and host immune responses in regulating intestinal inflammation3. Gut microbiota provides a series of immunostimulatory molecules and antigens, which can activate immune responses4. While the balance between effector T cells and regulatory T cells (Tregs) is critical in maintaining intestinal homeostasis, the excessive intestinal mucosal CD4+ T cell response to gut microbiota antigens contributes to intestinal inflammation5,6,7. As an immunodominant gut microbiota antigen, CBir1 flagellin has been related to the pathogenesis of human CD8,9. Furthermore, transfer of CBir1 TCR transgenic (Tg) T cells induces intestinal inflammation in immune-deficient mice6, closely resembling the human IBD, indicating that this T cell transfer model helps investigate the mechanisms of human IBD.
This work describes the detailed protocol of inducing colitis in Rag1-/- mice by adoptive transfer of CBir1 TCR Tg na&#970;ve CD4+ T cells and assessing disease severity. Besides, the anticipated results are shown, and the critical steps of the procedure and troubleshooting are discussed, which will help researchers investigate the mechanisms of pathogenesis of intestinal inflammation and test the potential drugs for treating IBD.

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		<tr>
			<td>0.22 &#181;m vacuum-driven disposable bottle top filter</td>
			<td>MilliporeSigma</td>
			<td>SCGPS05RE</td>
			<td></td>
		</tr>
		<tr>
			<td>100x Penicillin-Streptomycin</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>100-&#181;m strainer</td>
			<td>BD Biosciences</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>3-mL Transfer Pipette</td>
			<td>Fisherbrand</td>
			<td>13-711-9CM</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD16/32</td>
			<td>Biolegend</td>
			<td>101302</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD25-Percp/Cy5.5</td>
			<td>Biolegend</td>
			<td>102030</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD3-Percp/Cy5.5</td>
			<td>Biolegend</td>
			<td>100327</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD4 APC</td>
			<td>Biolegend</td>
			<td>100516</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD4 Magnetic Particles</td>
			<td>BD Biosciences</td>
			<td>551539</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD4-BV421</td>
			<td>Biolegend</td>
			<td>100544</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD62L-PE</td>
			<td>Biolegend</td>
			<td>104408</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse Foxp3-PE</td>
			<td>ThermoFisher</td>
			<td>12-5773-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse IFN&#947;-FITC</td>
			<td>Biolegend</td>
			<td>505806</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse IL-17A-PE/Cy7</td>
			<td>Biolegend</td>
			<td>506922</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated Cell Counter</td>
			<td>Bio-rad</td>
			<td>TC20</td>
			<td></td>
		</tr>
		<tr>
			<td>Brefeldin A</td>
			<td>BD Biosciences</td>
			<td>555029</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Fisher Bioreagents</td>
			<td>BP1600-1</td>
			<td></td>
		</tr>
		<tr>
			<td>C tube</td>
			<td>Miltenyi</td>
			<td>130-093-237</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Separation Magnet</td>
			<td>BD Biosciences</td>
			<td>552311</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>Sigma-Aldrich</td>
			<td>C5138</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissociator Machine</td>
			<td>Miltenyi</td>
			<td>130-096-427</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Corning</td>
			<td>46-034-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA (0.5 M, PH 8.0)</td>
			<td>Corning</td>
			<td>46-034-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>R&#38;D Systems</td>
			<td>S11550</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>BD Biosciences</td>
			<td>LSD Fortessa</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat Lamp</td>
			<td>CoverShield</td>
			<td>BR40</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin and Eosin (H&#38;E) Stain Kit</td>
			<td>Abcam</td>
			<td>ab245880</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin Syringes</td>
			<td>BD Biosciences</td>
			<td>329412</td>
			<td></td>
		</tr>
		<tr>
			<td>Ionomycin</td>
			<td>ThermoFisher</td>
			<td>I24222</td>
			<td></td>
		</tr>
		<tr>
			<td>Live/dead Fixable Near-IR Dead Cell Stain kit</td>
			<td>ThermoFisher</td>
			<td>L10119</td>
			<td></td>
		</tr>
		<tr>
			<td>MaxQ 6000 Incubated/Refrigerated Stackable Shakers</td>
			<td>ThermoFisher</td>
			<td>SHKE6000</td>
			<td></td>
		</tr>
		<tr>
			<td>NH4Cl</td>
			<td>Thermo Scientific</td>
			<td>A687-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>GE Healthcare</td>
			<td>17-0891-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Phorbol-12-myristate 13-acetate</td>
			<td>Sigma-Aldrich</td>
			<td>P8139</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium</td>
			<td>Cytiva HyClone</td>
			<td>SH3002702</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorter</td>
			<td>BD Biosciences</td>
			<td>Arial Fusion</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Automatic Processor</td>
			<td>ThermoFisher</td>
			<td>STP120</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Embedding/Processing Cassette</td>
			<td>Fisher Healthcare</td>
			<td>22048142</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Thermo Scientific</td>
			<td>BP154-1</td>
			<td></td>
		</tr>
		<tr>
			<td>True-Nuclear Transcription Factor Buffer Set (including Perm Buffer)</td>
			<td>Biolegend</td>
			<td>424401</td>
			<td></td>
		</tr>
	</tbody>
</table>

induction of intestinal inflammation by adoptive transfer of cbir1 tcr transgenic cd4+ t cells to immunodeficient mice

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable health and financial burden on individuals and society. Therefore, it is critical to investigate the mechanisms underlying the pathogenesis and development of IBD. Here, a gut microbiota antigen-specific T cell transfer colitis model is described. CBir1 flagellin has been recognized as the immunodominant gut bacterial antigen in experimental colitis and patients with Crohn&#39;s disease. CBir1 TCR transgenic na&#970;ve CD4+ T cells, specific to CBir1 flagellin, can induce chronic colitis after adoptive transfer into immune-deficient Rag1-/- mice. The disease severity is assessed by histopathology. The CD4+ T cell phenotypes in colonic lamina propria are also determined. This model closely resembles the development of IBD, which provides an ideal murine model for investigating the mechanisms driving the pathogenesis of IBD and testing the potential drugs for treating IBD.

Approximately 5 x 106 CBir1 TCR Tg na&#970;ve CD4+ T cells per spleen were isolated from an adult CBir1 TCR Tg mouse. Transfer of CBir1 TCR Tg na&#970;ve CD4+ T cells induced chronic colitis in recipient Rag1-/- mice. After cell transfer, clinical signs were monitored to evaluate the progression of intestinal inflammation, including weight loss, stool consistency, and hunched posture. As expected, mice began to lose weight around three weeks post cell transfer, and the...

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Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

In this protocol, a gut microbiota antigen-specific T cell adoptive transfer colitis model is described. CD4+ T cells are isolated from CBir1 TCR transgenic mice. These are specific for an immunodominant gut microbiota antigen CBir1 flagellin, which is transferred into recipient Rag1-/- mice, leading to intestinal inflammation.

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable ...

Inflammatory bowel disease, or IBD, imposes a considerable health and financial burden on individuals and the society. This colitis model is helpful to study the mechanisms of IBD and evaluate the treatments for IBD. This immunodominant Cbir flagellin T-cell-specific adoptive transfer colitis model provides insights into how gut bacteria antigen induces T-cell responses to induce colitis.After euthanizing the mouse, make an approximately one-centimeter left abdominal incision and pull the skin away from the abdominal muscle tissue. Then, make an approximately three-centimeter incision in the abdominal muscle tissue. Next, remove the spleen with sterile scissors and forceps.Place the spleen in a culture dish containing five milliliters of pre-cooled washing buffer. Grind the spleen with the rough surface of two sterile glass slides. Transfer the cell suspension into a 50-milliliter centrifuge tube by passing it through a 100-micrometer cell strainer.Rinse the glass slides and culture dish with five milliliters of pre-cooled washing buffer, and transfer the washing buffer into the tube. Centrifuge the cell suspension, discard the supernatant, and resuspend the cells with five milliliters of prewarmed Tris ammonium chloride lysis buffer per spleen. Incubate for 10 minutes at room temperature.Add 10 milliliters of pre-cooled washing buffer to the tube. Then, centrifuge the cell suspension, discard the supernatant, and resuspend the cells with 10 milliliters of pre-cooled isolation buffer. To count the cells, mix 10 microliters each of cell suspension and Trypan blue thoroughly, and load 10 microliters onto a slide.Then, insert the slide into the automated cell counter to obtain the viable cell number. Centrifuge the remaining cell suspension and discard all supernatant. Vortex the anti-mouse CD4 magnetic particles, directly add 50 microliters of the particles per 10 million cells, and mix with cell pellets thoroughly.Incubate the mixture for 30 minutes at four degrees Celsius. Transfer the cell particle suspension into a sterile collection tube. Add 3.5 milliliters of pre-cooled isolation buffer into the tube.Place the tube on the cell separation magnet for eight minutes at room temperature. Then, carefully aspirate off the supernatant with a three-milliliter transfer pipette. Remove the tube from the cell separation magnet.Then, resuspend the cells with 3.5 milliliters of pre-cooled isolation buffer and place the tube on the magnet for four minutes at room temperature. Carefully aspirate the supernatant with a three-milliliter pipette. Finally, resuspend the cells in one milliliter of pre-cooled FACS buffer.To transfer the cells into the recipient mice, resuspend the Cbir1 TCR transgenic-naive CD4-positive T-cells to 5 million cells per milliliter in 1x PBS. Warm the mice under a heat lamp and restrain the mice using a mouse restrainer. Intravenously inject 200 microliters of the cell suspension into the tail vein of the mice.After euthanizing the mouse, make an approximately one-centimeter ventral midline skin incision. Pull the skin away from the abdominal muscle tissue. Then, make a three-centimeter incision in the abdominal muscle tissue.Next, identify the coecum and remove the entire colon with sterile scissors and forceps. Wet the colon with pre-cooled PBS in a culture dish. Incise the colon lengthwise, and rinse it with pre-cooled PBS.Cut 1/3 of the colon longitudinally. Place the colon strip in a paper towel with the lumenal side facing upward. Perform Swiss-rolling using a toothpick.Place the colon Swiss into a cassette, and put the cassette in 10%buffered formalin for 24 hours. Dehydrate and paraffin-embed the colon with an automated processor. Then, cut five-micrometer tissue sections on a microtome.Mount the tissue on slides and perform hematoxylin and eosin staining. The Rag knockout mice began to lose weight around three weeks post-cell transfer, and their weight reached around 80 to 85%of their original weight six weeks post-cell transfer. Recipient mice showed short colon length six weeks post-cell transfer.The recipient mice demonstrated more cell infiltration in the intestinal lamina propria four weeks post-cell transfer. They showed goblet cell loss and intestinal epithelial cell hyperplasia five weeks post-cell transfer, as well as mucosal erosion and inflammatory cell infiltration in the submucosa of the colon six weeks post-cell transfer. The histopathological scores increased substantially from four to six weeks.Simultaneously, there was no inflammation in Rag knockout mice receiving PBS alone. Using this procedure, colonic cytokine levels could be determined by ELISA and qPCR.

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