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 can be used but should be at 8-12 weeks. The entire protocol is summarized in Figure 1.
1. Preparation of the recipient mice
<ol>
	<li>Prepare Rag1-/- mice on the C57BL/6 background, bred in the same specific pathogen-free animal facility. Calculate the number of mice per group by power analysis11. 
	NOTE: Rag1-/- mice do not have mature T cells and B cells10.</li>
	<li>Mark the mice by ear punch.</li>
	<li>Weigh the mice on the same day of T cell transfer.</li>
</ol>
2. Preparation of the reagents and solutions
NOTE: The reagents used are toxic, or biohazard and their handling need precautions and safety measures.
<ol>
	<li>Prepare Washing Buffer: add 5 mL of 100x Penicillin-Streptomycin into 500 mL of RPMI 1640 medium. Mix it thoroughly and store it at 4 &#176;C.</li>
	<li>Prepare Tris-NH4Cl Lysis Buffer.
	<ol>
		<li>Prepare Solution Part A. Dissolve 2.06 g of Tris base in 100 mL of double-distilled water (ddH2O) and adjust the pH value to 7.2 with HCl.</li>
		<li>Prepare Solution Part B. Dissolve 7.47 g of NH4Cl in 800 mL of ddH2O.</li>
		<li>Mix A and B thoroughly. Measure the pH and adjust it to 7.2 if not.</li>
		<li>Adjust the total volume to 1000 mL. Autoclave and then store it at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare the Isolation Buffer.
	<ol>
		<li>Add 2.5 g of BSA and 500 &#181;L of EDTA (0.5 M, pH 8.0) into 500 mL of 1x PBS. Mix it thoroughly.</li>
		<li>Filter the solution through a 0.22 &#181;m vacuum-driven disposable bottle top filter (see Table of Materials). Store it at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare FACS Buffer. Add 1 mL of FBS and 50 &#181;L of EDTA (0.5 M, pH 8.0) into 50 mL of Washing Buffer (prepared in step 2.1). Mix thoroughly and store it at 4 &#176;C.</li>
	<li>Prepare Complete Medium. Add 5 mL of FBS in 45 mL of Washing Buffer. Mix thoroughly and store it at 4 &#176;C.</li>
	<li>Prepare EDTA-PBS Buffer.
	<ol>
		<li>Calculate the volume of the EDTA-PBS Buffer needed. Volume (mL) = mouse number x 20.</li>
		<li>Add appropriate volume of FBS, EDTA, and HEPES in the PBS (2% of FBS, 0.5 mM of EDTA, 10 mM of HEPES in PBS) (see Table of Materials).</li>
		<li>Mix it thoroughly and pre-warm in a 37 &#176;C water bath.</li>
	</ol>
	</li>
	<li>Prepare the Digestion Buffer.
	<ol>
		<li>Calculate the volume of the Digestion Buffer needed. Volume (mL) = mouse number x 10.</li>
		<li>Add appropriate volume of FBS, Collagenase IV, and DNase I in Washing Buffer (2% of FBS, 0.5 mg/mL of Collagenase IV, and 10 U/mL of DNase I in Washing Buffer) (see Table of Materials).</li>
		<li>Mix it thoroughly and pre-warm in a 37 &#176;C water bath.</li>
	</ol>
	</li>
	<li>Prepare Percoll Solution.
	<ol>
		<li>Prepare 100% Percoll. Add 5 mL of 10x PBS in 45 mL of original Percoll (see Table of Materials).</li>
		<li>Prepare 2% FBS in Washing Buffer. Add 1 mL of FBS in 49 mL of Washing Buffer.</li>
		<li>Caulculate the volume of 40 % Percoll Solution and 75 % Percoll Solution. Volume of 40% Percoll Solution (mL) = mouse number x 4; Volume of 75% Percoll Solution (mL) = mouse number x 2. 
		NOTE: The reagents/solutions prepared in steps 2.1-2.5 will be used in steps 3-4, and those prepared in steps 2.6-2.8 will be used in step 9. All the reagents/solutions used in step 9 should be freshly prepared. Making 5 % extra Buffer is recommended for all the steps.</li>
	</ol>
	</li>
</ol>
3. Isolation of splenic CBir1 TCR Tg CD4+ T cells
<ol>
	<li>Euthanize CBir1 TCR Tg mouse/mice by a cervical dislocation with CO2 euthanasia (30%-70% gas-air displacement rate). Wet the mice with 70% ethanol.</li>
	<li>Perform a ~1 cm left abdomen incision, pull the skin away from the abdominal muscle tissue, make a ~3 cm incision in the abdominal muscle tissue, and remove the spleen with sterile scissors and forceps. Place the spleen in a culture dish containing 5 mL of pre-cold Washing Buffer (prepared in step 2.1).</li>
	<li>Grind the spleen with the rough surface of two sterile glass slides. Transfer the cell suspension into a 50 mL centrifuge tube by passing through a 100 &#181;m cell strainer (see Table of Materials). Rinse the glass slides and culture dish with 5 mL of pre-cold Washing Buffer and transfer the Washing Buffer into the tube.</li>
	<li>Centrifuge the cell suspension at 350 x g for 8 min at 4 &#176;C. Discard the supernatant and resuspend the cells with 5 mL of pre-warmed Tris-NH4Cl Lysis Buffer (prepared in step 2.2) per spleen. Incubate for 10 min at room temperature. Add 10 mL of pre-cold Washing Buffer to the tube.</li>
	<li>Centrifuge the cell suspension at 350 x g for 8 min at 4 &#176;C. Discard the supernatant and resuspend the cells with 10 mL of pre-cold Isolation Buffer (prepared in step 2.3).</li>
	<li>Count the cells. Mix 10 &#181;L of cell suspension with 10 &#181;L of trypan blue thoroughly. Load 10 &#181;L of the mixture onto a slide, insert the slide into the Automated Cell Counter (see Table of Materials) and obtain the viable cell number12. 
	NOTE: Approximately 1 x 108 cells can be obtained from one donor mouse in this step.</li>
	<li>Centrifuge the remaining cell suspension from step 3.6 at 350 x g for 8 min at 4 &#176;C. Discard all the supernatants.</li>
	<li>Vortex the anti-mouse CD4 Magnetic Particles thoroughly (see Table of Materials), directly add 50 &#181;L of the particles per 107 cells and mix with cell pellets thoroughly. Incubate for 30 min at 4 &#176;C. 
	NOTE: Any other commercial CD4+ T cell enrichment kit can be used here.</li>
	<li>Transfer the cell-particle suspension into a sterile collection tube. Add 3.5 mL of pre-cold Isolation Buffer into the tube.</li>
	<li>Place the tube on the Cell Separation Magnet (see Table of Materials) for 8 min at room temperature. Carefully aspirate off the supernatant using a 3 mL Transfer Pipette.</li>
	<li>Remove the tube from Cell Separation Magnet (see Table of Materials), resuspend the cells with 3.5 mL pre-cold Isolation Buffer, and place the tube to the Magnet for 4 min at room temperature. Carefully aspirate off the supernatant using a 3 mL Transfer Pipette.</li>
	<li>Repeat step 3.11.</li>
	<li>Resuspend the cells with 1 mL of pre-cold FACS Buffer (prepared in step 2.4).</li>
</ol>
4. Purification of CBir1 TCR Tg na&#970;ve CD4+ T cells
<ol>
	<li>Count the cells following step 3.6.</li>
	<li>If the cell concentration is &#62;107/mL, add a volume of FACS buffer to make sure the cell concentration is &#8804; 107/mL. 
	NOTE: ~1 &#215; 107 cells can be obtained from one donor mouse in this step.</li>
	<li>Stain the surface markers with 10 &#181;L of anti-mouse CD4-APC, 10 &#181;L of anti-mouse CD25-Percp/Cy5.5, and 10 &#181;L of anti-mouse CD62L-PE13,14 (see Table of Materials). Mix gently and incubate for 30 min at 4 &#176;C in the dark.</li>
	<li>Wash the cells with 2 mL of pre-cold FACS buffer. Centrifuge the cell suspension at 350 x g for 8 min at 4 &#176;C. Aspirate all the supernatantsusing a 3 mL Transfer Pipette.</li>
	<li>Repeat Step 4.4.</li>
	<li>Resuspend the cells to the concentration of 40 x 106/mL in pre-cold FACS buffer. 
	NOTE: To prevent the sorter from clogging, pass the cells through a 70 &#181;m strainer.</li>
	<li>Add 0.1 &#181;g/mL of DAPI. 
	NOTE: DAPI is used for excluding the dead cells.</li>
	<li>Prepare 15 mL centrifuge tubes containing 4 mL of Complete Medium (prepared in step 2.5) for collecting the sorted cells.</li>
	<li>Load the cells onto the sorter. Sort single viable na&#970;ve CD4+ T cells (DAPI- CD4+ CD25- CD62L+ cells) in purity mode (Nozzle size: 70 &#181;m; Pressure: 70 PSI; Event rate: 8000-12000 events/s; Efficiency: higher than 90%) (Figure 2). 
	NOTE: Na&#970;ve CD4+ T cells express high expression of CD62L and lack the activation marker CD2513,14.</li>
	<li>Centrifuge the cell suspension at 350 x g for 8 min at 4 &#176;C. Aspirate all the supernatants using a 3 mL Transfer Pipette.</li>
	<li>Resuspend the cells in 500 &#181;L of 1x PBS.</li>
	<li>Count cells following step 3.6 
	NOTE: ~5 &#215; 106 cells can be obtained from one donor mouse in this step.</li>
</ol>
5. Cell transfer into the recipient mice
<ol>
	<li>Resuspend the CBir1 TCR Tg na&#970;ve CD4+ T cells to the 5 x 106/mL concentration by adding 1x PBS.</li>
	<li>Warm the Rag-/- mice under a heat lamp (see Table of Materials) for 4 min, and restrain the mice using a mouse restrainer.</li>
	<li>Intravenously transfer 200 &#181;L of the cell suspension into the tail vein of Rag-/- mice using a 1 mL insulin syringe (27 G) (see Table of Materials). 
	NOTE: Cells from one donor mouse are enough to transfer to around five recipient mice.</li>
</ol>
6. Monitoring clinical signs during colitis progression
<ol>
	<li>Weigh the mice every week and increase the observation to twice a week once the mice start losing &#62;5% of original weights.</li>
	<li>Observe mice response/move when gently stimulated.</li>
	<li>Observe other clinical abnormalities. i.e., posture and stool consistency.</li>
</ol>
7. Colon collection and histopathological scoring
<ol>
	<li>Sacrifice the recipient mice by a cervical dislocation with CO2 at a time point of the weight loss &#62;20% of original weight or 6-weeks post cell transfer. Wet the mice with 70% ethanol.</li>
	<li>Perform a ~1 cm ventral midline skin incision, pull the skin away from the abdominal muscle tissue, make a ~3 cm incision in the abdominal muscle tissue, identify the cecum, and remove the entire colon with sterile scissors and forceps. Wet the colon with pre-cold PBS in a culture dish.</li>
	<li>Incise the colon lengthwise and rinse it with pre-cold PBS. Cut 1/3 of the colon longitudinally.</li>
	<li>Place the colon strip in a paper towel with the luminal side facing upward. Perform Swiss rolling using a toothpick15.</li>
	<li>Place the colon Swiss into a cassette and put the cassette in 10% buffered formalin for 24 h16, followed by dehydration&#160;using an automated processor (see Table of Materials) and paraffin embedding.</li>
	<li>Cut 5 &#181;m tissue sections on a microtome, mounted on slides, and perform the Hematoxylin and eosin (H&#38;E) stain17 (see Table of Materials).</li>
	<li>Determine the histopathological scores by combining the scores for each of the six parameters for a maximum of 12. Lamina propria inflammation (normal, 0; mild, 1; moderate, 2; severe, 3); goblet cell loss (normal, 0; mild, 1; moderate, 2; severe, 3); abnormal crypt (normal, 0; hyperplastic, 1; disorganization, 2; crypt loss, 3); crypt abscesses (absent, 0; present, 1); mucosal erosion and ulceration (normal, 0; mild, 1; moderate, 2; severe, 3); and submucosal change (none, 0; submucosa, 1; transmural, 2)18.</li>
</ol>
8. Isolation and staining of intestinal lamina propria cells
<ol>
	<li>After step 7.3, cut another 2/3 of the colon into 0.5-1 cm pieces and wash it with pre-cold PBS.</li>
	<li>Transfer the colon segments into 20 mL pre-warmed EDTA-PBS buffer in a 50 mL centrifuge tube. Incubate at 37 &#176;C with 250 rpm shaking for 30 min.</li>
	<li>Vortex the tube, discard the supernatants by passing it through a sterile sieve (diameter: 0.01 inches), and resuspend the colon segments in 20 mL of pre-cold PBS in the 50 mL tube.</li>
	<li>Repeat step 8.3 twice.</li>
	<li>Place the colon segments in a C tube (see Table of Materials) containing 10 mL of pre-warmed Digestion Buffer.</li>
	<li>Place the tube on a Dissociator machine (see Table of Materials) and incubate under the program of &#34;37C_m_LPDK_1&#34; for 25 min. 
	NOTE: &#34;37C_m_LPDK_1&#34; is a standard preset program in the Dissociator machine used for stirring the samples and keeping them at 37 &#176;C.</li>
	<li>Check if tissue is digested completely, which means that no piece of tissue is in the Digestion buffer. If not, repeat the program of &#34;37C_m_LPDK_1&#34;.</li>
	<li>Collect the supernatant by passing through a metal sieve and 100 &#181;L strainer. Rinse with 10 mL of pre-cold PBS.</li>
	<li>Centrifuge the cell suspension at 350 x g for 8 min at 4 &#176;C. Aspirate all the supernatants.</li>
	<li>Resuspend the cells in 4 mL of 40% Percoll Solution and mix it thoroughly (prepared in step 2.8).</li>
	<li>Transfer the resuspended cells to 2 mL of 75% Percoll solution in a 15 mL centrifuge tube.</li>
	<li>Centrifuge the cell suspension at 850 x g for 20 min at 20 &#176;C (Acceleration ramp: 0; Brake ramp: 0).</li>
	<li>Carefully remove fat on the top layer using a 3 mL Transfer Pipette and transfer the cell layer to 20 mL of Washing Buffer in a 50 mL tube.</li>
	<li>Centrifuge the cell suspension at 350 x g for 8 min at 4 &#176;C. Discard all the supernatants and resuspend cells in 1 mL of Completed Medium.</li>
	<li>Count the cells following step 3.6.</li>
	<li>Seed the cells in a 24-well plate, activate them with 50 ng/mL of Phorbol-12-myristate 13-acetate and 750 ng/mL of ionomycin for 2 h, followed by incubation with 5 &#181;g/mL of Brefeldin A for 3 h (see Table of Materials). 
	NOTE: The reagents used are toxic, and their handling needs precautions and safety measures.</li>
	<li>Transfer the cells into a FACS tube, add 2 mL of FACS Buffer, and centrifuge the cells at 350 x g for 8 min at 4 &#176;C. Discard the supernatant.</li>
	<li>Incubate the cells with 12.5 &#181;g/mL of anti-mouse CD16/3219 in (see Table of Materials) FACS buffer to block Fc receptors for 5 min at room temperature.</li>
	<li>Stain for live/dead and surface marker.
	<ol>
		<li>Wash the cells with 2 mL of FACS Buffer and centrifuge them at 350 x g for 8 min at 4 &#176;C. Discard the supernatant.</li>
		<li>Stain the cells with live dye and surface markers (i.e.,anti-mouse CD3 and anti-mouse CD4 antibodies)20 (see Table of Materials) in FACS Buffer at the optimized concentration for 30 min at 4 &#176;C in the dark. 
		NOTE: The reagents used are toxic, and their handling needs precautions and safety measures.</li>
	</ol>
	</li>
	<li>Perform the cellular and nuclear staining.
	<ol>
		<li>Add 2 mL of FACS Buffer and centrifuge the cell suspension at 350 x g for 8 min at 4 &#176;C. Discard the supernatant.</li>
		<li>Permeabilize and fix the cells by resuspending the cells with 200 &#181;L of Transcription Factor Fix working solution (see Table of Materials) for 40 min at room temperature.</li>
		<li>Add 2 mL of Perm buffer and centrifuge the cell suspension at 350 x g for 8 min at 4 &#176;C. Discard the supernatant.</li>
		<li>Incubate the cells with cellular and nuclear markers (i.e., anti-mouse IFN&#947;, anti-mouse IL-17A, and anti-mouse Foxp3 antibodies)20 in Perm buffer (see Table of Materials) for 30 min-1 h at room temperature.</li>
		<li>Add 2 mL of Perm buffer and centrifuge the cell suspension at 350 x g for 8 min at 4 &#176;C. Discard the supernatant.</li>
		<li>Add 1 mL of FACS Buffer and centrifuge the cell suspension at 350 x g for 8 min at 4 &#176;C. Discard the supernatant.</li>
	</ol>
	</li>
	<li>Resuspend cells with 200 &#181;L of FACS Buffer and run the samples on a flow cytometer (see Table of Materials).</li>
</ol>

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Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+murine+intestinal+inflammation+by+adoptive+transfer+of+effector+CD4++CD45RB+high+T+cells+into+immunodeficient+mice.">Induction of murine intestinal inflammation by adoptive transfer of effector CD4+ CD45RB high T cells into immunodeficient mice.</a> Journal of Visualized Experiments. (98), e52533 (2015).</li><li>Atale, N., Gupta, S., Yadav, U. C., Rani, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-death+assessment+by+fluorescent+and+nonfluorescent+cytosolic+and+nuclear+staining+techniques.">Cell-death assessment by fluorescent and nonfluorescent cytosolic and nuclear staining techniques.</a> Journal of Microscopy. 255 (1), 7-19 (2014).</li><li>Manichanh, C., Borruel, N., Casellas, F., Guarner, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+gut+microbiota+in+IBD.">The gut microbiota in IBD.</a> Nature Reviews Gastroenterology &amp; Hepatology. 9 (10), 599-608 (2012).</li><li>Sun, 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=Microbiota-derived+short-chain+fatty+acids+promote+Th1+cell+IL-10+production+to+maintain+intestinal+homeostasis.">Microbiota-derived short-chain fatty acids promote Th1 cell IL-10 production to maintain intestinal homeostasis.</a> Nature Communications. 9 (1), 3555 (2018).</li><li>Feng, 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=Th17+cells+induce+colitis+and+promote+Th1+cell+responses+through+IL-17+induction+of+innate+IL-12+and+IL-23+production.">Th17 cells induce colitis and promote Th1 cell responses through IL-17 induction of innate IL-12 and IL-23 production.</a> Journal of Immunology. 186 (11), 6313-6318 (2011).</li><li>Chiaranunt, P., Tometich, J. T., Ji, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> T Cell Proliferation and Colitis Are Initiated by Defined Intestinal Microbes. 201 (1), 243-250 (2018).</li><li>Feng, T., Cao, A. T., Weaver, C. T., Elson, C. O., Cong, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-12+converts+Foxp3++regulatory+T+cells+to+interferon-&#947;-producing+Foxp3++T+cells+that+inhibit+colitis.">Interleukin-12 converts Foxp3+ regulatory T cells to interferon-&#947;-producing Foxp3+ T cells that inhibit colitis.</a> Gastroenterology. 140 (7), 2031-2043 (2011).</li></ol>

No authors have conflicting financial, professional, or personal interests.

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 activation markers of T cells13,14. In this study, we first used anti-CD4 magnetic beads to isolate CD4+ T cells from spleens. Then we used the combination of anti-CD4, anti-CD62L, and anti-CD25 antibodies for isolation of na&#239;ve CD4+ T cells13,14. Researchers could use other markers for sorting the na&#239;ve CD4+ T cells. CD45RBhi is also marker of na&#239;ve T cells. CD45RBhi CD25- CD4+ T cells are commonly used as na&#239;ve T cells of wild-type non-TCR transgenic T cells23. Therefore, the techniques of sorting cells and intravenously injection of the T cells into the recipient mice are essential. Setting up the gating protocol as a template is helpful to speed up the experiments with fewer errors. To avoid cell death, cells should always be kept on ice. Besides, staining cells with DAPI is highly recommended for excluding dead cells because DAPI cannot transit across intact cell membranes, making it an excellent dead cell probe24. Warming the mice to stimulate dilation of the tail veins provides better vein visibility for intravenous injection. All procedures are recommended to be performed by trained researchers.
Many factors might impact the outcome of the colitis models, which needs to be paid attention. First, the recipient Rag1-/- mice should be age and gender-matched. In the CD45RBhi T cell transfer model, T cells from male and female donors can be transferred to male Rag-/- recipients, while only female donors can be used when using female recipients23. However, we do not see a significant difference between male and female recipients. The recipient mice could be either females or males, and T cells from male donors can also induce colitis in female recipients. Since weight change is a valuable indicator of colitis progression, it is recommended to use the recipient mice between 8-12 weeks to present a stable weight line. These recipient mice should be bred and kept in the same room of the animal facility because microbiota is critical in regulating colitis development25. The time to develop colitis varies when transferring different numbers of CBir1 TCR Tg na&#239;ve CD4 T cells. As expected, fewer cells require a longer time for induction of colitis, and a higher number of cells require a shorter time for induction of colitis. Using 1 x 106 cells per recipient mouse is recommended since the recipient mice demonstrate clinical signs of colitis ~2-3 weeks post cell transfer and develop relatively severe colitis ~6 weeks post cell transfer. In addition, compared with intraperitoneal injection, intravenous injection of cells into the tail vein induces more consistent colitis. For isolation of colonic lamina propria cells, the colon tissues must not get dry; otherwise, it would reduce cells&#39; yield and viability. One of the particular concerns is that the duration of the colitis would be changed if the recipient mice are transferred with genetic-modified CBir1 TCR Tg T cells or treated with drugs26. In addition, CBir1 TCR Tg T cells also induce intestinal inflammation in other immune-deficient mice, such as TCR&#946;/&#948;-/- mice, which lack T cells27.
As accumulating evidence indicates a crucial role of gut bacterial antigen-specific reactive T cells in the pathogenesis of IBD, using T cells specific for a defined gut bacterial antigen will provide insights into how gut bacterial antigen induce T cell responses to induce colitis. Gut microbiota antigen CBir1 flagellin is abundant in the gastrointestinal tract, which is related to the pathogenesis of IBD8,9. This colitis model resembles several critical characteristics of IBD, including diarrhea, weight loss, histopathological finding, and abnormal intestinal immune responses. Therefore, this colitis model is useful to study the mechanisms of human IBD and provides a tool to evaluate the treatments for IBD. Interestingly, the recent work of Chiaranunt et al. indicated that T cell specificity to the microbiota CBir 1 antigen alone might not be sufficient to induce T cell activation and colitis. This is evidenced by the wild-type CBir1 TCR Tg T cells induced colitis in Rag-/- recipients, whereas Rag-/- CBir1 T cells did not induce colitis in their animal facility, suggesting that gut T cells responding to specific gut bacterial antigen may require other interrelated commensal bacteria, for example, Helicobacter spp, to function as an adjuvant28. An exciting aspect of this model is that different T cell subsets, namely Th1, Th17, and Treg cells, are present in lamina propria of colitic recipient mice, which provides a unique opportunity for investigating the roles of not only effector T cells but also Tregs in the pathogenesis of colitis29.
However, as this colitis model is mediated by gut microbiota-specific T cells, one limitation for this colitis model is that the duration of induction of colitis may vary in different animal facilities depending on gut microbiota.

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.

<table><tbody><tr><td>0.22 &amp;micro;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-&amp;micro;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&amp;gamma;-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&amp;amp;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&amp;amp;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>NH&lt;sub&gt;4&lt;/sub&gt;Cl</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 weight reached around 80%-85% of original weight six weeks post cell transfer (Figure 3). Additionally, mice showed diarrhea around 3-4 weeks post cell transfer and demonstrated hunched posture when they developed severe colitis. Gross morphology of the colon was shown, and the colitis severity was assessed by the histopathological score when mice were sacrificed. Mice receiving CBir1 TCR Tg na&#970;ve CD4+ T cells showed short colon length 6 weeks post cell transfer (Figure 4A). The recipient mice demonstrated more cell infiltration in the intestinal lamina propria 4 weeks post cell transfer (Figure 4C), goblet cell loss and intestinal epithelial cell hyperplasia 5 weeks post cell transfer (Figure 4D), and mucosal erosion and inflammatory cell infiltration in the submucosa of the colon 6 weeks post cell transfer (Figure 4F). At the same time, there was no inflammation in Rag1-/- mice receiving PBS alone (Figure 4B). Besides, there is no inflammation in the small intestine, but the cecum has inflammation. In addition, CD4+ T cell phenotypes in colonic lamina propria were determined by flow cytometry. The gating strategy is shown (Figure 5A-E). CBir1 TCR Tg na&#970;ve CD4+ T cells developed into IFN&#947;+ Th1 cells, IL-17A+ Th17 cells, IFN&#947;+ IL-17+ CD4+ T cells (Figure 5F), and Foxp3+ Treg cells in intestinal lamina propria of Rag1-/- recipients (Figure 5G).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63293/63293fig01.jpg" /> 
Figure 1: The procedure of induction colitis and assessment of disease severity. Splenic CD4+ T cells were isolated from CBir1 TCR transgenic mice using magnetic beads, and then na&#970;ve T cells were purified by sorting. CBir1 TCR transgenic na&#970;ve T cells were then intravenously transferred into recipient Rag1-/- mice. When the mice were sacrificed around six weeks post cell transfer, colitis severity was assessed by histopathological scores. The CD4+ T cell phenotypes in colonic lamina propria were determined by flow cytometry. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63293/63293fig01large.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/63293/63293fig02.jpg" /> 
Figure 2: The gating strategy for sorting CBir1 TCR transgenic na&#970;ve T cells. Viable single CBir1 TCR transgenic na&#970;ve T cells were purified by excluding debris (A), non-single cells (B-C), dead cells (D), and activated cells (E-F). The subpopulation was shown in (G). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63293/63293fig02large.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/63293/63293fig03.jpg" /> 
Figure 3: The weight changes of Rag1-/- mice post T cell transfer. 1 x 106 CBir1 TCR transgenic na&#970;ve T cells were transferred into Rag1-/- mice, and Rag1-/- mice receiving PBS were used as controls. Mouse weights were recorded every week. Data were presented in mean &#177; SD; Student&#39;s t-test; ***p&#60;0.001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63293/63293fig03large.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/63293/63293fig04.jpg" /> 
Figure 4: The gross morphology and Hematoxylin and eosin staining of the colon from Rag1-/- mice receiving CBir1 TCR transgenic na&#970;ve T cells. (A) The recipient mice were sacrificed six weeks post cell transfer, and the gross morphology of the colon was shown. (B-E) The recipient mice were sacrificed at different time points, and Rag1-/- mice received PBS as controls. The colons were processed for Hematoxylin and eosin staining. Representative images of Hematoxylin and eosin staining of the colon are shown. Scale bar = 200 &#181;m. (F) Histopathological scores were determined. Data were presented in mean &#177; SD. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63293/63293fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63293/63293fig05.jpg" /> 
Figure 5: The CD4+ T cell phenotypes in colonic lamina propria of Rag-/- mice receiving CBir1 TCR transgenic na&#970;ve T cells. When the recipient mice were sacrificed 6 weeks post cell transfer, colonic lamina propria cells were isolated for staining CD4+ T cell phenotypes. (A-E) The gating strategy for analysis of T cell phenotypes. (F-G) (F) IL-17A+ CD4+ T cells, IFN&#947;+ CD4+ T cells, IL-17+ IFN&#947;+ CD4+ T cells, and (G) Foxp3+ Tregs were determined by flow cytometry. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63293/63293fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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 ...

induction-intestinal-inflammation-adoptive-transfer-cbir1-tcr

Nanyang Technological University

Research

JoVE Journal

Medicine

2.5K Views.  University of Texas Medical Branch. Inflammatoire darmziekte, of IBD, legt een aanzienlijke gezondheids- en financiële last op individuen en de samenleving. Dit colitismodel is nuttig om de mechanismen van IBD te bestuderen en de behandelingen voor IBD te evalueren. Dit immunodominante Cbir flagellin T-cel-specifieke adoptieve transfer colitis model geeft inzicht in hoe darmbacteriën antigeen T-cel reacties induceert om colitis te induceren.Maak na het euthanaseren van de muis een linker buikincisie van ongeveer een ce...

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

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Immunology and Infection

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.

We provide a reproducible method to induce type 1 diabetes (T1D) in mice within two weeks by the adoptive transfer of islet antigen-specific, primary CD4+ T cells.

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-&#947;)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.

An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy ...

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 an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...

Induction of Intestinal Graft-versus-host Disease and Its Mini-endoscopic Assessment in Live Mice

Acute graft-versus-host disease (GvHD) represents the most severe complication that patients previously undergoing allogeneic hematopoietic stem cell transplantation (allo-HCT) face and is frequently associated with a poor clinical outcome. While, for instance, GvHD manifestations of the skin are usually responsive to established immune-suppressive therapies and are, hence, not taking a fatal course, the presence and the intensity of intestinal GvHD, especially of the mid-to-lower parts of the gut, strongly influence the outcome and overall survival of patients with acute GvHD. Therapeutic options are essentially limited to the classic immune-suppressive agents yielding only moderate disease-mitigating effects. Hence, detailed knowledge about the tissue-resident immune cascade, changes in the intestinal microbiota, and the stromal response prior, upon, and after intestinal GvHD onset are urgently needed to understand the events and mechanisms underlying its pathogenesis and to develop innovative therapeutic options. Murine models of GvHD are frequently employed to identify and functionally assess molecules and pathways putatively driving intestinal GvHD. However, means to specifically monitor and evaluate intestinal inflammation over time are essentially lacking since established scores to assess and grade acute GvHD are routinely comprised of various parameters which rather reflect&#160;systemic GvHD manifestations. The detailed evaluation of intestinal GvHD has been restricted to studies using euthanized mice, thereby essentially excluding longitudinal (i.e., kinetic) analyses of the colonic compartment under a given experimental condition (e.g., antibody-mediated blockade of a proinflammatory cytokine) in live mice (i.e., in vivo). The mini-endoscopic in situ assessment of the distal colon of allo-HCT-treated mice described here allows a) a detailed macroscopic evaluation of different aspects of intestinal inflammation and b) the option to collect tissue samples for downstream analyses at various time points over the course of the observation period. Overall, the mini-endoscopic approach provides a major advance in preclinical noninvasive monitoring and assessment of intestinal GvHD.

Here, we present a protocol that describes allogeneic hematopoietic stem cell transplantation and allows repetitive mini-endoscopic evaluations of the distal colon in situ for the presence, characteristics, and severity of colonic inflammation within live mice suffering from intestinal graft-versus-host disease.

Acute graft-versus-host disease (GvHD) represents the most severe complication that patients previously undergoing allogeneic hematopoietic stem cell ...

Mechanistic Insight into the Development of TNBS-Mediated Intestinal Fibrosis and Evaluating the Inhibitory Effects of Rapamycin

Significant studies have been carried out to understand effective management of intestinal fibrosis. However, the lack of better knowledge of fibrosis has hindered the development of a preventative drug. Primarily, finding a suitable animal model is challenging in understanding the mechanism of Crohn&#39;s-associated intestinal fibrosis pathology. Here, we adopted an effective method where TNBS chemical exposure to mice rectums produces substantially deep ulceration and chronic inflammation, and the mice then chronically develop intestinal fibrosis. Also, we describe a technique where a rapamycin injection shows inhibitory effects on TNBS-mediated fibrosis in the mouse model. To assess the underlying mechanism of fibrosis, we methodically discuss a procedure for purifying Cx3Cr1+ cells from the lamina propria of TNBS-treated and control mice. This detailed protocol will be helpful to researchers who are investigating the mechanism of fibrosis and pave the path to find a better therapeutic invention for Crohn&#39;s-associated intestinal fibrosis.

In this study, we describe a detailed procedure of TNBS-mediated intestinal fibrosis, which exhibits comparable pathophysiology to Crohn&#39;s fibrosis. We also discuss this approach in light of rapamycin facilitated inhibitory effects on intestinal fibrosis.

Significant studies have been carried out to understand effective management of intestinal fibrosis. However, the lack of better knowledge of fibrosis has ...

Chronic Salmonella Infection Induced Intestinal Fibrosis

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and dysregulated repair. In inflammatory bowel disease (IBD), fibrosis leads to recurrent stricture formations for which there is no effective therapy other than surgical resection. Due to its late onset, the processes that drive fibrosis is less studied and largely unknown. Therefore, fibrotic complications represent a major challenge in IBD. In this protocol, a robust in vivo model of intestinal fibrosis is described where streptomycin pre-treatment of C57Bl/6 mice followed by oral gavage with vaccine grade Salmonella Typhimurium &#916;AroA mutant leads to persistent pathogen colonization and fibrosis of the cecum. Methodologies for preparing S. Typhimurium &#916;AroA for inoculation, quantifying pathogen loads in the cecum and spleen, and evaluating collagen deposition in intestinal tissues are explained. This experimental disease model is useful for examining host factors that either enhance or exacerbate CD-like intestinal fibrosis.

This protocol describes a mouse model of Salmonella driven intestinal fibrosis that resembles key pathological hallmarks of Crohn&#8217;s disease including transmural inflammation and fibrosis. This method can be used to evaluate host factors that alter fibrotic outcomes using mutant mice maintained on a C57Bl/6 genetic background.

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and ...

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.

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

Investigating Target Gene Function in a CD40 Agonistic Antibody-induced Colitis Model using CRISPR/Cas9-based Technologies

The immune system functions to defend humans against foreign invaders such as bacteria and viruses. However, disorders of the immune system may lead to autoimmunity, inflammatory disease, and cancer. The inflammatory bowel diseases (IBD)-Crohn's disease (CD) and ulcerative colitis (UC)-are chronic diseases marked by relapsing intestinal inflammation. Although IBD is most prevalent in Western countries (1 in 1,000), incident rates are increasing around the world. Through association studies, researchers have linked hundreds of genes to the pathology of IBD. However, the elaborate pathology behind IBD and the high number of potential genes pose significant challenges in finding the best therapeutic targets. Additionally, the tools needed to functionally characterize each genetic association introduce many rate-limiting factors such as the generation of genetically modified mice for each gene. To investigate the therapeutic potential of target genes, a model system has been developed using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease (Cas9)-based technologies and a cluster of differentiation 40 (CD40) agonistic antibody. The present study shows that CRISPR/Cas9-mediated editing in the immune system can be used to investigate the impact of genes in vivo. Limited to the hematopoietic compartment, this approach reliably edits the resulting reconstituted immune system. CRISPR/Cas9-edited mice are generated faster and are far less expensive than traditional genetically modified mice. Furthermore, CRISPR/Cas9 editing of mice has significant scientific advantages compared to generating and breeding genetically modified mice such as the ability to evaluate targets that are embryonic lethal. Using CD40 as a model target in the CD40 agonistic antibody-induced colitis model, this study demonstrates the feasibility of this approach.

Here, we describe the methodology to knock out a gene of interest in the immune system using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease (Cas9)-based technologies and the evaluation of these mice in a cluster of differentiation 40 (CD40) agonistic antibody-induced colitis model.

The immune system functions to defend humans against foreign invaders such as bacteria and viruses. However, disorders of the immune system may lead to ...

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

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