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

Inflammatory bowel diseases (IBD), mainly including Crohn'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 inflammation^5,6,7. As an immunodominant gut microbiota antigen, CBir1 flagellin has been related to the pathogenesis of human CD^8,9. 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 naϊve 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.

All animal procedures were performed according to the University of Texas Medical Branch'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

1. 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.
2. Mark the mice by ear punch.
3. Weigh the mice on the same day of T cell transfer.

2. Preparation of the reagents and solutions

NOTE: The reagents used are toxic, or biohazard and their handling need precautions and safety measures.

1. 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 °C.
2. Prepare Tris-NH₄Cl Lysis Buffer.
   1. 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.
   2. Prepare Solution Part B. Dissolve 7.47 g of NH₄Cl in 800 mL of ddH₂O.
   3. Mix A and B thoroughly. Measure the pH and adjust it to 7.2 if not.
   4. Adjust the total volume to 1000 mL. Autoclave and then store it at 4 °C.
3. Prepare the Isolation Buffer.
   1. Add 2.5 g of BSA and 500 µL of EDTA (0.5 M, pH 8.0) into 500 mL of 1x PBS. Mix it thoroughly.
   2. Filter the solution through a 0.22 µm vacuum-driven disposable bottle top filter (see Table of Materials). Store it at 4 °C.
4. Prepare FACS Buffer. Add 1 mL of FBS and 50 µL 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 °C.
5. Prepare Complete Medium. Add 5 mL of FBS in 45 mL of Washing Buffer. Mix thoroughly and store it at 4 °C.
6. Prepare EDTA-PBS Buffer.
   1. Calculate the volume of the EDTA-PBS Buffer needed. Volume (mL) = mouse number x 20.
   2. 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).
   3. Mix it thoroughly and pre-warm in a 37 °C water bath.
7. Prepare the Digestion Buffer.
   1. Calculate the volume of the Digestion Buffer needed. Volume (mL) = mouse number x 10.
   2. 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).
   3. Mix it thoroughly and pre-warm in a 37 °C water bath.
8. Prepare Percoll Solution.
   1. Prepare 100% Percoll. Add 5 mL of 10x PBS in 45 mL of original Percoll (see Table of Materials).
   2. Prepare 2% FBS in Washing Buffer. Add 1 mL of FBS in 49 mL of Washing Buffer.
   3. 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.

3. Isolation of splenic CBir1 TCR Tg CD4⁺ T cells

1. 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.
2. 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).
3. 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 µm 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.
4. Centrifuge the cell suspension at 350 x g for 8 min at 4 °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.
5. Centrifuge the cell suspension at 350 x g for 8 min at 4 °C. Discard the supernatant and resuspend the cells with 10 mL of pre-cold Isolation Buffer (prepared in step 2.3).
6. Count the cells. Mix 10 µL of cell suspension with 10 µL of trypan blue thoroughly. Load 10 µL 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.
7. Centrifuge the remaining cell suspension from step 3.6 at 350 x g for 8 min at 4 °C. Discard all the supernatants.
8. Vortex the anti-mouse CD4 Magnetic Particles thoroughly (see Table of Materials), directly add 50 µL of the particles per 10⁷ cells and mix with cell pellets thoroughly. Incubate for 30 min at 4 °C.
   NOTE: Any other commercial CD4⁺ T cell enrichment kit can be used here.
9. Transfer the cell-particle suspension into a sterile collection tube. Add 3.5 mL of pre-cold Isolation Buffer into the tube.
10. 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.
11. 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.
12. Repeat step 3.11.
13. Resuspend the cells with 1 mL of pre-cold FACS Buffer (prepared in step 2.4).

4. Purification of CBir1 TCR Tg naϊve CD4⁺ T cells

1. Count the cells following step 3.6.
2. If the cell concentration is >10⁷/mL, add a volume of FACS buffer to make sure the cell concentration is ≤ 10⁷/mL.
   NOTE: ~1 × 10⁷ cells can be obtained from one donor mouse in this step.
3. Stain the surface markers with 10 µL of anti-mouse CD4-APC, 10 µL of anti-mouse CD25-Percp/Cy5.5, and 10 µL of anti-mouse CD62L-PE^13,14 (see Table of Materials). Mix gently and incubate for 30 min at 4 °C in the dark.
4. Wash the cells with 2 mL of pre-cold FACS buffer. Centrifuge the cell suspension at 350 x g for 8 min at 4 °C. Aspirate all the supernatantsusing a 3 mL Transfer Pipette.
5. Repeat Step 4.4.
6. 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 µm strainer.
7. Add 0.1 µg/mL of DAPI.
   NOTE: DAPI is used for excluding the dead cells.
8. Prepare 15 mL centrifuge tubes containing 4 mL of Complete Medium (prepared in step 2.5) for collecting the sorted cells.
9. Load the cells onto the sorter. Sort single viable naϊve CD4⁺ T cells (DAPI^- CD4⁺ CD25^- CD62L⁺ cells) in purity mode (Nozzle size: 70 µm; Pressure: 70 PSI; Event rate: 8000-12000 events/s; Efficiency: higher than 90%) (Figure 2).
   NOTE: Naϊve CD4⁺ T cells express high expression of CD62L and lack the activation marker CD25^13,14.
10. Centrifuge the cell suspension at 350 x g for 8 min at 4 °C. Aspirate all the supernatants using a 3 mL Transfer Pipette.
11. Resuspend the cells in 500 µL of 1x PBS.
12. Count cells following step 3.6
    NOTE: ~5 × 10⁶ cells can be obtained from one donor mouse in this step.

5. Cell transfer into the recipient mice

1. Resuspend the CBir1 TCR Tg naϊve CD4⁺ T cells to the 5 x 10⁶/mL concentration by adding 1x PBS.
2. Warm the Rag^-/- mice under a heat lamp (see Table of Materials) for 4 min, and restrain the mice using a mouse restrainer.
3. Intravenously transfer 200 µL 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.

6. Monitoring clinical signs during colitis progression

1. Weigh the mice every week and increase the observation to twice a week once the mice start losing >5% of original weights.
2. Observe mice response/move when gently stimulated.
3. Observe other clinical abnormalities. i.e., posture and stool consistency.

7. Colon collection and histopathological scoring

1. Sacrifice the recipient mice by a cervical dislocation with CO₂ at a time point of the weight loss >20% of original weight or 6-weeks post cell transfer. Wet the mice with 70% ethanol.
2. 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.
3. Incise the colon lengthwise and rinse it with pre-cold PBS. Cut 1/3 of the colon longitudinally.
4. Place the colon strip in a paper towel with the luminal side facing upward. Perform Swiss rolling using a toothpick¹⁵.
5. Place the colon Swiss into a cassette and put the cassette in 10% buffered formalin for 24 h¹⁶, followed by dehydration using an automated processor (see Table of Materials) and paraffin embedding.
6. Cut 5 µm tissue sections on a microtome, mounted on slides, and perform the Hematoxylin and eosin (H&E) stain¹⁷ (see Table of Materials).
7. 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.

8. Isolation and staining of intestinal lamina propria cells

1. After step 7.3, cut another 2/3 of the colon into 0.5-1 cm pieces and wash it with pre-cold PBS.
2. Transfer the colon segments into 20 mL pre-warmed EDTA-PBS buffer in a 50 mL centrifuge tube. Incubate at 37 °C with 250 rpm shaking for 30 min.
3. 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.
4. Repeat step 8.3 twice.
5. Place the colon segments in a C tube (see Table of Materials) containing 10 mL of pre-warmed Digestion Buffer.
6. Place the tube on a Dissociator machine (see Table of Materials) and incubate under the program of "37C_m_LPDK_1" for 25 min.
   NOTE: "37C_m_LPDK_1" is a standard preset program in the Dissociator machine used for stirring the samples and keeping them at 37 °C.
7. Check if tissue is digested completely, which means that no piece of tissue is in the Digestion buffer. If not, repeat the program of "37C_m_LPDK_1".
8. Collect the supernatant by passing through a metal sieve and 100 µL strainer. Rinse with 10 mL of pre-cold PBS.
9. Centrifuge the cell suspension at 350 x g for 8 min at 4 °C. Aspirate all the supernatants.
10. Resuspend the cells in 4 mL of 40% Percoll Solution and mix it thoroughly (prepared in step 2.8).
11. Transfer the resuspended cells to 2 mL of 75% Percoll solution in a 15 mL centrifuge tube.
12. Centrifuge the cell suspension at 850 x g for 20 min at 20 °C (Acceleration ramp: 0; Brake ramp: 0).
13. 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.
14. Centrifuge the cell suspension at 350 x g for 8 min at 4 °C. Discard all the supernatants and resuspend cells in 1 mL of Completed Medium.
15. Count the cells following step 3.6.
16. 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 µg/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.
17. 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 °C. Discard the supernatant.
18. Incubate the cells with 12.5 µg/mL of anti-mouse CD16/32¹⁹ in (see Table of Materials) FACS buffer to block Fc receptors for 5 min at room temperature.
19. Stain for live/dead and surface marker.
    1. Wash the cells with 2 mL of FACS Buffer and centrifuge them at 350 x g for 8 min at 4 °C. Discard the supernatant.
    2. 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 °C in the dark.
       NOTE: The reagents used are toxic, and their handling needs precautions and safety measures.
20. Perform the cellular and nuclear staining.
    1. Add 2 mL of FACS Buffer and centrifuge the cell suspension at 350 x g for 8 min at 4 °C. Discard the supernatant.
    2. Permeabilize and fix the cells by resuspending the cells with 200 µL of Transcription Factor Fix working solution (see Table of Materials) for 40 min at room temperature.
    3. Add 2 mL of Perm buffer and centrifuge the cell suspension at 350 x g for 8 min at 4 °C. Discard the supernatant.
    4. Incubate the cells with cellular and nuclear markers (i.e., anti-mouse IFNγ, 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.
    5. Add 2 mL of Perm buffer and centrifuge the cell suspension at 350 x g for 8 min at 4 °C. Discard the supernatant.
    6. Add 1 mL of FACS Buffer and centrifuge the cell suspension at 350 x g for 8 min at 4 °C. Discard the supernatant.
21. Resuspend cells with 200 µL of FACS Buffer and run the samples on a flow cytometer (see Table of Materials).

Approximately 5 x 10⁶ CBir1 TCR Tg naϊve CD4⁺ T cells per spleen were isolated from an adult CBir1 TCR Tg mouse. Transfer of CBir1 TCR Tg naϊve 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 naϊve 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 naϊve CD4⁺ T cells developed into IFNγ⁺ Th1 cells, IL-17A⁺ Th17 cells, IFNγ⁺ IL-17⁺ CD4⁺ T cells (Figure 5F), and Foxp3⁺ Treg cells in intestinal lamina propria of Rag1^-/- recipients (Figure 5G).

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 naϊve T cells were purified by sorting. CBir1 TCR transgenic naϊve 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. Please click here to view a larger version of this figure.

Figure 2: The gating strategy for sorting CBir1 TCR transgenic naϊve T cells. Viable single CBir1 TCR transgenic naϊve 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). Please click here to view a larger version of this figure.

Figure 3: The weight changes of Rag1^-/- mice post T cell transfer. 1 x 10⁶ CBir1 TCR transgenic naϊve 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 ± SD; Student's t-test; ***p<0.001. Please click here to view a larger version of this figure.

Figure 4: The gross morphology and Hematoxylin and eosin staining of the colon from Rag1^-/- mice receiving CBir1 TCR transgenic naϊve 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 µm. (F) Histopathological scores were determined. Data were presented in mean ± SD. Please click here to view a larger version of this figure.

Figure 5: The CD4+ T cell phenotypes in colonic lamina propria of Rag^-/- mice receiving CBir1 TCR transgenic naϊve 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, IFNγ⁺ CD4⁺ T cells, IL-17⁺ IFNγ⁺ CD4⁺ T cells, and (G) Foxp3⁺ Tregs were determined by flow cytometry. Please click here to view a larger version of this figure.

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 naϊve CD4⁺ T cells to induce intestinal inflammation. We used spleens for the isolation of naïve CD4⁺ T cells instead of MLNs. Because the yield of naïve CD4⁺ T cells in MLNs is much lower than in spleens. CD62L is highly expressed in naïve T cells, and CD44 and CD25 are the activation markers of T cells^13,14. 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 naïve CD4⁺ T cells^13,14. Researchers could use other markers for sorting the naïve CD4⁺ T cells. CD45RB^hi is also marker of naïve T cells. CD45RB^hi CD25^- CD4⁺ T cells are commonly used as naïve 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 CD45RB^hi 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 naïve 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' 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 TCRβ/δ^-/- 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 IBD^8,9. 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.