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

Podsumowanie

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.

Streszczenie

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's disease. CBir1 TCR transgenic naϊve 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.

Wprowadzenie

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.

Protokół

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).

Wyniki

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...

Dyskusje

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 activa...

Materiały

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