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.
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.
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 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 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
2. Preparation of the reagents and solutions
NOTE: The reagents used are toxic, or biohazard and their handling need precautions and safety measures.
3. Isolation of splenic CBir1 TCR Tg CD4+ T cells
4. Purification of CBir1 TCR Tg naϊve CD4+ T cells
5. Cell transfer into the recipient mice
6. Monitoring clinical signs during colitis progression
7. Colon collection and histopathological scoring
8. Isolation and staining of intestinal lamina propria cells
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 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 naïve CD4+ T cells13,14. Researchers could use other markers for sorting the naïve CD4+ T cells. CD45RBhi is also marker of naïve T cells. CD45RBhi 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 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 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 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.
The authors have nothing to disclose.
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.
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 |