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

Summary

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.

Abstract

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.

Introduction

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 i....

Protocol

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 .......

Discussion

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.......

Materials

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