The overall goal of this antigen-driven colitis model is to determine whether CX3, CR1 positive mononuclear phagocytes interact with and activate CD4 positive T cells during antigen-specific colitis and whether this mononuclear phagocyte-dependent effector T cell expansion occurs within the intestinal lamina propria. This method can help answer key questions in the inflammatory bowel disease field, such as what types of interaction occur within antigen presenting cells and the effector T cells in the colonic lamina propria. The main advantage of this technique is that it allows the study of immune cells'interaction and the elements that lead to the pathogencity of inflammatory bowel disease.
This method uses the T cell transfer colitis model, in which the red fluorescent antigen-specific naive CD4 T cells are transferred to the immunodeficient host, expressing the green fluorescent protein in mononuclear cells. Cell transfer is followed with the oral gavage of the blue fluorescent antigen-expressing bacteria. The immune events taking place in the intestinal lamina propria of the hosts can be monitored at various stages of disease development.
Use dissection scissors to open the colon longitudinally and wash the tissue in a petri dish containing 25 milliliters of PBS to remove any debris and mucus. Cut the colon into five to eight millimeter pieces and place the resulting fragments in 20 milliliters of fresh DPBS without calcium and magnesium, supplemented with ten millimolar HEPES and five millimolar EDTA. Vortex the tube with the sample for 30 seconds on maximum speed.
Then, incubate at 37 degrees Celsius for ten minutes with gentle shaking to remove the epithelium. At the end of the incubation, Vortex again for 30 seconds. Then, discard the supernatant containing the epithelial cells.
Transfer the tissue pieces into a new tube containing 20 milliliters of fresh PBS, HEPES, and EDTA buffer, and repeat the incubation with the vortexing steps before and after. After the third repetition, all the epithelial cells are removed and the supernatant is clear. Wash the tissue fragments gently in PBS to remove any residual epithelial cells.
Cut the tissue fragments into two by two millimeter pieces and transfer the pieces for digestion in ten millimeters of RPI medium, supplemented with 0.5 milligrams per milliliter of collagenase type eight and ten units per milliliter of DNAse one. Vortex the samples for 30 seconds. Then, incubate the tissue pieces at 37 degrees Celsius with shaking for 30 to 35 minutes and vortex the tube every five minutes during the incubation.
When the tissue is digested, strain the resulting slurry through a 70 micron cell strainer into a 50 milliliter conical tube. Wash the cell strainer once and spin down the single cell suspension by centrifugation. Finally, wash the cells once in PBS and centrifuge.
Then, determine the number of viable cells by trypan blue exclusion. Cyan fluorescent protein ovalbumin protein producing e. coli activate interferon gamma production in OT-II cells in vitro in contrast to e.
coli that express CFP only. Ex vivo 3D confocal imaging of the colonic tissue of transgenic green fluorescent protein expressing mice, fed with e. coli PCFP ova demonstrates the presence of the fluorescent protein-expressing bacteria within the colonic crypts near the intestinal epithelial cells and co-localized with the host intestinal phagocytes.
In OT-II red animals, the CD4 positive T cells are characterized by their co-expression of red fluorescent protein and V beta five point one. When challenged with e. coli PCFP ova, BNT lymphocyte-deficient green fluorescent protein-expressing transgenic mice that have been adoptively transplanted with OT-II red CD4 positive CD62 ligand positive T cells rapidly lose body weight and develop clinical signs of colitis.
Seven days after cell transfer, only a few host phagocytes have sampled e. coli PCFP ova and OT-II red positive cells are not detected. 14 days after cell transfer, host phagocytes that have sampled the e.
coli PCFP ova are located in close proximity to the adoptively transferred OT-II red positive cells. By 21 days after cell transfer, a high number of host cells have sampled the e. coli PCFP ova and donor OT-II red positive cells, which are found dispersed throughout the colonic lamina propria, are in close contact with the host intestinal phagocytes.
When mastered, this technique can be completed in four hours if it is performed properly. Following this procedure, other methods like staining of the intestinal lymphocytes and serum cytokine analysis can be performed to answer additional questions about the activation state of CD4 T cells and to confirm the severity of the colitis. After each development, this technique paves the way for research in the field of and immunology to explore the little events that lead to inflammatory bowel disease in the mouse model.
After watching this video, you should have a good understanding of how to isolate colonic lamina propria immune cells for the downstream ex vivo analysis.
