Supplementary Materials

Tissue adaptation of regulatory and intraepithelial CD4+ T cells controls gut inflammation

Tomohisa Sujino, Mariya London, David P. Hoytema van Konijnenburg, Tomiko Rendon, Thorsten Buch, Hernandez M. Silva, Juan J. Lafaille, Bernardo S. Reis, Daniel Mucida

Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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  • Materials and Methods
  • Figs. S1 to S4
  • Captions for Movies S1 to S3
  • Reference (35)

Images, Video, and Other Media

Movie S1
TCRγδIELs localize mainly to the intraepithelial compartment. Time-lapse video (Z-stack) of intravital microscopy imaging of ileum villi in a TCRγδGFP mouse. Nuclei of intestinal epithelial cells (IECs) were stained with Hoechst prior to imaging. TCRγδIELs are shown in the green (GFP) channel, IECs shown in the blue channel. Each frame is approximately at a 30 second interval. TCRγδIELs were identified using co-localization of appropriate channel intensities and tracked using Imaris (Bitplane, UK) software with auto-regressive tracking algorithms at default settings. Z-stack video is representative of ± 50 villi from 3 different movies.
Movie S2
Tregs localize in the lamina propria and frequently visit the epithelium. Time-lapse video (Z-stack) of in vivo intravital microscopy imaging of ileum villi in a iFoxp3Tomato mouse one day post tamoxifen administration. Nuclei of intestinal epithelial cells (IECs) were stained with Hoechst prior to imaging. Foxp3+ (Tregs) are shown in the red (Tomato) channel, IECs shown in the blue channel. Each frame is approximately at a 30 second interval. Tomato+ cells were identified using co-localization of appropriate channel intensities and tracked using Imaris (Bitplane, UK) software with auto-regressive tracking algorithms at default settings. Z-stack video representative of ± 20 villi from 6 different movies.
Movie S3
ThPOKhigh and ThPOKlow cells display different localization and migration dynamics. Time-lapse video (Z-stack) of in vivo intravital microscopy imaging of ileum villi containing ThPOK(GFP)high and ThPOK(GFP)low cells. Sorted naïve CD4+ T cells from OT-II (RFP ThpokGFP) mice were transferred to Rag1???/??? recipient mice. Recipient animals were fed OVA-containing diet for 7 days before intravital imaging. Nuclei of intestinal epithelial cells (IECs) were stained with Hoechst prior to imaging. All transferred cells are in red (RFP) channel, IECs in blue channel, and ThPOK(GFP)high cells in green channel. Part one shows the villus with blue (IECs) and green (GFPhigh cells) only. Part two shows blue, green and red channel overlay allowing simultaneous visualization of GFPhigh (in yellow) and GFPlow (in red) cells in the same villus. Each frame is approximately at a 30 second interval. Z-stack video is representative of ± 50 villi from 4 different movies.