Dynamic Imaging of IEL-IEC Co-Cultures Allows for Quantification of CD103-Dependent T Cell Migration

Abstract

Intraepithelial lymphocytes (IEL) are widely distributed within the small intestinal epithelial cell (IEC) layer and represent one of the largest T cell pools of the body. While implicated in the pathogenesis of intestinal inflammation, detailed insight especially into the cellular cross-talk between IELs and IECs is largely missing in part due to lacking methodologies to monitor this interaction. To overcome this shortcoming, we employed and validated a murine IEL-IEC (organoids) ex vivo co-culture model system. Using livecell imaging we established a protocol to visualize and quantify the spatio-temporal migratory behavior of IELs within organoids over time. Applying this methodology, we found that IELs lacking CD103 (i.e., integrin alpha E, ITGAE) surface expression usually functioning as a retention receptor for IELs through binding to E-cadherin (CD324) expressing IECs displayed aberrant mobility and migration patterns. Specifically, CD103 deficiency affected the ability of IELs to migrate and reduced their speed during crawling within organoids. In summary, we report a new technology to monitor and quantitatively assess especially migratory characteristics of IELs communicating with IEC ex vivo. This approach is hence readily applicable to study the effects of targeted therapeutic interventions on IEL-IEC cross-talk.

Autologous organoid co-culture model reveals T cell-driven epithelial cell death in Crohn’s Disease

“…However, a continuum and a largely shared TCR repertoire between T cells of the lamina propria and the epithelium has recently been demonstrated in both CD4 and CD8 T cells ( 40 ). Furthermore, the fixed view of the position of the cells given by anatomopathological studies of the digestive mucosa, is challenged by recent dynamic studies of lymphoepithelial interactions that highlight the continuous mobility of lymphocytes in contact with the epithelium and within the matrix ( 41 ). Finally, while phenotypic characteristics differentiate intra-epithelial and lamina propria lymphocytes, it is important to keep in mind the important plasticity of mucosal T cells and their capacity to acquire surface markers and functional specificities linked to the inflammatory and cytokine context ( 42 , 43 ).…”

High-throughput Raman spectroscopy allows ex vivo characterization of murine small intestinal intra-epithelial lymphocytes (IEL)

“…Flow cytometry analysis confirmed the expected constellation with T cells detectable within the SI IEL compartment of mice previously receiving T cells while missing in untreated Rag1 −/− mice. The finding that a substantial fraction of the isolated and flow cytometrically evaluated cell types is either of non-immune cell origin or not viable, reflects the fact that involved cell types from this specific site are sensitive to rapid cell death ex vivo 52 underscoring that the applied procedure is rather an IEL enrichment than IEL isolation step.…”

Novel T cell/organoid culture system allows ex vivo modeling of intestinal graft-versus-host disease

“…Live imaging of the co-culture was performed on d1 on a Zeiss Spinning Disc Axio Observer Z1 microscope at the Optical Imaging Centre Erlangen (OICE) as described before ( 25 ). In short, B6 (allogeneic) CD3+ IELs were stained with 1 μM Cell Proliferation Dye eFluor 670 (eBioscience) according to the manufacturer’s protocol on d0 and co-cultures of labeled IELs and previously grown B6 (syngeneic)- or Balb/c (allogeneic)-derived SI organoids were plated in a µ-Slide 4 Well chamber slide (ibidi).…”

“…However, groundbreaking work showing that small intestinal (SI) organoids closely mimicking small intestinal epithelial cell structures found in vivo can be generated and expanded ex vivo from Lgr5 + intestinal stem cells ( 18 ) has paved the way for novel methodological approaches to study IEL biology. So progress by our group and others ( 17 , 19 , 20 ) demonstrating that SI organoids offer an appropriate microenvironment to stably culture IELs ex vivo has opened up new avenues to analyze IEL biology under physiological conditions, i.e. within the intestinal epithelial layer.…”