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Summary: Plasma cells secreting affinity-matured antibodies develop in germinal centers (GCs), where B cells migrate persistently and directionally over defined periods of time. How modes of GC B cell migration influence plasma cell development remained unclear. Through genetic deletion of the F-actin bundling protein Swiprosin-1/EF-hand domain family member 2 (EFhd2) and by two-photon microscopy, we show that EFhd2 restrains B cell speed in GCs and hapten-specific plasma cell output. Modeling the GC reaction reveals that increasing GC B cell speed promotes plasma cell generation. Lack of EFhd2 also reduces contacts of GC B cells with follicular dendritic cells in vivo. Computational modeling uncovers that both GC output and antibody affinity depend quantitatively on contacts of GC B cells with follicular dendritic cells when B cells migrate more persistently. Collectively, our data explain how GC B cells integrate speed and persistence of cell migration with B cell receptor affinity.

The aberrant regulation of the epithelial barrier integrity is involved in many diseases of the digestive tract, including inflammatory bowel diseases and colorectal cancer. Intestinal epithelial cell organoid cultures provide new perspectives for analyses of the intestinal barrier in vitro. However, established methods of barrier function analyses from two dimensional cultures have to be adjusted to the analysis of three dimensional organoid structures. Here we describe the methodology for analysis of epithelial barrier function and molecular regulation in intestinal organoids. Barrier responses to interferon-γ of intestinal organoids with and without epithelial cell-specific deletion of the interferon-γ-receptor 2 gene were used as a model system. The established method allowed monitoring of the kinetics of interferon-γ-induced permeability changes in living organoids. Proteolytic degradation and altered localization of the tight junction proteins claudin-2, −7, and − 15 was detected using confocal spinning disc microscopy with 3D reconstruction. Hessian analysis was used for quantification of re-localization of claudins. In summary, we provide a novel methodologic approach for quantitative analyses of intestinal epithelial barrier functions in the 3D organoid model. Keywords: Inflammatory bowel disease, Interferon-gamma, Intestinal organoids, 3D imaging, Permeability, Tight junction

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.