Thymocyte trafficking has an important role in thymic selection. Here we show that the Hippo homologue mst1 is required for thymocyte migration and antigen recognition by LFA-1 and ICAm-1 within the medulla. using two-photon imaging of thymic tissues, we found that highly motile mature thymocytes arrest and are activated in the vicinity of rare populations of Aire + ICAm-1 hi medullary thymic epithelia in a negatively selecting environment. notably, mst1 deficiency or blocking the cell adhesion molecules LFA-1 and ICAm-1 results in inefficient migration and antigen recognition of CD4 + thymocytes within the medulla. Consistent with these defects, thymocyte selection is impaired in Mst1 − / − mice, which display T cell-dependent inflammatory infiltrates in multiple organs and develop autoantibodies. our results suggest that mst1 has a key role in regulating thymocyte self-antigen recognition in the medulla.

Antigen-specific adhesion between T cells and antigen-presenting cells (APC) during the formation of the immunological synapse (IS) is mediated by LFA-1 and ICAM-1. Here, LFA-1–ICAM-1 interactions were measured at the single-molecule level on supported lipid bilayers. High-affinity binding was detected at low frequencies in the inner peripheral supramolecular activation cluster (SMAC) zone that contained high levels of activated Rap1 and kindlin-3. Rap1 was essential for T cell attachment, whereas deficiencies of ste20-like kinases, Mst1/Mst2, diminished high-affinity binding and abrogated central SMAC (cSMAC) formation with mislocalized kindlin-3 and vesicle transport regulators involved in T cell receptor recycling/releasing machineries, resulting in impaired T cell-APC interactions. We found that NDR1 kinase, activated by the Rap1 signaling cascade through RAPL and Mst1/Mst2, associated with and recruited kindlin-3 to the IS, which was required for high-affinity LFA-1/ICAM-1 binding and cSMAC formation. Our findings reveal crucial roles for Rap1 signaling via NDR1 for recruitment of kindlin-3 and IS organization.

Although the cell-to-cell contact between CD4+Foxp3+ regulatory T (Treg) and their target cells is important for the suppressor function of Treg cells, the regulation of this process is not well understood. Here we show that the Mst1 kinase plays a critical role in the suppressor function of Treg cells through regulation of cell contact dependent processes. Mst1 -/- Treg cells failed to prevent the development of experimental colitis and antigen-specific suppression of naïve T cells proliferation in vitro. Mst1 -/- Treg cells exhibited defective interactions with antigen-presenting dendritic cells (DCs), resulting in reduced down-regulation of costimulatory molecules. While wild-type CD4+ Foxp3+ Treg cells formed mobile immunological synapses on supported planar membrane, Mst1 -/- Treg cells did not exhibit ICAM-1 ring or central peptide-MHC clustering. Using two-photon imaging we showed that antigen-specific wild-type Treg cells exhibited dynamic mobile contacts with antigen-pulsed DCs bearing stably associated naïve T cells. In contrast, Mst1 -/- Treg had impairments in their interactions with DCs. Thus, Mst1 is required for Treg cells to mediate contact-dependent suppressor functions.

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