The regulation of lymphocyte adhesion and migration plays crucial roles in lymphocyte trafficking during immunosurveillance. However, our understanding of the intracellular signalling that regulates these processes is still limited. Here, we show that the Ste20‐like kinase Mst1 plays crucial roles in lymphocyte trafficking in vivo . Mst1−/− lymphocytes exhibited an impairment of firm adhesion to high endothelial venules, resulting in an inefficient homing capacity. In vitro lymphocyte adhesion cascade assays under physiological shear flow revealed that the stopping time of Mst1−/− lymphocytes on endothelium was markedly reduced, whereas their l‐selectin‐dependent rolling/tethering and transition to LFA‐1‐mediated arrest were not affected. Mst1−/− lymphocytes were also defective in the stabilization of adhesion through α4 integrins. Consequently, Mst1−/− mice had hypotrophic peripheral lymphoid tissues and reduced marginal zone B cells and dendritic cells in the spleen, and defective emigration of single positive thymocytes. Furthermore, Mst1−/− lymphocytes had impaired motility over lymph node‐derived stromal cells and within lymph nodes. Thus, our data indicate that Mst1 is a key enzyme involved in lymphocyte entry and interstitial migration.

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.

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.