Copyright by The Rockefeller University PressRecent observations using multiphoton intravital microscopy (MP-IVM) have uncovered an unexpectedly high lymphocyte motility within peripheral lymph nodes (PLNs). Lymphocyteexpressed intracellular signaling molecules governing interstitial movement remain largely unknown. Here, we used MP-IVM of murine PLNs to examine interstitial motility of lymphocytes lacking the Rac guanine exchange factor DOCK2 and phosphoinositide-3-kinase (PI3Kƴ), signaling molecules that act downstream of G protein–coupled receptors, including chemokine receptors (CKRs). T and B cells lacking DOCK2 alone or DOCK2 and PI3Kƴ displayed markedly reduced motility inside T cell area and B cell follicle, respectively. Lack of PI3Kƴ alone had no effect on migration velocity but resulted in increased turning angles of T cells. As lymphocyte egress from PLNs requires the sphingosine-1-phosphate (S1P) receptor 1, a Gi protein–coupled receptor similar to CKR, we further analyzed whether DOCK2 and PI3Kƴ contributed to S1P-triggered signaling events. S1P-induced cell migration was signifi cantly reduced in T and B cells lacking DOCK2, whereas T cell–expressed PI3Kƴ contributed to F-actin polymerization and protein kinase B phosphorylation but not migration. These fi ndings correlated with delayed lymphocyte egress from PLNs in the absence of DOCK2 but not PI3K, and a markedly reduced cell motility of DOCK2-defi cient T cells in close proximity to efferent lymphatic vessels. In summary, our data support a central role for DOCK2, and to a lesser extent T cell–expressed PI3Kƴ, for signal transduction during interstitial lymphocyte migration and S1P-mediated egress.Peer reviewed