The β2 integrin–kindlin-3 interaction is essential for T-cell homing but dispensable for T-cell activation in vivo

Abstract

Key Points• TTT-motif in beta2-integrin binds kindlin-3.• Mutation of TTT-motif affects T-cell homing not activation.Kindlin-3 is mutated in the rare genetic disorder, leukocyte adhesion deficiency type III, which is characterized by deficient integrin-mediated adhesion of leukocytes and platelets. However, the specific roles of kindlin-3-b2-integrin interactions in T-cell adhesion and homing and immune responses in vivo remain unclear. Here, we show that the TTT motif in b2 integrins controls kindlin-3 binding. Mutation of the kindlin-3 binding site in b2 integrins caused a loss of firm adhesion of T cells under both static and shear flow conditions and a reduction of T-cell homing to lymph nodes in vivo. However, atomic force microscopy studies of integrin-ligand bonds revealed that initial ligand binding could still occur, and 2-dimensional T-cell migration was reduced but not abolished by the TTT/AAA mutation in the b2 integrin. Importantly, dendritic cell-mediated T-cell activation in vivo was normal in TTT/AAA b2 integrin knock-in mice. Our results reveal a selective role of the kindlin-3-integrin association for lymphocyte functions in vivo; the integrin-kindlin-3 interaction is particularly important in adhesion strengthening under shear flow, and for T-cell homing to lymph nodes, but dispensable for T cell activation which occurs in a

Optimal T Cell Activation and B Cell Antibody Responses In Vivo Require the Interaction between Leukocyte Function–Associated Antigen-1 and Kindlin-3

“…The TTT/ AAA b 2 -integrin KI mouse line was described previously (11). The KI mice were crossed with OT-II mice (provided by Prof. Colin Watts, University of Dundee) to generate homozygote Itgb2 KI mice expressing the OT-II transgene.…”

“…Static adhesion assays were performed as described (11). Briefly, the integrin ligands ICAM-1 (6 mg/ml; R&D Systems), fibronectin (10 mg/ml), and VCAM (6 mg/ml; R&D Systems) were coated onto 96-well MaxiSorp plates (Nunc) by overnight incubation at 4˚C.…”

“…Previously, we generated a mutant mouse line in which the threonine triplet in the b 2 -integrin tail was substituted with alanine residues (TTT/AAA b 2 -integrin knock-in (KI) mouse). This mutation abolishes binding of the important integrin regulator kindlin-3 to the integrin cytoplasmic domain (11), resulting in impaired integrin activation to its high-affinity state and, therefore, reduced integrin function. We showed that polyclonal activation of TTT/AAA b 2 -integrin KI T cells in vitro with soluble anti-CD3 results in a reduction in T cell activation and proliferation compared with wild-type (WT) T cells, whereas activation in response to plate-bound anti-CD3 is unaffected by the KI mutation (11).…”

Interaction of kindlin-2 with integrin β3 promotes outside-in signaling responses by the αVβ3 vitronectin receptor

“…22,23 Moreover, interaction between kindlin-3 and b2 integrins seems to be required for T-cell homing in vivo. 24 Kindlin-2 is the predominant isoform in ECs, and kindlin-2 1/2 mice exhibit reduced tumor vessel density and growth, 25 suggesting a role for kindlin-2 in EC functions. However, because kindlins may also function in integrin-independent processes, 26 the extent to which any function of kindlin-2 in ECs is due to its direct interaction with integrin b cytoplasmic tails remains to be determined.…”

Adhesive Dynamics

“…These activated integrins bind with mechanical strength to cognate ligands, increasing the probability of firm adhesion, as predicted by the state diagrams described above. A number of biochemical analyses have identified the key molecular players that bind to integrin receptor cytoplasmic tails, inducing conversion to mechanically strong states, including RapL, talin, and kindlin [28]. This class of simulation is called integrated signaling adhesive dynamics (ISAD).…”