Productive activation of T lymphocytes is thought to require at least two signals: one delivered by the T cell receptor (TCR) complex after antigen recognition, and one provided by engagement of costimulatory receptors, such as CD28 and integrins. Although integrin family members such as the very late antigen 4 (VLA-4, α4β1) have been implicated in T cell costimulation, the precise mechanism is not well understood. Recent studies showed that both antigen receptors and integrins assemble signaling microclusters containing the SH2 domain-containing leukocyte protein of 76 kDa (SLP-76), a critical adapter protein that regulates T cell signaling and development. The formation and persistence of SLP-76 microclusters (SLP-76 MCs) are thought to initiate and sustain T cell signaling and functions. Furthermore, mutant analyses of SLP-76 indicated that the SH2 domain of SLP-76, which binds the adapter protein adhesion and degranulation-promoting adapter protein (ADAP), is critical for microcluster formation. ADAP functions downstream of the TCR and regulates integrin functions. Thus, I proposed that VLA-4 facilitates T cell costimulation by controlling SLP-76 MC movement and signaling through ADAP.

Here, we show that VLA-4-meditated T cell costimulation requires integrin-mediated outside-in signals. The role of integrin in cell adhesion is required but not sufficient to drive costimulation. Using multicolor dynamic imaging techniques to examine the effect of costimulatory VLA-4 on the behavior of SLP-76 MCs in live T cells, we showed that costimulation via VLA-4 immobilizes SLP-76 microclusters within the actin-rich peripheral regions of the cell contact. The immobilization of SLP-76 retains its interaction with its upstream kinase, zeta associated protein of 70 kDa (ZAP-70), which correlates with increased tyrosine phosphorylation of SLP-76 MCs. Furthermore, the immobilization of SLP-76 MCs is associated with the drastic reduction of retrograde actin flow, suggesting that microcluster movement is actin dependent. The actin cytoskeleton can associate with integrins, TCRs and components of SLP-76 MCs, suggesting the existence of a physical connection between these molecules. Therefore, we examined their localizations. We showed that VLA-4, TCR, and SLP-76 MC exist in three distinct but adjacent microclusters. Molecular characterization of these microclusters revealed that ADAP is a key molecule that links SLP-76 MCs to VLA-4. We showed that ADAP assembled and coordinately moved with SLP-76 MC in response to TCR stimulation. This movement inhibited by VLA-4 costimulation. When ADAP was knocked down by shRNAi, NF-AT responses were suppressed approximately 50 percent. This functional defect is consistent with the reduction of SLP-76 MC persistence. Therefore, we concluded that VLA-4 coligation prolongs the persistence of SLP-76 MCs and enhances downstream signaling that contributes to effective T cell activation.