SLP-76-ADAP adaptor module regulates LFA-1 mediated costimulation and T cell motility

Abstract

Although adaptor ADAP (FYB) and its binding to SLP-76 has been implicated in TcR-induced ''inside-out'' signaling for LFA-1 activation in T cells, little is known regarding its role in LFA-1-mediated ''outsidein'' signaling. In this study, we demonstrate that ADAP and SLP-76-ADAP binding are coupled to LFA-1 costimulation of IL-2 production, F-actin clustering, cell polarization, and T cell motility. LFA-1 enhancement of anti-CD3-induced IL-2 production was completely dependent on SLP-76-ADAP binding. Further, anti-CD3 was found to require CD11a ligation by antibody or ICAM1 to cause T cell polarization. ADAP augmented this polarization induced by anti-CD3/CD11a, but not by anti-CD3 alone. ADAP expression with LFA-1 ligation alone was sufficient to polarize T cells directly and to increase T cell motility whereas the loss of ADAP in ADAP؊/؊ primary T cells reduced motility. A mutant lacking SLP-76-binding sites (M12) blocked LFA-1 costimulation of IL-2 production, polarization, and motility. LFA-1-ADAP polarization was also dependent on src kinases, Rho GTPases, phospholipase C, and phosphoinositol 3-kinase. Our findings provide evidence of an obligatory role for the SLP-76-ADAP module in LFA-1-mediated costimulation in T cells.outside-in signaling ͉ T cell activation I ntegrins are transmembrane receptors that mediate cell adhesion, motility, proliferation, and differentiation. T lymphocytes express at least 12 integrins with ␣L␤2, leukocyte function-associated antigen-1 (LFA-1) also termed CD11aCD18, binding to intracellular adhesion molecule (ICAM)-1, -2, and -3 (1-3). Whereas LFA-1 is in a low-affinity state on resting cells, ligation by the antigen-receptor complex (TCR/CD3) generates ''inside-out'' signals that increase the avidity of binding by clustering (2, 3). Mediators involved in this process include CD4/CD8-Lck, IL2-inducible T cell kinase (ITK), the guanine nucleotide exchange factor Vav-1, phosphatidylinositol 3-kinase (PI 3K), Rap1 and its binding partner RapL (regulator of cell adhesion and polarization enriched in lymphoid tissues) or Riam (Rap1-interacting adaptor molecule), and adaptors SLP-76 (76-kDa src homology 2 domaincontaining leukocyte phosphoprotein), ADAP (adhesion and degranulation-promoting adaptor protein) [HUGO Gene Nomenclature: FYB-120/130 (Fyn-binding protein-120/130)], and SKAP1 [src kinase-associated phosphoprotein 1, also SKAP-55 (55-kDa src kinase-associated phosphoprotein)] (3, 4). SLP-76 is an immune cell adaptor with key N-terminal tyrosine residues and a C-terminal SH2 domain that binds to ADAP (5, 6). ADAP is also an adaptor with a unique N-terminal region, a canonical and a noncanonical SH3 domain, a putative Ena/VASP homology 1 (EVH1)-binding domain, and nuclear localization motifs (7,8). ADAP deficient T cells show defective LFA-1 clustering and adhesion (9, 10). SLP-76 SH2 domain binds to the 2 YDDV sites on ADAP (Y 595/651 DDV) (11-13). Mutation of the sites disrupts LFA-1 clustering and adhesion, localization at the periphery of the immunological synapse and T c...

The LAT Story: A Tale of Cooperativity, Coordination, and Choreography

“…Phosphorylated ADAP binds the SH2 domain of SLP-76 (da Silva et al 1997;Musci et al 1997). This interaction is crucial for adhesion and integrin function in T cells (Baker et al 2009;Horn et al 2009;Wang et al 2009). Tyrosine phosphorylated Shb also binds the SH2 domain of SLP-76 and promotes LAT, SLP-76, Vav, and PLC-g1 phosphorylation (Lindholm et al 1999;Lindholm et al 2002).…”

T Cell–Independent Modulation of Experimental Autoimmune Encephalomyelitis in ADAP-Deficient Mice

“…Following LFA-1 stimulation of T cells, the ADAP-dependent formation of a ringshaped actin reorganization called actin cloud was discovered (14). This LFA-1-mediated costimulation enhances IL-2 production, F-actin clustering, T cell polarization, and T cell motility (15). Besides its regulation of outside-in signaling in T cells, ADAP is also required for optimal CD11c integrin-mediated outside-in signaling in dendritic cells (DCs) (16).…”

Cytotoxic T lymphocyte lysis of HTLV-1 infected cells is limited by weak HBZ protein expression, but non-specifically enhanced on induction of Tax expression

“…In contrast, HBZ-specific CTLs did not kill a significant number of Tax-expressing cells; nor was any reduction in Tax MFI observed. We conclude that presentation of HBZ [26][27][28][29][30][31][32][33][34] by naturally-infected cells from HLA-A*A02 + individuals was not sufficient to efficiently trigger cytolytic functions at this E:T ratio. 7.…”

“…Therefore, any observed reduction in the rate of killing of naturally-infected cells by the HBZ-specific clone was due to the level of epitope processing or presentation by target cells, rather than lower CTL efficiency. To confirm that the HBZ [26][27][28][29][30][31][32][33][34] epitope is processed and presented naturally, we transfected cells with an expression plasmid encoding fulllength HBZ (HBZ-IRES-GFP) or GFP alone [13]: HBZ-transfected cells were selectively killed by HBZ-1 CTLs ( Figure 5B). 6.…”