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Coupling of the TCR to Integrin Activation by SLAP-130/Fyb

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Science  21 Sep 2001:
Vol. 293, Issue 5538, pp. 2263-2265
DOI: 10.1126/science.1063486

Abstract

SLAP-130/Fyb (SLP-76–associated phosphoprotein or Fyn-binding protein; also known as Fyb/Slap) is a hematopoietic-specific adapter, which associates with and modulates function of SH2-containing leukocyte phosphoprotein of 76 kilodaltons (SLP-76). T cells from mice lacking SLAP-130/Fyb show markedly impaired proliferation following CD3 engagement. In addition, the T cell receptor (TCR) in SLAP-130/Fyb mutant cells fails to enhance integrin-dependent adhesion. Although TCR-induced actin polymerization is normal, TCR-stimulated clustering of the integrin LFA-1 is defective in SLAP-130/Fyb–deficient cells. These data indicate that SLAP-130/Fyb is important for coupling TCR-mediated actin cytoskeletal rearrangement with activation of integrin function, and for T cells to respond fully to activating signals.

Following the engagement of lymphocyte surface receptors, adapter proteins play critical roles in regulating second messenger signaling cascades (1). T cell receptor (TCR) signaling (2) and murine thymocyte development (3, 4) require the presence of the hematopoietic adapter SLP-76 (SH2 domain containing Leukocyte Phosphoprotein of 76 kD). SLP-76 likely functions as a scaffold for multimolecular complexes that coordinate signaling. However, it is not fully understood how SLP-76 couples TCR-stimulated protein tyrosine kinase (PTK) activity with downstream signals. SLP-76 associates in a TCR-inducible fashion with SLAP-130/Fyb, another hematopoietic-specific adapter (5, 6). Although SLAP-130/Fyb has been implicated in T cell migration and rearrangement of the actin cytoskeleton (7, 8), overexpression studies seeking to define a functional role for SLAP-130/Fyb in TCR signaling have been inconclusive (5,6).

To address the role of SLAP-130/Fyb in TCR signaling, we generated SLAP-130/Fyb–deficient mice (9–12). SLAP-130/Fyb mutant animals are born at expected Mendelian frequencies; the mice are viable, fertile, and show normal growth. Hematopoietic cellularity is normal with the exception of modest thrombocytopenia, a 50% reduction in splenic T cells, and mildly decreased thymocyte number (13). We found no differences between wild-type and SLAP-130/Fyb–deficient mice in subsets of thymocytes expressing developmental markers or coreceptors (13). Thus, SLAP-130/Fyb, unlike SLP-76, appears dispensable for orderly progression of T cell development.

Because a SLP-76–deficient T cell line exhibits uncoupling of TCR-stimulated protein tyrosine kinases (PTKs) from signals critical for cellular activation (2), we examined TCR-stimulated biochemical events in purified SLAP-130/Fyb−/− lymph node and splenic T cells. The earliest detectable biochemical event following engagement of the TCR is activation of members of several families of PTKs. SLAP-130/Fyb−/− T cells exhibit TCR-induced phosphorylation of SLP-76 and PLCγ-1 (Fig. 1A) similar to control cells. Downstream of PTK activation, MAPK up-regulation and calcium elevation are required elements in TCR-dependent cellular activation. Unlike SLP-76–deficient Jurkat cells, SLAP-130/Fyb−/−-purified T cells display TCR-dependent MAPK phosphorylation with kinetics comparable to controls (Fig. 1B). We found no decrease in TCR-mediated calcium elevation either by ratiometric flow cytometric assay (Fig. 1C) or by microscopic digital imaging (14) of individual T cell fura-2 fluorescence (15). From these data, we conclude that SLAP-130/Fyb does not regulate TCR signaling either at the level of PTK activation or in the coupling of PTK activity with key cytoplasmic biochemical events.

Figure 1

Proximal signaling occurs in the absence of SLAP-130/Fyb, but early activation antigen expression is impaired. (A) Purified splenic T cells (9–12) were left unstimulated, or treated with soluble anti-CD3ɛ(500A2, 5 μg/ml, Pharmingen) for indicated minutes, or with pervanadate (PV) (1 mM NaVO4, 1% H2O2) for 2 min. Cellular lysates were prepared in 1% NP-40, and were subjected to immunoprecipitation with anti-SLP-76 or anti-PLCγ1 (Upstate Biotechnology), followed by Western blotting with indicated antisera. In (B), cells were left unstimulated or treated with anti-CD3ɛ (500A2, 5 μg/ml) for the indicated times. Resolved cell lysates were interrogated with anti-phospho-ERK (New England Biolabs). For (C), purified lymph node T cells (9) were loaded with Indo-1 and stained with anti-CD3ɛ at 30°C, followed by stimulation with goat anti-hamster (10 μg/ml). Intracellular calcium elevations after TCR stimulation (“anti-CD3”) or ionomycin (“Iono”) were detected by ultraviolet laser flow cytometry. (D) Purified splenic T cells were cultured with platebound anti-CD3ɛ (2C11, 5 μg/ml) for 18 hours, stained with anti-CD69 or anti-CD25 (Pharmingen), and analyzed by FACS.

TCR signaling results in transactivation of several gene products, including the surface antigens CD25 and CD69. We stimulated purified SLAP-130/Fyb−/− T cells with anti-CD3 for 18 hours, and assessed expression of both markers. Up-regulation of CD25, and to a lesser extent, of CD69, was markedly reduced in SLAP-130/Fyb−/− T cells compared with controls (Fig. 1D). This result suggested suboptimal activation response to TCR engagement, despite intact proximal signaling in SLAP-130/Fyb−/−cells.

We next examined proliferation and interleukin-2 (IL-2) production as longer term parameters of T cell activation. A profound defect in the proliferative response of purified SLAP-130Fyb−/− T cells to anti-CD3ɛ stimulus (Fig. 2A) was observed. Costimulation of the mutant T cells with anti-CD28 slightly augmented, but did not restore anti-CD3-stimulated proliferation to wild-type levels. Addition of phorbol myristate acetate (PMA), a receptor-independent co-stimulus, partially rescued the proliferation defect (Fig. 2A), and stimulation of the mutant cells with PMA plus calcium ionophore produced proliferation comparable with wild-type (15). Similarly, a marked decrease in IL-2 production by purified SLAP-130/Fyb−/− T cells was apparent after stimulation with anti-CD3, or with anti-CD3 plus anti-CD28 (Fig. 2B). Stimulation with combined PMA and calcium ionophore elicited equivalent IL-2 production in SLAP-130/Fyb−/− and control T cells (Fig. 2B), suggesting no intrinsic IL-2 synthesis defect in the mutant.

Figure 2

SLAP-130/Fyb−/− T cells exhibit impaired proliferation and IL-2 production. (A) 105 purified splenic T cells were left unstimulated, or were cultured with anti-CD3ɛ (2C11, plate bound, 0.75 μg/ml) with anti-CD3ɛ plus soluble anti-CD28 (1 μg/ml), or anti-CD3ɛ plus PMA (20 ng/ml) for 48 hours. Proliferation was measured by 3H-thymidine (ICN) uptake. (B) Purified T cells were stimulated under the indicated conditions for 40 hours (ionomycin, 200 ng/ml). IL-2 in culture supernatants was measured by ELISA (R&D Systems).

Lack of IL-2 production does not completely account for the proliferation defect in SLAP-130/Fyb−/− T cells, as addition of exogenous IL-2 only partially rescued CD3-induced proliferation. Interactions of integrins such as lymphocyte function-associated antigen–1 (LFA-1) with their cognate ligands have been shown to be critical for TCR-mediated responses (16). Moreover, SLAP-130/Fyb has been implicated in the regulation of integrin function (8). Accordingly, we explored a potential role for SLAP-130/Fyb in TCR-mediated activation of LFA-1–dependent adhesion to intercellular adhesion molecule–1 (ICAM-1) (17). Basal adhesion of unstimulated SLAP-130/Fyb−/− T cells to plate-bound ICAM-1 was comparable to that of wild-type cells (Fig. 3A). As in human T cells (17,18), engagement of the TCR on wild-type murine T cells resulted in a rapid increase in LFA-1–dependent adhesion to mouse ICAM-1. In contrast, no increase in adhesion was seen following TCR stimulation of SLAP-130/Fyb−/− T cells. However, treatment of the SLAP-130/Fyb−/− cells with either PMA (Fig. 3A), which bypasses proximal TCR signaling events (17, 18), or with Mn2+ (15), which enhances LFA-1 activity through direct induction of LFA-1 conformational changes (19), induced comparable adhesion of control and SLAP-130/Fyb−/− T cells to ICAM-1. These results show that LFA-1 expressed on SLAP-130/Fyb−/− T cells can mediate adhesion to ICAM-1, but that enhanced cell adhesion after TCR ligation is defective. Similar impairment of TCR-, but not PMA- or Mn2+-induced increases in adhesion to purified mouse vascular cell adhesion molecule–1 (VCAM-1), a ligand for the α4β1 and α4β7 integrins, was observed in SLAP-130/Fyb−/− T cells (15). The defect in TCR-induced adhesion of SLAP-130/Fyb−/− T cells to ICAM-1 is not due to altered LFA-1 expression, as similar levels of the CD11a (αL) subunit of LFA-1 are expressed on control and SLAP-130/Fyb−/− T cells (Fig. 3B).

Figure 3

SLAP-130/Fyb−/− T cells display defective TCR-induced adhesion to ICAM-1 and impaired LFA-1 lateral mobility. (A) Calcein-AM (Molecular Probes)–labeled B220-depleted splenocytes were exposed to anti-CD3ɛ (2C11, 2 μg/ml) and 1 μg/ml rabbit anti-hamster IgG or to PMA (10 ng/ml) and then incubated in 96-well plates coated with recombinant, murine ICAM-1 (provided by M. Mescher, University of Minnesota, and F. Takei, British Columbia Cancer Agency, Vancouver, Canada) for 10 min at 37°C. Adherent cell number was quantified by fluorescence intensity as previously described (28). The results are representative of six independent experiments. (B) T cells were stained with fluorochrome-conjugated CD11a (subunit of LFA-1) mAb (Pharmingen) and analyzed by FACS. (C) Purified splenic T cells were cultured overnight with anti-CD3ɛ (2C11, platebound, 5 μg/ml), rested for 5 hours at 37°C, then treated for 2 min with soluble anti-CD3ɛ, stained with fluorescein-phalloidin (Sigma), and assayed for cellular F-actin (polymerized), as described (18). (D) LFA-1 clustering on purified T cells was assessed and quantified as described (9–12). Typical micrographs of unstimulated and CD3 stimulated cells are shown, and results are expressed as percentages of imaged cells showing polarized LFA-1 distribution (graph). (E) Purified splenic T cells were incubated with control Ab- or anti-CD3 (2C11)–coated beads for 20 min, then stained and analyzed by fluorescence microscopy (9–12). Typical images of cell-bead contacts (arrows in photomicrographs) are shown. Beads are indicated by asterisks. Results are expressed as the percentage of cell-bead contacts positive for actin or LFA-1 caps (graphs).

Molecular events linking the TCR to LFA-1 activation include activation of phosphatidylinositol-3 kinase (PI-3K) and actin cytoskeletal rearrangement (20). We found no clear defect either in TCR-stimulated PI-3K enzymatic activity or in the phosphorylation of the PI-3K effector Akt in SLAP-130/Fyb−/− T cells (15). We also examined TCR-dependent increases in polymerized actin (21), and found no difference between SLAP-130/Fyb−/− and control T cells (Fig. 3C).

Integrin redistribution into concentrated clusters on T cells after activating stimuli has been reported (22, 23). We assessed TCR-stimulated membrane clustering of LFA-1. Purified T cells were left unstimulated or were stimulated with soluble anti-CD3, fixed, stained with fluorochrome-conjugated anti-LFA-1, then evaluated by confocal fluorescence microscopy for evidence of LFA-1 redistribution. Figure 3D (photomicrographs) shows typical “nonpolarized” and “polarized” LFA-1 staining patterns observed in unstimulated or CD3-stimulated, wild-type T cells, respectively. Following anti-CD3 mAb stimulation, we observed a marked increase in the percentage of wild-type T cells displaying the polarized LFA-1 pattern. In contrast, the SLAP-130/Fyb−/− T cells show no TCR-stimulated increase in percentage of cells with polarized LFA-1 (Fig. 3D, graph).

In addition to global redistribution of LFA-1 on the T cell membrane, recent work has suggested that LFA-1 is specifically recruited to a region concentric with aggregated TCR complexes and in proximity to a polymerized actin cap during the formation of an “immunologic synapse” (24, 25). To study actin and LFA-1 recruitment in response to a polarizing TCR stimulus, we exposed T cells to beads covalently linked either with anti-CD3 or with control antibody. Fixed cells were stained with phalloidin or with anti–LFA-1, then examined by fluorescence microscopy. We found that, when conjugated with control antibody-coated beads, less than 10% of resting T cells displayed concentration (capping) of polymerized actin or of LFA-1 (Fig. 3E, photomicrographs) at the cell-bead contact site. Contact with anti-CD3 linked beads induced actin or LFA-1 capping (typical cell-bead contacts seen in Fig. 3E, right panels) in greater than 40% of wild-type T cells. SLAP-130/Fyb−/− T cells displayed CD3-bead-specific induction of an actin cap comparable to wild-type cells, yet 50% fewer mutant T cells formed LFA-1 caps in response to bead contact (Fig. 3E, graphs). Taken together, these data indicate that loss of SLAP-130/Fyb uncouples TCR-dependent actin cytoskeletal reorganization from membrane redistribution of LFA-1.

Our data indicate that, although SLAP-130/Fyb is dispensable for the generation of mature T cells and for TCR coupling to proximal signaling events, it has a novel, specific role in the TCR-induced augmentation of integrin activity. Impaired LFA-1 clustering observed following CD3 stimulation of SLAP-130/Fyb–deficient T cells is consistent with previous work documenting a central role for avidity regulation in CD3-induced increases in LFA-1 functional activity on T cells (26). The adhesion deficit in SLAP-130/Fyb–deficient cells may also contribute to a failure of cells to proliferate ex vivo in response to a TCR stimulus, as T cell responses are impaired by antagonist antibodies to LFA-1 or ICAM-1, and mice made deficient in LFA-1 display T cell proliferative defects (27–29).

SLAP-130/Fyb–deficient T cells reveal that proximal signaling events such as MAPK activation and calcium elevation, while known to be required for TCR-dependent transcriptional activation, are not sufficient to mediate TCR signaling to integrins. Moreover, whereas these PTK-dependent biochemical events display an absolute requirement for the presence of SLP-76 and linker for activated T cells (LAT) (2, 30), recent work suggests that these adapters are dispensable for TCR-induced changes in integrin function (31). Collectively, these studies and our current observations suggest that SLAP-130/Fyb plays a key role in a TCR-activated pathway parallel with, yet distinct from, the signaling cascades that involve LAT or SLP-76. On the basis of these studies and other work implicating SLAP-130/FYB in vasoactive mediator release (32), we propose that SLAP-130/FYB be redesignated Adhesion and Degranulation promoting Adapter Protein (ADAP).

  • * To whom correspondence should be addressed. E-mail: koretzky{at}mail.med.upenn.edu

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