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Previous Article | Next Article 
Molecular and Cellular Biology, October 2007, p. 6863-6875, Vol. 27, No. 19
0270-7306/07/$08.00+0 doi:10.1128/MCB.00556-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Cell Signalling Section, Division of Immunology, Department of Pathology, Tennis Court Road, University of Cambridge, Cambridge CB2 1QP,1 Molecular Immunology Section, Department of Immunology, Imperial College London, Hammersmith Hospital, London W12 0NN, United Kingdom2
Received 29 March 2007/ Returned for modification 1 May 2007/ Accepted 17 July 2007
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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chains, the recruitment of ZAP-70 (zeta-associated protein-tyrosine kinase of 70 kDa), and the activation of TEC kinases ITK/RLK (interleukin-2-inducible/resting lymphocyte kinase) (1, 31-33, 41). Adaptor proteins possess binding sites and domains needed for complex-complex formation (31, 32, 41). Immune cell-specific adaptors include LAT (linker for activation of T cells), GADS (Grb-2-like adaptor downstream of Shc), SLP-76 (SH2 domain-containing leukocyte protein of 76 kDa), ADAP (adhesion- and degranulation-promoting adaptor protein; previously known as FYN T-binding protein/SLP-76-associated protein [FYB/SLAP]), and SKAP-55 (Src kinase-associated phosphoprotein of 55 kDa; also known as SCAP1) (3, 23, 24, 26, 31, 32, 40-42). Phosphorylation of LAT recruits phospholipase C
1, Grb-2, and GADS-SLP-76 and induces Ca2+ mobilization and cytokine transcription (32, 33, 41, 44).
Binding of leukocyte function-associated antigen 1 (LFA-1; also known as CD11/CD18 or 
Lß2) to intercellular adhesion molecules 1 and 2 (ICAM-1 and -2) on APCs mediates T-cell-APC conjugation (5, 8, 11, 21, 37). Following initial adhesion, TCR/CD3 ligation induces signals (i.e., "inside-out signaling") that further activate integrin adhesion (2, 5, 8, 11, 37). Conversion of LFA-1 to intermediate- or higher-affinity forms involves changes in conformation and receptor clustering (11, 37). Multiple signaling proteins mediate this process. They include the GTP-binding protein Rap-1, its ligand RapL (regulator of cell adhesion and polarization enriched in lymphoid tissues), RIAM (Rap1-GTP-interacting adaptor molecule), the guanine nucleotide exchange factor Vav-1, and the adaptors SLP-76, ADAP, and SKAP-55 (6, 10, 13, 15, 16, 17, 18, 20, 22, 27, 28, 34). The protein-tyrosine kinase ZAP-70 phosphorylates YESP sites in SLP-76, which allows binding to the Src homology 2 (SH2) domain of Vav-1 (29), while the SH2 domain of SLP-76 binds to two YDDV sites in ADAP (30, 39). T-cell lines lacking SLP-76 show impaired superantigen-induced conjugation (44).
ADAP is an immune cell-specific adaptor with a unique N-terminal region, a proline-rich region, a canonical and a noncanonical SH3 domain, one Ena/VASP homology 1 (EVH1) binding domain, and two putative nuclear localization motifs (3, 4, 19, 26, 31). ADAP is preferentially phosphorylated by the Src kinase p59fynT (4, 25) and can cooperate with p59fynT and SLP-76 in amplifying TCR-induced interleukin-2 (IL-2) transcription (30). The adaptor can up-regulate integrin-mediated adhesion in certain basophilic cell lines (9), while ADAP–/– T cells show profound defects in ß1 and ß2 integrin clustering and adhesion (10, 27). Binding to SLP-76 is needed, as evidenced by the fact that mutation of the SLP-76 binding sites in ADAP impairs conjugation, supramolecular activation cluster (SMAC) formation, and cytokine production (39). The current model proposes that p59fynT phosphorylates YDDV sites in ADAP that interact with SLP-76 SH2 domains, followed by complex recruitment to LAT via binding of the adaptor GADS to a noncanonical motif in SLP-76 (31, 32, 44).
SKAP-55, another immune cell adaptor, is the main binding partner for ADAP in T cells (14, 23, 24). It is enriched in T cells and possesses a unique N-terminal region followed by a pleckstrin homology domain and a C-terminal SH3 domain (23, 24). SKAP-55 binds with high stoichiometry to ADAP (4, 25), an interaction that is mediated by binding of the SKAP-55 SH3 domain to a proline-rich region in ADAP (23, 25) and by weaker binding of the ADAP SH3c domain to a tyrosine-based RKXXYXXY motif in SKAP-55 (7, 14). SKAP-55 may also interact with the D1 domain of the phosphatase CD45 (43). We have previously shown that SKAP-55 is capable of enhancing TCR-mediated activation of ß1 and ß2 integrins and of forming conjugates between T cells and APCs (40) and that small interfering RNA-mediated knockdown of SKAP-55 impairs conjugate formation and LFA-1 clustering in mouse T cells (13). This effect occurred under conditions of normal expression of the homologue SKAP-55-related (SKAP-55R) or SKAP-55Hom, indicating a nonredundant function for SKAP-55 in T cells (13). An interaction between ADAP and SKAP-55 is needed for adhesion, since the loss of the SKAP- 55 SH3 domain or the ADAP SH3c domain binding sites on SKAP-55 ablates adhesion and conjugate formation (7, 18, 40). ADAP-SKAP-55 may facilitate the translocation of Rap1 to membranes (18).
While ADAP-SKAP-55 comprises a module for integrin adhesion, it has been unclear whether ADAP and/or SKAP-55 is directly responsible for this function (10, 12, 18, 27). An important recent observation has been that ADAP modulates SKAP-55 protein turnover and degradation such that ADAP-deficient cells show a major loss of SKAP-55 expression (12). In this study, we report the phenotype of the SKAP-55-deficient mouse, where T cells lack SKAP-55 but retain ADAP expression. Despite this, SKAP-55-deficient T cells show major defects in integrin adhesion, cytokine production, and proliferation comparable to those for ADAP-deficient T cells. Dwell times of SKAP-55-deficient T cells in response to staphylococcal enterotoxin A (SEA) presented by dendritic cells (DCs) were shorter concurrent with reduced localization of the TCR/CD3 complex at the IS and reduced proliferation. However, the requirement for ADAP-SKAP-55 was not absolute. Adhesion assays and cell cycle analysis identified a subset of T cells that adhere without the need for the adaptors, a dependency that differs with the strength of the TCR signal. Our studies identify an effector function for SKAP-55 that operates independently of ADAP.
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MATERIALS AND METHODS |
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Cell culture and antibodies. Primary T cells from SKAP-55+/+, SKAP-55+/–, and SKAP-55–/– mice were cultured in RPMI 1640 medium with 10% (vol/vol) fetal calf serum (FCS), 5% (vol/vol) glutamine, 5% (vol/vol) penicillin-streptomycin, and 5 x 10–5 M 2-mercaptoethanol. CD4-positive T cells were purified using anti-CD4 beads as previously described (40). For the generation of bone marrow DCs (BMDCs), bone marrow was flushed from femurs and passed through a 200-µm mesh to remove fibrous tissue, and red blood cells were lysed using ammonium chloride lysis buffer. Cells were cultured at 106/ml in RPMI medium supplemented with 10% FCS, 2 mM glutamine, 50 µM 2-mercaptoethanol, 100 IU/ml penicillin, 100 µg/ml streptomycin, 20 ng/ml recombinant murine granulocyte-macrophage colony-stimulating factor, and 1,000 IU/ml IL-4. On day 5 of culture, floating cells were gently removed and fresh medium containing recombinant murine granulocyte-macrophage colony-stimulating factor was added. On day 7 of culture, BMDCs were induced to mature by addition of 1 µg/ml lipopolysaccharide to the cultures. After overnight incubation, nonadherent cells and loosely adherent proliferating BMDC aggregates were collected, washed, and replated for 1 h at 37°C to remove contaminating macrophages. DCs were then plated with various concentrations of SEA for 2 to 3 h prior to coculture with T cells for proliferation, cytokine, contact time, and imaging analysis. The contact time of individual T-cell-APC conjugates was followed over 1,800 s using MetaMorph imaging software.
Monoclonal antibodies against ADAP and SKAP-55 were purchased from Transduction Laboratories (San Diego, CA), while unconjugated or conjugated antibodies against CD11a (LFA-1, L chain), CD18 (LFA-1, ß2 chain), CD4, CD8, CD44, CD25, CD29 (anti-integrin ß1 antibody), CD69, DX5, TCR, and B220 were from Pharmingen (Oxford, United Kingdom). Unconjugated antibodies against mouse CD3 (2C11; hamster anti-mouse CD3) or phosporylated tyrosine (4G10) were prepared and purified by Bioexpress. Alexa Fluor 546- or Alexa Fluor 633-conjugated secondary antibodies were purchased from Molecular Probes (Eugene, OR). Five-domain murine ICAM-1 Fc was purchased from R&D Systems. Antiactin was from Sigma, and an antibody against extracellular signal-regulated kinases 1 and 2 (anti-ERK1/2) was purchased from Cell Signaling [New England Biolabs (UK) Ltd., Hertfordshire, United Kingdom]. Anti-phospho-ERK1/2 T202/Y204 or T185/Y187 was purchased from BioSource United Kingdom (Nivelles, Belgium).
Intracellular staining and CFSE labeling.
Intracellular staining and flow cytometry were used to measure the level of antibody against gamma interferon (anti-IFN-). After anti-CD3 stimulation, cells were fixed in 4% paraformaldehyde (Sigma) and permeabilized with 0.3% saponin (Sigma), followed by a second staining with allophycocyanin-tagged anti-IFN- in saponin containing phosphate-buffered saline (PBS)-bovine serum albumin (BSA). Cells were fixed and analyzed using a FACSCalibur flow cytometer. For carboxyfluorescein diacetate succinimide ester (CFSE) labeling, 5 x 106 cells were washed twice and resuspended in 250 µl of staining buffer (PBS supplemented with 0.1% BSA). A 250-µl volume of freshly diluted 2 µM CFSE (Molecular Probes) was added, and cells were incubated at room temperature for 10 min. An equal volume of FCS was added to stop labeling. After three washes, cells were activated with anti-CD3 for 72 h with a culture medium containing 10% FCS. Cells were harvested and stained with allophycocyanin-conjugated anti-CD4, followed by gating of CD4-positive cells for detection of CFSE fluorescence on a FACSCalibur system (BD Biosciences). Data were analyzed using CellQuest (BD Biosciences).
PCR. PCR amplification was performed in a 50-µl reaction mixture using 1 µl cDNA with 25 µl of 2x Taq master mix and reverse primer B with oligonucleotide primer LTR or primer A (0.5 µM each). The cycling parameters were as follows: 5 min at 95°C for 1 cycle; 1 min at 94°C, 1 min at 65°C, and 1 min and 30 s at 72°C for 10 cycles, with the annealing temperature decreasing 1°C in each cycle; 1 min at 94°C, 1 min at 55°C, and 1 min and 30 s at 72°C for 28 cycles; and a final extension at 72°C for 10 min. After PCR, the amplification reaction product was analyzed on a 2% agarose-ethidium bromide gel.
Immunoblotting. For immunobotting, 4 x 106 cells were lysed in ice-cold lysis buffer containing 1% Triton X-100 (vol/vol) in 20 mM Tris-HCl (pH 8.3), 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na4VO3, 10 mM NaF, and 1 mM Na4P2O7 as described previously (29). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were then blocked with 5% skim milk in TBS (10 mM Tris-HCl [pH 7.6], 150 mM NaCl) and incubated for 1 h with the indicated antibodies. Levels of bound antibody were then measured using a horseradish peroxidase-conjugated rabbit anti-mouse secondary antibody, followed by detection with enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech, Piscataway, NJ).
LFA-1 adhesion and clustering assays. Anti-CD3-stimulated or unstimulated T cells were added to plates coated with recombinant murine ICAM-1 (R&D Systems) and incubated for 30 min at 37°C. Nonadherent cells were gently washed off, and the remaining cells were counted as previously described (39). LFA-1 clustering/capping experiments were conducted as follows. Briefly, cells were incubated with 5 µg/ml anti-CD3 (clone 145-2C11) on ice for 30 min. Cells were then washed twice with cold medium and cross-linked by incubation with 10 µg/ml of a rabbit anti-hamster immunoglobulin G antibody (Jackson Immunoresearch) at 37°C for 30 min. Cells were fixed with 2% paraformaldehyde for 20 min, washed with PBS, and blocked with 3% BSA-5% FCS-PBS for 45 min. Samples were then stained for anti-CD11a and an isotype-specific Alexa Fluor 546-conjugated anti-rat antibody as described elsewhere (39). At least 50 T cells were counted for cap formation in each experiment. These experiments were conducted using four different SKAP-55 KO mice. Immunofluorescence microscopy was conducted as described previously (39).
IL-2 ELISA and T-cell proliferation assay. SKAP-55+/+, SKAP-55+/–, and SKAP-55–/– primary mouse cells (2 x 105/well) were cultured in 2 µg/ml anti-CD3 (145-2C11) applied to plates for 48 h. The supernatants were collected, and IL-2 was measured by an enzyme-linked immunosorbent assay (ELISA) with a rat anti-mouse IL-2 monoclonal antibody (ELISA capture) and a biotinylated rat anti-mouse IL-2 monoclonal antibody (ELISA detection). For the T-cell proliferation assay, cells were stimulated with 1 µg/ml plated anti-CD3 for various times (e.g., 48 h). To assess proliferation, cells were pulsed with 1 µCi of [3H]thymidine (Amersham Biosciences) for the last 12 h. Cells were collected on GSC filters (PerkinElmer, Wallace Dy, Finland). Radioactivity was measured in a microplate scintillation counter.
Statistical analysis. Results are given as means ± standard deviations. The difference between means was tested either by an unpaired Student t test (for two means) or by one-way analysis of variance with Dunnett's posttest (multiple test) using GraphPad Prism, version 3.02 for Windows (GraphPad Software, San Diego, CA). A P value of <0.05 was considered significant.
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Given the effect on conjugation, it was important next to determine whether loss of SKAP-55 influenced anti-CD3-induced integrin adhesion of primary T cells (Fig. 4). Isolated mesenteric lymph cells were stimulated with anti-CD3, followed by an assessment of fibronectin (FN) and ICAM-1 binding, as previously described (40). First, CD29 (i.e., ß1 FN receptor) expression was the same on SKAP-55+/+ and SKAP-55–/– T cells (Fig. 4a). Despite this, the SKAP-55-deficient cells showed low levels of adhesion to FN relative to those for wild-type cells (Fig. 4b). While anti-CD3 at 2 µg/ml increased wild-type cell adhesion from 4 to 32% of cells, only 15% of SKAP-55–/– T cells bound to FN (Fig. 4b, left). The loss of SKAP-55 therefore resulted in a >50% reduction in the number of cells capable of binding to FN. Examples of adherent cells on plates are shown in the right panels of Fig. 4b.
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production. While anti-CD3 increased IL-2 production from 70 to 630 pg/ml in SKAP-55+/+ cells at 48 h of culture, IL-2 production at this time was only 350 pg/ml for SKAP-55+/– and 280 pg/ml for SKAP-55–/– T cells (Fig. 6a). Similar decreases were observed at other time points (i.e., 36 and 60 h) (data not shown). IFN- production was also reduced in SKAP-55-deficient T cells (Fig. 6b). Intracellular staining was used to measure IFN- as described in Materials and Methods. On average, there was a 40 to 50% reduction in the percentage of T cells expressing IFN-. This reduction in adhesion and cytokine production was mirrored by reduced SKAP-55–/– T-cell proliferation (Fig. 6c). Proliferation was 50 to 75% lower for SKAP-55–/– T cells than for wild-type T cells. The reduced proliferation of SKAP-55+/– and SKAP-55–/– cells closely resembled that of ADAP+/– and ADAP–/– T cells. These observations indicate that SKAP-55 expression in primary cells is needed for optimal IL-2 and IFN- production and proliferation.
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We next assessed the numbers of cells in the thymi of SKAP-55+/+, SKAP-55+/–, and SKAP-55–/– mice (Fig. 8a). Although an occasional SKAP-55–/– mouse showed a reduction in thymic cellularity, this was not found to be statistically significant over five independent experiments. The expression of the antigen receptor (TCR) was normal for SKAP-55+/– and SKAP-55–/– thymocytes (Fig. 8b), as was the presence of CD4 and CD8 double-negative (DN), double-positive (DP), and CD4 or CD8 single-positive (SP) cells (Fig. 8c). Using CD44 and CD25 as markers, the DN1-to-DN4 transition appeared normal in SKAP-55+/– and SKAP-55–/– mice (Fig. 8d). A slight reduction in the CD44- and CD25-negative compartment was occasionally observed; but this was not a consistent observation. We also confirmed that normal ratios of T and B cells were observed in the lymph nodes and spleen when anti-CD3 and anti-B220 were used to stain cells (Fig. 9a), and normal ratios of peripheral mature CD4 SP and CD8 SP T-cell subsets were observed (Fig. 9b). Occasionally, SKAP-55–/– and SKAP-55+/– mice showed slight increases in the numbers of CD4+ CD8+ T cells; however, this was not reproducible. In addition, the compartments of /
T cells in the lymph nodes and spleen were equivalent in SKAP-55+/+, SKAP-55+/–, and SKAP-55–/– mice (Fig. 9c), as were those of NK cells as defined by CD3– DX5+ staining (Fig. 9d). These data confirm a normal composition of cells in the peripheral T-cell compartments of SKAP-55-deficient mice.
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DISCUSSION |
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cytokine production, and proliferation. In fact, SKAP-55–/– T cells showed the same general impairment of function as ADAP–/– T cells, indicating that SKAP-55 is an effector of the ADAP-SKAP-55 module. At the same time, the requirement for ADAP and SKAP-55 was not absolute, since a subset of peripheral T cells adhere without the need for either adaptor. Further, dependency on SKAP-55 or ADAP differed with the strength of the TCR signal. Higher concentrations of anti-CD3 enabled greater numbers of cells to bind ICAM-1 and proliferate in the absence of SKAP-55 or ADAP. Our findings identify a clear effector role for SKAP-55 in the up-regulation of LFA-1 adhesion in peripheral T cells and demonstrate that higher levels of TCR engagement can bypass the need for SKAP-55 and ADAP in the up-regulation of T-cell adhesion. ADAP and SKAP-55 form a module where they bind to each other with high stoichiometry, and immunoprecipitation with an anti-ADAP antibody depletes most SKAP-55 from cell lysates and vice versa (4, 25). Further, ADAP controls the degradation of SKAP-55 such that ADAP-deficient T cells have little if any SKAP-55 protein expression (12) (Fig. 1d). The question of whether SKAP-55 might also influence ADAP expression was addressed previously; small interfering RNA-mediated reduction of SKAP-55 levels in a mouse antigen-specific T-cell line markedly reduced integrin adhesion without altering ADAP expression (13). Similar findings have recently been reported with Jurkat cells (18). Our study has now extended this finding by demonstrating that SKAP-55 has no obvious role in regulating ADAP expression in normal primary peripheral T cells. ADAP therefore serves a unidirectional chaperone function for its binding partner within the module.
This observation, combined with the finding of impaired ß1- and ß2-mediated adhesion of SKAP-55–/– T cells, has, for the first time, identified an effector role for SKAP-55 within the ADAP-SKAP-55 module in primary T cells. SKAP-55-deficient T cells showed multiple defects, including defects in cell aggregation and subsequent cytokine production and proliferation in response to SEA (Fig. 2) and defects in anti-CD3-induced ICAM-1 adhesion, cytokine production, and proliferation (Fig. 6). Occasionally, ADAP–/– cells showed greater impairment of ICAM-1 binding; however, this was not a consistent observation. Instead, the level of defective function for SKAP-55-deficient T cells was remarkably similar to that for ADAP-deficient T cells. This was observed in multiple assays including assessment of cell aggregation, cytokine production, and proliferation in response to SEA (Fig. 2), ICAM-1 binding (Fig. 5b), anti-CD3-induced proliferation (Fig. 6c), and the abilities of high anti-CD3 concentrations to bypass the need for the adaptor (Fig. 7c and d). These findings introduce the intriguing question of whether the impaired function of ADAP-deficient mice is due primarily to the loss of SKAP-55 or whether ADAP can play an additional, independent role in adhesion. In the former scenario, SKAP-55 would be the sole effector of the ADAP-SKAP-55 module. ADAP would indirectly influence adhesion by altering SKAP-55 expression, or possibly its conformation or localization. In the more likely scenario, ADAP provides overlapping or alternate signals for adhesion. The presence of an EVH1 domain and G-actin binding motifs in ADAP would support this alternate role (19). The modulation of the cytoskeleton at the IS could facilitate more effective adhesion without directly affecting the status of LFA-1 on the surface of the cell. However, it is interesting that multiple mutations in the EVH1 motif had no obvious effect on the ability of T cells to form conjugates with APCs (39). In either case, ADAP acts to bridge SLP-76 with downstream SKAP-55. p59fynT phosphorylates YDDV sites in ADAP, leading to SLP-76 SH2 domain binding and possibly to recruitment of the ADAP-SLP-76-GADS complex to LAT (31, 32, 41). Mutation of YDDV sites in ADAP impairs conjugation, SMAC formation, and cytokine production (39). Further, loss of the SKAP-55 SH3 domain, loss of the ADAP SH3c domain, or over expression of a peptide which binds to the proline-rich region of ADAP ablates adhesion (7, 18, 39). These observations are consistent with the existence of a cascade linking SLP-76 to ADAP and ADAP to SKAP-55 in the control of adhesion. The data from this study now underscore the central role of SKAP-55 as a downstream effector in the cascade.
The loss of SKAP-55 impaired anti-CD3-induced adhesion to FN (Fig. 4) and ICAM-1 (Fig. 5). Generally ß1-mediated binding was impaired more than ß2-mediated binding, introducing the possibility that SKAP-55 may differentially regulate the different classes of integrins. In terms of response to antigens, the loss of SKAP-55 reduced SEA-induced T-cell aggregation (Fig. 2) and the dwell times of T cells with APCs (Fig. 3). The length of time that a T-cell spends attached to a DC will influence the level of antigen-receptor clustering and the intracellular signaling needed for proliferation. In this regard, while the difference in general cell aggregation induced by SEA did not correlate strictly with reduced proliferation, a correspondence between the reduction in the percentage of long-term contacts (i.e., >1,800 s), the reduction in the number of cells with the TCR/CD3 complex localized at the IS, and the level of IL-2 production and proliferation was observed (i.e., 40 to 50%) (Fig. 2 and 3). A small reduction in number of TCR/CD3 microclusters in SKAP-55–/– T cells was also observed (data not shown). TCR/CD3 and intracellular signaling proteins form microclusters at the IS, where signals are generated for proliferation (5, 8, 35). The reduction in conjugation time and TCR microcluster formation in turn correlated with an overall reduction in the tyrosine phosphorylation of many proteins in T cells (Fig. 3c). To date, a selective reduction in the phosphorylation of one protein relative to another has not been observed.
Interestingly, SKAP-55 also played a role in antigen-independent conjugate formation. Even in the absence of SEA, SKAP-55–/– T cells interacted with APCs for shorter periods than wild-type cells (Fig. 3a). The addition of SEA caused a shift to longer adhesion periods for both SKAP-55+/+ and SKAP-55–/– cells. The inability of SKAP-55–/– cells to achieve longer contact times appeared to be influenced by the fact that they already exhibited shorter contacts in an antigen-independent context. Only 40% of SKAP-55–/– T cells achieved the longer conjugation periods (i.e., >1,800 s) in response to SEA. Many SKAP-55–/–cells failed to respond to TCR ligation, while others formed conjugates of intermediate duration. Overall, our findings are consistent with a general role for SKAP-55 in antigen-dependent and antigen-independent conjugate formation.
One surprising observation was the existence of heterogeneity in the peripheral T-cell population with regard to their dependence on SKAP-55 and ADAP. A percentage of SKAP-55–/– T cells underwent normal adhesion, conjugation, and proliferation (Fig. 2 to 6). In fact, some 30 to 50% of SKAP-55- and ADAP-deficient T cells proliferated normally in response to SEA presented by DCs (Fig. 2), bound to ICAM-1 on plates (Fig. 5), produced IL-2 and IFN-, and proliferated (Fig. 6). Cell cycle analysis extended this observation by showing that a significant portion of SKAP-55-deficient T cells underwent multiple rounds of cell division (Fig. 7). Once committed to proliferation, these cells underwent the same number of subsequent cell divisions as wild-type cells. The defect was in the ability of a subset of T cells to enter the first cycle of division. This indicates that the "inside-out" pathway is more complex than previously appreciated and that SKAP-55 is not essential for the function of all peripheral T cells. Similar findings were made with ADAP-deficient T cells. A subset of T cells appears to have an alternate mechanism that can substitute for SKAP-55.
Further, it is noteworthy that the relative proportions of SKAP-55- and ADAP-dependent and -independent cells in the peripheral compartment are not fixed. Rather, they appear to reflect a heterogeneity in the population of T cells that can be altered by the strength of the TCR signal. An increase in the anti-CD3 concentration from 1 to 2 µg/ml (otherwise, 2 µg/ml was used throughout the study) and 10 µg/ml induced increasing numbers of cells to adhere to ICAM-1 and to proliferate without the need for SKAP-55 or ADAP (Fig. 7c and d). In fact, 10 µg of antibody/ml activated the entire population of T cells without the need for SKAP-55 or ADAP. This level of receptor cross-linking and this signal strength are probably higher than those that would normally be encountered under physiological conditions. Nevertheless, the observation is informative in that it indicates that SKAP-55 and ADAP are not obligatory components in the TCR signaling leading to adhesion. The strength of the signal can override the need for SKAP-55. In this context, the ADAP/SKAP-55 pathway may preferentially support low- to intermediate-affinity ligand stimulation of T cells. The degree to which the ADAP/SKAP-55 pathway supports physiological responses to peptides of different affinities and the nature of the alternate pathway remain to be determined. Another protein(s) may constitute a pathway that is distinct from SKAP-55, or the TCR complex may directly activate another mediator downstream of SKAP-55 in the same pathway. Possible candidates include the GTPase Rap1 and/or its binding partners RapL and RIAM (6, 15, 16, 22, 28, 34).
Last, a similarity between the phenotypes of the SKAP-55- and ADAP-deficient mice was also observed in the lack of a profound effect on thymic differentiation and the composition of the peripheral T-cell compartment. Both ADAP- and SKAP-55-deficient mice expressed a normal array of thymocytes (i.e., CD4, CD8, DN1 to DN4) and peripheral cell subsets (i.e., CD4 SP, CD8 SP, and TCR T cells; B-cells; and NK cells) (Fig. 8 and 9). These findings do not exclude the possibility that more-subtle alterations will eventually be observed in TCR-transgenic animals, as reported for ADAP–/– mice (42). We occasionally observed a decrease in SKAP-55–/– thymic cellularity; however, when averaged over six animals, this difference was not found to be statistically significant. This differs from the observations for ADAP–/– mice, which showed a moderate decrease in thymocyte numbers (10, 27, 43). In instances of mice with decreased numbers of thymocytes, the cells showed an increased propensity to undergo cell death in media (data not shown). Although occasionally SKAP-55–/– thymocytes showed slight increases in the percentages of DN2 and DN3 thymocytes with a reduction in the percentage of the DN4 subset, this also was not reproducibly different from findings for non-TCR-transgenic mice. Consistent with this, LFA-1-deficient mice show no apparent defects in thymic differentiation (36). Further studies will be needed to uncover whether ADAP- and SKAP-55-deficient T cells influence the activation of other pathways linked to the regulation of T-cell adhesion.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Published ahead of print on 23 July 2007.
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