Immobilization of glycosylphosphatidylinositol-anchored proteins inhibits T cell growth but not function

Mina D. Marmor, Martin F. Bachmann3, Pamela S. Ohashi1, Thomas R. Malek2 and Michael Julius

Department of Immunology, University of Toronto, and The Arthritis and Immune Disorder Research Centre, The Toronto Hospital, Toronto, Ontario M5G 2M9, Canada
1 Departments of Immunology and Medical Biophysics, University of Toronto and The Ontario Cancer Institute, The Toronto Hospital, Toronto, Ontario M5G 2M9, Canada
2 Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, FL 31336, USA

Correspondence to: M. Julius, c/o 610 University Avenue, 620 University Avenue, Room 700B, Toronto, Ontario M5G 2M9, Canada


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Accumulating evidence suggests that proteins tethered to the plasma membrane through glycosylphosphatidylinositol (GPI) anchors share common biological properties. In the present study we demonstrate that GPI-anchored proteins regulate T cell growth. Specifically, anti-TCR-induced proliferation was profoundly inhibited by co-immobilized mAb specific for Thy-1, CD48 and Ly6A/E. However, neither IL-2 production nor the effector function of cytotoxic T lymphocytes was impaired in these circumstances. Analysis of the IL-2 receptor (IL-2R) signaling pathway revealed that the association of IL-2R ß and {gamma} chains with the Janus kinases, JAK1 and JAK3, was not perturbed in the presence of mAb specific for GPI-linked proteins. However, in these conditions,

IL-2-mediated recruitment of IL-2R{alpha}, ß and {gamma} chains, resulting in the formation of the high-affinity hetero-trimeric IL-2R, was inhibited. The resulting phosphorylation of JAK1 and JAK3, indicative of their activation states, was correspondingly reduced. These results characterize a novel state of T cell physiology in which effector function is maintained, in the absence of clonal expansion. A physiological role for GPI-anchored proteins in the maintenance of cellular homeostasis and function is discussed.

Keywords: glycosylphosphatidylinositol-anchored proteins


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
While not affecting the specificity of T cell activation, an increasing number of membrane molecules have been shown to alter signals induced through the TCR. The modifications mediated by accessory activation molecules can be extreme, resulting in cell growth, anergy or death. Antibodies to glycosylphosphatidylinositol (GPI)-anchored proteins have been shown to function in this context. Specifically, Thy-1, Ly6A/E, CD48 and TSA-1 specific mAb can inhibit anti-TCR–CD3-induced T cell activation (14). GPI-anchored proteins have also been implicated in T cell activation. Proliferation and cytokine secretion follow direct mAb-mediated aggregation of Thy-1, Ly6A/E, Qa-2 and CD48 on murine lymphocytes (3,57). Further, aggregation of CD59, CD55 and CD73 on human lymphocytes has been shown to induce cellular growth (810). The distinct consequences of signaling through various GPI-linked proteins on the physiology of T lymphocytes in vitro appears to depend on the protocol used to ligate the molecules. Specifically, the addition of soluble mAb, compared with their deliberate aggregation, supports fundamentally distinct consequences on T cell physiology. While the central role of GPI-linked molecules in development is supported by evidence that targeted disruption of an enzyme central to the biosynthesis of the anchor moiety results in embryonic death (11), the role of GPI-linked molecules in cellular activation and growth control in physiological circumstances remains unknown.

Signaling through different GPI-anchored proteins induces qualitatively similar responses, consistent with the involvement of shared signaling pathways. Furthermore, the mitogenicity of mAb to different GPI-linked molecules, whose protein moieties are otherwise unrelated, suggests that the GPI anchor is critical. Consistent with this notion is the demonstration that antibody-mediated aggregation of transmembrane forms of Ly6A/E, CD55 or Qa-2 does not result in cellular growth (1214). One consequence that GPI linkage imparts upon membrane molecules is their localization to detergent-insoluble sub-domains of the plasma membrane (15), which have a unique lipid composition, and are enriched in Src family protein tyrosine kinases (PTK), hetero-trimeric and small G proteins, LAT, phospoinositides and phosphatidylinositol-3-kinase (1618). The localization of these signaling proteins to the sphingolipid microdomains is dependent upon their acylation (18,19). Src PTK have been implicated in signaling through GPI-anchored proteins as these are co-precipitated in non-ionic detergents (20) and as mAb-mediated aggregation of GPI-anchored proteins leads to a rapid increase in the tyrosine phosphorylation of intracellular substrates (21). Furthermore, association of GPI-linked proteins with Src PTK appears to predicate the capacity of mAb specific for the GPI-linked protein to induce cellular growth (13).

While of interest due to their capacity to activate T cells and to fundamentally alter signals generated through TCR–CD3, the conceptual difficulty associated with analyses of signal transduction through GPI-linked proteins stems from the fact that the majority of their physiological ligands remain uncharacterized. Putative ligands for GPI-linked molecules may be expressed on neighboring cells, on the same cell or in the extracellular milieu. Furthermore, the physiological role of certain GPI-linked molecules with known ligands remain unclear. For example, although CD48 has been shown to bind CD2 (22), the outcome of this interaction in vivo remains equivocal.

The question addressed in the present study is whether some common effector function can be attributed to any protein tethered to the plasma membrane through a GPI anchor. We assessed the effects of immobilizing various GPI-linked proteins on mAb-mediated T cell activation. The results demonstrated that mAb-mediated immobilization of GPI-anchored proteins on both primary CD8+ T cells, as well as on an IL-2-dependent, antigen-specific T cell clone, virtually ablated cellular growth induced through the TCR complex. However, in these same circumstances, neither IL-2 production and secretion nor cell-mediated cytotoxic effector function was impaired. The inability to utilize IL-2 to support cell growth demonstrated a lesion in IL-2–IL-2 receptor (IL-2R) signaling. Flow cytometric analysis revealed the absence of the IL-2Rß chain; however, forced expression of this chain did not revert the anti-GPI-induced lesion in cell growth. Further analyses revealed that when GPI-anchored proteins are immobilized, IL-2 failed to induce the recruitment of IL-2R{alpha}, ß and {gamma} chains, and the generation of the hetero-trimeric high-affinity IL-2R complex was inhibited. Consistent with this defect is that while mAb specific for GPI-linked proteins did not affect the stoichiometry of IL-2Rß- and {gamma}-associated JAK1 and JAK3 respectively, the latter did not become activated. These results highlight a novel mechanism through which T cells could mediate their effector functions in the absence of clonal expansion, and potential physiological counterparts reflecting these circumstances are discussed in terms of cellular homeostasis and function.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Primary T cells, clones and transfections
Primary CD8+ T cells were purified from the lymph nodes of 6- to 8-week-old C57Bl/6 mice, purchased from Jackson Laboratories (Bar Harbor, ME) as described previously (23). Clone 2.10 is an IL-2-dependent, Vß4-expressing, CD4 T cell clone specific for ovalbumin-derived peptide comprised of residues 143–157 in the context of I-Ab (24). Forced expression of IL-2Rß in clone 2.10 was achieved by electroporating 107 2.10 cells with 10 µg of the eukaryotic expression vector BCMGSneo (25) or with this vector containing cDNA encoding mouse IL-2Rß (26). Following selection in 1 mg/ml G418, cells were sorted for high expression of IL-2Rß on a FACStar Plus (Becton-Dickinson, Mountain View, CA). Levels of IL-2Rß expression remained stable when cells were propagated in the presence of G418. The CTLL-2 cell line was used in bioassay to detect the presence of IL-2 in culture supernatants and was obtained from ATCC (Rockville, MD).

Proliferation assays
All antibodies used were diluted to the indicated concentrations in HBSS without calcium or magnesium chloride. Antibody solution (50 µl) was added to wells of 96-well cluster flat-bottom tissue culture plates and plates were incubated for 1 h at 37°C. Following two washes with HBSS, 5x104 primary T cells or 2x104 2.10 T cells were added in 200 µl of serum-free culture medium as described previously (24). After 40 h, each culture was pulsed with 1 µCi of [3H]thymidine and harvested 6 h later. Thymidine uptake was assessed by liquid scintillation spectroscopy. The bioassay for IL-2 content was performed by adding 5x103 CTLL-2 cells to the dilution of culture supernatant indicated. Cultures were brought to a final volume of 200 µl of serum-free culture medium, pulsed with [3H]thymidine and harvested as described for proliferation assays.

Antibodies and flow cytometry
Antibodies used in this study include the mAb specific for Thy-1, 30H12 (27), and the rat IgG2b mAb isotype control, GK1.5, which is specific for CD4 (28); the Ly6A/E specific mAb D7 (29) and the rat IgG2a isotype control H129, which is also specific for CD4 (30). These mAb, as well as the IL-2-specific mAb, S4B6.34.1 (31), and the IL2R{alpha}-specific mAb, PC61 (ATCC), were purified from hybridoma supernatants on Sepharose conjugated with mouse anti-rat Ig{kappa}, MARK-1 (32). mAb specific for TCR Cß, H57-597 (33), and CD48, 5-8A10 (34), were purified on Protein A–Sepharose. Protein A–Sepharose purified normal hamster IgG (Jackson ImmunoResearch, West Grove, PA) was used as an isotype control for anti-CD48.

Flow cytometric analysis was performed following labeling of cells (1x105) in 100 µl of PBS containing 5% FCS with the indicated antibodies for 10 min on ice, followed by three washes in PBS/FCS. Prior to analysis on a FACSCalibur using Lysys software (Becton Dickinson), cells were resuspended in 1 µg/ml 7-amino-actinomycin-D (7-AAD; Sigma, St Louis, MO). Fluorescence data in all plots shown were collected as a list mode on 10,000 viable cells as determined by light scatter parameters and exclusion of 7-AAD. IL-2R expression was analyzed using mAb specific for IL-2R{alpha} (PC61) followed by FITC–mouse anti-rat IgG (Jackson ImmunoResearch); phycoerythrin-labeled mAb specific for IL-2Rß (PE-TMß); PharMingen, San Diego, CA); and mAb specific for IL-2R{gamma} [4G3 (35)] followed by FITC–mouse anti-rat IgG.

Cytotoxic T lymphocyte (CTL) assays
Lymph nodes cells were obtained from mice transgenic for a V{alpha}2/Vß8 TCR specific for a lymphocytic choriomeningitis virus (LCMV) glycoprotein-derived peptide, residues 33–41 (gp33), in the context of H-2Db (36). Cells (5x105) were stimulated in vitro for 2 days in the presence of 10–7 M gp33 and 4x106 irradiated syngeneic splenocytes in 24-well cluster tissue culture plates in serum-free medium. Viable cells were isolated on Lympholyte-M (Cedarlane, Hornby, Ontario, Canada) and cultured in 25 U/ml IL-2 for 5 days, after which cells were harvested and re-stimulated with gp33 for 3 days, as described above. Viable cells isolated at this point were used in subsequent assays. Cells were pre-incubated for 1 h at 37°C in flat-bottom 96-well cluster tissue culture plates which had been coated with 10 µg/ml of either anti-CD48 or normal hamster IgG. Following this pre-incubation, 104 51Cr (NEN/DuPont, Boston, MA)-labeled MC57G fibroblasts were added as target cells. Prior to their addition, the MC57G cells were pulsed with antigen by incubating 106 cells in 100 µl of 10–7 M gp33 for 1 h at 37°C followed by two washes in medium. The 51Cr release into the supernatant of cultures after a 6 h culture period at 37°C was quantitated using LumaPlates and a Topcount Counter (Canberra Packard, Meriden, CT). The percentage of specific lysis was calculated as (experimental release – spontaneous release)/(total release – spontaneous release)x100. The growth inhibitory effects of anti-Thy-1 and anti-CD48 on these cells was confirmed by performing proliferation assays in parallel. [3H]Thymidine uptake was assessed after 40 h of culture.

Northern blot analysis
The 2.10 T cells were untreated or cultured for 20 h in the presence of co-immobilized anti-TCR Cß and either anti-CD48 or normal hamster IgG. Total RNA was extracted using TRIzol (Gibco/BRL, Burlington, Ontario, Canada) as per the manufacturer's instructions. Briefly, cells were lysed in TRIzol and RNA was extracted by phenol–chloroform, precipitated in 50% isopropanol and washed in 75% ethanol. The ratio of optical densities of the RNA samples at 260 and 280 nm was consistently >1.8. Aliquots of 15 µg of each RNA sample were electrophoresed on a 1.2% agarose gel containing 3% formaldehyde, 0.02 M MOPS, 8 mM sodium acetate and 1 mM EDTA. RNA bands were transferred to a GeneScreen nylon membrane (Dupont) and cross-linked with UV light. The blots were pre-hybridized overnight at 42°C in 25 ml of 6xSSC, 50% formamide, 0.5% SDS, 10% dextran sulfate, 5xDenhardt's solution and 100 µg/ml herring sperm DNA, and then hybridized for 8 h with 25x106 c.p.m. of the indicated probe. Probes were prepared by radiolabeling the 1.3 kb EcoRI insert of pSI mIL-2R{alpha}; the EcoRI–NotI inserts of pSI mIL-2Rß (1.8 kb); or pSI mIL-2R{gamma} (1.2 kb), using a commercial kit (Pharmacia, Baie D'Urfe, Quebec, Canada). Labeled probes were separated from excess [32P]dCTP (Dupont) by chromatography on Sephadex G-50 columns (Pharmacia). After hybridization, membranes were washed twice with 2xSSC for 10 min at room temperature and then with 5xSSC, 1% SDS at 60°C. Results were visualized by autoradiography, and quantitative analysis was performed using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Blots were stripped by washing with 0.1xSSC and 1% SDS at 80°C for 1 h, and hybridization was carried out as above with 32P-labeled L32 ribosomal protein cDNA, which provided a loading control to which signals for the IL-2R chains were normalized (37).

Immunoprecipitations and immunoblotting
The 2.10 T cells were left untreated or cultured for 20 h in the presence of co-immobilized anti-TCR Cß and either anti-CD48 or normal hamster IgG. For determination of phosphotyrosine content of JAK1 and JAK3, cells were lysed in buffer containing: 50 mM HEPES, 1% Triton X-100, 150 mM NaCl, 10% glycerol, 1.5 mM MgCl2, 1 mM Na2VO4, 1 mM PMSF, and 10 µg/ml each of aprotinin and leupeptin, adjusted to pH 7.5. JAK1 and JAK3 were immunoprecipitated from post-nuclear fractions of lysates containing 3x106 and 1x106 cell equivalents respectively, using 5 µl of specific antisera (UBI, Lake Placid, NY). Proteins were resolved by SDS–PAGE and transferred to nitrocellulose. Immunoblotting was performed using the phosphotyrosine-specific mAb 4G10 (38), followed by horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Sigma) and revealed by enhanced chemiluminescence (Amersham, Oakville, Ontario, Canada). Membranes were stripped by incubating for 15 min at room temperature in buffer containing: 150 mM NaCl, 10 mM Tris–HCl, adjusted to pH 2.3, and levels of JAK1 and JAK3 were revealed by immunoblotting using anti-JAK1 (Transduction Laboratories, Lexington, KY) followed by goat anti-mouse IgG–HRP or JAK3-specific antisera (UBI) followed by Protein A–HRP (Amersham).

Immunoprecipitation of IL-2R chains was performed following lysis of cells in buffer containing: 0.5% NP-40, 10 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM Na2VO4, 1 mM PMSF, and 2.5 µg/ml each aprotinin and leupeptin, adjusted to pH 7.4. IL-2R ß and {gamma} chains were immunoprecipitated from post-nuclear fractions of lysate containing 1.5x107 cell equivalents using 0.5 ml 5H4 culture supernatant (26) or 20 µg 3E12 (35) respectively, bound to Protein A–Sepharose which had been pre-coated with rabbit anti-rat Ig (Jackson ImmunoResearch). Proteins were resolved by SDS–PAGE, transferred to nitrocellulose and immunoblotting was performed as indicated. Polyclonal anti-IL-2Rß and anti-IL-2R{gamma} chains were used for Western blot analysis (Santa Cruz Laboratories, Santa Cruz, CA).

IL-2 binding assays
Ligand–receptor binding studies were adapted from a previous description (39). Neo-IL-2Rß-2.10 cells stimulated overnight with co-immobilized anti-TCR Cß and either anti-CD48 or normal hamster IgG were incubated for 1 min in 10 mM sodium citrate and 150 mM NaCl adjusted to pH 4.0 to dissociate IL-2 bound to IL-2R. Following two washes in PBS containing 3% FCS and 0.1% sodium azide, cells were incubated with 5x10–10 M [125I]IL-2 (NEN, Boston, MA) for 30 min on ice. From this point, cells were maintained on ice to prevent the internalization of [125I]IL-2 bound to IL-2R. After two washes in PBS/0.1% sodium azide, cells were incubated at 2x107 cells/ml in 2 mM of the cross-linker DSS (Pierce, Rockford, IL) for 10 min on ice. Unreacted DSS was quenched by the addition of 5 mM ammonium acetate for 1 min. Cells were washed twice in PBS/0.1% sodium azide and lysed in buffer containing: 0.5% NP-40, 50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA with PMSF, and 2.5 µg each of leupeptin and aprotinin. IL-2R{alpha} was immunoprecipitated from post-nuclear fractions of lysate containing 5x106 cell equivalents using mAb 7D4 (40), and IL-2Rß and {gamma} chains were immunoprecipitated from post-nuclear fractions of lysate containing 5x107 cell equivalents with 5H4 and 3E12 respectively. Immune complexes were recovered using Protein A– Sepharose pre-coated with rabbit anti-rat Ig. Proteins were resolved by SDS–PAGE, gels were fixed in a solution of 40% methanol and 10% acetic acid, dried, and autoradiographed for 72 h at –70°C prior to developing.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Immobilization of GPI-linked proteins ablates T cell growth but not effector functions
To determine whether immobilization of GPI-linked proteins alters the consequences of signals emanating from the TCR–CD3 complex, we first assessed effects on primary T cells. CD8+ lymph node T cells were cultured on plates which had been pre-coated with mAb specific for TCR Cß in combination with mAb specific for Thy-1 (30H12) or an isotype control. As illustrated in Fig. 1Go(A), anti-TCR induced DNA synthesis was profoundly inhibited in the presence of anti-Thy-1, over a broad range of anti-TCR concentrations used to coat the culture wells. Further, while the coating concentration of the Thy-1-specific mAb used in this experiment was 10 µg/ml, titration of the coating concentrations for this mAb revealed that it mediated its effects from 100 to 3 µg/ml (not shown). Anti-TCR-induced DNA synthesis was unaffected by the isotype control (Fig. 1AGo) or mAb specific for other membrane molecules including CD45, CD8 and MHC class I (not shown). Further, the effects observed were not specific to mAb 30H12, as two other mAb specific for Thy-1 [M5/49 (ATCC) and 5-3.2.1 (PharMingen)] mediated identical effects (not shown). Importantly, immobilization of these mAb was required, as the addition of soluble mAb to plates coated with anti-TCR had no effect on the induction of T cell growth.



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Fig. 1. Antibodies to GPI-linked molecules inhibit anti-TCR Cß-induced T cell proliferation. Primary CD8+ T lymphocytes from lymph nodes (A) or the 2.10 T cell clone (B) were cultured in wells that had been coated with the indicated concentrations of anti-TCR Cß and 10 µg/ml of mAb specific for Thy-1 (circles) or a mAb isotype control (squares). After 40 h, each culture was pulsed with 1 µCi of [3H]Thymidine for 6 h, harvested and thymidine uptake assessed by liquid scintillation spectroscopy. (C) The proliferation of 2.10 cells in response to the optimal concentration of anti-TCR Cß (1 µg/ml), in the presence of 10 µg/ml Thy-1, Ly6A/E and CD48 specific mAb, or their isotype-matched control antibodies, was assessed as above. (D) Photographs (at x100 magnification) of 2.10 cells cultured for 20 h in the presence of immobilized anti-TCR Cß and anti-Thy-1 (left) or isotype-matched control mAb (right).

 
In aid of the further biochemical analysis of the signaling lesions in this system, we next determined whether the above observations could be extended to T cell clones. We have utilized the antigen-specific, IL-2-dependent T cell clone, 2.10 (24). As illustrated in Fig. 1Go(B), Thy-1-specific mAb inhibited anti-TCR-induced DNA synthesis of 2.10. Furthermore, mAb specific for CD48 and Ly6A/E mediate the same effects as anti-Thy-1 in this assay system, on both 2.10 (Fig. 1CGo) and primary CD8+ T cells (not shown). We have also assessed the effect of mAb specific for Qa-2 [695H1-1-2, 695H1-7-5 and 695H1-9-9 (41), TSA-1 [MTS35 (PharMingen)] and CD73 [TY/23 (42)], and observed less profound inhibition (40–60%), which in turn correlated with lower levels of membrane expression as assessed by flow cytometry (not shown). Thus, the phenomenon may be generalizable to GPI-anchored molecules.

Culture of primary T cells or clone 2.10 in plates coated with immobilized mAb specific for Thy-1 resulted in profound morphological changes. Within 1 h of culture at 37°C, 2.10 cells appeared flattened, with irregularities in cell shape and spindle-like projections. In contrast, cells proliferating in response to co-immobilized anti-TCR and control mAb have spherical morphology and grow in clusters (Fig. 1DGo). The photomicrographs presented in Fig. 1Go(D) are at the same final magnification, and thus emphasize the marked difference in the apparent size of the cells in the presence of immobilized isotype control and Thy-1-specific mAb. These changes in cell morphology are dependent upon the immobilization of mAb specific for Thy-1 and are not dependent on the presence of co-immobilized anti-TCR (not shown). Thus, Thy-1-mediated alterations in cell morphology are likely independent of signals generated upon TCR aggregation. These changes in cell morphology were also observed upon culture of cells in the presence of immobilized anti-CD48 or anti-Ly-6A/E, albeit to varying extents which may also be a reflection of the level of expression of the GPI-anchored protein. Anti-Thy-1 resulted in the most profound changes; cells cultured on plates coated with immobilized anti-Thy-1 were flattened and adherent such that they could not be harvested from the plates, even in the presence of trypsin. In contrast, cells cultured in the presence of mAb specific for CD48 or Ly6A/E could readily be harvested in PBS containing the divalent cation chelators EDTA and EGTA, and as such these conditions were employed in experiments described below which required cells in suspension, such as flow cytometric analysis. Changes in cell morphology were not observed with mAb specific for the transmembrane proteins CD8 or MHC class I (not shown).

Viability of clone 2.10 is strictly dependent on IL-2. The presence of this cytokine, provided exogenously or produced endogenously following antigen receptor stimulation, results in cell growth. Thus, it was striking to observe that the cells were not dying in conditions in which DNA synthesis was ablated by mAb specific for GPI-anchored proteins. We therefore assessed whether anti-TCR-induced IL-2 production was perturbed in these circumstances. As illustrated in Fig. 2Go(B), supernatant from growth inhibited cultures contained more IL-2 than those from cultures containing isotype control mAb, consistent with the fact that it was not being utilized in support of proliferation. Proliferation of the indicator cell line was shown to be mediated by IL-2, as thymidine uptake was inhibited by anti-IL-2 (Fig. 2BGo), but not by mAb specific for IL-4 or IL-5 (not shown). These results are identical to those obtained with primary T cells (Fig. 2AGo). Again, immobilization of these mAb was required to aid generating this phenotype. Thus, while immobilization of GPI-anchored proteins, with concomitant activation through the antigen receptor complex, ablated cell growth, cytokine-mediated effector functions remained intact.



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Fig. 2. Anti-TCR-induced IL-2 secretion is not inhibited by mAb specific for GPI-linked molecules. Supernatants from primary CD8+ T cells (A) or clone 2.10 (B) cultured for 20 h with anti-TCR and anti-Thy-1 (circles) or an isotype control (squares) were diluted as indicated, and IL-2 content was assessed by culture with the IL-2-dependent cell line CTLL-2. After 40 h, cultures were pulsed with 1 µCi of [3H]thymidine for 6 h, harvested and thymidine uptake assessed by liquid scintillation spectroscopy. Closed symbols indicate pre-incubation of the respective supernatants with 10 µg/ml anti-IL-2 prior to the addition of CTLL-2.

 
To assess the effects of this treatment on cell-mediated effector function, we determined whether mAb specific for CD48 perturbed CTL activity. We utilized lymph node T cells from mice transgenic for a LCMV-specific TCR (36). Lymph node cells from these transgenic animals were stimulated with gp33 peptide (residues 33–41) from LCMV glycoprotein in vitro and CTL activity assessed in the presence of anti-CD48 or isotype control. As illustrated in Fig. 3Go(A), the CTL activities were indistinguishable in these two culture conditions, over the indicated titration of E:T ratios. The effect of immobilized anti-Thy-1 was not assessed in these experiments as the target cells expressed this molecule and thus we would not be able to rule out any effects of antiThy-1 on target cells.



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Fig. 3. The cytotoxic T cell response is not affected by anti-CD48. (A) In vitro activated lymph nodes cells from TCR transgenic mice specific for LCMV glycoprotein-derived peptide gp33 (36) were incubated for 1 h in plates coated with anti-CD48 (circles) or normal hamster IgG (squares). 51Cr-labeled target cells (MC57G fibroblasts) incubated with gp33 (open symbols) or not (closed symbols) were then added and 51Cr release into the supernatant was assessed 6 h later. The growth inhibitory effect of anti-CD48 and anti-Thy-1 on these cells was confirmed by assessing thymidine uptake, after 40 h of culture, in response to 25 U/ml mIL-2 (B) or 1 µg/ml co-immobilized anti-TCR (C).

 
To ensure that the effects of immobilizing GPI-linked molecules on these CTL paralleled those observed in primary CD8+ T cells and clone 2.10, we determined the affect of anti-CD48 and anti-Thy-1 on proliferation assays set up in parallel with the CTL assays. These effector cells were derived from antigen-stimulated cultures and IL-2-supported proliferation was observed in the absence of further TCR stimulation. This growth was inhibited in the presence of either anti-CD48 or anti-Thy-1 (Fig. 3BGo). Furthermore, anti-TCR-induced proliferation was also inhibited by mAb specific for GPI-linked proteins (Fig. 3CGo). Thus, as for T cell effector functions supported by cytokines, a cell-mediated T cell function was not perturbed by the GPI-mediated growth inhibition.

Immobilization of GPI-linked proteins inhibits IL-2Rß expression
Notwithstanding the production of IL-2 by T cells stimulated with co-immobilized mAb specific for TCR and GPI-anchored proteins, the T cells exhibit a growth-inhibited phenotype. This suggests a lesion in IL-2R signaling. To determine the presence of the IL-2R chains necessary for IL-2-mediated T cell growth, we assessed the levels of membrane expression of IL-2R components. As illustrated in Fig. 4Go(A), the level of IL-2R{alpha} was in fact higher in the presence of anti-CD48 than in the presence of isotype control. Further, the level of IL-2R{gamma} was indistinguishable in the two culture conditions. In marked contrast, membrane expression of IL-2Rß was virtually undetectable in cultures containing anti-CD48. Steady-state levels of mRNA for each of the three chains were assessed by Northern blot analysis. Phosphor imaging of Northern blots revealed a 4-fold increase in the level of IL-2R{alpha} mRNA and no change in levels of IL-2R{gamma} mRNA when cells were cultured in the presence of anti-CD48 relative to isotype control (Fig. 4BGo). Further, only a 2-fold decrease in IL-2Rß mRNA was observed in these circumstances (Fig. 4BGo). Thus, the profound decrease in membrane expression of IL-2Rß is not accounted for by comparable changes in steady-state levels of mRNA, which in turn suggests regulation at the level of translation, internalization or indeed the capacity to detect the protein at the membrane of cells treated with immobilized mAb specific for GPI-linked proteins.



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Fig. 4. Antibodies to GPI-linked molecules alter IL-2R expression. The 2.10 cells were untreated, or cultured for 20 h in the presence of immobilized anti-TCR Cß (3 µg/ml) and co-immobilized anti-CD48 or normal hamster IgG. (A) Cells were analyzed by flow cytometry for the presence of IL2R{alpha}, ß and {gamma} chains. Open histograms represent staining with secondary antibodies alone, while shaded histograms represent specific staining. Numbers in the upper right corner represent the fold increase in mean fluorescence intensity over background. (B) Northern blot analysis of total RNA was performed with 32P-labeled murine IL-2R{alpha}, ß and {gamma} cDNA (35). The bottom panel shows Northern blot analysis of the membranes stripped and re-probed with 32P-labeled L32 ribosomal protein cDNA as a loading control (37). Sizes of RNA standards are indicated.

 
Forced expression of exogenous IL-2Rß does not revert GPI-mediated growth inhibition
If the growth-inhibited phenotype mediated by immobilization of GPI-anchored proteins was secondary to the lack of membrane expression of IL-2Rß, the prediction follows that forced expression of exogenous IL-2Rß should rescue growth. Towards testing this hypothesis, clone 2.10 was transfected with a construct encoding IL-2Rß. Cells were selected in G418 and populations expressing increased levels of IL-2Rß were isolated using a FACStar Plus. The resulting population (neo-IL-2Rß 2.10) expressed 3- to 4-fold higher levels of IL-2Rß compared to those expressed by either clone 2.10 or variants of 2.10 which had been transfected with the `empty' vector (neo-2.10) and selected in G418 (Fig. 5AGo, upper panel), without effect on the cell surface expression of IL-2R{alpha} and IL-2R{gamma} (not shown). Furthermore, the exogenous IL-2Rß was functional, as over-expressing cells displayed increased sensitivity to limiting concentrations of IL-2 (not shown). The question follows whether forced expression of this IL-2R chain reverts the phenotype mediated by immobilizing GPI-anchored proteins.



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Fig. 5. Forced expression of IL-2Rß does not rescue anti-TCR-induced proliferation in the presence of mAb specific for GPI-linked proteins. The 2.10 cells were transfected with a vector encoding the neor gene, or neor as well as the IL-2Rß gene. (A) The expression of IL-2Rß on G418 resistant cells was assessed by flow cytometry as in Fig. 4Go(A). Numbers in the upper right corner represent the fold increase in mean fluorescence intensity over background. (B) Thymidine uptake by untransfected 2.10 (circles), neo-IL-2Rß 2.10 (triangles) and neo-2.10 (squares) in response to co-immobilized anti-TCR Cß and either anti-CD48 (closed symbols) or normal hamster IgG (open symbols) was assessed as in Fig. 1Go. (C) IL-2Rß was immunoprecipitated from the indicated cell lines following culture for 20 h in the presence of immobilized anti-TCR Cß (3 µg/ml) and co-immobilized anti-CD48 or normal hamster IgG. Proteins were resolved by SDS–PAGE, material transferred to nitrocellulose and immunoblotting was performed using polyclonal rabbit anti-IL-2Rß.

 
Flow cytometric analysis revealed that, as was seen in untransfected clone 2.10, membrane expression of IL-2Rß on neo-IL-2Rß-2.10 was decreased in anti-TCR-stimulated cells in the presence of anti-CD48, relative to controls (Fig. 5AGo). Nonetheless, the expression of IL-2Rß in these circumstances was comparable to its expression on untransfected cells in control cultures (Fig. 5AGo, compare upper left and middle lower panels). Notwithstanding the maintenance of levels of IL-2Rß expression comparable to those observed in cells which respond to IL-2 with growth, cells stimulated with co-immobilized anti-TCR and anti-CD48 exhibited a growth inhibited phenotype (Fig. 5BGo). Thus, the lack of membrane IL-2Rß expression per se does not account for the observed lack of IL-2-mediated growth.

In order to determine whether IL-2Rß was still present in the cell in circumstances where it cannot be detected on the cell membrane by immunofluoresence, IL-2Rß was immunoprecipitated from cells cultured with anti-TCR and anti-CD48 or normal hamster IgG. As illustrated in Fig. 5Go(C), IL-2Rß was revealed in immunoprecipitates of neo-2.10 and neo-IL-2Rß-2.10 cells cultured in the presence of CD48-specific mAb, despite undetectable or decreased levels of cell surface expression.

Immobilization of GPI-linked molecules correlates with the lack of Janus kinase activation
The lesion in IL-2R signaling in this system was further analyzed by assessing the status of the Janus kinases, JAK1 and JAK3, previously demonstrated to be central in IL-2-induced cellular growth (43). Towards this end, cellular JAK1 and JAK3 were immunoprecipitated from lysates of clone 2.10 which had been cultured in the presence of co-immobilized anti-TCR and either anti-CD48 or an isotype control. Precipitates were resolved by SDS–PAGE, transferred to membranes, immunoblotted with phosphotyrosine-specific mAb and signals quantitated by scanning densitometry on a Storm PhosphorImager (Molecular Dynamics). As illustrated in Fig. 6Go, the phosphotyrosyl content of JAK1 and JAK3, indicative of their activation states (43), was reduced by factors of 10 and 5 respectively when cells were cultured in the presence of anti-CD48 relative to isotype control. Identical effects were mediated by immobilized mAb specific for Thy-1 (not shown). To ensure that IL-2 was not limiting in these cultures, despite the observed increase in levels of JAK tyrosine phosphorylation in control cultures, some cultures were supplemented with 1000 U/ml of exogenous IL-2. As illustrated in Fig. 6Go, even this concentration of IL-2 did not rescue the increase in JAK1/3 phosphotyrosyl content observed in the presence of isotype control mAb. To ensure equivalent loading, immunoblots were stripped and reprobed with either JAK1- or JAK3-specific antisera.



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Fig. 6. Antibodies to GPI-linked molecules inhibit IL-2-induced JAK activation. The 2.10 cells were untreated or cultured for 20 h in the presence of co-immobilized anti-TCR Cß and either anti-CD48 or normal hamster IgG. JAK1 and JAK3 were immunoprecipitated from cell lysates using specific antisera, and immunoblotting was performed using the phosphotyrosine-specific mAb (4G10). Lanes 4 and 5 are lysates from cells that had been pulsed with 1000 U/ml of recombinant mIL-2 (64) for 15 min prior to the end of the 20 h culture period. Membranes were stripped, and levels of JAK1 and JAK3 were revealed by immunoblotting.

 
Identical results were obtained using primary CD8+ T cells and variants of clone 2.10 overexpressing IL-2Rß (not shown). These results suggest that the IL-2R signaling lesion is membrane proximal in that coupling to the first second messenger generating system known to be operative in IL-2-mediated signal transduction is perturbed. While demonstrating a reduction in the activation of cellular JAK kinases, these results leave open the possibility that the stoichiometry of JAK1 and JAK3 association with IL-2Rß and {gamma} chains respectively is altered as a consequence of immobilizing GPI-linked proteins. The absence or reduction of constitutively associated JAK kinases with IL-2R could account for the observed phenotype.

Immobilization of GPI-linked proteins inhibits IL-2 induced formation of the hetero-trimeric IL-2R complex
To establish whether the defect in IL-2R-mediated JAK kinase activation could be due to the disruption of the association with their respective IL-2R chains, IL-2Rß and {gamma} chains were immunoprecipitated from lysates of neo-IL-2Rß-2.10 cells stimulated with co-immobilized anti-TCR and either CD48-specific mAb or an isotype control mAb. Precipitates were resolved by SDS-PAGE, transferred to membranes and immunoblotted with JAK1 or JAK3 specific antisera. As illustrated in Fig. 7Go(A), the stoichiometry of neither JAK1/IL-2Rß nor JAK3/IL-2R{gamma} association appeared altered in cells from anti-CD48-containing cultures compared to cells from cultures containing the isotype control mAb. Again, immobilized mAb specific for Thy-1 mediated effects which were identical to those observed using anti-CD48 (not shown). To ensure equivalent loading, these immunoblots were stripped and re-probed with IL-2Rß- and {gamma}-specific antisera (Fig. 7AGo). This result obviates the possibility that the absence of Janus kinase activation in this system is due to their lack of constitutive association with their respective IL-2R chains. Thus, the basal composition of the IL-2R chains in this context appeared poised to respond to IL-2, yet did not.



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Fig. 7. Neo-IL-2Rß cells were cultured for 20 h in the presence of co-immobilized anti-TCR Cß and either anti-CD48 or normal hamster IgG. (A) IL-2Rß or {gamma} chains were immunoprecipitated, proteins resolved by SDS–PAGE, material transferred to nitrocellulose and immunoblotting was performed as indicated. (B) Cells were incubated with 125I-labeled IL-2. After two washes, bound IL-2 was cross-linked to cell-surface proteins. Following lysis, IL-2R{alpha}, ß and {gamma} were immunoprecipitated, and proteins resolved by SDS–PAGE. Gels were fixed, dried and visualized by autoradiography.

 
The activation of JAK1 and JAK3 is thought to be mediated by IL-2-induced hetero-trimerization of the IL-2R chains. Ligand-mediated aggregation of these chains would result in the juxtaposition of associated kinases, leading to their transphosphorylation and activation (43). Since we have demonstrated that GPI-mediated growth inhibition is not due to the disrupted association of these kinases with their respective IL-2R chains, it is plausible that the impaired activation of JAK kinases reflects the inability of IL-2 to recruit the IL-2R chains, resulting in defective hetero-trimerization and absence of high-affinity IL-2R.

This question was addressed by taking advantage of the capacity of the anti-IL-2R{alpha} mAb 7D4 to efficiently immunoprecipitate IL-2 cross-linked to either IL-2R{alpha}, ß or {gamma} as a stable multi-subunit complex. The variant of clone 2.10 in which IL-2Rß was over-expressed was used to ensure the presence of sufficient levels of membrane receptor. Thus, neo-IL-2Rß-2.10 cells derived from cultures containing co-immobilized anti-TCR and either anti-Thy-1 or isotype control mAb were incubated with 125I-labeled IL-2. Cell-bound IL-2 was chemically cross-linked to membrane proteins using DSS, followed by lysis and the immunoprecipitation of IL-2R chains from post-nuclear fractions of lysates. In cells stimulated with anti-TCR and an isotype control, immunoprecipitation of IL-2R{alpha} revealed three bands. The mobility of these bands corresponded to that expected for IL-2 complexed with each of IL-2R{alpha}, {gamma} and ß (Fig. 7BGo). Immunoprecipitation with anti-IL-2Rß and {gamma} confirmed the identity of the two upper bands (Fig. 7BGo). In marked contrast, immunoprecipitation from lysates of anti-Thy-1 growth-inhibited cells with anti-IL-2R{alpha} revealed a dominant band migrating with an apparent mol. wt of 70 kDa and thus corresponding to IL-2 complexed with IL-2R{alpha}. The bands consistent with IL-2 complexed with IL-2Rß and {gamma} chains were not observed in anti-IL-2R{alpha} precipitates from lysates of these cells. As illustrated in Fig. 7Go(B) and consistent with a profound reduction in the generation of the high-affinity hetero-trimeric IL-2R in the presence of immobilized anti-Thy-1, direct precipitation with mAb specific for IL-2Rß and {gamma} did not reveal bands consistent with IL-2 complexed with either of these IL-2R chains.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
A GPI anchor imparts functional attributes upon membrane molecules, including localization to glycosphingolipid membrane microdomains and increased lateral mobility in the membrane. Furthermore, as many GPI-anchored proteins have been implicated in T cell signaling and, moreover, have been shown to mediate similar effects, the GPI anchor may impart signaling functions. The aims of this study were to address whether GPI-linked proteins mediate a common function in regulating T cell growth and to characterize the mechanism through which this regulation occurs. The approach taken was to determine the effects of immobilized mAb specific for a panel of GPI-anchored proteins on T cells stimulated with co-immobilized anti-TCR. The effect of immobilized mAb may be to impede the mobility of GPI-linked proteins in the membrane, to prevent their interaction with ligands or to induce signaling events.

We demonstrated that in both primary CD8+ T cells and T cell clones, anti-TCR-induced proliferation was profoundly inhibited in the presence of co-immobilized mAb specific for the GPI-linked proteins Thy-1, CD48 and Ly6A/E. Other transmembrane molecules expressed on the surface of these T lymphocytes, including CD45, CD8 and MHC class I, did not alter proliferative responses. Thus, inhibitory effects on T cell proliferation may be generalizable to GPI-anchored molecules and may reflect the perturbation of common signaling pathways. Src family PTK, associated with GPI-linked molecules in glycosphingolipid membrane microdomains and implicated in signaling through these proteins, may be critical in growth inhibition. Importantly, there is a precedent for Src PTK in inhibition of growth, as the anti-proliferative effects of IFN-{alpha} in T cells have been shown to require p56lck (44).

Immobilized mAb specific for GPI-linked molecules resulted in rapid and profound morphological changes in T cells. In contrast to the spherical shape and growth in clusters which was observed in control cultures, mAb-mediated ligation of GPI-anchored proteins resulted in cell flattening and irregularities in cell shape, such as the generation of spindle-like projections. These morphological changes may reflect re-organization of the actin cytoskeleton and may also involve activation of Src family PTK, which have been implicated in cytoskeletal changes (45). Consistent with this hypothesis, similar morphological changes have been observed in response to immobilized anti-CD45 (46) and are blocked by inhibitors of actin polymerization, as well as by a PTK inhibitor. Moreover, the phosphoproteins induced by immobilized anti-CD45 are co-immunoprecipitated with anti-p56lck (46). Further, a GPI-linked protein (uPAR) and Src PTK have been co-precipitated with the ß2 integrin (CR3) (47), and other GPI-linked proteins undergo lateral associations with integrins (48), presenting another mechanism through which signaling and cytoskeletal organization can be effected by proteins tethered to the membrane through a GPI anchor.

A hallmark of the growth inhibited phenotype described herein is that T cell effector functions are retained. Thus, despite the growth inhibition induced by co-immobilized mAb for TCR and GPI-anchored proteins, both primary cells as well as the IL-2-dependent 2.10 T cell clone, remained viable. This was particularly striking for the latter, as its viability is strictly IL-2 dependent. Assessment of the amount of IL-2 in culture supernatants of growth-inhibited T cells revealed the presence of more IL-2 than in control cultures, consistent with the lack of utilization of this cytokine in support of proliferation. Further, cell-mediated cytolysis proceeded unimpaired in circumstances of growth inhibition. Consistent with these observations is that while Ly6A-deficient cells from knockout mice exhibit dysregulated anti-TCR-induced proliferative responses, cytolytic functions were not perturbed (49).

It needs to be emphasized that the GPI-mediated state of T cell physiology reported here required the immobilization of mAb specific for the various GPI-linked proteins. Other methods of ligating these molecules result in a distinct T cell phenotype. Corresponding with previous reports (1,3,50), when T cell activation was achieved through the addition of soluble anti-TCR, the addition of soluble mAb specific for GPI-anchored proteins, in the presence of FcR-bearing accessory cells, inhibited both anti-TCR-induced proliferation and IL-2 secretion (not shown). Further, recent reports have implicated GPI-anchored proteins in the potentiation of early signals generated through the TCR (51) and, specifically, soluble anti-CD48 was shown to enhance TCR-induced tyrosine phosphorylation of intracellular substrates (52). Thus, mAb specific for GPI-linked proteins can affect membrane proximal signals emanating from the TCR–CD3 complex. Signaling through TCR results in the recruitment of detergent-insoluble membrane microdomains or `rafts' which are enriched in GPI-anchored proteins and signaling molecules (52,53). GPI-anchored proteins may affect TCR-induced signaling by modulating the association of TCR–CD3 with these membrane microdomains. While we have not specifically addressed the effect of co-immobilized mAb specific for GPI-anchored proteins on lipid rafts, it is unlikely that TCR-induced signaling is disrupted, as signaling sequelae resulting in IL-2 secretion are intact. We have characterized a system whereby mAb specific for GPI-anchored proteins do not interrupt TCR signaling, but rather affect downstream events to inhibit IL-2-induced proliferation. Thus, the role of GPI-linked molecules in T cell activation may be 2-fold, affecting both early and late activation events.

It has been demonstrated that inhibition of T cell cytokine secretion and cell growth by soluble mAb specific for Ly6A/E and Tsa-1 (Sca-2) was independent of the GPI anchor, as transmembrane forms of these molecules mediated the same effects as wild-type molecules (4,54). However, we cannot infer that the GPI anchor is dispensable in mediating the effects described in this study and it remains to be determined whether transmembrane forms of Ly6A/E or Tsa-1 can mediate the growth-inhibited phenotype supported by their GPI-linked counterparts. Furthermore, it is plausible that the proteinaceous portions of Ly6A/E and Tsa-1 are involved in the inhibition of T cell activation, independently and in addition to functions imparted by the GPI anchor. Importantly, and in contrast to the dispensability of the GPI anchor in processes supporting the inhibition of cytokine secretion and cellular growth, the activation of T cells following aggregation of GPI-linked proteins is dependent upon the GPI anchor. Thus, aggregation of transmembrane forms of Ly6A/E, CD55 or Qa-2 does not result in cell growth (1214). The demonstration in the present study that all the GPI-anchored proteins tested to date mediated the same phenotype strongly suggests that the GPI anchor is critical.

Towards elucidating the basis for GPI-mediated growth inhibition, notwithstanding the presence of IL-2, we assessed the levels of membrane expression of IL-2R components. Immobilized mAb specific for GPI-linked proteins resulted in perturbations in the expression of the hetero-trimeric IL-2 receptor. While IL-2R{alpha} and {gamma} expression was increased or unchanged, membrane expression of IL-2Rß was virtually undetectable in cultures containing anti-CD48. This result was not reflected in steady-state mRNA levels, as Northern blot analysis revealed only a modest decrease in IL-2Rß mRNA. IL-2Rß expression may be regulated at the level of translation or by receptor internalization. Alternatively, the ability to detect IL-2Rß at the cell surface by flow cytometry may be impaired as a result of conformational changes or membrane alterations in cells treated with immobilized mAb specific for GPI-linked proteins. It is of note in this context that despite undetectable levels of expression of membrane IL-2Rß by immunofluorescence, the protein was readily detectable in lysates of growth inhibited cells. However, the lack of membrane IL-2Rß expression per se did not account for the observed lack of IL-2-mediated growth. Forced expression of exogenous IL-2Rß did not rescue anti-TCR induced proliferation, despite levels of expression in the presence of GPI-specific mAb comparable to untransfected cells in control cultures. Thus, membrane expression of all three of the IL-2R components in circumstances of growth inhibition is consistent with disruption of IL-2R signaling.

The protein tyrosine kinases JAK1 and JAK3 are constitutively associated with the IL-2Rß and {gamma} chains respectively, and are critical in IL-2R signal transduction. We demonstrate that these interactions are not disrupted in the presence of mAb specific for GPI-linked molecules. Nonetheless, IL-2 failed to induce increases in the phosphotyrosyl content of these associated kinases, which is indicative of their activation. This result suggests that the uncoupling of IL-2-mediated JAK kinase activation may be due to a block in ligand-mediated receptor heterotrimerization. A defect in the aggregation of the receptor chains would result in the failure to juxtapose JAK1 and JAK3, and in turn, their impaired transphosphorylation and activation. IL-2 binding assays were performed using clonal variants overexpressing IL-2Rß to ensure the ability to immunoprecipitate IL-2R chains. IL-2-mediated receptor aggregation was indeed perturbed in circumstances of growth inhibition. Immunoprecipitation of IL-2R{alpha} revealed three bands corresponding to IL-2 cross-linked to IL-2R{alpha}, {gamma} and ß. In contrast, immunoprecipitation of IL-2R{alpha} from cells stimulated in the presence of anti-Thy-1 revealed a predominant band corresponding to IL-2 cross-linked to IL-2R{alpha}. Furthermore, bands visualized following the immunoprecipitation of IL-2Rß and {gamma} were decreased in these circumstances. Thus, and possibly reflected in the profound changes in cell morphology induced by these culture conditions, IL-2 induced redistribution of membrane IL-2R chains was inhibited and subsequent growth responses to IL-2 proportionately reduced. The impaired generation of the high-affinity hetero-trimeric IL-2R complex in these circumstances suggests that the mobility of some of the chains may be restricted by the treatment. Of note in this context is that IL-2Rß, but not IL-2R{alpha}, is localized within detergent-insoluble subdomains of the plasma membrane (55).

These results characterize a novel mechanism through which T cells could mediate their effector functions in the absence of clonal expansion. There are data consistent with a physiological role for GPI-anchored proteins in the maintenance of cellular homeostasis and function. Specifically, insight is derived from analyses of patients suffering from paroxysmal nocturnal hemoglobinuria (PNH), an acquired hematopoietic stem cell disorder characterized by a lack of GPI-anchored proteins due to a mutation in the Pig-A gene, which is required for anchor biosynthesis (56). As several complement regulatory molecules are GPI-linked, PNH is characterized by complement-mediated hemolysis. In PNH patients, a stem cell clone with a somatic mutation in Pig-A expands such that a large proportion of hematopoietic cells harbors this mutation. This suggests that the lack of expression of GPI-anchored proteins imparts a growth advantage (57). Consistent with this notion, following in vitro stimulation of peripheral blood cells from patients with PNH, the proportion of GPI-deficient T lymphocytes increased relative to GPI+ T cells from the same patient (58). Further, a recent report describing the bone marrow transplantation of a PNH patient with material derived from a healthy identical twin demonstrated that GPI cells in the recipient had a survival advantage (59).

Studies using targeted gene knockout mice also support a role for GPI-linked proteins in regulating T cell growth in vivo. Mice lacking GPI expression on T lymphocytes were generated as a result of a T cell-specific disruption of the Pig-A gene (60) and have demonstrated that GPI-anchored proteins are not critical in the initiation of T cell activation, as had been previously suggested (51,61,62). These mice do not have defects in T cell development and peripheral responses to superantigen. However, the role of GPI-anchored proteins in growth control was not assessed by extensive characterization of T cell responses. Thymocytes from Thy-1-deficient mice show modest increases in responsiveness to stimulation through the antigen receptor and the differentiation of thymocytes from the double-positive to the single-positive stage is decreased, perhaps due to increased TCR signaling and negative selection (63). Further, T cells lacking Ly6-A expression display prolonged proliferation following stimulation through the antigen receptor complex (49). These results suggest that in the absence of any given GPI-linked protein, subtle defects in growth control occur; however, signaling mediated by the remaining GPI-anchored proteins may compensate to maintain growth regulation. In vitro, the outcome of anti-TCR-induced activation is profoundly altered using immobilized antibodies specific for a given GPI-anchored molecule; however, in vivo, all GPI-anchored proteins expressed on the surface of a cell may contribute to the regulation of T cell growth. In support of this, mAb specific for Thy-1 and CD48 inhibit the proliferation of CD8+ lymph node T cells from Ly6-deficient animals (M. D. Marmor and M. Julius, unpublished observations), demonstrating that other GPI-linked molecules can modify anti-TCR-induced proliferation of Ly6A-deficient cells. The extent to which a GPI-anchored protein contributes to the regulation of cell growth may depend upon its level of expression. mAb specific for Qa-2, CD73 and TSA-1 mediated less profound inhibition of anti-TCR-induced proliferation than Thy-1, CD48 or Ly6A/E, which in turn correlated with lower levels of membrane expression as assessed by flow cytometry.

A plausible physiological paradigm consistent with the observations presented in this study characterizes a role for GPI-anchored membrane proteins in regulating clonal expansion. Effector T cells specifically localizing to sites of inflammation could be confronted with an array of previously described and/or as yet uncharacterized ligands for GPI-anchored membrane proteins expressed on the surrounding inflamed tissues, which in turn could effect the circumstances which we have created in vitro. The consequences would ensure that the peripheral pool of T cells is not unduly diluted, while effector function towards clearance of the insult would proceed unimpaired.


    Acknowledgments
 
We thank Drs W. Stanford and P. Flood for providing the Ly-6A-deficient mice; H. Reiser for the 5-8A10 hybridoma; B. Drucker for the 4G10 mAb; F. Fitch for the D7 hybridoma, L. Thompson for the TY/23 hybridoma, and D. Sachs for hybridomas 695H1-1-2, 1-7-5 and 1-9-9. This work was supported by the Medical Research Council of Canada. M. D. M. is supported by an MRC Studentship. T. R. M. is supported by the NIH (CA45957 and AI40114).


    Abbreviations
 
7-AAD7-amino-actinomycin-D
CTLcytotoxic T lymphocyte
GPIglycosylphosphatidylinositol
HRPhorseradish peroxidase
IL-2RIL-2 receptor
LCMVlymphocytic choriomeningitis virus
PEphycoerythrin
PNHparoxysmal nocturnal hemoglobinuria
PTKprotein tyrosine kinase

    Notes
 
3 Present address: Basel Institute for Immunology, Basel, CH 4005, Switzerland Back

Transmitting editor: J. F. Kearney

Received 3 February 1999, accepted 10 May 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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