β-Galactoside-Binding Protein (βGBP) Alters the Cell Cycle, Up-Regulates Expression of the α- and β-Chains of the IFN-γ Receptor, and Triggers IFN-γ-Mediated Apoptosis of Activated Human T Lymphocytes¹

Acomplex interplay between growth-promoting and growth-inhibiting signals controls the clonal expansion of Ag-activated T lymphocytes. Among these factors, cytokines are of critical importance for T lymphocyte survival and progression through the cell cycle (1). Often their effect appears double-edged, since they may trigger either proliferation or apoptosis of T lymphocytes, depending on the presence or absence of concomitant environmental signals (2), the phases of the cell cycle (3, 4), or the intensity of the expression of their receptors on the cell membrane, as has been shown for IL-2 and IFN-γ (3, 5, 6).

A more precise understanding of the mechanisms controlling T lymphocyte response can be sought through the characterization of other factors involved in its regulation. β-galactoside-binding protein (βGBP),³ a 15-kDa protein encoded by the LGALS1 gene (7, 8, 9), is a negative regulator of cell cycle that blocks the transition from S to G₂ phase (7). βGBP is physiologically released by fibroblasts (7), and its structure places it in the galectin family, of which the members are animal lectins characterized by their affinity for β-galactoside residues (9, 10). βGBP exists both as a monomer (7) and a homodimer (11, 12). These two molecular forms are in a state of equilibrium (13) and bind β-galactoside residues on the cell membranes (14). Through its retention of associated cell surface β-galactoside residues, the homodimer form (11, 13) displays a wide range of biologic activities involving cell adhesion and immune regulation (9, 10, 11, 12, 13, 14, 15, 16). Besides the binding to β-galactosides residues, additional functions have been attributed to monomeric form. It has been reported, in fact, that the monomer interacts with a high affinity cell surface receptor on target cells, since its biologic activity is maintained even when the saccharide-binding site is masked by a glycan complex (7).

In this paper, the effects of monomeric βGBP on the cell cycle progression and expansion of activated normal and malignant human T lymphocytes were examined. Even in the presence of lactose, βGBP markedly inhibited their proliferation by arresting them in the S and G₂/M phases. In addition, by up-regulating the expression of both the α- and β-chains of their membrane IFN-γR, it rendered them sensitive to IFN-γ-induced apoptosis (6).

These findings suggest that βGBP plays an important immunoregulatory role by switching off T lymphocyte effector functions. Moreover, they provide the first evidence of up-modulation of IFN-γR expression, particularly that of its β-chain, on T lymphocytes by a negative cell growth regulator.

RPMI 1640, FCS, l-glutamine, penicillin, streptomycin, gentamicin, and trypan blue dye were from Life Technologies (Grand Island, NY); PHA, paraformaldehyde, RNase A, Tween 20, lactose, DMSO, and propidium iodide (PI) were from Sigma (St. Louis, MO); PBS, BSA, ethanol, and sodium azide were from Merk Chemicals (Milan, Italy); phycoerythrin (PE)-conjugated streptavidin, biotin-conjugated rabbit anti-mouse Ig, mouse IgG1, and mouse IgG2a control Ab were from Dako (Milan, Italy); mouse anti-IFN-γRα IgG1 (γR99) and rIFN-γ were a gift from Dr. G. Garotta (Human Genome Sciences, Rockville, MD); mouse anti-IFN-γRβ IgG2a (C.11) was a gift from Dr. S. Pestka (Piscataway, NJ); cell culture glassware was from Corning Costar (Cambridge, MA).

The cDNA enclosing the full length coding sequence (L. Mallucci and V. Wells, international patent application W092/07938) of the human βGBP protein was isolated by screening a human lung fibroblast cDNA library in λgt 11 (Stratagene, Cambridge, U.K.) using a full length murine βGBP cDNA (7). The human cDNA was first subcloned into puc 19 vector using a Kpn-SstI fragment. The sequence encoding the human βGBP cDNA was then amplified by PCR using forward primer 5′-G TCA ATC ATG GCT TGT GGT CTG GTC-3′ and reverse primer 5′-GT TCA GTC AAA GGC CAC AC-3′ and directly cloned into the eukaryotic cloning vector pCR3.1 using Escherichia coli strain TOP 10F′. The extracted plasmid was transfected in COS1 cells and the recombinant protein purified by immunoaffinity chromatography following procedures described previously (7). The competing sugar (100-mM lactose) was used after preincubation with the βGBP solutions for 20 min at room temperature.

Human PBL from heparinized venous blood from healthy donors were isolated by Lymphoprep gradient (Ficoll Type 400, Pharmacia, Uppsala, Sweden) centrifugation and stimulated (1 × 10⁶/ml) with 2.5 μg/ml PHA (Sigma). The cells resulting after 3 days of culture (94–98% CD3⁺) received 50 U/ml r-IL2 (EuroCetus, Milan, Italy). After a further 3 days, they were treated with βGBP, and r-IL2 was added. Cells were examined every 24 h.

ST4 (CD1⁺, CD2⁻, CD3⁻, CD4⁻, CD8⁺, CD25⁻), PF382 (CD1⁺, CD2⁻, CD3⁻, CD4⁻, CD8⁺, CD25⁻), and Jurkat (CD1⁺, CD2⁺, CD3⁺, CD4⁺, CD8⁻, CD25⁻) are previously described human T cell lines (5).

PHA-activated T lymphocytes (1 × 10⁶/ml) were cultured in complete medium supplemented or not with βGBP at different doses. At 24, 48, and 72 h, a small aliquot of the cell suspension was removed; 50 μl of this was mixed with 10 μl of trypan blue dye, and viable cells were counted. The results are expressed as the arithmetic mean of cell numbers from triplicate cultures. The experiments were performed with six donors and representative experiments are shown.

PHA-activated T lymphocytes (1 × 10⁶/ml) were cultured in triplicate in round-bottom 96-well plates in the presence or absence of 400 ng/ml βGBP. After 66 h, the cultures were pulsed with 1 μCi [³H]TdR (Amersham, Milan, Italy). After 6 h, the cells were harvested on a glass fiber filter and [³H]TdR uptake was evaluated by Matrix-96 beta counter (Canberra-Packard, Milan, Italy). The results are expressed as the arithmetic mean ± SD of total cpm from triplicate cultures.

DNA staining for cell cycle analysis was performed as previously described (17). Briefly, 1 × 10⁶ cells were resuspended in 0.875 ml of cold PBS and 0.125 ml of cold 2% paraformaldehyde solution, then incubated for 1 h on ice. The fixed cells were washed and gently resuspended in 1 ml of 0.2% Tween 20 in PBS at room temperature. The mixture was incubated for 20 min at 37°C. One milliliter of PBS supplemented with 2% FCS and 0.1% NaN₃ (PBS-azide) was added, and the suspension was spun for 5 min at 1300 rpm. After the supernatant was decanted, DNA was stained by incubating the cells in 1 ml of PBS-azide containing 10 μg/ml PI and 11.25 Kunitz U of RNase for at least 30 min in the dark. DNA content was analyzed by flow cytometry.

Cell surface expression of the α- and β-chains of IFN-γR was assessed by flow cytometry analysis. The α-chain was detected with the murine IgG1 mAb γR99, which is specific for this chain (5, 6). The β-chain was detected by using a murine IgG2a mAb (6). Staining was followed by biotinylated rabbit anti-mouse Ig and streptavidin-PE. Membrane Ag expression was analyzed with a FACScan flow cytometer (Becton Dickinson, Milan, Italy). Each analysis represents the results from 10,000 events.

Apoptosis was evaluated by fluorochrome labeling of DNA strand breaks by means of the terminal deoxynucleotidyl transferase (TdT) assay, using the Apo-Direct kit from PharMingen (San Diego, CA). This procedure allows the detection of apoptotic cells simultaneously with their DNA content (18). Fixation and staining were performed according to the manufacturer’s instructions. Briefly, 1 × 10⁶ cells per sample were suspended in 0.5 ml of PBS. Cell suspensions were supplemented with 5 ml 1% paraformaldehyde in PBS and placed on ice for 15 min. Cells were then washed twice in 5 ml PBS, and 5 ml of ice-cold 70% ethanol was added. The samples were stored at −20°C until use. Each sample was incubated for 60 min at 37°C with TdT and FITC-dUTP in a reaction buffer. The cells were then washed, resuspended in 1 ml of PI and RNase solution, and incubated for 30 min at room temperature. Samples were analyzed by flow cytometry within 3 h of staining.

PHA-activated T lymphocytes (94–98% CD3⁺) from six healthy donors were cultured in medium containing 50 U/ml IL-2 and βGBP (4–400 ng/ml) for 72 h. A dose-dependent βGBP inhibition of IL-2-induced proliferation was observed when their expansion was evaluated by direct cell counting (Fig. 1 A). The IL-2-independent proliferation of three malignant T cell lines, ST4, PF382 and Jurkat, was also inhibited by βGBP (data not shown).

The kinetics of both cell viability and apoptosis in βGBP-treated T lymphocytes was evaluated to look for the mechanism of this inhibition. Increasing doses (4–400 ng/ml) of βGBP had no effect on the percentage of viable (Fig. 1,B) and apoptotic (Fig. 2 A) T cells.

The effect of 400 ng/ml of βGBP on IL-2-dependent T cell proliferation and cell viability was not altered when its sugar-binding sites were saturated with 100-mM lactose (Figs. 1, A and B).

Since 400 ng/ml (27 × 10⁻⁹ M) βGBP abolished T lymphoblast proliferation but had no effect on viability, its influence on the cell cycle was next evaluated through cytofluorometric analysis of DNA content by PI staining (Fig. 2,A). At the beginning of the cultures established in medium supplemented with IL-2, few PHA-activated T lymphoblasts were in the S and G₂/M phases (cycling cells), the remainder being in the G₀/G₁ phases. The numbers of cycling cells increased progressively after 24 and 48 h. After 72 h, they dropped to the start levels (Fig. 2,A, upper panels). This pattern suggests that T lymphoblasts responded to IL-2 by expanding 3.5-fold (Fig. 1,A) and then returned to a steady state with most of them in G₀/G₁ phases. The presence of 400 ng/ml βGBP induced a dramatic inhibition in cell number but no striking differences in DNA content for the first 48 h of culture. (Fig. 2 A, lower panels).

The ST4, PF382, and Jurkat cell cycle was also arrested in S/G₂ phase by βGBP (data not shown).

Saturation with 100 mM lactose did not change the cell cycle in the presence or absence of 400 ng/ml βGBP (Fig. 2 B), showing that its blocking effect does not depend on its sugar-binding properties.

Negative regulation of cell proliferation by βGBP is due to its ability to arrest cells between the S and G₂ phases (7). The inhibition of cell expansion due to the presence of βGBP strongly suggests that βGBP impairs the growth of T lymphocytes by accumulating them in S and G₂/M phases, as previously reported for fibroblasts (7). The discrepancy in S and G₂/M distribution observed after 72 h between T lymphocytes cultured in the presence (42%) or absence (24%) of βGBP (Fig. 2 A, 72 h, upper vs lower panel) was not due to enhanced growth, but to arrest in S/G₂ phases. To confirm that the increase in numbers of cells in S/G₂ was due to arrest by βGBP, the [³H]TdR uptake of IL-2-responding T lymphocytes cultured in the presence or absence of βGBP was evaluated. [³H]TdR uptake measures cells in the S phase, so T lymphocytes cultured with βGBP could display an enhancement of this value after 72 h as the result of their accruing in S/G₂ phases.

When DNA synthesis was evaluated, a >60% increase in [³H]TdR uptake was evident only in the presence of 400 ng/ml βGBP (Fig. 3). The increase induced by 400 ng/ml βGBP was not affected by saturation of its sugar-binding sites with 100 mM lactose (Fig. 3).

We have previously shown that the intensity of the IFN-γR α- and β-chains’ expression on the T cell membrane decides whether IFN-γ induces proliferation or apoptosis (6). Therefore, the ability of βGBP to modulate IFN-γR chains was evaluated next. Cytofluorometric analysis with the anti-IFN-γRα γR99 mAb and the anti-IFN-γRβ C.11 mAb indicated that PHA-activated T lymphocytes cultured for 72 h in the absence of 400 ng/ml βGBP expressed high α-chain and barely detectable β-chain levels (Fig. 4,A, left panels). The presence of βGBP further enhanced the α-chain expression and elicited a marked expression of the β-chain (Fig. 4 A, right panels).

The kinetics of the fluorescence mean of α- and β-chain-positive cells also shows that βGBP up-regulates the expression of both chains (Fig. 4 B). This increase began after 24 h and peaked after 72 h. Once again, saturation with 100-mM lactose had no effect. Similar results were obtained with the ST4, PF382, and Jurkat lines (data not shown).

The binding of IFN-γ to T lymphocytes expressing high amounts of IFN-γR β-chain induces their apoptosis (6, 19). Since βGBP induced a substantial β-chain increase, the extent to which normal and malignant T cell apoptosis was enhanced by the addition of IFN-γ was investigated.

PHA-activated normal T lymphocytes were first cultured for 72 h in the presence or absence of 400 ng/ml βGBP and then for a further 48 h in complete medium, with or without 1000 U/ml IFN-γ. No apoptosis was detected by evaluating the induction of DNA strand breaks with the TdT technique and PI staining when cells were cultured in medium only (Fig. 5, upper left), with IFN-γ alone (Fig. 5, upper right panel), or with βGBP alone (Fig. 5, lower left), whereas it was dramatically evident in the presence of βGBP and IFN-γ (Fig. 5, lower right).

Further evidences of βGBP-induced sensitivity to IFN-γ-mediated apoptosis came from experiments with ST4 malignant T cells cultured in the presence or absence of 400 ng/ml of βGBP for 48 h. DNA content analysis showed an expected block in S/G₂ phase induced by βGBP (Fig. 6, left). Cells were then washed and cultured for a further 48 h in complete medium supplemented (Fig. 6, right) or not (Fig. 6, middle) with 1000 U/ml of IFN-γ, and apoptosis was evaluated. No changes in apoptosis or cell cycle were observed when cells previously cultured in the absence of βGBP (Fig. 6, upper left) were recultured in the presence or absence of IFN-γ (Fig. 6, upper middle and right panels). ST4 cells previously cultured in the presence of βGBP were blocked in S/G₂ phases (Fig. 6, lower left) and restarted their normal cycle when washed and recultured in medium only (Fig. 6, lower middle). This indicated that the βGBP effect on cell cycle arrest disappears following its removal. By contrast, the number of apoptotic cells soared to 60% when they were recultured in medium supplemented with IFN-γ (Fig. 6, lower right). Similar results were obtained with the PF382 and Jurkat lines (data not shown).

These findings demonstrate that βGBP inhibits the proliferation of both malignant and normal activated T lymphocytes by blocking them in the S/G₂ phases. This βGBP-induced increase of arrest of T lymphocytes impaired their ability to exit from G₂ phase and move to G₀/G₁ phases and dramatically inhibited their numeric expansion. βGBP-related perturbation of cell cycle up-modulates α- and β-chain IFN-γR expression and thus favors their bias toward IFN-γ-mediated apoptosis (6, 19).

The βGBP arrest of T cell cycle may have a broader physiologic consequence. Its effect on the cycle of PHA-activated T lymphoblasts and malignant T cell lines is a block at the S/G₂ checkpoint. This block is no longer evident on T lymphoblasts 96 to 120 h of culture (data not shown). Further evidence of the reversibility of this arrest was provided by its disappearance in ST4 cells when they were washed and βGBP was removed (Fig. 6).

The discrepancy between the S and G₂/M distribution and [³H]TdR uptake values observed in T lymphocytes cultured in the presence of βGBP is only apparent. The observation that βGBP induces the accumulation of T cells in S/G₂ phases and thus blocks their progress through the cycle mirrors its control of the S/G₂ checkpoint previously reported on fibroblasts, where it acts in the late S phase and arrests them in G₂ (7). The increased [³H]TdR uptake of activated T cells in the presence of βGBP appears to be the result of temporary accumulation of T cells in S/G₂ phases as a consequence of their inability to exit from G₂ and proceed into M phase.

The DNA content of resting T cells was unaffected (data not shown), presumably because βGBP controls the S/G₂ phase transition and thus can act only on proliferating cells (PHA-activated T lymphocytes or T cell lines), whereas resting T cells are nearly all in G₀/G₁.

Up-modulation of IFN-γR α- and β-chain expression associated with the βGBP-mediated cell cycle inhibition appears to have important consequences for the fate of T lymphocytes.

We have previously shown that induction of proliferation, differentiation (20), or apoptosis (5, 6, 19) of human T lymphocytes by IFN-γ depends on differences in the expression of the IFN-γR α- and β-chains on their membrane. High expression of the α-chain, in fact, favors only growth-promoting signals (5, 20, 21), and high expression of both chains favors apoptotic signals (6, 19).

In normal and neoplastic T lymphocytes, high IFN-γR chain expression is induced by serum (5) and IL-2 deprivation (6, 19), TCR ligation (6), and exposure to x-rays (5) or chemotherapeutic drugs (22). Since most of these treatments induce arrest in S and G₂/M (23, 24, 25), making T lymphocytes susceptible to IFN-γ-mediated apoptosis (5, 6, 19), our findings for βGBP effects suggest that up-regulation of IFN-γR is a general event related to T cell cycle arrest.

The βGBP-dependent up-regulation of the two IFN-γR chains and the subsequent bias of T cells toward IFN-γ-mediated apoptosis opens up a new, molecularly defined way in which the fate of T cells encountering IFN-γ is decided. Indeed, βGBP, can be seen as a factor concurring in the IFN-γ switch of the T cell program from proliferation to apoptosis. Control of this switch may be of importance in many immunologic scenarios, especially in autoimmune diseases, where the tendency of Th lymphocytes to differentiate into Th1 cells and produce large amounts of IFN-γ has already been indicated as a major pathogenic mechanisms (26). The IFN-γR complex is functional in differentiated human Th1 and Th2 clones, with IFN-γ mediating their autocrine or paracrine apoptosis only when its β-chain is highly expressed on their membrane (19). The ability of βGBP to convert IFN-γ signals from proliferative to apoptotic could be a new way of switching off sustained activation of Th1 lymphocytes in autoimmune diseases.

Modulation of β-chain is a critical event during Th1/Th2 differentiation (27, 28, 29). The antiproliferative effect of IFN-γ on Th2 cells (30) is due to their ability to express β-chain. Lack of this expression would seem to make Th1 cells resistant to IFN-γ by preventing transduction of its signals (27, 28). Present data suggest that βGBP may play a physiologic role in Th1/Th2 differentiation by favoring the IFN-γ-mediated apoptosis of Th cells with high levels of β-chain expression.

These biologic effects of βGBP position it as a new member of the cytokine network. Cytokines act at low concentration and through specific receptor interaction. The concentration range (27 × 10⁻¹¹ −27 × 10⁻⁹ M) at which βGBP functions independently from the sugar binding we used is close to the physiologic concentration at which cytokines act (10⁻⁹ −10⁻¹² M) (31), rather than those of lectinic proteins (12, 32). In addition, our data showed that the effects of βGBP are due to a specific receptor binding distinct from a more general carbohydrate interaction, since the effects of βGBP are not affected by the addition of saturating amounts of lactose. These data obtained on T lymphocytes confirm those previously reported on fibroblasts showing that βGBP-induced inhibition of the exit of cells from G₀ and G₂ is not due to its lectin properties, since it binds with high affinity (K_d of 10⁻¹⁰ M) to ∼5 × 10⁴ receptor sites per cell through molecular domains other than those that link saccharide determinants (7).

In conclusion, the present data suggest that the LGALS1 gene product has different mechanisms of action. It exists as both a dimer and a monomer in a dose-dependent equilibrium (13). The dimeric form is expressed in peripheral lymphoid tissues and the thymus (11, 12, 32) and may be therapeutically active against autoimmune diseases (15, 16) through direct deletion of reactive T cells (16). The finding that this form directly induces the apoptosis of activated T lymphocytes (12) and thymocytes (32) further supports this possibility. In contrast, we demonstrated that the monomer does not directly induce the apoptosis of activated T cells, but biases them to an apoptotic response to IFN-γ. Perillo et al., too, have shown that the dimer induces apoptosis in T cells at a concentration 1 × 10⁵-fold higher than that we used to obtain the βGBP-induced cell cycle arrest (12). Our dose was one at which the equilibrium shifts toward the monomeric form. The 400 ng/ml concentration (corresponding to 27 × 10⁻⁹ M) is 4000-fold lower than the estimated K_d of the dimer to monomer (7 × 10⁻⁶ M) (13). Therefore, our data strongly suggest that the monomeric form, although it retains sugar-binding ability (13), can act in a cytokine-like manner.

The immunomodulatory effects of the LGALS1 gene product may be finely controlled in vivo through regulation of the equilibrium between the cytokine-like monomeric form and the lectin-like dimeric form (14).

These findings thus provide further evidence that IFN-γR expression is modulated by a negative regulator of cell growth and point the way to the elaboration of new therapeutic strategies for manipulating the growth of autoimmune T cells by using βGBP to force their IFN-γ-mediated apoptosis.

We thank Dr. J. Iliffe for critically reading the manuscript.