2 INSERM U526, Laboratoire de Virologie, Faculté de Médecine, avenue de Valombrose, 06107 Nice Cedex 2, France; 3 The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA, 92037; 4 Service de Médecine Interne, Hôpital Archet 1, Route de Saint Antoine de GinestiÉre, 06200, Nice, France; 5 UCLA Department of Pathology and Laboratory Medicine, UCLA School of Medicine, 10833 Le Conte Avenue, Los Angeles, CA 90095
Received on November 29, 2002; revised on August 4, 2003; accepted on August 5, 2003
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Abstract |
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Key words: cell death / core 2 O-glycan / lactosa-mine / poly-N-acetyllactosamine / T cells
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Introduction |
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We and others have shown that activation of T lymphocytes results in altered glycosylation of cell surface glycoproteins (Chervenak and Cohen, 1982; Gallego et al., 2001
; Galvan et al., 1998
; Kaufmann et al., 1999
; Landolfi and Cook, 1986
; Piller et al., 1988
). These changes include loss of sialic acid and an increase in the number of core 2 O-glycans (Piller et al., 1988
; Sportsman et al., 1985
); such changes in glycan structure would promote extension of lactosamine sequences on cell surface oligosaccharides (Fukuda et al., 1986
). Recent reports have indicated that in the mouse, core 2 O-glycans may be important for the differentiation of effector and memory CD8 cells (Harrington et al., 2000
), as well as for the loss of CD8 cells in vivo (Priatel et al., 2000
). Moreover, an increased fraction of both CD4 and CD8 peripheral T cells from HIV-1-infected patients express core 2 O-glycans (Sportsman et al., 1985
), and the level of core 2 O-glycan expression correlates with the level of CD4 cell loss in patients with AIDS (Giordanengo et al., 1999
).
These types of glycosylation changes (i.e., loss of sialic acid, increase in core 2 O-glycans and elongation of lactosamine sequences) all promote oligosaccharide binding to galectin-1 (Galvan et al., 2000b; Leffler and Barondes, 1986
), an endogenous human lectin widely expressed in a variety of tissues (Perillo et al., 1998
). Galectin-1 binding induces cell death of activated lymphocytes (Perillo et al., 1995
) as well as immature thymocytes (Perillo et al., 1997
) by binding to the oligosaccharides on specific cell surface glycoproteins, including CD45, CD43, and CD7 (Nguyen et al., 2001
; Pace et al., 1999
, 2000
). However, the role of galectin-1 in elimination of HIV-1-infected T cells has not been examined.
Here we show that galectin-1 induces cell death of latently HIV-1-infected CEM cells and of peripheral lymphocytes from HIV+ patients. Although less than 0.1% of peripheral blood mononuclear cells derived from HIV+ patients harbor detectable integrated HIV-1 provirus (Simmonds et al., 1990), 1240% of peripheral blood lymphocytes (PBLs) derived from HIV+ patients died in response to galectin-1. These results imply that altered cell surface glycosylation of T cells associated with HIV-1 infection increases susceptibility to galectin-1-induced cell death, a mechanism that could contribute to T cell loss in AIDS.
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Results |
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To more precisely characterize the glycosylation changes in CEM cells latently infected with HIV-1LAI (CEMLAI/NP), we performed lectin flow cytometry using a panel of plant lectins that recognize specific glycan structures (Figure 1A). We confirmed the loss of O-linked sialic acid on the CEMLAI/NP cells, as shown by increased binding of PNA, that recognizes unsialyated galactose ß1,3N- acetylgalactosamine (Galß1,3GalNAc) (core 1 O-glycan) as well as by decreased binding of Maackia amurensis agglutinin (MAA), that recognizes N-acetylneuraminic acid 2,3Gal (NAN
2,3Gal), that is, sialic acid
2,3Gal. In addition, infected cells demonstrated an increase in lactosamine (Galß1,4N-acetylglucosamine) (Galß1,4GlcNAc) sequences, demonstrated by increased binding of Erythrina cristagalli agglutinin (ECA). Importantly, HIV-1-infected cells also demonstrated an increase in the expression of polylactosamine sequences {(Galß1,4GlcNAc)n}, detected by increased binding of Lycopersicon esculentum agglutinin (LEA) (Figure 1A), compared to uninfected cells. Increased expression of polylactosamine sequences was also observed on CEMLAI/NP cells and PBLs from HIV+ patients (Figure 1B) using an anti-i blood group antibody that recognizes linear polylactosamine extensions (Gooi et al., 1984
).
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To definitively address the glycosylation changes on O-glycans associated with HIV-1 infection, we analyzed CD43 O-glycans immunoprecipitated from HIV-1-infected and noninfected T cells. Oligosaccharides from CD43 were chemically analyzed after [3H] glucosamine incorporation by CEM and CEMLAI/NP cells. As shown in Figure 2A, CD43 oligosaccharides from parental CEM cells yielded peaks 1, 2, and 3 on Bio-Gel P-4 gel filtration. Using standard reference oligosaccharides, these peaks were identified as disialosyl core 2 and core 1 branched O-glycans (Figure 2A, peak 1), monosialylated core 2 branched and core 1 branched O-glycans (Figure 2A, peak 2), and asialo core 2 branched O-glycans (Figure 2A, peak 3) (Figure 2E). After desialylation, the same sample yielded Galß1, 4GlcNAcß1,6(Galß1,3)GalNAcOH (Figure 2B, peak 2), sialic acid (Figure 2B, peak 3), Galß1,3GalNAcOH (Figure 2B, peak 4), and GalNAcOH (Figure 2B, peak 5). As shown in Figure 2E, more than 97% of the CD43 O-glycans from uninfected CEM cells were sialylated. Importantly, no polylactosamine sequences were detected on CD43 oligosaccharides from uninfected CEM cells (Figure 2E). Although parental CEM cells produced a peak that eluted at the void volume, this peak represents N-glycan contaminants.
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Galectin-1-induced cell death of latently HIV-1-infected CEM cells
CD45 and CD43 are important T cell surface receptors for galectin-1, and both of these glycoproteins regulate susceptibility to galectin-1-induced cell death. In addition, the glycosylation changes that we observed on CEMLAI/NP cells would be predicted to increase binding of and susceptibility to galectin-1. The binding of galectin-1, tested at various concentrations, was more important on CEMLAI/NP cells, in comparison with parental CEM cells (Figure 1C). A natural form of galectin-1 (galaptin), which is recognized by the rabbit antiserum raised against recombinant human galectin-1 (Baum et al., 1995), was also tested and showed a binding equal to the recombinant form (not shown).
To directly compare the susceptibility of CEM and CEM-LAI/NP cells to galectin-1-induced cell death, cells were incubated with galectin-1 or buffer control for 5 h, and the level of cell death was assessed by cell loss as determined by forward versus side scatter and by annexin V binding and propidium iodide permeability. Galectin-1 induced cell death of a significant fraction of both CEM and CEMLAI/NP cells (Figure 3A). As shown in Figure 3B, the effect of galectin-1 was directly correlated to its concentration in the reaction mix. The role played by galectin-1 in the induction of cell death was controlled using lactose (10 mM) to block its activity while the disaccharide cellobiose (10 mM) was unable to interfere (Figure 3C). Besides, because the dimerization of galectin-1, which is maintained by dithiothreitol (DTT), is essential for warranting the activity of the lectin and leads to the development of aggregated cells, we have verified that the phenomenon of aggregation was not able to induce cell death by itself. As shown in Figure 4, the plant lectin Wisteria floribunda agglutinin (WFA), which recognizes -GalNAC residues, strongly aggregates CEMLAI/NP cells, much more than galectin-1, and to a lesser degree parental CEM cells, but was unable to induce cell death (Figure 3D).
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The cell death induced by galectin-1 was evaluated by annexin V staining, which evidences exposure of phosphatidylserine at the surface of the cell. To verify that the changes exhibited by annexin V in our system corresponded to irreversible membrane damages leading to cell death, cultures of galectin-1-induced CEMLAI/NP cells were conserved for 2 days at 37°C to control for their loss of viability. Indeed, although CEMLAI/NP cells maintained in the presence of DTT alone showed a growth rate equivalent to controls, galectin-1 clearly induced growth inhibition and extensive cell destruction, especially when used in association with DTT. Hence, galectin-1 was indeed able to dramatically interfere with the viability of CEMLAI/NP cells and to kill them (Figure 4B).
PBLs from HIV-1-infected patients are susceptible to galectin-1-induced cell death
We and others have shown that PBLs from AIDS patients express increased levels of core 2 O-glycans, as detected by the T305 mAb (48.5% T305+ for AIDS patients versus 22.5% T305+ healthy donors) (Gallego et al., 2001; Giordanengo et al., 1999
; Sportsman et al., 1985
). In addition, the results in Figure 1B demonstrate that an increased fraction of PBLs from AIDS patients bear polylactosamine sequences (mean 15%) compared to healthy donors (mean 6%), as detected by the anti-i antibody. Because core 2 O-glycans and polylactosamine sequences are both known to regulate galectin-1 binding and cell death, we wished to determine if PBLs from AIDS patients demonstrated altered susceptibilty to galectin-1 death.
We obtained PBLs from 47 patients with AIDS, all of whom were undergoing highly active antiretroviral therapy (HAART), and 32 healthy donors. Freshly isolated cells were tested directly for susceptibility to galectin-1 death, without mitogen or antigen stimulation. As shown in Figure 5A (lower panel), galectin-1 treatment resulted in a significant loss of PBLs from AIDS patients, as detected by forward versus side scatter. In contrast, we observed virtually no cell death in unstimulated PBLs from healthy donors (Figure 5A, upper panel). The resistance of unstimulated PBLs from healthy donors to galectin-1 is consistent with our previous report describing that only activated T cells are susceptible to galectin-1. However, the marked susceptibility of unstimulated PBLs from AIDS patients is consistent with previous reports of hyperactivation of peripheral T cells, as well as increased expression of activation markers (such as T305) in HIV-1 infection (Gallego et al., 2001; Giordanengo et al., 1999
; Sportsman et al., 1985
).
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Because death of CD4 cells is a critical event in the pathogenesis of AIDS, we examined the relationship between the degree of galectin-1 cell death and CD4 percentage in the 47 HIV-1 infected patients. As shown in Figure 5B, there was an inverse correlation (P < 0.001) between the percent of cell death and CD4 cell count. This correlation suggested that CD4 cells may be susceptible to galectin-1 death.
Knowing that only <0.1% of PBLs from HIV-1 patients are typically infected with virus (Simmonds et al., 1990), our results, which showed relative high percents of these PBLs susceptible to galectin-1-induced cell death, could be very likely explained by a chronic synergistically associated activation status of PBLs from HIV+ patients but not by a direct triggering from HIV-1 expression. To recall our previous results (Perillo et al., 1995
), PBLs from 12 healthy donors were stimulated by Phaseolus vulgaris leucoagglutinin (PHA-L) followed by interleukin-2 (IL-2) for 5 days. As shown in Figure 5D, PBLs activated via a non-HIV mechanism are significantly susceptible to galectin-1.
As already noticed, the AIDS patients examined in this study were all undergoing HAART. For a majority of the patients, one of the antiretroviral drugs used was zidovudine (better known as AZT) that has been shown to dramatically induce changes in glycoconjugates synthesis (Steet et al., 2000; Yan et al., 1995
). The principal effects of this drug are decreased incorporation of sialic acid and inhibition of synthesis of complex oligosaccharides, such as poly-N-acetyllactosamines (Steet et al., 2000
). Hence, an important question was whether AZT, which is known to participate almost in part in the restoration of the CD4 cell count, might paradoxically contribute to increase galectin-1-induced cell death. This question is particularly pertinent because CEMLAI/NP cells, which are preferentially galectin-1 sensitive versus parental CEM cells, are also hyposialylated. To address this question, the binding of galectin-1 was evaluated on parental CEM cells priorly incubated with AZT (50 µM) for 3 days. As expected, treated CEM cells were reactive with PNA (not shown), thus demonstrating a decreased incorporation of sialic acid. As shown in Figure 1C, the binding of galectin-1 was not significantly modified by AZT. Besides, cell death was not significantly modified (not shown).
To directly determine which T cell subsets from HIV-1-infected patients were susceptible to galectin-1-induced death, we specifically examined the loss of CD4 and CD8 cells after treatment with galectin-1. PBLs from 21 AIDS patients were stained with anti-CD4-fluorescein isothiocyanate (FITC) and anti-CD8-FITC after treatment with galectin-1 or buffer control. Viable cells were gated by forward versus side scatter, and the absolute number of viable cells was calculated for total PBLs and CD4+ and CD8+ subsets. To take into account the wide spread in the data between the different subjects HIV-infected or not, especially because of the variability of the response of HIV+ patients to HAART, statistical analysis was done using matched-pair data, subject per subject. As shown in Figure 5C, the absolute number of viable cells in both the CD4 and CD8 subsets decreased after galectin-1 treatment, demonstrating that galectin-1 induced death of both T cell subsets.
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Discussion |
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These types of glycan modifications, namely, reduced cell surface sialic acid, increased amounts of core 2 O-glycans and extension of polylactosamine sequences, were all present on latently HIV-1-infected CEM T cells and PBLs from AIDS patients (Figures 1 and 2). The sum of the amounts of exposed lactosamine residues + polylactosamine chains, recognized by galectin-1, are of 36.5%, and only 2.6% for CD43 from HIV-1-infected CEM T cells and parental CEM cells, respectively. Only 4% polylactosamine sequences were detected in CD43 oligosaccharides from HIV-1-infected CEM T cells. Nevertheless, the special distribution of repeated lactosamine units along polylactosamine chains, capable of linking multiple dimeric galectin-1 molecules (Cho and Cummings, 1997), is particularly favorable to promote cross-linking of glycoproteins that bear this kind of chain thus leading to highly amplified signals. The increased expression of polylactosamine sequences on T cell populations infected with HIV-1 that we observed is reminiscent of early studies demonstrating decreased proliferation and increased cell death of PBLs from asymptomatic HIV-1-infected subjects in response to pokeweed mitogen, a lectin that recognizes polylactosamine chains (Groux et al., 1991
, 1992
; Hofmann et al., 1989
; Irimura and Nicolson, 1983
).
Importantly, only about 12% of the cells in the CEMLAI/NP cultures are infected with virus (Lefebvre et al., 1994a), but the glycosylation changes on CEMLAI/NP cells observed in Figure 1 affected the entire culture population. Likewise, although <0.1% of PBLs from HIV-1 patients are typically infected with virus (Simmonds et al., 1990
), we observed binding of the anti-i antibody to 430% of patient PBLs with a mean of 15% (Figure 1B). Thus the population of T cells in vivo that would display altered cell surface glycosylation and would be predicted to be susceptible to galectin-1 is in vast excess of the fraction of infected cells. It is essential to consider that each of these glycosylation changes may contribute to enhancing galectin-1 sensitivity, so that although individual patients may have differences in the extent of glycosylation changes, the changes may synergize to increase galectin-1 sensitivity.
The results in Figures 3 and 5 demonstrate that both latently infected CEMLAI/NP cells and PBLs from AIDS patients demonstrated a significant increase in galectin-1 sensitivity, compared to uninfected CEM cells or PBLs from healthy donors. Again, our finding that >40% of CEMLAI/NP cells died in response to galectin-1 suggests that noninfected cells bearing the appropriate cell surface glycans and with an intact intracellular death pathway, are susceptible to galectin-1. Similarly, the level of cell death observed with patient samples, 1240%, is in vast excess of the number of cells that would be predicted to be infected with virus. Indeed, again we have to notice that <0.1% of PBLs from HIV-1 patients are typically infected with virus (Simmonds et al., 1990). In fact, the level of cell death for unstimulated PBLs tested directly after isolation from the patients is striking, given our previous observation that activation of PBLs from healthy donors is required to render the cells susceptible to galectin-1 (Perillo et al., 1995
) (Figure 5D). We and others have found that T cell activation inversely correlates with CD4 count in AIDS patients (Giordanengo et al., 1999
; Leng et al., 2001
; Muro-Cacho et al., 1995
). In the present study, we also find that galectin-1 susceptibility of PBLs inversely correlates with CD4 count in AIDS patients (Figure 5B). These findings are consistent with recent studies indicating that homeostatic elimination of activated, uninfected T cells contributes to T cell loss in AIDS (reviewed in McCune, 2001
), and suggests that galectin-1-induced death may contribute to this process.
Altogether, these results highlight the important contribution that uninfected cells bring in the T cell depletion along the clinical course of AIDS and recall the interest of understanding the mechanisms of immunological disorders that underlie the disease. Indeed, in contrast to Figure 5B, we found no correlation between PBLs susceptibility to galectin-1 and plasma viral load in individual patients (data not shown), again implying that viral infection is not a prerequisite for cell death (Leng et al., 2001; Muro-Cacho et al., 1995
). Because galectin-1 is widely expressed in virtually every organ, including lymph nodes and spleen, and is expressed by macrophages and dendritic cells (Perillo et al., 1998
), it is likely that in vivo activated T cells will encounter galectin-1 at sites where cell death occurs in HIV-1 infection (Bofill et al., 1995
; Finkel et al., 1995
; Muro-Cacho et al., 1995
). Because susceptibility to galectin-1 is regulated at the level of the T cell by differential glycosylation (Galvan et al., 2000b
; Nguyen et al., 2001
; Perillo et al., 1995
), once these glycosylation changes occur, galectin-1 may eliminate all T cells, infected or uninfected, that present the appropriate oligosaccharide on the required cell surface glycoprotein.
The T cell glycoprotein receptors that bind galectin-1 and transduce the death are also critical for regulating susceptibility to death. Although several glycoproteins can bear the oligosaccharide structures recognized by galectin-1, galectin-1 binds selectively to a subset of these glycoproteins, including CD2, CD3, CD4, CD7, CD43, and CD45 (Pace et al., 1999, 2000
; Walzel et al., 2000
). Importantly, both CD43 and CD45 have been shown to regulate susceptibility to galectin-1 death (Nguyen et al., 2001
; unpublished data). In the present study, we demonstrate that HIV-1 infection results in dramatic remodeling of the oligosaccharides on both CD43 and CD45. The oligosaccharides expressed on CD43 and CD45 after HIV-1 infection would promote galectin-1 binding and receptor cross-linking and segregation, a critical step in triggering death (Nguyen et al., 2001
; Pace et al., 1999
).
The AIDS patients examined in this study were all undergoing HAART. Because AZT was part of the associated drugs for several patients which were testedand knowing that AZT has been shown to profoundly modify glycosylationwe verified a possible role of this molecule in the susceptibilty of treated cells to galectin-1. No significant change in the binding of galectin-1 nor in the cell death induced by galectin-1 was observed, thus excluding any undesirable effect of AZT at this level. HAART has recently been shown to reduce both T cell proliferation and cell death (Mohri et al., 2001). A reduction in T cell proliferation would also be expected to reduce the number of cells expressing the oligosaccharide structures recognized by galectin-1. HAART may also directly block cell death triggered by a variety of mechanisms. However, although the response to HAART can be variable (Autran et al., 1999
; Carcelain et al., 2001
), it is clear from the data in Figure 5 that a significant fraction of PBLs from AIDS patients on HAART remained susceptible to galectin-1. These results suggest that multiple mechanisms may control T cell death in vivo. Identifying these mechanisms is a crucial step in controlling T cell loss in AIDS.
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Materials and methods |
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Cells, cultures, and HIV-1 strain
The parental CCRF-CEM cell line (ATCC CCL-119) and the virus-nonproducing CEM cell line latently infected by HIV-1 strain Lai (CEMLAI/NP) have been described elsewhere (Lefebvre et al., 1994a, 1994b
). PBLs from patients and healthy donors were obtained by Ficoll- Hypaque (Pharmacia, Uppsala, Sweden) gradient centrifugation and removing of monocytes by adherence to plastic. In some cases, PBLs from healthy donors were activated for two days by PHA-L (2 µg/ml) (Sigma, St. Louis, MO), then propagated for 3 days in the presence of IL-2 (10 U/ml) (Sigma).
Viability assay
To verify the induction of cell death by galectin-1, CEMLAI/NP cells were distributed in a 96-well plate (5.104 cells/well) with or without galectin-1 (20 µM) and/or DTT (1.2 mM) and maitained at 37°C for 2 days. Thereafter, trypan blue was gently added without dispersing the cells. After a 15-min period of incubation, the cell layers were photographed.
Preparation of recombinant galectin-1
Human galectin-1 was purified from Escherichia coli transformed with the expression vector pT7IML-1 as previously described (Couraud et al., 1989). Galectin-1 was stored in 8 mM DTT at -70°C. A natural form of galectin-1 (also termed galaptin) purified from bovine spleen (Sigma) was also used.
Antibodies and reagents
FITC-conjugated anti-human CD8 and CD4 mAbs were purchased from Beckman Coulter (Villepinte, France). The anti-i blood group mAb was purchased from Oxytele (Versailles, France), and anti-CD45 (725D3) that recognizes all CD45 isoforms was kindly provided by J. Vives (Alsinet et al., 1990). The T305 mAb that recognizes core 2 O-glycan has been described elsewhere (Piller et al., 1991
). The rabbit antiserum directed to galectin-1 has been raised as previously described (Baum et al., 1995
). PNA-, ECA-, LEA-, MAA-FITC and MAA-, GNA-, LEA-, ECA-, SNA, GS-I-B4-, STA-, and WFA-agarose or -FITC were purchased from E-Y Laboratories (San Matteo, CA). AZT, 4-O-ß-D-glucopyranosyl-D-glucose (cellobiose), and galaptin were purchased from Sigma.
Flow cytometry analysis
Phenotypic analyses with fluorescein-conjugated lectins or antibodies were performed as previously described (Lefebvre et al., 1994a). Stained cells were analyzed on a FACScan flow cytometer (Becton-Dickinson, San Jose, CA).
Metabolic radiolabeling and lectin-precipitation of immunopurified CD45 molecules
Cells were labeled for 18 h in SO42-free RPMI 1640 medium containing 35SO42-methionine (ICN Biomedicals, Costa Mesa, CA) (1 mCi/ml) and 5% dialyzed fetal calf serum, as previously described (Lefebvre et al., 1994a). CD45 molecules were precipitated from cells lysates using protein A Sepharose 4B beads precoated with the 72-5D3 mAb. CD45 molecules were eluted and reprecipitated using various lectin-coated agarose beads as previously described (Lefebvre et al., 1994a
). Precipitates were resuspended in sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE) sample buffer (2% SDS, 1.7 M 2-mercaptoethanol) and boiled for 3 min. Samples were resolved by 7.5% SDSPAGE.
Separation of O-linked oligosaccharides isolated from CD43
CEM and CEMLAI/NP cells were metabolically labeled with [3H]-glucosamine as previously described (Piller et al., 1991). CD43 was immunoprecipitated using T305 antibody, and the sample was digested with pronase. Glycopeptides were then applied to a column (1.0 x 120 cm) of Sephadex G-50 (super fine) eluted with 0.1 M NH4HCO3. Glycopeptides eluted at the void volume and containing O-linked oligosaccharides were then subjected to alkaline borohydride (0.05 M NaOH, 1 M NaBH4) treatment at 37°C for 36 h. The sample was neutralized by addition of acetic acid in methanol and evaporated under nitrogen stream. The sample was then subjected to Sephadex G-50 gel filtration under the same conditions described. Released O-linked oligosaccharides were then subjected to Bio-Gel P-4 gel filtration in 0.1 M ammonium acetate buffer (pH 6.7) (Hiraoka et al., 1999
).
Measurement of cell death by annexin V
PBLs or CEM cells (104) were incubated at 37°C in phosphate buffered saline (PBS) containing 1.2 mM DTT with or without 20 µM galectin-1 for 5 h. In some experiments, cells were incubated with AZT (50 µM) for 3 days before testing galectin-1. All samples were assayed in triplicate. All cells were resuspended in 0.1 M ß-lactose for 30 s at room temperature to disperse aggregates, washed twice with ice-cold PBS, and stained with 100 µl annexin VFITC (R&D Systems, Minneapolis, MN) plus propidium iodide for 15 min in the dark on ice, according to the manufacturer's recommendations. Cells were analyzed on a FACScan flow cytometer (Becton-Dickinson). To analyze loss of CD4 and CD8 subpopulations, cells were incubated with galectin-1 alone. Aliquots were stained with either CD4-FITC or CD8-FITC mAbs before and after incubation with galectin-1. The absolute number of viable cells (total, CD4+, or CD8+, labeled in separate tubes) after treatment with galectin-1 or buffer control was determined by forward vs. side scatter gating as previously described (Pace et al., 2000).
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Acknowledgements |
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Footnotes |
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Abbreviations |
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References |
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