Altered T cell surface glycosylation in HIV-1 infection results in increased susceptibility to galectin-1-induced cell death

Marion Lantéri2, Valérie Giordanengo2, Nobuyoshi Hiraoka3, Jean-Gabriel Fuzibet4, Patrick Auberger2, Minoru Fukuda3, Linda G. Baum5 and Jean-Claude Lefebvre1,2

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


    Abstract
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 Abstract
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 Results
 Discussion
 Materials and methods
 References
 
The massive T cell death that occurs in HIV type 1 (HIV-1) infection contributes profoundly to the pathophysiology associated with AIDS. The mechanisms controlling cell death of both infected and uninfected T cells ("bystander" death) are not completely understood. We have shown that HIV-1 infection of T cells results in altered glycosylation of cell surface glycoproteins; specifically, it decreased sialylation and increased expression of core 2 O-glycans. Galectin-1 is an endogenous human lectin that recognizes these types of glycosylation changes and induces cell death of activated lymphocytes. Therefore we studied the possible contribution of galectin-1 in the pathophysiology of AIDS. O-glycan modifications were investigated on peripheral lymphocytes from AIDS patients. Oligosaccharides from CD43 and CD45 of CEM cells latently infected with HIV-1 were chemically analyzed. Consistent with our previous results, we show that HIV-1 infection results in accumulation of exposed lactosamine residues, oligosaccharides recognized by galectin-1 on cell surface glycoproteins. Both latently HIV-1-infected T cell lines and peripheral CD4 and CD8 T cells from AIDS patients exhibited exposed lactosamine residues and demonstrated marked susceptibility to galectin-1-induced cell death, in contrast to control cultures or cells from uninfected donors. The fraction of cells that died in response to galectin-1 exceeded the fraction of infected cells, indicating that death of uninfected cells occurred. Altered cell surface glycosylation of T cells during HIV-1 infection increases the susceptibility to galectin-1-induced cell death, and this death pathway can contribute to loss of both infected and uninfected T cells in AIDS.

Key words: cell death / core 2 O-glycan / lactosa-mine / poly-N-acetyllactosamine / T cells


    Introduction
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
The loss of T cells is a major factor in the pathophysiology of Acquired Immune Deficiency Syndrome (AIDS) (Gougeon et al., 1991Go; Groux et al., 1991Go). While specific HIV type 1 (HIV-1) polypeptides, such as vpu, nef, and vpr, can directly induce death of infected T cells, excess death of uninfected "bystander" T cells likely accounts for most of the T cell loss of both CD4 and CD8 cells in patients infected with HIV-1 (reviewed in Badley et al., 2000Go). The Fas/FasL pathway has been implicated in the death of uninfected activated T cells early in HIV-1 infection. However, the range of factors contributing to increased T cell depletion during the early stages of HIV-1 infection are not well understood.

We and others have shown that activation of T lymphocytes results in altered glycosylation of cell surface glycoproteins (Chervenak and Cohen, 1982Go; Gallego et al., 2001Go; Galvan et al., 1998Go; Kaufmann et al., 1999Go; Landolfi and Cook, 1986Go; Piller et al., 1988Go). These changes include loss of sialic acid and an increase in the number of core 2 O-glycans (Piller et al., 1988Go; Sportsman et al., 1985Go); such changes in glycan structure would promote extension of lactosamine sequences on cell surface oligosaccharides (Fukuda et al., 1986Go). 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., 2000Go), as well as for the loss of CD8 cells in vivo (Priatel et al., 2000Go). 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., 1985Go), and the level of core 2 O-glycan expression correlates with the level of CD4 cell loss in patients with AIDS (Giordanengo et al., 1999Go).

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., 2000bGo; Leffler and Barondes, 1986Go), an endogenous human lectin widely expressed in a variety of tissues (Perillo et al., 1998Go). Galectin-1 binding induces cell death of activated lymphocytes (Perillo et al., 1995Go) as well as immature thymocytes (Perillo et al., 1997Go) by binding to the oligosaccharides on specific cell surface glycoproteins, including CD45, CD43, and CD7 (Nguyen et al., 2001Go; Pace et al., 1999Go, 2000Go). 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., 1990Go), 12–40% 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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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
HIV-1 infection results in modification of O-glycans on T cell surface glycoproteins
CD43 and CD45 are the two major cell surface glycoproteins of T cells, accounting for approximately 30% of the total cell surface proteins. Both CD43 and CD45 are heavily O-glycosylated, and CD45 also bears numerous N-glycans. We previously found that HIV-1 infection of CEM T cells dramatically reduces the level of sialylation of both CD45 and CD43, as demonstrated by changes in electrophoretic mobility and increased binding of the plant lectin peanut agglutinin (PNA) (Lefebvre et al., 1994aGo, 1994bGo). The association of hyposialylation with HIV-1 infection was established in vivo by the detection of autoantibodies directed against partially sialylated CD43 in HIV+ individuals (Ardman et al., 1990Go; Giordanengo et al., 1995Go). HIV-1 infection of CEM T cells also results in increased binding of the T305 monoclonal antibody (mAb), that detects core 2 O-glycans on CD43 (Giordanengo et al., 1999Go).

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 {alpha}2,3Gal (NAN{alpha}2,3Gal), that is, sialic acid {alpha}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., 1984Go).



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Fig. 1. Glycosylation changes on HIV-1-infected cell populations. (A) Binding of FITC-conjugated plant lectins PNA (Galß1,3GalNAc), ECA (Galß1,4GlcNAc), MAA (NAN{alpha}2,3Gal) and LEA (poly-LacNAc), to parental CEM and CEMLAI/NP cells. The glycans recognized by the different lectins are indicated in the right column. (B) Expression of the i-antigen (linear polylactosamine) on parental CEM and CEMLAI/NP cells, and on PBLs from HIV+ subjects and HIV- controls. The percent positive for the sample is shown followed by the range for 45 AIDS patients in parentheses. (C) Binding of galectin-1 at various concentrations on parental CEM cells pretreated or not with AZT (50 µM) and CEMLAI/NP cells. (D) Two-step precipitation of [35S]-methionine labeled CD45 from CEM and CEMLAI/NP cells. CD45 was precipitated first with mAb 72–5D3 (lane 7), then eluted and reprecipitated with a panel of lectins of the indicated saccharide specificity: GS-I-B4 (Gal{alpha}1,3Gal), SNA (NAN{alpha}2,6Gal), STA (polylactosamine), PNA, LEA, and GNA (Man{alpha}1,3Man).

 
CD45 and CD43 from HIV-1-infected cells bear O-glycans with reduced sialic acid and increased extension of polylactosamine
Because the plant lectins and anti-i antibody all recognize saccharides independent of the polypeptide backbone, we wished to determine whether the glycosylation changes detected in Figure 1A (especially the increase in polylactosamine) occurred specifically on CD45 and CD43. We immunopurified CD45 from parental CEM and CEMLAI/NP cells and precipitated the purified CD45 with plant lectins. As shown in Figure 1D, CD45 from CEMLAI/NP cells bound increased levels of PNA and decreased levels of SNA, which recognizes the NAN{alpha}2,6Gal sequence, compared to CD45 from CEM cells, confirming the loss of sialic acid on CD45 from HIV-1-infected T cells. We also found that two lectins that specifically recognize polylactosamine sequences, Solanum tuberosum agglutinin (STA) and LEA, precipitated a dramatically increased fraction of CD45 from CEMLAI/NP cells, compared to CD45 from CEM cells (Figure 1D, lanes 3, 5), demonstrating that there is increased expression of polylactosamine sequences on CD45 from HIV-1-infected cells. As a positive control, CD45 was precipitated with the lectin Galanthus nivalis agglutinin (GNA), specific for the disaccharide mannose {alpha}1,3mannose (Man{alpha}1,3Man) on N-glycans, and no difference was observed between CD45 from infected or uninfected cells (Figure 1D, lane 6). To demonstrate specificity of lectin binding, we observed no precipitation of CD45 with the lectin Griffonia simplicifolia I isolectin B4 (GS-I-B4), which recognizes a disaccharide (Gal{alpha}1,3Gal) not expressed on human cells (Figure 1D, lane 1).

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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Fig. 2. Separation of O-linked oligosaccharides isolated from CD43 molecules immunoprecipitated from parental and CEMLAI/NP cells lysates. Core 2 O-glycans are included in peaks 1 + 2 + 3. CD43 oligosaccharides from CEM cells have a major peak of sialylated glycans (panels A and E, peak 1), whereas the major peak in CD43 oligosaccharides from CEMLAI/NP cells is nonsialylated (panels C and E, peak 3). Core 2 O-glycans elongated with poly-LacNAc chains, apparent after chemical desialylation, are present only on CEMLAI/NP cells (panels D and F, peak 1).

 
CD43 oligosaccharides from CEMLAI/NP cells, on the other hand, yielded abundant asialo core 2 branched O-glycans (Figure 2C, peak 3) and monosialyl GalNAc (Figure 2C, peak 4). Of note, core 2 branched O-glycans with N-acetyllactosamine repeat(s) (Figure 2C, peak 1) were present in CD43 from CEMLAI/NP cells and were not detected in CD43 from CEM cells. On desialylation, CD43 from CEMLAI/NP cells yielded large amounts of core 2 branched O-glycans (Figure 2D, peak 2) and N-acetylgalactosamine (Figure 2D, peak 5), as well as poly-N-acetyllactosaminyl core 2 branched O-glycans (Figure 2D, peak 1) and core 1 branched O-glycans (Figure 2D, peak 4). It is noteworthy that the sample from parental CEM cells released much more sialic acid than that from CEMLAI/NP cells (compare peak 3 in Figures 2B and 2D). These results demonstrate that CEMLAI/NP cells synthesize hyposialylated core 2 branched oligosaccharides, compared to parental CEM cells, as predicted by the pattern of lectin binding in Figure 1. Moreover, CD43, like CD45, from CEMLAI/NP cells contains increased levels of poly-N- acetyllactosaminyl O-glycans, compared with CEM cells.

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., 1995Go), 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 {alpha}-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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Fig. 3. Galectin-1 induces cell death of latently HIV-1-infected CEM cells. (A) CEM and CEMLAI/NP cells (104) were incubated with galectin-1 or buffer control for 5 h. Viable cells were gated by forward versus side scatter, and cells were analyzed by binding of annexin V–FITC and propidium iodide uptake. (B) Effects of galectin-1 at various concentrations on parental CEM and CEMLAI/NP cells. (C) Inhibition of galectin-1-induced cell death by lactose (10 mM) and not by cellobiose (10 mM). (D) WFA does not induce cell death.

 


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Fig. 4. Agglutination and viability assays. (A) Galectin-1 and WFA agglutinate parental CEM and CEMLAI/NP cells. Parental CEM (1, 2, 3) and CEMLAI/NP (4, 5, 6) cells were incubated for 5 h with PBS alone (1, 4), PBS added wih 20 µM galectin-1 and 1.2 mM DTT (2, 5), or 10 µg/ml of WFA (3, 6). (B) CEMLAI/NP cells were incubated for 2 days with or without galectin-1 (20 µM) and/or DTT. Cells were thereafter photographed without any preparation (panel 1) or after gentle addition of trypan blue without dispersing the cells (panel 2).

 
CEMLAI/NP cells were much more sensitive to galectin-1 than parental CEM cells, with 46% and 21% annexin V+, propidium iodide- cells, respectively. These results demonstrated that the glycosylation changes observed after HIV-1 infection correlate with increased susceptibility to galectin-1-induced cell death. The relative high percentage of parental CEM cells susceptible to galectin-1 can be explained by the constitutive activation status of these cells certified by their high content in core 2 O-glycans (Piller et al., 1988Go), (Figure 2E, peaks 1 + 2 + 3), expressed by uninfected lymphoblastoid cells, which have been shown to be susceptible to galectin-1 (Baum et al., 1995Go).

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., 2001Go; Giordanengo et al., 1999Go; Sportsman et al., 1985Go). 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., 2001Go; Giordanengo et al., 1999Go; Sportsman et al., 1985Go).



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Fig. 5. Galectin-1 induces cell death of unstimulated PBLs from AIDS patients. (A) Galectin-1 induces cell death of PBLs from AIDS patients. PBLs from 47 HIV+ or 32 healthy subjects were treated with galectin-1 or buffer control. Representative samples from an AIDS patient and a healthy control are shown. In this sample, cell loss is shown by forward versus side scatter, as described in Materials and methods. (B) Inverse correlation between the percent of annexin V+ and propidium iodide- cells after galectin-1 treatment and the percent of circulating CD4+ cells in 47 AIDS patients (P < 0.001). The average percent of annexin V+ and propidium iodide- cells from 32 healthy controls is shown (dotted line). (C) Absolute number of viable cells after treatment with galectin-1 or buffer control, determined by gating on forward versus side scatter. For enumeration of CD4 and CD8 cells, CD4-FITC or CD8-FITC was added after galectin-1 or buffer treatment, and the number of viable cells was calculated for the indicated population. Numbers are mean ± SD for 21 AIDS patients. (D) Activation with PHA-L/IL-2 was prerequisite for resting PBLs from healthy donors (N = 12) to be susceptible to galectin-1-induced cell death.

 
Using statistical comparison of mean values, PBLs from AIDS patients (N = 47) were significantly (P < 0.001) more susceptible to galectin-1-induced cell death (22.3%, {sigma} = 6.48), compared to PBLs from healthy donors (9.24%, {sigma} = 4.21, N = 32) (Figure 5B). However, the degree of galectin-1 cell death for PBLs from AIDS patients was variable, ranging from 12% to 40%, thus reflecting usual observations that AIDS patients under HAART can restore their immune system with variable efficacy (Autran et al., 1999Go).

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., 1990Go), 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., 1995Go), 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., 2000Go; Yan et al., 1995Go). 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., 2000Go). 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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The precise mechanism of galectin-1 death is not known. One report has described caspase activation in galectin-1-induced cell death (Rabinovich et al., 2002Go), we have determined that caspase activation is not required (Hahn et al., unpublished data). However, it is clear that T cell susceptibility to galectin-1 can be regulated at the level of glycosylation (Galvan et al., 2000aGo, 2000bGo; Nguyen et al., 2001Go; Perillo et al., 1995Go). Addition of sialic acid to cell surface glycoproteins blocks galectin-1 death (Galvan et al., 2000aGo), whereas cells that have reduced cell surface sialic acid, such as effector T cells (Galvan et al., 1998Go; Priatel et al., 2000Go), are susceptible to galectin-1 (Perillo et al., 1995Go). In addition, core 2 O-glycans that provide sites for polylactosamine extension are required for galectin-1-induced T cell death (Galvan et al., 2000bGo; Nguyen et al., 2001Go). Lactosamine extensions on N-glycans may also promote galectin-1 binding and signaling (Demetriou et al., 2001Go).

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, 1997Go), 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., 1991Go, 1992Go; Hofmann et al., 1989Go; Irimura and Nicolson, 1983Go).

Importantly, only about 12% of the cells in the CEMLAI/NP cultures are infected with virus (Lefebvre et al., 1994aGo), 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., 1990Go), we observed binding of the anti-i antibody to 4–30% 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, 12–40%, 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., 1990Go). 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., 1995Go) (Figure 5D). We and others have found that T cell activation inversely correlates with CD4 count in AIDS patients (Giordanengo et al., 1999Go; Leng et al., 2001Go; Muro-Cacho et al., 1995Go). 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, 2001Go), 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., 2001Go; Muro-Cacho et al., 1995Go). 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., 1998Go), 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., 1995Go; Finkel et al., 1995Go; Muro-Cacho et al., 1995Go). Because susceptibility to galectin-1 is regulated at the level of the T cell by differential glycosylation (Galvan et al., 2000bGo; Nguyen et al., 2001Go; Perillo et al., 1995Go), 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., 1999Go, 2000Go; Walzel et al., 2000Go). Importantly, both CD43 and CD45 have been shown to regulate susceptibility to galectin-1 death (Nguyen et al., 2001Go; 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., 2001Go; Pace et al., 1999Go).

The AIDS patients examined in this study were all undergoing HAART. Because AZT was part of the associated drugs for several patients which were tested—and knowing that AZT has been shown to profoundly modify glycosylation—we 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., 2001Go). 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., 1999Go; Carcelain et al., 2001Go), 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Patients
All patients tested were at the AIDS stage under HAART. All samples were obtained from patients and healthy volunteers with respect for Helsinki Accord.

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., 1994aGo, 1994bGo). 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., 1989Go). 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 (72–5D3) that recognizes all CD45 isoforms was kindly provided by J. Vives (Alsinet et al., 1990Go). The T305 mAb that recognizes core 2 O-glycan has been described elsewhere (Piller et al., 1991Go). The rabbit antiserum directed to galectin-1 has been raised as previously described (Baum et al., 1995Go). 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., 1994aGo). 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., 1994aGo). 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., 1994aGo). Precipitates were resuspended in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) sample buffer (2% SDS, 1.7 M 2-mercaptoethanol) and boiled for 3 min. Samples were resolved by 7.5% SDS–PAGE.

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., 1991Go). 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., 1999Go).

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 V–FITC (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., 2000Go).


    Acknowledgements
 
This work was supported by institutionanl grants from the Institut National de la Santé et de la Recherche Médicale (INSERM), grant CA33000 from NCI, and a grant from the Jonsson Comprehensive Cancer Center at UCLA. We thank Josette Lesimple for expert technical assistance, Jean-François Peyron for critical reading of the manuscript, and Mabel Pang and Moira Donnell for preparation of galectin-1.


    Footnotes
 
1 To whom correspondence should be addressed; e-mail: jean-claude.lefebvre{at}unice.fr Back


    Abbreviations
 
AIDS, Acquired Immune Deficiency Syndrome; CEMLAI/NP, CEM cell line latently infected by HIV type 1 strain Lai; DTT, dithiothreitol; ECA, Erythrina cristagalli agglu tinin; FITC, fluorescein isothiocyanate; GNA, Galanthus nivalis agglutinin; GS-I-B4, Griffonia simplicifolia I isolectin B4; HAART, highly active antiretroviral therapy; HIV-1, HIV type 1; IL-2, interleukin-2; LEA, Lycopersicon esculentum agglutinin; MAA, Maackia amurensis agglutinin; mAb, monoclonal antibody; NAN, N-acetylneuraminic acid; PBL, peripheral blood lymphocyte; PBS, phosphate buffered saline; PHA-L, Phaseolus vulgaris leucoagglutinin; PNA, peanut agglutinin; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; SNA, Sambucus nigra agglutinin; STA, Solanum tuberosum agglutinin; WFA, Wisteria floribunda agglutinin


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