Trypanosoma cruzi surface mucin TcMuc-e2 expressed on higher eukaryotic cells induces human T cell anergy, which is reversible

Pablo F. Argibay1,2, Javier M. Di Noia3, Alejandra Hidalgo2, Esteban Mocetti4, Mariana Barbich2, Alicia S. Lorenti2, Daniel Bustos5, Monica Tambutti6, Sung H. Hyon2, Alberto C.C. Frasch3 and Daniel O. Sánchez3

2Instituto de Ciencias Básicas y Medicina Experimental,Hospital Italiano de Buenos Aires, Gascón 450, (1181) Buenos Aires, Argentina; 3Instituto de Investigaciones Biotecnológicas, Instituto Tecnológico de Chascomús CONICET, Universidad Nacional de General San Martín, Pcia. de Buenos Aires, Argentina; 4Laboratory of Molecular Pathology, Hospital Italiano de Buenos Aires, Gascón 450, (1181) Buenos Aires, Argentina; 5Laboratory of Clinical Immunology, Hospital Italiano de Buenos Aires, Gascón 450, (1181) Buenos Aires, Argentina; and 6Laboratory of Immunogenetics from Hospital Italiano de Buenos Aires, Gascón 450, (1181) Buenos Aires, Argentina

Received on May 9, 2001; revised on June 27, 2001; accepted on August 17, 2001.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Chagas’ disease is a chronic, debilitating, multisystemic disorder that affects millions of people in Latin America. The protozoan parasite Trypanosoma cruzi, the etiological agent of Chagas’ disease, has a large number of O-glycosylated Thr/Ser/Pro-rich mucin molecules on its surface (TcMuc). These mucins are the main acceptors of sialic acid and have been suggested to play a role on various host–parasite interactions, such as adhesion to macrophages, protection from complement lysis, and immunomodulation of the immune response mounted by the host. To observe the immunologic effect obtained by the heterologous expression of a TcMuc gene in higher eukaryotic cells exposed to xenogeneic lymphocytes, we developed a strategy based on the transfection of a known T. cruzi mucin gene (TcMuc-e2) into Vero cells. In contrast to the brisk proliferation and activation of human lymphocytes observed at 3, 4, and 5 days induced by normal Vero cells, neither proliferation nor signicant activation of human lymphocytes was observed with TcMuc-e2-transfected Vero cells. This TcMuc-e2 mucin-induced suppression of T cell response can be reversed by the addition of exogenous IL-2. In addition it was demonstrated that the immunosuppressive reaction was not related to the induction of an important degree of apoptosis in human lymphocytes. Posttranslational modification are required for the inhibitory effect that TcMuc-e2 exerts when transfected to Vero cells. O-glycosylation and sialylation are required to obtain the immunomodulatory effect as assessed by O-sialoglycoprotease and neuraminidase treatments. These results are consistent with other studies showing that surface glycoconjugates from T. cruzi and mammalian cells can induce an inhibition of the immune response.

Key words: immunomodulation/mucins/T. cruzi


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Trypanosoma cruzi–associated mucins are highly O-glycosylated glycoproteins that serve as the main acceptors for sialic acid in a reaction catalyzed by the parasite’s enzyme trans-sialidase (Previato et al., 1985Go; Schenkman et al., 1993Go). The family of sialylated T. cruzi mucins were shown to be involved in both adhesion to the host cells (Schenkman et al., 1992Go) and in protection of the parasite from the immune response. The latter function is achieved by shielding the trypanosome from lytic antibodies (Pereira-Chioccola et al., 2000Go) and by inducing anergy in peripheral blood mononuclear cells (PBMCs) (Kierszenbaum et al., 1999Go). There are two kinds of developmentally regulated mucin-like molecules in T. cruzi, and both are complex mixtures of heterogeneous molecules. Two large gene families that encode at least part of these molecules were described in the past for our group (Di Noia et al., 1995Go, 2000; Pollevick et al., 2000Go). The TcMuc family contains about 500 members divided into two subfamilies according to the presence or absence of tandem repeats in the central region of the genes. One group of these genes encode products with the central region composed of tandem repeats with the sequence T8KP2 were demonstrated to be mucins in vivo in the parasite (Pollevick et al., 2000Go). These kind of genes also yield a highly O-glycosylated product in Vero cells (Di Noia et al., 1996Go), as predicted by the NetOGlyc software (Hansen et al., 1998Go), that indicates several higher eukaryotic cell mucin-type O-glycosylation sites in TcMuc genes.

In the present study we have designed a strategy with the objective of assessing the utility of expressing a single T. cruzi mucin gene in higher eukaryotic cells and to evaluate its immunomodulatory activity on a xenogeneic response in vitro. The gene chosen for this purpose (TcMuc-e2) encodes for a T. cruzi mucin that is expressed in the blood-circulating stage of the parasite. The rationality of that strategy was based on the following:

1. The mucins that coat the surface of T. cruzi are potentially responsible for some of the parasite’s capacity to circumvent the vertebrate host response (Kierszenbaum et al., 1999Go).

2. Despite the predictable ocurrence of differences in the pattern of glycosylation, the organisation of TcMuc genes resembles those present in mammalian cells (central domains, rich in codons for Thr and/or Ser and Pro residues made up of a variable number of repeat units in tandem; Di Noia et al., 1995Go).

3. Mucin-type glycoproteins expressed in higher eukaryotic cells can mediate pathological interactions among leukocytes modulating the immune response (Kim et al., 1999Go).

4. The above-mentioned software prediction (Hansen et al., 1998Go).

In addition, in previous experiments we showed that when expressed on Vero cells, TcMuc-e2 inhibits lymphocytic proliferation in Balb C mice, both in vivo and in vitro (Argibay et al., 2000Go). In this work, we found that mammalian cells expressing the TcMuc-e2 mucin induce a nonresponding phenotype in human lymphocytes in vitro. In addition, we elucidated some of the mechanisms involved in this effect, such as downregulation of IL-2 receptor CD25 and the necessity of glycosylation on TcMuc-e2 to acquire immunomodulatory effects.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Expression of a T. cruzi mucin in higher eukaryotic cells
A T. cruzi mucin gene (TcMuc-e2), with its signal peptide replaced by the human IgE endoplasmic reticulum import signal (Figure 1A) was cloned into the eukaryotic plasmid vector pCDNA3.1. After transfection into Vero cells, several clones were obtained by selection with Neomycin®. Transcription of the TcMuc-e2 gene was assessed by reverse transcription polymerase chain reaction (RT-PCR) (Figure 1B) and the homogeneous expression of the product in the cell clones was evidenced by immunohistochemistry (Figure 1C, D). Three different sera were used, raised against proteins and synthetic peptides (see Materials and methods). A markedly positive reaction on mucin-transfected Vero cells (Figure 1C) and in porcine hepatocytes transfected with TcMuc-e2 (Figure 2A) was obtained when antibodies from mice immunized with the plasmid pCTcMUCe2/IgEsp were used. Vero cells and porcine hepatocytes transfected with the vector alone were used as controls (Figure 1D and Figure 2B, respectively).



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Fig. 1. Expression of the T. cruzi mucin TcMuc-e2 in Vero cells. (A) In scale scheme of the deduced product from TcMuc-e2 gene with the leader peptide of IgE (IgEsp). The signal peptide cleavage site localized between IgEsp (shaded), and TcMuc-e2 sequences was predicted using SignalIP software (Nielsen et al., 1997Go) and is indicated by asterisks. Vertical bars indicate potential higher eukaryotic cell mucin-type O-glycosylation sites, as predicted using NetOGlyc software (Hansen et al., 1998Go). The arrow indicates the addition site of the glycosylphosphatidil inositol anchor used in T. cruzi, and the open arrowhead indicates a consensus N-glycosylation site. (B) Reverse transcription from mRNA of E2-Vc (lane 1) and pC-Vc (lane 2) was done using a primer from the 3' end of TcMuc-e2. The product was submitted to two subsequent PCRs using a primer from the 5' end of the gene and two nested oligonucleotides from the 3' region. Lane 3, control PCR on nonreverse transcribed RNA from E2-Vc. A size marker of the product expected length is indicated on the left. (C) Monolayers of Vero cells transfected with pCTcMuce2/IgEsp (left) or pCDNA (right) were stained using anti-TcMuc antibodies obtained from mice immunized with the plasmid pCTcMUCe2/IgEsp. The study was developed with peroxidase-antiperoxidase and hematoxilin counterstaining (50x, bars indicate 10 µm).

 


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Fig. 2. Expression of TcMuc-e2 on porcine hepatocytes. Porcine hepatocytes obtained through perfusion with collagenase cultured and transfected with pCTcMuce2/IgEsp (A) or pCDNA (B) were stained using anti-TcMuc antibodies obtained from mice immunized with the plasmid pCTcMUCe2/IgEsp. The study was developed with peroxidase-antiperoxidase and hematoxilin counterstaining (50x, bars indicate 10 µm)

 
Vero cells expressing TcMuc-e2 inhibit the proliferation of human T cells
To evaluate the effect of TcMuc-e2 expression on the modulation of a xenogeneic proliferative response, we used a mixed culture setting of irradiated Vero cells and normal human T cells as stimulator and responder cells, respectively. After 3, 4, and 5 days of mixed culture, a strong inhibition of the proliferation of human lymphocytes was observed by coculture with Vero cells expressing TcMuc-e2 mucin gene (E2-Vc), as compared with the results obtained with Vero cells transfected with the vector alone (pC-Vc) or with nontransfected Vero cells (NT-Vc) (Figure 3). To evaluate if human T cells retain their ability to secrete cytokines following Vero cell stimulation despite their impaired capacity to proliferate, we collected the supernatants from the proliferation assay plate. Interferon-{gamma} (IFN-{gamma}) was present in all of the cultures (data not shown), showing that human lymphocytes retain the ability to secrete IFN-{gamma} even in a state of anergy following xenogeneic stimulation from E2-Vc. These results are consistent with other studies showing anergy with unaltered capacity to secrete cytokines (Mayumi et al., 1996Go).



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Fig. 3. Expression of TcMuc-e2 on Vero cells led to inhibition of human T cell proliferation. Normal human lymphocytes were cocultured with irradiated Vero cells expressing TcMuc-e2 (E2-Vc), irradiated Vero cells transfected with pCDNA vector (pC-Vc), or irradiated untransfected Vero cells (NT-Vc). After 3, 4, or 5 days of coculture, proliferation was assessed by [3H]thymidine incorporation. (–): Resting human T cells. Each group was set up in a replicate of three wells. Each experiment was made by triplicate. The data are shown as mean cpm of the replicate wells ± SD. P < 0.001 for differences observed by days 3, 4, and 5 (analysis of variance test).

 
Human T cells exposed to E2-Vc showed an altered expression of cell activation markers
Because the above results indicated that exposure to E2-Vc induced an anergic state in human lymphocytes, some relevant markers of cell activation were analyzed. Figure 4 shows the phenotypic expression of CD25 and CD69 in human CD3(+) T cells after 3, 4, and 5 days of exposure to xenogeneic cells, as evaluated by flow cytometry. Human T cells exposed to either E2-Vc or pC-Vc showed expression of CD69 (Figure 4A), although some delay in the expression was observed in the first case. On the other hand, expression of the IL2 receptor CD25 by human T lymphocytes was absent after exposure to mucin-transfected E2-Vc, but not after coculture with pC-Vc or NT-Vc control cells (Figure 4B).



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Fig. 4. Differential effects of TcMuc-e2 Vero cells on the phenotypic expression of T cell activation makers. Surface antigens from human lymphocytes cocultured with the indicated control or transfected cells were quantified by flow cytometry. Experiments were done by triplicate in each case and expressed as means ± SD. (A) CD69 expression in resting lymphocytes cocultured with E2-VC or pC-VC was similar (P = 0.2, Student t test), except at day 3 (P = 0.04, Student t test). (B) CD25 levels in the same cells showed a significant decrease when exposed to E2-Vc (P = 0.004, P = 0.008, P = 0.04 at days 3, 4, and 5, respectively). (–): Resting human lymphocytes; (+): phytohemagglutinin-stimulated human lymphocytes; pC-Vc: Vero cells transfected with the pCDNA3.1 vector without insert; E2-Vc: Vero cells expressing TcMuc-e2. All experiments were repeated with lymphocytes exposed to nontransfected Vero cells that show levels of the two markers similar to the pC-Vc exposed ones (data not shown).

 
Exposure to up to 200 µg/ml of purified TcMuc-e2 recombinant protein (rTcMuc-e2) expressed in bacteria had no effect on the expression of CD25 antigens in PHA activated (0.2 µg/ml) human lymphocytes (Figure 5A). On the other hand, T cells exposed to an extract from E2-Vc containing the posttranslationally modified TcMuc-e2 (sTcMuc-e2), evidenced a significant decrease in the expression of these antigens as observed with the corresponding intact cells (Figure 5A). These results suggested that if a direct mechanism involving TcMuc-e2 mucins was causing the effect, then posttranslational modifications were required. In this regard, treatment of E2-Vc with O-sialoglycoprotein endopeptidase (OSGPE) rendered them able to induce activation of human T cells in a range comparable with the controls (Figure 5B). In addition the treatment with OSGPE abolishes the recognition by the anti-TcMuc-e2 antibodies (Figure 6). This result suggests that the TcMuc-e2 product is being not only highly O-glycosylated but also sialylated, as deduced from the specificity of the enzyme used (Abdullah et al., 1992Go). To elucidate the role of sialic acid on the immunomodulatory effect, transfected Vero cells were exposed to a Clostridium perfringens neuraminidase. This treatment induced a significant level of activation when compared to nonsialidase-treated transfected Vero cells (Figure 5B).



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Fig. 5. (A) Requirement of posttranslational modifications to achieve the anergic effect. Human lymphocytes stimulated with 0.2 µg/ml of phytohemagglutinin were exposed to purified TcMuc-e2 recombinant protein (rTCMuc-e2) or to a purified extract from TcMuc-e2 transfected Vero cells (sTcMuc-e2). The difference of the expression of the CD25 antigen between both populations was significant (P < 0.001, Student t test). (+) Positive control: phytohemagglutinin-stimulated human lymphocytes; (–) negative control: resting nonstimulated human lymphocytes. (B) Different populations of Vero cells monolayers were either mock-treated or treated with OSGPE (indicated by an asterisk) or sialidase I (indicated by double asterisk). Cells were washed, human T cells were added, and leukocyte activation was assessed after 3 days of coculture by measuring CD25 using flow cytometry as in Figure 3. Means ± SE are plotted. Difference between E2-Vc and E2-Vc* was significant (P < 0.001, Student t test). Difference between E2-Vc and E2-Vc** was significant (P < 0.05, Student t test)

 


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Fig. 6. Recognition by anti-TcMuc-e2 antibodies was abolished after treatment with OSGPE. TcMuc-e2 transfected Vero cells were either treated (A) or mock-treated (B) with OSGPE. Cells were washed and stained using anti-TcMuc antibodies obtained from mice immunized with the plasmid pCTcMUCe2/IgEsp. The study was developed with peroxidase-antiperoxidase and hematoxilin counterstaining (50x, bars indicate 10 µm).

 
TcMuc-e2 mucin-mediated downregulation of human lymphocytes is reversible
To determine whether TcMuc-e2 mucin-induced inhibition of human T cell proliferation could be affected exogenously, IL-2 was added to T lymphocytes previously exposed to TcMuc-e2 Vero cells. We found that the addition of exogenous human IL-2 to lymphocytes induces a proliferative activity in culture, independently of a previous exposure to TcMuc-e2 Vero cells (Figure 7).



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Fig. 7. The inhibition of human lymphocyte proliferation is reversible by IL-2. Normal human lymphocytes were cocultured with E2-Vc, as described in Figure 2. After the initial culture, IL-2 (50 U/ml) was added to one of the culture plates (E2-Vc*). After this treatment the culture plate was set up as previously described. Inhibition of proliferation seen in Figure 2 and in the nontreated plate (E2-Vc) in this experiment was reversed in the presence of IL-2. The data are shown as means of cpm from three replicate wells ± SD. The experiment was repeated three times, and data from one representative experiment are shown (P < 0.01 between E2-Vc and E2-Vc* for days 3, 4, and 5, Student t test). (–): Negative controls: resting nontreated human lymphocytes; (+): positive controls: human lymphocytes treated as in E2-Vc*, not cocultured with Vero cells.

 
TcMuc-e2 expressed on Vero cells does not induce apoptosis in human lymphocytes
Our described experiments showed that expression of TcMuc-e2 mucin on Vero cells inhibits human lymphocytes proliferative response and that this inhibition could be reversed by the addition of exogenous human IL-2. To evaluate if the lack of proliferation was also related to a significant degree of apoptosis being induced by the TcMuc transfected cells, a sample of human lymphocytes exposed to either E2-Vc or pC-Vc was fixed in formaldehyde and studied by the TUNEL method. Staining for apoptosis in human T cells exposed to either E2-Vc or pC-Vc was almost undetectable by days 3 and 4 (Figure 8A). Only scattered (5%) preapoptotic lymphocytes (Figure 8B) were observed by day 5 in human T cells exposed to E2-Vc compared to 2% in those exposed to pC-Vc controls (Figure 8A).



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Fig. 8. Apoptosis is not responsible for the inhibition of proliferation in human lymphocytes incubated with E2-Vc. Samples of lymphocytes from the experiment shown in Figure 3B were stained for apoptosis by the TUNEL method. Positive cells in five fields with 300 cells each were recorded. (A) Mean ± SD of apoptotic cells at each day of coculture is indicated. (P > 0.05 between lymphocytes exposed to TcMuc-e2 transfected Vero cells (Tc-Muc) and lymphocytes exposed to Vero cells transfected with pCDNA vector (pCDNA), analysis of variance test. (B) In situ TUNEL assay of T lymphocytes cocultured with E2-Vc at day 5. The lymphocytes from E2-Vc cocultures show dense, sharply defined clumps of perinuclear chromatin associated with the nuclear membrane, characteristic of early apoptotic stages (200x, the bar indicates 10 µm).

 

    Discussion
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
In addition to strategies for evading the humoral immune response, T. cruzi possesses the (at least partial) capacity to induce a state of cellular immunosupression both in vitro and in vivo (Tarleton, 1988aGo,b). This state was explained by the action of several different molecules present on the surface of the parasite (Kierszenbaum et al., 1990Go; Millar et al., 1999Go; Leguizamon et al., 1999Go).

We know that the O-glycosylation, both in parasites and in mammalian cells can be different. However, there are interesting functional analogies between these two systems that could be utilized to study the immunomodulatory role of a particular T. cruzi gene encoding a heavily O-glycosylated surface mucin (Pollevick et al., 2000Go) when expressed in mammalian cells. Mucins are involved in immunomodulatory effects both in mammals and in lower organisms (Rudd et al., 1998Go; Kierszenbaum et al., 1999Go). In recent years, in vitro studies have revealed not only that T. cruzi mucins are the most abundant glycoproteins on the surface of the parasite but also that they play a relevant role in the mechanisms displayed by the parasite for cell invasion and evasion from the immune system (Schenkman et al., 1992Go; Pereira-Chioccola et al., 2000Go). On the other hand, it was shown that the inefficient host immune response to cancer antigens is at least in partly modulated by the presence of carcinoma-associated mucins (Gimmi et al., 1996Go; Agrawal et al., 1998Go; Kim et al., 1999Go).

In consistency with those studies, our study shows that the expression of the T. cruzi gene TcMuc-e2 in mammalian cells can inhibit the human T cell proliferative response to a xenogeneic stimulus. The mechanism involved in such anergy presents two sides:

1. On the side of the stimulus, it is known that the TcMuc products are O-glycosylated in T. cruzi (Pollevick et al., 2000Go) as well as in mammalian cells (Di Noia et al., 1996Go). In this regard, our results with the apomucin expressed in bacteria suggest that some posttranslational changes are needed to achieve the immunomodulatory effect. The total abolition of the anergy-inducing effect of E2-Vc by OSGPE treatment and partially by neuraminidase treatment, points to O-glycosylation and sialic acid.

2. On the lymphocytes side, previous studies regarding the induction of unresponsiveness either by cancer mucins or glycoconjugates from T. cruzi have focused on two potential mechanisms: anergy and apoptosis. It is known that tumor-infiltrating lymphocytes express the early activation marker CD69 but express low levels of the IL-2 receptor (Alexander et al., 1995Go). This anergy phenotype associated with cancer mucins is compatible with our results. We found that human lymphocytes exposed to xenogeneic cells, both TcMuc-e2 positive or controls, express the CD69 antigen despite the fact that this expression presents some delay in the case of T cells exposed to E2-Vc. But, as is the case in anergy associated with cancer mucins, T cells exposed to E2-Vc remain as resting lymphocytes with no expression of the CD25 antigen. This mechanism of anergy coincides with the fact that T. cruzi has a selective suppressive effect on IL-2 gene expression (Soong and Tarleton, 1992Go), and with the recent results showing that lymphocytes cocultured with a mucin from T. cruzi show a significant decrease in their capacity to express IL-2 receptor molecules (Kierszenbaum et al., 1999Go).

In concordance with other communications regarding anergy (Sad and Mosmann, 1995Go), we found that E2-Vc-inhibited human lymphocytes retained their capacity to secrete cytokines in response to a xenogeneic challenge. Therefore, partial anergy was achieved, so that cell proliferation and upregulation of CD25 were inhibited, but the T helper 1 cytokine pattern was unaffected.

In the present work, we found only a low degree of early apoptosis in lymphocytes exposed to E2-Vc by day 5, but not before. This process could not explain the early inhibition of proliferation seen by days 3 and 4, and it is probably an associated phenomenon. T cell reversible anergy (Beverly et al., 1992Go) and lack of stimulation through the IL-2 pathway could be a mechanism to explain the TcMuc-e2 mucin-induced inhibition of T cell proliferation and poor expression of the CD25 antigen. In further support of this hypothesis, the addition of exogenous human IL-2 overcame TcMuc-e2 mucin-induced inhibition of proliferation.

Regardless of the mechanism, the fact that TcMuc-e2 mucin expressed on Vero cells can cause such profound inhibitory effects on human lymphocytes is compatible with experimental studies suggesting an immunosuppressive effect of T. cruzi–associated mucins. Furthermore, our results are compatible with clinical studies showing that hosts acutely infected with T. cruzi develop manifestations of immunosuppression, including functional alterations in lymphocytes and other cells relevant to mount an immune response. However, although suggestive, the similarities between the anergic effects of TcMuc-e2 positive Vero cells and those elicited by natural T. cruzi–derived factors must be taken with caution. The structure of posttranslational modifications, in particular O-glycosylation, of both the Vero expressed TcMuc-e2 product and the natural parasite molecule are still not well defined. So, the immunomodulatory effects reported for the native parasite mucins and those observed herein might be achieved by different pathways or due to different effectors. Further work is needed to clarify if any mucin-type molecule expressed in Vero or other cell types would have the same effect, or if there is some particular feature to the T. cruzi apomucin that facilitates the induction of anergy.

The model here described might find, in addition to any possible application to Chagas’ disease, a diversity of applications ranging from parasitology to clinical glycoimmunology. In particular, the field of transplant glycoimmunology could be enriched.


    Materials and methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Culture, transfection, and expression of TcMuc-e2 in higher eukaryotic cells
Vero E-6 cells (kidney fibroblasts from African green monkeys), from ATCC (USA), supplied by a local cell bank (Asociación Banco Argentino de Células), were utilized. Cells were incubated at 37°C in a humidified atmosphere supplemented with 5% CO2. In all cases, Dulbecco’s modified Eagle medium (DMEM) (Life Technologies, Long Island, NY) was used as culture medium, supplemented with 10% fetal bovine serum (Life Technologies).

The gene TcMuc-e2 (GeneBank accession number AF036409) encoding a T. cruzi mucin (Di Noia et al., 1998Go), with its signal peptide replaced by the human IgE signal peptide, was cloned into the eukaryotic vector pCDNA3.1 (Invitrogen, San Diego, CA). The resulting plasmid pCTcMuce2/IgEsp was purified using Qiagen columns (Qiagen, Valencia, CA) and transfected into higher eukaryotic cells by the calcium phosphate procedure (Graham and Eb, 1973Go). After 10 days of drug selection, neomycin-resistant colonies were obtained. The presence of the TcMuc-e2 mRNA was detected by RT-PCR, using TcMuc-e2-specific primers. Expression of the protein TcMuc-e2 on the surface of two different transfected cell types (Vero cells and primary culture of porcine hepatocytes) was ascertained by immunohistochemistry, using three polyclonal antibodies obtained (1) from a rabbit immunized with the synthetic peptide KP2T8KP2; (2) from a rabbit immunized with a recombinant protein having five T8KP2 tandem repeats and the conserved TcMuc C-terminus fused to Schistosoma mansoni glutation-S-transferase; and (3) from a serum obtained from a mouse immunized with the plasmid pCTcMuce2/IgEsp. Vero cells expressing TcMuc-e2 were named E2-Vc. A tagged version of a T. cruzi mucin homologous to TcMuc-e2 has been expressed in Vero cells and has shown to yield a highly O-glycosylated mucin (Di Noia et al., 1996Go). Because our present objective was the study of immunomodulation, the presence of an immunogenic tag not belonging to the original TcMuc product was avoided in these experiments. pC-Vc and NT-Vc were used as controls throughout the experiments.

Preparation of T cells from PBMCs
Fresh blood drawn from healthy donors using heparinized syringes was centrifuged through Histopaque-1077 (Sigma Diagnostics, St. Louis, MO). The PBMCs collected at the interface were washed twice and resuspended in DMEM. Enriched T cell populations were purified by using nylon wool columns described elsewhere (Agrawal et al., 1996Go). Cell viability, determined by trypan blue exclusion, was above 90%.

Mixed xenogeneic Vero cells–lymphocytes culture
Vero cells, both TcMuc-e2 transfected or controls, were used as stimulators and human lymphocytes as responder cells. One hundred microliters of a suspension of T cells (1.5 x 106) from healthy individuals were incubated with 100 µl of a suspension of cobalt-irradiated (1200 Rads) Vero cells. Culture media were supplemented with 20% human fresh serum from a pool of healthy donors. Every experiment with lymphocytes from the same donor was done by triplicate and PBMCs from three different donors were used. Proliferation assessment of human lymphocytes was done at days 3, 4, and 5. [3H]Thymidine (1 µCi, New England Nuclear, Boston, MA) was added to each well 6 h before culture withdrawal. Cells were harvested on filter paper and cpm determined in a liquid scintillation counter. In a different experiment after the initial culture, lymphocytes exposed to TcMuc-e2 Vero cells were treated with human IL-2 (Sigma Diagnostics). After addition of IL-2 (50 U/ml), the well plate cultures were set up as described previously, and proliferation assessment was done.

Detection of IFN-{gamma} by ELISA
Nunc enzyme-linked immunosorbent assay (ELISA) plates coated with mouse anti-human IFN-{gamma} (Genzyme, Cambridge, MA) were used according to manufacturer instructions. The plates were read in an ELISA reader in the kinetic mode.

Detection of apoptosis
Lymphocytes were washed (phosphate buffered saline [PBS], pH 7.2) and fixed with neutral buffer formaldehyde for 24 h. Antigen recuperation was done in a microwave oven. Endogenous peroxidases were blocked with PBS:H2O2 (70:30), and protein blockade was done with normal calf serum. TUNEL assay (Apoptag peroxidase kit, Intergen, Oxford, UK) was utilized according to the instructions supplied by the manufacturer.

Flow cytometry
Analyses were done on an Ortho Cytoron Flow Cytometer (Ortho Diagnostics, Raritan, NJ). To evaluate the expression of CD25, CD69 antigens on CD3(+) lymphocytes, relative fluorescence intensity was measured using anti-CD25 (Immunotech, Westbrook, ME) and anti-CD69 (Becton-Dickinson, Mountain View, CA) antibodies.

Recombinant protein expression and purification
The insert from a genomic clone of TcMuc gene isolated from an expression library (Reyes et al., 1994Go) was cloned in pGEX-1{lambda}T vector (Pharmacia Biotech, Uppsala, Sweden). The resulting recombinant protein contained the hypervariable region, the central amino acid repeats and the conserved C-terminus as C-terminal fusion with glutathione-S-transferase. The glutathione-S-transferase fusion protein was induced and purified by affinity chromatography on glutathione-sepharose beads as described (Di Noia et al., 1996Go). Purity was assessed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and quantified using Bradford reagent (Bio-Rad Laboratories, Richmond, CA).

O-syaloglycoprotease and sialidase treatment
Transfected Vero cells (106) were treated with OSGPE (8 µg/ml, Cedarlane Laboratories, Hornby, Ontario, Canada) in DMEM for 1 h at 37°C. The supernatant was collected and cells washed with PBS. Human lymphocytes (106) were added to the treated or mock-treated monolayers and cocultured for 3 days, after which T cells were harvested and prepared for flow cytometry as indicated. The same procedure was done with Sialidase I from C. perfringens (Glyco, Novato, CA).

Statistical analysis
Numerical data were processed using the Primer of Biostatistics statistical package (Stanton D. Glantz, McGraw-Hill, 1992) and expressed as percent or mean with the corresponding standard deviation. The comparative statistical level of significance was calculated accordingly. In every experiment we considered an alpha of 0.05 and a power of 0.8.


    Acknowledgments
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
This work was partially supported by grants from the Hospital Italiano de Buenos Aires; the UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases; the Agencia Nacional de Promoción Científica y Tecnológica; and the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina. The work of A.C.C.F. was partially supported by an International Research Scholar award from the Howard Hughes Medical Institute. A.C.C.F. and D.O.S. are researchers, and J.D.N. was a postdoctoral fellow of the CONICET during this work.


    Abbreviations
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
DMEM, Dulbecco’s modified Eagle medium; E2-Vc, Vero cells expressing TcMuc-e2; ELISA, enzyme-linked immunosorbent assay; IFN, interferon; NT-Vc, nontransfected Vero cells; OSGPE, O-sialoglycoprotein endopeptidase; PBMC, peripheral blood monnuclear cell; PBS, phosphate buffered saline; pC-Vc, Vero cells transfected with pCDNA3.1; RT-PCR, reverse transcription polymerase chain reaction.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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