A GRA1 DNA Vaccine Primes Cytolytic CD8+ T Cells To Control Acute Toxoplasma gondii Infection
Department of Toxoplasmosis, Pasteur Institute of Brussels, 1180 Brussels, Belgium6vk, http://www.100md.com
Received 2 June 2002/ Returned for modification 27 July 2002/ Accepted 4 October 20026vk, http://www.100md.com
ABSTRACT6vk, http://www.100md.com
Protective immunity against Toxoplasma gondii is known to be mediated mainly by T lymphocytes and gamma interferon (IFN-). The contribution of CD4+ and CD8+ T-lymphocyte subsets to protective immune responses against T. gondii infection, triggered by a GRA1 (p24) DNA vaccine, was assessed in this study. In vitro T-cell depletion experiments indicated that both CD4+ and CD8+ T-cell subsets produced IFN- upon restimulation with a T. gondii lysate. In addition, the GRA1 DNA vaccine elicited CD8+ T cells that were shown to have cytolytic activity against parasite-infected target cells and a GRA1-transfected cell line. C3H mice immunized with the GRA1 DNA vaccine showed 75 to 100% protection, while 0 to 25% of the mice immunized with the empty control vector survived challenge with T. gondii cysts. In vivo T-cell depletion experiments indicated that CD8+ T cells were essential for the survival of GRA1-vaccinated C3H mice during the acute phase of T. gondii infection, while depletion of CD4+ T cells led to an increase in brain cyst burden during the chronic phase of infection.
INTRODUCTIONhh1-i, 百拇医药
In immunocompetent individuals, Toxoplasma gondii generally induces a mild asymptomatic infection that is associated with the rapidly dividing tachyzoite form of the parasite. Resolution of the infection in the host occurs through induction of strong and persistent cell-mediated immunity that results in the control of T. gondii tachyzoites (11, 46). In humans, this relatively benign infection may reactivate under conditions of immunosuppression, resulting in toxoplasma encephalitis and other complications (11). A primary T. gondii infection and subsequent transplacental transmission during pregnancy can result in miscarriage or in severe disease in the infant (26). These pathological consequences associated with congenital toxoplasmosis not only represent a threat to humans but also are a cause of economic losses due to abortions in farm animals (17). Therefore, a vaccine capable of controlling the tachyzoite multiplication associated with the acute primary infection is important and has been the subject of study in our laboratory.
Reports on DNA vaccination against experimental T. gondii infection in mice have been accumulating, and the antigens that have been tested now include T. gondii membrane-associated surface antigen SAG1 (1, 33), excreted-secreted dense granule proteins GRA1 (43), GRA4 (16), and GRA7 (43), and rhoptry proteins ROP1 (23) and ROP2 (30, 43). For all these antigens, immunity was associated with Th1-type responses, which are characterized by production of gamma interferon (IFN-). It was reported previously that DNA vaccination with three distinct immunogenic Toxoplasma antigens (GRA1, GRA7, and ROP2) induced partial immunity in C3H/HeN mice and that the responses were associated with a Th1-type profile (43). The GRA1 antigen (p24), a product of T. gondii tachyzoites and bradyzoites, is a promising vaccine candidate (3, 7, 18, 43). This antigen induces humoral and cellular immune responses in mice and humans in the chronic phase of the infection (7, 19). Vaccination with GRA1 has been shown to be protective in two animal models of infection (18, 38). Adoptive transfer of T cells from rats vaccinated with GRA1-expressing vaccinia virus partially protected nude rats against lethal challenge with the virulent T. gondii RH strain (18). In addition, immunization of sheep with recombinant Mycobacterium bovis BCG producing and secreting GRA1 resulted in specific, partially protective cellular immune responses characterized by the production of IFN- (38).
It is well established that IFN- and T cells play a central role in host resistance to T. gondii during both the acute and chronic phases of infection (11, 46). However, the concerted interplay of several other cytokines may be necessary to maintain the delicate balance between protection and immunopathology caused by excessive inflammation (22, 32, 35). Another crucial interaction for protection against a T. gondii infection is the one between CD8+ and CD4+ T cells (21). Although some reports have suggested that CD8+ T cells mediate their effect through IFN- production (5, 14, 27), CD8+ T cells with cytotoxic activity against infected cells have been described in humans (9, 31, 34, 45) and mice (8, 12, 25, 27, 37).if7t, 百拇医药
In the present study we compared the contributions of CD4+ and CD8+ T cells in the protection conferred by the pVR1020-GRA1 DNA vaccine. Both CD4+ and CD8+ T cells produced IFN-, and the CD8+ T-cell subset had T. gondii-specific cytolytic activity. Our results suggest that although CD4+ T cells are the strongest producers of IFN-, CD8+ T cells are the major effectors of the vaccine-induced protection against acute toxoplasmosis.
MATERIALS AND METHODS);, 百拇医药
Plasmid construction. The DNA construct used for vaccination was based on the plasmid vector VR1020, obtained from Vical, Inc. (San Diego, Calif.). The gene encoding GRA1 was amplified by PCR from cloned DNA fragments and inserted into pVR1020 as described previously (43). Briefly, sense and antisense primers were designed to contain a BamHI restriction site. The gene was cloned into the BamHI site of the expression vector pVR1020 to generate an in-frame fusion with the vector-encoded signal sequence of human tissue plasminogen activator. All plasmids were propagated in Escherichia coli DH1. DNA for vaccination was purified by anion-exchange chromatography (EndoFree plasmid giga kits; Qiagen GmBH, Hilden, Germany) and was dissolved in sterile endotoxin-free phosphate-buffered saline (PBS) (BioWhittaker Europe, Verviers, Belgium). Plasmid integrity was checked by agarose gel electrophoresis after digestion with appropriate restriction enzymes. The DNA concentration was determined by absorbance at 260 nm.
Vaccination of experimental animals. Female inbred C3H/HeN mice (H-2k) that were 6 to 8 weeks old were purchased from Harlan (Horst, The Netherlands). The C3H/HeN mouse model is relevant for investigation of the induction of protective anti-T. gondii immune responses because the mice are moderately resistant to acute infection and develop T. gondii brain cysts and progressive toxoplasma encephalitis (39). These features allow workers to monitor the acute phase as well as the chronic phase of infection in response to vaccination. The mice received three injections of 100 µg of pVR1020-GRA1 DNA (separated by 2-week intervals) in both tibialis anterior muscles administered with a 0.3-ml syringe (BD Biosciences, San Diego, Calif.). Mice injected with the empty vector pVR1020 were used as negative controls. Three weeks after the third DNA injection, the vaccinated mice were bled from the tail vein, and sera were analyzed with a GRA1-specific enzyme-linked immunosorbent assay (ELISA) as described previously (43). All mice vaccinated with pVR1020-GRA1 were shown to have GRA1-specific antibodies after the third injection. This study was conducted in compliance with the regulations concerning the use of laboratory animals at the Pasteur Institute, Brussels, Belgium.
Parasite and antigen preparation. The T. gondii type II IPB-G and IPB-M strains (43) were isolated from the placentas of women who gave birth to infants with congenital toxoplasmosis. Tachyzoites of the IPB-M and IPB-G strains were obtained from the ascites of C3H mice infected by intraperitoneal injection and subcutaneously treated with hydrocortisone acetate (25 µg; Laboratoires Roussel, Paris, France) at 2-day intervals for 1 week. Expression of GRA1 in both strains was confirmed by Western blotting.a4#{i^3, 百拇医药
To prepare toxoplasma lysate (TLA), tachyzoites from the virulent T. gondii RH strain were obtained from the peritoneal fluid of infected Swiss mice as described previously (43). The material was passed twice through a 26-gauge needle. The parasites were washed, resuspended in PBS, and sonicated (1-min burst, 1 min of cooling, 150 W) with an Ultrasonic disintegrator (MSE, Leicester, United Kingdom). The protein concentration of TLA was determined by the Bio-Rad DC protein assay (Bio-Rad Laboratories, Hercules, Calif.).
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was carried out as described by Laemmli (28) by using a 12% polyacrylamide gel and the Bio-Rad minigel system (Bio-Rad Laboratories). The BenchMark prestained protein ladder (Life Technologies, Grand Island, N.Y.) was used for molecular weight standards. Electrophoretic transfer onto nitrocellulose membranes (Hybond-C; Amersham Biosciences, Uppsala, Sweden) was done with a mini Trans-Blot electrophoretic cell system (Bio-Rad Laboratories) as instructed by the manufacturer. The membrane was blocked by incubation with 3% bovine serum albumin (Merck, Darmstadt, Germany) in Tris-buffered saline containing 0.1% Tween 20 for 1 h at room temperature. For detection of recombinant GRA1 in BW-Sp3 transfectants, a polyclonal pool of sera from five T. gondii-infected C3H mice, diluted 1:200, was used as the primary antibody; it was incubated overnight at 4°C and subsequently with a peroxidase-labeled rat anti-mouse immunoglobulin G (IgG) (Amersham Biosciences) as the secondary antibody for 1 h at room temperature. The chemiluminescent compound Supersignal (Pierce, Rockford, Ill.) was used as the substrate according to the manufacturer's instructions. For detection of native GRA1 in T. gondii IPB-M- and IPB-G-derived TLA, a monoclonal antibody (MAb) against GRA1, MAb BATO 35 (36), was used at a dilution of 1:1,000, and pools of sera from three seropositive pVR1020-GRA1-vaccinated C3H mice or pVR1020-vaccinated C3H mice, diluted 1:300, were also used. These primary antibodies were incubated overnight at 4°C with the membrane strips. A peroxidase-labeled rat anti-mouse IgG (Sigma, St. Louis, Mo.) was used as the secondary antibody for 1 h at room temperature. The chromogenic reaction was performed with 4-chloro-naphthol substrate tablets (Sigma) according to the manufacturer's instructions. The reaction was stopped by washing the preparations in water.
In vitro spleen cell cultures. Animals were sacrificed 2 months after the third DNA injection. Single-cell suspensions of splenocytes were prepared, and red blood cells were lysed with RBC Lysing buffer (Sigma). The cell suspension recovered was washed in RPMI 1640 (GIBCO, Life Technologies, Paisley, United Kingdom) and plated on RPMI 1640 supplemented with 10% fetal calf serum (FCS), 2 mM glutamine 1640 (GIBCO, Life Technologies), 0.05 mM 2-mercaptoethanol (Sigma), 1x nonessential amino acids (GIBCO, Life Technologies), 1 mM sodium pyruvate (GIBCO, Life Technologies), and 100 U of penicillin-streptomycin (Life Technologies) per ml. Splenocytes (3 x 106 cells/ml) were stimulated with TLA (25 µg/ml) and cultured for 4 days in 24-well plates (Nunc, Roskilde, Denmark). The optimal TLA concentration (25 µg/ml) and the optimal time of culture were determined previously on the basis of a kinetics experiment (data not shown).j., http://www.100md.com
In vitro depletion of CD4+ and CD8+ T cells and IFN- measurement. For in vitro and in vivo depletion experiments, culture supernatants of the anti-mouse CD4-producing GK1.5 hybridoma (American Type Culture Collection) and the anti-mouse CD-8{alpha}
-producing H35-17.2 clone (kindly provided by Anja Geldhof, Free University of Brussels) were obtained and concentrated by passage through protein G-Sepharose columns (Amersham Biosciences) by using an Econopump and a UV monitor (Bio-Rad Laboratories). The concentrates were exhaustively dialyzed against PBS, and the protein content was measured by the Bio-Rad DC protein assay (Bio-Rad Laboratories). The MAbs were divided into aliquots and stored at -80°C until they were used. Selective in vitro depletion of CD4+ or CD8+ T cells was achieved by incubating splenocytes (2 x 107 cells) either with 20 µg of the anti-mouse CD4 MAb per ml or with 20 µg of the anti-mouse CD8{alpha}vyp?, http://www.100md.com
MAb per ml at 4°C for 30 min. The cells were washed, the cell concentration was adjusted to the original concentration in medium containing 5% FCS, and each preparation was incubated by using a ratio of sheep anti-rat IgG M-450 dynabeads (Dynal Biotech, Oslo, Norway) to cells of 4:1 for 1 h at 37°C with constant rotation. CD4+ and/or CD8+ T cells were removed by magnetic sorting. Finally, the cells were washed twice and resuspended at a concentration of 3 x 10 54 cells/ml in complete medium (RPMI 1640 containing 2 mM glutamine, 1x nonessential amino acids [GIBCO, Life Technologies], 1 mM sodium pyruvate [GIBCO, Life Technologies], 100 U of penicillin/ml, 100 µg of streptomycin/ml, and 50 µM 2-mercaptoethanol). The efficiency of depletion of each T-cell subset was monitored by cytofluorometric analysis before and after depletion, as well as after the 4 days of in vitro stimulation with TLA, and was found to be at least 90% for each T-cell subset. Both T-cell subsets remained at these levels on day 4 of the experiment (data not shown), when the supernatants were harvested. The IFN-(T. Scorza S. D'Souza . Laloup J. Dewit J. De Braekeleer H. Verschueren M. Vercammen K. Huygen and E.)
Received 2 June 2002/ Returned for modification 27 July 2002/ Accepted 4 October 20026vk, http://www.100md.com
ABSTRACT6vk, http://www.100md.com
Protective immunity against Toxoplasma gondii is known to be mediated mainly by T lymphocytes and gamma interferon (IFN-). The contribution of CD4+ and CD8+ T-lymphocyte subsets to protective immune responses against T. gondii infection, triggered by a GRA1 (p24) DNA vaccine, was assessed in this study. In vitro T-cell depletion experiments indicated that both CD4+ and CD8+ T-cell subsets produced IFN- upon restimulation with a T. gondii lysate. In addition, the GRA1 DNA vaccine elicited CD8+ T cells that were shown to have cytolytic activity against parasite-infected target cells and a GRA1-transfected cell line. C3H mice immunized with the GRA1 DNA vaccine showed 75 to 100% protection, while 0 to 25% of the mice immunized with the empty control vector survived challenge with T. gondii cysts. In vivo T-cell depletion experiments indicated that CD8+ T cells were essential for the survival of GRA1-vaccinated C3H mice during the acute phase of T. gondii infection, while depletion of CD4+ T cells led to an increase in brain cyst burden during the chronic phase of infection.
INTRODUCTIONhh1-i, 百拇医药
In immunocompetent individuals, Toxoplasma gondii generally induces a mild asymptomatic infection that is associated with the rapidly dividing tachyzoite form of the parasite. Resolution of the infection in the host occurs through induction of strong and persistent cell-mediated immunity that results in the control of T. gondii tachyzoites (11, 46). In humans, this relatively benign infection may reactivate under conditions of immunosuppression, resulting in toxoplasma encephalitis and other complications (11). A primary T. gondii infection and subsequent transplacental transmission during pregnancy can result in miscarriage or in severe disease in the infant (26). These pathological consequences associated with congenital toxoplasmosis not only represent a threat to humans but also are a cause of economic losses due to abortions in farm animals (17). Therefore, a vaccine capable of controlling the tachyzoite multiplication associated with the acute primary infection is important and has been the subject of study in our laboratory.
Reports on DNA vaccination against experimental T. gondii infection in mice have been accumulating, and the antigens that have been tested now include T. gondii membrane-associated surface antigen SAG1 (1, 33), excreted-secreted dense granule proteins GRA1 (43), GRA4 (16), and GRA7 (43), and rhoptry proteins ROP1 (23) and ROP2 (30, 43). For all these antigens, immunity was associated with Th1-type responses, which are characterized by production of gamma interferon (IFN-). It was reported previously that DNA vaccination with three distinct immunogenic Toxoplasma antigens (GRA1, GRA7, and ROP2) induced partial immunity in C3H/HeN mice and that the responses were associated with a Th1-type profile (43). The GRA1 antigen (p24), a product of T. gondii tachyzoites and bradyzoites, is a promising vaccine candidate (3, 7, 18, 43). This antigen induces humoral and cellular immune responses in mice and humans in the chronic phase of the infection (7, 19). Vaccination with GRA1 has been shown to be protective in two animal models of infection (18, 38). Adoptive transfer of T cells from rats vaccinated with GRA1-expressing vaccinia virus partially protected nude rats against lethal challenge with the virulent T. gondii RH strain (18). In addition, immunization of sheep with recombinant Mycobacterium bovis BCG producing and secreting GRA1 resulted in specific, partially protective cellular immune responses characterized by the production of IFN- (38).
It is well established that IFN- and T cells play a central role in host resistance to T. gondii during both the acute and chronic phases of infection (11, 46). However, the concerted interplay of several other cytokines may be necessary to maintain the delicate balance between protection and immunopathology caused by excessive inflammation (22, 32, 35). Another crucial interaction for protection against a T. gondii infection is the one between CD8+ and CD4+ T cells (21). Although some reports have suggested that CD8+ T cells mediate their effect through IFN- production (5, 14, 27), CD8+ T cells with cytotoxic activity against infected cells have been described in humans (9, 31, 34, 45) and mice (8, 12, 25, 27, 37).if7t, 百拇医药
In the present study we compared the contributions of CD4+ and CD8+ T cells in the protection conferred by the pVR1020-GRA1 DNA vaccine. Both CD4+ and CD8+ T cells produced IFN-, and the CD8+ T-cell subset had T. gondii-specific cytolytic activity. Our results suggest that although CD4+ T cells are the strongest producers of IFN-, CD8+ T cells are the major effectors of the vaccine-induced protection against acute toxoplasmosis.
MATERIALS AND METHODS);, 百拇医药
Plasmid construction. The DNA construct used for vaccination was based on the plasmid vector VR1020, obtained from Vical, Inc. (San Diego, Calif.). The gene encoding GRA1 was amplified by PCR from cloned DNA fragments and inserted into pVR1020 as described previously (43). Briefly, sense and antisense primers were designed to contain a BamHI restriction site. The gene was cloned into the BamHI site of the expression vector pVR1020 to generate an in-frame fusion with the vector-encoded signal sequence of human tissue plasminogen activator. All plasmids were propagated in Escherichia coli DH1. DNA for vaccination was purified by anion-exchange chromatography (EndoFree plasmid giga kits; Qiagen GmBH, Hilden, Germany) and was dissolved in sterile endotoxin-free phosphate-buffered saline (PBS) (BioWhittaker Europe, Verviers, Belgium). Plasmid integrity was checked by agarose gel electrophoresis after digestion with appropriate restriction enzymes. The DNA concentration was determined by absorbance at 260 nm.
Vaccination of experimental animals. Female inbred C3H/HeN mice (H-2k) that were 6 to 8 weeks old were purchased from Harlan (Horst, The Netherlands). The C3H/HeN mouse model is relevant for investigation of the induction of protective anti-T. gondii immune responses because the mice are moderately resistant to acute infection and develop T. gondii brain cysts and progressive toxoplasma encephalitis (39). These features allow workers to monitor the acute phase as well as the chronic phase of infection in response to vaccination. The mice received three injections of 100 µg of pVR1020-GRA1 DNA (separated by 2-week intervals) in both tibialis anterior muscles administered with a 0.3-ml syringe (BD Biosciences, San Diego, Calif.). Mice injected with the empty vector pVR1020 were used as negative controls. Three weeks after the third DNA injection, the vaccinated mice were bled from the tail vein, and sera were analyzed with a GRA1-specific enzyme-linked immunosorbent assay (ELISA) as described previously (43). All mice vaccinated with pVR1020-GRA1 were shown to have GRA1-specific antibodies after the third injection. This study was conducted in compliance with the regulations concerning the use of laboratory animals at the Pasteur Institute, Brussels, Belgium.
Parasite and antigen preparation. The T. gondii type II IPB-G and IPB-M strains (43) were isolated from the placentas of women who gave birth to infants with congenital toxoplasmosis. Tachyzoites of the IPB-M and IPB-G strains were obtained from the ascites of C3H mice infected by intraperitoneal injection and subcutaneously treated with hydrocortisone acetate (25 µg; Laboratoires Roussel, Paris, France) at 2-day intervals for 1 week. Expression of GRA1 in both strains was confirmed by Western blotting.a4#{i^3, 百拇医药
To prepare toxoplasma lysate (TLA), tachyzoites from the virulent T. gondii RH strain were obtained from the peritoneal fluid of infected Swiss mice as described previously (43). The material was passed twice through a 26-gauge needle. The parasites were washed, resuspended in PBS, and sonicated (1-min burst, 1 min of cooling, 150 W) with an Ultrasonic disintegrator (MSE, Leicester, United Kingdom). The protein concentration of TLA was determined by the Bio-Rad DC protein assay (Bio-Rad Laboratories, Hercules, Calif.).
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was carried out as described by Laemmli (28) by using a 12% polyacrylamide gel and the Bio-Rad minigel system (Bio-Rad Laboratories). The BenchMark prestained protein ladder (Life Technologies, Grand Island, N.Y.) was used for molecular weight standards. Electrophoretic transfer onto nitrocellulose membranes (Hybond-C; Amersham Biosciences, Uppsala, Sweden) was done with a mini Trans-Blot electrophoretic cell system (Bio-Rad Laboratories) as instructed by the manufacturer. The membrane was blocked by incubation with 3% bovine serum albumin (Merck, Darmstadt, Germany) in Tris-buffered saline containing 0.1% Tween 20 for 1 h at room temperature. For detection of recombinant GRA1 in BW-Sp3 transfectants, a polyclonal pool of sera from five T. gondii-infected C3H mice, diluted 1:200, was used as the primary antibody; it was incubated overnight at 4°C and subsequently with a peroxidase-labeled rat anti-mouse immunoglobulin G (IgG) (Amersham Biosciences) as the secondary antibody for 1 h at room temperature. The chemiluminescent compound Supersignal (Pierce, Rockford, Ill.) was used as the substrate according to the manufacturer's instructions. For detection of native GRA1 in T. gondii IPB-M- and IPB-G-derived TLA, a monoclonal antibody (MAb) against GRA1, MAb BATO 35 (36), was used at a dilution of 1:1,000, and pools of sera from three seropositive pVR1020-GRA1-vaccinated C3H mice or pVR1020-vaccinated C3H mice, diluted 1:300, were also used. These primary antibodies were incubated overnight at 4°C with the membrane strips. A peroxidase-labeled rat anti-mouse IgG (Sigma, St. Louis, Mo.) was used as the secondary antibody for 1 h at room temperature. The chromogenic reaction was performed with 4-chloro-naphthol substrate tablets (Sigma) according to the manufacturer's instructions. The reaction was stopped by washing the preparations in water.
In vitro spleen cell cultures. Animals were sacrificed 2 months after the third DNA injection. Single-cell suspensions of splenocytes were prepared, and red blood cells were lysed with RBC Lysing buffer (Sigma). The cell suspension recovered was washed in RPMI 1640 (GIBCO, Life Technologies, Paisley, United Kingdom) and plated on RPMI 1640 supplemented with 10% fetal calf serum (FCS), 2 mM glutamine 1640 (GIBCO, Life Technologies), 0.05 mM 2-mercaptoethanol (Sigma), 1x nonessential amino acids (GIBCO, Life Technologies), 1 mM sodium pyruvate (GIBCO, Life Technologies), and 100 U of penicillin-streptomycin (Life Technologies) per ml. Splenocytes (3 x 106 cells/ml) were stimulated with TLA (25 µg/ml) and cultured for 4 days in 24-well plates (Nunc, Roskilde, Denmark). The optimal TLA concentration (25 µg/ml) and the optimal time of culture were determined previously on the basis of a kinetics experiment (data not shown).j., http://www.100md.com
In vitro depletion of CD4+ and CD8+ T cells and IFN- measurement. For in vitro and in vivo depletion experiments, culture supernatants of the anti-mouse CD4-producing GK1.5 hybridoma (American Type Culture Collection) and the anti-mouse CD-8{alpha}
-producing H35-17.2 clone (kindly provided by Anja Geldhof, Free University of Brussels) were obtained and concentrated by passage through protein G-Sepharose columns (Amersham Biosciences) by using an Econopump and a UV monitor (Bio-Rad Laboratories). The concentrates were exhaustively dialyzed against PBS, and the protein content was measured by the Bio-Rad DC protein assay (Bio-Rad Laboratories). The MAbs were divided into aliquots and stored at -80°C until they were used. Selective in vitro depletion of CD4+ or CD8+ T cells was achieved by incubating splenocytes (2 x 107 cells) either with 20 µg of the anti-mouse CD4 MAb per ml or with 20 µg of the anti-mouse CD8{alpha}vyp?, http://www.100md.com
MAb per ml at 4°C for 30 min. The cells were washed, the cell concentration was adjusted to the original concentration in medium containing 5% FCS, and each preparation was incubated by using a ratio of sheep anti-rat IgG M-450 dynabeads (Dynal Biotech, Oslo, Norway) to cells of 4:1 for 1 h at 37°C with constant rotation. CD4+ and/or CD8+ T cells were removed by magnetic sorting. Finally, the cells were washed twice and resuspended at a concentration of 3 x 10 54 cells/ml in complete medium (RPMI 1640 containing 2 mM glutamine, 1x nonessential amino acids [GIBCO, Life Technologies], 1 mM sodium pyruvate [GIBCO, Life Technologies], 100 U of penicillin/ml, 100 µg of streptomycin/ml, and 50 µM 2-mercaptoethanol). The efficiency of depletion of each T-cell subset was monitored by cytofluorometric analysis before and after depletion, as well as after the 4 days of in vitro stimulation with TLA, and was found to be at least 90% for each T-cell subset. Both T-cell subsets remained at these levels on day 4 of the experiment (data not shown), when the supernatants were harvested. The IFN-(T. Scorza S. D'Souza . Laloup J. Dewit J. De Braekeleer H. Verschueren M. Vercammen K. Huygen and E.)