VIRAL IMMUNOLOGY Volume 5, Number 2, 1992 Mary Ann Lieben, Inc., Publishers Pp. 93-103
Herpes Simplex Virus-1-Specific Human Cytotoxic T Lymphocytes Are Induced in Vitro by Autologous Virus-Infected Mononuclear Cells
RITA
MACCARIO,1
M. GRAZIA REVELLO,2 PATRIZIA GIUSEPPE GERNA2
COMOLI,'
and
ABSTRACT
technique for in vitro activation of cytotoxic T lymphocytes (CTLs) specific for herpes simplex virus type 1 (HSV-1) is described. Autologous phytohemagglutinin (PHA)-activated, HSV-1-infected peripheral blood mononuclear cells (PBMC) were used, after fixation with 1% paraformaldehyde, to activate virus-specific CTLs in short-term cultures. The same unfixed PBMC were used as target cells in the cytotoxicity assay. By using this technique high levels of HSV-1-specific cytotoxic activity (50.06 ± 16.76% at 30:1 effectontarget ratio) were repeatedly obtained in 24 experiments using PBMC from 16 HSV-1 antibody-positive healthy donors, while no cytotoxic activity was observed using PBMC from 3 HSV-1 antibodynegative donors. HSV-1-induced CTLs were shown to be virus-specific as they did not lyse autologous, PHA-activated PBMC infected with influenza A virus or autologous EpsteinBarr virus (EBV) lymphoblastoid cell line (LCL), while they were able to lyse both HSV-1-infected, autologous PHA-activated PBMC and EBV-LCL. HSV-1-specific cytotoxicity was mediated by T lymphocytes, since depletion of CD3-positive cells from the effector population completely removed the killing of HSV-1-infected target cells. CD8-positive CTLs were primarily involved in the killing of HSV-1-infected targets since depletion of CD8positive cells caused a strong reduction of virus-specific cytotoxic activity while elimination of CD4-positive lymphocytes increased killing capacity. Finally, this technique has proven to be highly reproducible, easy to perform, and thus suitable for clinical investigations. A
new
virus type 1 (HSV-1) is a common and widespread human pathogen. Clinical manifestations of HSV-1 infection depend on several factors such as age, host immune status, and anatomic site of the infection. Human neonates and immunocompromised patients are at risk of developing
Herpes simplex
'Department of Pediatrics and 2Virus Policlinico S. Matteo, Pavia, Italy.
Laboratory,
Institute of Infectious Diseases,
93
University
of Pavia, IRCCS
MACCARIO ET AL. HSV infections more frequently than healthy subjects ( 1,6,7). Virus-specific cytotoxic T lymphocytes (CTLs) play a protective role against HSV infections in animal models (15,16,27) and their functioning is supposed to be essential for recovery from virus infections in humans. Several authors have demonstrated the presence of circulating precursor cells that are able to generate HSV-specific human CTLs in vitro. The in vitro activation of HSV-specific CTLs was achieved by stimulation of peripheral blood mononuclear cells (PBMC) with soluble viral antigens (14,25,26,28,29,31-33,35,36) or virus-infected autologous fibroblasts (34). In the present study, the activation of HSV-1-specific CTLs was obtained by short-term in vitro culture of PBMC with autologous, phytohemagglutinin (PHA)-stimulated PBMC, which were infected with HSV-1 and then fixed with 1% paraformaldehyde before use as stimulators. The same infected cells, without fixation, were employed as target cells. By testing a group of healthy HSV-1-immune or nonimmune adults we demonstrated that autologous, HSV-1-infected mononuclear cells are able to induce strong and highly reproducible virus-specific CTL activity only in immune donors. severe
MATERIALS AND METHODS Virus strains. HSV-1 reference strain (Mclntyre), originally obtained from the American Type Culture Collection (ATCC, Rockville, MD), was propagated in Vero cell cultures. When the cytopathic effect was 100%, the virus stock was harvested, clarified by low-speed centrifugation, aliquoted, and stored at 80°C until use. For control experiments, influenza A virus, strain A/Port Chalmers/1 /73, also obtained from ATCC, was propagated in primary African green monkey kidney cell cultures, harvested as a cell culture medium when the hemagglutination titer was > 1:160, and stored at -80CC. HSV-1 isolation from PHA-activated, HSV-1-infected PBMC was performed in Vero cell cultures and virus identification was achieved by immunofluorescence using type-specific monoclonal antibodies as reported (23). Donors and cell separation. Heparinized peripheral blood was collected once or more from 19 healthy volunteers and PBMC were obtained by centrifugation over a Ficoll-Hypaque gradient as describedby Boyum (3). The donors' immune status to HSV-1 was determined by enzyme-linked immunosorbent assay (23). Establishment of lymphoblastoid cell line (LCL). PBMC were incubated with Epstein-Barr virus (EBV)-containing supernatant (1:4) from B95.8 marmoset cell line (ATCC), in the presence of 600-800 ng/ml of cyclosporin A, in complete medium (RPMI 1640 medium supplemented with 2 mM L-glutamine, 50 u.g/ml gentamicin, and 10% fetal calf serum). EBV-LCL were maintained by serial passages in complete medium. Preparation of stimulator and target cells. PBMC were cultured in the presence of PHA (M Form, Gibco Ltd, Paisley, Scotland; 1:100) at a concentration of 5 x 105 cells/ml, in 25 cm2 flasks or in 24-well plates using complete medium. Incubation, at 37°C in a 5% C02 humidified atmosphere was continued for 48-72 hr. For HSV-1 infection, PHA-activated PBMC, or EBV-LCL, were extensively washed and the cell pellet was incubated with HSV-1 suspension (MOI: 10-50) for 1 hr at 37°C. After 2 further washings, the infected cells were cultured 18 hr at 37°C. For influenza virus infection, PHA-activated PBMC were incubated at 37CC for 2 hr with influenza A virus and washed twice before use. PHA-activated, virus-infected PBMC to be used as stimulator cells were fixed with 1 % paraformaldehyde in phosphate-buffered saline (pH 7.2) for 10 min and extensively washed with complete medium. The same fixed cells were stored at 4°C and then used to restimulate effector cells following 7-day culture. Proliferation assay. PBMC were added to U-shaped microwells at a concentration of 5 x 105 cells/ml and virus-infected paraformaldehyde-fixed stimulator cells were added at a concentration ranging from 5 x 104 to 5 x 105 cells/ml in a final volume of 0.2 ml of RPMI 1640 medium supplemented with 2 mM L-glutamine, 50 u-g/ml gentamicin, and 10% pooled human sera obtained from HSV-1-negative blood donors. Controls included PBMC incubated with medium alone or with mock-infected stimulator cells. Cell cultures were incubated for 7 days at 37°C in a humidified 5% C02 atmosphere. [3H]TdR incorporation was measured during the last 21 hr of incubation by adding 0.5 p,Ci/well of [3H]TdR (specific activity: 2 Ci/mmol) to each well in a volume of 25 u.1. Cultures were harvested onto glass fiber filter disks and [3H]TdR incorporation was measured in a liquid scintillation counter. 94
HSV-1-SPECIFIC CTLs Activation of virus-specific cytotoxic cells. The same protocol was followed to activate HSV-1- and influenza A-specific cytotoxic activity. PBMC were cultured at the concentration of 106 cells/ml in 25-cm2 flasks or in 24-well plates in RPMI 1640 medium supplemented with 2 mM L-glutamine, 50 p-g/ml gentamicin, and 10% pooled human serum obtained from HSV-1-negative volunteers. Autologous, virus-infected, paraformaldehyde-fixed stimulator cells were added at a concentration of 5 x 105/ml. Controls included PBMC incubated with mock-infected stimulator cells. After 7-day culture, effector cells were restimulated with 5 x 105/ml autologous virus-infected fixed stimulator cells and, 3 days later, tested in the cytotoxicity assay. In the experiments designed to obtain influenza A virus-specific effector cells, 10 U/ml of recombinant interleukin-2 (RIL-2, Hoffmann-La Roche Inc., Nutley, NJ) was added, together with stimulator cells, following 7-day culture. Cytotoxicity assay. Target cells included autologous PHA-activated PBMC uninfected and infected with HSV-1 or influenza A virus, autologous EBV-LCL, and HSV-1-infected autologous EBV-LCL. Target cells were labeled with 100 u.Ci 5lCr (New England Nuclear Corp., Boston, MA) for 1-2 hrat 37°C and extensively washed before use. Cytotoxicity was assayed in U-shaped microwells; 0.5-1 x 104 target cells in 0.1 ml of complete medium were added to each well. Different concentrations of effector cells in 0.1 ml of complete medium were added to the wells to achieve an effector to target (E:T) ratio of 100:1, 30:1, and 10:1. Microplates were centrifuged at 200g for 5 min and incubated for 6 hr at 37°C. Following incubation, 0.1 ml of the supernatant was collected from each well and counted for 1 min in a gamma counter. Maximal release was determined by freezing and thawing target cells; spontaneous release was determined by adding 0.1 ml of complete medium to 0.1 ml of target cells. The percentage of specific release was calculated by the formula „
. _
release spontaneous release total release spontaneous release
experimental
„
—
—
Spontaneous release from the target cells was consistently less than 10% for EBV-LCL and less than 25% for PHA-activated PBMC. Results were expressed as percentage of specific lysis or as the number of lytic units (LU)/106 effector cells. An LU was defined as the number of cells required to produce 30% specific cytotoxicity using 5 x 103 labeled target cells.
Surface marker analysis. Monoclonal antibodies used to characterize effector cell populations were anti-Leu4 (CD3), purified and FITC-conjugated anti-Leu3a (CD4), purified and phycoerythrin (PE)conjugated anti-Leu2a (CD8), anti-IL-2R (CD25), anti-HLA-DR, anti-Leullc (CD16), and anti-Leu7 (CD57) (Becton Dickinson, Mountain View, CA). To characterize stimulator cells, the following monoclonal antibodies were employed: anti-HLA-class I (Dakopatts A/S, Glostrup, Denmark) and anti-HLA-DR (Becton-Dickinson). The percentage of cells infected with HSV-1 was evaluated by using type-specific monoclonal antibodies recognizing viral glycoproteins (11) on the surface of infected cells (Syva Co., Palo Alto, CA). Similarly, influenza A virus-infected cells were identified and counted by using a pool of monoclonal antibodies (CDC-A1 and CDC-A3) directed against broadly reactive type-specific antigens present on the cell membrane and developed by A. Kendall at CDC (Atlanta, GA). The immunofluorescence staining was performed as previously described (18). Cytofluorographic analysis of cell populations was performed by means of direct or indirect immunofluorescence on a FACScan flowcytometer. Depletion of T lymphocyte subsets by the magnetic microsphere technique. The same procedure was used to remove CD3-positive, CD4-positive, or CD8-positive lymphocytes from the effector population: 107 cells obtained after 10-day culture were incubated, for 30 min in ice, with 0.3 ml of complete medium, in the presence of 10 u,g of each monoclonal antibody (Leu4, Leu3a, Leu2a, Becton Dickinson). After two washings the cell pellet (labeled cells) was resuspended in 1 ml of PBS (pH 7.4) supplemented with 0.1% bovine serum albumin (PBS-BSA). Magnetic microspheres (4 x 108), coated with goat anti-mouse IgG (Dynabeads M-450, Dynal AS, Oslo, Norway), were washed four times with PBS-BSA and then resuspended in 1 ml of the same medium. Equal volumes of magnetic microspheres and labeled cells were mixed, incubated at 4°C for 25 min, and placed on a magnetic particle concentrator (Dynal MPC-6, Dynal AS, Oslo, Norway). After 2 min, supernatant was recovered and cells were washed twice in complete medium before use. After depletion procedures, CD3-positive cells were
Herpes Simplex Virus-1-Specific Human Cytotoxic T Lymphocytes Are Induced in Vitro by Autologous Virus-Infected Mononuclear Cells
RITA
MACCARIO,1
M. GRAZIA REVELLO,2 PATRIZIA GIUSEPPE GERNA2
COMOLI,'
and
ABSTRACT
technique for in vitro activation of cytotoxic T lymphocytes (CTLs) specific for herpes simplex virus type 1 (HSV-1) is described. Autologous phytohemagglutinin (PHA)-activated, HSV-1-infected peripheral blood mononuclear cells (PBMC) were used, after fixation with 1% paraformaldehyde, to activate virus-specific CTLs in short-term cultures. The same unfixed PBMC were used as target cells in the cytotoxicity assay. By using this technique high levels of HSV-1-specific cytotoxic activity (50.06 ± 16.76% at 30:1 effectontarget ratio) were repeatedly obtained in 24 experiments using PBMC from 16 HSV-1 antibody-positive healthy donors, while no cytotoxic activity was observed using PBMC from 3 HSV-1 antibodynegative donors. HSV-1-induced CTLs were shown to be virus-specific as they did not lyse autologous, PHA-activated PBMC infected with influenza A virus or autologous EpsteinBarr virus (EBV) lymphoblastoid cell line (LCL), while they were able to lyse both HSV-1-infected, autologous PHA-activated PBMC and EBV-LCL. HSV-1-specific cytotoxicity was mediated by T lymphocytes, since depletion of CD3-positive cells from the effector population completely removed the killing of HSV-1-infected target cells. CD8-positive CTLs were primarily involved in the killing of HSV-1-infected targets since depletion of CD8positive cells caused a strong reduction of virus-specific cytotoxic activity while elimination of CD4-positive lymphocytes increased killing capacity. Finally, this technique has proven to be highly reproducible, easy to perform, and thus suitable for clinical investigations. A
new
virus type 1 (HSV-1) is a common and widespread human pathogen. Clinical manifestations of HSV-1 infection depend on several factors such as age, host immune status, and anatomic site of the infection. Human neonates and immunocompromised patients are at risk of developing
Herpes simplex
'Department of Pediatrics and 2Virus Policlinico S. Matteo, Pavia, Italy.
Laboratory,
Institute of Infectious Diseases,
93
University
of Pavia, IRCCS
MACCARIO ET AL. HSV infections more frequently than healthy subjects ( 1,6,7). Virus-specific cytotoxic T lymphocytes (CTLs) play a protective role against HSV infections in animal models (15,16,27) and their functioning is supposed to be essential for recovery from virus infections in humans. Several authors have demonstrated the presence of circulating precursor cells that are able to generate HSV-specific human CTLs in vitro. The in vitro activation of HSV-specific CTLs was achieved by stimulation of peripheral blood mononuclear cells (PBMC) with soluble viral antigens (14,25,26,28,29,31-33,35,36) or virus-infected autologous fibroblasts (34). In the present study, the activation of HSV-1-specific CTLs was obtained by short-term in vitro culture of PBMC with autologous, phytohemagglutinin (PHA)-stimulated PBMC, which were infected with HSV-1 and then fixed with 1% paraformaldehyde before use as stimulators. The same infected cells, without fixation, were employed as target cells. By testing a group of healthy HSV-1-immune or nonimmune adults we demonstrated that autologous, HSV-1-infected mononuclear cells are able to induce strong and highly reproducible virus-specific CTL activity only in immune donors. severe
MATERIALS AND METHODS Virus strains. HSV-1 reference strain (Mclntyre), originally obtained from the American Type Culture Collection (ATCC, Rockville, MD), was propagated in Vero cell cultures. When the cytopathic effect was 100%, the virus stock was harvested, clarified by low-speed centrifugation, aliquoted, and stored at 80°C until use. For control experiments, influenza A virus, strain A/Port Chalmers/1 /73, also obtained from ATCC, was propagated in primary African green monkey kidney cell cultures, harvested as a cell culture medium when the hemagglutination titer was > 1:160, and stored at -80CC. HSV-1 isolation from PHA-activated, HSV-1-infected PBMC was performed in Vero cell cultures and virus identification was achieved by immunofluorescence using type-specific monoclonal antibodies as reported (23). Donors and cell separation. Heparinized peripheral blood was collected once or more from 19 healthy volunteers and PBMC were obtained by centrifugation over a Ficoll-Hypaque gradient as describedby Boyum (3). The donors' immune status to HSV-1 was determined by enzyme-linked immunosorbent assay (23). Establishment of lymphoblastoid cell line (LCL). PBMC were incubated with Epstein-Barr virus (EBV)-containing supernatant (1:4) from B95.8 marmoset cell line (ATCC), in the presence of 600-800 ng/ml of cyclosporin A, in complete medium (RPMI 1640 medium supplemented with 2 mM L-glutamine, 50 u.g/ml gentamicin, and 10% fetal calf serum). EBV-LCL were maintained by serial passages in complete medium. Preparation of stimulator and target cells. PBMC were cultured in the presence of PHA (M Form, Gibco Ltd, Paisley, Scotland; 1:100) at a concentration of 5 x 105 cells/ml, in 25 cm2 flasks or in 24-well plates using complete medium. Incubation, at 37°C in a 5% C02 humidified atmosphere was continued for 48-72 hr. For HSV-1 infection, PHA-activated PBMC, or EBV-LCL, were extensively washed and the cell pellet was incubated with HSV-1 suspension (MOI: 10-50) for 1 hr at 37°C. After 2 further washings, the infected cells were cultured 18 hr at 37°C. For influenza virus infection, PHA-activated PBMC were incubated at 37CC for 2 hr with influenza A virus and washed twice before use. PHA-activated, virus-infected PBMC to be used as stimulator cells were fixed with 1 % paraformaldehyde in phosphate-buffered saline (pH 7.2) for 10 min and extensively washed with complete medium. The same fixed cells were stored at 4°C and then used to restimulate effector cells following 7-day culture. Proliferation assay. PBMC were added to U-shaped microwells at a concentration of 5 x 105 cells/ml and virus-infected paraformaldehyde-fixed stimulator cells were added at a concentration ranging from 5 x 104 to 5 x 105 cells/ml in a final volume of 0.2 ml of RPMI 1640 medium supplemented with 2 mM L-glutamine, 50 u-g/ml gentamicin, and 10% pooled human sera obtained from HSV-1-negative blood donors. Controls included PBMC incubated with medium alone or with mock-infected stimulator cells. Cell cultures were incubated for 7 days at 37°C in a humidified 5% C02 atmosphere. [3H]TdR incorporation was measured during the last 21 hr of incubation by adding 0.5 p,Ci/well of [3H]TdR (specific activity: 2 Ci/mmol) to each well in a volume of 25 u.1. Cultures were harvested onto glass fiber filter disks and [3H]TdR incorporation was measured in a liquid scintillation counter. 94
HSV-1-SPECIFIC CTLs Activation of virus-specific cytotoxic cells. The same protocol was followed to activate HSV-1- and influenza A-specific cytotoxic activity. PBMC were cultured at the concentration of 106 cells/ml in 25-cm2 flasks or in 24-well plates in RPMI 1640 medium supplemented with 2 mM L-glutamine, 50 p-g/ml gentamicin, and 10% pooled human serum obtained from HSV-1-negative volunteers. Autologous, virus-infected, paraformaldehyde-fixed stimulator cells were added at a concentration of 5 x 105/ml. Controls included PBMC incubated with mock-infected stimulator cells. After 7-day culture, effector cells were restimulated with 5 x 105/ml autologous virus-infected fixed stimulator cells and, 3 days later, tested in the cytotoxicity assay. In the experiments designed to obtain influenza A virus-specific effector cells, 10 U/ml of recombinant interleukin-2 (RIL-2, Hoffmann-La Roche Inc., Nutley, NJ) was added, together with stimulator cells, following 7-day culture. Cytotoxicity assay. Target cells included autologous PHA-activated PBMC uninfected and infected with HSV-1 or influenza A virus, autologous EBV-LCL, and HSV-1-infected autologous EBV-LCL. Target cells were labeled with 100 u.Ci 5lCr (New England Nuclear Corp., Boston, MA) for 1-2 hrat 37°C and extensively washed before use. Cytotoxicity was assayed in U-shaped microwells; 0.5-1 x 104 target cells in 0.1 ml of complete medium were added to each well. Different concentrations of effector cells in 0.1 ml of complete medium were added to the wells to achieve an effector to target (E:T) ratio of 100:1, 30:1, and 10:1. Microplates were centrifuged at 200g for 5 min and incubated for 6 hr at 37°C. Following incubation, 0.1 ml of the supernatant was collected from each well and counted for 1 min in a gamma counter. Maximal release was determined by freezing and thawing target cells; spontaneous release was determined by adding 0.1 ml of complete medium to 0.1 ml of target cells. The percentage of specific release was calculated by the formula „
. _
release spontaneous release total release spontaneous release
experimental
„
—
—
Spontaneous release from the target cells was consistently less than 10% for EBV-LCL and less than 25% for PHA-activated PBMC. Results were expressed as percentage of specific lysis or as the number of lytic units (LU)/106 effector cells. An LU was defined as the number of cells required to produce 30% specific cytotoxicity using 5 x 103 labeled target cells.
Surface marker analysis. Monoclonal antibodies used to characterize effector cell populations were anti-Leu4 (CD3), purified and FITC-conjugated anti-Leu3a (CD4), purified and phycoerythrin (PE)conjugated anti-Leu2a (CD8), anti-IL-2R (CD25), anti-HLA-DR, anti-Leullc (CD16), and anti-Leu7 (CD57) (Becton Dickinson, Mountain View, CA). To characterize stimulator cells, the following monoclonal antibodies were employed: anti-HLA-class I (Dakopatts A/S, Glostrup, Denmark) and anti-HLA-DR (Becton-Dickinson). The percentage of cells infected with HSV-1 was evaluated by using type-specific monoclonal antibodies recognizing viral glycoproteins (11) on the surface of infected cells (Syva Co., Palo Alto, CA). Similarly, influenza A virus-infected cells were identified and counted by using a pool of monoclonal antibodies (CDC-A1 and CDC-A3) directed against broadly reactive type-specific antigens present on the cell membrane and developed by A. Kendall at CDC (Atlanta, GA). The immunofluorescence staining was performed as previously described (18). Cytofluorographic analysis of cell populations was performed by means of direct or indirect immunofluorescence on a FACScan flowcytometer. Depletion of T lymphocyte subsets by the magnetic microsphere technique. The same procedure was used to remove CD3-positive, CD4-positive, or CD8-positive lymphocytes from the effector population: 107 cells obtained after 10-day culture were incubated, for 30 min in ice, with 0.3 ml of complete medium, in the presence of 10 u,g of each monoclonal antibody (Leu4, Leu3a, Leu2a, Becton Dickinson). After two washings the cell pellet (labeled cells) was resuspended in 1 ml of PBS (pH 7.4) supplemented with 0.1% bovine serum albumin (PBS-BSA). Magnetic microspheres (4 x 108), coated with goat anti-mouse IgG (Dynabeads M-450, Dynal AS, Oslo, Norway), were washed four times with PBS-BSA and then resuspended in 1 ml of the same medium. Equal volumes of magnetic microspheres and labeled cells were mixed, incubated at 4°C for 25 min, and placed on a magnetic particle concentrator (Dynal MPC-6, Dynal AS, Oslo, Norway). After 2 min, supernatant was recovered and cells were washed twice in complete medium before use. After depletion procedures, CD3-positive cells were
