VIRAL IMMUNOLOGY Volume 4. Number 4, 1991 Mary Ann Liebert, Inc., Publishers
Suppression of Interleukin-2 Production and Activity by Factor(s) Released by Peripheral Blood Mononuclear Cells during Papillomavirus Infections VIMLARANI
CHOPRA1 and STEPHEN K. TYRING12
ABSTRACT
Supernatant fluids from cultured peripheral blood mononuclear cells (PBMC) obtained from patients with extensive papillomavirus infections such as condyloma acuminatum (CA) and epidermodysplasia verruciformis (EV) depressed the proliferative responses of T cells to phytohemagglutinin-P (PHA-P) and the production of interleukin-2 (IL-2) from those preparations. Fluids from the same cultures also inhibited the mitogenic activity of IL-2 on CTLL-2 cells as IL-2-dependent target cells. These soluble suppressor factors (SSF) from PBMC were present in significantly higher concentrations in fluids from cultured PBMC from patients in comparison to healthy controls. A soluble suppressor factor was characterized also from cultured rabbit PBMC after the rabbits had been infected with Shope papillomaviruses. This suppressor factor likewise inhibited IL-2 production and IL-2 activity.
Many oncogenic
and
nononcogenic viral agents alter immune functions and thus augment the develop-
(3, 5, 12, 13, 17, 19, 23, 28, 34, 35). It is not clear whether the tumor grows of because blocking factors, blocking antigens, autoimmune antibodies, or antigen-antibody progressively of a defect(s) in the host's cellular or humoral immunity (20, 27). A variety of because or (2) complexes factors have been identified in human and murine tumors that downregulate immune functions suppressor (31). The source of these suppressor elements could be lymphoid cells (10, 15), macrophages (6, 18), or possibly the tumor itself, which antagonizes the host immune response (31). A depressed immune response has been observed in patients with human papillomavirus (HPV) infections associated with diseases such as condyloma acuminatum (CA) (7) and epidermodysplasia verruciformis (EV) (20). Impairment of these accessory cell functions in HPV infections could be associated with a regulatory substance referred to as suppressor factor(s). In this study, we investigated the presence of suppressor factor(s) in the supernatant fluids of cultured PBMC from humans and from rabbits with extensive papillomavirus infections. These fluids were tested for their effects of T-cell proliferation, production of IL-2, and mitogenic activity of IL-2 on target cells. The results suggest that cultured PBMC from these viral-infected humans and animals release factors that ment of tumors
From the Departments of
'
Microbiology and 2Dermatology, University of Texas Medical Branch Galveston, TX 77550 237
CHOPRA AND TYRING
production and IL-2 activity. The biologic significance characterization and purification of these factors is in progress. influence IL-2
of these factors is unknown. The
MATERIALS AND METHODS The sources of reagents were as follows: RPMI 1640 medium, L-glutamine, antibiotic-antimycotic solution. Hanks buffered salt solution, fetal bovine serum (FBS) (Gibco Laboratories, Grand Island, NY); hydroxyethyl piperazin-N'-2-ethane sulfonic acid (HEPES), ethylenediamine tetraacetic acid (EDTA), trypsin, soybean trypsin inhibitor, and phenylmethylsulfonylfloride (PMSF) (Sigma Chemical Co., St. Louis. MO); proteinase K (Boehringer Mannheim, Germany); Ficoll-Hypaque (Pharmacia Fine Chemicals,
Piscataway, NJ); phytohemaagglutinin-P (PHA-P);
3H-thymidine
(6.7 Ci/mmol) (New England Nuclear,
Boston, MA); 0.2 pim filters (Gelman, Acrodisc, Millipore, Bedford, MA); IL-2 ELISA kit (Genzyme Corporation, Boston, MA), recombinant IL-la and human IL-2 (Cistron Biotechnology, Pinebrook, NJ); and fluoroscein isothiocyanate (FITC)-conjugated Tac orT9 antibody (Coulter Immunology, Hialeah, FL). Patients and controls. After obtaining informed consent, venous blood was collected from eight patients between 20 and 50 years of age with persistent or treatment-resistant large condylomas (3= 1 cm3 of lesions). Blood was collected also from one patient with EV who had widespread papillomas and multiple primary cutaneous squamous-cell carcinomas. All patients were otherwise healthy and had not received any previous
immunotherapy or known immunosuppressive treatment. Ten sex- and age-matched normal adults were used controls. Blood samples were collected from patients and controls for the separation of PBMC. Animals. Dutch belted rabbits (6-8 weeks old, weighing 1-2 kg) were obtained from The Hop Stop Rabbitry (Splendora, TX), and New Zealand white rabbits (8 weeks old, weighing 4 kg) were obtained from Ray Nicholas Rabbitry (Lumberton, TX). BALB/c mice, 4-6 weeks old, were obtained from Harlan Sprague Dawley (Indianapolis, IN). Preparation and culture of mononuclear cells. Heparinized blood was collected from the patients,
as
infected rabbits, normal adults, and control rabbits. The PBMC were isolated from the interface after Ficoll-Hypaque centrifugation and washed 3x with RPMI 1640. The PBMC were cultured at a density of 1 x 10ft/ml in 25-cm2 flasks in RPMI 1640 with or without 10% FBS containing 2 mM L-glutamine and 1 % (vol/vol) antibiotic-antimycotic solution. The supernatant fluids were collected after incubation for 48 h, centrifuged at 1500 rpm for 20 min, and filtered through 0.2-p.M filters. The fluids were used as the source of suppressor factor. T-Cell proliferation assays. The PBMC were separated by Ficoll-Hypaque centrifugation, washed 3x with RPMI 1640, and incubated for 2 to 3 h in RPMI 1640 supplemented with 10% FBS. The nonadherent cells were then separated and passed over nylon wool columns. The T cells thus purified were used in mitogen (PHA-P, 1 p.g/mD-induced proliferation assays in the presence or absence of suppressor factor. The viability of PBMC and T cells were tested by trypan blue exclusion. The T cells or PBMC (5 x 104/ml) were cultured in 0.2 ml of serum-supplemented RPMI 1640 medium at 37°C for 3 to 5 days in flat-bottom 96-well microtiter plates in the presence of 5% C02 Six to eight hours before termination of the cultures, 1 p.Ci of H-thymidine was added to each well, and the incorporated radioactivity was measured. The counts per minute (cpm) for cultures with both mitogen and supernatant fluids (containing suppressor factor) were then compared with those of cultures stimulated with mitogen alone. The per cent of inhibition caused by the addition of suppressor factor was expressed as follows:
100 X cpm cultures with mitogen and supernatant cpm cultures with mitogen alone IL-2 production and assay. The PBMC ( 1 x 106/ml) were cultured in PHA-P 1 p-g/ml for 24 h, and the medium was tested for IL-2 activity by using the CTLL-2 cell line according to the method of Gillis et al. ( 11 ). Units of IL-2 were determined by the quantitative IL-2 ELISA method (Genzyme). 238
IL-2 IN PAPILLOMAVIRUS INFECTION was performed on PBMC (1 x 10ft ml) by using FITC-conjugated antibodies to Tac or T9 (Coulter Immunology, Hileah, FL). At least 200 cells were counted by using a fluorescence microscope (Zeiss USA) to determine the percentage of positive cells stained with FITC-conjugated anti-Tac or -T9 antibodies. Physicochemical characterization of suppressor factor. Serum-free supernatant fluids in RPMI 1640 were treated with trypsin 100 p-g/ml for 30 min at 37°C, and the reaction was stopped with soybean trypsin inhibitor 400 p.g/ml. Proteinase K treatment was performed using a concentration of 100 p-g/ml at 37CC for 30 min, and the reaction was stopped with PMSF. The enzyme-treated supernatant fluids were dialyzed against phosphate-buffered saline (PBS) (pH 7.5). To examine the heat stability of the suppressor factor, supernatant fluids were heated in a waterbath at 56°C for 1 h or at 80°C for 10 min. To study the effect of pH on the suppressor factor, the fluids were dialyzed overnight against the following buffers: 0.3 M acetate (pH 2,3,4, and 5), 0.3 M phosphate (pH 6 and 7), 0.3 M Tris (pH 8 and 9), and 0.3 M carbonate pH 11.5 and then against PBS (pH 7.5) overnight and tested for their abilities to suppress PHA-P-induced T-cell proliferation. Statistical analysis. Control and test values were analyzed by calculating arithmetic means and standard deviations for each set of data and comparing paired data using Student's / test.
Immunofluorescence. Direct immunofluoroscence
RESULTS Effect of SSF-H from cultured PBMC of HPV-infected patients on mitogen-induced T-Iymphocyte The T-cell proliferation in response to PHA-P was markedly suppressed when 50% of the medium consisted of supernatant fluid from the cultured PBMC of the patients with EV or CA (soluble suppressor factor-human; SSF-H). In contrast, PHA-P-induced proliferation of T cells cultured in the presence of medium conditioned with supernatant fluid from cultured PBMC of human control subjects did not demonstrate any change (Table 1). The T-cell proliferation was also inhibited with supernatant fluids collected from cultured PBMC of Shope papillomavirus (SPV)-infected Dutch belted rabbits (DBR) and New
proliferation.
Table 1. Effect of Supernatant Fluids from Cells of Papillomavirus Infected Subjects on Mitogen—Induced T-Lymphocyte Proliferation PHA-P
Human T cells T cells T cells T cells T cells Rabbits T cells T cells T cells T cells T cells
+ EV fluid + CA fluid + control fluid
Proliferation (3H-Tdr Incorporation, cpmfb 64,328 ±4,891'
+ + + +
2,847 ± 782d 1,984 + 594d 58,816 + 9,218 121 ± \2d
-
+ NZW fluid + DBR fluid + control fluid
+ + + +
58,343 + 2,108 4,289 ± 318a 2,087 ± 574 62,731 ± 7,314 96+15''
-
Proliferation after a 4-day incubation was expressed as total 3H-tdr incorporated during 8-h pulse labeling period of T cells collected from control subjects. bT cells (5 X 104/ml) were cultured with PHA-P (1 Mg/ml) in RPMI 1640 medium alone or supplemented with 50% (vol/vol) of each supernatant fluid. 'Each value represents data from five experiments. dThese values are significantly less than control (T + PHA-P and T + PHA-P + control supernatant; P2 days) T cells + fluid*
29,751 ± 962
T cells +
31,724
+
754
+ 1200
Fluid
37,120
+ 990
31,820 ± 1100
+ 981
47,812+111
32,911 + 1109
20,782 ± 621
29,870 ± 987
37,115
mitomycinc 42,867
(>2 days) T cells + fluid
+ 987
All values represent data from three experiments. Reversibility of proliferative response was examined after incubation of T cells with or without supernatant fluid for 2 days in presence of PHA-P ( 1 /ug/ml) in a 4-day culture. The cells were washed befor the PHA-P was added after 2 days' incubation. 'Induction of suppressor-T cells by suppressor factor was studied after incubation of T cells with or without suppressor factor for 2 days and studying proliferation in presence of mitomycin-c (30 /ug/30 min) and autologous T cells. "
b
241
CHOPRA AND TYRING
c
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Concentration of
Supernatant
of supernatant fluids from papillomavirus-infected humans or rabbits on the FIG. 3. Effect of increasing inhibition of IL-2 production (Panel A) and IL-2 activity (Panel B). PBMC ( l06/ml) from patients with CA (•—•) or EV (o—o), SPV-infected Dutch belted rabbits (a—A), and human control subjects (o—D) and uninfected rabbits (A—a) were incubated without mitogen for 48 h. Supernatant fluids from each of these cultures were harvested and incubated at concentrations of 10%, 25% and 50% (vol/vol) for 4 days with fresh PBMC from control subjects along with PHA-P ( 1 u-g/ml). The IL-2 was assayed by using murine cytotoxic T cells (Panel A). Inhibition of IL-2 activity by the supernatant fluids was assayed on murine cytotoxic-T cells in presence of exogenous IL-2 (Panel B). Inhibition was calculated as percentage of control. Data represent mean ± SE of six experiments. amounts
supernatant fluids from cultured PBMC of papillomavirus-infected subjects on IL-2 production and activity. To test the effects of supernatant fluids on production of IL-2, T cells ( I x 106/ml) were cultured with PHA-P ( 1 p.g/ml) in medium alone or in a medium supplemented with 50% supernatant fluid from cultured PBMC of papillomavirus-infected subjects. The CTLL-2 bioassay was used to demonstrate the effect of supernatant fluids on the production of IL-2. Supernatant fluids from cultures of PBMC from infected patients and rabbits (i.e., EV, CA, and DBR) markedly depressed IL-2 production (0.4 ± 0.2 U/ml, n 6) compared with controls (8.2 ± 1.4 U/ml, n 6). These units were measured by using ELISA reagents. It can be seen from Figure 3A that as the concentration of supernatant fluid from cultured PBMC from infected subjects was increased, the inhibition of IL-2 production by the normal PBMC Effect of
=
=
increased proportionally. The IL-2 produced in the presence of supernatant fluid from cultured PBMC of human control subjects or uninfected rabbits was not affected in the presence of PHA-P. The effect of the supernatant fluid on IL-2 activity was measured by the CTLL-2 bioassay in the presence of exogenous IL-2 and the supernatant fluid from cultured PBMC of papillomavirus-infected patients or rabbits. These fluids affected the IL-2 activity on CTLL-2 cells in a dose-dependent manner, as shown in Figure 3B. The inhibition of IL-2 activity was seen with the supernatant fluid from infected subjects but not with the fluid from normal healthy controls (40,384 ±1210 cpm, n 6). This suppressive effect on IL-2 CTLL-2 the cells did not lose the proliferative as effect on to attributable a not was toxic cells, activity ± incubation with the supernatant fluid for 24 of IL-2 984 in the after 4) (45,384 cpm,« response presence h and after washing (2x) with RPMI 1640 compared with the proliferation of untreated CTLL-2 cells in 6). Also, if the suppression of lymphocyte response to human recombinant IL-2 (47,482 ±1184 cpm, n fluids reduced was to the secondary production of IL-2, then its effect should be proliferation by supernatant reversed by the addition of exogenous IL-2. However, the proliferation of T lymphocytes cultured with PHA-P and fluid from cultured PBMC of a patient with EV at 50% concentration did not increase significantly with the addition of recombinant IL-2 40 U/ml (Fig. 4). Effect of supernatant fluids from cultured PBMC of an EV patient on IL-2 receptor expression and the interaction of IL-2 with its receptor. Experiments were conducted also to determine whether =
=
=
242
IL-2 IN PAPILLOMAVIRUS INFECTION
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100
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=
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25%
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Supernatant Supernatant FIG. 4. Effect of IL-2 on papillomavirus-induced suppression of T-lymphocyte proliferation. The T cells (5 x l()4in 0.2 ml) were cultured with PHA-P ( l p.g/ml) and 0, 25 or 50% EV supernatant fluid in presence (D) or absence ( ) of IL-2 (40 U/ml final concentration) for4 days. Resulting T-cell proliferation was measured as 'H-thymidine incorporation. Data are mean ± SE of total cpm for four experiments. supernatant fluid from cultured PBMC from an EV patient affected IL-2 receptor generation and subsequently interaction with its receptor. The inability of IL-2 to overcome the inhibitory influence of the supernatant fluid
suggested that this factor has an additional effect on IL-2 responsiveness. To address this question, we tested for the presence of IL-2 receptors on PBMC by Tac expression. The percentage of Tac+ T lymphocytes enumerated with fluorescence microscopy was not different whether the cells were cultured for l day with PHA-P in the presence or absence of supernatant fluid (37 ± 12% v 42% ± 9%, respectively; /; 3). However, it is evident from Figure 5 that after 3 days of culture with PHA-P, fewer lymphocytes expressed Tac antigen in the presence of supernatant fluid than in medium alone ( 18 ± 10% v 62 ± 15%, respectively; n 3). This difference was even more marked after incubation for 5 days. The decrease in the number of =
=
fluorescent cells reflects the decrease in the number of IL-2 receptors or a decrease in the percentage of activated cells. The presence of activated cells was studied by staining the transferrin (Ty) receptor, another activation antigen, and was quantitated by using the fluorescence microscope. When T cells were cultured with PHA-P for 3 days, there were fewer Tt,+ cells in cultures containing 50% supernatant fluid from an HPV-infected patient (22 ± 7%) than the medium alone (52 ± 12%, n 3; P < 0.003) (data not shown). Because PHA-P-activated T cells were unresponsive to IL-2 after 24 h of exposure to supernatant fluids, experiments were conducted to determine whether an associated decline in Tac expression was observed. The T lymphocytes stimulated with PHA-P for 3 days contained (72 ± 9%) Tac+ cells. On incubation of T cells with 50% supernatant fluids from cultured PBMC from HPV-infected persons for 24 h, the percentage of Tac cells was unchanged (80 ± 12%) as determined by fluorescence microscopy. Although the supernatant fluids inhibited T-cell proliferation with no significant change in Tac antigen expression, it might have blocked the binding of IL-2 to its receptor. To evaluate this possibility, the effect of supernatant fluid on IL-2-induced proliferation of activated T cells was determined. The PBMC from infected patients were incubated with PHA-P ( 1 p-g/ml) for 6 days to produce PHA-P blasts. The resulting blasts produced insignificant amounts of IL-2 (not measurable by CTLL bioassay) in the presence of PHA-P, but most of them (67 ± 12%) expressed IL-2 receptors, and hence they proliferated in response to exogenous recombinant IL-2 (1 U/ml). This proliferation was minimally suppressed by supernatant fluids from cultures of PBMC from HPV-infected =
243
CHOPRA AND TYRING
100 80 w
Ö O
60
+
Ü
a
H
40
0
Day FIG. 5.
(5
x
1
Day
3
Effect of PBMC supernatant fluid from HPV-infected persons
Day on
5
expression
of Tac
antigen. The
T cells
104in0.2ml) were cultured in microtiter wells withPHA-P(l u,g/ml) in serum-supplemented RPMI 1640(D)alone
with 50% supernatant fluid (0). On various days, cells were tested for Tac expression via analysis on fluorescence anti-Tac antibody. Average of four experiments show that fluids from cultures of cells from HPV-infected persons significantly decreased Tac expression on day 3 and 5 (P < 0.02).
or
microscope using
persons (Table 3). In contrast, the same supernatant fluid at a 50% concentration markedly inhibited the proliferation of PHA-P blasts in response to PHA-P (90 ± 13%; P < 0.03) (Table 3). The immunosuppressive effect of the supernatant fluids from cultured PBMC from HPV-infected patients therefore is not primarily attributable to the blockage of the binding of IL-2 to its receptor but rather to a decrease in Tac expression from cultured PBMC of normal control subjects in the presence of the fluid (Fig. 5). Properties of PBMC supernatant fluids collected from an EV patient and infected Dutch belted rabbits. The suppressor factor(s) present in fluids from cultured PBMC retained its inhibitory activity after repeated freezing and thawing (Fig. 6). Heating the supernatant fluid at 80°C for 1 h or lowering the pH to 2.0 or increasing it to 11.5 destroyed most of the suppressor activity. The inhibitory activity was stable from pH 3.0 to pH 9.0. The soluble suppressor factor(s) (SSF) was sensitive to proteinase K treatment and was partially sensitive to trypsin treatment. This factor is being further purified and characterized.
DISCUSSION Viruses may cause immunosuppression by a variety of mechanisms. For example, suppression may result from viral triggering of an imbalance in immune regulation causing overreactivity of suppressor-T cells. Table 3. Effect of PBMC Supernatant Fluids from HPV-Infected Subjects on PHA-P-or IL-2-Induced Proliferation of PHA-P Blasts"
Proliferation (3H Tdr, cpm) -
Stimulus
In Medium
PHA-P IL-2
10,678 + 942 6,732 ± 1704
In 50%
Supernatant Fluid
979+124 4750 ±79
Percent
Inhibition 90+13 30+12
"All values represent data from three experiments. ^Inhibition of PHA-P-induced response was significantly greater than that of IL-2-induced response (P u
Suppression of Interleukin-2 Production and Activity by Factor(s) Released by Peripheral Blood Mononuclear Cells during Papillomavirus Infections VIMLARANI
CHOPRA1 and STEPHEN K. TYRING12
ABSTRACT
Supernatant fluids from cultured peripheral blood mononuclear cells (PBMC) obtained from patients with extensive papillomavirus infections such as condyloma acuminatum (CA) and epidermodysplasia verruciformis (EV) depressed the proliferative responses of T cells to phytohemagglutinin-P (PHA-P) and the production of interleukin-2 (IL-2) from those preparations. Fluids from the same cultures also inhibited the mitogenic activity of IL-2 on CTLL-2 cells as IL-2-dependent target cells. These soluble suppressor factors (SSF) from PBMC were present in significantly higher concentrations in fluids from cultured PBMC from patients in comparison to healthy controls. A soluble suppressor factor was characterized also from cultured rabbit PBMC after the rabbits had been infected with Shope papillomaviruses. This suppressor factor likewise inhibited IL-2 production and IL-2 activity.
Many oncogenic
and
nononcogenic viral agents alter immune functions and thus augment the develop-
(3, 5, 12, 13, 17, 19, 23, 28, 34, 35). It is not clear whether the tumor grows of because blocking factors, blocking antigens, autoimmune antibodies, or antigen-antibody progressively of a defect(s) in the host's cellular or humoral immunity (20, 27). A variety of because or (2) complexes factors have been identified in human and murine tumors that downregulate immune functions suppressor (31). The source of these suppressor elements could be lymphoid cells (10, 15), macrophages (6, 18), or possibly the tumor itself, which antagonizes the host immune response (31). A depressed immune response has been observed in patients with human papillomavirus (HPV) infections associated with diseases such as condyloma acuminatum (CA) (7) and epidermodysplasia verruciformis (EV) (20). Impairment of these accessory cell functions in HPV infections could be associated with a regulatory substance referred to as suppressor factor(s). In this study, we investigated the presence of suppressor factor(s) in the supernatant fluids of cultured PBMC from humans and from rabbits with extensive papillomavirus infections. These fluids were tested for their effects of T-cell proliferation, production of IL-2, and mitogenic activity of IL-2 on target cells. The results suggest that cultured PBMC from these viral-infected humans and animals release factors that ment of tumors
From the Departments of
'
Microbiology and 2Dermatology, University of Texas Medical Branch Galveston, TX 77550 237
CHOPRA AND TYRING
production and IL-2 activity. The biologic significance characterization and purification of these factors is in progress. influence IL-2
of these factors is unknown. The
MATERIALS AND METHODS The sources of reagents were as follows: RPMI 1640 medium, L-glutamine, antibiotic-antimycotic solution. Hanks buffered salt solution, fetal bovine serum (FBS) (Gibco Laboratories, Grand Island, NY); hydroxyethyl piperazin-N'-2-ethane sulfonic acid (HEPES), ethylenediamine tetraacetic acid (EDTA), trypsin, soybean trypsin inhibitor, and phenylmethylsulfonylfloride (PMSF) (Sigma Chemical Co., St. Louis. MO); proteinase K (Boehringer Mannheim, Germany); Ficoll-Hypaque (Pharmacia Fine Chemicals,
Piscataway, NJ); phytohemaagglutinin-P (PHA-P);
3H-thymidine
(6.7 Ci/mmol) (New England Nuclear,
Boston, MA); 0.2 pim filters (Gelman, Acrodisc, Millipore, Bedford, MA); IL-2 ELISA kit (Genzyme Corporation, Boston, MA), recombinant IL-la and human IL-2 (Cistron Biotechnology, Pinebrook, NJ); and fluoroscein isothiocyanate (FITC)-conjugated Tac orT9 antibody (Coulter Immunology, Hialeah, FL). Patients and controls. After obtaining informed consent, venous blood was collected from eight patients between 20 and 50 years of age with persistent or treatment-resistant large condylomas (3= 1 cm3 of lesions). Blood was collected also from one patient with EV who had widespread papillomas and multiple primary cutaneous squamous-cell carcinomas. All patients were otherwise healthy and had not received any previous
immunotherapy or known immunosuppressive treatment. Ten sex- and age-matched normal adults were used controls. Blood samples were collected from patients and controls for the separation of PBMC. Animals. Dutch belted rabbits (6-8 weeks old, weighing 1-2 kg) were obtained from The Hop Stop Rabbitry (Splendora, TX), and New Zealand white rabbits (8 weeks old, weighing 4 kg) were obtained from Ray Nicholas Rabbitry (Lumberton, TX). BALB/c mice, 4-6 weeks old, were obtained from Harlan Sprague Dawley (Indianapolis, IN). Preparation and culture of mononuclear cells. Heparinized blood was collected from the patients,
as
infected rabbits, normal adults, and control rabbits. The PBMC were isolated from the interface after Ficoll-Hypaque centrifugation and washed 3x with RPMI 1640. The PBMC were cultured at a density of 1 x 10ft/ml in 25-cm2 flasks in RPMI 1640 with or without 10% FBS containing 2 mM L-glutamine and 1 % (vol/vol) antibiotic-antimycotic solution. The supernatant fluids were collected after incubation for 48 h, centrifuged at 1500 rpm for 20 min, and filtered through 0.2-p.M filters. The fluids were used as the source of suppressor factor. T-Cell proliferation assays. The PBMC were separated by Ficoll-Hypaque centrifugation, washed 3x with RPMI 1640, and incubated for 2 to 3 h in RPMI 1640 supplemented with 10% FBS. The nonadherent cells were then separated and passed over nylon wool columns. The T cells thus purified were used in mitogen (PHA-P, 1 p.g/mD-induced proliferation assays in the presence or absence of suppressor factor. The viability of PBMC and T cells were tested by trypan blue exclusion. The T cells or PBMC (5 x 104/ml) were cultured in 0.2 ml of serum-supplemented RPMI 1640 medium at 37°C for 3 to 5 days in flat-bottom 96-well microtiter plates in the presence of 5% C02 Six to eight hours before termination of the cultures, 1 p.Ci of H-thymidine was added to each well, and the incorporated radioactivity was measured. The counts per minute (cpm) for cultures with both mitogen and supernatant fluids (containing suppressor factor) were then compared with those of cultures stimulated with mitogen alone. The per cent of inhibition caused by the addition of suppressor factor was expressed as follows:
100 X cpm cultures with mitogen and supernatant cpm cultures with mitogen alone IL-2 production and assay. The PBMC ( 1 x 106/ml) were cultured in PHA-P 1 p-g/ml for 24 h, and the medium was tested for IL-2 activity by using the CTLL-2 cell line according to the method of Gillis et al. ( 11 ). Units of IL-2 were determined by the quantitative IL-2 ELISA method (Genzyme). 238
IL-2 IN PAPILLOMAVIRUS INFECTION was performed on PBMC (1 x 10ft ml) by using FITC-conjugated antibodies to Tac or T9 (Coulter Immunology, Hileah, FL). At least 200 cells were counted by using a fluorescence microscope (Zeiss USA) to determine the percentage of positive cells stained with FITC-conjugated anti-Tac or -T9 antibodies. Physicochemical characterization of suppressor factor. Serum-free supernatant fluids in RPMI 1640 were treated with trypsin 100 p-g/ml for 30 min at 37°C, and the reaction was stopped with soybean trypsin inhibitor 400 p.g/ml. Proteinase K treatment was performed using a concentration of 100 p-g/ml at 37CC for 30 min, and the reaction was stopped with PMSF. The enzyme-treated supernatant fluids were dialyzed against phosphate-buffered saline (PBS) (pH 7.5). To examine the heat stability of the suppressor factor, supernatant fluids were heated in a waterbath at 56°C for 1 h or at 80°C for 10 min. To study the effect of pH on the suppressor factor, the fluids were dialyzed overnight against the following buffers: 0.3 M acetate (pH 2,3,4, and 5), 0.3 M phosphate (pH 6 and 7), 0.3 M Tris (pH 8 and 9), and 0.3 M carbonate pH 11.5 and then against PBS (pH 7.5) overnight and tested for their abilities to suppress PHA-P-induced T-cell proliferation. Statistical analysis. Control and test values were analyzed by calculating arithmetic means and standard deviations for each set of data and comparing paired data using Student's / test.
Immunofluorescence. Direct immunofluoroscence
RESULTS Effect of SSF-H from cultured PBMC of HPV-infected patients on mitogen-induced T-Iymphocyte The T-cell proliferation in response to PHA-P was markedly suppressed when 50% of the medium consisted of supernatant fluid from the cultured PBMC of the patients with EV or CA (soluble suppressor factor-human; SSF-H). In contrast, PHA-P-induced proliferation of T cells cultured in the presence of medium conditioned with supernatant fluid from cultured PBMC of human control subjects did not demonstrate any change (Table 1). The T-cell proliferation was also inhibited with supernatant fluids collected from cultured PBMC of Shope papillomavirus (SPV)-infected Dutch belted rabbits (DBR) and New
proliferation.
Table 1. Effect of Supernatant Fluids from Cells of Papillomavirus Infected Subjects on Mitogen—Induced T-Lymphocyte Proliferation PHA-P
Human T cells T cells T cells T cells T cells Rabbits T cells T cells T cells T cells T cells
+ EV fluid + CA fluid + control fluid
Proliferation (3H-Tdr Incorporation, cpmfb 64,328 ±4,891'
+ + + +
2,847 ± 782d 1,984 + 594d 58,816 + 9,218 121 ± \2d
-
+ NZW fluid + DBR fluid + control fluid
+ + + +
58,343 + 2,108 4,289 ± 318a 2,087 ± 574 62,731 ± 7,314 96+15''
-
Proliferation after a 4-day incubation was expressed as total 3H-tdr incorporated during 8-h pulse labeling period of T cells collected from control subjects. bT cells (5 X 104/ml) were cultured with PHA-P (1 Mg/ml) in RPMI 1640 medium alone or supplemented with 50% (vol/vol) of each supernatant fluid. 'Each value represents data from five experiments. dThese values are significantly less than control (T + PHA-P and T + PHA-P + control supernatant; P2 days) T cells + fluid*
29,751 ± 962
T cells +
31,724
+
754
+ 1200
Fluid
37,120
+ 990
31,820 ± 1100
+ 981
47,812+111
32,911 + 1109
20,782 ± 621
29,870 ± 987
37,115
mitomycinc 42,867
(>2 days) T cells + fluid
+ 987
All values represent data from three experiments. Reversibility of proliferative response was examined after incubation of T cells with or without supernatant fluid for 2 days in presence of PHA-P ( 1 /ug/ml) in a 4-day culture. The cells were washed befor the PHA-P was added after 2 days' incubation. 'Induction of suppressor-T cells by suppressor factor was studied after incubation of T cells with or without suppressor factor for 2 days and studying proliferation in presence of mitomycin-c (30 /ug/30 min) and autologous T cells. "
b
241
CHOPRA AND TYRING
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g
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Concentration of
Supernatant
of supernatant fluids from papillomavirus-infected humans or rabbits on the FIG. 3. Effect of increasing inhibition of IL-2 production (Panel A) and IL-2 activity (Panel B). PBMC ( l06/ml) from patients with CA (•—•) or EV (o—o), SPV-infected Dutch belted rabbits (a—A), and human control subjects (o—D) and uninfected rabbits (A—a) were incubated without mitogen for 48 h. Supernatant fluids from each of these cultures were harvested and incubated at concentrations of 10%, 25% and 50% (vol/vol) for 4 days with fresh PBMC from control subjects along with PHA-P ( 1 u-g/ml). The IL-2 was assayed by using murine cytotoxic T cells (Panel A). Inhibition of IL-2 activity by the supernatant fluids was assayed on murine cytotoxic-T cells in presence of exogenous IL-2 (Panel B). Inhibition was calculated as percentage of control. Data represent mean ± SE of six experiments. amounts
supernatant fluids from cultured PBMC of papillomavirus-infected subjects on IL-2 production and activity. To test the effects of supernatant fluids on production of IL-2, T cells ( I x 106/ml) were cultured with PHA-P ( 1 p.g/ml) in medium alone or in a medium supplemented with 50% supernatant fluid from cultured PBMC of papillomavirus-infected subjects. The CTLL-2 bioassay was used to demonstrate the effect of supernatant fluids on the production of IL-2. Supernatant fluids from cultures of PBMC from infected patients and rabbits (i.e., EV, CA, and DBR) markedly depressed IL-2 production (0.4 ± 0.2 U/ml, n 6) compared with controls (8.2 ± 1.4 U/ml, n 6). These units were measured by using ELISA reagents. It can be seen from Figure 3A that as the concentration of supernatant fluid from cultured PBMC from infected subjects was increased, the inhibition of IL-2 production by the normal PBMC Effect of
=
=
increased proportionally. The IL-2 produced in the presence of supernatant fluid from cultured PBMC of human control subjects or uninfected rabbits was not affected in the presence of PHA-P. The effect of the supernatant fluid on IL-2 activity was measured by the CTLL-2 bioassay in the presence of exogenous IL-2 and the supernatant fluid from cultured PBMC of papillomavirus-infected patients or rabbits. These fluids affected the IL-2 activity on CTLL-2 cells in a dose-dependent manner, as shown in Figure 3B. The inhibition of IL-2 activity was seen with the supernatant fluid from infected subjects but not with the fluid from normal healthy controls (40,384 ±1210 cpm, n 6). This suppressive effect on IL-2 CTLL-2 the cells did not lose the proliferative as effect on to attributable a not was toxic cells, activity ± incubation with the supernatant fluid for 24 of IL-2 984 in the after 4) (45,384 cpm,« response presence h and after washing (2x) with RPMI 1640 compared with the proliferation of untreated CTLL-2 cells in 6). Also, if the suppression of lymphocyte response to human recombinant IL-2 (47,482 ±1184 cpm, n fluids reduced was to the secondary production of IL-2, then its effect should be proliferation by supernatant reversed by the addition of exogenous IL-2. However, the proliferation of T lymphocytes cultured with PHA-P and fluid from cultured PBMC of a patient with EV at 50% concentration did not increase significantly with the addition of recombinant IL-2 40 U/ml (Fig. 4). Effect of supernatant fluids from cultured PBMC of an EV patient on IL-2 receptor expression and the interaction of IL-2 with its receptor. Experiments were conducted also to determine whether =
=
=
242
IL-2 IN PAPILLOMAVIRUS INFECTION
cT i
100
o V
X
g c
.2
X
o c
(3 .2 t; *-
=
80
a
60
rJr
o
a o
40
X
Ü
Ç
'Í
¡2
20
Í 0
^
2 Medium alone
25%
-V7
y¿. 50%
Supernatant Supernatant FIG. 4. Effect of IL-2 on papillomavirus-induced suppression of T-lymphocyte proliferation. The T cells (5 x l()4in 0.2 ml) were cultured with PHA-P ( l p.g/ml) and 0, 25 or 50% EV supernatant fluid in presence (D) or absence ( ) of IL-2 (40 U/ml final concentration) for4 days. Resulting T-cell proliferation was measured as 'H-thymidine incorporation. Data are mean ± SE of total cpm for four experiments. supernatant fluid from cultured PBMC from an EV patient affected IL-2 receptor generation and subsequently interaction with its receptor. The inability of IL-2 to overcome the inhibitory influence of the supernatant fluid
suggested that this factor has an additional effect on IL-2 responsiveness. To address this question, we tested for the presence of IL-2 receptors on PBMC by Tac expression. The percentage of Tac+ T lymphocytes enumerated with fluorescence microscopy was not different whether the cells were cultured for l day with PHA-P in the presence or absence of supernatant fluid (37 ± 12% v 42% ± 9%, respectively; /; 3). However, it is evident from Figure 5 that after 3 days of culture with PHA-P, fewer lymphocytes expressed Tac antigen in the presence of supernatant fluid than in medium alone ( 18 ± 10% v 62 ± 15%, respectively; n 3). This difference was even more marked after incubation for 5 days. The decrease in the number of =
=
fluorescent cells reflects the decrease in the number of IL-2 receptors or a decrease in the percentage of activated cells. The presence of activated cells was studied by staining the transferrin (Ty) receptor, another activation antigen, and was quantitated by using the fluorescence microscope. When T cells were cultured with PHA-P for 3 days, there were fewer Tt,+ cells in cultures containing 50% supernatant fluid from an HPV-infected patient (22 ± 7%) than the medium alone (52 ± 12%, n 3; P < 0.003) (data not shown). Because PHA-P-activated T cells were unresponsive to IL-2 after 24 h of exposure to supernatant fluids, experiments were conducted to determine whether an associated decline in Tac expression was observed. The T lymphocytes stimulated with PHA-P for 3 days contained (72 ± 9%) Tac+ cells. On incubation of T cells with 50% supernatant fluids from cultured PBMC from HPV-infected persons for 24 h, the percentage of Tac cells was unchanged (80 ± 12%) as determined by fluorescence microscopy. Although the supernatant fluids inhibited T-cell proliferation with no significant change in Tac antigen expression, it might have blocked the binding of IL-2 to its receptor. To evaluate this possibility, the effect of supernatant fluid on IL-2-induced proliferation of activated T cells was determined. The PBMC from infected patients were incubated with PHA-P ( 1 p-g/ml) for 6 days to produce PHA-P blasts. The resulting blasts produced insignificant amounts of IL-2 (not measurable by CTLL bioassay) in the presence of PHA-P, but most of them (67 ± 12%) expressed IL-2 receptors, and hence they proliferated in response to exogenous recombinant IL-2 (1 U/ml). This proliferation was minimally suppressed by supernatant fluids from cultures of PBMC from HPV-infected =
243
CHOPRA AND TYRING
100 80 w
Ö O
60
+
Ü
a
H
40
0
Day FIG. 5.
(5
x
1
Day
3
Effect of PBMC supernatant fluid from HPV-infected persons
Day on
5
expression
of Tac
antigen. The
T cells
104in0.2ml) were cultured in microtiter wells withPHA-P(l u,g/ml) in serum-supplemented RPMI 1640(D)alone
with 50% supernatant fluid (0). On various days, cells were tested for Tac expression via analysis on fluorescence anti-Tac antibody. Average of four experiments show that fluids from cultures of cells from HPV-infected persons significantly decreased Tac expression on day 3 and 5 (P < 0.02).
or
microscope using
persons (Table 3). In contrast, the same supernatant fluid at a 50% concentration markedly inhibited the proliferation of PHA-P blasts in response to PHA-P (90 ± 13%; P < 0.03) (Table 3). The immunosuppressive effect of the supernatant fluids from cultured PBMC from HPV-infected patients therefore is not primarily attributable to the blockage of the binding of IL-2 to its receptor but rather to a decrease in Tac expression from cultured PBMC of normal control subjects in the presence of the fluid (Fig. 5). Properties of PBMC supernatant fluids collected from an EV patient and infected Dutch belted rabbits. The suppressor factor(s) present in fluids from cultured PBMC retained its inhibitory activity after repeated freezing and thawing (Fig. 6). Heating the supernatant fluid at 80°C for 1 h or lowering the pH to 2.0 or increasing it to 11.5 destroyed most of the suppressor activity. The inhibitory activity was stable from pH 3.0 to pH 9.0. The soluble suppressor factor(s) (SSF) was sensitive to proteinase K treatment and was partially sensitive to trypsin treatment. This factor is being further purified and characterized.
DISCUSSION Viruses may cause immunosuppression by a variety of mechanisms. For example, suppression may result from viral triggering of an imbalance in immune regulation causing overreactivity of suppressor-T cells. Table 3. Effect of PBMC Supernatant Fluids from HPV-Infected Subjects on PHA-P-or IL-2-Induced Proliferation of PHA-P Blasts"
Proliferation (3H Tdr, cpm) -
Stimulus
In Medium
PHA-P IL-2
10,678 + 942 6,732 ± 1704
In 50%
Supernatant Fluid
979+124 4750 ±79
Percent
Inhibition 90+13 30+12
"All values represent data from three experiments. ^Inhibition of PHA-P-induced response was significantly greater than that of IL-2-induced response (P u
