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Cancer Investigation, 10(3), 201-208 (1992)
Immunosuppression Derived from Human B-Lymphoblastoid and Melanoma Cell Lines Charlene J. Repique, M.S.,* James D. Kettering, Ph.D.,* and Daila S. Gridley, Ph.D.*-f Departments of *Microbiology and ?Radiation Sciences, Section of Radiation Oncology Lorna Linda University Schod of Medicine Lorna Linda, California 92350
ABSTRACT
Previous work conducted by the authors, using a murine model, suggested that soluble factors secreted by tumor cells suppress lymphocpe responses. To apply this premise to human tumors, the effects of UC729-6 (lymphoblastoid B-cell) and M2I-HPB (malignant melanoma) conditioned media (CM)on normal lymphocyteproliferation, as well as on rumor cell growth in autologous CM was studied. m e CM was collected at 2-5 day intervalsfrom cultures of UC729-6 and M21-HPB cells in serumfree media. Phytohemagglutinin- and concanavalin A-stimulated mononuclear peripheral blood cells from healthy human donors shaved decreased PHIthymidine ([3H]Tdr)uptake in the presence of each CM when compared with controls. In assays using 100%CM, mitogen stimulation was 68-85% less than that of controls and 40-50% less using 50% CM. l l e suppression was more pronounced with UC729-6 CM than with M21-HPB CM. In mixed lymphocyte cultures (MLC), &ition of 50% CMffom either tumor cell line resulted in 40-50% reduction in pH]Tdr uptake by lymphocytes. Incubation of UC72P6 cells in 5 % to 100% of UC72P6 f CM (filter-concentrated) produced a decrease in pH]Tdr uptake which was directly proportional to the amount offCMpresent. In contrast, M21-HPB cell growth in autologousf CM w s dependent on cell number, as well as on the amount off CM used. Treatment of the UC729-6f CM using acid @H4.5). trypsin (I 00 pg/ml), and heat (56°C) did not restore mitogen-stimulated lymphoproliferation. However, the inhibition observed with UC729-6f CM was partially reversed ajer dialysis with membranes having M, limits of 2.5 x I @ , 1.5 X I @ , or I x I@. 201 Copyright 0 1992 by Marcel Dekker, Inc.
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INTRODUCTION The progression of neoplasia and its escape from host immune mechanisms remain an enigma. This is especially confounding when the tumor is immunogenic and the host is, at least initially, immunocompetent. The impairment of host defenses in tumor-bearing subjects has been demonstrated in numerous studies. For example, the depression of lymphoproliferation, immunoglobulin synthesis, and macrophage and neutrophil functions have been reported. The fact that immunosuppression can be reversed upon reduction of the tumor load (1) implicates the malignant tissue itself as a possible source of the inhibitory effects. Recently, interest in tumor-derived immunosuppressive factors has greatly increased because of the development of new immunological techniques for cancer therapy (2-4). Studies of lymphokineactivated killer (LAK) cells and tumor-infiltrating lymphocytes (TILs) show that despite the close association of immune cells with tumor tissue, these cells retain potential antitumor activity (5). Generation of LAK cells and TILs for cancer therapy can be inhibited by factors secreted by glioblastoma, melanoma, and colorectal cancer cells (6,7). These factors may contribute to the failure of adoptive immunotherapy in a significant number of patients. It has also been demonstrated that factors produced by malignant cells can exhibit autocrine, as well as paracrine, activity (8-11). A wide range of neoplasms such as leukemias, glioblastoma, colorectal cancer, melanoma, and small-cell lung carcinoma have been studied in this respect. Investigators have found that different regulatory factors can be found in tumor effusions and cell culture supernatants, as well as in sera from tumor-bearing subjects (12-14). An important aspect of these studies is that malignant cells may produce factors that are selfstimulatory, but inhibitory to potentially immunocompetent cells, thus giving the tumor tissue a growth advantage within the host. These findings have generated interest in the development of this investigation. The purpose of this study was to determine if cell culture supernatants obtained from a human malignant melanoma, M21-HPB, and a human lymphoblastoid B-cell line, UC729-6, could inhibit normal T lymphocyte activity. In addition, the effects of the autologous supernatants on the growth of the tumor cells themselves and preliminary characterization of the immunosuppressive activity derived from UC729-6 cells are also reported here.
MATERIALS AND METHODS Tumor Cell Lines UC729-6 is a 6-thioguanine-resistant human lymphoblastoid B-cell line obtained from Dr. Mark Glassy at University of California, San Diego, CA. It was originally developed by treating WIL-2 lymphoblastoid cells with 25 pM 6-thioguanine (15,16). This nonantibodyproducing cell line has the potential for human hybridoma and monoclonal antibody production (17). M21-HPB (Hybritech Incorporated, San Diego, CA) is a human melanoma cell line derived from a secondary metastasis of malignant melanoma (18). It is tumorigenic in nude mice and has been used in our laboratories in previous studies (19,20). Both cell lines were grown and maintained in Roswell Park Memorial Institute (RPMI) 1640medium (Irvine Scientific, Santa Ana, CA) supplemented with 10% heat-inactivated fetal calf serum (FCS) @vine Scientific; Hyclone Laboratories Incorporated, Logan, UT), 2 pg/ml fungizone (E. R. Squibb and Sons, Incorporated, Princeton, NJ), and 50 pglml gentamicin sulfate (Sigma Chemical Company, St. Louis, MO) at 37°C in 5% C 0 2 . The UC729-6 cells grew in suspension culture, whereas the M21-HPB cells produced monolayers. Both cell lines were found to be free of mycoplasma contamination after testing with the Mycotrim Triphasic Culture System (Hana Biologics, Incorporated, Alameda, CA).
Conditioned Media (CM) UC729-6 cultures at moderate to heavy growth and M21-HPB cultures at approximately 75 % to complete confluency in 75 cm2 tissue culture flasks (Falcon, Oxnard, CA; Coming Glass Works, Coming, NY) were washed three times with serum-free RPMI. Fresh serumfree medium was added and the cells were allowed to grow for 2 to 3 h at 37 "C in 5 % COz . This medium was again replaced with fresh serum-free RPMI and the cultures were reincubated for 48 h. The CM was then collected and clarified by centrifugation at 200 g for 10 min. At the time of CM collection, cell viability was > 95% as determined by trypan blue exclusion. The CM from each cell line was pooled, aliquoted, and stored at -20 "C until immediately before use.
Mitogen-Induced Lymphoproliferation Whole blood from 5 healthy donors, 3 males and 2 females, aged 25 to 46 years, was collected into tubes
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with sodium heparin as the anticoagulant. One individual was tested 4 times, another 3 times, and a third subject was tested twice for a total of 9 specimens. Each sample was diluted 1:10 with RPMI or with each of the CM. The RPMI medium was supplemented with 10% FCS, fungizone, gentamicin, 10 mM HEPES buffer (Calbiochem Corporation, La Jolla, CA), and 5 X mM mercaptoethanol (Sigma). Phytohemagglutinin (PHA; Burroughs Wellcome, Triangle Park, NC) and concanavalin A (ConA; Sigma), both previously titrated to determine maximum stimulatory concentrations, were diluted with either RPMI or with CM. Cells and mitogens were dispensed into 96-well flat bottom microtiter plates (Falcon; Coming) to yield 50 or 100% of each CM in a total volume of 200 pl. Control wells contained cells and medium or CM, but no mitogen. Assays which included CM were also compared with simultaneously performed control assays which contained RPMI and no CM. The microtiter plates were incubated at 37 "Cin 5 % C 0 2 for 3 days and labelled with 1 pCi of [3H]Tdr/well (specific activity = 46 Ci/mmol; ICN Biomedicals, Costa Mesa, CA) for the final 5 h of incubation. The cells were harvested onto glass microfiber filters with an automated cell harvester (Brandel Incorporated, Gaithersburg, MD). The radioactivity incorporated into cell DNA was counted in disintegrations per minute (dpm) for 1 min/sample in a liquid scintillation counter (Beckman Instruments, Incorporated, Fullerton, CA). All samples were tested in quadruplicate.
CM from both UC729-6 and M21-HPB cultures were concentrated to one tenth of the original volumes using a lo00 M, filter in an Amicon filtration system (Amicon Division W. R. Grace and Company, Danvers, MA). The CM were filtered using 55 to 60 lbs. of positive pressure and continuous mixing with magnetic stirrers at a flow rate of approximately 55 ml/h at 4°C to produce filterconcentrated CM (fCM) at one-tenth of the original volume. The tested fraction contained molecules of > lo00 M,. Isolated mononuclear cells (2 X l@/well) were incubated in 50% and 100% fCM from UC729-6 or M21-HPB cells, 100% filter-concentrated RPMI medium, and nonconcentrated RPMI. The cells were tested for viability by trypan blue exclusion after 24 h and after 72 h.
Mixed Lymphocyte Cultures
Tumor Cell Growth in Autologous fCM
Whole blood was collected from 6 healthy donors, 3 males, and 3 females, aged 25 to 46 years. Three individuals were tested twice for a total of 9 separate specimens. Mononuclear cells were obtained using standard Ficoll-Hypaque (Pharmacia LKB Biotechnology, Incorporated, Piscataway ,NJ), density gradient centrifugation procedures. After isolation the mononuclear cells were washed twice and resuspended in RPMI to a concentration of 2 X 106 cells/ml. Each suspension was divided into two fractions: responders and stimulators. Stimulator cells were irradiated with 2750 rad from a 137Cssource (Model 143 Irradiator, J. L. Sheperd and Associates, San Fernando, CA) to prevent DNA replication when mixed with allogenic responders. Next, 50 pl of stimulator cells and 50 pl of responder cells were dispensed into 96-well flat bottom microtiter plates to establish mixed cultures. Autologous controls in which stimulatorsand responders from the same individualswere plated together were included. Cell controls consisting of
stimulator or responder cells alone were used to screen for spontaneous or unexplained proliferation of the lymphocytes. Each cell sample was treated with 100 pl of either of the CM or with RPMI as an additional control. The cultures were incubated at 37 "C in 5 % COz for 5 days. Each well was then labelled with 1 pCi of [3H]Tdr for an additional 18 h and the cells were harvested (Brandel). The radioactivity incorporated into cell DNA was counted in dpm for 1 min/sample in a liquid scintillation counter. All samples were tested in triplicate.
Preparation and Toxicity Testing of Filter-Concentrated CM (fCM)
UC729-6 and M21-HPB cells were dispensed into 96-well flat bottom microtiter plates in 100 pl at concentrations of 105, 104, and 103 per well each. Various dilutions of each fCM were prepared with RPMI. Volumes of 100plcontaining 100%, 50%, l o % , and5% fCMor 100% ,50 % ,and 10% CM were plated with each cell type at each cell concentration. The cultures were incubated for 54 h at 37°C in 5% C02. Each well was then labelled with 1 pCi rH]Tdr for 18 h of additional incubation. The cells were trypsinized, if necessary, and harvested with an automatic cell harvester. The incorporated radioactivity was counted in a liquid scintillation counter for 1 min/sample. The samples were tested in triplicate.
Characterization of the Immunosuppressive Activity in UC729-6 fCM Because of rapid, abundant growth and the capacity for high supernate volume generation, focus was placed on
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the UC729-6 cell line. Aliquots of the fCM were heattreated in a H20 bath at temperatures of either 56 "C or 37 "C (control) for 1 h. Other aliquots of fCM were adjusted to pH 4.5 with 1N HC1 for 1 h at room temperature. The pH was readjusted to 7.4 with 3N NaOH before use in assays. Additional fCM samples were treated with 100 pg/ml of trypsin for 1 h at 37 "C. Soybean trypsin inhibitor (Sigma; 250 pg/100 pg of trypsin) was added to stop the action of the enzyme. Finally, portions of each of the two fCMs were dialyzed against serum-freeRPMI using Spectrapor membrane tubings (Spectrum Medical Industries, Incorporated, Los Angeles, CA) with M, cutoffs of 1 X 104, 1.5 x 104, and 2.5 x 104. These dialyzed fCM were filter-sterilized with 0.22 pm pore size filters and added to mitogen stimulation lymphoproliferation assays at a final concentration of 50%per well. The results were compared with those obtained with control RPMI which had been concentrated and treated in the same manner as the fCM. Each sample was tested in triplicate.
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Figure 1. The amount of lymphoproliferationafter mitogen-induced stimulationin the presence of UC729-6CM or M21-HPB CM. The data are expressed as a percentage of mean values found for control cultures incubated with RPMI medium. Each bar represents the results of 11 samples obtained from 5 healthy volunteers. Asterisks above bars indicate significant difference (p < 0.05) from the RPMI control for each mitogen. PHA = W, ConA = a.
Statistical Analysis
Effect of CM on One-way MLC
One-way analysis of variance and the nonparametric Kruskal-Wallis test were used to assess the data and to calculate the significance of differences among groups. A p value of < 0.05 indicated significance.
In these assays the effects of UC729-6 and M21-HPB CM at 50%concentration were tested on allogeneic lymphoproliferative responses (Table 1). The presence of either CM significantly suppressed the uptake of pH]Tdr by responder cells, whereas no effect was seen on the
RESULTS
Table 1
Effect of CM on Mitogen-Stimulated Lymphoproliferation CMs from UC729-6 and M21-HPB tumor cell lines were tested in microculture lymphoblastogenesis assays which used PHA and ConA as the mitogens (Fig. 1). Significant reductions in r3H]Tdr uptake were observed in the presence of either CM when compared with their respective RPMI controls. However, the effect was more striking with UC729-6 CM than with M21-HPB CM. The mean dpm values were as follows: RPMI assay control, 34.7 X lo3 (PHA) and 5.5 X 1 6 (ConA); 50% UC729-6 CM, 23.7 x 1@ (PHA) and 3.0 x lo3 (ConA); 100%UC7294 CM, 6.4 X 16 (PHA) and 1.8 X 1@ ( C o d ) ; 50% M21-HPB CM, 20.5 X 16 (PHA) and 3.2 x lo3 (ConA); 100% M21-HPB CM, 10.1 x lo3(PHA) and 1.7 X 1 6 (ConA). No significant variations were seen in control wells containing CM, but no mitogen. The dpm values ranged from 5.4 x 102 to 7.6 x 102.
lhe Effects of Supernatantsfrom UCR9-6 and M21 -HPB Twnor Cell Lines on Mixed Lymphocyte Cultures Mediuma Mixed lymphocyte culture RPMI control UC729-6 CM M21-HPB CM Autologous culturesd RPMI control UC729-6 CM M21-HPB CM
Mean dpm ( X 103)
% Inhibition
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8.5 f 3.7b 5.0 f 0.7' 3.5 1.8c
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1.7 f 0.7 1.7 f 0.6 1.5 f 0.5
0.0 0.0 11.8
'50% CM was present in cultures containing tumor cell CM. bEach mean f SEM was obtained from 9 samples of isolated mononuclear cells from six different donors. 'Significantly different from the respective RPMI controls (p c 0.05).
dCultures in which only responder cells were present gave values similar to those obtained with autologous cultures in which responder and stimulator cells for the same individual were incubated together.
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proliferation of responders incubated with stimulator cells from the same donor in autologous control cultures. Control which contained only responder cells gave dpm values similar to those of the autologous cultures. Minimal incorporation of isotope was observed with irradiated stimulators alone.
A
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Toxicity Testing of fCM There was some decrease in viability with time when normal blood mononuclear cells were incubated for 3 days. However, no significant differences were observed with fCM from UC729-6 or from M21-HPB cells when compared with filter-concentrated RPMI medium or to nonconcentrated RPMI.
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UC729-6 and M21-HPB cells at concentrations of l@, 104, and 103/well each were incubated in 5% to 100% of their own respective fCM (Fig. 2). Significant dosedependent suppression in UC729-6 cell growth was observed with increasing amounts of fCM, especially when 104 or lo3cells were used (panel A of Fig. 2). The growth of M2 1-HPB cells in the presence of their f CM was more variable in that enhancement, as well as suppression, of proliferation was seen (panel B of Fig. 2). The effect was dependent on both the cell concentration and the amount of M21-HPB fCM present. Control dpm values for UC729-6 cells incubated in medium with no fCM were 70.8 X 106 (105 cells), 3.9 X 105 (104 cells) and 5.5 X 104 (107 cells). Similar controls for the M21-HPB cells were 11.2 x 106 (105 cells), 27.8 x 105 (lo4 cells), and 4.3 X 104 (lo3 cells) dpm.
Preliminary Characterization of the fCM from UC729-6 Cells The fCM from UC729-6 cells was treated with acid, trypsin, heat, or dialysis with membranes having M, cutoffs of 2.5 X 104, 1.5 x 104, or 1 X lo" (Table 2). As expected, fCM values were significantly lower compared with corresponding RPMI results. Acid, trypsin, or heat did not significantly affect the inhibitory activity of the f CM in mitogen-induced lymphoproliferation assays when compared with the untreated fCM control. However, partial reversal of suppression was noted with all three dialyzed fractions. Use of the > 1 X 104 M, membrane resulted in the lowest amount of inhibitory activity.
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5 10 50 AMOUNT OF M21-HPB fCM (%)
100
Figure 2. The growth of UC729-6 and It421-HPBcells in the presence of autologous fCM obtained from the two tumor cell lines. Data are expressed as a percentage of mean values found for control cultures incubated with no fCM. Asterisks above bars indicate significant difference (p < 0.05) from the controls obtained at each respective cell concentration. 1 6 cells = I@ cells = m, I@ cells = EI.
.,
DISCUSSION The production of immunosuppressive factors by tumors is a potential escape mechanism from the host's immune system (2 1,22). Recent reports indicate that certain melanomas and lymphomas of animals may produce such factors. However, there are relatively few studies describing such factors from human B-cell lymphomas. This study was undertaken to determine whether the supernates from two human tumor cell lines, UC 729-6 B-cell lymphoma and M21-HPB malignant melanoma, are
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Table 2 Characterization of the Immunosuppressive Activity Found in UC729-6 Tumor Cell Supernutants Using Lymphoproliferation Assays
Mean dpm ( x
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Treatmenta Control (untreated) RPMI fCMC pH 4Sd RPMI fCM 56 'Ce RPMI fCM Trypsinf RPMI fCM
Cancer Investigation, 10(3), 201-208 (1992)
Immunosuppression Derived from Human B-Lymphoblastoid and Melanoma Cell Lines Charlene J. Repique, M.S.,* James D. Kettering, Ph.D.,* and Daila S. Gridley, Ph.D.*-f Departments of *Microbiology and ?Radiation Sciences, Section of Radiation Oncology Lorna Linda University Schod of Medicine Lorna Linda, California 92350
ABSTRACT
Previous work conducted by the authors, using a murine model, suggested that soluble factors secreted by tumor cells suppress lymphocpe responses. To apply this premise to human tumors, the effects of UC729-6 (lymphoblastoid B-cell) and M2I-HPB (malignant melanoma) conditioned media (CM)on normal lymphocyteproliferation, as well as on rumor cell growth in autologous CM was studied. m e CM was collected at 2-5 day intervalsfrom cultures of UC729-6 and M21-HPB cells in serumfree media. Phytohemagglutinin- and concanavalin A-stimulated mononuclear peripheral blood cells from healthy human donors shaved decreased PHIthymidine ([3H]Tdr)uptake in the presence of each CM when compared with controls. In assays using 100%CM, mitogen stimulation was 68-85% less than that of controls and 40-50% less using 50% CM. l l e suppression was more pronounced with UC729-6 CM than with M21-HPB CM. In mixed lymphocyte cultures (MLC), &ition of 50% CMffom either tumor cell line resulted in 40-50% reduction in pH]Tdr uptake by lymphocytes. Incubation of UC72P6 cells in 5 % to 100% of UC72P6 f CM (filter-concentrated) produced a decrease in pH]Tdr uptake which was directly proportional to the amount offCMpresent. In contrast, M21-HPB cell growth in autologousf CM w s dependent on cell number, as well as on the amount off CM used. Treatment of the UC729-6f CM using acid @H4.5). trypsin (I 00 pg/ml), and heat (56°C) did not restore mitogen-stimulated lymphoproliferation. However, the inhibition observed with UC729-6f CM was partially reversed ajer dialysis with membranes having M, limits of 2.5 x I @ , 1.5 X I @ , or I x I@. 201 Copyright 0 1992 by Marcel Dekker, Inc.
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INTRODUCTION The progression of neoplasia and its escape from host immune mechanisms remain an enigma. This is especially confounding when the tumor is immunogenic and the host is, at least initially, immunocompetent. The impairment of host defenses in tumor-bearing subjects has been demonstrated in numerous studies. For example, the depression of lymphoproliferation, immunoglobulin synthesis, and macrophage and neutrophil functions have been reported. The fact that immunosuppression can be reversed upon reduction of the tumor load (1) implicates the malignant tissue itself as a possible source of the inhibitory effects. Recently, interest in tumor-derived immunosuppressive factors has greatly increased because of the development of new immunological techniques for cancer therapy (2-4). Studies of lymphokineactivated killer (LAK) cells and tumor-infiltrating lymphocytes (TILs) show that despite the close association of immune cells with tumor tissue, these cells retain potential antitumor activity (5). Generation of LAK cells and TILs for cancer therapy can be inhibited by factors secreted by glioblastoma, melanoma, and colorectal cancer cells (6,7). These factors may contribute to the failure of adoptive immunotherapy in a significant number of patients. It has also been demonstrated that factors produced by malignant cells can exhibit autocrine, as well as paracrine, activity (8-11). A wide range of neoplasms such as leukemias, glioblastoma, colorectal cancer, melanoma, and small-cell lung carcinoma have been studied in this respect. Investigators have found that different regulatory factors can be found in tumor effusions and cell culture supernatants, as well as in sera from tumor-bearing subjects (12-14). An important aspect of these studies is that malignant cells may produce factors that are selfstimulatory, but inhibitory to potentially immunocompetent cells, thus giving the tumor tissue a growth advantage within the host. These findings have generated interest in the development of this investigation. The purpose of this study was to determine if cell culture supernatants obtained from a human malignant melanoma, M21-HPB, and a human lymphoblastoid B-cell line, UC729-6, could inhibit normal T lymphocyte activity. In addition, the effects of the autologous supernatants on the growth of the tumor cells themselves and preliminary characterization of the immunosuppressive activity derived from UC729-6 cells are also reported here.
MATERIALS AND METHODS Tumor Cell Lines UC729-6 is a 6-thioguanine-resistant human lymphoblastoid B-cell line obtained from Dr. Mark Glassy at University of California, San Diego, CA. It was originally developed by treating WIL-2 lymphoblastoid cells with 25 pM 6-thioguanine (15,16). This nonantibodyproducing cell line has the potential for human hybridoma and monoclonal antibody production (17). M21-HPB (Hybritech Incorporated, San Diego, CA) is a human melanoma cell line derived from a secondary metastasis of malignant melanoma (18). It is tumorigenic in nude mice and has been used in our laboratories in previous studies (19,20). Both cell lines were grown and maintained in Roswell Park Memorial Institute (RPMI) 1640medium (Irvine Scientific, Santa Ana, CA) supplemented with 10% heat-inactivated fetal calf serum (FCS) @vine Scientific; Hyclone Laboratories Incorporated, Logan, UT), 2 pg/ml fungizone (E. R. Squibb and Sons, Incorporated, Princeton, NJ), and 50 pglml gentamicin sulfate (Sigma Chemical Company, St. Louis, MO) at 37°C in 5% C 0 2 . The UC729-6 cells grew in suspension culture, whereas the M21-HPB cells produced monolayers. Both cell lines were found to be free of mycoplasma contamination after testing with the Mycotrim Triphasic Culture System (Hana Biologics, Incorporated, Alameda, CA).
Conditioned Media (CM) UC729-6 cultures at moderate to heavy growth and M21-HPB cultures at approximately 75 % to complete confluency in 75 cm2 tissue culture flasks (Falcon, Oxnard, CA; Coming Glass Works, Coming, NY) were washed three times with serum-free RPMI. Fresh serumfree medium was added and the cells were allowed to grow for 2 to 3 h at 37 "C in 5 % COz . This medium was again replaced with fresh serum-free RPMI and the cultures were reincubated for 48 h. The CM was then collected and clarified by centrifugation at 200 g for 10 min. At the time of CM collection, cell viability was > 95% as determined by trypan blue exclusion. The CM from each cell line was pooled, aliquoted, and stored at -20 "C until immediately before use.
Mitogen-Induced Lymphoproliferation Whole blood from 5 healthy donors, 3 males and 2 females, aged 25 to 46 years, was collected into tubes
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with sodium heparin as the anticoagulant. One individual was tested 4 times, another 3 times, and a third subject was tested twice for a total of 9 specimens. Each sample was diluted 1:10 with RPMI or with each of the CM. The RPMI medium was supplemented with 10% FCS, fungizone, gentamicin, 10 mM HEPES buffer (Calbiochem Corporation, La Jolla, CA), and 5 X mM mercaptoethanol (Sigma). Phytohemagglutinin (PHA; Burroughs Wellcome, Triangle Park, NC) and concanavalin A (ConA; Sigma), both previously titrated to determine maximum stimulatory concentrations, were diluted with either RPMI or with CM. Cells and mitogens were dispensed into 96-well flat bottom microtiter plates (Falcon; Coming) to yield 50 or 100% of each CM in a total volume of 200 pl. Control wells contained cells and medium or CM, but no mitogen. Assays which included CM were also compared with simultaneously performed control assays which contained RPMI and no CM. The microtiter plates were incubated at 37 "Cin 5 % C 0 2 for 3 days and labelled with 1 pCi of [3H]Tdr/well (specific activity = 46 Ci/mmol; ICN Biomedicals, Costa Mesa, CA) for the final 5 h of incubation. The cells were harvested onto glass microfiber filters with an automated cell harvester (Brandel Incorporated, Gaithersburg, MD). The radioactivity incorporated into cell DNA was counted in disintegrations per minute (dpm) for 1 min/sample in a liquid scintillation counter (Beckman Instruments, Incorporated, Fullerton, CA). All samples were tested in quadruplicate.
CM from both UC729-6 and M21-HPB cultures were concentrated to one tenth of the original volumes using a lo00 M, filter in an Amicon filtration system (Amicon Division W. R. Grace and Company, Danvers, MA). The CM were filtered using 55 to 60 lbs. of positive pressure and continuous mixing with magnetic stirrers at a flow rate of approximately 55 ml/h at 4°C to produce filterconcentrated CM (fCM) at one-tenth of the original volume. The tested fraction contained molecules of > lo00 M,. Isolated mononuclear cells (2 X l@/well) were incubated in 50% and 100% fCM from UC729-6 or M21-HPB cells, 100% filter-concentrated RPMI medium, and nonconcentrated RPMI. The cells were tested for viability by trypan blue exclusion after 24 h and after 72 h.
Mixed Lymphocyte Cultures
Tumor Cell Growth in Autologous fCM
Whole blood was collected from 6 healthy donors, 3 males, and 3 females, aged 25 to 46 years. Three individuals were tested twice for a total of 9 separate specimens. Mononuclear cells were obtained using standard Ficoll-Hypaque (Pharmacia LKB Biotechnology, Incorporated, Piscataway ,NJ), density gradient centrifugation procedures. After isolation the mononuclear cells were washed twice and resuspended in RPMI to a concentration of 2 X 106 cells/ml. Each suspension was divided into two fractions: responders and stimulators. Stimulator cells were irradiated with 2750 rad from a 137Cssource (Model 143 Irradiator, J. L. Sheperd and Associates, San Fernando, CA) to prevent DNA replication when mixed with allogenic responders. Next, 50 pl of stimulator cells and 50 pl of responder cells were dispensed into 96-well flat bottom microtiter plates to establish mixed cultures. Autologous controls in which stimulatorsand responders from the same individualswere plated together were included. Cell controls consisting of
stimulator or responder cells alone were used to screen for spontaneous or unexplained proliferation of the lymphocytes. Each cell sample was treated with 100 pl of either of the CM or with RPMI as an additional control. The cultures were incubated at 37 "C in 5 % COz for 5 days. Each well was then labelled with 1 pCi of [3H]Tdr for an additional 18 h and the cells were harvested (Brandel). The radioactivity incorporated into cell DNA was counted in dpm for 1 min/sample in a liquid scintillation counter. All samples were tested in triplicate.
Preparation and Toxicity Testing of Filter-Concentrated CM (fCM)
UC729-6 and M21-HPB cells were dispensed into 96-well flat bottom microtiter plates in 100 pl at concentrations of 105, 104, and 103 per well each. Various dilutions of each fCM were prepared with RPMI. Volumes of 100plcontaining 100%, 50%, l o % , and5% fCMor 100% ,50 % ,and 10% CM were plated with each cell type at each cell concentration. The cultures were incubated for 54 h at 37°C in 5% C02. Each well was then labelled with 1 pCi rH]Tdr for 18 h of additional incubation. The cells were trypsinized, if necessary, and harvested with an automatic cell harvester. The incorporated radioactivity was counted in a liquid scintillation counter for 1 min/sample. The samples were tested in triplicate.
Characterization of the Immunosuppressive Activity in UC729-6 fCM Because of rapid, abundant growth and the capacity for high supernate volume generation, focus was placed on
Repique, Kettering, and Gridley
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the UC729-6 cell line. Aliquots of the fCM were heattreated in a H20 bath at temperatures of either 56 "C or 37 "C (control) for 1 h. Other aliquots of fCM were adjusted to pH 4.5 with 1N HC1 for 1 h at room temperature. The pH was readjusted to 7.4 with 3N NaOH before use in assays. Additional fCM samples were treated with 100 pg/ml of trypsin for 1 h at 37 "C. Soybean trypsin inhibitor (Sigma; 250 pg/100 pg of trypsin) was added to stop the action of the enzyme. Finally, portions of each of the two fCMs were dialyzed against serum-freeRPMI using Spectrapor membrane tubings (Spectrum Medical Industries, Incorporated, Los Angeles, CA) with M, cutoffs of 1 X 104, 1.5 x 104, and 2.5 x 104. These dialyzed fCM were filter-sterilized with 0.22 pm pore size filters and added to mitogen stimulation lymphoproliferation assays at a final concentration of 50%per well. The results were compared with those obtained with control RPMI which had been concentrated and treated in the same manner as the fCM. Each sample was tested in triplicate.
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100%
UC 729-6
Figure 1. The amount of lymphoproliferationafter mitogen-induced stimulationin the presence of UC729-6CM or M21-HPB CM. The data are expressed as a percentage of mean values found for control cultures incubated with RPMI medium. Each bar represents the results of 11 samples obtained from 5 healthy volunteers. Asterisks above bars indicate significant difference (p < 0.05) from the RPMI control for each mitogen. PHA = W, ConA = a.
Statistical Analysis
Effect of CM on One-way MLC
One-way analysis of variance and the nonparametric Kruskal-Wallis test were used to assess the data and to calculate the significance of differences among groups. A p value of < 0.05 indicated significance.
In these assays the effects of UC729-6 and M21-HPB CM at 50%concentration were tested on allogeneic lymphoproliferative responses (Table 1). The presence of either CM significantly suppressed the uptake of pH]Tdr by responder cells, whereas no effect was seen on the
RESULTS
Table 1
Effect of CM on Mitogen-Stimulated Lymphoproliferation CMs from UC729-6 and M21-HPB tumor cell lines were tested in microculture lymphoblastogenesis assays which used PHA and ConA as the mitogens (Fig. 1). Significant reductions in r3H]Tdr uptake were observed in the presence of either CM when compared with their respective RPMI controls. However, the effect was more striking with UC729-6 CM than with M21-HPB CM. The mean dpm values were as follows: RPMI assay control, 34.7 X lo3 (PHA) and 5.5 X 1 6 (ConA); 50% UC729-6 CM, 23.7 x 1@ (PHA) and 3.0 x lo3 (ConA); 100%UC7294 CM, 6.4 X 16 (PHA) and 1.8 X 1@ ( C o d ) ; 50% M21-HPB CM, 20.5 X 16 (PHA) and 3.2 x lo3 (ConA); 100% M21-HPB CM, 10.1 x lo3(PHA) and 1.7 X 1 6 (ConA). No significant variations were seen in control wells containing CM, but no mitogen. The dpm values ranged from 5.4 x 102 to 7.6 x 102.
lhe Effects of Supernatantsfrom UCR9-6 and M21 -HPB Twnor Cell Lines on Mixed Lymphocyte Cultures Mediuma Mixed lymphocyte culture RPMI control UC729-6 CM M21-HPB CM Autologous culturesd RPMI control UC729-6 CM M21-HPB CM
Mean dpm ( X 103)
% Inhibition
0.0
8.5 f 3.7b 5.0 f 0.7' 3.5 1.8c
41.0
+
58.8
1.7 f 0.7 1.7 f 0.6 1.5 f 0.5
0.0 0.0 11.8
'50% CM was present in cultures containing tumor cell CM. bEach mean f SEM was obtained from 9 samples of isolated mononuclear cells from six different donors. 'Significantly different from the respective RPMI controls (p c 0.05).
dCultures in which only responder cells were present gave values similar to those obtained with autologous cultures in which responder and stimulator cells for the same individual were incubated together.
Immunosuppression by B-Lymphoblasts
205
proliferation of responders incubated with stimulator cells from the same donor in autologous control cultures. Control which contained only responder cells gave dpm values similar to those of the autologous cultures. Minimal incorporation of isotope was observed with irradiated stimulators alone.
A
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Toxicity Testing of fCM There was some decrease in viability with time when normal blood mononuclear cells were incubated for 3 days. However, no significant differences were observed with fCM from UC729-6 or from M21-HPB cells when compared with filter-concentrated RPMI medium or to nonconcentrated RPMI.
5
0
10
50
100
AMOUNT OF UC729-6 fCM (%)
Growth of Tumor Cells in Autologous fCM
*
190 -I
180
B
UC729-6 and M21-HPB cells at concentrations of l@, 104, and 103/well each were incubated in 5% to 100% of their own respective fCM (Fig. 2). Significant dosedependent suppression in UC729-6 cell growth was observed with increasing amounts of fCM, especially when 104 or lo3cells were used (panel A of Fig. 2). The growth of M2 1-HPB cells in the presence of their f CM was more variable in that enhancement, as well as suppression, of proliferation was seen (panel B of Fig. 2). The effect was dependent on both the cell concentration and the amount of M21-HPB fCM present. Control dpm values for UC729-6 cells incubated in medium with no fCM were 70.8 X 106 (105 cells), 3.9 X 105 (104 cells) and 5.5 X 104 (107 cells). Similar controls for the M21-HPB cells were 11.2 x 106 (105 cells), 27.8 x 105 (lo4 cells), and 4.3 X 104 (lo3 cells) dpm.
Preliminary Characterization of the fCM from UC729-6 Cells The fCM from UC729-6 cells was treated with acid, trypsin, heat, or dialysis with membranes having M, cutoffs of 2.5 X 104, 1.5 x 104, or 1 X lo" (Table 2). As expected, fCM values were significantly lower compared with corresponding RPMI results. Acid, trypsin, or heat did not significantly affect the inhibitory activity of the f CM in mitogen-induced lymphoproliferation assays when compared with the untreated fCM control. However, partial reversal of suppression was noted with all three dialyzed fractions. Use of the > 1 X 104 M, membrane resulted in the lowest amount of inhibitory activity.
140 130
40 50
0
5 10 50 AMOUNT OF M21-HPB fCM (%)
100
Figure 2. The growth of UC729-6 and It421-HPBcells in the presence of autologous fCM obtained from the two tumor cell lines. Data are expressed as a percentage of mean values found for control cultures incubated with no fCM. Asterisks above bars indicate significant difference (p < 0.05) from the controls obtained at each respective cell concentration. 1 6 cells = I@ cells = m, I@ cells = EI.
.,
DISCUSSION The production of immunosuppressive factors by tumors is a potential escape mechanism from the host's immune system (2 1,22). Recent reports indicate that certain melanomas and lymphomas of animals may produce such factors. However, there are relatively few studies describing such factors from human B-cell lymphomas. This study was undertaken to determine whether the supernates from two human tumor cell lines, UC 729-6 B-cell lymphoma and M21-HPB malignant melanoma, are
Repique, Kettering, and Gridley
206
Table 2 Characterization of the Immunosuppressive Activity Found in UC729-6 Tumor Cell Supernutants Using Lymphoproliferation Assays
Mean dpm ( x
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Treatmenta Control (untreated) RPMI fCMC pH 4Sd RPMI fCM 56 'Ce RPMI fCM Trypsinf RPMI fCM
