Immunology 1978 34 137
Isolation and partial characterization of an antibody enhancing factor from leukaemic owl monkey cell cultures
A. B. MACDONALD, C. E. 0. FRASER* & A. S. RUBINt *New England RegionalPrimate Research Center, Southboro, Massachusetts, U.S.A., and t University of Texas at Austin, Department of Microbiology, Austin, Texas 78712, U.S.A.
Received 15 July 1976; accepted for publication 25 April 1977
been demonstrated that some owl monkeys infected with H. saimiri develop high titres of antibodies to the virus. The antibody titres approach 1 x 106 (Fraser, MacDonald & Melendez, 1974) as demonstrated by fixed cell fluorescence and 1 x 105 as measured by neutralization in the presence of high titres of complement (Fraser, MacDonald, Melendez & Daniel, 1974). The high titres of antibody coincide with increased levels of serum globulins as determined by electrophoresis, i.e. the serum globulin fraction approaches 100 mg/ml in infected monkeys compared to 13 mg/ml in non-infected control animals (Fraser et al., 1974). There is a growing body of evidence that the lymphoid cells involved in cell-mediated immunity (CMI) and humoral immunity can be regulated by soluble mediators released from specificially (or mitogenically) activated thymus-derived lymphocytes (T cells). These effector molecules have been categorized as 'lymphokines'. Those factors with a defined role in CMI reactions include migration inhibitory factor, lymphotoxin, and chemotactic factor (Remold, 1972), while the lymphokines involved in the regulation of the humoral immune response have been shown to alter the synthesis of antibody by bone marrow-derived lymphocytes (B cells) against heterologous erythrocytes. Both positive (enhancer) and negative (suppressor) regulatory factors have been described (Rubin & Coons, 1971; Thomas, Roberts & Talmage, 1975).
Summary. Herpesvirus saimiri inoculated into owl monkeys (Aotus trivirgatus) causes leukaemia and lymphoma. Peripheral lymphocytes from leukaemic monkeys grown in culture are predominantly T lymphocytes. The supernatants from these cultures contain a factor which enhances the antibody response of murine B cells to sheep red blood cells (SRBC). The factor has been partially characterized by ammonium sulphate precipitation, DEAEchromatography, gel filtration over Sephadex G-150 and disc-gel electrophoresis. The enhancing factor is a protein with a molecular weight of approximately 40,000. INTRODUCTION Herpesvirus saimiri is an endogenous viral agent of squirrel monkey (Saimiri sciureus). The virus was first isolated by Melendez et al. and was shown to cause malignant lymphoma and leukaemia in owl monkeys (Aotus trivirgatus) (Melendez, Hunt, Daniel, Blake & Garcia, 1971), and other New World primates (Melendez, Hunt, Daniel, Garcia & Fraser, 1969; Melendez, Hunt, Daniel, Fraser, Gracia & Williamson, 1970; Rabin, 1971; Hunt, Melendez, King & Garcia, 1972). Recently, it has Correspondence: Dr A. B. MacDonald, Department of Microbiology, Harvard University School of Public Health, Boston, Massachusetts 02115, U.S.A.
137
138
A. B. MacDonald, C. E. 0. Fraser & A. S. Rubin
Recently, we reported the isolation and partial characterization of an immuno-enhancing factor, derived from supernatants of an allogeneic mixture of human lymphocyte cell lines, which was capable of non-specifically augmenting the anti-sheep erythrocyte (SRBC) plaque-forming cell (PFC) response of normal mouse spleen cells in vitro (Rubin & MacDonald, 1976). The high titres of anti-H. saimiri antibody, increased concentrations of serum globulin, and the observation that H. saimiri-induced leukaemia is of T cell origin (Wallen, Neubauer, Rabin & Cicmanec, 1973; Wallen, Neubauer & Rabin, 1974) might indicate that lymphocyte cultures derived from the peripheral blood of leukaemic owl monkeys may be predominantly T cells. In addition, an immunoenhancing factor may be released by these aberrant cells into the supernatant fluid in which they are cultured. The propagation of lymphoid cells from leukaemic owl monkeys in culture, the release of a soluble factor that amplifies antibody synthesis from these cells, and the partial characterization of this mediator are described in this communication.
MATERIALS AND METHODS Long-term culture of leukaemic cells An owl monkey inoculated with H. saimiri developed leukaemia 4 months later and remained leukaemic until it was killed nearly 12 months after. At the time of death, peripheral blood was harvested using preservative-free heparin (100 U/ml). The lymphocytes were separated from the erythrocytes and plasma by centrifugation on a Ficoll-Hypaque gradient and were washed twice with RPMI 1640 supplemented with 20% IgG-free foetal calf serum and antibiotics (100 U/ml penicillin and 100.pg/ml streptomycin). The cells were then layered on sterile Gel-Foam (Upjohn) and maintained in 75 cm2 Falcon tissue culture flasks. After 9 days, the GelFoam containing lymphocytes in various stages of growth was transferred to another 75 cm2 plastic flask containing a monolayer of owl monkey kidney cells. The owl monkey cells used subsequently as a feeder layer were either OMK 210 or OMK 637 cell lines which were established in our laboratories. After 2 weeks, the lymphocytes were clearly replicating in the Gel-Foam/feeder layer cultures and could be seen in the culture medium both as single
cells and as clumps of cells. The medium of these cultures was changed every 2-3 days. The growth of lymphocytes over a feeder layer of permissive owl monkey cells apparently activates the repressed virus genome present in leukaemic lymphocytes. This results in the cytolytic destruction of the feeder layer. The lymphocytes were transferred to a new monolayer of OMK cells whenever the feeder layer was destroyed. Long-term cultures of normal lymphocytes have been achieved by utilizing this same technique. The cells have been maintained in this manner for 19 months. Evaluation of supernatant enhancing activity Owl monkey supernatants were assayed for their ability nonspecifically to increase the anti-sheep erythrocyte (SRBC) plaque-forming cell (PFC) response of normal mouse spleen cell cultures by methods used in previous reports (Rubin, MacDonald & Coons, 1973, 1974). Briefly, spleen cell cultures from normal 2-4-month-old DBA/2 female mice were prepared as before by a modification of the method of Mishell & Dutton, except that cells were maintained at a density of 1-5-20 x 107 cells/ ml (0 5 ml volumes) in Costar-24 tissue culture plates (Costar, Cambridge, Mass.). In some experiments, cells were maintained at the same density in 1 ml volumes in Falcon 35 x 10 mm dishes, as described previously. Cultures were stimulated with 3 million SRBC on day 0. Filtered test supernatants, diluted in fresh medium, were added to such cultures on day 2. Direct '19 S' plaques against SRBC in individual cultures were enumerated by the Jerne plaque technique on day 5. The degree of enhancement is expressed as the percentage difference between the numbers of plaques produced in cultures that received diluted test supernatants compared to the PFC response of similar cultures that received diluted, control supernatants from cultures of uninfected monkey lymphoid cells.
Preparation of antisera Anti-human T cell antiserum was kindly provided by Dr Michael Fanger, Case Western Reserve Medical School, Cleveland, Ohio. The preparation of this antiserum has been previously reported (Owen & Fanger, 1974). An anti-owl monkey immunoglobulin was produced in goats from owl monkey immunoglobulin. Lipid was removed from owl monkey serum by centrifugation at 40 and filtered through glass wool. Immunoglobulins
Purification of owl monkey enhancing Jactor were precipitated from a solution of 18% sodium sulphate followed by precipitation from a 14 % sodium sulphate solution. The precipitate fraction was subjected to ion exchange chromatography over DEAE cellulose. The front fraction was concentrated and 3 mg were emulsified in Freund's complete adjuvant and injected intramuscularly into a goat. Three weeks later a similar injection was made and 1 week later the serum was harvested.
Conjugation of antisera by fluorescein isothiocyanate Immunoglobulins were precipitated from an 18% solution of sodium sulphate and dialysed against 0-06 M NaHCO3 buffer, pH 9 5. Fluorescein isothiocyanate was added in a ratio of 0 025 mg FITC per mg protein at 250. The reaction was allowed to proceed for 5 min. Unbound FITC was removed by gel filtration over Sephadex G-25. The eluate containing the conjugated protein was precipitated from 14% sodium sulphate. The precipitate was washed twice with 14% sodium sulphate, then redissolved and dialysed against borate buffered saline (BSB) (0-01 M borate, pH 8-0, 0-154 M sodium chloride). Immunoglobulins conjugated in this manner lose little or no antibody titre compared to unconjugated antisera. Ammonium sulphate fractionation Supernatants from cultured cells of an owl monkey (240-72) with leukaemia were pooled. These supernatants represented passage numbers 13-28. Ammonium sulphate was added to 428 ml of supernatant at 40 to a final concentration of 70%. The mixture was centrifuged at 34,000 g at 4°. The precipitate was dissolved in 23 ml of BSB and dialysed against phosphate-buffered saline (PBS) 0-15 M, pH 7-2 prior to testing for OMEF activity. DEAE chromatography The 70% ammonium sulphate precipitate fraction was dialysed against 0-02 M phosphate buffer at pH 6 9. After dialysis, the fraction was subjected to chromatography over a 40 ml diethylaminoethylcellulose (DEAE) column and eluted with 0-02 M phosphate buffer, pH 6-9. When the absorbance at 280 nm returned to baseline levels ( < 0 05), a linear gradient from 0-02 M to 0-3 M phosphate buffer, pH 6 9-8 0 (200 ml each) was initiated and aliquots collected. Fractions were dialysed vs PBS prior to addition to assay cultures.
139
Gel filtration over Sephadex G-150 A Sephadex G-150 column (1-5 x 87 cm) was prepared in BSB and eluted in an ascending direction. The column was standardized with four molecular weight markers: (a) rabbit F (ab)2 conjugated with fluorescein isothiocyanate (FITC) (105,000 Daltons); (b) 125I-labelled bovine serum albumin (62,000 Daltons); (c) Ovalbumin (40,000 Daltons); and (d) cytochrome c (12,500 Daltons). The fractions from the DEAE column that contained most of the enhancing activity were pooled, concentrated 12-fold and dialysed against BSB. The concentrated fraction was then subjected to molecular filtration over Sephadex G-150 at 4'. Column fractions, dialysed against PBS, were tested for enhancing factor activity as described above.
Acrylamide gel electrophoretic purification of OMEF The OMEF-rich G-150 column fraction was subjected to disc electrophoresis on a Pharmacia GE-4 electrophoresis system (Pharmacia Fine Chemicals, Piscataway, N.J.). The gels used contained 7-5% acrylamide in Tris-glycine buffer, pH 8-3, at 25°. All gels were pre-run prior to sample application. The 15-20ul samples were then subjected to electrophoresis at 2 4 mA per gel. Each gel was cut into four sections. Equivalent sections from five gels were pooled, homogenized, suspended in 5 ml of PBS (0-15 M, pH 7-2) and centrifuged (35,000g, 10 min, 40) to remove acrylamide. Each resulting supernatant was dialysed for 48 h against 1000 volumes of PBS at 4° before testing for OMEF by the usual method. A gel run in parallel was fixed and stained with 0-25% Coomassie Blue R in 10% acetic acid, then destained with Dowex 1-X8 anion exchange resin beads (J. T. Baker, Phillipsburg, N.J.). The gel absorbance at 570 nm was read on a Gilford 640 recording spectrophotometer coupled with a linear transport system (Model 2410-S). Enzyme treatment of OMEF Active column fractions of partially-purified OMEF were tested for their sensitivity to enzymatic degradation with deoxyribonuclease I (DNase, Worthington Biochemical Corp., Freehold, N.J.), ribonuclease A (RNase, type IIIA, Sigma Chemical Co, St. Louis, Mo.) or protease (type VI, Sigma Chemical Co.). Column fractions were divided into three aliquots. DNase, RNase or protease were added to two of the aliquots at a final concentration of 10 ,pg/ml. One aliquot was not treated. Active
A. B. MacDonald, C. E. 0. Fraser & A. S. Rubin
140
Table 1. Percent of peripheral cells from owl monkeys staining with specific antiserum
Cell type
Peripheral lymphocytes
Passage number
Anti-human-T-cell (%)
Anti-owl monkey Ig
Disease
14 3
Leukaemia (H. saimiri) Normal
63 25
0 0
fraction-enzyme mixtures were stirred continuously (1 h, 370) to ensure adequate contact between enzyme and available substrate. Treated and untreated aliquots were then tested for OMEF activity by the method outlined above except that baseline control cultures received SRBC and diluent (complete medium) on days 0 and 2, respectively. OMEF activity on T cell depleted B cell enriched cultures DBA/2 splenic B cells were separated from T cells by the nylon wool column method of Handwerger & Schwartz (1974). B cell-enriched (T cell-depleted) populations contained 90-950% immunoglobulinpositive cells as determined by standard immunofluorescent techniques using an FITC-conjugated rabbit anti-mouse immunoglobulin serum to stain column eluate B cells. B cells were maintained at a density of 1 x 107 cells/ml (in 0 5 ml volumes) in Costar-24 plates in complete medium supplemented with 5 x 10-5 M 2-mercaptoethanol to functionally replace the adherent population of macrophages removed by the nylon wool separation procedure. Cultures were stimulated with SRBC on day 0, and with diluted, partially-purified OMEF on day 2. Control B cell cultures received only SRBC and diluent. IgM PFC vs SRBC were enumerated on day 5. Mitogenic activity of OMEF Whole human blood was drawn into heparin (preservative free, Connaught Laboratories, Toronto, Canada) at 100 U/ml. The blood was then added to 6% dextran (M.W. 2 x 106) in a ratio of 6: 1 blood to dextran and incubated for 1 h at 37°. The leucocytes were removed and separated from the plasma by centrifugation at 200 g at room temperature for 5 min. The cells were washed twice in Plus 1 Hepes Buffer (IBL, Rockville, Maryland), and supplemented with 100 fg/ml of streptomycin. Each well of a microtitre plate (Microbiological
(%)
Associates, Walkersville, Maryland) received 01 ml of the cell suspension and 01 ml of phytohaemagglutinin (PHA-P) (Burroughs Wellcome, Greenville, North Carolina) at a concentration of 25,ug/ml or 0-1 ml of the OMEF active fraction from the Sephadex, G-150 column which was previously dialysed against PBS. The OMEF was added in dilutions of 1 :100, 1: 200 and 1: 500. The cultures were incubated 3 or 5 days at 37° in an atmosphere of 5 % CO2 (in air). Four microcuries of [3H] thymidine (6 7Ci/mmol specific activity) (New England Nuclear, Boston, Massachusetts) were added per culture well for the final 8 h of the incubation period. The cells were harvested on filter paper through a Mash II cell harvester (Microbiological Associates, Walkersville, Maryland). The filters were dried in air at room temperature and each spot containing the cells was placed in liquid scintillation fluid and the radioactivity determined in a Beckman Scintillation Counter Model LS-200. Triplicate test samples were counted; controls were run in quadruplicate. RESULTS Source and activity of owl monkey enhancing factor
(OMEF) Peripheral blood lymphocytes (PBL) from owl monkeys can be cultured in an in vitro culture system utilizing Gel-Foam and an owl monkey kidney cell feeder layer. However, the cells cultured from monkeys inoculated with H. saimiri and suffering leukaemia and lymphoma are different from cells cultured from normal owl monkeys. The leukaemic cells appear to be predominantly of T lymphocyte lineage as indicated by immunofluorescence labelling with a specific anti-human T cell antiserum. These results are given in Table 1. The preponderance (63 %) of cells from leukaemic monkeys, maintained in vitro, stain with antiserum directed against T cell markers (Owen & Fanger, 1974), whereas only 25 %
141
Purification of owl monkey enhancing factor Table 2. Enhancement of the anti-SRBC PFC response of mouse spleen cells with supernatants from cultured peripheral lymphocytes from owl monkeys
Supernatant no. Cell origin
Passage no.
Leukaemia (H. saimiri)
14 14 14 +15 3 +4 14 14 14 +15 3 +4
(2) (8) (11)
Normal Leukaemia (H. saimiri)
(2) (8) (11)
Normal
Supernatant % Enhancement final vs controlt PFC ± s.d.* dilution 1:100 1:100
1:200 1:200
290 ± 14 405 ±92 815 ±50 123 ±40 505 ±92 345 ±35 420 ±28 170 ±40
P value:
Isolation and partial characterization of an antibody enhancing factor from leukaemic owl monkey cell cultures
A. B. MACDONALD, C. E. 0. FRASER* & A. S. RUBINt *New England RegionalPrimate Research Center, Southboro, Massachusetts, U.S.A., and t University of Texas at Austin, Department of Microbiology, Austin, Texas 78712, U.S.A.
Received 15 July 1976; accepted for publication 25 April 1977
been demonstrated that some owl monkeys infected with H. saimiri develop high titres of antibodies to the virus. The antibody titres approach 1 x 106 (Fraser, MacDonald & Melendez, 1974) as demonstrated by fixed cell fluorescence and 1 x 105 as measured by neutralization in the presence of high titres of complement (Fraser, MacDonald, Melendez & Daniel, 1974). The high titres of antibody coincide with increased levels of serum globulins as determined by electrophoresis, i.e. the serum globulin fraction approaches 100 mg/ml in infected monkeys compared to 13 mg/ml in non-infected control animals (Fraser et al., 1974). There is a growing body of evidence that the lymphoid cells involved in cell-mediated immunity (CMI) and humoral immunity can be regulated by soluble mediators released from specificially (or mitogenically) activated thymus-derived lymphocytes (T cells). These effector molecules have been categorized as 'lymphokines'. Those factors with a defined role in CMI reactions include migration inhibitory factor, lymphotoxin, and chemotactic factor (Remold, 1972), while the lymphokines involved in the regulation of the humoral immune response have been shown to alter the synthesis of antibody by bone marrow-derived lymphocytes (B cells) against heterologous erythrocytes. Both positive (enhancer) and negative (suppressor) regulatory factors have been described (Rubin & Coons, 1971; Thomas, Roberts & Talmage, 1975).
Summary. Herpesvirus saimiri inoculated into owl monkeys (Aotus trivirgatus) causes leukaemia and lymphoma. Peripheral lymphocytes from leukaemic monkeys grown in culture are predominantly T lymphocytes. The supernatants from these cultures contain a factor which enhances the antibody response of murine B cells to sheep red blood cells (SRBC). The factor has been partially characterized by ammonium sulphate precipitation, DEAEchromatography, gel filtration over Sephadex G-150 and disc-gel electrophoresis. The enhancing factor is a protein with a molecular weight of approximately 40,000. INTRODUCTION Herpesvirus saimiri is an endogenous viral agent of squirrel monkey (Saimiri sciureus). The virus was first isolated by Melendez et al. and was shown to cause malignant lymphoma and leukaemia in owl monkeys (Aotus trivirgatus) (Melendez, Hunt, Daniel, Blake & Garcia, 1971), and other New World primates (Melendez, Hunt, Daniel, Garcia & Fraser, 1969; Melendez, Hunt, Daniel, Fraser, Gracia & Williamson, 1970; Rabin, 1971; Hunt, Melendez, King & Garcia, 1972). Recently, it has Correspondence: Dr A. B. MacDonald, Department of Microbiology, Harvard University School of Public Health, Boston, Massachusetts 02115, U.S.A.
137
138
A. B. MacDonald, C. E. 0. Fraser & A. S. Rubin
Recently, we reported the isolation and partial characterization of an immuno-enhancing factor, derived from supernatants of an allogeneic mixture of human lymphocyte cell lines, which was capable of non-specifically augmenting the anti-sheep erythrocyte (SRBC) plaque-forming cell (PFC) response of normal mouse spleen cells in vitro (Rubin & MacDonald, 1976). The high titres of anti-H. saimiri antibody, increased concentrations of serum globulin, and the observation that H. saimiri-induced leukaemia is of T cell origin (Wallen, Neubauer, Rabin & Cicmanec, 1973; Wallen, Neubauer & Rabin, 1974) might indicate that lymphocyte cultures derived from the peripheral blood of leukaemic owl monkeys may be predominantly T cells. In addition, an immunoenhancing factor may be released by these aberrant cells into the supernatant fluid in which they are cultured. The propagation of lymphoid cells from leukaemic owl monkeys in culture, the release of a soluble factor that amplifies antibody synthesis from these cells, and the partial characterization of this mediator are described in this communication.
MATERIALS AND METHODS Long-term culture of leukaemic cells An owl monkey inoculated with H. saimiri developed leukaemia 4 months later and remained leukaemic until it was killed nearly 12 months after. At the time of death, peripheral blood was harvested using preservative-free heparin (100 U/ml). The lymphocytes were separated from the erythrocytes and plasma by centrifugation on a Ficoll-Hypaque gradient and were washed twice with RPMI 1640 supplemented with 20% IgG-free foetal calf serum and antibiotics (100 U/ml penicillin and 100.pg/ml streptomycin). The cells were then layered on sterile Gel-Foam (Upjohn) and maintained in 75 cm2 Falcon tissue culture flasks. After 9 days, the GelFoam containing lymphocytes in various stages of growth was transferred to another 75 cm2 plastic flask containing a monolayer of owl monkey kidney cells. The owl monkey cells used subsequently as a feeder layer were either OMK 210 or OMK 637 cell lines which were established in our laboratories. After 2 weeks, the lymphocytes were clearly replicating in the Gel-Foam/feeder layer cultures and could be seen in the culture medium both as single
cells and as clumps of cells. The medium of these cultures was changed every 2-3 days. The growth of lymphocytes over a feeder layer of permissive owl monkey cells apparently activates the repressed virus genome present in leukaemic lymphocytes. This results in the cytolytic destruction of the feeder layer. The lymphocytes were transferred to a new monolayer of OMK cells whenever the feeder layer was destroyed. Long-term cultures of normal lymphocytes have been achieved by utilizing this same technique. The cells have been maintained in this manner for 19 months. Evaluation of supernatant enhancing activity Owl monkey supernatants were assayed for their ability nonspecifically to increase the anti-sheep erythrocyte (SRBC) plaque-forming cell (PFC) response of normal mouse spleen cell cultures by methods used in previous reports (Rubin, MacDonald & Coons, 1973, 1974). Briefly, spleen cell cultures from normal 2-4-month-old DBA/2 female mice were prepared as before by a modification of the method of Mishell & Dutton, except that cells were maintained at a density of 1-5-20 x 107 cells/ ml (0 5 ml volumes) in Costar-24 tissue culture plates (Costar, Cambridge, Mass.). In some experiments, cells were maintained at the same density in 1 ml volumes in Falcon 35 x 10 mm dishes, as described previously. Cultures were stimulated with 3 million SRBC on day 0. Filtered test supernatants, diluted in fresh medium, were added to such cultures on day 2. Direct '19 S' plaques against SRBC in individual cultures were enumerated by the Jerne plaque technique on day 5. The degree of enhancement is expressed as the percentage difference between the numbers of plaques produced in cultures that received diluted test supernatants compared to the PFC response of similar cultures that received diluted, control supernatants from cultures of uninfected monkey lymphoid cells.
Preparation of antisera Anti-human T cell antiserum was kindly provided by Dr Michael Fanger, Case Western Reserve Medical School, Cleveland, Ohio. The preparation of this antiserum has been previously reported (Owen & Fanger, 1974). An anti-owl monkey immunoglobulin was produced in goats from owl monkey immunoglobulin. Lipid was removed from owl monkey serum by centrifugation at 40 and filtered through glass wool. Immunoglobulins
Purification of owl monkey enhancing Jactor were precipitated from a solution of 18% sodium sulphate followed by precipitation from a 14 % sodium sulphate solution. The precipitate fraction was subjected to ion exchange chromatography over DEAE cellulose. The front fraction was concentrated and 3 mg were emulsified in Freund's complete adjuvant and injected intramuscularly into a goat. Three weeks later a similar injection was made and 1 week later the serum was harvested.
Conjugation of antisera by fluorescein isothiocyanate Immunoglobulins were precipitated from an 18% solution of sodium sulphate and dialysed against 0-06 M NaHCO3 buffer, pH 9 5. Fluorescein isothiocyanate was added in a ratio of 0 025 mg FITC per mg protein at 250. The reaction was allowed to proceed for 5 min. Unbound FITC was removed by gel filtration over Sephadex G-25. The eluate containing the conjugated protein was precipitated from 14% sodium sulphate. The precipitate was washed twice with 14% sodium sulphate, then redissolved and dialysed against borate buffered saline (BSB) (0-01 M borate, pH 8-0, 0-154 M sodium chloride). Immunoglobulins conjugated in this manner lose little or no antibody titre compared to unconjugated antisera. Ammonium sulphate fractionation Supernatants from cultured cells of an owl monkey (240-72) with leukaemia were pooled. These supernatants represented passage numbers 13-28. Ammonium sulphate was added to 428 ml of supernatant at 40 to a final concentration of 70%. The mixture was centrifuged at 34,000 g at 4°. The precipitate was dissolved in 23 ml of BSB and dialysed against phosphate-buffered saline (PBS) 0-15 M, pH 7-2 prior to testing for OMEF activity. DEAE chromatography The 70% ammonium sulphate precipitate fraction was dialysed against 0-02 M phosphate buffer at pH 6 9. After dialysis, the fraction was subjected to chromatography over a 40 ml diethylaminoethylcellulose (DEAE) column and eluted with 0-02 M phosphate buffer, pH 6-9. When the absorbance at 280 nm returned to baseline levels ( < 0 05), a linear gradient from 0-02 M to 0-3 M phosphate buffer, pH 6 9-8 0 (200 ml each) was initiated and aliquots collected. Fractions were dialysed vs PBS prior to addition to assay cultures.
139
Gel filtration over Sephadex G-150 A Sephadex G-150 column (1-5 x 87 cm) was prepared in BSB and eluted in an ascending direction. The column was standardized with four molecular weight markers: (a) rabbit F (ab)2 conjugated with fluorescein isothiocyanate (FITC) (105,000 Daltons); (b) 125I-labelled bovine serum albumin (62,000 Daltons); (c) Ovalbumin (40,000 Daltons); and (d) cytochrome c (12,500 Daltons). The fractions from the DEAE column that contained most of the enhancing activity were pooled, concentrated 12-fold and dialysed against BSB. The concentrated fraction was then subjected to molecular filtration over Sephadex G-150 at 4'. Column fractions, dialysed against PBS, were tested for enhancing factor activity as described above.
Acrylamide gel electrophoretic purification of OMEF The OMEF-rich G-150 column fraction was subjected to disc electrophoresis on a Pharmacia GE-4 electrophoresis system (Pharmacia Fine Chemicals, Piscataway, N.J.). The gels used contained 7-5% acrylamide in Tris-glycine buffer, pH 8-3, at 25°. All gels were pre-run prior to sample application. The 15-20ul samples were then subjected to electrophoresis at 2 4 mA per gel. Each gel was cut into four sections. Equivalent sections from five gels were pooled, homogenized, suspended in 5 ml of PBS (0-15 M, pH 7-2) and centrifuged (35,000g, 10 min, 40) to remove acrylamide. Each resulting supernatant was dialysed for 48 h against 1000 volumes of PBS at 4° before testing for OMEF by the usual method. A gel run in parallel was fixed and stained with 0-25% Coomassie Blue R in 10% acetic acid, then destained with Dowex 1-X8 anion exchange resin beads (J. T. Baker, Phillipsburg, N.J.). The gel absorbance at 570 nm was read on a Gilford 640 recording spectrophotometer coupled with a linear transport system (Model 2410-S). Enzyme treatment of OMEF Active column fractions of partially-purified OMEF were tested for their sensitivity to enzymatic degradation with deoxyribonuclease I (DNase, Worthington Biochemical Corp., Freehold, N.J.), ribonuclease A (RNase, type IIIA, Sigma Chemical Co, St. Louis, Mo.) or protease (type VI, Sigma Chemical Co.). Column fractions were divided into three aliquots. DNase, RNase or protease were added to two of the aliquots at a final concentration of 10 ,pg/ml. One aliquot was not treated. Active
A. B. MacDonald, C. E. 0. Fraser & A. S. Rubin
140
Table 1. Percent of peripheral cells from owl monkeys staining with specific antiserum
Cell type
Peripheral lymphocytes
Passage number
Anti-human-T-cell (%)
Anti-owl monkey Ig
Disease
14 3
Leukaemia (H. saimiri) Normal
63 25
0 0
fraction-enzyme mixtures were stirred continuously (1 h, 370) to ensure adequate contact between enzyme and available substrate. Treated and untreated aliquots were then tested for OMEF activity by the method outlined above except that baseline control cultures received SRBC and diluent (complete medium) on days 0 and 2, respectively. OMEF activity on T cell depleted B cell enriched cultures DBA/2 splenic B cells were separated from T cells by the nylon wool column method of Handwerger & Schwartz (1974). B cell-enriched (T cell-depleted) populations contained 90-950% immunoglobulinpositive cells as determined by standard immunofluorescent techniques using an FITC-conjugated rabbit anti-mouse immunoglobulin serum to stain column eluate B cells. B cells were maintained at a density of 1 x 107 cells/ml (in 0 5 ml volumes) in Costar-24 plates in complete medium supplemented with 5 x 10-5 M 2-mercaptoethanol to functionally replace the adherent population of macrophages removed by the nylon wool separation procedure. Cultures were stimulated with SRBC on day 0, and with diluted, partially-purified OMEF on day 2. Control B cell cultures received only SRBC and diluent. IgM PFC vs SRBC were enumerated on day 5. Mitogenic activity of OMEF Whole human blood was drawn into heparin (preservative free, Connaught Laboratories, Toronto, Canada) at 100 U/ml. The blood was then added to 6% dextran (M.W. 2 x 106) in a ratio of 6: 1 blood to dextran and incubated for 1 h at 37°. The leucocytes were removed and separated from the plasma by centrifugation at 200 g at room temperature for 5 min. The cells were washed twice in Plus 1 Hepes Buffer (IBL, Rockville, Maryland), and supplemented with 100 fg/ml of streptomycin. Each well of a microtitre plate (Microbiological
(%)
Associates, Walkersville, Maryland) received 01 ml of the cell suspension and 01 ml of phytohaemagglutinin (PHA-P) (Burroughs Wellcome, Greenville, North Carolina) at a concentration of 25,ug/ml or 0-1 ml of the OMEF active fraction from the Sephadex, G-150 column which was previously dialysed against PBS. The OMEF was added in dilutions of 1 :100, 1: 200 and 1: 500. The cultures were incubated 3 or 5 days at 37° in an atmosphere of 5 % CO2 (in air). Four microcuries of [3H] thymidine (6 7Ci/mmol specific activity) (New England Nuclear, Boston, Massachusetts) were added per culture well for the final 8 h of the incubation period. The cells were harvested on filter paper through a Mash II cell harvester (Microbiological Associates, Walkersville, Maryland). The filters were dried in air at room temperature and each spot containing the cells was placed in liquid scintillation fluid and the radioactivity determined in a Beckman Scintillation Counter Model LS-200. Triplicate test samples were counted; controls were run in quadruplicate. RESULTS Source and activity of owl monkey enhancing factor
(OMEF) Peripheral blood lymphocytes (PBL) from owl monkeys can be cultured in an in vitro culture system utilizing Gel-Foam and an owl monkey kidney cell feeder layer. However, the cells cultured from monkeys inoculated with H. saimiri and suffering leukaemia and lymphoma are different from cells cultured from normal owl monkeys. The leukaemic cells appear to be predominantly of T lymphocyte lineage as indicated by immunofluorescence labelling with a specific anti-human T cell antiserum. These results are given in Table 1. The preponderance (63 %) of cells from leukaemic monkeys, maintained in vitro, stain with antiserum directed against T cell markers (Owen & Fanger, 1974), whereas only 25 %
141
Purification of owl monkey enhancing factor Table 2. Enhancement of the anti-SRBC PFC response of mouse spleen cells with supernatants from cultured peripheral lymphocytes from owl monkeys
Supernatant no. Cell origin
Passage no.
Leukaemia (H. saimiri)
14 14 14 +15 3 +4 14 14 14 +15 3 +4
(2) (8) (11)
Normal Leukaemia (H. saimiri)
(2) (8) (11)
Normal
Supernatant % Enhancement final vs controlt PFC ± s.d.* dilution 1:100 1:100
1:200 1:200
290 ± 14 405 ±92 815 ±50 123 ±40 505 ±92 345 ±35 420 ±28 170 ±40
P value:
