Proc. Nati. Acad. Sci. USA Vol. 73, No. 10, pp. 3676-3679, October 1976

Immunology

Polyclonal activation of bone-marrow-derived lymphocytes from human peripheral blood measured by a direct plaque-forming cell assay (pokeweed mitogen/lipopolysaccharide/hemolysis/gel/inhibitor)

ANTHONY S. FAUCI AND KAREN R. PRATT Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20014

Communicated by Alexander G. Bearn, July 20,1976

system to become established as a feasible approach to the study of human B lymphocyte activation, it must be adaptable to the most easily accessible lymphoid organ, peripheral blood. The present study describes the precise conditions necessary for the successful and reproducible culture and PFC assay of lymphocytes from human peripheral blood stimulated with polyclonal B cell activators. MATERIALS AND* METHODS Cell Suspensions. Heparinized venous blood (60-100 ml) was drawn from 15 normal adults, and purified mononuclear cell suspensions (lymphocytes and monocytes) were obtained by standard Hypaque-Ficoll density centrifugation (13). Culture Conditions. Cells were suspended for culture in RPMI-1640 medium ssupplemented with 1% trypticase soy broth, 0.02 M L-glutamine, penicillin, 100 units/ml, streptomycin sulfate, 100,gg/ml, and 10% pooled human AB serum. The human AB serum was absorbed in-the cold multiple times before use in order to remove the antibody against sheep erythrocytes present in variable concentrations in most lots of human serum. Varying densities of cells ranging from 1.5 to 10 X 106 lymphocytes in 2 ml of medium were cultured either in multi-well, flat-bottom plastic plates (1.5 cm diameter wells) or in 17 X 100 mm round-bottom, standup plastic tubes. Cultures were stimulated with varying concentrations of either pokeweed mitogen or Eschertchia coli 0128:B12 lipopolysaccharide. Also, separate cultures were stimulated with various concentrations of sheep erythrocytes (0.1 to 5 X 106 per culture). Cultures were incubated for 1-10 days on a rocker platform (7 cycles per min) at 370 in 5% CO2 in air at 100% humidity. At the end of the culture period, cells were harvested from the plates by gentle scraping with a rubber policeman, transferred to plastic tubes, washed twice in cold RPMI-1640, and brought to the appropriate concentration for the plaque assay. Cultures done in the standup tubes were washed in the same culture tubes. Assay for PFC. Single cell antibody production directed against sheep erythrocytes were measured by a direct PFC assay using a previously described modification (12) of the original Jerne-Nordin hemolysis-in-gel method (14). Briefly, 0.7 ml of 0.5% Noble agar (heated to boiling) in Hank's balanced salt solution containing 0.05% DEAE-dextran (to overcome the anticomplementary effect of agar) was added to 12 X 75 mm plastic tubes and kept at 46°. Thereafter, 0.05 ml of indicator sheep erythrocytes diluted 1:5 in Hank's solution (20% suspension), 0.2 ml of the lymphocyte suspension in the appropriate concentration, and 0.05 ml of guinea pig serum (previously absorbed with sheep erythrocytes) diluted 1:4 in Veronal-buffered saline was added to each tube. The mixture was then plated by placing three separate 0.2-ml drops on a plastic

A culture and assay system for the stimulation ABSTRACT of human peripheral blood lymphocytes with polyclonal activators of bone-marrow-derived lymphocytes (B cells), such as pokeweed mitogen and Escherichia coil lipopolysaccharide, and subsequent measurement of single cell antiboy production by a hemolysis-in-gel direct plaque-forming cell assay against sheep erythrocytes has been established. The critical culture requirements have been delineated and a new highly sensitive ultrathin gel assay method has been described. Under these conditions a substantial and highly reproducible plaque-forming cell response was detected in normal human peripheral blood. This system can be readily used to explore the complex events associated with activation of human B cells.

Knowledge of the complex mechanisms and events associated with activation of bone-marrow-derived (B) lymphocytes is crucial for our understanding of the various disorders of overor underproduction of antibody in man. In the mouse model, sophisticated probing of the precise mechanisms of B cell activation has been made possible by the availability of simple and reproducible plaque-forming cell (PFC) assays for the measurement of single cell antibody production after primary antigenic stimulation in vitro (1) as well as stimulation by polyclonal B cell activators (2, 3). This type of methodology has generally been lacking in human studies in which immunoglobulin production by lymphocytes in culture is usually measured by radioimmunoassay of culture supernates (4) or direct visualization of intracytoplasmic immunoglobulin by fluorescent anti-immunoglobulin techniques (5). Although a few studies have appeared that have reported successful activation of human B lymphocytes in vitro particularly from tonsil, by soluble or erythrocyte antigens and measurement of single cell antibody production in a PFC assay (6-10), it has generally been difficult, and in most cases impossible, to consistently reproduce these findings. Moreover, it has recently been pointed out how artifactual plaques may have accounted for much of the reported responses of human lymphocyte cultures to primary stimulation in vitro with sheep erythrocytes (11). Until recently there have been no reproducible studies in which single cell antibody production by human lymphocytes measured by a PFC assay after activation of cultures with polyclonal B cell activators has been demonstrated. However, we have recently developed a system for culture and PFC assay of human tonsillar lymphocytes stimulated either with sheep erythrocytes or polyclonal activators (12). Although this system has made available a simple methodology for study of human B cell activation, it was consistently successful only with tonsillar and not with peripheral blood lymphocytes. In order for such a simple and convenient culture and assay Abbreviations: B cell, bone-marrow-derived lymphocyte; PFC, plaque-forming cell; Ig, immunoglobulin.

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Fauci and Pratt

Proc. Natl. Acad. Sci. USA 73 (1976)

Table 1. Effect of cell density and concentration of supplemental serum on the PFC response of peripheral blood lymphocytes to stimulation by pokeweed mitogena Exp. 1

Exp. 2

(PFC per 106 lymphocytes)

3677

250 0

° 200

S

150

G

Cell density per mlb 1 X 106 2.5 x 106 5 X 106 10 X 106 Serum concentration (%)c 1 5 10 20

C100

69 78 138 8

30 160 240 2

C.)

UA. a0

11

50 0

8 120 138 65

-

a Experiments

done in round-bottom tubes. Cell density experiments were done at a serum concentration of 10%. c Pooled human AB serum absorbed three times with sheep erythrocytes was used in every experiment. Serum concentration experiment was done at a cell density of 5 x 106 per ml.

1

2

3 4 5 6 7 DURATION OF CULTURE (days)

8

9

10

FIG. 1. Typical time curve of PFC response of lymphocyte culture, stimulated with pokeweed mitogen, from a single individual. This particular culture peaked at seven days. Depending on the particular individual, peak PFC responses were seen anywhere from day 5 to day 8. (0) Unstimulated and (v) stimulated cultures.

b

petri dish (100 mm diameter). A 22 X 32 mm glass coverslip was immediately placed on each drop, which then spread into an ultrathin layer beneath the coverslip. Plates were incubated for 3 hr at 370, and plaques were counted on a dissecting microscope.

Inhibition of PFC. In some experiments cells were cultured for 5 days and then killed by heating at 56° for 30 min prior to assay. In other experiments complement was not added to the assay system. In still another group of experiments, 24 hr before harvesting, 10 Aig/ml of puromycin or 10 ,g/ml of cycloheximide was added to the cultures. RESULTS Culture conditions Experiments illustrating optimal cell density as well as optimal serum concentration are shown in Table 1. Optimal cell density proved to be 5 X 106 cells per 2 ml both for the flat-bottom wells and the standup tubes. However, numbers of PFC per 106 lymphocytes were usually 4-fold or more greater in roundbottom standup tubes than in flat-bottom plates. In five experiments in which PFC responses to stimulation by pokeweed mitogen were simultaneously compared in flat-bottom plates and round-bottom tubes, the mean (+SEM) response was 28(+9) PFC per 106 lymphocytes in plates and 150(+32) PFC per 106 lymphocytes in tubes. This is contrary to cultures of tonsillar lymphocytes in which there is little if any difference in numbers of PFC between plates and tubes. Serum was absolutely necessary, since cells did not survive in serum-free medium. Optimal serum concentration was 10%, with concentrations of 20% or greater resulting in suppression of PFC (Table 1). As emphasized previously (12), it was absolutely essential to absorb the supplemental human AB serum used in the cultures multiple times with sheep erythrocytes. This removes antibody against sheep erythrocytes (present in most lots of human serum), which blocks the anti-sheep erythrocyte response of lymphocytes polyclonally stimulated by B cell activators such as pokeweed mitogen and E. coli lipopolysaccharide (Table 2). In addition, it has also been shown that this antibody against sheep erythrocytes present in serum is responsible for the appearance of artifactual plaques in lym-

phocyte cultures antigenically stimulated with sheep erythrocytes (11, 12). The data shown in Table 2 represent cultures stimulated with polyclonal activators pokeweed mitogen or lipopolysaccharide and not sheep erythrocytes. Hence, artifactual plaques do not appear in these cultures containing nonabsorbed serum. Since there was a great deal of variability from person to person in the optimal stimulatory dose of pokeweed mitogen, it was necessary to use a wide dose range in each experiment from 1:20 through 1:10,000 final dilution. In cultures stimulated with lipopolysaccharide, the concentrations used were 0.5-500 ug/1ml of culture. In most experiments, peak PFC responses were seen at pokeweed mitogen concentrations of 1:20 to 1:1000 and lipopolysaccharide concentrations of 5 and 50 gg/ml. Stimulation of cultures with phytohemagglutinin did not result in a significant number of PFC above background. Very few, if any, PFC were detected through day 3 of culture. Substantial numbers were detected by day 4, with a sharp peak by day 5. Maximal numbers of PFC were seen from days 5 to 8 (varying from individual to individual) with a marked decline by day 10. A typical time curve for the pokeweed mitogen response is shown in Fig. 1. Assay for PFC As previously described, the technique of an ultrathin gel layer beneath a glass coverslip was very well suited for the human PFC assay. Plaques were sharp and clearly contrasted in the gel and a mononuclear cell was identifiable in the center of each Table 2. Effect of absorption of supplemental serum with sheep erythrocytes on the PFC response of lymphocytes from human peripheral blood

Nonabsorbeda serum

Stimulus None (background) Pokeweed mitogen Lipopolysaccharide a Human

Absorbed serum

(PFC per 106 lymphocytes) 0 8 1

0 182 63

AB serum (10%) was used to supplement cultures in all experiments. Nonabsorbed serum was used after heat inactivation (560 for 45 min). Absorbed serum was heat-inactivated and then absorbed with sheep erythrocytes three times at 4°.

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Immunology: Fauci and Pratt 250 r

Proc. Nati. Acad. Sci. USA 73 (1976) Table 4. Inhibition of PFC response of peripheral blood lymphocytes to stimulation by pokeweed mitogen

I

C,) w

200

Exp. 1

Exp. 2

0

x I

150

Conditions s' u

100

Normal Complement withheld from assay Lymphocytes heat-killed

lo

w

C-

2

50

oL

UNSTIMULATED

(560, 30nmin)a

LPS STIMULATED

PWM

plaque. They appeared by 30 min and reached maximal numbers and clarity by 2-3 hr. The mean peak PFC responses of 10 subjects whose lymphocyte cultures were stimulated with either pokeweed mitogen or lipopolysaccharide in standup tubes is shown in Fig. 2. Background PFC in unstimulated cultures was almost always 0. It is clear that pokeweed mitogen is much more potent a stimulus of PFC against sheep erythrocytes than is the lipopolysaccharide used in this study. There was a wide range of responses from individual to individual, but generally the same subjects were consistent in the magnitude of their responses measured on different days (Table 3). In addition, simultaneous assays on the same blood sample were generally reproducible within 10%. There were very few, if any, PFC above background in cultures stimulated with sheep erythrocytes. Plaque formation required live cells and complement. Also, de novo synthesis of antibody was necessary for demonstration of PFC since puromycin and cycloheximide added to cultures 24 hr before harvest in concentrations (10 ,ug/ml) that did not decrease cell viability resulted in up to 90% suppression of numbers of PFC (Table 4). DISCUSSION The present study demonstrates a simple and highly reproducible system for the stimulation of lymphocytes from human peripheral blood with polyclonal B cell activators such as pokeweed mitogen and lipopolysaccharide, and subsequent measurement of single cell antibody production by a direct Table 3. Day-to-day and person-to-person variability in the PFC response of peripheral blood lymphocytes to stimulation by pokeweed mitogen Experimenta

2

1

A B C D E

3

(PFC per 106 lymphocytes)

249 18 173 73 258

(1:200)b (1:1000) (1:100) (1:2000) (1:100)

140 78 135 60 125

(1:200) (1:2000) (1:100) (1:200) (1:100)

120 (1:20) 34 (1:200) 140 (1:100) 96

(1:200)

65 (1:200)

Three separate experiments were done at least a week apart on five different subjects (A through E). b Numbers in parentheses are the optional stimulatory dilution of pokeweed mitogen in each experiment. a

173

90

0

0

1

0

9

15

16

8

Puromycin (10 yg/ml)

FIG. 2. PFC responses of peripheral blood lymphocyte cultures after stimulation with either pokeweed mitogen (PWM) or lipopolysaccharide (LPS). Data represent the mean (±SEM) responses of 10 normal subjects. Cultures were harvested on day 5 to 6.

Subject

(PFC per 106 lymphocytes)

addedb

Cycloheximide (10 Mg/ml)

addedb

Lymphocytes were cultured as usual for 6 days and then heatkilled and washed once immediately before the assay for PFC. b Both puromycin and cycloheximide were added to cultures 24 hr prior to harvest and assay. This concentration of inhibitor did not significantly affect cell viability. a

hemolysis-in-gel PFC assay against sheep erythrocyte targets. The convenience, simplicity, and importance of a PFC assay to study the mechanisms of B cell activation is well established (15). Its use in mouse spleen cell cultures to study antigenic stimulation (1) as well as polyclonal B cell activation (2, 3) has been invaluable in our understanding of the complex mechanisms, cellular requirements, and interactions associated with B cell activation in this system. Unfortunately, attempts to establish such a model in human lymphocyte cultures, particularly of peripheral blood, have generally met with failure. The success of the present study depended on rather simple but nonetheless critical factors, including the culture requirements of optimal cell density, medium volume, tube size and shape, concentration of stimulus, and duration of culture. Of utmost importance was the serum supplement. It became obvious during the study that the most critical factor was the multiple absorptions of the human AB serum with sheep erythrocytes prior to culture. This removes a suppressor factor which is probably the antibody against sheep erythrocytes present in most human sera. This factor blocks the anti-sheep erythrocyte response of lymphocytes stimulated with polyclonal B cell activators. It is also possible that the absorption procedure releases into the serum some adjuvant-like material from the sheep erythrocytes used in the absorption which then enhances the anti-sheep erythrocyte response of polyclonally stimulated cultures without increasing background PFC. This latter possibility has not as yet been ruled out. In addition, most studies have been done attempting to stimulate peripheral blood lymphocytes directly with sheep erythrocytes and measure PFC against sheep erythrocyte targets. These studies have consistently met with failure, and indeed in the present study direct sheep erythrocyte stimulation of cultures resulted in very few, if any, PFC against sheep erythrocytes. The use of B cell mitogens or, more appropriately termed, B cell activators (3) has been extremely informative in our understanding of the complexities of B cell stimulation (2, 3, 16). The measurement of polyclonal B cell activation by a PFC assay against sheep erythrocytes is based on the understanding that polyclonal B cell activators stimulate lymphocytes to secrete antibodies with broad ranges of specificities, including those that react with antigenic determinants on the sheep erythrocytes. It is clear from this study that this system is also applicable to lymphocytes from human peripheral blood.

Immunology: Fauci and Pratt The present model can be used to explore the numerous complex factors required for human B cell activation in the normal immune response, and hopefully some of the defects in humoral immunodeficiency states as well as the defective regulatory mechanisms in diseases characterized by aberrant production of antibody. We thank Ms. Gail Whalen for expert technical assistance. 1. Mishell, R. I. & Dutton, R. W. (1967) J. Exp. Med. 126, 423442. 2. Gronowicz, E. & Coutinho, A. (1975) Scand. J. Immunol. 4, 429-437. 3. Coutinho, A. & M6ller, G. (1975) Adv. Immunol. 21, 113-236. 4. Waldmann, T. A., Durm, M., Broder, S., Blackman, M., Blaese, R. M. & Strober, W. (1974) Lancet ii, 609-613. 5. Cooper, M. D., Lawton, A. R. & Bochman, D. E. (1971) Lancet ii, 791-795. 6. Mann, P. L. & Falk, R. E. (1973) in Proceedings of the Seventh

Proc. Nati. Acad. Scd. USA 73 (1976)

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Leukocyte Culture Conference, ed. Daguillard, F. (Academic Press, New York), pp. 39-48. 7. Geha, R. S., Schneeberger, E., Rosen, F. S. & Merler, E. (1973) J. Exp. Med. 138,1230-1247. 8. Hoffman, M. K., Schmidt, D. & Oettgen, H. F. (1973) Nature 243,408-410. 9. Watanabe, T., Yoshizaki, K., Yaguro, T. & Yamamura, Y. (1974) J. Immunol. 113,608-616. 10. Dosch, H. & Gelfand, E. (1976) J. Immunol. Methods, in press. 11. Muchmore, A. V., Koshi, I., Dooley, N. & Blaese, R. M. (1976) J. Immunol. 116, 1016-1019. 12. Fauci, A. S. & Pratt, K. R. (1976) J. Exp. Med. 144,674-684. 13. B6yum, A. (1968) Scand. J. Clin. Lab. Invest. (Suppl.) 21, 7789. 14. Jerne, N. K. & Nordin, A. A. (1963) Science 140, 405. 15. Jerne, N. K., Henry, C., Nordin, A. A., Fuji, H., Koros, A. M. C. & Lefkovits, I. (1974) Transplant. Rev. 18, 130-191. 16. Janossy, G. & Greaves, M. (1975) Transplant. Rev. 24, 177236.

Polyclonal activation of bone-marrow-derived lymphocytes from human peripheral blood measured by a direct plaque-forming cell assay.

Proc. Nati. Acad. Sci. USA Vol. 73, No. 10, pp. 3676-3679, October 1976 Immunology Polyclonal activation of bone-marrow-derived lymphocytes from hum...
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