Soluble suppressor factor produced by pokeweed mitogen-stimulated human peripheral blood leukocytes

CELLULAR

IMMUNOLOGY

55,

20-31 (1980)

Soluble Suppressor Factor Produced by Pokeweed Mitogen-Stimulated Human Peripheral Blood Leukocytes’ FLORENCEHOFMAN,~ CELSA A. SPINA,NEELAM Department

of Microbiology and Immunology, Los Angeles, California

Received

September

RAND,ANDJOHN

L. FAHEY

UCLA School of Medicine, 90024

12, 1979; Accepted January

15, 1980

Human peripheral blood leukocytes were exposed to either PWM or Con A mitogens. Cells activated by both these mitogens were able to depress proliferation in an MLC, and to inhibit the generation of spontaneous killer cell (SK) and induced T-cell cytotoxic activity. PWM-activated cells incubated in media for 48 hr were able to elaborate a soluble factor in vitro. This factor suppressed cytotoxicity, and was active only when present at the initiation of MLC cultures. In contrast, cells exposed to Con A were able to suppress immune responsiveness, but this population did not release a soluble factor which could inhibit cytotoxicity. PWM induction appears to be dependent on phagocytic cells, while Con A activation is less dependent on this adherent population. An enriched adherent cell population, stimulated with PWM, was able to suppress cytotoxicity. Thus, the PWMstimulated system of suppression is mediated through a soluble factor and is dependent on adherent cells.

INTRODUCTION Suppression of immune responsiveness has been shown to be mediated by adherent cells (1, 2), activated lymphocytes (3, 4), and soluble factors derived from these cells (5, 6). Lymphocytes stimulated by Con A3 have demonstrated inhibition of several immune parameters. Con A-activated cells are of T-cell origin and do not require the presence of adherent cells (7). They depress MLC reactivity, mitogen activation, cell-mediated cytolysis, and antibody production. In the murine system, Con A-activated cells or allogeneically stimulated cells suppress directly (3) or via a soluble factor (6). The factors derived from these stimulated T cells were reported to act directly on T lymphocytes or on a nonlymphocyte subpopulation. In humans there is some evidence of Con A suppressor factors from Con A-stimulated cord blood lymphocytes, but supemates from mitogeninduced normal peripheral blood leukocytes demonstrate activity in less than i Supported by NC1 Grant CA 09120 (F.H.), NIAID Grant AI 07126 (C.A.S.), and NC1 Grant CA 12800. 2 Present address: Department of Pathology, USC School of Medicine, Los Angeles, Califomia90033. 3 Abbreviations used: PWM, pokeweed mitogen; Con A, concanavalin A; MLC, mixed leukocyte culture; PBL, peripheral blood leukocytes; PBS, phosphate-buffered saline; B-LCL, B-lymphoblastoid cell lines; EBV, Epstein-Barr virus; SK, spontaneous killer. 20 0008-8749/80/130020-12$02.00/O Copyright 8 1980by AcademicPress,Inc. A11 rights of reproductionin any form reserved.

SOLUBLE

FACTOR

DERIVED

FROM PWM-STIMULATED

CELLS

21

40% of the individuals tested (8). Thus, the possibility of a soluble suppressor factor from induced human leukocytes is still under investigation (9). In the work presented here we have compared cells stimulated with PWM to those activated by Con A. Cells stimulated with both mitogens were able to suppress proliferation and cytotoxicity. However, we have demonstrated that the mechanism of suppression by PWM-stimulated cells is through a soluble factor. Furthermore, the adherent cell is probably the critical cell involved in PWMinduced suppression. MATERIALS

AND METHODS

Isolation of peripheral blood leukocytes (PBL). Leukocytes were isolated from peripheral blood of normal healthy donors. The blood was diluted 1:l with phosphate-buffered saline (PBS) and then layered on Ficoll-Hypaque according to the technique of Boyum (10). Cells at the interface were recovered and washed two to three times with PBS (pH 7.2). The cells were examined for viability by trypan blue dye exclusion and resuspended to the proper concentration. Preparation of mitogen-stimulated cells. Peripheral blood leukocytes were washed and then resuspended to 4 x lo6 cells/ml of medium in plastic culture tubes (16 x 25 mm). Con A (Sigma Chemical Co., St. Louis, MO.) was added to cultures at 10 pg/ml. The PWM (Grand Island Biological Co., Grand Island, N.Y.) was added at 1000 pg/ml. The medium consisted of 10% pooled human sera (heat inactivated at 56°C for 30 min) in RPM1 (Gibco) supplemented with 25 rnJ4 Hepes buffer. The cells were incubated at 37°C for 18-20 hr in 5% COz in air. After the incubation period, the cells were washed three times in PBS and resuspended in the proper concentration to be used for culture. Preparation of mitogen-activated cell supernates. The peripheral blood leukocytes were prepared as described above. Following the last of the three washes, 1.0 ml of complete medium with serum was added to the culture tubes. The cells were incubated for 72 hr. The supernatant fluid was then collected, sterile filtered (0.45 pm) (Millex filter) and stored at -20°C until used. Preparation of mixed leukocyte cell cultures. Responding lymphocytes at 2 x lo6 per culture were incubated with the stimulating cells from human Blymphoblastoid cell lines (B-LCL) using a modification of a described technique (11). Briefly, the B-LCL were treated with 50 Fg/rnl of mitomycin C for 30 min at 37”C, washed, and resuspended to the proper concentration in medium supplemented with 10% human serum. The ratio of responder to stimulator cells was 15:l at a total volume of 2.0 ml per tube. The B-LCL lines used in this study were LA-237, LA-328, both EBV transformed B-cell lines derived from a normal individual, and IM-9, a B-cell line derived from a patient with multiple myeloma. Cell cultures were incubated for 5 days at 37°C in 5% CO, in air. Adherent cell isolation and stimulation. Peripheral blood leukocytes at lo25 x lo6 cells/3 ml of medium (RPM1 1640) and serum were placed in a culture dish (Lux, 60 x 15 mm). The plates were centrifuged at 600 rpm for 5 min and then allowed to incubate at 37°C for 30 min. The plates were shaken gently approximately 20 times. The supernates were removed and the plates were washed twice with media. Lidocaine (2.5 ml) at a final concentration of 12 mM was added to each plate. The dishes were incubated at 25°C for lo-15 min. The

22

HOFMAN

ET AL.

plates were then washed vigorously to remove most of the adherent cells. This population was washed, aliquoted into tubes at the proper concentration, and exposed to the particular mitogen. Using criteria of esterase staining (22) and morphology, we consistently observed 80% of the cells to be macrophages. Phagocyte depletion of PBL. PBL at 50 x lo6 in a total volume of 10 ml of 20% pooled human sera in RPM1 1640 were exposed to autoclaved carbonyl iron powder (GAF Corporation, New York, N.Y.) (0.5 mg/ml). The cells and iron were gently rotated and incubated for 30 min at 37°C. The carbonyl iron was then removed by a magnet and transferred to a new culture tube. This was done three times. The PBL were then washed twice in PBS and resuspended to the proper concentration. The depleted PBL were examined for monocyte/ macrophage contamination using differential morphology with crystal violet and esterase staining. The residual monocyte/macrophage population remaining ranged between 0 and 4%, the rest of the cells being lymphocytes. Proliferative response in MLC. After 5 days in culture, the cells were counted and aliquots of 2 x lo4 cells/O.1 ml were deposited into each well of a roundbottom tissue culture multiwell plate. Two microcuries of E3H]thymidine (specific activity = 6.7 mCi/mole) (AmershamSearle Corp., Arlington Heights, Ill.) were added to each well and incubated at 37°C for 4 hr. Following the incubation, the cells were collected on a MASH cell harvester (Microbiological Associates, Walkersville, Md.) and the number of counts determined using scintillating spectometry. Cytotoxicity assay. The assay used to measure cytotoxicity was a modification of a standard 3-hr 51Cr release assay (12). The target cells, either normal Bcell lines derived from normal subjects, or K-562 and Molt were incubated with 200 &i of 51Cr (New England Nuclear, Boston, Mass.) for 1.5 hr. The labeled cells were then washed and resuspended to lo5 cells/ml. The effector and target cells were combined at a ratio of 2O:l (unless otherwise specified) in a total volumn of 0.2 ml in round-bottom microtitre plates. After a 3-hr incubation at 37°C the plates were centrifuged at 1200 rpm for 6 min; 0.1 ml of media was removed from each well and counted by gamma emission spectometry.‘Maximum release was established by adding 0.1 ml of 1% Triton detergent to the target wells. Spontaneous release was determined by adding 0.1 ml of media to the target well. All samples were done in triplicate. The percentage cytotoxicity was calculated as follows: cpm with experiment culture - cpm of spontaneous target cell release x loo. maximum release cpm - cpm of spontaneous target cell release

RESULTS Suppression of Spontaneous and Induced Cytotoxicity Mediated by PWM- and Con A-Stimulated Isogeneic Cells The effect of PWM- and Con A-stimulated cells on the proliferation of MLC and the induction of cytotoxicity was examined. PBL were exposed to the mitogen for 20 hr, washed, counted, and added to cultures of isogeneic PBL plus mitomycin-treated stimulator cells. Table 1 shows the results when MLC cultures

SOLUBLE FACTOR DERIVED FROM PWM-STIMULATED

23

CELLS

TABLE I Inhibition of MLC and Cytotoxicity Mediated by PWM-Stimulated Isogeneic Cells” Percentage cytotoxicity Stimulated PBL A. (1) (2) (3) (4) (5) (6) (7) (8) (9) (10)

+Media +PWM cells (1 x 106) +PWM cells (0.5 x 106) +PWM cells (0.25 x 106) +PWM cells (0.05 x 106) +Con A cells (1 x 106) +Con A cells (0.5 x 106) +Con A cells (0 25 x 10”) +Con A cells (0105 x IO’) +PBL (cultured) (1 x 106)

B. (1) PBL (2) PBL (3) PBL (4) PBL (5) PBL (6) PBL (7) PBL (8) PBL

+ + + + + + +

IM-9, + media IM-9, + PWM cells IM-9, + Con A cells IM-9, + PBL(cultured) PWM cells Con A cells PBL (cultured)

cpm [3H]Thymidine

K-562

Percentage suppression

IM-9

28,340 2,477 6,965 12,050 17,929 4,619 8,991 15,798 22,845 27,473

60.8 + 3.9 + 13.7 + 25.1 k 35.7 c 2.0 2 12.7 k 22.3 k 55.1 + 63.2 k

2.0 0.4 1.0 1.4 5.4 1.1 2.5 2.3 2.0 2.2

53.4 + 0.1 ” 5.4 k 18.3 + 39.3 2 15.4 + 23.4 + 36.3 + 51.2 2 55.8 +

4.6 0.8 1.7 0.8 0.7 0.7 1.9 3.6 2.7 1.4

N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D.

1.7 z!z1.1 66.6 k 2.3 8.9 2 0.9 16.3 + 1.5 63.6 2 1.5 5.7 k 0.1 0.6 +- 0.0 1.9 -c 1.2

-0.9 + 20.4 2 1.2 t 8.4 k 18.2 k 0.5 t 0.8 2 -1.5 2

1.4 4.5 1.6 2.0 1.3 2.2 0.2 0.6

0 94 77 59 41 97 79 63 10 0

B (A) PBL at 2 x 10” cells per culture were incubated with the mitomycin C-treated B-cell line IM-9 in mixed leukocyte cultures. Media, mitogen-stimulated cells, or untreated cells were added to the MLCs. Similar results were obtained in five different experiments. (B) 0.5 x lo6 mitogen-stimulated cells or cultured PBL were added to the mixed leukocyte cultures or to PBL alone. Cytotoxicity is expressed as a mean and standard deviation oftriplicate samples. Percentage suppression is expressed with respect to the K-562 target.

were exposed to isogeneic cells treated with PWM- and Con A-induced ceils. After 5 days in vitro, both PWM- and Con A-treated cells were able to depress proliferation, as measured by [3H]thymidine incorporation, and cytotoxicity as determined by 51Cr release. Different target cells were used to distinguish spontaneous killer (SK) cytolysis from T-cell-mediated cytotoxicity. The K-562 target is sensitive to T-cell cytotoxicity and both induced and noninduced SK killing (13). The IM-9 cells serve as a specific target for the T-cell-mediated cytotoxicity. In the experiments reported here, both SK and T cell cytotoxicity were depressed. Unstimulated PBL exposed to PWM or Con A cells showed no change in activity (Table lB), indicating that little if any mitogen was carried over with the treated cells. PBL exposed to mitogens alone show increase in cytotoxicity to the targets tested (14). Effect of Supernatant Factors Derivedfrom Induction of Cytotoxicity

Mitogen-Stimulated

Cells on in Vitro

Since the mitogen-stimulated cells were active in suppression, we wanted to test whether this suppression could be mediated through a soluble factor released by these activated cells. Cells were exposed to PWM or Con A for 24 hr; follow-

24

HOFMAN ET AL.

EFFECTOR : TARGET

FIG. 1. Comparison of PWM and Con A supernatant activity at various effector:target ratios. (A) Target cell is K-562; (B) target cell is IM-9. The mitogen-activated cells and supernates were added to MLC cultures at initiation. After 5 days the cells were assayed at a fourfold effector to target ratio. The effector cell count was based on the number of lymphocytes relative to target cells.

ing this incubation, the cells were washed and resuspended in fresh medium for 72 hr. The supernate collected from cultures of PWM- or Con A-stimulated cells showed differential effects on the effector plus stimulator cultures. Figure 1 demonstrates that cytotoxicity is depressed by the cells activated with either mitogen; however, only the supernatant derived from the PWM cells shows an inhibitory effect on cytotoxicity. The suppression of reactivity by the PWM-stimulated cells and supernates is observed over a fourfold effector to target ratio, and for both SK and specific T-cell cytotoxicity. PWM-stimulated factor was able to suppress cytotoxicity induced by a variety of mitomycin-treated stimulator cell lines, while the supemates from Con Aactivated cells exhibited no suppressive activity in either of the systems tested. Table 2 shows results using the B lymphoblastoid cell line LA-328. The target TABLE 2 The Effect on Cytotoxicity of Supemates Derived from Mitogen-Stimulated Cells” Molt (1) PBL (2) PBL (3) PBL (4) PBL (5) PBL (6) PBL

+ + + + + +

media LA-328, + media LA-328, + PWM supemate LA-328, + Con A supemate PWM supernate Con A supemate

1.0 k 75.6 2 1.7 k 59.6 k 23.9 k 3.6 +

LA-328 1.7 5.5 0.8 2.0 1.2 1.3

2.1 2 1.9 66.7 ” 0.7 6.7 + 1.8 58.9 5 2.2 17.1 k 1.8 1.6 -r- 1.6

a Responding lymphocytes were stimulated with the B-lymphoblastoid cell line LA-328. The target cell lines were the 5*Cr-labeled MOLT, a T-cell line, and LA-238, a B-cell line. 1.0 ml of mitogenactivated supernatant factor was added to each of the designated cultures.

SOLUBLE FACTOR DERIVED FROM PWM-STIMULATED I00

CELLS

25

t

PERCENT OF WiM

SUPERNATANT

FIG. 2. Titration of PWM supernatant activity in culture. PWM-stimulated supernatant factor was added to MLC cultures of PBL plus mitomycin C-treated stimulator IM-9 cells. The total volume per culture tube remained at 2.0 ml. Percentage suppression was calculated by taking maximum stimulation as 0% suppression. The solid line (-) represents the IM-9 target; the broken line (- - -) represents the K-562 target.

cells used were MOLT, a T-cell line of malignant origin which is known to be selectively lysed by SK cells, and LA-328, the stimulating cell line, which is lysed by cytotoxic T cells (13). The activity of the PWM supernatant was demonstrated to be dependent on its relative concentration in the cell culture medium. PWM supernatant comprising 0 to 50% of the total culture volume were examined for activity. The data shown in Fig. 2 suggest that the suppression of the generation of both types of cytotoxicity is directly dependent on the concentration of PWM-induced factor. Greater than 50% inhibition of both SK and specific T-cell-mediated cytotoxicity was detected with 0.5 ml of PWM-induced supernates/2.0 ml of culture medium. Exposing PBL to PWM for 24 hr is sufficient to activate suppressor cells in the PBL population. We then determined the amount of time required for the elaboration of the potent PWM-induced supernatant factor. Cells that have been treated with mitogen and washed were maintained in media for an additional 24, 48, or 72 hr. The supernates were then collected, filter sterilized, and tested in mixed leukocyte cultures. Table 3 demonstrates that 24-hr supernates show minimal suppressive activity. However, by 48 hr, suppression of cytotoxicity is greater than 50%. This indicates that the active soluble factor requires some time to be elaborated by the mitogen-stimulated cells. Unstimulated PBL exposed to PWM supernates from the various days exhibited no significant change in cytotoxicity. In several experiments, PBL alone were incubated with PWMinduced soluble factor. There was no increase in proliferation, indicating that PWM was not carried over in the supemates (data not shown). Mitogens like PWM, PHA, and Con A have been shown to cause enhancement of cytotoxicity (13). The viability and recovery of all cultures was greater than 90% suggesting that the PWM supernate factor was not cytotoxic to the responding population. The question was then asked whether the PWM supernates had to be present at the initiation of the cell cultures for suppression to be detected, or could

26

HOFMAN ET AL. TABLE 3 Elaboration of PWM Supernatant Factor Activity in Vitro” Percentage cytotoxicity

(1) PBL (2) PBL (3) PBL (4) PBL (5) PBL (6) PBL (7) PBL (8) PBL (9) PBL (10) PBL (11) PBL (12) PBL

+ + + + + + + + + + + +

media IM-9, + media IM-9, + PWM supernatant (24 hr) IM-9, + PWM supematant (48 hr) IM-9, + PWM supematant (72 hr) IM-9, + PWM cells IM-9, + PBL cells PWM supematant (24 hr) PWM supematant (48 hr) PWM supematant (72 hr) PWM cells PBL cells

K-562

IM-9

0.1 ” 0.6 55.2 h 1.2 42.1 k 0.2 22.5 2 0.4 16.9 2 0.0 4.3 t 0.9 61.6 f 3.8 8.5 z!z0.4 0.8 k 0.3 0.1 2 0.4 0.8 f 0.1 -0.4 2 0.1

0.1 f 0.2 17.1 2 1.3 11.8 f 0.2 7.6 k 0.7 4.9 + 0.5 0.7 2 0.5 20.6 rf: 1.3 3.2 2 0.1 0.7 f 0.1 1.1 2 0.8 0.8 2 0.2 0.9 k 0.7

a PBL were incubated with PWM overnight. The stimulated cells were then washed and exposed to media for 24, 48, or 78 hr. The supernatant was subsequently collected and added at 1.0 ml to the MLC at initiation of culture. The isogeneic PWM-stimulated cells or untreated cells (at 0.5 x lo6 cells per culture) were added to PBL or to the MLC. The PWM-induced supematant factor was derived from a donor allogeneic to the mitogen-stimulated cells and effector cells of the MLC.

suppression be mediated by addition of PWM supernates at any time throughout the 5-day culture period. PWM supernate was added daily to cultures of responders plus stimulators beginning on Day 0. The results shown in Table 4 clearly demonstrate that suppression by PWM supernatant was effective only if present at the initiation of the MLC. After 24 hr, the cultures were resistant to the suppressive activity of PWM supernates. Comparison of Allogeneic and Isogeneic PWM Supernatants In all the experiments reported thus far, the cells that were induced with PWM for supernatant collection were allogeneic to both the responder and stimulating cells of the MLC. We wanted to examine whether HLA differences between the cells which produce PWM supernate and the cells that respond in the MLC are important in the suppression of cytotoxicity. PBL from donor A was used for both preparation of PWM-activated supernatant and as the responding population. PWM-activated supernatant from donor B was prepared in parallel. When the cell cultures were exposed to the PWM-activated supematant from donor A and B, cytotoxicity was inhibited by both supernates (Table 5). To eliminate the possibility that donors A and B are HLA-identical, their lymphocytes were typed: donor A-(A-2, ll), (Bw-15.1, 22.2), (Cw-1, 3), (DrW-1, 4); donor B(A-2, 9), (Bw-15.1, 38), (Cw-1) (DrW-not detectable, therefore not 1, 2, 3, 4, 5, or 7). Mixed lymphocyte reactions between donors A and B showed that each could stimulate and respond to the other in a proliferation assay. The evidence indicates that they are not HLA identical. Thus, the capacity of PWM-induced supernatant from allogeneic cells is as effective as that from isogeneic cells in suppressing cytotoxicity.

SOLUBLE FACTOR DERIVED FROM PWM-STIMULATED

27

CELLS

TABLE 4 Addition of PWM Supematant Factor on Consecutive Days after Initiation of MLC” Percentage cytotoxicity K-562 (1) (2) (3) (4) (5) (6) (7)

PBL PBL PBL PBL PBL PBL PBL

+ + + + + + +

IM-9, IM-9, IM-9, IM-9, IM-9, IM-9, IM-9,

+ + + + + + +

media PWM supernatant PWM supernatant PWM supernatant PWM supernatant PWM supernatant PWM supernatant

(Day (Day (Day (Day (Day (Day

0) 1) 2) 3) 4) 5)

67.8 29.0 68.8 69.0 67.8 60.0 68.6

+ t t t f k t

1.2 1.9 1.2 2.3 4.5 2.1 1.6

IM-9 61.0 24.9 60.6 71.7 64.9 44.1 49.6

2 2.1 -r- 0.5 t 3.3 t 3.1 2 1.4 + 1.5 t 0.5

a PWM-stimulated supernates, collected after 72 hr post-mitogen activation, were added to the MLC on Day 0 and on successive days following initiation of culture. 1.0 ml of supematant was added to each culture, bringing the total volume of 2.0 ml per tube. On Day 5 the supernatant factor was added to the MLC 2 hr before assay time.

The Leukocyte Subpopulation Involved in the Stimulation by PWM To determine which cell population was activated by the mitogens, we first depleted the PBL of phagocytic cells using carbonyl iron. The remaining cells were then stimulated with PWM or Con A. The data shown on Table 6 indicate that phagocyte depletion has little effect on the Con A-stimulated population. In contrast, the PWM stimulation was sensitive to phagocyte depletion. Line 4 shows that the removal of the phagocytes caused a marked change in the ability of PWM-stimulated cells to suppress the generation of both SK and specific Tcell-mediated cytotoxicity. Thus the PWM versus Con A cell populations show relative differences in their dependency on the presence of phagocytes; removal of 96 to 99% of the phagocytic cells reduces PWM-stimulated cell activity, but does little to affect the Con A-activated cells. TABLE 5 Effect of Supemates from Cells Isogeneic or Allogeneic to the Responding Lymphocytes” Percentage cytotoxicity LA-237

(1) PBL, (2) PBL(*, + LA-237, (3) PBL(*, + LA-237, + PWM supernate (A) (4) PBL,,, + LA-237, + PWM supemate (B)

-0.2 c 19.6 + 1.4 t 1.7 r

0.4 0.6 0.2 0.6

K-562 0.4 67.7 24.9 25.5

2 k k ?

0.4 1.7 0.5 0.6

a PBL from normal donors A and B were used to produce PWM-activated supernatant factor. PWM-induced supemates A and B were added to the mixed leukocyte culture where the responding cell was of donor A origin. 1.0 ml of the supemate was added to the cultures. Similar results have been obtained using three different donors in comparing the activity of the supernatant factor in the isogeneic versus allogeneic systems.

28

HOFMAN ET AL. TABLE 6 The Differential Effect of Phagocyte Depletion on PWM versus Con A-Treated Cells” Percentage cytotoxicity LA-237 (1) PBL (2) PBI. (3) PBL (4) PBL (5) PBL

+ + + + +

LA-237, LA-237, LA-237, LA-237, LA-237,

+ + + + +

media PWM cells PWM (depleted) cells Con A cells Con A (depleted) cells

19.6 ? 2.7 t 11.9 ” 2.6 k 3.2 k

0.6 0.3 0.4 0.2 1.7

K-562 67.7 2 25.7 2 55.9 2 19.7 2 21.7 2

1.7 1.2 2.6 2.1 1.5

a PBL were depleted of phagocytic cells using the carbonyl iron method. These cells were then exposed to PWM or Con A. Mitogen-stimulated cells at 0.5 x lo6 lymphocytes were added to each of the mixed leukocyte cultures. Cytotoxicity was measured after 5 days. These results are representative of three experiments.

Since phagocytic cells, probably nonlymphoid, appeared to be important in the PWM-generated suppression, we examined the adherent cells alone for their ability to be stimulated by the mitogen. Cells adherent to plastic were isolated and recovered as described under the Methods section. These cells were then exposed to PWM or Con A and quantitatively added to the MLC at the time of initiation of culture. The results shown in Table 7 demonstrate that a low number of adherent cells can be stimulated by PWM to become potent suppressors of cytotoxicity. These adherent cells at a ratio of 2O:l responder to suppressor cells could cause greater than 75% suppression. As shown in Table 1, when whole PBL stimulated with PWM was measured at a ratio of 8:l responder to suppressor cells, inhibition was 60%. Thus, the adherent cells are potent suppressors of cytotoxicity, and the activity of the adherent population is not likely due to a possible 20% contamination by lymphocytes. Con A-stimulated adherent cells demonstrated little, if any, effect, while the Con A-activated PBL did suppress activity. These data strongly suggest that it is the adherent cells which are involved in the induction of suppression by PWM. DISCUSSION The work presented in this communication demonstrates that in humans, PWMactivated PBL mediate the suppression of DNA synthesis in MLR, and the generation of cytotoxic effector cells in culture. We have compared certain characteristics of PWM-stimulated suppression to Con A-activated inhibition. The most striking difference between the PWM- and Con A-induced suppressive effects appears to be the cell population activated by the mitogen. Adherent cells, greater than 80% macrophage as determined by staining and morphology, are activated by the PWM whereas all evidence indicates that lymphocytes are the population activated by Con A. At a 2O:l responder to adherent cell ratio, the PWM-activated adherent cells are potent suppressors. Adherent cells similarly treated with Con A have no effect on cytotoxicity (Table 7). Evidence from the murine system shows that Con A stimulates the T-cell population to become suppressive (3). Elimination of 8 sensitive cells completely abrogates this suppressor population. Macrophages may be involved in the stimulation by the

SOLUBLE FACTOR DERIVED FROM PWM-STIMULATED

29

CELLS

TABLE 7 Direct Activation of Adherent Cells with PWM and Con A Mitogens” Percentage cytotoxicity A. (1) MLC (2) MLC (3) MLC (4) MLC (5) MLC (6) MLC (7) MLC (8) MLC (9) MLC (10) MLC

+ + + + + + + + +

PWM cells (0.5 x 106) adherent (PWM) cells (0.4 x adherent (PWM) cells (0.2 x adherent (PWM) cells (0.1 x Con A cells (0.5 x 106) adherent (Con A) cells (0.4 x adherent (Con A) cells (0.2 x adherent (Con A) cells (0.1 x adherent cells (0.4 x 106)

B. (1) MLC (2) MLC (3) MLC (4) MLC (5) MLC (6) MLC (7) MLC (8) MLC (9) MLC

+ + + + + + + +

PWM cells (0.5 x 106) PWM cells (0.1 x lOti) PWM cells (0 05 x 10”) PWM cells (0101 x 106) adherent (PWM) cells (0.1 x 106) adherent (PWM) cells (0.05 x 106) adherent (PWM) cells (0.01 x 106) adherent PBL cells (0.1 x 10”)

106) 106) 106) 106) 106) 106)

Percentage suppression

67.5 + 5.9 + 15.9 + 16.0 + 15.9 + 20.3 + 59.0 + 53.1 * 70.7 + 61.3 +

1.2 0.1 0.8 0.4 0.7 2.6 0.2 2.2 2.2 1.2

0 91 76 76 76 70 13 21 0 9

75.5 + 36.8 t 38.8 + 45.6 2 60.4 k 40.1 t 43.6 ” 57.2 2 60.2 k

5.4 2.8 3.2 1.7 2.4 0.9 0.5 1.1 1.4

0 51 49 40 20 47 43 24 20

rr PBL were enriched for adherent cells using adhesion to plastic. These adherent cells were recovered and exposed to PWM or Con A for 24 hr. Following this incubation the cells were quantitated and added to the MLC. (A) The suppressive effect of adherent cells and peripheral blood leukocytes stimulated with either Con A or PWM was compared. (B) PWM-stimulated cells and adherent cells, exposed to PWM, were added to the MLC at various concentrations. Cytotoxicity is measured using the K-562 target. Percentage suppression is expressed based on the chromium release data.

mitogen-induced cells (15), but this has been controversial (16). Thus, both PWMand Con A-stimulated cells suppress, but the cell populations activated by these mitogens are probably distinct. The PWM-activated cells elaborate a factor into the supernatant which mediates suppression of cell-mediated cytotoxicity. This factor is produced in sufficient quantities after 48 hr of cell culture to effect inhibition of cytotoxic responsiveness (Table 3). The PWM-stimulated supernatant must be present within the first 24 hr of cell culture for any depression of the response to be observed (Table 4). This indicates that the supernatant factor is involved during the initial stages of activation, possibly at the initiation of DNA synthesis, as has been reported in other suppression systems (6). The PWM suppression is not attributable to general cytotoxicity since the time requirement is so crucial. Furthermore, the recovery and viability of all cultured cells is greater than 90%. Cytotoxicity was measured against the SK-sensitive targets K-562 and MOLT, and induced Tcell-sensitive targets IM-9 and LA-328. There appeared to be no significant difference in the percentage inhibition using the different target cells. Perhaps a more sensitive technique than 51Cr release may be required to demonstrate differential suppression if it does exist.

30

HOFMAN

ET AL.

Several factors derived from Con A-stimulated cells and allosensitized cells in the mouse have been described (3, 6, 17, 18). Truitt et al. (15) have reported at least two distinct supernatant factors derived from T-cell cultures of in vivo allogeneically stimulated mice. One factor suppresses mixed leukocyte reactions (MLR-TsF), while the other depresses cytotoxicity (CTL-TsF). The MLR-TsF inhibits [3H]thymidine incorporation when added throughout the culture period and requires subregion homology of the H-2. In contrast, the CTL-TsF is not genetically restricted and must be present during the first 72 hr of culture. Their CTL-TsF has similar characteristics to the PWM-induced supernates. As demonstrated in Table 4, PWM supernatant factor must be added to the MLC on the first day of culture for suppression to be achieved. Table 5 indicates that the suppressor factor and responding cells do not have to be HLA identical for suppresion to be measured. Donors A and B were HLA typed and found to differ at the DrW locus. In mouse studies, it appears that the genetic restriction in the interaction between suppressor factor and responder cells is linked to the I-c/S region (23). Since these and several other donors tested were not matched for DrW antigens, the PWM supernate does not seem to require DrW homology for activity to be observed. The particular donors described in Table 5 did share the common A-2 and B-15.1 loci. In other responder cell and supernate culture combinations, where both donors shared no HLA-detectable antigens, the PWM supernatant factor was also suppressive (data not shown). Thus the PWM-induced supernate described here may stimulate a subpopulation of cells with particular suppressive qualities similar to those observed in the mouse system. The cells which are activated by the PWM are shown to be adherent (Table 7). It is not clear from the studies presented here whether these adherent cells are suppressive, or whether they in turn stimulate suppressor lymphocytes in the effector population. In the mouse system, the CTL-TsF (15) is derived from a stimulated T cell and acts on an accessory non-T cell, thus demonstrating a lymphocyte-accessory cell interaction in suppression. We are currently characterizing the target population of the PWM-induced soluble factor. Con A and allosensitized inducible soluble factors have been readily produced from mouse cells. However, human cell cultures have been less amenable to factor production. It has been reported that in the human system, cell-to-cell contact is perhaps necessary for inhibition, or that a tightly bound soluble factor mediates suppression (9). In our system as well, the supernatant from Con Astimulated cells has little or no effect on cytotoxicity (Fig. 1). To demonstrate which cell population is responsible for elaborating the PWM active supematant, we have exposed adherent cells to PWM. These cells were active in suppression (Table 7). However, we could not obtain an active supernatant from these cultures. This is most likely due to the poor viability of the adherent cells after 48 hr when cultured alone, without other peripheral blood cells. Supernates from phagocyte depleted PWM-stimulated cells have demonstrated variable effects in cultures. This may be due to incomplete removal of phagocytic cells using carbonyl iron treatment. Work is in progress to define clearly the leukocyte subpopulation which is responsible for the production of suppressor factor. The balance of stimulation versus suppression of the immune system may play a critical role in the disease situation. A lack of suppressor cells has been reported

SOLUBLE

FACTOR

DERIVED

FROM PWM-STIMULATED

CELLS

31

in SLE patients (19) and in GVH disease (20). In cancers of the head and neck, adherent cells have been implicated in abnormally high levels of suppression in proliferation and cytotoxicity. It has been suggested that such populations are indicative of advanced disease (21). Further investigation is necessary to relate these disease situations to the control mechanisms in the normal individual. ACKNOWLEDGMENTS We wish to express our appreciation to Drs. William Clark and Susan Kanowith-Klein critical and constructive comments regarding this manuscript.

for their

REFERENCES 1. Folch, H., and Waksman, B. H., J. Immunol. 113(l), 140, 1974. 2. Spina, C. A., and Hofman, F., Fed. Proc. 38(3), 5567, 1979. 3. Peavy, D. L., and Pierce, C. W., J. Exp. Med. 140, 356, 1974. 4. Shoo, L., Schwartz, S. A., and Good, R. A., J. Exp. Med. 143, 1100, 1976. 5. Rich, R. R., and Pierce, C. W., J. Immunol. 112(4), 1360, 1974. 6. Rich, R. R., and Rich, S., J. Immunol. 114(3), 1112, 1975. 7. Sakane, T., and Green, I., J. Immunol. 119(3), 1169, 1977. 8. Williams, R. C., Jr., and Korsmeyer, S. J., Clin. Immunol. Immunoparhol. 9, 335, 1978. 9. Finerman, S. M., Mudawwar, F. B., and Geha, R. S., Cell. Immunol. 45, 120, 1979. 10. Boyum, A., &and. J. Clin. Lab. Invest. 21(Suppl. 97), 1968. 11. Svedmyr, E., Wigzell, H., and Jondal, M., Stand. J. Immunol. 3, 499, 1974. 12. Brunner, K. T., Manuel, J., Cerottini, J. C., and Chapuis, B., Immunology 14, 181, 1968. 13. Jondal, M., Spina, C. A., and Targan, S., Nnture (London) 272, 62, 1978. 14. Targan, S., Manuscript in preparation, 1980. 15. Truitt, G. A., Dennison, D. K., Rich, R. R., and Rich, S. S., J. Immunol. 123(2), 745, 1979. 16. Maynes, B. F., and Fauci, A. S., J. Immunol. 121(2), 559, 1978. 17. Tadakuma, T., and Pierce, C. W., J. Immunol. 120(2), 481, 1978. 18. Truitt, G. A., Richland, R. R., and Rich, S. S., J. Immunol. 121(3), 1045, 1978. 19. Abdou, N. I., Sagawa, A., Pascual, E., Herbert, J., and Sadeghee, S., Clin. Immunol. Immunopathol. 6, 192, 1976. 20. Reinherz, E. L., Parkman, R., Rappeport, J., Rosen, F. S., and Schlossman, S. F., N. Engl. J. Med. 300(19), 1061, 1979. 21. Zighelboim, J., Dorey, F., Parker, N. H., Calcaterra, T., Ward, P., and Fahey, J. L., Cancer 44, 117, 1979. 22. Tucker, S. B., Pierre, R. V., and Jordon, R. E., J. Immunol. Methods 14, 267, 1977. 23. Rich, S. S., and R. R. Rich, J. Exp. Med. 143, 672, 1976.