Activation of suppressor T cells by low-molecular-weight factors secreted by spleen cells from tumor-bearing mice

CELLULAR

IMMUNOLOGY

93, 364-374 (1985)

Activation of Suppressor T Cells by Low-Molecular-Weight Factors Secreted by Spleen Cells from Tumor-Bearing Mice BARBARA

L. POPE

Department of Pharmacology, Dalhousie University, Sir Charles Tupper Medical Building, Hal&x, Nova Scotia B3H 4H7, Canada Received November 5. 1984; accepted February 13, 1985

The spleens of mice bearing large M-l fibrosarcomas have been shown to contain several populations of cells which nonspecifically suppress antibody synthesis by cocultured normal spleen cells. It has now been shown that the spleensof tumor-bearing mice also contain inducer cells which secrete soluble factors capable of activating suppressor T cells from unprimed precursor cells. The activated suppressor cells are Thy I+, Lyt 1+2+ and secrete a soluble suppressive factor. They inhibit the in vitro generation of antibody-forming cells by cocultured normal spleen cells stimulated by T-cell-dependent antigens. They do not, however, suppress the antibody responseto T-cell-independent antigens and do not inhibit antibody synthesis by cocultured nude mouse spleen cells cultured with T-cell-dependent antigens and exogenous helper factors. In addition, suppression is blocked if conditioned medium containing T-cell growth factors is added to the suppressorcell assays.These data suggestthat cells in the spleens of tumor-bearing mice secrete inducing factors which activate suppressorcells. These activated suppressor cells in turn secrete soluble suppressor factors which inhibit antibody synthesis, possibly by interfering with the synthesis or release of T-cell growth factors. o 1985 Academic Press. Inc.

INTRODUCTION Both tumor-specific and generalized immunosuppression have been well documented in hosts with progressively growing tumors (1). Antigen-specific suppression of the immune response to tumor antigens has been described in experimental tumor models and antigen nonspecific immunosuppression has been frequently associatedwith advanced tumor growth in both experimental animals and in human cancer patients. Suppressor cells, which have attracted a great deal of attention in the past years as important mediators of suppression, have been demonstrated in tumor-bearing individuals (2). These suppressor cells have been subdivided into specific and nonspecific effector cells, have been identified as macrophages (3-5), T cells (58), and B cells (9), and have been shown to inhibit a variety of immune responsesby a number of different mechanisms. The complexities of suppressor cell activation during tumor growth may be due to the initiation of several different levels of immune regulation during the persistent growth of neoplastic cells. For example, antigen specific suppressor cell circuits, which have been described in tumor-bearing animals (lo- 12), closely resemble pathways activated during immune responses to more conventional antigens 364 0008-8749/85 $3.00 Copyright 0 1985 by Academic Press. Inc. All rights of reproduction in any form reserved.

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(12, 13). In both cases inducer T cells respond to antigenic stimulation with the subsequent release of soluble factors. The inducer factors act on acceptor T cells or pre-T cells which then activate or differentiate into effector suppressor T cells. Primed B cells (14, 15), primed T cells ( 16-1 S), and antigen-antibody complextreated cells (19, 20) have all been shown to be inducers of antigen-specific feedback suppression. As a possible second level of suppression, B-cell blasts (2 I), non-T cells from tumor-bearer spleens (22), and tumor-activated T cells (23) have also been shown to induce nonspecific suppressor cells. This may reflect a level of suppression superimposed on the antigen-specific circuits or may be an amplification or distortion of a nonspecific component of antigen-specific suppressor cells (24, 25). Third, as an additional level of regulation, suppressor cell circuits may be initiated by autochoids released from proliferating tumor cells or from lymphoid cells responding to the tumor. Nonspecific suppressor cells have been shown to be activated by prostaglandins (26, 27), leukotrienes (28), and histamine (29). We have studied the suppressor cells associated with the growth of M-l fibrosarcomas in DBA/2J mice (30, 31). Suppressor T cells from the spleens of tumorbearing mice selectively inhibit the antibody response of normal spleen cells stimulated by the T-cell-dependent antigen, sheep red blood cells (SRBC). In this study we demonstrate that the spleens of tumor-bearing mice also contain inducer cells which secretesoluble factors capable of activating suppressor effector cells from unprimed precursor cells. The in vitro activated suppressor cells resemble one of the populations of suppressor cells found in the spleens of tumor-bearing mice. MATERIALS

AND METHODS

Mice. Male DBA/U mice (Jackson Laboratory, Bar Harbor, Maine) were used at 2 to 4 months of age. Male, Hsd BALB/c AnBom Nu/Nu mice were obtained from Sprague-Dawley (Harlan Industries, Madison, Wise.) at 4 to 5 weeks of age. They were maintained in sterilized cages placed in a laminar flow hood in an isolated room and were used at 2 to 4 months of age. Tumor. The tumor used in these experiments was the transplantable fibrosarcoma which we previously named the M-l tumor (32). The M-l tumor was originally induced at the Jackson Laboratory by subcutaneous injection of methylcholanthrene into DBA/2J mice but is now maintained by the NC1 tumor bank (Frederick, Md.) where it is cataloged as the SAD2 fibrosarcoma. The tumor line currently in use in this laboratory was obtained 3 years ago from the NC1 bank as frozen tumor fragments which were injected subcutaneously into DBA/2J mice. The tumor which grew from these fragments has since been maintained by continuous in vivo passages and short term in vitro cultures have been established periodically as a source of homogeneous tumor cells. Source of inducer cells. Spleen cells capable of secreting soluble factors which activate suppressor cells (inducer cells) were obtained from tumor-bearing mice 3 to 4 weeks following the subcutaneous injection of lo5 cultured M-l tumor cells, (a concentration of tumor cells which kills 100% of the mice in 5 weeks). Preparation of spleen cell suspensions. Spleen cells were teased into culture medium (RPM1 1640 medium (pH 7.4) containing penicillin (100 IU/ml), streptomycin (100 pg/ml), 10% fetal calf serum (GIBCO Canada, Burlington, Ont.), 5 X 1O-5 M 2-mercaptoethanol, and 2 mM glutamine). The suspension was filtered

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L. POPE

through sterile gauze, centrifuged for 7 min at 2OOg,and the cells obtained were resuspended in the culture medium described above. Depletion of T-cell subpopulations. Spleen cells were suspended at a concentration of 5 X lo6 cells/ml in culture medium containing a 1O-3 dilution of monoclonal anti-Thy 1.2, anti-Lyt 1.1, or anti-Lyt 2.1 (New England Nuclear Corp., Boston, Mass.) and a 1:4 dilution of guinea pig complement (C’). As a control, a separate suspension of cells was treated with C’ alone. Following an incubation of 45 min at 37°C the cells were washed twice with phosphate-buffered saline (PBS). Cell recoveries following treatment with antisera and complement were approximately 50% with anti-Thy 1.2, 60% with anti-Lyt 1.1, and 65% with anti-Lyt 2.1. Activation of suppressor cells. The suppressor cells were activated in vitro in periscopic Marbrook vessels (Bellco Glass, Vineland, N.J.). The inducer and precursor cells were cultured in separate chambers such that only soluble factors could pass between the two populations. The Marbrook chambers were prepared by attaching a strip of dialysis tubing to the inner chamber (IC) by an elastic ring. The chambers were soaked in distilled water overnight, filled with phosphatebuffered saline, autoclaved, and emptied just prior to use. The IC of each vessel was filled with 1 ml culture medium containing 5 X lo6 normal spleen cells (suppressor cell precursors). The outer chamber (OC) was filled with 10 ml of culture medium containing either 5 X 10’ spleen cells from tumor-bearing mice as a source of inducer cells or an equal number of cells from aged-matched normal mice as a control. The vesselswere incubated at 37°C in 5% CO2 for 24 hr, after which the cells were removed from the IC, washed once with PBS, and assayedfor suppressor cell activity. Suppressor cell assay (T-cell-independent antibody synthesis). In vitro activated suppressor cells were assayed for the ability to inhibit the in vitro stimulation of antibody synthesis by the T-cell-independent antigen dinitrophenylated-lipopolysaccharide (DNP-LPS). Responder spleen cells from normal mice were cultured in 24-well plates (Falcon #3008) at 2 X lo6 cells/well in 1 ml culture medium. Cell populations to be tested for suppressor cell activity (cells from the IC of the Marbrook vessels)were added to replicate wells at a concentration of lo6 cells per well. All cultures were incubated with 1 pg DNP-LPS per well for 2 days at 37°C in 5% COZ. Anti-DNP synthesized by the DNP-LPS-stimulated cells was assayed using the cellular enzyme linked immunosorbant assay (ELISA) assay of Kelly et al. (33). Using this system the ELISA can detect antibodies secreted by small numbers of cultured cells. Ninety-six-well EIA trays (Flow Laboratories, Mississauga, Ont.) were coated with antigen by preincubation for 24 hr with 100 &well of dinitrophenylated-keyhole limpet hemocyanin (DNP-KLH) freshly diluted to 5 pg/ ml in carbonate buffer (pH 9.6). Cultured cells to be assayedfor anti-DNP synthesis were removed from the multiwell plates, washed once with PBS, resuspended in culture medium (1 ml/well), and plated (0.25 ml cell suspension/well) in the precoated EIA trays which had been washed three times with PBS to remove unbound DNP-KLH. Following a 24-hr incubation of the cells in the EIA trays, the plates were washed three times with PBS-Tween buffer (pH 7.4) in order to remove all cells and unbound antibodies. The trays were incubated at room temperature for 2 hr with 100 &well of a 1:500 dilution of alkaline phosphataseconjugated rabbit anti-mouse immunoglobulin, washed three times with PBSTween buffer, and incubated for 1 hr with 100 &well substrate (p-nitrophenyl

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367

phosphate at 1 mg/ml in 10%diethanolamine buffer). The absorbance was measured using a Multiscan plate reader (Flow Laboratories). The intensity of color was proportional to the amount of antibody bound to the plastic on the EIA tray. Data are presented as the mean absorbance f the standard deviation for triplicate wells containing the test cells and the antigen, DNP-KLH. Background absorbances for a single well containing the test cells without antigen (PBS in EIA wells) were subtracted prior to averaging. Suppressor cell assay (T-cell-dependent antibody synthesis). Suppressor cells were tested for the ability to inhibit the in vitro generation of antibody-forming cells following stimulation by the antigen sheep red blood cells. In this assay, responder spleen cells were cultured in 96-well microtrays (Falcon #3042) at a concentration of 1.25 X lo6 cells per well in a total volume of 0.25 ml culture medium. Nu/Nu spleen cells were cultured at 1.5 X 1O6cells per well and the medium was supplemented with 20% exogenously produced helper factors. Suppressor cells were added to triplicate wells at a concentration of 5 X lo5 cells per well. All wells were stimulated with 5 X lo5 SRBC for 5 days at 37°C in 5% COz. Anti-SRBC antibody production by the cultured cells was assessedusing the plaque-forming cell (PFC) assay of Cunningham and Szenberg (34) in which the contents of triplicate wells for each test group of cells were removed from the wells, pooled, diluted with PBS, centrifuged for 10 min at 2OOg,and resuspended in PFC medium (RPM1 1640 medium supplemented with penicillin (100 IU/ml), streptomycin (100 pg/ml), 2 mM glutamine, and 10% FCS). An aliquot of this suspension was removed, mixed with SRBC and guinea pig complement to give final concentrations of 5% SRBC and 10%complement. Aliquots of this mixture were incubated in triplicate Cunningham slides for 1 hr at 37°C. Data are presented as the PFC f standard deviation per test culture. Production of exogenous helper factors. Spleen cells from normal mice were cultured in 24-well microtrays at a concentration of 5 X lo6 cells per ml in culture medium containing 4 pg/ml concanavalin A (Con A). Following a 24-hr incubation at 37°C in 5% COZ, the supernatant was removed, centrifuged at 2008 for 10 min, filter sterilized, and diluted 1:4 in culture medium. This medium is referred to as Con A-induced supernatant-conditioned medium (Con A-CM). Commercial T-cell growth factors were purchased from Sigma Chemical Company (St. Louis, MO.). Assay for soluble suppressorfactors. Suppressor cells were tested for the ability to secrete suppressive factors capable of passing through dialysis tubing. In these experiments, the putative suppressor cells (or control populations) were placed in the OC of Marbrook vesselsat a concentration of 5 X lo6 cells in 10 ml culture medium. Responder cells from normal mice, cultured in the IC at a concentration of 5 X lo6 cells in 1 ml, were stimulated with 1 X lo6 SRBC. The vessels were incubated at 37°C in 5% CO2 for 5 days. At this time the responder cells were removed from the IC with a Pasteur pipet, washed once with PBS, and assayedfor anti-SRBC PFC as described previously. RESULTS Activated suppressor cells inhibit T-cell-dependent antibody synthesis. In order to determine whether the spleensof tumor-bearing mice contained inducer cells capable of activating suppressor cells (SC) we cultured spleen cells from normal and tumor-

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BARBARA L. POPE

bearing mice in separatechambers of Marbrook vessels.Following a 24-hr incubation, the normal cells were removed from the IC and assayedfor the ability to suppress T-cell-dependent antibody synthesis by cocultured normal spleen cells. Suppression was estimated by comparing the PFC responses of the cocultured cells to the response of cells cultured alone (no SC). Table 1 contains data from 10 typical experiments in which in vitro activated suppressor cells were tested for the ability to inhibit the anti-SRBC response of cocultured normal spleen cells. This series is representative of a large number of experiments in which activated suppressor cells have been shown to inhibit the PFC responsesby 20 to 80%. Control cells (obtained from Marbrook vessels containing spleen cells from normal mice in the OC) did not significantly decrease the PFC response in any experiment and frequently increased the response above that seen with responder cells alone. Activated suppressor cells do not inhibit T-cell-independent antibody synthesis. Previous investigations of the in vivo activated suppressor cells (3 1) have shown that suppressor macrophages from M-l tumor-bearing mice were capable of suppressing the DNP-LPS-stimulated antibody response of normal spleen cells, but that a suppressor-T-cell population from the same mice was not inhibitory in this assay. Table 2 shows two experiments in which the in vitro activated suppressor cells were tested for the ability to inhibit the anti-DNP response of cocultured normal spleen cells. In these, as well as in other experiments, the activated suppressor cells did not inhibit the antibody response significantly more than did the control cells. In contrast, using the same assay system, suppressor cells activated in vivo by growth of the M-l tumor (spleen cells from tumor-bearing mice) were significantly more TABLE I Suppressor Cells Inhibit T-Cell-Dependent Antibody Responses PFC/culture of responder cells” Expt 1 2 3 4 5 6 I 8 9 10

No SCb 2923 + 1002 2 1064 + 1115 + 1502 f 1111 + 2092 f 1561 2 1403 + 2166 f

11 54 83 67 58 67 206 179 32 194

Control cells’

Activated SCd

3369 + 196 (115) 1509 ?I 52 (151) 1292 + 91 (121) 1829 f 134 (164) 1333 + 127 (89) 1341 + 48 (120) 3250 f 336 (155) 1778 k 12 (114) 1341 +. 48 (96) 1938 ic 139 (90)

1523 + 32 (52) 503 f 32 (50) 676 f 83 (64) 883 zk 57 (79) 623 f 106 (41) 414 f 23 (37) 458 + 38 (22) 889 AZ 85 (57) 552 f 39 (39) 1345 + 71 (62)

’ PFC responseof 1.25 X lo6 normal responder spleen cells stimulated in vitro with SRBC and cultured alone (no SC) or with an additional 5 X lo5 control or activated suppressor cell (SC) populations. Values in parentheses indicate percentages of normal response (i.e., [(PFC of responder cells + SC) + PFC of responder cells alone] X 100). ’ No control or SC added to the responder cells. ‘Control cells were normal spleen cells cultured in the IC of Marbrook vesselsand exposed for 18 hr to dialyzable factors from 5 X 10’ normal spleen cells cultured in the OC. dActivated suppressor cells were normal spleen cells cultured in the IC of Marbrook vessels and exposed for 18 hr to dialyzable factors from 5 X 10’ tumor-bearer spleen cells cultured in the DC.

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FACTORS

TABLE 2 Activated Suppressor Cells Do Not Inhibit T-Cell-Independent Antibody Synthesis Anti-DNP response(OD)’ Activation of SC In vilrob

No SC

Control cells Activated SC In viva’

Expt 1

Expt 2

0.642 + 0.062 0.679 + 0.122 (106) 0.612 + 0.056 (95)

0.327 + 0.034 0.292 +- 0.062 (89) 0.287 k 0.055 (88)

0.827 + 0.063 1.027 _+0.047 (124) 0.475 f 0.020 (57)

0.864 k 0.074 0.734 + 0.039 (85) 0.337 rtr 0.058 (39)

Suppressor cells

No SC

Normal spleen Tumor-bearer

0 The anti-DNP response of 2 X IO6 normal responder cells stimulated in vitro with DNP-LPS and cultured alone (no SC) or with an additional 1 X IO6control cells or activated SC. Values in parentheses indicate percentagesof normal response. b In vitro activated SC were prepared in Marbrook vesselsas described in the notes to Table 1. cIn vivo activated SC were spleen cells obtained directly from tumor-bearing mice. Spleen cells obtained from normal, age-matched mice were used as a control population.

suppressive than were control cells (spleen cells from normal mice). The latter data demonstrate that this assay is sufficiently sensitive to demonstrate suppression by the in viva activated suppressor cells, but that the in vitro activated suppressor cells are not inhibitory. Activated suppressor cells are Thy I’, Lyt 1+2+. In order to determine whether the activated suppressor cells expressed T-cell-differentiation markers, we treated cells from the IC with anti-Thy 1, anti-Lyt 1, or anti-Lyt 2 and complement before assaying for suppressor cell activity. The data in Table 3 demonstrate that the suppressor cell activity was completely lost following depletion of Thy l+ cells. In contrast suppressor cells treated with complement alone retained the ability to inhibit antibody synthesis. Control cells treated with either complement alone or anti-Thy 1 and complement were not inhibitory in this assay. Similarly, the

TABLE 3 In Vitro Activated SuppressorCells are Thy 1.2’

PFC/culture of responder cells Suppressor cells

Treatment”

Expt 1

Expt 2

None

None

1115_+ 67

1099 * 12

Control

C’ Anti-Thy 1.2 + C’

1790 z!z107 (161) 1747 * 44 (157)

1548 AZ89 (141) 1591 + 42 (145)

Activated

C’ Anti-Thy 1.2 + C’

549 f 16 (49) 1092 + 149 (98)

772 _+94 (70) 1427 _+47 (130)

’ Control and SC populations were treated with C’ or anti-Thy 1.2 + C’ following removal from the IC of the Marbrook vessels.Treated cells were counted and 5 X IO5 viable cells were assayed for SC activity.

370

BARBARA L. POPE TABLE 4 In Vitro Activated Suppressor Cells are Lyt 1.1+2.1+

PFC/culture of responder cells Suppressor cells

Treatment’

Expt 1

Expt 2

None

None

1140+

Control

C’ Anti-Lyt 1.1 + C’ Anti-Lyt 2.1 + C’

1915 * 59 (168) 1482 !z 32 (130) 980 f 46 (86)

2599 f 129 (135) 1794 f 97 (93) 1392 f 81 (72)

Activated

C’ Anti-Lyt 1.1 + C’ Anti-Lyt 2.1 + C’

544 -c 34 (48) 2554 f 180 (224) 2114 f 91 (185)

851 f 83 (44) 1442 + 170 (75) 2323 + 163 (120)

32

1932 f

83

’ Control and SC populations were treated with C’ or anti-Lyt + C’ following removal from the IC of the Marbrook vessels.Treated cells were counted and 5 X 10’ viable cells were assayedfor SC activity.

treatment of activated suppressor cells with either anti-Lyt 1 or anti-Lyt 2 and complement depleted the cells from the IC of suppressor cell activity (Table 4). Activated suppressor cells secrete a soluble suppressor factor. Suppressor T cells from the spleens of tumor-bearing mice were previously found to secrete a soluble suppressor factor which would cross a dialysis membrane and suppress the T-celldependent antibody responseof normal spleen cells. The data in Table 5 demonstrate that the in vitro activated suppressor cells were also capable of secreting a suppressor factor. Responder cells cultured in the IC of Marbrook vessels with SRBC were consistently inhibited in their ability to produce antibodies if activated suppressor cells were cultured in the OC. In contrast, control cells did not decrease, and frequently enhan
synthesis while having no effect on the T-cell-independent antibody response to DNP-LPS. A possible reason for this selectivity was that the mechanism of TABLE 5 In Vitro Activated Suppressor Cells Secrete a Soluble Factor

PFC/IC of Marbrook vesselb Cells in OC of Marbrook vessela

Expt 1

Expt 2

Medium Control cells Activated SC

1911 f 57 2231 + 118 (117) 1433 k 80 (75)

2166 f 174 2685 + 253 (124) 1241 + 219 (58)

a SC, activated in vitro in Marbrook vesselsas described previously, were transferred to the OC of New Marbrook vesselsat a concentration of 5 X lo6 cells per chamber. b Normal responder cells (5 X lo6 cells per ml) were cultured in the IC of Marbrook vesselswith 1 X IO6 SRBC. Following a 5day incubation period, the cells were removed from the IC and assayedfor anti-SRBC PFC.

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FACTORS

suppression involved the inhibition of helper-T-cell activity. One approach used to test this hypothesis was to assay for the ability of the suppressor cells to inhibit the antibody response of nude mouse spleen cells stimulated by SRBC in the presence of exogenously synthesized helper factors. The data in Table 6 show that activated suppressor cells were not capable of inhibiting the PFC response of spleen cells from nude mice in experiments in which they were clearly suppressive for spleen cells from normal mice. Suppressor cell activity is reversed by the addition of T-cell growth factors. As shown in the previous sections, the activated suppressor cells inhibited T-celldependent but not T-cell-independent antibody synthesis. In addition, the synthesis of anti-SRBC antibodies by nude mouse spleen cells was not inhibited. This led us to the hypothesis that the suppressor cells act by inhibiting the production of T-cell growth factors in the mixing assay. In order to address this question, we tested Con A-CM and commercially available T-cell growth factors for the ability to overcome the suppressor cell activity. As can be seen from the data in Table 7, both of these preparations were capable of reversing suppression. DISCUSSION We have shown previously that the spleens of mice bearing M-l fibrosarcomas contain suppressor T cells which inhibit T-cell-dependent antibody synthesis via secreted soluble suppressor factors. This study provides the first evidence for a suppressor cell circuit in the activation of the suppressor cells. In this paper we describe inducer cells which secrete factors capable of crossing dialysis membranes and activating suppressor T cells from unprimed precursor cells. The activated suppressor cells are Thy 1+, Lyt 1+2+, secrete a soluble suppressor factor, and selectively inhibit antibody synthesis against T-cell-dependent antigens. The facts that T-cell-independent antibody synthesis and antibody synthesis by Nu/Nu spleen cells cultured with exogenous helper factors are not inhibited are consistent with the possibility that the suppressorcells act by blocking the production of lymphokines by helper T cells.

TABLE 6 Suppressor Cells Do Not Inhibit Antibody Synthesis by Spleen Cells from Nude Mice PFC/culture of responder cells” Responder cells

Suppressor cells

Expt I

Normal

No SC Control cells Activated SC

1502 f 58 1333 f 127 (89) 565 + 48 (38)

2166 + 194 1938 f 139 (90) 1345 f 71 (62)

Nu/Nu

No SC Control cells Activated SC

1138k 947 f 838 f

1915 IL 68 2006 + 230 (105) 3146 f 189 (164)

14 35 (83) 49 (74)

Expt 2

’ PFC response of 1.25 X lo6 normal spleen cells or 1.5 X lo6 spleen cells from Nu/Nu mice which were stimulated with SRBC and cultured alone (no SC) or with an additional 5 X 10’ control or activated SC. Nu/Nu spleen cells were cultured in Con A-CM.

372

BARBARA L. POPE TABLE 7 Suppression Is Reversed by Exogenous T-Cell Growth Factors (TCGF) Expt

Suppressor cells? No SC Control cells

Activated SC

No SC Control cells Activated SC

TCGF preparation b None Medium 10% Con 20% Con Medium 10% Con 20% Con

A-CM A-CM A-CM A-CM

None Medium Sigma TCGF Medium Sigma TCGF

PFC/culture’ 830 AI 23 992 -c 39 (120) 1015 f 56 (122) 939 f 72 (113) 439 f 39 (53) 801 + 58 (96) 877 f 46 (106) 823 f 679 f 744 + 245 f 557 +

59 22 (83) 44 (90) 42 (30) 51 (68)

’ Control and activated suppressor cells from the IC of Marbrook vesselswere assayed for the ability to suppressthe PFC responseof cocultured normal spleen cells. * TCGF preparations were added to the mixing assaysat time 0. Sigma TCGF was used at a dilution giving 2 units of IL-2 activity. ’ Anti-SRBC PFC response of I .25 X IO6normal spleen cells cultured alone (No SC) or with 5 X lo5 cells of control cells or activated SC. Values in parenthesesindicate percentagesof normal response.

There are a number of possible explanations for the inducer cells in the spleens of tumor-bearing mice. Antigen-specific inducer T cells may be activated in vivo by circulating soluble tumor antigen or antigen-antibody complexes. The “activated” antigen-specific inducer cells may, when transferred to the Marbrook vessels,release inducer factors which activate suppressor T cells which suppress nonspecifically. Alternatively, activated T or B cells from the spleens of tumor-bearing mice may secrete lymphokines which activate nonspecific suppressor cells. Last, the inducer cells may secrete low-molecular-weight substances such as prostaglandins which activate suppressor cells. Nepom et al. ( 16) have described a tumor antigen-specific circuit in which inducer cells secrete factors which act on unprimed presuppressor T cells and activate them to become effector suppressor T cells. In this case the induction is antigen specific but the effector cells are nonspecific. It is possible that the spleens of mice bearing large M-l tumors contain antigen-specific inducer cells which are already activated by soluble tumor antigens or antigen-antibody complexes in the circulation and that these “activated” inducer cells secrete factors which activate suppressor cells in the Marbrook vessels. Although this mechanism is theoretically possible, it seems unlikely for two reasons.First, the inducing factor passesthrough dialysis membranes. Although antigen-specific suppressor factors have been variable in molecular weight, they generally exceed 12,000 Da and would not likely be dialyzable (35). In addition, although tumor antigen may be present in the tumor-bearer spleen cells, it is unlikely that antigen is present in the chamber containing the precursor cells. It is possible, then, that activated lymphoid cells from tumor-bearing mice secrete inducing lymphokines which activate suppressor cells. Ingenito and Calkins (23) have described a suppressor cell circuit in mice bearing a methylcholanthrene-

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induced lymphoma. The inducer cells are activated T cells which generate effector cells from unprimed spleen cells. The major difference with their circuit is that the inducer cells are present in the spleen and other lymphoid organs 7 days after the injection of tumor cells and have disappeared by the 15th day of tumor growth. In contrast, we find inducer cells late in the growth of the tumors and do not seethem in the first 2 weeks of tumor growth. Caufield and Cerny (22) have also described non-T, non-B inducer cells in the spleens of mice bearing Moloney leukemias. In both tumor systems, however, the inducer cells have been cocultured with the precursor cells and it is therefore unknown if inducer lymphokines are released. The third possibility is that the inducer cells secreteprostaglandins which activate suppressor cells. Prostaglandin synthesis has been shown to be important in the mechanism of suppression by M- 1 tumor-bearer spleen cells (36) and therefore a role in the induction process is possible. Nonspecific suppressor cell circuits in which prostaglandins (PG) activate effector cells have been described by Fulton and Levy (26) and Goodwin and Webb (27). Rogers et al. (37) have demonstrated that prostaglandin E (PGE)-activated suppressor cells secrete a suppressive factor. It is possible that the effector cell activated in our study is identical to the one activated by PGE, however there is at least one difference between the two populations. The PGE-activated suppressor cells appear to act directly on B cells since T-cellindependent antibody synthesis is suppressed.Our effector cells do not suppressthe response to DNP-LPS or the response of spleen cells from nude mice. The selective suppression of T-cell-dependent antibody synthesis may be due to a blockade of the production of helper factors. A similar mechanism has been described for the suppressorcells activated during delayed hypersensitivity responses. Malkovski et al. (38) have shown that inducer cells release soluble factors which activate acceptor cells which, in turn, release suppressive factors. Although the induction process is antigen specific, the suppressive factor is antigen nonspecific and acts by depressing the production of interleukin 2 (IL-2). Prostaglandins have also been shown to directly inhibit both the release and action of T-cell growth factors which are necessaryfor cytotoxic-T-cell development (39, 40). The fact that several different preparations of T-cell growth factors blocked the activity of the suppressor cells in our study suggeststhat the mechanism of suppression may be the inhibition of the synthesis or release of IL-2 or another lymphokine required for T-cell-dependent antibody synthesis. The activity of T-cell growth factors in the assay is apparently not blocked since the antibody synthesis by nude or normal mouse spleen cells was normal in the presence of exogenously added T-cell growth factors. ACKNOWLEDGMENT This work was supported by a grant from the National Cancer Institute of Canada.

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Oehler, J. R., Campbell, D. A., Jr., and Herberman, R. B., Cell. Immunol. 28, 355, 1977. Eggers,A. E., and Wunderlich, J. R., J. Immunol. 114, 1554, 1975. Elgert, K. D., and Fat-tar, W. L., J. Immunol. 120(4), 1345, 1978. Gershon, R. K., Mokyr, M. B., and Mitchell, M. S., Nature (London) 250, 594, 1974.

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