Vol. 23, No. 2

INFECTION AND IMMUNITY, Feb. 1979, p. 360-365 0019-9567/79/02-0360/06$02.00/0

Adjuvants in the Induction of Suppressor Cells RUTH NETAl* AND S. B. SALVIN2

Department ofMicrobiology, University of Notre Dame, Notre Dame, Indiana 46556,' and Department of Microbiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 152612 Received for publication 15 November 1978

The effect of different mycobacterial adjuvants on the parameters of delayed hypersensitivity was investigated in strain 13 guinea pigs. The composition of the tubercle bacilli and the type of vehicle in which the antigen was presented determined the presence and extent of suppressor cell activity. When antigen was introduced in complete Freund adjuvant, both adherent and nonadherent cells had suppressive properties, with the suppressive effect demonstrable in vitro from 1 to 5 weeks after sensitization. Suppressor activity was indicated in vivo by a reduction of delayed footpad hypersensitivity in animals presensitized with complete Freund adjuvant.

The expression of delayed hypersensitivity has been associated with the activation of thymus-derived lymphocytes. In the regulation of this phenomenon, several factors have been implicated, such as B- or T-suppressor cells, macrophages, and enhancing antibody (3, 5, 11, 19). In this regard, the route and vehicle for introduction of the antigen have been emphasized. Attempts have been made to determine what components of the immunogenic inoculum are most influential in the activation of stimulatory or suppressor cells. The structure of the antigen, the dose of the antigen, and the route of injection have all been effective in activation of suppressor cells (7; D. Turkin and E. E. Sercacz, Fed. Proc. 35:571, 1976). In addition, the vehicle for the antigen has been found to affect the activation of suppressor cells (10, 11). When guinea pigs were inoculated with low doses of antigen in incomplete Freund adjuvant (IFA), specific suppression of delayed skin reactions was demonstrated. This delayed hypersensitivity was shortlived in that it lasted for only about 4 days before the suppressor-cell activity became manifest. This suppressor effect was transferred adoptively with splenic lymphocytes to sensitized recipients (11). Transfer of lymphoid cells from suppressed animals could reduce delayed skin responses and in parallel reduce responses of cells replicating in the presence of antigen. The active cells had characteristics of B-lymphocytes, in that they had immunoglobulin receptors on their surfaces, did not adhere to plastic surfaces, and did not form rosettes with rabbit erythrocytes (12). Because sensitization with antigen in Freund adjuvant containing dead tubercle bacilli produces a long-lasting state of delayed skin hypersensitivity, complete Freund adjuvant (CFA)

was compared with IFA as to the capacity to induce suppressor activity. In this paper results are presented which indicate that suppressorcell activity is stimulated by inclusion of CFA in the inoculum and is influenced both by the form of tubercle bacilli and by the type of vehicle in the adjuvant mixture. Several parameters of delayed hypersensitivity were measured, and their presence was correlated with the extent of suppressor activity. MATERIALS AND METHODS Animals. Strain 13 guinea pigs weighing 250 to 450 g were used.

Antigen. Living Mycobacterium bovis BCG was grown in a modified Dubos-Middlebrook medium for 7 to 11 days, concentrated by centrifugation, and suspended in phosphate-buffered saline to the desired concentration, and the cell density was determined with a Klett colorimeter. Suspensions of 107 cells were used per animal. BCG cell walls (cw) and other purified mycobacterial fractions were obtained through the courtesy of E. Ribi, Rocky Mountain Laboratory, Hamilton, Mont. (17). Dead, whole M. tuberculosis (Jamaica strain) cells were maintained in a lyophilized form. Purified hen egg albumin (HEA) was purchased from K & K Laboratories, Inc., Jamaica, N. Y., and was sterilized in solution by membrane filtration before measurement in a Beckman spectrophotometer at 277 ,tm (6). Adjuvants. Lyophilized cell walls, dried whole bacteria, or other dried bacterial products were placed in a 15-ml tissue grinder. A light mineral oil, Drakeol6/VR (Pennsylvania Refining Co., Butler, Pa.) (0.12 ml), was added, and the mixture was ground to a smooth paste. A 10-ml amount of 0.85% NaCl containing 0.2% Tween 80 was added to the paste, and the grinding was continued. A 6.7-ml amount of Tween 80 saline diluent was then added. A purified "cord factor" (P3) and HEA were dissolved in chloroform-methanol at a 94:6 ratio. After evaporation in a stream of nitrogen, the resulting ingredients were dispersed in the 360

VOL. 23, 1979

ADJUVANTS AND SUPPRESSOR CELLS

appropriate adjuvant (17). Thus, 1 mg of cell wall, 1 to 2 mg of dead whole bacteria, or 150 ,ug of P3 was injected into each animal. IFA was purchased from Difco Laboratories, Inc., Detroit, Mich. To form the complete adjuvant, 1 or 2 mg of dried tubercle bacilli, depending on the experiment, was added to the inoculum for each animal. Concanavalin A was purchased from Miles Laboratories, Inc., Kankakee, Ill., with 1 ug dispensed with cells in 0.2 ml of medium. Sensitization and skin tests. Guinea pigs were sensitized in all four footpads with 5 Iug of HEA in one of several adjuvants. Animals were subsequently skin tested on the flanks with 5 fug of HEA in physiological saline. Measurement of MIF. Migration inhibitory factor (MIF) was measured in vitro by the migration-on-agar technique (16), with 200 ug of HEA as antigen. Determination of lymphocyte replication. Spleens, draining lymph nodes, and oil-induced peritoneal cells were obtained from HEA-sensitized guinea pigs at different times after sensitization. The tissues were teased and suspended in RPMI 1640 containing 5% fetal calf serum, and distributed in microtiter plates (Mash II, Microbiological Associates), so that each well contained 4 x 105 lymph node cells, 4 x 105 lymph node cells plus 4 x 105 splenic cells, 4 x 105 splenic cells, or 8 X 105 lymph node cells in 0.2 ml with either 50 ,ug of HEA or 1 jig of concanavalin A. Each sample was assayed in triplicate, in addition to appropriate controls which did not contain a stimulating agent. The samples were incubated for 72 h at 370C, after which 1 ,uCi of [3H]thymidine was added. Additional incubation for 18 h was followed by harvesting of the samples on glass fiber filters (Reeves Angel grade 934AH), and the radioactivity of each sample was then measured in a Packard scintillation counter (12).

W

ANTIGEN

CONCANAVALIN A

x

'a-&

z

0

RESULTS Influence of different adjuvants on the induction of splenic suppressor activity. Guinea pigs were sensitized with 5 ,ug of HEA in one of several vehicles, namely, IFA, CFA (2 mg of tubercle bacilli per animal), a saline suspension of living BCG (107 organisms), or a Drakeol-Tween 80 oil-in-water emulsion containing either CW (1.0 mg per animal) or trehalose dimycolate (P3) (150 ,ug per animal). Splenic cells were harvested 2 weeks after sensitization, mixed in microcultures with 4 x 105 lymph node cells, and incubated for 72 h at 370C in the presence of 50 Mg of specific antigen or of 1 Mg of concanavalin A. The cell mixtures were pulsed with 1 ,uCi of [3H]thymidine for 18 h at 370C, and the extent of suppression was determined (Fig. 1). In the presence of either 50 Mug of specific antigen or of 1 Mug of concanavalin A, cell mixtures produced a marked suppression of cell replication when the splenic cells were from animals sensitized with antigen in CFA or IFA

B

QX g

Separation of cells. Suspensions of 2 x 107 splenic cells per ml were incubated at 370C for 30 min in petri dishes containing minimum essential medium. The supernatant cells were decanted and similarly reincubated. The supernatant suspension was again decanted and used as a nonadherent population. On microscopic examination, the original adherent population contained 90 to 95% macrophages. The adherent and nonadherent cells were washed and diluted in RPMI 1640 to a concentration of 4 x 106 cells per ml.

A

0

361

u _i 2J

i

to

>

n

2:

Q XJ

0

500-

I0

100 FIG. 1. Influence of splenic suppressor cells on responses of sensitized lymph node cells, as measured by [3Hlthymidine uptake and in the presence of 50 pg of specific antigen or I pg of concanavalin A. Suppression throughout the studies was calculated according to the formula: [1 - (obtained counts in cell mixture/sum of counts of individual cell suspensions)] x 100%. -

362

NETA AND SALVIN

INFECT. IMMUN.

(Fig. 1). Suppressor activity was greater when the splenic cells were obtained from animals sensitized with antigen in CFA. In contrast, splenic cells did not display suppressor activity when they were derived from guinea pigs sensitized with antigen (i) in Drakeol-Tween 80 emulsion containing CW or "purified cord factor" (trehalose dimycolate, or P3) or (ii) with living BCG. Delayed skin hypersensitivity in animals sensitized to HEA in each of the foregoing adjuvants was detected at the time the cells were harvested (Table 1). Duration of suppressor cell activity. When guinea pigs were inoculated with a protein antigen in IFA, suppressor activity reached a peak on about day 7 after sensitization and then rapidly declined (11). Experiments were initiated in guinea pigs to determine the influence of an inoculum containing CFA on the time of onset and on the duration of suppressive activity (Fig. 2). In such animals, suppression by splenic cells could be detected 1 week after sensitization, persisted at a high level for an additional 3 weeks, and then slowly declined for another 6 weeks. The suppressor activity of splenic cells from animals sensitized with HEA in CFA was thus more extensive and prolonged in comparison with cells from animals sensitized with IFA. Characterization of suppressor cells by adherence. Because macrophages have been implicated as suppressor cells (19) and because sensitization with the antigen in CFA is associated with infiltration of macrophages, macrophages were assayed for their suppressor activity. Splenic cells were obtained from animals 2 weeks after sensitization with antigen in CFA, and 1 week after sensitization in IFA were sepTABLE 1. Delayed skin responses of strain 13 guinea pigs sensitized to HEA in different adjuvants on days 12 and 22 after sensitization Induration diam (mm) at:

Adjuvanta

TB/Fr

Day 12 17 x 19

19x21 CW/Dr CW/Fr

24 x 30 27x31 27 x 28

20x22 23 x 20 24x29

Day 22

19 x 24 23x21

arated according to their properties for adherence to plastic surfaces and assayed for suppressive activities. Unseparated splenic cells from animals sensitized with antigen in either CFA or IFA showed marked suppressor activity (Fig. 3). In contrast, adherent cells from guinea pigs sensitized with IFA did not have detectable suppressor properties, although adherent splenic

9e

z~~~~~~ 64O 40 Z S

.~~~~~~~~

-

20

-

2 IN WEI4 AFE 6ENmTIO8 .2. pei TIME (iIN WEEKS AFTER SENSITIZATION ) FIG. 2. Extent of suppressive activity of splenic cells in donors sensitized with 5 ug of HEA in CFA.

WITH SIPECIFIC ANTIGEN

~Iw z

_

w

b~~~~I

t

II

22 x 24

27x23 21 x 22

25x30 TB/Dr 28 x 27 23x25 22 x 24 Living BCG 23 x 24 19x21 27x26 a TB/Fr, Tubercle bacilli in Freund adjuvant; CW/Dr, CW in Drakeol-Tween 80 adjuvant; CW/Fr, CW in Freund adjuvant; and TB/Dr, tubercle bacilli in Drakeol-Tween 80 adjuvant.

i QK 4 FIG. 3. Suppressive activity of adherent and nonadherent cells in different adjuvant mixtures.

VOL. 23, 1979

cells from animals sensitized with CFA had some suppressive activity. Nonadherent cells from animals sensitized with either IFA or CFA were most suppressive, with the cells from the CFAsensitized animals being more inhibitory. Splenic cells (i) from guinea pigs sensitized with a Drakeol-Tween 80 emulsion containing mycobacterial CW or P3, or (i) from normal guinea pigs did not have detectable suppressive properties. Thus, (i) nonadherent splenic cells from IFA-sensitized animals and (ii) both nonadherent and adherent cells from CFA-sensitized animals exhibited suppressor activity on the proliferation of sensitized lymph node cells. Influence of carrier vehicle. The addition of tubercle bacilli to Freund adjuvant containing a soluble protein antigen increased the extent of suppressor activity in the splenic cells of the injected guinea pig, whereas the addition of CW of M. bovis to an emulsion of Drakeol-Tween 80 containing protein antigen did not. The adjuvant effect of the CW in the latter emulsion was pronounced, as seen by positive skin tests and enhanced cell replication. Experiments were therefore initiated to determine the relative effects of vehicle versus the form of mycobacterium in the activity of suppressor cells. Groups of five to seven guinea pigs in three separate experiments were sensitized with 5 tug of HEA in one of the following vehicles: (i) 1 mg per animal, whole tubercle bacilli in Freund adjuvant (paraffin oil-Arlacel); (ii) 1 mg per animal, whole tubercle bacilli in Drakeol-Tween 80; (iii) 1 mg per animal, CW in Freund adjuvant; (iv) 1 mg per animal, CW in Drakeol-Tween 80; and (v) IFA. The suppressive effect of spleen cells excised from these animals on days 7, 14, and 21 after sensitization on the replication of sensitized lymph node cells was examined (Table 2). Comparison of the two mycobacterial forms in the two vehicles indicates that the combination of whole, dead tubercle bacilli and Freund adjuvant (water in oil) is most active as an inducer of suppressor activity, more so than CW in Freund adjuvant. Comparison of the two forms of tubercle bacilli in Drakeol-Tween 80 as a vehicle showed that sensitization of an animal with whole tubercle bacilli resulted in the splenic cells enhancing proliferation more than splenic cells from animals sensitized with CW. Thus, both the form of mycobacterium and the vehicle are influential in the induction of suppressor cells. Effect of adjuvants on production of MIF. Suppressor cell activity can affect some parameters of delayed hypersensitivity, such as cell replication, and not others, such as delayed skin reactions (12, 13). The influence of carrier vehi-

363

ADJUVANTS AND SUPPRESSOR CELLS

cle and mycobacterial form was investigated as to their effect on the production of MIF (Table 3). In direct assays in vitro of oil-induced peritoneal-exudate cells, similar MIF activity was produced by three of the four combinations, namely, whole tubercle bacilli in Freund adjuvant and the purified CW in either Freund adjuvant or Drakeol-Tween 80. The fourth combination, i.e., tubercle bacilli in Drakeol-Tween adjuvant, did not induce significant MIF production. Thus, the capacity of whole tubercle bacilli to stimulate production of MIF varied with the type of vehicle. Significance of suppressor cells in vivo. Because delayed skin hypersensitivity developed in the presence of splenic suppressor cells, the question arose as to what significance suppressor cells would have in the intact animal. Guinea pigs were preinoculated in the footpads with either (i) CFA or (ii) CW in Drakeol-Tween 80. After 2 weeks, these animals plus a third uninoculated group were sensitized with 5.0 ,tg of HEA in CFA. The guinea pigs TABLE 2. Effect of addition of splenic cells (from animals sensitized with different forms of tubercle bacilli and carrier vehicle) on the replication of lymph node cells Spleen cell sources

% Suppression' at:

Day 7

Day 14

Day 21

-52 -31 -67 TB/Frc 70 58 120 TB/Dr -22 -73 -14 CW/Fr 20 27 -24 CW/Dr -6 +58 -54 IFA a Cells were obtained at three different times after sensitization. See footnote a of Table 1 for abbreviations. b Each figure is the average of pools of cells from two to four guinea pigs in each of four separate experiments. - indicates suppression; + indicates enhancement; and days refer to time after sensitization. 'In this series of experiments, 1 mg of dead whole TB per animal was used as a comparison with 1 mg of CW per animal.

TABLE 3. Production of MIF in guinea pigs sensitized with 50 ytg of HEA in one of several vehicles Vehicle

TB/Frb

% Inhibition at day after sensitization: 27 14a 20 31 33 32 4 9 7 41 36 20 26 35 28

TB/Dr CW/Fr CW/Dr a Average of four experiments. bSee footnote a, Table 1, for abbreviations.

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NETA AND SALVIN

were skin tested 8 to 10 days later with 5 Itg of HEA in saline. Of the three groups studied involving 85 animals in four separate experiments, the control guinea pigs inoculated with CFA had erythema and induration 25 to 30 mm in diameter. Animals presensitized with CW in Drakeol-Tween 80 showed little, if any, decrease in the erythema and induration. Animals, however, presensitized with CFA, showed a 30 to 50% reduction in the erythema and induration after skin-testing. Thus, treatment of guinea pigs with CFA 2 weeks before sensitization with a protein antigen in CFA resulted in a suppression of delayed skin hypersensitivity.

DISCUSSION A strong suppressor activity has been demonstrated in vitro in the spleens of guinea pigs after footpad injection of antigen in CFA. Two distinct variables are present, however, each of which may influence the degree and duration of suppressor activity, i.e., (i) the nature of the vehicle and (ii) the composition of the mycobacterium. The suppressive effect was more severe and more prolonged when the animals were sensitized with CFA rather than IFA. When CW were included in the sensitizing antigen-Freund adjuvant emulsion, less of a suppressive effect of donor splenic cells on replicating lymph node cells was observed. When the same two mycobacterial components were included in Drakeol-Tween emulsion, not only was the suppressor activity on cell replication absent, but an enhancement of cell replication was induced by the splenic cells. Therefore, the composition of the tubercle bacilli and the nature of the vehicle determined the extent of induction of suppressive activity. The suppressor activity was primarily associated with nonadherent cells, when IFA was used in the sensitizing inoculum. However, when tubercle bacilli were added to the adjuvant, adherent cells displayed suppressive activity, although the extent of their activity was less than that of nonadherent cells. Suppressor activity has been demonstrated to be functional in a number of immune processes, such as in formation of antibody (2) and in such cell-mediated processes as allergic encephalomyelitis (18) and tumor rejection (4). In the present experiments, suppressor activity was demonstrated in splenic cells from HEA-CFAsensitized guinea pigs by their effect on replication of sensitized lymph node cells in the presence of specific antigen or of concanavalin A. The suppressor activity was primarily in the spleen. However, the extent of the suppressor effect on the various immune processes is still

INFECT. IMMUN.

not completely known. For example, MIF is produced in animals under conditions which favor suppressive activity. In fact, when the antigen is incorporated in incomplete adjuvant, neither suppressor activity nor MIF is detectable at 3 weeks after sensitization. Also, when hypersensitivity is induced with an adjuvant containing whole tubercle bacilli in Drakeol-Tween 80, enhancing instead of suppressive activity is detected by experiments involving cell replication, and significant production of MIF does not develop. Thus, conditions of sensitization that enhance suppressor activity, as measured by cell replication in vitro, do not alter the animal's capacity to produce MIF; conditions in which splenic cells increase proliferative activity of lymph node cells do not show production of MIF by peritoneal-exudate cells (Tables 2 and 3). Thus far, suppressor cell activity had been measured in vitro; experiments involving presensitization were conducted to determine suppressor cell activity in vivo. When delayed skin hypersensitivity was assayed after sensitization with specific antigen (HEA) in different vehicles, equally strong skin responses to HEA could be detected (Table 1). Presensitization with CFA alone interfered with development of delayed skin reactions to the HEA in animals subsequently sensitized with HEA in CFA. Presensitization with mycobacterial CW in DrakeolTween 80 emulsion did not have such an effect on animals sensitized with HEA in CFA. Similar suppressive effects of CFA were described (i) when presensitization of guinea pigs with CFA 10 days before injection of antigen interfered with delayed skin responses (8), (ii) when donors or recipients pretreated with CFA were shown to transfer or develop less delayed skin reactivity, respectively, than nonpretreated animals (1), and (iii) when sensitization with antigen in CFA resulted in less cytotoxicity to specific target cells because of the presence of suppressor cells (14). Delayed skin responses, cell replication, and MIF formation have been used, sometimes interchangeably, as parameters of delayed hypersensitivity or cell-mediated immunity. In fact, delayed hypersensitivity has been considered to be an important component of cell-mediated resistance (9). Yet, although good delayed skin hypersensitivity or MIF production follows injection of antigen in CFA, markedly enhanced resistance does not develop (15). The question then arises as to the significance of the suppressor cell on the cell-mediated immunity. Is the splenic suppressor cell that inhibits proliferation of lymph-node cells the same cell that affects cell-mediated resistance or, after presensitiza-

ADJUVANTS AND SUPPRESSOR CELLS

VOL. 23, 1979

tion with CFA, delayed skin hypersensitivity to subsequently presented antigen? If the suppressor cell influences these two types of immune responses, then the selection of an adjuvant should provide a means for possible manipulation of cell-mediated resistance.

9.

ACKNOWLEDGMENTS

10.

This work was supported by the U.S.-Japan Cooperative Medical Science Programs administered by the Public Health Service grant AI-08528 from the National Institute of Allergy and Infectious Disease. We thank D. B. Wasson for his outstanding and valuable assistance in the laboratory.

11.

LITERATURE CITED 1. Allwood, G. G., and G. L. Asherson. 1971. Depression of delayed hypersensitivity by pretreatment with Freund type adjuvants. II. Mechanism of the phenomenon. Clin. Exp. Immunol. 9:259-266. 2. Baker, P. J., P. W. Stashak, D. F. Amsbaugh, and B. Prescott 1974. Regulation of the antibody response to Type III Pneumococcal polysaccharide. fI. Mode of action of thymic derived suppressor cells. J. Immunol. 112:404-409. 3. Feldman, J. D. 1972. Immunological enhancement: a study of blocking antibodies. Adv. Immunol. 15:

8.

12. 13. 14. 15. 16.

167-214. 4. Fujimoto, S., M. I. Greene, and A. H. Sehon. 1976. Regulation of the immune response to tumor antigens. I. Immunosuppressor cells in tumor-bearing hosts. J. Immunol. 116:791-799. 5. Gershon, R. K. 1974. T-cell control of antibody production. Contemp. Top. Immunobiol. 3:1-40. 6. Gitlin, D. 1949. Use of ultraviolet absorption spectroscopy in the quantitative precipitin reaction. J. Immunol. 62:

437-451. 7. Ha, T. Y., and B. H. Waksman. 1973. Role of the thymus

17.

18. 19.

365

in tolerance. X. "Suppressor" activity of antigen-stimulated rat thymocytes transferred to normal recipients. J. Immunol. 110:1290-1299. Jankovic, B. D. 1962. Impairment of immunological reactivity in guinea pigs by prior injection of adjuvant. Nature (London) 193:789-790. Mackaness, G. G. 1964. The immunologic basis of acquired cellular resistance. J. Exp. Med. 120:105-120. Neta, R., and S. B. Salvin. 1973. Specific depression of delayed hypersensitivity to purified proteins with relation to production of circulating antibody. Cell. Immunol. 9:242-250. Neta, R., and S. B. Salvin. 1974. Specific suppression of delayed hypersensitivity: the possible presence of a suppressor B-cell in the regulation of delayed hypersensitivity. J. Immunol. 113:1716-1725. Neta, R., and S. B. Salvin. 1976. T and B lymphocytes in the regulation of delayed hypersensitivity. J. Immunol. 117:2014-2020. Neta, R., A. Winkelstein, S. B. Salvin, and H. Mendelow. 1977. The effect of cyclophosphamide on suppressor cells in guinea pigs. Cell. Immunol. 33:402-411. Reinish, C. L, N. A. Gleiner, and S. F. Schlossman. 1976. Adjuvant regulation of T-cell function. J. Immunol. 116:710-715. Salvin, S. B., and R. Neta. 1975. A possible relationship between delayed hypersensitivity and cell-mediated immunity. Am. Rev. Respir. Dis. 111:373-377. Salvin, S. B., and J. Nishio. 1969. In vitro cell reactions in delayed hypersensitivity. J. Immunol. 103:138-141. Salvin, S. B., E. Ribi, D. L. Granger, and J. S. Youngner. 1975. Migration inhibitory factor and Type II interferon in the circulation of mice sensitized with mycobacterial components. J. Immunol. 114:354-359. Swierkosz, J. E., and R. H. Swanborg. 1975. Suppressor cell control of unresponsiveness to experimental allergic encephalomyelitis. J. Immunol. 115:631-633. Veit, B. C., and J. D. Feldman. 1976. Altered lymphocyte functions in rats bearing syngeneic Moloney sarcoma virus. J. Immunol. 117:655-660.

Adjuvants in the induction of suppressor cells.

Vol. 23, No. 2 INFECTION AND IMMUNITY, Feb. 1979, p. 360-365 0019-9567/79/02-0360/06$02.00/0 Adjuvants in the Induction of Suppressor Cells RUTH NET...
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