Suppression and augmentation of the primary in vitro immune response by different classes of antibodies

CF.LLITLAR

IMM~JNOLOGY

Suppression

15. 392-402

(1975)

and Augmentation Response

of the Primary

by Different

Rccrivcd

Classes

Marrh

in Vitro

immune

of Antibodies

lo,1974

The capacity of purified rG, and rG, anti-sheep red blood cell (SRBC) antibodies to exert antigen-specific feedback regulations on the primary in zdtro immune response to SRBC was studied. Antibodies were administered to the culture in the native form, as sheep erythrocyte-antibody complexes or as pepsin-derived F(ab’)s antibody fragments. Marked differences in the feedback regulatory effects of rG1 and rGz antibodies were found. Antibodies of the -yG, class suppressed the immune response to SRBC, whereas +yG, antibodies isolated from the same serum exerted an augmenting effect on antibody synthesis. These opposing feedback effects on in vitro antibody synthesis were immunologically specific, relatively insensitive to changes in antigen concentrations, and could be elicited by either adding antibodies and antigen separately to the culture or as preformed antigen-antibody complexes. Experiments comparing the activities of the F(ab’)z antibody fragments with the parent rG1 and ?G, antibodies suggested that the Fc fragments may be involved in these regulatory effects on the immune response. It is concluded that the antigen-specific suppressive and augmenting effects on antibody synthesis shown here are determined by the antibody class. In addition, we suggest that these opposing antibody-mediated feedback effects may represent one of the important elements of the immune response.

INTRODUCTION The effects of passively administered antibody on the immune response have been extensively studied (1). Although the possibility of antibody-mediated feedback regulation of the immune response was suggested over a decade ago (2)) only more recent evidence has shown that this mechanism is a physiological one (3, 4). The regulatory effects of antibody have subsequently been readily demonstrable in several systems (5-S). The most frequently observed effect of passively administered antibody is specific inhibition (9, 10) ; however, in certain circumstances it has been shown to enhance the immune response (11-13). While much is known about the suppressive effects of passive antibody, very little is understood about the nature of its enhancing activity. In some recent test systems employing multideterminant antigens, the ability of passively administered antibodies to suppress the immune response to determinants to which they could bind while concomitantly enhancing the response to other unrelated antigenic determinants of the same molecule (14) or cell (15) has been 1 Present

address:

Department

of Immunology,

Mayo 392

Copyright All rights

2

1975 by Academic Press, Inc. reproduction in any form reserved.

Clinic,

Rochester,

MN

55901.

REGULATION

OF THE

ANTIBODY

RESPONSE

393

demonstrated. However, in other similar studies (16, 17), it has been clearly shown that passively administered antibody can also suppress the immune response to unrelated antigenic determinants. Therefore, in certain situations an antibody to one antigen may influence events which will lead to a suppressed or enhanced response to a different antigen depending on such factors as the ratio of antibody to antigen and the physical association which exists between the antigens. Another form of antibody-mediated regulation of the immune response exists which is distinguishable from the above-mentioned phenomena in that it is immunologically specific and is related to the properties of particular classes of antibodies and not to the net effect of the antiserum from which they are derived. Mtiller and Wigzell (18) studied the suppressive effect of 75 and 19s antibodies separated from the same antiserum and found 19s antibody far less effective than iS in suppression. Henry and Jerne (13), using purified yM and yG antibody preparations, demonstrated that passively administered yM antibody to sheep red blood cells (SRBC) could specifically enhance the primary yM antibody response to suboptimal doses of antigen (SRBC), while yG antibodies always suppressed, even in very low doses. Primary yG antibody responses were also shown to be augmented by passively administered yM antibody and suppressed by yG antibodies (19). The immunoregulatory properties of different classes of yG-related (yG1, yGza, and yGP,,) antibodies were recently studied by Murgita and Vas (20), who found that yG, antibody most effectively inhibited antibody synthesis, while yG, antibody isolated from the same antiserum specifically augmented the immune response in &to to optimal antigen doses. These findings lead to the suggestion that certain classes of serum yG antibodies might act to regulate the normal immune response by exerting both positive and negative antigen-specific feedback control of antibody synthesis. In the present study, the effects of yG, and yGP antibodies, their pepsin-derived F(ab’) 2 fragments, and antigen-antibody complexes on in vitro antibody synthesis were evaluated. This report confirms and extends our previous findings (20, 21) that marked differences exist in the immunoregulatory properties of various classes of serum yG antibodies. MATERIALS

AND

METHODS

Aninzals and Antigens Ten- to 16-week-old male and female CBA (Jackson Laboratories, Bar Harbor, ME) and C57BL mice (McIntyre Animal Center, McGill University, Montreal) were used in all experiments. Sheep red cells (SRBC) were supplied by the Institut de Microbiologic et Hygiene, Montreal, and were always obtained from the same animal. Chicken red cells (ChRBC) were purchased from a local poultry shop. The bloods were collected and stored for up to 2 weeks at 4°C in Alsever’s solution. Fresh human red cells (HuRBC) were used in the absorption studies.

Antisera to SKBC were produced in C57BL, mice of 5 X lOa SRRC in 0.5 ml of saline. Hyperimmune mice injected weekly with SRBC for a total of four after the last injection. The sera were separated from tion and stored at -20°C.

by intraperitoneal injections antisera were obtained from injections and bled 10 clays clotted blood by centrifuga-

394

GORDON

Isolation of Anti-SKBC

AND~~URGITA

Antibodies

Hyperimmune C57BL sera to SRBC were fractionated to obtain 7S yG1 and yG, anti-SRBC antibodies by a combination of zone electrophoresis and density gradient isoelectric focusing as previously described (22). Briefly, whole immune sera were fractionated by Pevikon-Geon block electrophoresis using barbital buffer, pH 8.5. The crude y-globulin fraction obtained was further purified using LKB SlOO ampholine electrofocusing equipment (LKB-Producter AB S-161 Bromma 1, Sweden). Selected fractions containing only fast migrating yG1 and slow migrating yGz y-globulins were pooled after completion of the isoelectric separation. The homogeneity of the antibody preparation was ascertained by immunoelectrophoresis and gel diffusion against class-specific antisera as previously described (20). Preparation of F (ab’) 2 Fragwents Pepsin-derived F(ab’), fragments were prepared from yG, and yG:! globulins according to the method of Nisonoff (23). Half of each y-globulin preparation (dialyzed against 0.1 M acetate buffer, pH 4.1) was digested with pepsin at a concentration of 2% by weight for 18 hr at 37°C. The other half of each preparation served as a control and was incubated without pepsin. In order to ensure complete digestion of the Fc fragments, the above procedure was repeated once. The preparations were then dialyzed against 0.05 M Tris buffer, pH 8.0, to stop proteolysis and to remove dialyzable peptides from the Fc region. No Fc determinants in the pepsin-digested yG, and yGa globulin preparations were detected by Ouchterlony gel diffusion analysis using heavy chain-specific antisera, and no difference in the susceptibility to digestion of these two classesof antibodies was noted. In addition, the antibody-binding hemagglutinin activities of the antibody preparations were not noticeably affected by the digestion procedure. Measurement

of Hmaagylutinin Activity

(HA)

Titrations of hemagglutinin activity were carried out by the Microtiter method as previously described (20). Titers were expressed as log2 of the reciprocal of final dilutions showing HA observed macroscopically. The yG1 and yG2 antibody preparations and their corresponding F(ab’)2 fragments were adjusted to a comparable HA titer of 4. Quantitative Estimation of Inwwzoglobulin

and Antibody Concentration

Immunoglobulin concentration of the isolated yG1 and yG2 preparations were determined by the single radial immunodiffusion method (24). Purified myeloma globulins corresponding to the yG1 (yF), yGaa (YG), and YG~~ (yH) classeswere isolated and used as standard reference proteins. Estimation of specific anti-SRBC antibody content was performed by the quantitative antibody-binding method of Nash and Heremans (25). The total y-globulin concentration of each of the yG1 and yGa preparations was 1 mg/ml, and the specific anti-SRBC antibody content was estimated to be approximately 25% (0.25 mg/ml) of the y-globulin concentrations.

REGULATION

OF THE

ANTIBODY

TABLE EFFECT

Expt No.

395

RESPONSE

1

OF ANTIGEN DOSE ON YG~-MEDIATED SUPPKESSION AND yG2-IM~~~~~~~ ADGMENTATIOK OF THE IN VITRO IMMUNE RESPONSE No. of PFC suppressed/culture by 1 pg rG

Antigen dose

No. of PFC augmented/culture

by 0.2 erg YGP

1

20 x 10 x 2 x

106 106 106

0 180 4.50

300 320 240

2

2 x 1 x 1 x

100 106 103

378 167 39 71

230 462 425 77

0

Cell Culture The culture method used was that of Marbrook (26). The establishment of optimal conditions for this method of in z&o culture of mouse spleen cells has been described elsewhere (27). Fifteen to 20 x lo6 CBA spleen cells were cultured with 2 X lo6 SRBC in Medium 1066 (Grand Island Biological Co., NY) containing 15% fetal calf serum. The cultures were incubated at 37°C in an atmosphere of 10% COz, and 90% compressedair for 5 days. The number of direct (,M) PFC were enumerated after 5 days of culture according to the method of Jerne and Nordin (28). The number of PFC recorded represent averages of triplicate cultures. RESULTS Iwnune Responseto a Fixed Dose of Antigen in the Presence of Var&zg Concentrations of yG, and yG2 Antibodies In these experiments the effect of various concentrations of yG1 and yG, antibodies on the in vitro antibody responseto a predetermined (27) optimal immunizing dose of SRBC were studied. Spleen cells were incubated with 2 x lo6 SRBC and varying amounts of antibodies extended over a thousandfold dilution range. Illustrated in Fig. 1 are the results of a typical experiment in which the effects of equivalent concentrations of yG, and yG, antibodies present for the entire S-day period of culture were compared. The control level is represented as the 100% value on the ordinate, and the PFC responsesin antibody-treated cultures are expressed as a percent of the control value. The results show the yG, antibody was immunosuppressive at all concentrations tested, with maximal inhibition occurring at a concentration equal to or greater than 1 pg of yG1 globulin. In contrast, yGa antibody augmented the PFC responsewith a definite peak of enhancement evident between 0.1 and 1 pg of yG,. These results are strikingly similar to previously reported in viva experiments (20) in which the effects of these same two classesof antibodies were compared.

396

GORDON

AND

MURGlT.4

200

I60

160 0

140 d

5

120

8

0

08

-0

A

100

l3 0 9 0

.------.

60

60

/

40 .-.A 20

10

t

DOSE

(pg

total

.Oi

.I

#G)

FIG. 1. Immune response to a fixed dose of antigen in the presence of varying concentrations of rGl and rG* antibodies. The control PFC level is represented as the 100% value on the ordinate, and the PFC response in rG1 (solid circles) and rG2 (open circles) treated cultures are expressed as a percent of the control value.

Ejject of Variation in the Antigen Dose of yG,-Mediated Mediated Augmentation of the Isvnune Response

Suppression and vG,-

The effect of varying the antigen dose from the optimal immunizing concentration of 2 x lo6 SRBC on yGl-mediated suppression and rGZ-mediated augmenta,,

SRBC

fl

ChRBC

PFC PFC

02 TREATMENT

FIG. 2. Specificity of inhibition rGz anti-SRBC antibodies.

and augmentation

of the PFC response (ordinate)

by $3, and

REGULATION

OF THE

ANTIBODY

TABLE SPECIFICITY

397

RESPONSE

2 BY yGz

OF AUGMENTATION

yG% antibody added to culturesO

Mean Expt

ANTIBODY

No.

PFC/culture

1

Expt

& SE

2

Expt

3

-.~.~ Not absorbed Absorbed with human RBC Absorbed with sheep RBC

1218 2318 2695 1331

a Aliquots of ?Gz anti-SRBC antibody erythrocytes. The absorbed supernatants taining 2 X lo6 SRBC and 15 X lo6 unabsorbed, were tested at a concentration

f f f f

16 2 63 62

433 681 546 368

* zt f f

12 24 7 8

853 1269 1201 906

f f f f.

18 10 33 36

were incubated with an equal volume of sheep or human were assayed for augmenting activity in cultures conspleen cells. The antibody preparations, absorbed and of 0.05 rg in Expts 1 and 2, and 0.1 pg/culture in Expt 3.

tion is shown in Table 1. The results demonstrate that rGz-mediated augmentation was remarkably insensitive to changes in antigen concentration, differentiating this phenomenon from the previously described enhancing effects of yM antibody (13, 19). The augmentation effect did not diminish when the antigen dose was reduced a thousandfold below, or increased tenfold above the optimal antigen concentration of 2 X lo6 SRBC/culture, which is especially noteworthy in view of the marked dose dependence of the phenomenon relative to yG, concentration (Fig. 1). In contrast, the degree of inhibition by a constant amount of vG1 antibody decreased with decreasing concentration of antigen and was overcome by a tenfold increase in SRBC per culture. Specificity

of Antibody-Mediated

Suppression

and Aupenfation

A high degree of immunological specificity of yG1-mediated suppression and yGz-mediated augmentation was observed in the previously reported in VZUOexperiments (20). In those experiments specificity was demonstrated in animals doubly immunized with two non-cross-reacting antigens, where it was shown that both suppression and augmentation were specific for the corresponding antigen, and in comparison of the responses of animals that received yG1 and YGP antibodies isolated from normal and immune sera. Similarly, in the present in vi&o study, both inhibition and augmentation were shown to be immunologically specific. The TABLE ANTIR~DY

Complex 2 X 1O’SRBC 2 X 10” SRBC (2 mcg) 2 X lo6 SRBC (0.2 mcg)

RESP~NSIC

added

f

ELICITIW

3

BY ANTIHODY-ANTIGEN

PF(./culture

yG1

+ yGr

579 i 257 f

+ SE 18 28

789 z!z 9

a Two million SRBC were incubated alone (control) antibody for 4 hr at 4YI. The complexes, washed three spleen cells.

Conwmmw PFC

lost

(-)

or gained

(+)

nil -322 +210

or with either 2 pg of rG1 or 0.2 fig lGz times, were cultured with 15 X lo6 CBA

398

GORDON

AND

MURGITA

TABLE EFFECT

Antibody” concentration

(9g)

4

UP INTACT ~GI AND F(ab’)z FRAGMENTS DEKIVELI yGl ON THE IN VITRO IMMUNE RESPONSE F(ab’)z

61 PFC/culture

f

499 f ND* 12 f 136 f 249 f 290 f

D The concentration intact antibody. b ND = not done.

PFC/culture

f

SE -

SE Expt

nil 10 5 2.5 1.25 0.625

FROM

25

499 i 25 403 f 79 1069 f 45 970 f 19 ND ND

5 5 48 8

of the F(ab’),

fragment

Expt

1

is expressed

624 zt ND ND 759 f 721 f 705 f as rg

2

Expt

25

563 f ND ND 550 f 535 f 1096 f

28 114 7

equivalent

of the

3 25

19 8 180

undigested

data presented in Fig. 2 illustrate that the PFC response to ChRBC was not affected by either class of anti-SRBC antibodies, whereas in parallel cultures a response to SRBC was suppressed or augmented by yG1 and yGa antibodies. Since, however, specificity of yG2-mediated augmentation was not shown in one of three separate experiments, the specificity was further substantiated by the absorption experiments shown in Table 2. Aliquots of yG, antibody were incubated with equal volumes of packed sheep or human erythrocytes for 30 min at 4°C. The erythrocytes were then removed by centrifugation and the supernates were tested for augmentation in vitro. The data in Table 2 clearly show that absorption of yGz antiSRBC antibody with SRBC removed the augmenting activity whereas absorption with HuRBC did not. Suppression Complexes

and Azlgrnentation

Elicited In Vitro by Preformed

Antigen-Antibody

These experiments were performed to determine whether preformed antigenantibody complexes made with yG1 and yG2 antibodies would depress or elevate, TABLE EFFECT

Antibodya concentration

5

OF INTACT yGz AND F(ab’)* FRAGMENTS DEMWD yGe ON THE IN VITRO IMMUNE RESPONSE Expt

1 PFC/cultures

Z!Z SE

Expt

FROM

2 PFC/cultures

f

SE

(pg)

62

F (ab’)z ____

nil 2.5 1.25 0.62 0.31 a The concentration intact antibody. * ND = not doue.

497 f 27 ND* 760 f 44 1214 f 154 697 f 67 of the F(ab’)t

497 f 957 f 898 zt 489 f 495 zk fragment

27 130 62 13 11

is expressed

9% 437 f ND 39s * 709 zt 466 f as pg equivalent

F (ab’)z 10 15 12 9

437 432 442 536 676

f f f f f

10 10 9 10 14

of the undigested

REGULATIOK

OF THE

ANTIBODY

399

RESPOKSE

respectively, the PFC responseas they have been shown to do when added to cultures separately. Therefore, 2 x 106 SRBC were incubated for 4 hr at 4°C with concentrations of yG1 and yGP antibodies which had elicited strong suppressive and augmenting activities when added to cultures unbound to antigen. The washed complexes were cultured with 15 x lo6 CBA spleen cells. The results presented in Table 3 show that a dose of antigen which gives an optimal PFC response ilt vitro can be modified to give a suppressedresponseif precoated with yG1 antibody and an augmented response if precoated with yG, antibody. It may be implied from this data that specific antibodies of the yG1 and yGZ classeshave appreciable antigen-binding affinities since they both readily combined with antigenic determinants on the SRBC molecules. Efect of Removal Antibodies

of the Fc

Fragments from Suppressing

and Augmenting

The necessity for the involvement of the Fab portions of the yG1 and yG1 antibody molecules in eliciting suppression and augmentation is dictated by the immunological specificity of these activities. The following experiments were carried out to determine whether the Fc portions were involved in these effects on the immune response. Shown in Tables 4 and 5 are the comparisons of the feedback properties of pepsin-derived F( ab’) 2 fragments of yG, and yGP antibodies and equivalent amounts (based on their HA activities) of the parent antibody molecules tested at several different concentrations. The results presented in Table 4 demonstrate clearly that removal of the Fc fragment from yG, antibody abolishes its suppressive activity on the in z&o antibody response. With regard to the Fc dependency of yGp-mediated augmentation, the interpretation of the data (shown in Table 5) was made difficult by the fact that the PFC responsesin some of the cultures were augmented by the F(ab’), fragments. The reason for the enhancement sometimes seen with F (ab’) 2 is not clear since it seemedto occur randomly at various antibody dilutions and was obtained with antibody fragments derived from both yG1 and yGz molecules. Perhaps more favorable antigen presentation occurs in culture when the SRBC molecules are complexed with bivalent F (ab’)z fragments. Data obtained for the two lowest antibody dilutions in Expt 1, in which the F (ab’)z fragments did not augment the PFC response, suggest that removal of the Fc from yGz antibody eliminated its augmenting activity. DISCUSSION The purpose of this investigation was to provide information concerning the comparative efficiencies of antibodies of different immunoglobulin classesin the feedback regulation of antibody synthesis. These findings demonstrate that serum antibodies of the yG1 and yG, classes can exert positive and negative feedback effects on the immune response in z&o as well as in v&o. Attempts to clemonstrate yM antibody-mediated augmentation in vifro have failed (unpublished observations; also, 29). These in vitro studies show suppression rather than the augmentation effect with the same antibody preparations used in v&o, indicating that the enhancement by yLT is dependent upon inducti\-e mechanisms which are on14 operative in z&o. Although the mechanisms involved in antibody-mediated regulation of the immune responsehave been extensively studied, it is still not clear whether antibody

400

GORDON

AND

MURGITA

exerts feedback control through a central mechanism, acting directly on immunocompetent lymphoid cells, or via a peripheral (afferent) mechanism by binding the potentially immunogenc sites of the antigen. It is also possible that both of these mechanisms may operate concomitantly in viva, as recently suggested (20) ; however, one may be of more physiological significance in the overall homeostasis of the immune response. While the data presented in this investigation offer no direct support for a given mechanism, some evidence is presented which is more compatible with a central effect. If antibody interacts directly with a cell(s) in exerting feedback control, such interaction may take place through the Fc portion of the antibody molecule, which is responsible for its cytophilic properties (30), and a corresponding Fc receptor on the cell. The demonstration of opposing feedback effects by equivalent amounts of yG, and yG, antibodies presented here and elsewhere (20) is presumptive evidence for a central action, since the difference in their activities may be ascribed to the Fc fragments which differentiate yG1 from yG2 immunoglobulins. This contention is further supported by our finding that the suppressive activity of yG1 antibody is completely eliminated on papain digestion. Furthermore, it is of interest to note that membrane-associated Fc receptors for antibody have been recently described in mice (31) which are present on all B lymphocytes and have a marked propensity for the yG, class. We propose that central suppression occurs when the Fc portion of the yGl antibody molecule undergoes conformational changes by combining with antigen which allows a stable interaction between antigen-yG1 antibody complexes and the cell bearing the yG1 Fc receptor, resulting in the transmission of a negative feedback signal. The augmentation effect of yGz antibody may be related to the specific cytophilic property of this class of mouse antibody to macrophages (32). Antigen bound to yG, antibody attached by cytophilic action to the surface of macrophages may be protected from the usual degradation by lysosomal hydrolases following endocytosis, resulting in an increased availability of processed antigen. Such a protective effect for immunogen by antibody cytophilically attached to macrophages has recently been proposed (33). Another finding reported here which would tend to support a central mechanism of immunoregulation by antibody, or direct interaction of antibody with responding cells, is the marked dependence of augmentation on antibody concentration and its remarkable insensitivity to variation in antigen concentration over a 4 log scale. Therefore, our results show that suppression and augmentation are not strictly dependent on ratios of antibody to antigen, but indicate instead that these effects are related to distinct biological properties of the two antibody classes. There are certain inherent difficulties in making comparisons of the immunoregulatory effects of different classes of antibodies directed against multideterminant antigens. Consideration must be given to possible differences in antigen-binding affinities and specificities of the yG1 and yGg antibodies. Although no attempts were made to measure directly the affinities of the yG1 and yG, antibodies, the experiments with preformed, washed antigen-antibody complexes (Table 3) established that antibodies responsible for the suppressing and augmenting effects could do SO by first combining with antigen. The antibodies used in these experiments were always obtained from the same serum collected at a given point in time following immunization which should tend to minimize the occurrence of major differences in affinities, since there is evidence in other species (34) that yG1 and yG2 antibodies isolated from the same serum generally have very similar antigen-binding affinities. Furthermore, yG anti-SRBC antibodies isolated from early (day 4)

REGULI\TION

OF THE

i\NTTROI)Y

401

RESPONSE

primary response immune serum, which is presumably of relatively low affinity, exert strong feedback suppressive effects on in vitro antibody synthesis (unpubTherefore, even if significant differences do exist in the lished observations). antigen-binding affinities of yG1 and YG~ anti-SRBC antibodies, it is unlikely that such disparities could account for their different biological activities in this system. With respect to antigenic specificity, the possibility that yG1 and yG2 antibodies to SRBC are each directed against different determinants on the erythrocyte molecule cantlot be discounted, although Finkelstein and 1Jhr (35) have emphasized that it is not unreasonable to assume that antibodies of different classes contain specific activity towards similar antigenic determinant sites on macromolecular antigens. The fact that we have now obtained consistent results in both in viva and in vitro studies including four different strains of mice with yG1 and yG2 antibodies isolated from several pools of homologous antisera would seem to support this assumption. The question of affinity differences and specificities will be examined further in studies currently in progress employing a hapten-antihapten system where the regulatory effects of different classes of antibodies having the same antigen-binding affinities and directed against the same haptenic determinants are being compared. The results presented here are in general agreement with similar experiments performed in zrivo and support the premise that yG1 and yG% antibodies can act as one of the specific regulatory elements in an immunie response by exerting opposing feedback effects through Fc-dependent interactions which may occur at the surface of one or more of the cells involved in the immune response. It is not yet known whether the mechanism of yG1-mediated suppression and YGz-mediated augmentation are directly interrelated or independent phenomena. The finding that animals treated with equal mixtures of yG1 and yG2 antibodies had immune responses intermediate between those of animals which received either antibody separately suggested that the two antibodies act in a competitive manner (36). There are a number of other studies which emphasize the importance of the Fc fragment of antibody in feedback regulation as well as the ability of certain classes of antibodies to influence the outcome of an immune response. It has been shown that the F(ab’)2 portion of anti-SRBC antibodies are much less efficient than intact 7s antibodies in suppressing the response to SRBC in viva (37) and in vitro (29, 38). Several investigators have shown that rejection or enhancement of tumor allografts may depend to a large extent on the class of alloantibody injected into the tumor recipients prior to grafting (39, 40). Furthermore, while enhaucement depends on the presence of a blocking antibody (41)) there are other antibodies which appear to inhibit the activity of the blocking antibody in viva (42) as well as in vitro (43). An interesting speculation which stems from these studies concerns the possible relationship between the suppressiug and augmenting antibodies shown here to exist in anti-erythrocyte antisera and the blocking and deblocking antibodies present in tumor-bearing animals which appear to determine whether tumor growth is suppressed or enhanced. ACKNOWLEDGMENTS We would like to express our appreciation to Dr. S. their help and discussion throughout this investigation.

I. k’as

REFERESCES and MGller, G., A&IJ~. I~~t+~z~rr~ol. 8, 81, 1968. 2. Uhr, J. W., and Baumann, J. B., J. Exp. Med. 113, 935, 1961. 1. Uhr,

J. W.,

and

Dr.

T.

B. Tomasi,

Jr,,

for

402

GORDON

AKD

MURGITA

3. Britton, S., and Mijller, G., J. Immun~ol. 100, 1326, 1968. 4. Graf, M. W., and Uhr, J. W., /. Exp. &fed. 130. 1175. 1969. 5. Dixon, F. J., Jacot-Guillarmod. FT., and McConahey, I’. J., J. li.rp. Med. 125, 1119, 1967. 6. Horibata, K., and Uhr, J. W., J. Zm*)rnno/. 98, 972. 1967. 7. Mijller, G., /. Nat. Cancer Id. 30, 1153, 1963. 8. Brody, N. I., Walker, J. G., and Siskind, G. W., J. &p. Med. 126, 81, 1967. 9. Rowley, D. A., and Fitch, F. W., J. Exp. Afed. 120, 987, 1964. 10. Wigzell, H., J. Exp. Med. 124, 953, 1966. 11. Stoner, R. O., and Terres, G., J. Imnznnol. 91, 761, 1960. 12. Pearlman, D. S., J. Exp. Med. 126, 127, 1967. 13. Henry, C., and Jerne, N. K., J. Exp. Med. 128, 133, 1968. 14. Pincus, C. S., Lamm, M. E., and Nussenzweig, V., J. Exp. Med. 134, 987, 1971. 1.5. McBride, R. A., and Schierman, L. W., J. Exp. Med. 134, 833, 1971. 16. Henney, C., and Ishizaka, K., J. Zmmnrzol. 101, 986, 1968. 17. Henney, C. S., and Ishizaka, K., J. Z?rzmz~rtol.104, 1540, 1970. 18. MGller, G., and Wigzell, H., J. Exp. Med. 121, 969, 1965. 19. Dennert, G., J. Immunol. 106, 951, 1971. 20. Murgita, R. A., and Vas, S. I., Immunology 22, 319, 1972. 21. Murgita, R. A., and Tokuda, S., Life Sci. 6, 2185, 1967. 22. Murgita, R. A., and Vas, S. I., J. Zmnzz~nol. 104, 514, 1970. 23. Nisonoff, A., Wissler, F. C., Lipman, L. N., and Woernley, D. L., Arch. Biochem. Biofihys. 89, 230, 1960. 24. Fahey, J. L., and McKelvey, E. M., J. Immunol. 94, 84, 1965. 25. Nash, D. R., and Heremans, J. F., Immunology 17, 685, 1969. 26. Marbrook, J., Lancet 2, 1279, 1967. 27. Tan, T., and Gordon, J., J. Exfi. Med. 133, 520, 1971. 28. Jerne, N. K., and Nordin, A. A., Science 140,405, 1963. 29. Wason, W. M., J. Zmmunol. 110, 1245, 1973. 30. Berken, A., and Benacerraf, B., J. Exp. Med. 123, 119, 1966. 31. Baster-i, A., Warner, N. L., and Mandel, T., J. Exb. Mrd. 135, 627, 1972. 32. Parish, W. W., Nature (London) 208, 594, 1965. 33. Bamford, D. R., and Black, S., Cell. Zmmunol. 4, 175, 1972. 34. Siskind, G. W., Paul, W. E., and Benacerraf, B., J. Ex$. Med. 135, 673, 1966. 35. Finkelstein, M. S., and Uhr, J. W., J. Immmaol. 91, 565, 1966. 36. Murgita, R. A., and Vas, S. I., Fed. Can. Biol. Sot. 13, 640, 1970. 37. Sinclair, N. R. Stc., J. Exp. Med. 129, 1183, 1969. 38. Lees, R. K., and Sinclair, N. R. Stc., Immzrrzology 24, 735, 1973. 39. Tokuda, S., and McEntee, P. F., Transplantatio~a 5, 606, 1967. 40. Voisin, G. A., Kinsky, R. G., and Due, H. T., J. Exp. Med. 135, 1185, 1972. 41. Hellstrom, I., and Hellstrom, K. E., Znt. J. Cancer 5, 195, 1970. 42. Bansal, S. C., and Sjagren, H. O., Nature (London) 233, 76, 1971. 43. Bansal, S. C., and Sjogren, H. O., Znt. J. Catzcrr 9, 490, 1972.