Modulation of the immune response toward allografts in vivo

CELLULAR IMMUNOLOGY 13, 52-65 (1974)

Modulation

of the Immune Response Toward Allografts

in Vivo

I. Selective Suppression of the Development of Cell-Mediated Immunity by Soluble Alloantigens 1 BEN JAMIN BONAVIDA AND JACOB ZIGHELBOIM Department of Microbiology a& Immunology, School of Medicine, University of California at Los Angeles, Los Angeles, California 90024 Received November 241973 Mice injected with single or multiple doses of soluble alloantigen extracts (SAE) derived from H-2b cells failed to develop thymus dependent cell-mediated immunity (CMI) upon subsequent immumzation with EL-4 (H-2b) tumor allograft. ,By contrast, the humoral response was only slightly affected. Nevertheless, the mice rejected the tumor allograft with no sign of enhancement. Suppression of cell-mediated immunity (SCMI) was obtained with SAE prepared from either tumor or spleen cells and was shown to be specific, i.e., SAE derived from sources histoincompatible with the allografted cells had no effect. The best conditions used to demonstrate specific SCM1 were the following: (A) the alloantigen must be soluble; (B) the SAE must be administered prior to tumor inoculation: (C) multiple injections of SAE were more effective than single injection; (D) it was best observed with suboptimal tumor dose inoculums. Mice treated with a single dose ‘of SA’E showed appr.eciable blocking activity in their serum prior to tumor inoculation. Blocking, however, was not detected in the serum of mice injected with multiple injections of soluble alloantigen. These findings offered possible mechanisms for SCM1 (afferent and/or central blocking). We conclude from these results that SAE preferentially suppress the formation of T-cytotoxic cells but not the T-helper cells. This preferential effect is discussed in view of its importance in promoting graft survival.

INTRODUCTION Both tumor cells and soluble antigen extracts (SAE)2 from cells and tissues absorb alloantibodies produced specifically against the H-Z histocompatibility antigens. Tumor cells and SAE, however, behave differently if administered into allogenic recipients. While tumor allograft induces strong cell-mediated cytotoxicity (CMC) and complement dependent cytotoxic antibody (CDCA), SAE 1 This investigation was supported by Grant No. ‘CA 12800 from the National Cancer Institute, National Institute of Health, Bethesda, MD, U.S.A. a Abbreviations used in this Paper: CDCA, complement dependent cytotoxic antibodies ; CMC, cell-mediated cytotoxicity ; CMI, cell-mediated immunity; E: T, effector to target cell ratio; MEM, minimal essential medium; PEC, peritoneal exudate cells ; SAE : EL-4, SAE: C57BL/6 and SAE : CBA; soluble antigen extracts prepared by 3M KC1 from EL-4 tumor cells, ,C57BL/6 (H-29 spleen cells and CBA (H-2’) spleen cells respectively; SCMI, suppression of cell-mediated immunity. 52 All rights of reproduction in any form reserved.

Copyright (B 1974 by Academic Press, Inc.

ANTIGEN

INDUCED

SUPPRESSION

OF

CMC

53

prepared by 3M KC1 extraction induce high titers of blocking activity and little or no detectible cell-mediated immunity or cytotoxic antibody (1). The reported effects of SAE administration into recipients of allografts (skin, kidney, tumor) have been contradictory. While some investigators reported accelerated rejection of skin grafts (Z-5) others reported prolonged survival (6-9). Similarly, enhancement of tumor growth in mice pretreated with SAE was observed (10-12). The exact mechanism by which soluble transplantation antigens exert their immunological effect is not yet known. The ability of soluble transplantation alloantigens to induce a state of immunological tolerance against histocompatible grafts has not been successfully accomplished. Clearly, such an effect would greatly aid in the field of clinical transplantation. The present studies were undertaken to reevaluate the immunological behavior of SAE using well-defined in vitro assay systems for measuring humoral and cellular responses against subsequent grafts. The results showed that animals injected with SAE and later challenged with tumor allografts of the same H-2 genotype as the SAE elicited weak or no cell-mediated immune response and a normal humoral antibody response. MATERIALS

AND METHODS

Mice

C57BL/6J, BALB/ c and CBA male mice, 8-12 weeks old, were purchased from the Jackson Laboratory, Bar Harbor, ME. Mice were also made available through the Mammalian Genetics and Animal Production Section, Cancer Chemotherapy National Service Center, National Cancer Institute. Tumor Cells

The mouse tumor EL-4 was originally obtained from Dr. R. Herbermann, NIH, and maintained in ascites form in C57BL/6J mice. Preparation

of Membranes and Soluble Antigens

(A) Crude membrane preparations were obtained by homogenizing the cells using 18 gauge needle. The suspension was then centrifuged at 2009 for 10 min in the cold and the sediment was discarded. The supernatant was centrifuged at 90,OOOgusing Beckman Ultracentrifuge, model II. The precipitate was suspended in phosphate buffer saline, the total protein content was estimated, and aliquots were frozen at -20°C until used. (B) Soluble membrane antigens were prepared by 3M KCl extraction as described by Reisfeld et al., (13). The total protein content was determined by the Lowry method using bovine serum albumin as standard (14). Soluble alloantigens and membranes were tested for the presence of serologically defined histocompatible antigens by their ability to inhibit the cytotoxic activity of BALB/c anti-EL-4 serum on 51Cr-EL4 cells (Fig. 1). The inhibition was specific as soluble antigens prepared from immunologically unrelated cells were not inhibitory in this system.

54

BONAVIDA

AND

ZIGHELBOIM

M EL-4 CELLS -EL-4 MEMBRANE SAE: EL-4 MSAE: CBA

AMOUNT OF INHIBITOR (rug protein)

FIG. 1. Inhibitory activity of extracts prepared from EL-4 or CBA spleen on the cytotoxic activity of anti-EL-4 serum on Yr-EL-4. The assay was done as described in the Materials and Methods section. The anti-EL-4 serum dilution used was 1: 2000. Each point represents the mean of tests performed in triplicate.

Antisera Hyperimmune antiserum to EL-4 was prepared by immunizing BALB/c mice with 4-6 weekly ip injections of 2 X lo7 cells. Animals were bled from the retroorbital plexus and sera were separated following storage of the whole blood at 37°C for 1 hr and then at 4°C for 24 hr; the sera were stored at -15” and heated to 56” C for 30 min prior to use. Cytotoxic Assays (A) Cell-mediated cytotoxicity. In vitro cytotoxicity against EL-4 was determined by the Vr release technique as already described (15, 16) and modified by substituting the petri dishes with disposable, rectangular trays, Model 96 WS (Limbro Chemical Co., Inc., New Haven, CT). Essentially, 0.2 ml containing the desired amount of sensitized cells and 0.1 ml containing 5 X lo4 61Cr-labeled cells are mixed together and incubated in humidified chamber on a rocker platform in an atmosphere of 5% CO2 and 95% air for 3 hr. The amount of radioactivity released is measured in. a Nuclear Chicago gamma counter. The medium used was MEM containing 10% fetal calf serum. The results are expressed as the per cent specific 51Cr released using the following formulation : Percent Specific Lysis =

Experimental counts - Background counts x 1.00. Maximum 51Cr release - Background counts

Background counts = counts released by the target cells in the presence of nonsensitized cells. Maximum WZr release = counts obtained by lysis of target cells with freezing and thawing (approximately 80% of total release was obtained). (B) Complement dependent cytotoxic activity (CDCA). The cytotoxic titer of immune sera were determined by incubating 0.1 ml serum dilution with lo5 51-CrEL-4 cells present in 0.1 ml for 30 min at 37°C. The cells are washed once and suspended in 0.2 ml medium containing 10% rabbit serum used as a source of complement. The tests are incubated for another 30 min at 37”C, 1 ml of medium

ANTIGEN

to z

lO%C4

IO7 EL-4

2x107Ek4 3 v iiz B g

55

INDUCED SUPPRESSION OF CMC

JO-

*O-

UJ ‘I

IO-

W 0 ;

200

loo

so

LOG

200 I

100 I

60

200

100

50

EFFECTOR TARGET

FIG. 2. BALB/c mice were injected ip with a single dose of 100 pg SAE: EL-4 (0 --- 0), control mice were injected with saline ( l --- l ), with 10 pg SAE:EL-4 (D----D), and 10 days later were injected with EL-4 tumor cells. The spleens were obtained 10 days after sensitization and tested for cell mediated cytolysis against Yr-EL-4 cells. The percent specific lysis was estimated in 3 hour incubation period and each point represents the mean lysis of two individual spleens. Each test was performed in triplicate.

is added, the tubes are centrifuged and the supernatant is measured for the presence of radioactivity. Each dilution of antiserum was run in triplicate. The percent specific lysis was calculated as in CMC above except that normal mouse serum was used for estimating the background counts. (C) Blocking of cell mediated cytolysis. Sensitized lymphocytes were derived from spleen cells of BALB/c mice inoculated with 2 X lo7 EL-4 ip 10 days before. The spleens were teased and a cell suspension was prepared in MEM containing 10% fetal calf serum. Antiserum blocking of cell mediated cytolysis was done by adding 0.1 ml of antiserum dilution to the Yr-labeled target cell, incubating for 30 min at 37” and then the lymphocytes were added and the test was performed as in (A) above, The results are expressed as percent inhibition of cytolysis as follows: Percent Inhibition

= 100 X

y0 Specific lysis in the presence of inhibitor y0 Specific lysis in the absence of inhibitor > ’ RESULTS

I. IMMUNE

RESPONSE TO ALLOGENEIC EL-4 CELLS IN BALB/c WITH A SINGLED• SEOF SAE:EL-4

MICE INJECTED

(A) Cellular Response Groups of BALB/c mice were injected with a single dose of SAE: EL-4 (100 pg) ip and 10 days later challenged with various doses of EL-4 tumor cells ip. At various times following challenge the mice were bled and their spleen cells were evaluated for CMC ire vitro. Cell-mediated cytotoxicity against Vr-EL-4 cells was considerably decreased in mice inoculated previously with SAE: EL-4. This effect, however, depended on the strength of tumor challenge. A representative experiment performed 10 days after tumor inoculation is shown in Fig, 2. Although for a large tumor inoculum (20 X lo8 cells) the cellular response in antigen treated mice was the same as controls, for lower tumor inoculums (10 x 108 or less), a significant depression of CMC was observed. For all lower inoculums, this suppres-

56

BONAVIDA

ZIGHELBOIM

AND

2xlO7EL-41

I:20

1:loa

I06 a-4

IO’ EL-4 I

1:4cm 110

l.mo

ANTISERUM

l:4ou

I:20

I:100

I:400

DILUTION

FIG. 3. Mice were injected with 100 gg SAE : EL-4 ( 0 - -- 0 ), 10 pg SAE : EL-4 (0 --- q ) or saline ( 0 --- 0 ) and 10 days later were injected with EL-4 cells. The mice were bled 10 days after tumor inoculation and the sera were tested for cytotoxic antibodies in the presence of complement. Each serum was assayed in triplicate, and each point represents the mean of two sera.

sion of cell-mediated immunity (SCMI) was noticed at all E : T ratios investigated. Identical results were obtained when CMC was tested at Days 8, 14 and 17 after tumor challenge. Inoculation of soluble antigen iv resulted in similar findings. Also, in experiments where mice were injected with SAE (10-100 pg), and later injected with EL-4 (> 2 x lo’), SCM1 was observed. (B) Humoral Response The sera of the antigen treated mice were tested for complement dependent cytotoxic antibodies (CDCA) (Fig. 3). A decrease in the titer was observed in TABLE

1

IMMUNE RESPONSE AGAINST EL-4 CELLS IN BALB/c TREATED WITH SAE : CBA

Amount of SAE: CBA injected (@da

Dose of EL-4b Cells

100 10

107 107

-

100 10

107

2 x 106 2 x 106 2 x 106

MICE PREVIOUSLY

Percent specific lysis Cellular responseC (E:T) Spleen PEC 25:1 1OO:l so:1 so:1 16.5 f 13.2 f 16.2 f 15.4 f 12.4 f 14.3 f

4 2 6 5 2 1

8.2 f 11.2 f 12.4 f 12.2 f 10.3 f 11.2 f

2 3 3 6 4 2

52.4 f 56.4 f 57.4 f 52.4 f 54.3 f 55.3 f

2 4 3 4 6 4

44.2 i 46.3 f 54.6 i 44.3 f 46.2 f 46.2 f

Humoral responsed

2 5 1 2 3 4

40.2 f 10 49.5 f 7 52.4 f 8 4.5 f 10 52.4 i 6 40.2 i 8

0 BALB/c mice were injected ip or iv with SAE:CBA and control mice were injected with saline. b Antigen treated mice were injected with EL-4 cells 10 days after antigen administration. c Cell mediated cytotoxicity was done at 11 days after EL-4 injection using spleen and peritoneal exudate cells (PEC) as effecters. The numbers represent the mean f SD specific lysis of two mice used in each experiment. d The humoral response was tested for complement dependent cytotoxic antibodies as described in the Materials and Methods Section. The numbers represent the percent specific lysis obtained at a 1:600 final dilution of antiserum. This dilution was chosen because it represented a point on the declining portion of the titration curve.

ANTIGEN

v,

50.

2xl05EL-4

200

57

INDUCED SUPPRESSION OF CMC

- 5d06EL-4

loo

50

25

200

PEC SPLEEN r-2 SAE:C57Sl/6-= -SAE:CBA m =SALINE .-.

100

50

25

LOG EFFECTOR TARGET

FIG. 4. Mice were injected daily for 5 days with soluble antigen and 10 days after the last inoculation were inoculated with 2 X 10’ EL-4 cells (left) or 5 X 10“ EL-4 cells (right). Spleen and PEC were obtained 11 days after sensitization and tested for CMC.

mice receiving 108 tumor cells, whereas with higher tumor inoculums no significant change in the humoral response was detected, although significant SCM1 was observed (Fig. 2).

The specificity of the immune depression obtained treated mice was assessed by inoculating mice with SAE It was found that SAE: CBA treated mice responded treated mice. Neither CMC nor CDCA was affected. These findings indicated that SCM1 is observed only cells share major histocompatibility antigens. II. IMMUNERESPONSETOALLOGENEIC

EL4 CELLSIN WITH SAE : C57BL,/6

with SAE: EL-4 (H-2b) : CBA ( H-2k) (Table 1). to EL-4 like control unwhen SAE and the tumor

BALB/c

MICE IN JECTED

In these experiments, the mice were given daily injections of SAE: C57BL/6 for five days, and ten days later were challenged with whole EL-4 tumor cells. Figures 4 and 5 summarise the results obtained in assays testing CMC and CDCA titers, respectively. Mice pretreated with SAE : C57BL/6 and subsequently challenged with 2 x l(46 or 5 X lo6 EL-4 cells showed poor CMC response (by both the spleen and PEC). The humoral response, however, was partially suppressed in the groups of mice that were sensitized with 2 x 108 EL-4 (Fig. 5). These results were found to be comparable to those observed in section I using soluble antigen derived from EL-4 tumor cells demonstrating that other sources of transplantation antigens such as C57BL/6 spleen cells could be used for inducing SCMI. III.

EFFECTOFMULTIPLEDOSES

OF SAE: EL-4 ON THE IMMUNERESPONSE

When the number of SAE: EL-4 inoculations was increased (10 injections administered in the space of 2 weeks), a complete abrogation of CMC was observed upon challenge with 5 X lo8 EL-4 (Fig. 6). Unlike the results shown above, both

58

0 5 Q 5 E

BONAVIDA

AND

2x106EL-4

r

ZIGHELBOIM

605040-

~SAE:C57Bw6 -SAE:CBA -SALINE

30-

5 Y

20-

5 0.

IO-

I:20

lz!oo

moo

1~20

l4opoo

ANTISERUM

l200

l2OOO l:lopoo

DILUTION

FIG. 5. C’omplement-dependent cytotoxic activity in the sera of antigen treated mice (injected daily for 5 days ip with 50 pg soluble antigen and 10 days later inoculated with tumor cells) obtained 11 days after EL-4 tumor inoculation. The dilution of antiserum shown in the figures represent the final dilution present in the reaction mixture. Each point represents the mean of lysis of two individual serums and each serum was assayed in triplicate.

the spleen and PEC were unable to mediate cytolysis of EL-4 cells even at high E: T ratios (ZOO: 1) while the CDCA titer was not altered (Table 2). These results suggested that the mechanism by which the soluble antigen suppresses the T-dependent CMC is different from that which affects the T-helper cells that cooperate with B cells to produce antibodies.

v, I v)

3

7

days

60

t

LOG

EFFECTOR TARGET

FIG. 6. Mice were injected repeatedly ip. 5 times per week for 2 weeks with 50 pg SAE: EL4. Ten days after the last injection, the mice were inoculated with 5 X IO8 EL-4 cells and both the spleens and PEC were assayed for CMC. ( l --- l ), spleen of mice pretreated with saline; ( 0 --- O), spleen of antigen treated mice; ( [7 - -- 0 ), PEC of saline treated mice; ( W --- n ), PEC of antigen treated mice. Cell-mediated cytotoxicity was tested at Days 7 (left) and 10 (right) after tumor inoculation.

ANTIGEN

INDUCED

SUPPRESSION

TABLE

59

OF CMC

2

COMPLEMENTDEPENDENT CYTOTOXICACTIVITY IN SERUMOF MICE PRETREATEDWITH MULTIPLE DOSESOF 50 pg SAE:EL-4 AND INJECTEDWITH 5 X 106 EL-4 CELLS Treatment

Day of bleeding0

Saline SAE : EL-4 Saline SAE : EL-4

7 7 10

Percent specific lysisb Antiserum Dilution 1:500 1:lOO 1:20 43 45 60 68

10

35 40 54 62

19 20 45 45

a Serum was obtained 7 and 10 days after tumor inoculation. * The numbers represent the mean of specific lysis obtained from 2 serums. IV.

EFFECT OF SAE: EL-4 WHEN ADMINISTERED AT VARIOUS TIMES FOLLOWING TUMOR IMMUNIZATION

When mice were injected simultaneously with SAE: EL-4 cells, little or no significant suppression of CMC was detected (Table 3, Groups 1 and 2). Similar results were obtained using single injections of SAE: EL-4 at Days 4 or 8 (Table 3, Groups 3 and 4), or multiple injections (at Days 7, 8 and 9) following tumor inoculation (Table 3, Groups 5 and 6). These results demonstrated that the soluble antigen must be injected prior to tumor inoculation for suppression of CMC to occur. It appears that once the immunocompetent precursor cytotoxic cells have TABLE

3

THE EFFECT OF TIME OF SAE:EL-4 ADMINISTRATIONON THE SUBSEQUENT IMMUNE RESPONSETO EL-4 CELLS Group

1 2 3 4 5 6

Soluble antigen” Nature Dose Time (rd (days) of administration SAE : CBA SAE : EL-4 SAE:CBA SAE : EL-4 SAE:CBA SAE : EL-4 SAE:CBA SAE : EL-4 SAE:CBA SAE:EL-4 SAE:CBA SAE:EL-4

100 100 100 100 50 50 50 50 50 50 50 50

7, 7, 7, 7,

1 1 1 1 4 4 8 8 8, 8, 8, 8,

EL-4 Inoculum

5 5 2 2 5 5 5 5 9 9 9 9

5 5 2 2

x x x x

106 106 106 106

X 106 X lo6

x x x x x x

106 106 106 106 106 106

Cell-mediated cytotoxicityb Percent Specific Lysis Spleen PEC 1OO:l so:1 10:1 1:l

63.4 56.5 38.6 43.2 54.4 54.5 56.02 46.5

51.3 51.3 42.3

40.1

46.3 40.2 25.4 28.5 44.3 48.4 47.3 39.9 49.2 48.2 35.4 36.2

54.6 69.5 65.4 59.6 54.6 51.0 59.6 49.8 56.3 49.1 49.84 49.12

44.4 47.1 39.2 42.4 14.15 15.1 9.4 5.4 14.9 12.7 15.9 18.2

= The soluble antigen was injected ip at various times indicated. Day 1 means the soluble antigen was administered simultaneously with EL-4 on the same day. b Cell mediated cytotoxicity was measured 11 days following antigen injections. The results are expressed as percent specific lysis and represent the mean ratios of two experimental mice for each group. CMC were assayed at different ratios of effector to target cells,

60

BONAVIDA AND ZIGHELBOIM

O-OS!LINE

EFFECTOR TARGET

LoG

wMEMBRbNE

/lo6EL-4

zE&,,,

)107EL-4

ANTISERUM DILUTION

FIG. 7. In these experiments mice were injected with 50 pg membrane prepared from EL-4 and 10 days later were injected with EL-4 cells. Ten days after sensitization, spleen and PEC were tested for CMC against Yr-EL-4 (figure to the left) and the serum was tested for CDCA (figure to the right).

begun their sensitization the sensitization phase.

by whole EL-4 cells, soluble antigens could not reverse

V. BLOCKINGACTIVITYINTHE

SERAOF MICE IN JECTEDWITH

SAE:EL-4

Sera obtained from mice prior to tumor inoculation were found to show blocking activity following a single injection of SAE: EL-4 (Table 4, Group 1). The blocking activity was specific in that serum from mice treated with SAE: CBA did not block CMC against EL-4. Nevertheless, in experiments where multiple injections of SAE : EL-4 were given (Table 4, Group 2)) no blocking activity was detected in the serum prior to tumor inoculation. Also, serum obtained from mice following SAE : EL-4 tumor inoculation was found to be devoid of blocking activity (Table 4, Group 3). TABLE BL~CKINGACTIVITY

Group 10

26 3”

INSERA OF MICE INJECTED WITH SOLUBLE ANTIGEN

Treatment Saline SAE:CBA SAE:EL-4 SAE:EL-4 SAE: CBA SAE : EL-4 SAE:EL-4 SAE:EL-4

4

(100 pg) (100 pg) (100 rg) (50 pg) (50 pg) (100 ,ug) (100 pg)

Percent specific lysis 42.9 f 45.4 i 30.98 f 35.22 f 38.2 f 41.5 f 42.89 f 41.82 f

2.0 3.2 3.8 1.85 2.05 3.2 2.1 1.5

Percent blocking

P<

0 28.4 f 3.2 18.7 f 2.4 4.3 f 2.5 0 0 0

NS 0.005 0.005 NS NS NS NS

a For these experiments, the serum was obtained 10 days after antigen administration and was tested for blocking activity as described in Materials and Methods. The antiserum dilution used was 1: 10. b In these experiments, the mice were given 5 SAE injections per week for 2 weeks and bled 10 days after the last injection. The serum obtained was tested for blocking activity. c The sera used were obtained 10 days after tumor inoculation (5 X 106) in mice which were previously treated with antigen.

ANTIGEN

INDUCED

SUPPRESSION

TABLE

61

OF CMC

5

PROLIFERATION OF TIJMOR CELLS IN THE PERITONEAL CAVITY OF MICE PREVIOUSLY TRGATED WITH SOLUBLE ANTIGEN EXTRACTS”

3

EL-4 Cells in peritoneal 6

x X x X

10’ 10’ 107 10’

Dose of EL-4

Treatmentb

2 2 5 5

SAE : EL-4 SAE:CBA SAE : EL-4 SAE: CBA

x x x x

106 106 106 106

1.08 2.03 3.59 3.62

5.2 6.1 9.7 8.4

x X x x

10’ 10’ 10’ 10’

cavity 7

1.2 1.4 2.08 2.3

x x X X

at daysC 10 6.08 2.05 2.6 1.05

108 108 lOa lo8

X x X x

10” 107 lo6 10’

a In this experiment, the number of tumor cells present in the peritoneal cavity was calculated. Macrophages were excluded from the counts by using neutral red as a marker for distinguishing tumor from macrophages. * Mice were injected with 50 pg SAE five times daily for one week and injected 7 days after the last injection with EL-4 cells. c The numbers in the table represent the mean number of tumor cells present in two mice.

We concluded from these results that serum blocking activity induced by SAE: EL-4 may represent one of the mechanisms by which suppression is obtained (tierent or central). The absence, however, of detectable blocking activity in sera of mice treated with multiple doses of SAE: EL-4 indicated that other mechanisms are responsible for the effects observed. VI.

IMMUNE

RESPONSE AGAINST WITH

MEMBRANES

EL-4 CELLS IN BALB/c PREPARED FROM EL-4

MICE

INJECTED

In order to determine whether the suppression of the immune response to EL-4 by SAE: EL4 could also be induced by antigenic preparations which are not in soluble form, membranes derived from EL-4 cells were tested for their immunological effect. Mice that received (SO-100 pg membranes equivalent to 150-300 ,ug SAE : EL-4) prior to tumor inoculation ( lo6 or 10’ cells) responded like controls (Fig. 7). The immune response was not altered for either CMC or CDCA. Thus, these results demonstrated that the antigen preparation must be in a soluble form for inducing SCMI. VII. TUMOR GROWTH IN MICE TREATED WITH SAE :EL-4 Experiments were done to test the growth curve of EL-4 in the peritoneal cavity of antigen treated mice and of control mice following EL-4 inoculation. As shown in Table 5, there was no striking difference in the number of tumor cells observed between experimental and control groups. These results showed that there was no accelerated rejection of EL-4 cells in antigen treated mice which would have been expected if the allograft rejection mechanism had been primed by the soluble antigens. DISCUSSION Evidence is provided for the first time that the development of CMC toward allograft could be selectively decreased or abrogated by prior treatment of the animals with SAE of the same H-2 genotype as the tumors. Our experiments showed that administration of SAE: EL-4 (H-Zb) into BALB/c (H-Zd) mice

62

BONAVIDA

AND

ZIGHELBOIM

prior to sensitization with EL-4 cells (H-2b) suppressed, and in some instances completely abrogated, the appearance of cytotoxic cells in both the spleen and peritoneal cavity. The effect on the development of cytotoxic cells by the SAE was not always parallelled by a decrease in the titer of cytotoxic antibodies in most tumor doses tested. However, a decrease in antibody response and CMC was observed at low tumor doses ( lo6 -2 x 106). The CMC activity studied here is a T-dependent lymphocyte function, and evidence for this has been well-documented elsewhere (17). The decrease in CM1 observed in our studies was not due to a shift in the kinetics of the immune response development, since evaluations done at various times after tumor inoculation (7-18) have all shown SCMI. This decrease was not due to putative blocking factors, either, since spleen cells were washed extensively before testing. The possibility that the EL-4 tumor cell extracts prepared by 3M KC1 contained immunosuppressive factors responsible for the observed phenomenon was ruled out by the demonstration that extracts prepared from C57BL/6 spleen cells (also H-2b) were also suppressive. The specificity of the H-2b antigens in SCM1 was demonstrated by the fact that SAE: CBA (H-2k) antigens did not alter the immune response to EL-4. These results indicated clearly that transplantation antigens present in the crude soluble antigen preparation were the components responsible for the suppressive effects observed. It is difficult, however, to ascertain the exact molecular form of the H-2 antigens responsible for the phenomenon and it is not known whether their association with other moieties render these antigens tolerogenie. The use of purified and well characterized transplantation antigens will undoubtedly determine better their in viva biological role. Several observations were made in this study. It was shown that multiple inoculations of antigen were more effective than a single injection. Whereas, with one injection a significant decrease of CMC was noted, complete abrogation was achieved with multiple injections (Fig. 6). These results are similar to those reported for the induction of tolerance for antibody in neonatal animals (18). The dose of tumor cells used for sensitization in the antigen treated mice was critical for demonstrating SCMI. Thus, under the conditions used in our studies, we were not able to demonstrate immune suppression with tumor doses greater than 1 X lo7 cells. These results coincided with the dose response curve obtained in mice sensitized against EL-4 which demonstrate maximum CMC activity with doses greater than lo7 tumor cells. It was demonstrated that suppression of CMC could only be obtained provided the mice had been treated with SAE prior to EL-4 sensitization (Table 3). There was no effect when the SAE was given simultaneously with EL-4 or at various times after. These results are similar to those obtained with tolerance for humoral antibody response and suggested that the mechanisms involved might be the same. Weigle et al. (19) have reported that helper T cells became tolerogenie 24 hr following antigen administration. In our case the exact time required for induction of tolerance for cytotoxic T cells by SAE is not yet known. The observation that membrane preparations from EL-4, which contained H-2 antigenic activity, were not inhibitory in our system suggested that solubility of the antigen is mandatory for obtaining the suppressive effect. Several studies have demonstrated that the induction of immunological tolerance to antigens other than tissue antigens could be achieved with soluble antigen devoid of aggregates (18).

ANTIGEN

INDUCED

SUPPRESSION

OF cMc

63

The preferential induction or suppression of cellular and/or humoral response has been shown by many investigators using sources of antigens other than histocompatibility antigens (see reviews 20, 21). In those studies, several investigators demonstrated preferential induction of cellular tolerance as measured by delayed hypersensitivity tests. Our studies are similar to those described with the exception that soluble H-2 antigens were used and our assessment for CM1 was measured in vitro by cell-mediated cytotoxic assays. With SAE we were not able to demonstrate a selective suppression of humoral response while retaining a normal cellular response. Such a phenomenon, however, was demonstrated in other systems (20, 22,23). The mechanism by which SAE inhibits the immune response is not clear. We have demonstrated that serums from mice which received a single injection of lymphocytes. SAE : EL-4 blocked CMC against 51Cr-EL-4 by immune BALB/c The presence in the serum of blocking activity prior to tumor inoculation might have influenced the development of a cell,-mediated response to EL-4 by an afferent type of inhibition. The role of blocking activity in enhancement of tolerance has been demonstrated by Voisin (24). The mechanism of afferent inhibition does not explain why the titer of cytotoxic antibodies was not altered. It has been clearly demonstrated that antibody production for tumor allografts is a thymus dependent response and requires T helper cells (25). It may be possible that the T cells mediating CMC are a different subset from the T helper cells and each carry receptors for different antigenic specificities, i.e., cytotoxic T cells have receptors for H-2 antigens and helper T cells have receptors for carrier antigens which may or may not be the same as the H-2 antigens. The absence of blocking activity in the serum of mice that received multiple SAE injections indicated, however, that other mechanisms were involved in the SCMI, namely central inhibition or decrease in the number of specific immune competent cells in the circulating pool. Central inhibition by antigen or antigen-antibody complexes in studies dealing with tolerance for antibody production has been demonstrated ( 18,21, 26). We have demonstrated previously that cytotoxic T cells can be inhibited from mediating cytolysis of target cells by SAE derived from the target cells (17). The latter results suggested that the cytotoxic cells bear receptors that recognize soluble alloantigens. It is likely that the precursor cells carry also such receptors which could interact with antigen alone or antigen-antibody complexes and are thus prevented from maturing into cytotoxic cells in the presence of sensitizing tumor cells. Preliminary studies done in vitro demonstrated that SAE inhibit the generation of cytoxic cells in a one-way mixed lymphocyte culture (Bonavida, B., unpublished). SCM1 could have resulted if one postulates that SAE administration decreases the number of specific immunocompetent precursor cells which otherwise were available to interact with the sensitizing antigens. Such a mechanism has been suggested by Rowley et al. (21). Mice which did not demonstrate CMC in vitro were able, nevertheless, to reject the tumor allografts. Thus, the absence of CMC in in vitro tests does not necessarily reflect an immune deficiency in rejection systems because other immune mechanisms such as antibody dependent cellular cytotoxicity, cytotoxic antibodies, etc., exist which can act alone or synergistically with CMC in tivo in tumor allograft rejection (27,ZS). Our results with soluble transplantation antigens offer new approaches in the modulation of the immune response toward allografts and offer new avenues for

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transplantation tolerance studies. Earlier studies on transplantation tolerance by Billingham et al., (29) demonstrated that when allogeneic cells were introduced into fetal or neonatal mice tolerance to the appropriate skin grafts was observed and the mechanism implied was a central failure of the immune response. Law et al. (30) injected mice at birth and for several weeks thereafter with soluble H-2 antigens. When skin-grafted at adulthood, these mice showed only a depression of antibody formation and normal cellular response (normal rejection of skin allografts). The failure to detect cellular tolerance by these investigators might have been due to the strength of the antigenic stimulus induced by the skin. Studies done in kidney transplants in rats demonstrated that prolongation of survival was obtained if recipients were given donor antigen in the form of lymphoid cells one day before surgery. Antigen given more than one day after surgery had no effect (21, 31, 32). Although these authors did not study in vitro the cellular immune response of the treated animals, their results are consistent with our interpretation that a defect in cellular immunity could have been responsible for the observed phenomena. In conclusion, the results reported here demonstrating a specific and selective suppression of CMC by SAE point to its clinical application. Thus, the use of soluble alloantigens might reduce the need for nonspecific immunosuppressive drugs and also offer new means for manipulating host immune responses against allografted organs and cells. ACKNOWLEDGMENTS The authors thank Professor John L. Fahey for reviewing the manuscript. We also thank Miss Ellen Harper and Mr. Gary Mayman for excellent technical assistance.

REFERENCES 1. 2. 3. 4. 5. 6.

Zighelboim, J., Bonavida, B., Rao, V. S., and Fahey, J. L., J. Immunol. 112, 433, 1974. Graff, R. S., Mann, D. L., and Nathenson, S. G., Transfhntation 10, 50, 1970. Graff, R. S., Nathenson, S. G., Transplant. Proc. 3,249, 1971. Strober, S., Appella, E., and Law, L. W., Proc. Nat. Acad. Sci., USA 67, 76.5,1970. Kahan, B. D., and Reisfeld, R. A., Proc. Sot. Exp. Biol. Med. 130, 765, 1969. Koene, R., McKenzie, C. F. C., Panek, D., Lacho, D. H., Winn, H. J. and Russell, P. S., Transplant. Proc. 3, 237, 1971. 7. Rosenberg, E. B., Mann, D. L., Hill, J. J., and Fahey, J. L., Transplantation 12, 402, 1971. 8. Rao, V. S., Bonavida, B., Zighelboim, J., and Fahey, J. L., Truns~lantation (in press). 9. Von Haegen, U., Wren, S. F. G., Shaipanich, T., Martins, A. C. P., Busch, G. J., and Wilson, R. E., Transplantation 16, 295, 1973. 10. Law, L. W., Appella, E., Wright, P. W., and Strober, S., Proc. Nat. Acad. Sci., USA 68, 3078, 1971. 11. Rosenberg, E. B., Hill, J., Ferrari, A., Herberman, R. B., Ting, C., Mann, D. L., and Fahey, J. L., J. Nat. Cancer Inst. 50, 1453, 1973. 12. Hilgert, I., Koubek, K., and Kristofova, H., In “Proc. Symp. Immunogenetics of the H-2 system, Liblice-Prague,” pp. 211-223. Karge, Basel, 1971. 13. Reisfeld, R. A., Pellgrino, M. A., and Kahan, R. D., Science 172, 1134, 1972. 14. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., J. Biol. Chem. 193, 265, 1951. 15. Brunner, K. T., Mauel, J., Cerottini, J. C., and Chapuis, B., Inzmultology 14, 181, 1968. 16. Canthy, T. G., and Wunderlich, J. R., J. Nat. Cancer Inst. 45, 671, 1970. 17. Bonavida, B., J. Immwol. 112, 926, 1974. 18. Landy, M., and Brawn, W., In “Immunological tolerance. A reassessment of the mechanisms of the immune response,” p. 53. Academic Press, New York, 1969.

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19. Weigle, W. O., Chiller, J. M., and Habicht, G. S., In “Progress in Immunology” (B. Amos, Ed.), p. 311. Academic Press, New York, 1971. 20. Parish, C. R., Transplant. Rev. 1.3,35, 1972. 21. Rowley, D. A., Fitch, F. W., Stuart, F. P., Lobler, H., and Cosenza, H., Science 181, 1133, 1973. 22. Janicki, B. W., Schechter, G. P. and Schultz, K. E., I. Immunol. 105, 527, 1970. 23. Thompson, K., Harris, M., Benjamin, E., Mitchell, G., and Noble, M., Nature New Riol. 238, 20, 1972. 24. Voisin, G., Cell. Immunol. 2, 670, 1971. 25. Cerottini, J. C., Nordin, A. A., and Brunner, K. T., Nature (Londorz) 228, 1308, 1970. 26. Diener, E., and Feldman, M., Transplant. Rec. 8, 76, 1972. 27. Perlman, P., and Holm, G., Advan. Immunol. 11, 117, 1969. 28. Perlman, R., Perlman, H., and Wigzell, H., Transplant. Rev. 13: 3, 1972. 29. Billingham, R. E., Brent, L., and Medawar, P. B., Phil. Trans. Prog. Sot. (London) SetYes B 239, 357, 1956. 30. Law, L. W., Apella, E., Strober, S., Wright, P., and Fischetti, T., Proc. Nat. Acad. Sci. USA 69, 1858, 1972. 31. Stuart, F. P., Saitoh, T., and Fitch, F. W., Science 160, 1463, 1968. 32. Lucas, Z. J., Markeley, J., and Travis, M., Fed. Proc. 29, 2041, 1970.