specific immunosuppressive activity of epiglycanin, a mucin-like

(CANCER RESEARCH 51, 1170-1176, February 15, 1991]

Specific Immunosuppressive Activity of Epiglycanin, a Mucin-like GlycoproteinSecreted by a Murine Mammary Adenocarcinoma (TA3-HA)1

Peter Y. S. Fung and B. Michael Longenecker2

Department of Immunology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7 [P. Y.S.F., B.M.L.], and Biomira, Inc., Research Centre One,Edmonton Research and Development Park, Edmonton, Alberta, Canada T6N1E5 fB.M.L.J

ABSTRACT

Epiglycanin (Epi) is a mucin-like glycoprotein carrying multiple Thomson Freidenreich (TF) and Tn determinants secreted by a murine mammary adenocarcinoma, TA3-Ha. As an attempt to further characterizeimmunoregulatory networks in the TA3-Ha animal model, we testedwhether Epi causes active suppression of the cell-mediated immuneresponse against TF determinants. In this study, we show that (a) s.c.injection of epiglycanin emulsified in either complete Freund's adjuvant

or Ribi adjuvant containing trehalose dimycolate and monophosphoryllipid A elicits a classical specific delayed-type hyperactivity response toTF haptens either naturally expressed on epiglycanin or as synthetichaptens conjugated to a protein carrier; (b) i.v. injection of as little as500 ng of Epi induces specific immunosuppression to anti-Epi and untili synthetic antigen delayed-type hyperactivity responses; (<â€¢)this im

munosuppression can be abrogated by i.v. injection of cyclophosphamideprior to immunizations; (il) Epi-induced specific immunosuppression canbe adoptively transferred by nylon wool-nonadherent cells 6 days following i.v. injection of Epi; (e) pretreatment of suppressor cell populationswith anti-Thy-1, anti-L3T4, anti-Lyt-1, or anti-I-Jk but not anti-Lyt-2

monoclonal antibodies plus complement prior to adoptive transfer abolished immunosuppression; (/) i.v. injection of immunosuppressiveamounts of Epi on Days 2 and 6 after transplantation of TA3-Ha cellsincreased the lethality of the tumor transplant. These results suggest thatEpi-induced specific immunosuppression in the TA3-Ha animal model ismediated by Thy-l+, L3T4*, Lyt-l'f2", and I-Jk+ suppressor cells. The

results are also consistent with the suggestion that this immunosuppression may enhance TA3-Ha tumor growth.

INTRODUCTION

We have recently (1-4) developed an animal model for ASI3using the murine mammary adenocarcinoma cell line TA3-Ha(5, 6). The TA3-Ha cell line, derived originally from a spontaneous tumor arising in an A/J mouse, produces a glycocalyxcontaining a mucin called epiglycanin (7, 8). Epi has an approximate molecular weight of 500,000 and is composed of 75-80% carbohydrate containing repeating TF (9) and Tn determinants. The TA3-Ha model has many similarities to humanadenocarcinomas (4) including the secretion into body fluids ofmucins which carry altered carbohydrate determinants including the TF antigen (10, 11) and its immediate precursor, Tn.

Many investigators have suggested that Epi associated with

Received 9/10/90; accepted 12/5/90.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by a grant from the National Cancer Institute of

Canada and by Biomira, Inc.2To whom requests for reprints should be addressed, at Department of

Immunology, 860 Medical Sciences Building, University of Alberta, Edmonton,Alberta, Canada T6G 2H7.

3The abbreviations used are: ASI, active specific immunotherapy; Epi, epiglycanin; TF, Thomsen Friedenreich; S-TAG, synthetic tumor-associated glyco-conjugate; BSA, bovine serum albumin; kl .11,keyhole limpet hemocyanin; HSA,human serum albumin; TF-a-KLH, /3Gal(l-3)aGalN Ac-keyhole limpet hemocyanin; TF-o-HSA, /3Gal(l-3)aGalN Ac-human serum albumin; DTH, delayed-type hypersensitivity; CFA, complete Freund's adjuvant; CY, cyclophosphamide.

the TA3-Ha tumor is responsible for its extremely high virulence (12). The TA3 mammary carcinoma arose spontaneouslyin a female A/HeHa mouse (13) and the strain-nonspecificsubline, TA3-Ha, was derived from a strain-specific subline,TA3-St (5, 12, 13). The ability of TA3-Ha tumor cells to groweven in foreign, H-2-incompatible mouse strains has been attributed to the presence of a thick glycocalyx composed primarily of Epi which has been shown to block the binding ofanti-H-2 antibodies to the cell surface of TA3-Ha cells (12, 14-16). Although there was no previous evidence for an immunosuppressive role of shed, circulating Epi, others have suggestedthat serum Epi may form blocking factors (17) in allogeneicmice. In support of this possibility it was observed that cell-freeascites fluid of TA3-Ha cells growing in mice of foreign strainsstimulates the growth of this tumor in normal mice (18).

In our ASI model (4) we have used S-TAGs containing TFhaptens as the main immunogenic epitopes conjugated to thecarrier protein KLH emulsified in Ribi adjuvant to immunizeTA3-Ha tumor-bearing mice. This basic "vaccine" formulation

provided approximately 25% long-term survival in groups ofmice bearing an otherwise 100% lethal tumor transplant. Lesseffective formulations which used purified Epi or X-irradiatedTA3-Ha tumor cells emulsified in Ribi adjuvant provided noprotection (4). However, when administration of the basic "vaccine" formulation was preceded by treatment with low dose Cy

up to 90% long-term survival was achieved (4). On the basis ofthese observations we have postulated (4) that Epi secreted bygrowing TA3-Ha tumors might induce a class of suppressorcells which render most attempts to achieve successful ASIrelatively ineffective. Direct evidence demonstrating a potentialimmunomodulatory effect of cancer-associated mucins is lacking, although high levels of serum mucins have been shown tocorrelate with poor prognosis in human cancer patients (19-21). In the present report, we show that i.v. injections of lowdoses of Epi induce a state of specific immunosuppression forDTH reactions to Epi and its associated TF epitopes. Evidenceis also presented that Epi induces a population of cyclophos-phamide-sensitive T-suppressor cells. Finally, it was demonstrated that i.v. injections of immunosuppressive quantities ofEpi can enhance the lethality of TA3-Ha tumor transplants.

MATERIALS AND METHODS

Mice. Eight- to 10-week-old pathogen-free female CAF1/J micepurchased from The Jackson Laboratory were used throughout thisinvestigation.

Tumor Cell Line. The TA3-Ha tumor cell line was originally providedby Dr. J. F. Codington (Massachusetts General Hospital, Boston, MA).The tumor cells were grown in vivo by weekly passage (i.p.) in CAF1/Jmice.

S-TAG and Control Antigens. TF-hapten [0-Gal(l-3)aGalNAc] conjugated to KLH or HSA were obtained from Biomira, Inc., Edmonton,Alberta, Canada. KLH was purchased from Calbiochem (La Jolla, CA).BSA and HSA was purchased from Sigma Chemical Co. (St. Louis,MO).

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IMMUNOSUPPRESSIVE ACTIVITY OF EPIGLYCANIN

PurifÃ¬cationof Epi. Ascites from BALB/c mice bearing TA3-Ha i.p.tumors was collected and cells were removed by centrifugation. Epifrom ascites was extracted on a peanut agglutinin lectin column asdescribed previously (1). The Epi preparation was characterized as asingle peak by fast protein liquid chromatography and the Epi activitywas detected and standardized with '"Â¡-labeled monoclonal antibody,49H.8 (22), and 125I-labeledpeanut agglutinin in a direct as well as a

sandwich radioimmunoassay (11). The lot of Epi used in these studieshad a carbohydrate:protein ratio of approximately 79:21 as determinedby the procedure of Lowry et al. (23), for the protein estimate and thephenolsulfuric acid method of Dubois et al. (24) for the carbohydratecontent. The amino acid composition of our preparation of Epi determined by the method of Moore (25) was found to be very similar to thepublished amino acid composition (12) of Epi containing approximately50% serine and threonine residues and approximately 9% proline,characteristic of O-linked mucin-like molecules. The amount of Epiused in this study was estimated using a quantitative sandwich radioimmunoassay (11).

Induction of DTH Responses to Epi and S-TAGs. Mice were giveninjections of 450 ng Epi or 50 Â¿igS-TAG emulsified in 50% CFA or0.2 ml Ribi adjuvant (containing trehalose dimycolate and monophos-phenyl lipid A) purchased from Ribi Immunochem Research, Inc.,Hamilton, MT. The emulsion was distributed equally among two s.c.sites in the upper belly and one site at the base of the tail. Seven daysafter immunization DTH was evaluated [as described previously (1)]following injection into the footpad of 450 ng Epi or 50 ^g S-TAG orcontrol antigens. Footpad thickness was measured with a vernier cali-per. The results, expressed as net footpad swelling, were calculated asthe difference in footpad thickness of test antigen and sterile saline-

injected pads at various time intervals minus the difference in footpadthickness of test antigen and sterile saline-injected pads before footpad

challenge.Induction of Immunosuppression. Mice were given i.v. injections of

various concentrations of Epi dissolved in sterile saline. Six to 7 dayslater, the mice were immunized with Epi + CFA or Epi + Ribi adjuvant,or S-TAGs emulsified in Ribi adjuvant in the manner described aboveand 7 days later were footpad tested for DTH responsiveness to Epi orS-TAGs. To test the immunosuppression induced by control antigenssuch as BSA or KLH, separate groups of mice were given i.v. injectionsof either 200 ng BSA or KLH in sterile saline. These animals wereimmunized and footpad tested with Epi, BSA, or KLH in a similarmanner.

Adoptive Transfer of Suppressor Cells. Spleens were collected frommice 6 to 7 days after i.v. injection of Epi. Single cell suspensions wereprepared by pressing tissues through a 100 mesh stainless steel screenin phosphate buffered saline. Spleen cells were treated with ammoniumchloride-Tris-buffered solution to lyse the erythrocytes. The cells werewashed three times, counted, and resuspended in phosphate bufferedsaline at a concentration of 6.4 x IO7cells/ml.

To determine the effects of suppressor cells on the induction of DTHresponses, adoptive transfers were done on the day of immunization ofthe recipients. These animals were tested for DTH responsiveness 7days later.

Antisera Treatment of Cells. Anti-Thy-1.2, anti-L3T4, anti-Lyt-2,and anti-I-Jk monoclonal (diluted 1:4) antibodies (kindly provided by

Dr. D. Green, Department of Immunology, University of Alberta) andlow toxicity rabbit complement (diluted 1:10; Cedarlane Laboratory,Hornby, Ontario, Canada), were used for cytotoxic depletion of cellsfrom splenic populations. Anti-Lyt-1.2 monoclonal antibody was purchased from Cedarlane Laboratory and was used at a final dilution of1:10. Enrichment for T-lymphocytes on nylon wool columns was performed according to previously described procedures (26).

CY Treatment. In an attempt to reverse Epi-induced immunosuppression, Epi- or saline-treated mice were treated i.v. with 100 mg/kg CY(Sigma) prior to immunization with appropriate antigens.

Statistical Analysis. Student's t test was used to test differences in

DTH reactivity among groups and modified Wilcoxon was used toevaluate survival differences.

RESULTS

Induction and Specificity of DTH Responses Elicited by Epi-glycanin and S-TAGs. We performed studies to establish thekinetics of a direct DTH reaction to epiglycanin following s.c.immunizations with Epi emulsified in appropriate adjuvants(Fig. 1). Maximum DTH reactions occurred between 24 and30 h in Epi + CFA- or Epi + Ribi adjuvant-immunized mice.No obvious early peak of DTH reactivity was observed between2 and 6 h after antigen challenge as was observed in otherantigenic systems (27). The DTH reactivities were still high at48 h and by 72 h they started to subside. By 96 h, the swellingsdecreased to an undetectable level. The kinetics of this DTH isdifferent from that of an antibody-mediated Arthrus reactionin which a single peak swelling is observed 4-10 h after antigenchallenge and similar to our previous data using an adoptivetransfer system for DTH (1) to Epi and S-TAGs. Mice immunized with nonspecific control antigens in CFA or Ribi adjuvantalways gave negligible footpad swelling (data not shown). Inseparate experiments (data not shown) we showed that DTHreactions to Epi are transferable with immune spleen T-cellswhich is in agreement with our previous studies (1).

Table 1 shows that Epi-induced DTH reactions can be specifically directed to TF (a) epitopes, again confirming ourprevious specificity studies using an adoptive transfer system(1). Mice immunized with Epi s.c. were able to mount a strongDTH response to Epi or the TF-a-HSA glycoconjugate, butnot to the carrier protein HSA. Using the tumor-associatedglycoconjugate (TF-a-KLH) as an immunizing antigen, we canalso generate a specific DTH reaction to the TF-a determinant(Table 2).

Induction of Immunosuppression. The next series of experiments were designed to investigate the effect of i.v. administration of Epi on DTH responses. Fig. 2 shows that mice receivingi.v. preinjections of Epi had a dose-dependent reduction in their

1 0.6

o-o Epi-CFA_â€¢ Epi-Ribi

0 2 6 12 24 30 48 72 96Hours after loot pad challenge

Fig. 1. Kinetics of DTH reactivity of CAF1/J mice to Epi 7 days afterimmunization with 450 ng Epi emulsified in either CFA or Ribi adjuvant. Footpadtesting antigen = 450 ng Epi in sterile saline. Each point represents an averageobtained from 3-4 mice. Bars, SD of the mean.

Table 1 Direct DTH responsiveness to TF-u determinants

TreatmentsEpi

+CFAEpi

+ RibiFootpad

antigenEpi

TF-a-HSAHSAEpiTF-a-HSA

HSANet

footpad swelling(mm Â±SD)"24h0.53

Â±0.040.43 Â±0.180.03 Â±0.030.33 Â±0.040.40

Â±0.010.02 Â±0.0348h0.1

5 Â±0.070.30 Â±0.210.03 Â±0.060.18Â±0.040.38

Â±0.110.03 Â±0.03

" Average of three mice. Each animal was immunized s.c. with 450 ng Epi

emulsified in either CFA or Ribi adjuvant. Mice were footpad tested for DTHresponsiveness to TF-Â«determinants 7 days after immunization.

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Table 2 Direct DTH responsiveness lo the TF-a hapten following immunizationwith TFa-KLH

Treatments50

^g TFn-KLH emulsifiedin 0.2 ml Ribi adjuvantFootpad

antigenTF-n-KLH

KLHTF-a-HSAHSAEpiNet

footpad swelling(mm Â±SD)"24

h0.38

Â±0.100.71 Â±0.200.33 Â±0.050.02 Â±0.020.35 Â±0.0548

h0.27

Â±0.110.42 Â±0.100.32 Â±0.030.00 Â±0.000.25 Â±0.05

" Average of three mice. All mice were footpad tested for DTH 7 days after

immunization.

Uniti; Of Epi

NS (n=4)

1.000

5,000

11.000

Salina n=5)

0.0 0.1 0.2 0.3

Mean DTH Response (mm +/- SEM)

Fig. 2. Dose response of immunosuppressive effect of epiglycanin on the DTHresponse to Epi. Mice were given i.v. injections of various concentrations of Epi(1 unit of Epi = I ng) or saline. Six days later all mice were immunized with Epiplus Ribi adjuvant and 7 days later they were footpad tested for DTH to Epi.n = number of mice/group. **'*. P < 0.0001; ***. P < 0.0005; *'. P < 0.005:*,P< 0.01; A'Snot significant Â»hencompared to saline injected control group.

DTH reactivities to Epi. It was found that as little as 500 units(=500 ng) still produced significant suppression. The nextexperiment examines the specificity of induction of immuno-suppression by unrelated antigens. Control mice which weregiven i.v. injections of either sterile saline, BSA, or KLH andlater immunized with Epi plus Ribi adjuvant s.c. all elicited astrong DTH reaction at 24 h (Fig. 3). When mice were givenBSA or KLH i.v. and subsequently immunized with the homologous antigens their DTH responses to the same antigenwere also significantly depressed (Groups 7 and 10) but suchtreatments had no effect on the induction of DTH responses toEpi (Groups 8 and 11).

Adoptive Transfer of Immunosuppression by Spleen Cells. Toinvestigate the possibility that Epi can induce active suppression, we collected spleen cells from donor mice given i.v.injections of 1.8 ng Epi 6-7 days earlier. The cells were transferred to normal syngeneic mice at the same time the recipientswere immunized. Seven days later, the recipient animals werefootpad challenged, and footpad thicknesses were measured 24h after challenge. Cells capable of suppressing the DTH response following adoptive transfer were detected in the spleensof Epi-treated mice while spleen cells from control mice hadno significant effect on the DTH response of the recipients(Table 3).

Specificity Studies of Cells Mediating Active Suppression. Toassess the specificity of active suppression, spleen cells from

Group1

2

3

4

5

6

7e91011Treatment

otmicetreatment

(Day0)Saline

0.9Â«|Ep.

1 Bug Epi

Saline

1 8 â€ž¿�9Ep.

Saline

200 M9 BSA

200 ug BSA

Saline

200 |ig KLH

200 U9 KLHwith

Ag+Rilx/CFA(Oil6)Epi.CFA

Epi.CFA

Epi.CFA

Epi*Ribi

Epi.Ribi

BSA.RiDi

BSA.Ribi

Epi.Ribi

KLH.Ribi

KLH.Rir*

Epi. HitÂ«DTH

responses 10 immunizingantigen24

h reading of mean increasefootpad thickness

(10-2mm) *SO (Day13)0

10 20 30 4050HZH

>H>Hâ€¢yiâ€”

iHZHH%

Suppression81.1

905900100.0

11.1825

11.1p

Value<

0001e

<0001e<001'Â«.005Â«

NS'<.005'

NS<

Fig. 3. Specificity of induction of immunosuppression of the DTH responseby unrelated antigens. (Ag). a, mice given injections of either sterile saline orantigen in a volume of 0.4 ml; 3 mice/group. A, mice immunized s.c. with either450 ng Epi or 50 ^g antigen emulsified either in 0.2 ml Ribi adjuvant or equalvolume of CFA; c, compared with group \;d. compared with Group 4; e, footpadtested with 50 fig BSA, compared with Group 6; / footpad tested with 50 figKLH. compared with Group 9. NS, nonsignificant.

Table 3 Adoptive transfer of immunosuppression of DTH responsiveness byspleen cells

DTH test (Day 13):Spleen cells Net footpadtransferred s.c. swelling % of

i.v. treatment (xlO7) immunization (mm Â±SD)' reduction(Day 0) (Day 6) (Day 6)" after 24 h(P)0.4

ml saline 6.41.8 jig Epi in 0.4 ml 6.4

salineEpi

+ CFA 0.37 Â±0.11Epi 4- CFA 0.05 Â±0.07 86.5

(<0.001)Â°Mice were immunized s.c. with 450 ng Epi emulsified in CFA.* Average of five mice. All mice were footpad tested for DTH responsiveness

to Epi (450 ng/footpad) 7 days after immunization.

Intravenoustreatments

(Day0)0

4 mlSalinei

8 nfl Ep.in 0 4 mlSalmeNWN

cells3transferred

(Day6)2x107

2 HO7Subcutaneous

(Day6)TFoKLH+Ribi

TFaKLH+RibiFool

pad Ags(50â€ž¿�g)TFaHSA

HSATFaKLHKLHTFaHSA

HSATFaKLH

KLHMean

increase footpad0thickness (IO'2 mm * S.D.)

(Day13)0

10 20 30 40SOHZHHH.

HH%Suppression(p

Value)NS47%

(p<005)NS

Fig. 4. Adoptive transfer of active suppression induced by epiglycanin. a,NfVN, nylon wool-nonadherent T-lymphocytes. b. average of three mice, DTHresponses read at 24 h. NS, nonsignificant; Ags, antigens.

Epi-treated mice were adoptively transferred to naive mice atthe time of immunization with TF(a)-KLH-t-Ribi. Seven days

after immunization, each group of mice was footpad challengedwith the homologous antigen, the carrier protein as well as withTF antigen conjugated on HSA, a different protein carrier. Fig.4 shows that nylon wool-enriched T-cells from mice treatedwith Epi inhibited the DTH response to TF-a-HSA by 47% (P< 0.05) whereas the same cell population exerted no effect onDTH response to the protein carrier KLH.

Characterization of the Cells Mediating Active Suppression. Data from Fig. 4 show that the Epi-induced spleen cellsmediating suppression were present in the nylon wool-non-adherent fraction which presumably contain T-cells. To further

characterize these cells, experiments were done to establishtheir surface phenotype by a cytotoxic antibody depletion technique. Recipients of untreated or antibody-treated cells wereimmunized on the day of cell transfer and footpad challenged7 days later. Treatment of cells before adoptive transfer with

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complement alone or anti-Lyt-2 plus complement had no effecton the ability of Epi-induced spleen cells to inhibit the DTHresponse (Fig. 5, Groups 3 and 7), whereas treatment with anti-Thy-1, anti-L3T4, anti-Lyt-1.2, or anti-I-JK antibodies pluscomplement abolished the soppressivi.- activity of the cells

(Groups 4, 5, 6, and 8 compared with Groups 2 and 3). Theseresults indicate that the cells mediating active suppression inthis system are Thy-l+, L3T4+, Lyt-r2", and I-J+ T-lympho-

cytes.Abrogation of Immunosuppression following CY Treatment.

CY has been shown to inhibit the development of activesuppression mediated by T-cells in experimental systems,

thereby allowing the host to generate an augmented level ofimmunity to a variety of antigens. Thus we investigated whetherCY pretreatment would restore the DTH response in Epi-treated mice. CY was injected i.v. at a dose of 100 mg/kg l dayprior to immunization with appropriate antigens. The animalswere then footpad challenged in the usual manner 7 days afterimmunization. Table 4 shows that the Epi-induced DTH responses in mice given i.v. injections of Epi were significantlysuppressed (82.4 and 81.9% suppression, P < 0.005), whereasEpi-treated mice which were subsequently given injections ofCY 24 h prior to immunization recovered their DTH reactivities(19 and 47.6% enhancement).

Effect of i.v. Epi Injections on Survival of TA3-Ha Tumor-bearing Mice. We used our new radioimmunoassay (11) tomonitor the presence of Epi in the circulation of TA3-Ha-bearing mice. CAF1 mice were given i.p. injections of approx-

Group1

2

3

4

5

6

7eTreatment

olmicei.v

â€¢¿�treatment(Day0)Saline

Epi

Epi

Epi

Epi

Epi

Epi

EpiTreatment0

of donorcells pnor to

transferC'

Thy.UC1

L3T4Â»C-

Lyt1.2Â»Cp

Lvt2*C'

W+C'Immunized

with450 nÃ§.Epi

S.C. (Day6)+

+DTH

responses toEpic24

h reading of mean increasefootpad thickness

(10 2mm) Â«S.D. (Day13)0

10 20 30 40 50|

1O

CZHh-1râ€”

1HOH%

Suppression89.3

82225.082.214.3P"

Value<.050

<.050NS<050

NS

Fig. 5. Characterization of Epi-induced spleen cells mediating active suppression, a, mice given injections of either sterile saline or 1.8 ng Epi in a volume of0.4 ml; 3 mice/group, b, 6 days after i.v. treatments, spleen cells pooled fromeach group of animals and 0.5-1.0 x 10* cells were treated with monoclonalantiserum plus complement (C') or complement alone prior to cell transfer.Percentage of cytotoxicity of cells after antiserum treatment with Thy-1, L3T4,Lyt-1.2, Lyt-2, and I-Jk were 40, 23, 20, 23, and 24%, respectively, c, all micefootpad tested with 450 ng Epi; d. /"compared to Group 1. NS, nonsignificant.

Table 4 Effect of cyclophosphamide treatment on immunosuppression

Intravenoustreatments(Day

0)Saline0.9

MgEpi0.9

Â»gEpi1.8

Â»igEpi1.8/igEpiSalinei.v.

CY/salinetreatments(Day

6)SalineSalineCYSalineCYCYs.c.

immunizationwith450 ngEpi(Day

7)Epi

+CFAEpi+CFAEpi

+CFAEpi

+CFAEpi

+CFAEpi

+ CFAMean

increasefootpadthickness(mm

Â±SD)Â°(Day

14)0.21

Â±0.080.04Â±0.020.25

Â±0.090.04

Â±0.020.31

Â±0.180.21

Â±0.05%ofsuppression

(â€”)/enhancement(+)(P)-82.4(<0.005)+

19.0(NS)*-81.9(<0.005)+47.6(NS)

â€¢¿�All mice were footpad tested with 450 ng Epi, 24-h readings.

CY, cyclophosphamide, 100 mg/kg body weight. Three mice/group.* NS, nonsignificant.

imately 700 TA3-Ha cells and bled on Days 3, 8, and 14. Therewas no detectable Epi in the blood on Days 3 or 8 but by Day14 all 5 mice tested had over 28.0 pg Epi/ml of blood (data notshown). We then investigated whether i.v. injection of Epi attimes when no detectable levels were apparent (Days 2 and 6)would accelerate the development of the tumor. Fig. 6 showsthat mice which had received i.v. treatments with Epi hadsignificantly accelerated death rates as compared to controlmice which had been given injections of saline or BSA.

DISCUSSION

Many studies have shown that tumor-associated mucins canbe detected in the sera of most adenocarcinoma patients (19,20, 28-32) and that high levels of cancer-associated mucinsappear to correlate with poor prognosis in some human cancerpatients (20, 21). Steck and Nicolson (33) observed that theconcentration of a mucin-like cell surface glycoprotein (A/r580,000) was correlated with the metastatic potential in a seriesof clones of rat mammary adenocarcinoma cells. The expressionof a sialomucin on rat mammary tumor cells has been correlatedwith natural killer cell susceptibility (34). In breast cancerpatients increased expression of TF antigen has been correlatedwith tumor progression and metastatic spread (35). Many additional studies suggest that TF antigen expression is associatedwith poor prognosis or malignant change (36-43). These andother findings suggested to us that circulating mucins in generaland TF-bearing antigens in particular might inhibit the host's

anti-tumor immune response. This prompted us to investigatewhether mucins might inhibit cell-mediated immune responsesin our TA3-Ha model. The TA3-Ha tumor produces a glyco-calyx containing a mucin called epiglycanin (7,8), a M, 500,000glycoprotein composed of 75-80% carbohydrate. Epi containsmultiple TF and Tn determinants which are also expressed onover 90% of human adenocarcinomas (9) as well as on mucin-like molecules in body fluids of adenocarcinoma patients (10,11). Epiglycanin is also secreted into body fluids (17). In orderto address the question of how the presence of mucins in bodyfluids might affect the immune function of the host, we injectedi.v. various concentrations of epiglycanin purified from TA3-Ha tumor ascites fluid and assessed the effect on subsequentDTH responses elicited against the homologous epiglycanin

S? 40-

I.V.EPI

I.V.BSA

Treatment

Days After TA3Ha Tumor Cell Challenge

Fig. 6. Effect of i.v. epiglycanin treatments on survival of TA3-Ha tumor-bearing mice. All animals (8 mice/group) were first given i.p. injections of 700TA3-Ha tumor cells on Day 0. One group of animals was treated i.v. with 1.8 jigEpi on Days 2 and 6, whereas the other group was either left untreated or treatedi.v. with 100 iig BSA on the same days. Survival of the Epi-treated group wassignificantly decreased compared to both the untreated group (P < 0.0005) andthe BSA-treated group (/>< 0.001, modified Wilcoxon).

1173




antigen or TF haptens conjugated to a protein carrier. We havefound that i.v. injected Epi induces specific immunosuppressionas well as cells capable of specifically inhibiting DTH reactionsin response to both Epi and the TF-Â«epitope. The cells mediating active suppression were detectable in the spleen 6 daysafter Epi injection. Immunosuppressive activity in this systemwas shown to be hapten specific since adoptive transfer of thespleen cells mediating suppression inhibited the induction ofthe DTH response to the TF-a hapten but had no effect on theresponse to the protein carrier KLH. However, when controlantigens such as BSA or KLH at a tolerogenic dose wereadministered i.v. into mice that were immunized s.c. with Epi+ Ribi 6 days later, no suppression of the Epi-induced DTHresponses was observed, indicating that the induction of immunosuppression of the DTH response observed in this studywas mucin induced and antigen specific.

In order to determine the nature of the cells mediating activesuppression we passed the splenic cell pools over nylon woolcolumns to remove the majority of B-cells and macrophagesand to enrich for T-cells prior to cell transfer. Using thisprocedure, we ascertained that the nylon wool-nonadherent cellpopulations from Epi-treated mice contained the suppressorcells. These results suggested that the cells responsible for thesuppression were T-lymphocytes. When the suppressive spleniccell populations were treated with anti-Thy-1 antibody pluscomplement prior to adoptive cell transfer, the suppressiveactivity was ablated, confirming that T-lymphocytes were required for the activity. Abrogation of the suppressive activityby treatment of the spleen cells with anti-L3T4, anti-Lyt-1.2,or anti-I-JK antibody plus complement but not with complementalone or those treated with anti-Lyt-2 and complement wasobserved, indicating that the cells mediating the suppressionwere L3T4+, Lyt-T2- and I-J+ T-cells.

Antigen-specific T-cells which suppress humoral and cell-mediated immune responses (44, 45) in many other systemshave been described. Such suppressor cell systems include thosethat regulate the DTH responses to azobenzene arsenale (46)and those that regulate the DTH and/or the antibody responsesto 4 hydroxynitrophenylacetyl (47, 48) and sheep erythrocytes(44). Suppressor T-cells can also be demonstrated to inhibitantitumor immunity (49, 50). Recently, Takahashi et al. (51)demonstrated that in B16 melanoma system, suppressor T-cellsgenerated by the soluble form of a tumor-associated antigenwere able to block the induction as well as the effector phase ofcytotoxic T-lymphocytes. Analysis of known antigen-specificsuppressor cells revealed that at least two populations of suppressor T-lymphocytes are involved in immune regulation (51).Induction-phase suppressor T-cells (TS1), which can be generated by antigen alone has the phenotype of L3T4+, Lyt-l*2~,and I-J*, whereas the effector-phase T-suppressors (TS2), which

depend for their generation on both antigen and TS1, wereL3T4~, Lyt-2+l", and I-J+. The suppressor T-lymphocytes iden

tified in this study, shown to have a suppressive effect on theinduction phase of specific DTH response to Epi and TF-aepitopes, were ThyT, L3T4+, Lyt-T2~, and I-JK+ T cells and

thus resembled the TS1 suppressor described in other antigen-specific suppressor cell systems (44, 45).

Codington et al. (10) demonstrated that many patients withhigh concentrations of epiglycanin-like antigen suffer fromadvanced metastatic cancer. Furthermore, it has been reportedthat the metastatic potential in various TA3-Ha ascites variantswas proportional to the epiglycanin concentration (16). Othershave suggested that shed Epi might be immunosuppressive (17,

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18). Our data show that Epi molecules are highly immunosuppressive for specific DTH reactions following their introductioninto blood circulations. This suggested to us that tumor-associated mucins which are shed into the blood circulation mightaccelerate the disease. In support of this hypothesis we havefound that i.v. injections of immunosuppresive amounts of Epiincreased the lethality of the TA3-Ha tumor transplant.

Early studies in mice have shown that doses of antigen aboveor below the usual immunizing range tend to induce high andlow zone tolerance, respectively (52-54). Injecting antigen i.v.has also been shown to be a way of providing a tolerogenicsignal (55). A dose of 10-40 Â¿igBSA and those of 5 mg orabove were effective in inducing a low and high zone tolerancein mice, respectively (52). In the present studies, we havedemonstrated that i.v. injection of a very low dose of Epi (500ng) is immunosuppressive for DTH reactions to Epi and itsassociated TF epitopes. Because of previous work demonstrating the immunosuppressive properties of heterologous proteinsinjected i.v. (52-55) it might be argued that there is nothingunique about the immunosuppressive properties of Epi. However, we believe that the present work should be viewed in thecontext of the important biological questions which are beingasked. Do mucin molecules which are normally sequestered insecretory granules or on mucosa! surfaces but which are secretedby most adenocarcinomas in altered form into the blood circulation have an effect on the immune response to the importantcancer-associated carbohydrate epitopes that they carry? Thisquestion assumes even greater importance if one considers thathigh mucin levels and/or high TF antigen expression oftencorrelates with poor prognosis or rapid tumor progression (10,20, 21, 33-43) despite the fact that TF on various natural orsynthetic glycoconjugates can be highly immunogenic (1-4, 9,56-61) and that cancer-associated carbohydrate antigens canapparently serve as effective targets for the immune destructionof tumor cells which carry those antigens ( 1-4,62-65). A logicalconclusion is that the immune response to this class of cancer-associated antigens is ineffective and/or suppressed. The present communication provides a rationale for the latter conclusionbut does not rule out the former.

In another study of active specific immunotherapy of micebearing TA3-Ha tumor transplants (4) we failed to provide anyprotection of animals which had received subsequent activeimmunizations with Epi or irradiated whole TA3-Ha tumorcells emulsified in Ribi adjuvant. We suggested that this failuremay have been due to an active immunosuppression induced bycirculating epiglycanin shed from the growing tumor and thepresent report supports that hypothesis. The suggested mechanism predicts that pretreatment with CY, an agent which atan appropriate dosage can preferentially inhibit the development of suppressor T-cells (66-68), should provide relief ofimmunosuppression in the tumor-bearing animals. In fact inthe same study we were able to show that tumor-bearing animalswhich were treated with low dose CY followed by active immunizations with TF-cv-KLH were able to achieve up to 90%long-term survival. In the present study, pretreatment of epi-glycanin-treated animals with CY also led to a successful recovery of their DTH reactivities to the immunizing agent. Thisadds further support to the potential therapeutic effect of CYin the active specific immunotherapy model. Our previous work(1) demonstrated that effector cell populations with a T-helperphenotype which showed a strong DTH reactivity to TF epitopes also gave specific antitumor effects when tested with TA3-Ha tumor cells in a Winn-type assay. At present we are studying



IMMUNOSUPPRESSIVE ACTIVITY OF EP1GLYCAN1N

these effector T-cell populations at the clonal level and plan toinvestigate the effect of suppressor cell populations on theirexpression.

ACKNOWLEDGMENTS

We thank T. Tan and M. Hajek for excellent technical assistance.We are grateful to Dr. D. Green for helpful suggestions as well as forreviewing this manuscript.

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1991;51:1170-1176. Cancer Res Peter Y. S. Fung and B. Michael Longenecker Adenocarcinoma (TA3-HA)Mucin-like Glycoprotein Secreted by a Murine Mammary Specific Immunosuppressive Activity of Epiglycanin, a

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