SEROTHERAPY OF AVIAN RETICULOENDOTHELIOSIS VIRUS-INDUCED TUMORS

PART VL. SPECIFIC IMMUNOTHERAPY (ANTIBODIES ) FOR TREATMENT OF CANCER

SEROTHERAPY OF AVIAN WTICULQENDOTHELIOSIS VIRUS-INDUCED TUMORS *

Cheng-po H u t and T. Juhani Linna

Department of Microbiology and Immunology Temple University School of Medicine

Philadelphia, Pennsylvania I9140

INTRODUCTION

Current concepts suggest that thymus-dependent cell-mediated immunity may play an important role in host defense against antigenic tumors.'* The role of humoral immunity in malignancy is less well defined. In vitro evidence indicates that serum fatcors may act to block the activity of immune lym- phocytes, thereby allowing the tumor to circumvent the rejection mechanism of the host.3 These factors have been suggested to be blocking antibodies4 or antigen-antibody complexes.6* However, recent findings indicate that the tumor-protective effect of serum is mediated by circulating antigens shed from tumor cell membrane or by antigen-antibody complexes in antigen

Humoral immunity may play a host-protective role in malignancy. It has been shown in many tumor systems that antibodies and complement are cytotoxic to tumor cells in vitro.lo* l1 Antibodies can also kill tumor cells by interaction with certain nonimmune lymphoid cells in vitro in antibody-dependent cell-mediated cytotoxicity assays.1214 The ability of passively administered antiserum to provide protection against tumor growth in vivo has been observed in several experimental systems, Perhaps the first convincing demon- stration of the importance of antiserum in tumor rejection was presented by Gorer and Amos15 in studies of the allogeneic rejection of the EL4 leukemia in mice. Studies of the protective role of antibodies have subsequently been extended to well-defined syngeneic tumor models wherein the antiserum was directed at virally induced antigens on the cell surface.16 Tumors induced by murine sarcoma virus (MSV) have been particularly susceptible to the activity of immune serum. It has been shown that immune serum against Moloney sarcoma can prevent tumor growth 17, l8 and induce tumor regres~ion.'~ With passive administration of the antiserum against PVGc rat leukemia, HerseyZ0 showed a good correlation between the in vitro activity of antisera in antibody-dependent cell-mediated cytotoxicity ( ADCC) against tumor cells and their in vivo protective effect. Although quite impressive results have been observed on effects of antisera on tumor development, in most of the cases, antibodies were only effective when given before tumor inoculation. Success- ful serotherapy of established tumors has been relatively rare.

The present study demonstrates that the passive administration of im-

*Supported by Grant CA 13347 from the National Cancer Institute, by Grant IN-88F from the American Cancer Society, and by a National Institutes of Health General Research Support grant to Temple University.

f Present address: Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md. 20910.

634

Hu & Linna: Reticuloendotheliosis Virus & Tumors 635

mune serum has a significant protective effect on already detectable tumors caused by avian reticuloendotheliosis virus, strain T (RE virus). RE virus is an avian RNA tumor virus outside the avian leukosis-sarcoma complex that produces reticuloendothelial tumors (RE tumors) mainly in the liver and spleen of infected chicken^.^^-^^ When RE virus is injected locally into the wing web, tumors of large mononuclear cells are RE virus can cause high mortality in a short period of time. This virus-induced malignancy was selected for our studies because of the high and predictable oncogenicity and the relatively short course of disease. Our previous studies have shown that not only thymectomy 24, 2B but also bursectomy, either surgical or chem- i ~ a 1 , ~ ~ ~ 25 significantly increased tumor mortality in chickens infected with RE tumors. Regression of tumors occurred less frequently in bursectomized than in control birds. These data and similar findings with other, unrelated tumors indicate that the bursa-dependent antibody-forming system may have a host- protective function in RE tumor development and also in the development of other tumors. The present paper shows that the passive administration of immune serum obtained from animals having undergone regression of R E tumors can induce regression of established RE tumors and reduction of tumor mortality, thus further substantiating the host-protective role of the antibody-forming system in this malignancy.

MATERIALS AND METHODS

Animals

Hy-line WC line chickens were used in all of these studies. These chickens are isogenic for the major histocompatibility blood group Iocus, carrying the BZ/B2 genotype. The numbers of animals in each experimental or control group can be found in the Tables.

Thymectomy (Tx) and Bursectomy (Bx)

Surgical thymectomy was performed on the day of hatching as described by Peterson et aLZ6 Chemical destruction of the bursa and the bursa-dependent system was performed by injecting chicks intraperitoneally with 4 mg of cyclophosphamide daily for the first 3 days of life. Such treatment has been demonstrated to lead to profound agammaglobulnemia and a lack of antibody- producing capacity in a vast majority of the birds without affecting the cell- mediated immunologic functions, when the chickens are evaluated at 1 month of age or later.27

Virus Preparation from Infected Spleens

The avian reticuloendotheliosis virus, strain T (RE virus), was initially obtained from Dr. R. G . Fischer, University of North Dakota, in the form of infected spleen tissue. Stock suspensions of virus preparation were made from infected spleen tissue in the following way. Spleens from moribund chickens infected with RE virus were collected and homogenized in Hanks' balanced

636 Annals New York Academy of Sciences

salt solution (HBSS) with a Waring blender. The homogenate was diluted to a 25% (w/v) suspension in HBSS and was centrifuged at 2000g for 20 min. The supernatant was collected and frozen in aliquots at -70" C. This preparation was used for tumor induction.

Virus Preparation from Cell Culture

Chick embryo fibroblasts were maintained in Eagle's minimal essential medium (MEM) with 2% heat-inactivated fetal calf serum, supplemented with 100 U of penicillin and 100 pg of streptomycin per milliliter of medium. The monolayer was infected with an RE virus preparation from infected spleens. After 72 hr, the culture fluid was collected and centrifuged at 2000g for 20 min to remove cellular debris. The supernatant was then centrifuged at 80,OOOg for 2 hr; the resultant pellets were used as the source of virus for absorption studies.

Virus Administration

Normal and Tx chickens were inoculated with 0.5-2 LD,, of RE virus into the wing web at 3 days of age. The cyclophosphamide-Bx chickens and their controls were infected with 1 LD,, of RE virus at 1 month of age, when the immune defect was specific for humoral immunity.

Immunization Procedures

Three-day-old chickens were immunized with a sublethal dose (0.1 LD,,) of RE virus into the wing web. Four weeks later, chickens were challenged with a high dose (5 LD,,s) of RE virus, which killed 90% of the nonim- munized birds of the same age. The sera of the immunized chickens that were resistant to this challenge were collected 6 weeks after the last inoculation, pooled, and stored at -70" C until use.

Serum Administration

Passive administration of immune serum was started 6 days after RE virus inoculation. At that time, approximately 60% of the birds had de- veloped local tumors at the inoculation site. A few days later, virtually all infected chickens were tumor bearing. One half milliliter of the immune serum was injected intravenously to experimental groups of chickens every other day with a total of nine injections. Control animals were injected with the same dose of virus and treated with control sera or remained untreated, as specified under RESULTS and in the Tables.

Cross-Protection Study

A study was performed to evaluate whether the protective effect of immune serum was tumor specific. For this purpose, three groups of chickens

Hu & Lima: Reticuloendotheliosis Virus & Tumors 637

were injected with the JMV strain of Marek's disease (originally obtained through the courtesy of Dr. M. Sevoian, University of Massachusetts) intraperitoneally on the day after hatching. One group of chickens received 0.5 ml of RE immune serum on the fourth, sixth, eighth, and 10th days after virus inoculation. Two control groups were treated with normal chicken serum or remained untreated.

Absorption of Immune Serum with RE Virus

To remove antiviral antibodies, the immune serum was absorbed with RE virus, Virus was obtained from tissue culture fluid of chick embryo fibroblasts infected with virus preparation from infected spleens. Absorption of immune serum by RE virus was performed by mixing 1 vol of serum with the pellets obtained from centrifugation at 80,OOOg of 2 vol of RE virus-containing culture fluid. The mixture was incubated at 37" C for 1 hr and then at 4" C overnight. After incubation, the serum was centrifuged at 80,OOOg for 2 hr to remove viruses and virus-antibody complexes. After each absorption, counter immunoelectrophoresis was used to detect the presence of antiviral antibodies contained in the serum. This absorption procedure was repeated five times until the precipitin line found between virus and nonabsorbed antiserum could no longer be detected.

Absorption of lmtnune Serum with RE Tumor Cells

A volume of immune serum was absorbed with an equal volume of packed RE tumor cells obtained from the spleens of infected animals. The serum and cell mixture were kept at 4" C for 1 hr with occasional shaking. After incubation, the suspension was centrifuged, and the supernatant serum was absorbed twice more in the same way but with a 2:l ratio (v/v) of serum to packed cells. After the third absorption, the suspension was centrifuged at 2000g for 20 min, and the supernatant serum was then centrifuged at 80,OOOg for 2 hr.

Fractionation of y Globulins

A 7-globulin fraction was obtained by (NH,),SO, precipitation at 35% saturation according to HeberLzX

Evaluation of Tumor Development and Tumor Mortality

Local wing web tumors started to appear 3 4 days after virus administration. Tumor development was followed by measuring local tumor sizes and by tumor mortality. The moribund and dead chickens were examined at autopsy for RE tumors. Tumors in the liver and the spleen were determined macroscopically. In questionable cases, the tissue samples of liver, spleen, and local tumors were fixed in 10% phosphate-buffered formalin and processed by standard histologic techniques for microscopic examination. Animals that died of nonspecific causes were excluded from evaluation. Differences be-

63 8 Annals New York Academy of Sciences

tween experimental and control animals in terms of tumor regression and survival were evaluated by Fisher’s exact test with fourfold tables.20

RESULTS AND THEIR INTERPRETATION

Eflect of Immune Serum on Development of RE Tumors in Normal Chickens

When immune serum or normal serum was administered to normal RE tumor-bearing recipients, significantly more immune serum-treated (94%, or %e) than normal serum-treated (19%, or 946) animals showed local tumor regression and survived. Among the untreated birds, local tumor regression occurred in 38% (%a) of the animals, and 25% (4113) of them were cured from the tumor and survived (TABLE 1). These results show clearly that immune serum causes regression of RE tumors and protects chickens from tumor death, with statistically significant differences between experimental and control groups.

TABLE 1 EFFECT OF IMMUNE SERUM ON DEVELOPMENT OF RE

TUMORS IN NORMAL CHICKENS

Regression of Local Tumors Survivors

Treatment (% 1 P (% 1 P Immuneserum 94 (15/16) <0.01 * <0.002t 94 (15/16) <0.002 * t Normalserum 19 (3/16) 19 (3/16) None 38 (6/16) 24 (4/16)

* Comparison with no treatment. t Comparison with normal serum treatment.

Effect of Immune Serum on Development of RE Tumors in Bursectomized Chickens

To exclude the contribution of humoral immunity of the host on tumor cure in the above study, serotherapy was performed in cyclophosphamide-Bx recipients. Local tumor regression occurred in only 53 % (%e) of the normal serum-treated group and in 50% (946) of the untreated group. In contrast, the tumors regressed in 90% (%o) of the immune serum-treated animals. In terms of survival rate, significantly more animals survived in the immune serum- treated group than in control groups (90 vs 37 and 12% ) (TABLE 2). There- fore, treatment with immune serum resulted in significant regression of already detectable tumors and in marked reduction of tumor mortality in animals deficient in the bursa-dependent system and antibody-forming capacity. Fur- thermore, immune serum was equally effective in curing Bx and normal tumor-bearing animals (TABLE 2). Therefore, it seems that the B-cell system


TABLE 2

CYCLOPHOSPHAMIDE-BURSECTOMIZED ( Bx) CHICKENS EFFECT OF IMMUNE SERUM ON DEVELOPMENT OF TUMORS IN


Group (% 1 P (% 1 P Bx, immune serum

Bx, normal serum treated 53 (10/19) 37 (7/19)

Bx, untreated 50 (8/16) 12 (2/16) Normal, immune

serum treated 94 (17/18) 94 (17/18)

* Comparison with Bx, normal serum-treated or untreated.

treated 90 (18120) <0.01 * <0.002 t 90 (18/20) <0.01 *

and humoral immunity of the host do not significantly contribute to the mechanism of serotherapy.

Effect of Immune Serum on Development of RE Tumors in Thymectomized Chickens

To study the possibility that host T celIs may contribute to the serotherapy mechanism, Tx chickens were treated with immune serum. The local tumors regressed in 53% ('940) of the untreated Tx animals, and 43% (%o) survived, whereas in the immune serum-treated Tx group, 71% (1%7) showed local tumor regression and survived. A group of sham-Tx chickens was included in this experiment. After treatment with immune serum, 95% (%3) of the sham-operated animals showed tumor regression and were cured (TABLE 3). It appears from the data shown in TABLE 3 that immune serum is able to contribute to host defense to some extent in Tx hosts. It is capable of increasing animal survival from 42 to 71% but not to the 95% survival level obtained in the immune serum-treated group with an intact thymus-dependent system.

TABLE 3 EFFECT OF IMMUNE SERUM ON DEVELOPMENT OF RE TUMORS

IN THYMECTOMIZED (Tx) CHICKENS


Group (% 1 (% 1 Tx, immune serum treated 71 (12117) 71 (12/17) Tx, untreated 53 (10/19) 42 (8/19) Sham-Tx, immune serum treated 95 (12/13) 95 (12/13)


TABLE 4 LACK OF PROTECTION IN JMV-INFECTED CHICKENS BY IMMUNE SERUM

Treatment Tumor Mortality

( % I RE immune serum 67 (10/15) Normal serum 93 (13/14) None 73 (11/15)

Lack of Protection in JMV-Infected Chickens by Immune Serum Raised Against RE Tumors

To study whether the protective effect of RE immune serum was tumor specific, chickens infected with the JVM strain of Marek's disease were treated with RE immune serum. Control chickens were treated with normal chicken serum or were untreated. TABLE 4 shows the lack of protection in JMV- infected chicks by immune serum raised against RE tumors. Seventy-three percent (%a) of the untreated birds and 67% (l%) of the immune serum- treated birds died of JMV. The normal serum-treated animals had a somewhat, but not significantly, higher tumor mortality (93%, or %4). Thus, the curative effect has at least some degree of specificity.

EBect of RE Virus-Absorbed Immune Serum on Development of RE Tumors

In a study that compared the effectiveness of RE virus-absorbed immune serum and nonabsorbed immune serum, tumor regression occurred in significantly more chickens treated with nonabsorbed immune serum than in untreated animals, as could be expected. RE virus-absorbed immune serum was at least as effective as nonabsorbed immune serum, because 80% (%s) showed local tumor regression, and 73% (%a) survived (TABLE 5 ) . These results show that the immune serum, from which antiviral antibodies were

TABLE 5 EFFECT OF RE VIRUS-ABSORBED IMMUNE SERUM ON

DEVELOPMENT OF RE TUMORS


Treatment (% 1 P (% 1 P RE virus absorbed

Nonabsorbed immuneserum 65 (11/17) 59 (10/17)

None 23 (3/13) 15 (2/13)

immuneserum 80 (12/15) <0.02 * 73 (11/15) <0.05 *

* Comparison with no treatment.

Hu & Linna: Reticuloendotheliosis Virus & Tumors 64 1

absorbed, was as effective as the nonabsorbed serum. Thus, the curative effect does not seem to be mediated by antiviral antibodies to any significant extent.

Effect of RE Tumor Cell-Absorbed Serum on Development of RE Tumors

From the above study, it seemed that the protective effect of the immune serum may be due to antibodies against tumor-associated transplantation antigens. If so, absorption of the immune serum with tumor cells should abolish its curative effect. As expected, chickens that received nonabsorbed immune serum had a high frequency of local tumor regression and survival (94%, or l%). Both normal serum-treated and untreated groups had significantly lower frequency of regression (63 and 45%, respectively) and survival ( 5 8 and 40%, respectively). Animals that received RE tumor cell-absorbed immune serum also had significantly lower percentage of tumor regression (60%, or %o) and survival (55%, ' M o ) . Thus, the curative effect of immune serum

TABLE 6 EFFECT OF RE TUMOR CELL-ABSORBED IMMUNE SERUM ON

DEVELOPMENT OF RE TUMORS


Treatment (%I P (%I P Non absorbed immune serum 94 (17/18) 94 (17/18) Normal cell absorbed

immune serum 82 (14/17) 82 (14/17) RE tumor cell-absorbed

immune serum 60 (12/20) <0.05 * 55 (11/20) <0.05 * Normal serum 63 (12119) 58 (11/19) None 45 (9/20) 40 (8/20)

* Comparison with nonabsorbed immune serum.

was abolished after absorption with RE tumor cells, whereas normal spleen cell-absorbed immune serum still retained a significant curative effect [TABLE 6). Consequently, the removal of curative activity of immune serum is not due to nonspecific absorption by spleen cells. Therefore, the therapeutic activity seems to be specific against tumor-associated transplantation antigens.

Effect of Serum Globulins on Development of RE Tumors

To demonstrate that the active factor of antiserum was, indeed, in the antibody-containing fraction, y globulins of immune serum or of normal serum were used to treat tumor-bearing animals. TABLE 7 shows that significantly more immune y globulin-treated than normal globulin-treated chickens survived (85 vs 45%). The effect of immunoglobulin fraction on tumor therapy is as potent as whole immune serum. Therefore, it seems that the active factor in tumor cure is in t.he antibody-containing fractions.


DISCUSSION

The present work has attempted to study the effect of antibodies on the development of RE virus-induced tumors. The data show that the passive administration of immune serum specific to RE tumors has a strong protective effect on tumor-bearing animals. RE virus-induced tumor presents an excellent model for investigation of host immune defenses involved in neoplasia. Among the characteristic features of tumors induced by RE virus are a predictable and rapid development of tumors and strong immunogenicity. Furthermore, when injected with suitable doses of virus into the wing web, chickens develop both systemic and local tumors that may regress spontaneously. Progression or regression of tumors can be easily influenced by virus dosage and by manipula- tion of the immunologic competence of the host. These findings indicate that host immunity plays an important role in affecting tumor growth.

Our previous studies have shown that either Tx or Bx, or a combination of Tx and Bx, significantly increased RE tumor growth and tumor mortality in 30 These results suggest that not only thymus-dependent im-

TABLE 7 EFFECT OF SERUM y GLoBULINS ON DEVELOPMENT OF TUMORS


Treatment ( % I P (% 1 P Immune y globulin 85 (17/20) 85 (17120) <o.02 Normal y globulin 50 (10/20) <0*05 45 (9/20) Immune serum Normal serum

75 (15/20) 17 (3/18) <0.002 75 (15/20)

11 (2/18) <0.002

None 40 (8/20) 40 (8/20)

munity but also the bursa-dependent antibody-forming systems may contribute to host protection in the development of RE tumors. Findings that suggest a host-protective function for both cell-mediated immunity and for the antibody-forming system have been obtained with similar experimental protocols in two unrelated malignancies: a Rous sarcoma virus-induced tumor 31 and a chemical carcinogen-induced transplantable tumor line.a2 The convergent findings in these tumor models of different genesis make it tempting to suggest that not only cell-mediated immunity but also the antibody-forming system may play a host-protective role in malignancy in general.

To further study the nature of this bursa-dependent host-protective function in RE virus-induced malignancy and the efficacy of immune serum in tumor therapy, passive transfer of immune serum or its components was performed in RE tumor-bearing animals. Immune serum treatment caused complete tumor regression in a high percentage of tumor-bearing animals when administered as late as 6 days after virus inoculation (TABLE 1). Serotherapy of other tumor systems in mice and rats has also shown that immune serum can prevent tumor development and, in some cases, be effective against established ~ m o r s . ~ ~ - ~ ~ ~ 3s

Hu & L i m a : Reticuloendotheliosis Virus & Tumors 643

In avian tumor systems, passively administered antibody has been reported to have a beneficial influence on the incidence and course of Marek‘s disease, although it did not prevent tumor development.3‘ Administration of the immunoglobulin fraction of immune serum to antibody-free chicks has a host- protective effect on Marek’s disease lesion d e ~ e l o p m e n t . ~ ~

To study the possibility that the bursa-dependent immunity of the host may influence tumor development during serotherapy, serum treatment was performed in cyclophosphamide-Bx tumor-bearing animals. In this case, RE tumors also completely regressed in significantly more immune serum-treated animals than in control animals (TABLE 2 ) . These results show that in animals deficient in B cells and antibody-forming capacity, treatment with immune serum results in regression of the established tumors and decreased tumor mortality. Moreover, immune serum protects Bx tumor-bearing animals as well as it protects normal tumor-bearing animals, because tumor regression occurred in about the same percentage (90% or more) of the chickens in both groups (TABLE 2) . Therefore, it seems that host B cells and humoral immunity do not significantly contribute to the mechanism of serotherapy. Thus, if tumor cell lysis in these animals is accomplished by a mechanism equivalent to antibody-dependent cell-mediated cytotoxicity ( ADCC) , the effector cell would be a non-B cell, a finding well in accordance with studies on K cells in

To study whether host T cells participate functionally in serotherpy, RE tumor development was followed in Tx chickens treated with immune serum. Twice as many untreated Tx as immune serum-treated Tx chickens died of RE tumors ( l%9 vs. 5/47; TABLE 3 ) . Thus, immune serum also seems to be able to work in animals with impaired thymic function in this malignancy. How- ever, the protective effect of immune serum in Tx animals is not as strong as in sham-operated animals. The lack of a more complete protection by immune serum in the Tx animals may well be due to the absence of thymus-dependent “surveillance” itself, which was previously shown to be involved in this sys-

However, the possibility of cooperation of T cells with immune serum cannot be excluded.

One difficulty in studying effects of regressor/survivor serum on RNA tumor virus-induced malignancies is that immune serum taken from regressor animals infected with tumor virus may contain both antiviral antibodies and antitumor antibodies. It may be argued that the therapeutic effect obtained with serum antibodies in this system was not due to the destruction of neoplastic cells but rather to neutralization of virions that otherwise would have infected and transformed new cells. To study the contribution of antiviral antibodies on RE tumor regression, the immune serum was extensively absorbed with RE virus. Although we have not demonstrated the complete absence of neutralizing antibodies after absorption, we have shown by counter immunoelectrophoresis a clear decrease of antiviral antibodies in the absorbed serum. This absorbed serum was still as effective as nonabsorbed immune serum in accomplishing tumor regression and host survival (TABLE 5) . Thus, the curative effect of RE immune serum was not mediated to any extent by antiviral antibodies.

Then, the alternative remaining is that the therapeutic effect is mediated by antibodies to transplantation-type antigens on tumor cells. If so, absorption of immune serum with tumor cells should abolish the host-protective effect. The results of this study showed that the curative effect of immune serum decreased significantly after extensive absorption with RE tumorous spleen cells (TABLE 6). Thus, the therapeutic effect of immune serum on established


RE tumors seems to be mainly mediated by antiserum specific against tumor- associated transplantation antigens. A somewhat related finding was reported by Dougherty et al.,37 who demonstrated that virus-neutralizing antibody to Rous sarcoma virus in chickens had little effect on growth rate of Rous sarcomas, and regressions did not appear to be related to antiviral immune re- sponse. Our findings also confirm the results in the Moloney sarcoma virus system. There is a very poor correlation of presence of virus-neutralizing antibodies with tumor regression, and mice with progressing tumors have been found to have antibody titers in the same range as those in many regressors.1l Therefore, the therapeutic effect of regressor sera to syngeneic mice with growing Moloney sarcomas does not appear to be related to virus-neutralizing antibody but rather to depend on direct killing of tumor cells by antibodies to Moloney sarcoma cells through the mechanism of complement-dependent cytotoxicity lo, l1 or antibody-dependent cell-mediated cytoto~icity.~~* 38

The active factor of antiserum was further shown to reside in the antibody- containing fraction, because immunoglobulins were as effective as whole serum in tumor protection. The fact that RE immune serum did not have any effect on tumors induced by the JMV strain of Marek's disease virus indicates that the action of immune serum, at least, has a certain degree of specificity.

From the above evidence, the therapeutic effect of RE immune serum seems to be mediated by antibodies specific against RE tumor-associated transplantation antigens. The exact mechanism by which protection by antibodies is af- forded is not clear. Tumor regression could be due to the cytotoxic effect of antibodies on tumor cells in the presence of complement. The interaction of antibodies with lymphoid cells in tumor cell killing also seems possible. Our data indicate that the effector cell in that case would be a non-B cell.

SUMMARY

Immune serum, obtained from animals that had survived sublethal challenge with RE virus, was very effective in achieving tumor cures in chickens infected with this virus. The therapeutic effect could also be obtained by the immunoglobulin fraction of immune serum. The serum did not influence the development of an unrelated malignancy. Serotherapy studies in Bx and Tx recipients indicate that the B-cell or T-cell system of the host does not significantly contribute to the curative activity of immune serum. Absorption studies show that the curative effect is mediated by antibodies to tumor-associated transplantation antigens on tumor cells, not by antiviral antibodies.

ACKNOWLEDGMENTS

We thank Ms. Annsofi Holst and Mr. Robert Kalwinsky for skillful technical assistance.

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DISCUSSION

QUESTION: Did you ever see enhanced tumor growth in the chickens? DR. Hu: No, we’ve never seen enhancement but always a strong curative

effect.


QUESTION: How complete was your adsorption? Did you test your adsorbed serum for virus-neutralizing activity? DR. Hu: We didn’t test virus neutralization, but before adsorption, we

could show a precipitin line by immunoelectrophoresis against a virus preparation. After each adsorption, the line became fainter, until after the fifth adsorption, it was no longer seen.

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SEROTHERAPY OF AVIAN RETICULOENDOTHELIOSIS VIRUS-INDUCED TUMORS