DISCUSSION PAPER: MODALITIES OF IMMUNOTHERAPY FOR VIRALLY INDUCED MURINE NEOPLASMS * Joseph G. Sinkovics Section of Clinical Tumor Virology and Immunology Department of Medicine The University of Texas System Cancer Center M . D . Anderson Hospital and Tumor Institute Houston, Texas 77025

INTRODUCTION The main motivation for immunotherapy of mouse leukemias and sarcomas has always derived from the expectation that treatment designs appropriate for the human counterparts of these diseases will be found. This report briefly reviews the stages of development of this concept in our laboratories. ACTIVEIMMUNIZATION WITH ATTENUATED MOUSELEUKEMIA VIRUS When the Rauscher mouse leukemia virus was maintained in tissue cultures of Timco Swiss mouse thymus and spleen, the release of immunogenic but not leukemogenic virus particles was observed. Supernatant fluids of these cultures were capable of immunizing Timco Swiss mice against challenge with lo1.' to 10' LD,,, mouse passage line leukemogenic Rauscher virus.l A time interval of more than 1 week was necessary between immunizations and challenge for protection. Undiluted attenuated virus and highly diluted virulent virus given 1 ) . This latter within 24 hours paradoxically increased leukemogenesis (TABLE phenomenon resembles multiplicity reactivation. Loss of genetic material (the oncogene) from the attenuated virus with retention of genetic material necessary for the replication of viral structural proteins (the virogene), but inadequate for the induction of malignant transformation, is one possible interpretation. On the contrary, during its replication the virulent virus would reproduce the oncogene in excess so that the leukemogenicity of attenuated virions would be restored. Whether or not viral attenuation, i.e., loss of oncogene with the retention of the virogene, can occur in leukemia virus preparations passaged in vivo is not known. However, recovery from proven viral leukemia occasionally occurred in Timco Swiss mice and was accompanied by production of leukemia virus-neutralizing and leukemia cell-lysing antibodies.2

* These studies were supported by U.S.P.H.S. Research Grants CA-6529 and CA-7923 from the National Institutes of Health. 557

558

Annals New York Academy of Sciences

TABLE1 ACTIVEIMMUNIZATION WITH LIVE A ~ E N U A T E MOUSE D LEUKEMIA VIRUS AND ACCELERATED LEUKEMOGENESIS UPON SIMULTANEOUS INOCULATION OF THE ATTENUATED VIRUS WITH HIGHLYDILUTEDVIRULENT VIRUS Results

Pretreatment Attenuated virus Attenuated virus Attenuated virus Attenuated virus Attenuated virus Attenuated virus Healthy METC fluid Healthy METC fluid None None

Days Between Pretreatment and Virulent Virus

Virulent Virus, Dilution

-

%

Leukemia *

1

25 100

Life Span (days) From Inoculation of Virulent Virus to Death 180 55

103.7 10-

12

-

-

103.' 1W7

12

lo3.'

12

15

10-

12

100

-

22

166 172 64 176 108 62 102

100

52

1

~

1

7

10-

38 8 88 10

METCkmouse embryo tissue culture. * 10-32 mice per groups. Results published in part previously.'

NONSPECIFICIMMUNOSTIMULATION

A dichotomy of action became evident very early. For example, H.pertussis vaccine given i.v. to newborn AKR mice reduced the appearance of leukemia 10-fold by 10 months of age,3 but the same vaccine promoted tumor growth in young adult Timco Swiss. mice bearing a Rauscher virus-induced but celltransplanted l y m p h ~ m a .The ~ effect of erythroagglutinin-free phytohemagglutinin on the growth of virally induced murine tumors depended on the biological behavior of the tumor: the growth of an immunosensitive diploid lymphoma was retarded, but an immunoresistant tetraploid lymphoma grew faster in phytohemagglutinin-treated mice.5 Intraperitoneally inoculated fibrosarcomas carrying Rauscher virus (or induced by the Rauscher virus?) were retarded in phytohemagglutinin-treated mice, but this treatment did not alter the growth rate of subcutaneously inoculated fibrosarcomas. An L-asparaginase-resistant mouse lymphoma showed accelerated growth in L-asparaginase-treated mice because of the immunosuppressive effects of the enzyme.6 PASSIVEIMMUNIZATION Rabbit immune sera produced against murine lymphomas carrying the Rauscher virus retained antibodies that significantly suppressed the growth of lymphoma cells in mice after absorption of the immune sera with tissues of

559

Sinkovics: Immunotherapy for Virally Induced Neoplasms

healthy mice; in a few mice cure was achieved. In other mice, however, the growth of immune serum-resistant lymphomatous tumors occurred.' ADOPTIVEPREVENTIVE IMMUNIZATION Timco Swiss mice immunized with formalin-killed or photodynamically inactivated Rauscher mouse leukemia virus produced antibodies with leukemia virus-neutralizing and leukemia cell-lysing properties. The spleen cells of such mice conveyed protection against challenge with lo2 LD,,, Rauscher virus in nonimmunized Timco Swiss mice (TABLE 2 ) . Spleen and lymph node cells of immunized mice exerted protective and immunotherapeutic effect, resulting in cure in some mice, against cell-transplanted, Rauscher virus-carrier murine sarcomas; the immune lymphocytes congregated around cultured sarcoma cells and retarded their attachment and growth." REACTION GRAFTVERSUSLEUKEMIA

Timco Swiss mice exhibit great susceptibility, whereas C57B1 mice are relatively resistant to Rauscher viral leukemia. Newborn Timco Swiss mice inoculated with spleen, lymph node, and bone marrow cells of adult C57B1 mice succumb to runt (homologous or allogeneic) disease. In Timco Swiss mice with chronic allogeneic disease of this type, the rate of replication, as determined by retitration of Rauscher virus inoculated into these mice, was significantly suppressed, and the characteristic histological and clinical picture of leukemia was delayed.I0 FOLLOWED BY ADOPTIVEIMMUNIZATION LETHALX-IRRADIATION

Lethal x-ray irradiation (700-800 R to whole body) substantially reduces the number of leukemic cells in leukemic Timco Swiss mice without affecting the leukemia virus. These mice, if left without reconstitution, rapidly succumb

TABLE2 TRANSFER OF ANTILEUKEMIA IMMUNITY WITHSPLEENCELLSFROMIMMUNIZED TO SUSCEPTIBLE RECIPIENTS DONORS

%L

Survival (average in days) of Leukemic Mi=

100 17

144

10' Spleen

Cells per Recipient i.v. & i.p.

Non-immune Immune Immune

L Leukemia Virus Days BeInoculated LDm i.p. tween Inocula Mice 10'

1w

-

3 3

-

Similar results published previously.8 L=leukemia.

7/7

5/30 0/11

-

77

-

5 60

Annals New York Academy of Sciences

to Pseudomonas sepsis. If reconstituted with hemato- and lymphopoietic cells of nonimmune donors, these mice succumb to recurrent (virally reinduced) leukemia. Large groups of Timco Swiss mice were inoculated with Moloney or Rauscher leukemia viruses and upon appearance of splenomegaly and/ or leukemic cells in blood, were given lethal total body x-irradiation. Within 3 hours and once again 3 days after the irradiation, these mice were given i.v. and i.p. los nucleated spleen, lymph node, and bone marrow cells harvested from healthy Timco Swiss mice that were actively immunized previously with leukemia virus vaccines and circulated antibodies neutralizing the leukemia virus or cytolytic (in the presence of complement) to leukemic cells. Preventive treatment with gentamicin was also given. Long-term leukemia-free survival occurred in 8% of the previously leukemic Rauscher virus-inoculated mice and in 25% of the previously leukemic Moloney virus-inoculated mice l1 (TABLE 3). Leukemia virus extracted from mice with Rauscher leukemia recurrent after lethal x-irradiation and adoptive immunization with immune hemato- and lymphopoietic cells showed decreased avidity to leukemia virus-neutralizing antibodies l2 but could be fully neutralized with anti-Rauscher virus immune sera after two passages in mouse embryo cells.l3 CROSS-TOLERANCE AND ADOPTIVEIMMUNIZATION AGAINSTVIRALLEUKEMIA Protection against viral leukemia, as measured by a spleen focus assay, could be achieved without allogeneic disease in mutually tolerant donor-recipient systems. Newborn and suckling BALB/c and Timco Swiss mice were mutually exposed to healthy cells of the other allogeneic strain. Young adult BALB/c mice tolerant to Timco Swiss cells were inoculated i.v. with Rauscher leukemia virus. Sixteen hours after the inoculation of the virus, the BALB/c mice were inoculated i.v. (and i.p.) with viable spleen and lymph node cells of Timco Swiss mice. The effect of donor Timco Swiss mouse spleen cells on viral leukemogenesis in BALB/c mice was determined by counting leukemic spleen foci in killed BALB/c mice 8 days later. Donor Timco Swiss mouse cells were ineffective when (1 ) they were immune to the leukemia virus; (2) derived from donors tolerant toward the recipients, but (3) were inoculated into recipients not tolerant toward the donors; or (4) when mutual tolerance between donor and recipient existed, but the donor cells were not immune to the leukemia virus (TABLE 4). Donor Timco Swiss cells were effective in reducing the number of spleen foci when (1) they were immune to the leukemia virus; (2) were inoculated into recipients tolerant toward the donor, disregarding (3) whether or not the donors were tolerant toward the recipients (TABLE 4). Therefore, antileukemia protection by adoptive immunization was possible without the criteria of allogeneic disease. ONCOLYTIC VIRUSES It was possible to superinfect a murine sarcoma with a neurotropic Newcastle disease virus strain. In adult mice, the growth of the virus-carrier sarcoma cells was significantly retarded, but the mice succumbed to Newcastle virusinduced encephalopathy. In order to prevent this complication, anti-Newcastle

-

700 R

700 R

-

800R

-

800 R 800R

-

Irradiation

Immune cells* Immune cells * 1 (189) 1 (163)

2 (205) 0

-

-

3 (131) 7 (191) 5 (159)

0

1 (202)

Immune cells * Immune cells * Normal cells

-

Adoptive Immunization

0

7 (218)

0 0 0 0

3 (244)

0 0

9

25 16 10 28

22

17 38

35

Number of Mice per Group

25 %

8%

LeukemiaFree Survival

* lo* spleen, lymph node, and bone marrow cells of donor mice immunized actively with leukemia virus vaccines; cells given 3 hours and 3 days after irradiation i.v. and i.p. Average day of death was 60 in group inoculated with Rauscher virus and 100 in group inoculated with Moloney virus; only late deaths or survivals are shown here. Similar tabulations were published previously."* 12

Rauscher Rauscher Rauscher Rauscher Rauscher Moloney Moloney Moloney Moloney

Type Of Leukerma

Number of Mice With Late Deaths With Without Leukemia Leukemia (days) (days)

TABLE 3 SURVIVAL OF LEUKEMIC MICE TREATED WITH LETHALX-RAY LATELEUKEMIC DEATHSAND LONGLEUKEMIA-FREE IRRADIATION AND ADOPTIVEIMMUNIZATION WITH IMMUNE DONOR CELLS

2

c

'0,

3 0

B

E

Li a

E G

2

z

5! s

2 v

3

C

3

F

?

5.

0

5 ' 7?

cn

Annals New York Academy of Sciences

5 62

virus immune serum was given to mice bearing sarcomas superinfected with this virus; however, in these mice enhanced tumor growth occurred.’.’

TETRAPLOID LYMPHOMA IMMUNORESISTANT,

A diploid, immunosensitive, Rauscher virus-induced, cell-passaged murine lymphoma yielded a subline that consisted of immunoresistant, tetraploid lymphoma cells.fi Similar tetraploid lymphoma cells could be obtained in the

TABLE4 REDUCTIONOF VIRALLY INDUCED SPLEENCOLONIES IN RECIPIENTS SUSCEPTIBLE TO THE LEUKEMIA VIRUS BUT TOLERANT TO DONORCELLS BY ADOF’TIVE VIRUS AND IMMUNIZATION WITH DONORCELLS IMMUNE TO THE LEUKEMIA TOLERANT (OR NOT TOLERANT) TO THE RECIPIENTS

Recipient BALB/c mice

Spleen colonies aver: Donor Timco age in Swiss mice group

A Not tolerant * Tolerant t Immune t B Not tolerant * C Tolerant

*

D Tolerant *

E Tolerant

*

Tolerant t Immune t Not tolerant t. Immune t Tolerant t Not immune t

p Value

No effect.

30 41.5

vs. C 0.004 VS.

4S

2 4o

Interpretation

B 0.002

vs. E 0.001 vs. B 0.001 vs. E 0.001

-

Base line controls. Significant reduction in number of leukemic spleen cell colonies in recipients. No effect.

* To donor. P To recipient. t: To leukemia virus.

Recipients were inoculated with lo-? dil. Rauscher leukemia virus at age 4 weeks, 16 hours before administration of donor cells. Donor cells derived from 8-week-old mice. Approximately 10s donor cells per recipient were given i.v. Number of spleen colonies were determined 8 days later. Preliminary results were published elsewhere.*

peritoneal cavity of mice inoculated with the immunosensitive, diploid lymphoma cells and with spleen cells of mice immune to this 1yrnph0ma.l~ The cultured tetraploid lymphoma cells produced deformed leukemia virus particles and immune globulins which very strongly neutralized the Rauscher leukemia l8 The interpretation was that cell-fusion occurred between the diploid lymphoma cells and antibody-producing plasmacytoid cells.1G The resulting tetraploid cells acquired immunoresistance by concealing budding viral antigenic sites on the cell membrane. Thus, the leukemia virus-producing lymphoma cell through fusion with a plasma cell, which was producing specific antiviral antibody, achieved the state of “self-enhancement.”

Sinkovics: Immunotherapy for Virally Induced Neoplasms

563

DISCUSSION These early, elementary observations forecast difficulties with antileukemia immunization in man. If a human leukemia-inducing oncornavirus will be available for vaccine production, attenuated live virus o r inadequately inactivated virus preparations can possibly regain leukemogenic potency through phenomena related to multiplicity reactivation. Nonspecific immunostimulation certainly will intensify the host’s general immune status, but the danger of tumor enhancement will not be eliminated until the mechanism of action of nonspecific immunostimulants is better understood. Adoptive immunization in outbred human populations is hindered by the induction of graft versus host and host versus graft reactions. Induction of mutual tolerance between donor and recipient may circumvent this complication without sacrificing the effectiveness of the procedure. The tetraploid, immunoresistant murine lymphoma cells may have an analogy in human pathology. Lymphoma cells of primary Burkitt’s tumors are diploid, but tumors recurrent in the immunoreactive host are often of tetraploid modes. If antibody-production by the neoplastic cell itself is capable of concealing neoantigens (viral or virally coded antigens) on the cell surface, the host is deprived of the target for an effective immune reaction. However, macrophages can attack antibodytoated cells-hence, the “starry sky” appearance of these types of tumors. The murine lymphoma described above also displays the “starry sky” histological pattern, thus resembling Burkitt’s tumor.” ACKNOWLEDGMENT The technical assistance in these studies of Barbara Bertin, Celia Bonney, Frances Ervin, Doris Gaines, Glenda Groves, Francine Mikulik, and Elaine Thornell is gratefully appreciated. The author is grateful to Dr. Herman Friedman for his invitation to submit this paper for publication in the volume of the Conference. REFERENCFS J. G., B. A. BERTIN& C. D. HOWE. 1966. Occurrence of low ~.SINKOVICS, leukemogenic but immunizing mouse leukemia virus in tissue culture. Nat. Cancer Inst. Monograph 2 2 349-367. J. G., F. G. GROVES, B. A. BERTIN& C. C. SHULLENBERGER. 1969. 2. SINKOVICS, A system of tissue cultures for the study of a mouse leukemia virus. J. Infect. Diseases 119: 19-38. J. G., M. J. AHEARN, E. SHIRATO & C. C. SHULLENBEROER. 1970. 3. SINKOVICS, Viral leukemogenesis in immunologically and hematologically altered mice. J. ReticuloendothelialSOC.8: 474-492. C. C. SHULLENBERGER & C. D. HOW. 1967. 4. HIRANO,M.,J. G. SINKOVICS, Murine lymphoma: Augmented growth in mice with pertussis vaccine-induced lymphocytosis. Science 158 1061-1064. J. G., J. R. PIENTA, J. M. TRUJILLO & M. J. AHEARN.1970. Leuke5 . SINKOVICS, mogenesis in mice immunologically altered at birth and immunocompetence of leukemic lymphoblasts. I n 4th International Conference on Cancer, “Immunity and Tolerance in Oncogenesis.” L.Severi, Division of Cancer Research, Perugia, Italy, June 26-July 1, 1969 :975-987.

5 64

Annals New York Academy of Sciences

6. SINKOVICS, J. G. 1973. Spleen focus assays with a mouse leukemia virus in mice treated with actinomycin D, L-asparaginase or rifampicin. In 5th International Symposium on Comparative Leukemia Research, 197 1, “Unifying Concepts of Leukemia.” Bibliotheca Haematol. 39: 377-380. S. Karger. Basel & New York. 7. SHIRATO, E., J. G. SINKOVICS & E. W. THORNELL. 1972. Passive immunization against murine lymphoma. Oncology 26( 1) :80-86. 8. SINKOVICS, J. G., B. A. BERTIN& C. D. HOWE. 1965. Some properties of the photodynamically inactivated Rauscher mouse leukemia virus. Cancer Res. 2 5 624-627. 9. SINKOVICS, J. G., J. R. PIENTA,J. M. TRUJILLO,I. M. AHEARN& F. M. MIKULIK. 1970. Activities of immune lymphoid cells against leukemia virus carrier murine malignant cells. In 4th International Symposium on Comparative Leukemia Research, Cherry Hill, N.J. Sept. 21-25, 1969. Bibliotheca Haematol. 36: 618-623. S. Karger. Basel & New York. 10. SINKOVICS,J. G., C. C. SHULLENBERGER & C. D. HOWE. 1965. Immunological functions of homologous spleen cells in viral mouse leukemia. Texas Rep. Biol. Med. 23 1: 94-109. 11. SINKOVICS, J. G. 1967. The causative viruses of murine leukemia and their identification through immune responses of the host. In Collected Papers of the 20th Annual Symposium on Fundamental Cancer Research, 1966, “Carcinogenesis: A Broad Critique” of the University of Texas M. D. Anderson Hospital and Tumor Institute : 157-175. Williams & Wilkins Co. Baltimore, Md. M. FIORENTINO & B. A. BERTIN. 1967. Neutrali12. SINKOVICS, J. G., R. J. PIENTA, zation of sublines of a mouse leukemia virus with murine antibody, as measured by the spleen focus assay. Cancer Res. 27: 88-94. 13. SINKOVICS. J. G. 1967. Project M23/gm8: The etiologic agents, immunology and spread of mouse leukemia. University of Texas M. D. Anderson Hospital and Tumor Institute, Houston, Texas. Research Report 1967 : 304-313. University of Texas, Printing Div. Austin, Texas. 14. SINKOVICS, J. G. & C. D. Howe. 1969. Superinfection of tumors with viruses. Experientia 2 5 733-734. IS. SXNKOVICS, J. G., B. DREWINKO& E. W. THORNELL. 1970. Immunoresistant tetraploid lymphoma cells. Lancet 1: 139-140. J. G., J. M. TRUJILLO,R. J. PIENTA& M. J. AHEARN.1970. Leuke16. SINKOVICS, mogenesis stemming from autoimmune disease. In Collected Papers of the 23d Annual Symposium on Fundamental Cancer Research, 1969, “Genetic Concepts and Neoplasia” of the University of Texas M. D. Anderson Hospital and Tumor Institute : 138-190. Williams & Wilkins. Baltimore, Md. J. G., J. R. PIENTA, J. M. TRUJILLO & M. J. AHEARN. 1969. An 17. SINKOVICS, immunological explanation for the starry sky histological pattern of a lymphoma. J. Infect. Diseases 1 2 0 250-254.

Discussion paper: modalities of immunotherapy for virally induced murine neoplasms.

DISCUSSION PAPER: MODALITIES OF IMMUNOTHERAPY FOR VIRALLY INDUCED MURINE NEOPLASMS * Joseph G. Sinkovics Section of Clinical Tumor Virology and Immuno...
382KB Sizes 0 Downloads 0 Views