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Src-Specific Immune Regression of Rous Sarcoma Virus-Induced Tximors1

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Src-Specific Immune Regression of Rous Sarcoma Virus-Induced Tximors1 (CANCER RESEARCH 53. 915-920. February 15. IW] src-specific Immune Regression of Rous Sarcoma Virus-induced TXimors1 Irwin H. Gelman2 and Hidesaburo Hanafusa Department nf Micmhioliigy, Mount Sinai Medical Center. New York. New York 1002V ¡I.H. GJ, and Department of Molecular Oncology, The Rockefeller University. New York, New York 10021-6)W IH. H.¡ ABSTRACT photyrosine substrates (19). as defined by monoclonal antibodies, as well as common induced cellular products such as those of the 9E3 We have developed an oncogene-specific tumor regression system in family (23). Thus, tumors expressing specific activated oncogenes chickens. Injection s.c. of chicken wing webs with either rASV1702 or might share 2 classes of TRAs: induced cellular products and onco- i ASVI57, mutants of Rous sarcoma virus (RSV) that express nonmyris- gene-encoded epitopes resulting from activating mutations. toylated src product containing novel N-terminal domains, results in non- Previously, we developed a .v/r-specific tumor regression model in invasive fibrosarcomas that regress fully. The ability of challenge infec tions with wild-type RSV to form tumors is suppressed. This protective chickens. This system is based on the finding that infection of chick effect was shown to be specific for determinants encoded or induced by ens with either rASV157 or rASV1702. mutants of RSV that encode \-src (I. Gelman and H. Hanafusa, J. Virol., 61: 2461-2468, 1989). In the nonmyristoylated src containing both novel N-terminal and active current study, we used SC chickens, inbred for major histocompatability PTK domains (24. 25), results in small, noninvasive tumors that complex Class I haplotype U-VIl-'.to investigate whether this protection regress fully. Chickens preinfected with either mutant virus were results from active immunity. Preinoculation of chickens with either rep protected against challenge infection with RSV but not against chal lication-defective rASV1702 virus or non-virus-producing syngeneic lenge infections with either Fujinami sarcoma virus encoding v-/p.s (a chicken embryo fibroblasts expressing 17(12src conferred protection related, .vrc-family kinase ( 17)] or a chimeric virus encoding RSV gag, against challenge infections with RSV. Thus, viremia was not required for pol, env, and either \-fps or polyomavirus middle T-antigen [which this protection. Splenic lymphocytes from rASV1702-infected donors activates endogenous p6O"src (26, 27)]. Thus, the protection was could transfer protective immunity against RSV tumor challenge to naive chickens. These lymphocytes were cytotoxic in vitro against RSV- or specific for m--encoded or -induced determinants and not for deter rASV1702-infected SC-chicken embryo fibroblasts, but not against S( '- minants encoded by either retroviral replication functions or other chicken embryo fibroblasts infected with helper virus, suggesting a spec oncogenes. The main evidence implicating .s/r-induced TRAs in our ificity for src-encoded or -induced determinants. In contrast, splenic lym regression system is that preinfection with 1702/c-src. encoding src phocytes from RSV-infected chickens transferred protective immunity product with low PTK activity (i.e., the I702.vrc N-terminal domain poorly and exhibited low in vitro cytotoxic potential for src determinants, fused to the PTK domain from c-.vrc), is much less capable of inducing suggesting possible suppression mechanisms. Finally, murine cell lines srr-specific protection than rASV1702 (which contains the PTK do expressing 157sn or \702src produced tumors in nude mice that failed to main from v-src). Thus, induction of .vw-specific TRAs in this system, regress. Thus, although cells expressing I57vrc or 1702-vrc are inherently although influenced by the 1702.w N-terminal domain, seems to tumorigenic, the tumors they induce most likely regress due to immune mechanisms. These results suggest that I702\rr and ISlsrc induce src- require potent src PTK activity. specific tumor Ag that potently prime an oncogene-specific protective In this study, we apply this tumor regression system to SC strain cellular immunity. chickens, inbred for the major histocompatability complex Class I haplotype B2/B2 in order to investigate whether: (a) the rASV1702- induced protection requires viremia; (b) this protection is based on INTRODUCTION active cellular immunity; and (<•)whether cells transformed with An approach central to cancer immunotherapy has been to identify 157.STCor1702m- are inherently tumorigenic as assayed in nude mice. novel TAs' that can function as TRAs by priming immunity capable Our results indicate that infection with rASV17()2 induces a cellular of rejecting tumors that express these TAs ( 1-7). However, this field immune response capable of rejecting tumor cells expressing 1702.W- of research has been confounded by the finding that TAs, though or v-.v«--inducedTA determinants. relatively easy to identify, are often not shared on tumors of a similar class or different tumors induced by the same carcinogen (4, 8-11). MATERIALS AND METHODS There is increasing evidence correlating the expression of activated oncogenes with the onset of specific cancers in humans (12-15). Cell Culture. SC-CEF were made from fertili/.ed SC strain (huplotype Mutations that activate the transforming and tumorigenic potential of B:/B2) eggs (HYLINE, Ames. IA) as described previously (27). SC-CEF were oncogenes result in aberrant signaling pathways such as increased grown in F-IO complete media (GIBCO, Bethesda, MD). which contains 10% protein tyrosine kinase activity for src (16, 17) and deregulated TPB. 5% CS, and 1% heat-inactivated chicken serum. NIH3T3 and BALB/ GTPase activity for ras (12). These tumor cells share common in c3T3 (American Type Culture Collection CRL1658 and CCL163. respectively) duced and modified cellular proteins (1, 18-22). For example, src- were grown in DEM media (GIBCO) supplemented with \(Wr heat-inactivated calf serum. Q4dh (28). an avian retrovirus packaging cell line (kindly provided transformed chicken, mouse, and human cells contain common phos- by A. Stoker), was grown in medium 199 supplemented with W7c TPB. 4°/c fetal calf serum, and \% chicken serum. CEF-conditioned media were pro Received 9/8/92; accepted 12/2/92. duced from primary cultures of SC-CEF grown 7 days in F-IO complete media, The costs of publication of this article were defrayed in part hy the payment of page then filtered through a sterile 0.45-um filter (Gelman Sciences. Ann charges. This article must therefore he herehy marked ntlvrriisetm-nl in accordance with Arbor, MI). 18 U.S.C. Section 1734 solely to indicale this fact. 1This work was supported hy USPHS Grant CA44356 from the National Cancer Viruses. SR-RSV-A and SR-RSV-B have been described (27). tdl07 is a Institute, and Postdoctoral Fellowship PF-3024 from the American Cancer Society RSV-based helper virus (29), and UK2AV is a UR2-based helper virus (30). (I. H. G.). Stocks of rASVI57 and rASVI702 were produced from CEF transfecled with 2 To whom requests for reprints should he addressed, at Box 1124, Department of Microbiology, Mount Sinai Medical Center, Annenberg Building. Room 16-08. One pS 157 or pS 1702 plus UR2AV helper virus DNA as described previously (24). Gustave L. Levy Plaza. New York, NY 10029. Production of r</(1702) Virus and NPÃœ702) Cells. The cloned copy of 1The abbreviations used are: TA. tumor antigen; TRA, tumor rejection antigen; RSV, the rASVI7()2 genome, pS1702 (24). is deleted for the env function (Fig. I); Rous sarcoma virus; CTL, cytotoxic T-]ymphocyte; CEF. chicken embryo fibrohlast; TPB, tryptose phosphate broth; CS, calf serum; DEM, Dulbecco's modified Eagle's medium; n/(1702) virus was produced hy cotransfecting Q4dh avian retrovirus packag ing cells with pS1702 (i>m~) and pSV2ne«(31). Virus was harvested 40 h SR-RSV-A, wild-type RSV. Schmidt-Ruppin strain subgroup A; SR-RSV-B. wild-type RSV, Schmidt-Ruppin strain subgroup B; PTK. protein tyrosine kinase; ffu. focus-forming posttransfection. Tilers ranged from IO2 to IO5 ffu/ml. These virus stocks are unit; NP, non-virus producer; PBS. phosphate-buffered saline; rii. replication-defective. defined as nl( 1702). NP( 1702) cells were produced hy infecting SC-CF.F with 915 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1993 American Association for Cancer Research. m-SPF.CIFIC TUMOR REGRESSION W/11702), overlaying the cultures with F-10 media containing 0.4% agar Colony Formation by Infected SC-CEF. SC-CEF were infected with (F-IO/soft agar). and incubating for 7-10 days. Foci were picked and grown either SR-RSV-B. rASVI702, r</(l702), or no virus (mock). The cells were in F-10 supplemented with 25% CEF-conditioned media. Clones expressing trypsiniz.ed and grown in soft agar as described (34). appropriate 1702.vrc product (M, 56.000) were identified by immuno- Adoptive Transfer of Splenic Lymphocytes or Serum. One-week-old SC precipitation/autophosphorylation assay (32) with MAb327. specific for p60sri chickens (6 chickens/experiment) were given injections of one wing web with (33). Of the 8 clones studied, none produced focus-forming titers, i.e.. they IO'1ffu of virus ¡n0.1-0.2 ml. After 2 weeks, spleens were removed, minced, were NP. after 2 months of growth. Clone NP(17()2)-#7 was expanded and and then passed through sterile wire mesh (Thomas Scientific. Swedesboro. used in the experiments described in Fig. 2B. NJ) to disperse clumped cells. The cells were washed 3 times with sterile PBS, Tumor Production and Analysis in Chickens. Two hundred ul of virus with gentle centrifugaron between each wash; 1()7spleen cells (predominantly (representing IO6 ffu. except in the case of re/(1702). which was 10'-104 ffu) lymphocytes, by microscopic observation) were injected into the major wing was injected s.c. into chicken wing webs on one side. Challenge injections. 14 vein of 2-week-old SC chickens.
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