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 Â¡I.H. GJ, and Department of Molecular Oncology, The Rockefeller University. New York, New York 10021-6)W IH. H.Â¡

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 (<â€¢)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-.vÂ«--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. 4Â°/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 rgenome, 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 pSV2neÂ«(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

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, ramino acid 76 (35). The c/v-.v/r domain consists of c-.v/r fused to the C-terminal 33 amino 25%. depending on the target cells. acids of V-.Ã‡/T.

'(1/6)

Fig. 2. .v/r-specific tumor protection elicited by preinfection with rASV1702 or rd( 1702) viruses, or NP( 1702) cells. A. I-week-old SC chicks were preinfected s.c. in one wing weh with 0.2 ml of rASVI7()2 virus supernatant. After 14 days, the chicks were challenge-infected with 0.2 ml of SR-RSV-B in the other wing web (A). Control 10 15 20 10 15 20 chickens were infected with either rASV1702 (X) Days post infection Days post infection or SR-RSV-B â€¢¿H. l-week-old SC chicks were preinoculated s.c. with 10" mock-infected SC-CEF (â€¢) or NP(I7()2) cells (â€¢) and then challenged with 0.2 ml of SR-RSV-B on day 14, or inoculated alone wilh NP( 1702) cells (O). mock-infected SC- CEF (X). or SR-RSV-B virus (â€¢). Numbers in 13 piirenÃ¬heses. \ of 6 whose SR-RSV-B-induced tu 12 mors failed to regress. C. I-week-old SC chicks 11 10 were preinfected s.c. with ri/(l7()2) virus and then 8 challenge-infected with SR-RSV-B on day 14 (Ex Tumor 8 periment 1. H; Experiment 2. A) or infected with size 7 cither rd( 1702) (O) or SR-RSV-B (â€¢). (Â¡nmm) 6

4 3 2 1 0

Days post infection

the NP(1702) cells suppressed the tumor-forming ability of SR-RSV challenge infection (Fig. 2, B and C). Sera taken I month after inoculating chickens with either ri/(1702) or NP(1702) contained nei ther focus-forming virus titers nor helper virus capable of blocking focus formation following infection with subgroup A RSV (data not shown). Thus, tumor protection can be induced by components of a localized 1702-induced tumor (e.g., .vrr-induced TAs). Adoptive Transfer of rASV1702-induced Tumor Protection. In rASV1702-preinfected chickens, the time interval required to induce protection against RSV-induced tumor challenge was 10-14 days (27). This suggests that the protection involves a cellular rather than hu moral immune mechanism. We attempted to transfer the protective Fig. 3. Tumors produced 2 weeks after s.c. injection of an SC chicken wing web with 0.2 ml (5.6 X |()-' t't'u) of rd(1702) virus. Arrow, cluster of 2-mm tumors. immunity from rASV1702-infected SC chickens. Fig. 5 shows that splenic lymphocytes from rASV1702-infected chickens partially pro tected against RSV challenge. These lymphocytes induced greater Analysis of Murine Cell Lines Expressing src Variants. The construc protection after being incubated for 4 days with UV-irradiated RSV- tion and characterization of NIH3T3 and BALB/c3T3 cell lines expressing infected SC-CEF (stimulators), which should facilitate the in vitro v-src, c-src, n02src, and so on, is described in detail elsewhere.4 Focus formation was performed by CaPO4 cotransfection (36) of 2.5 X IO5 NIH3T3 activation and expansion of CTLs specific for Arc-induced TAs. Splenic lymphocytes from helper virus-infected or uninfected chicks or BALB/C3T3 with 1 ug of pSV2neo plus 5 ug of a ligation mixture containing Sail cut pBH-REP (37) and either Sail cut pXD2 (v-src), p5H (c-src), pS1702, were not protective. Although some rASV1702 virus was transferred pSI57, pl702/c-src, pl57/c-src, or pBr322 control (24, 27, 38). The cells were with the activated splenic cells from rASV1702-infected donors (titers trypsinized and seeded at a 1:10 dilution 40 h after transfection, and then <10' ffu/ml), these titers were not sufficient to elicit protection (data incubated in DEM/10% CS supplemented with 10 RIM /V-2-hydroxyethyl- not shown) (27). In addition, previous results indicated that intervals piperazine-W-2-ethanesulfonic acid, pH 7.0, with changes of media every 2 of only 2 days between preinfection and challenge were insufficient to days. Foci were counted after 2 weeks. Focus formation values represent the elicit protection, even with much higher titers of rASVI702 (27). average of 3 independent transfection experiments. Growth in soft agar was Interestingly, lymphocytes from RSV-infected donors were only par determined by plating triplicates of IO5 transfected cells (described above) in tially protective, even after in vitro coincubation with irradiated stim 10-cm dishes. After incubating overnight, the cells were overlaid with DEM/ ulator cells. Finally, pooled sera taken 2 weeks after infection with 10% CS supplemented with 0.5% agar. Colony number and size were deter mined after 2 weeks of incubation. Tumorigenicity was assayed by injecting helper virus, rASV1702, or RSV were not protective. In contrast, BALB/c nude mice (Taconic Farms, Germantown, NY) s.c. with 5 X IO5 pooled sera taken 2 months after helper virus infection induced some transfected cells (described above). Tumors were measured by palpation every protection, presumably because of virus neutralization by anti-env 3 days for 2 weeks, and then daily for another 2 weeks to monitor for possible antibodies (data not shown). Thus, rASV 1702-induced tumor protec regression. tion can be transferred with splenic lymphocytes and not with serum antibodies. RESULTS Cytotoxic Activity of rASV1702-induced Splenic Lymphocytes. Splenic lymphocytes from mock-, rASV 1702-, or RSV-infected Regression of RSV-induced Tumors. Fig. 2A shows that tumors chickens were then tested for their cytotoxic potential in vitro against induced by rASV1702 typically grow to about 2-3 mm in diameter syngeneic target cells infected with helper virus. rASV 157, and then regress over the next 1-2 weeks. The growth of tumors rASV 1702, or RSV. Fig. 6 shows that effector lymphocytes from induced by SR-RSV-B, however, is progressive. We showed previ rASV1702-infected 7- or 58-day-old chickens that were activated and ously that infection with rASV1702 suppresses the tumor-forming expanded in vitro killed SR-RSV-infected target cells. However, these ability of an RSV-induced tumor challenge in a ire-specific manner effectors failed to kill RSV helper virus-infected target cells (tdl07), (38). Fig. \A shows that the relative growth of RSV-induced challenge which express RSV-type gag, pol, and env. This failure is most likely tumors in rASV1702-preinfected chicks is regressive. due to the fact that the rASV 1702 helper virus (UR2AV) encodes gag, Induction of Tumor Protection by rd(1702) Virus. To determine pol, and env products immunologically distinct from those of tdl07 whether viremia is involved in this rASV1702-induced tumor sup (which is derived from RSV). Thus, the rASV 1702-induced cytotox- pression, we developed both rd virus expressing the I702.W, icity against RSV-infected targets was specific for .w-induced deter rd(\702), as well as syngeneic NPchicken embryo fibroblasts infected minants. In contrast, the cytotoxic potential of effector lymphocytes with rd(llQ2) virus, NP(1702), as described in "Materials and Meth from SR-RSV-infected chickens against RSV-infected targets was ods." Although /â€¢Â£/(!702)virus titers were relatively low, they induced mostly in the context of helper virus-encoded determinants (compare small tumors averaging 2-3 mm (Fig. 3) in diameter, which regressed effectors: SR-RSV-A [58d] versus targets: tdl()7, to effectors: SR- fully after 3^4 weeks (Table 1). HistolÃ³gica! examination showed these to be noninvasive, well-differentiated fibrosarcomas containing Table 1 Transforming ami tutnorigenic potential of nifi702} vint* in O.T many areas of lymphocytic infiltration (Fig. 4). The rd(\102) virus also transformed CEF with characteristics typical of rASV1702-in- VirusUR2AVrASV virus"<0.00010.40.2O.X5Colonysize(mm)''02-32-33-5MorphologyFlatFusiformFusiformRoundTimiori-gcnicity'+/-+/-+++ duced transformation, namely, fusiform morphology and production of small colonies in soft agar (Table 1). Thus, the transformation and 1702r

Fig. 4. Hisiological examination of rd(1702)-induced tumor in SC chicken wing web. Thin sections of the rf/n?02)-induced tumors shown in Fig. 3 were stained with H&E. The tumor was examined 2 weeks postinfection, at which point regression was apparent. Note the differentiated, noninvasive fibro sarcoma in the center (7), surrounded by patches of infiltrating mononuclear lymphs (dark-staining nu clei) and occasional polymorphonucleocytes.

â€¢¿J'..'â€¢¿â€¢^â€¢''â€¢Ã•V?'*â€¢Â«Ll' ^MlPM&A

30 absence of T-cell-mediated immunity 1702src-induced tumors would grow progressively, we transfected NIH3T3 and BALB/C-3T3 cell lines with plasmid DNA encoding \-src, 1702.vrc, or other src variants, and injected them into athymic, nude mice. Table 2 shows that murine cells expressing 1702m- or \51src were partially transformed as mon itored by focus formation and growth in soft agar. Moreover, these cells produced progressively growing tumors in nude mice, although the growth rate was slower than tumors induced by \-src. Replace ment of the relatively activated kinase domains of 1702.src and \51src with that of c-src, whose kinase activity is roughly 10-fold less, decreased but did not abrogate the transforming and tumorigenic activities of these src mutants. These results suggest that active T-cell- mediated immunity is responsible for the regression of rASV 1702- induced tumors.

DISCUSSION

Previously, we developed a tumor regression system in chickens based on the finding that infection with rASV 157 or rASV 1702, which encode nonmyristoylated src products containing novel N-ter-

10 20 minal domains, results in tumors that regress fully (25, 27). Challenge Days post injection infection of these animals with tumor virus encoding v-src resulted in Fig. 5. Transfer of rASVI702-induced tumor protection with splenic lymphocytes. tumors that likewise regress (27). rASV 157 and rASV 1702 are unique Splenic lymphocytes ( IO7) pooled from 6 SC chicks infected with UR2AV (A). rASV 1702 among nonmyristoylated src mutants in that their src products asso (A), or SR-RSV-A (D) were donated to groups of 6 naive SC chicks and then challenged with SR-RSV-B. as described in "Materials and Methods." In addition, tumor-specific ciate with intrinsic plasma membrane structures, namely, the adhesion CTLs were activated and expanded in vitra hy incubating 4 days with syngeneic stimulator plaques. It is conceivable that this interaction allows their activated cells (UV-irradiated SC-CEF infected with SR-RSV-B). Naive SC chicks given injec protein tyrosine kinase domains to interact with cellular phosphoty- tions of IO7 amplified (Amp) lymphocytes from UR2AV- (O), rASV1702- (â€¢),and SR-RSV-A-infected (â€¢)donors were challenged with SR-RSV-B virus. SC chicks in rosine substrates critical for transformation and tumorigenesis ( 19, 25, fected with SR-RSV-B alone (x) are shown as a control. 39). Thus, their novel N-terminal domains potentiate the transforming activity and tumorigenicity of the attached activated src kinase do RSV-A [58d] versus targets: SR-RSV-B). Finally, both rASV 1702- main. The preliminary results of our rASV-induced tumor regression and SR-RSV-induced effectors were cytotoxic against rASV 1702- system implied that these novel domains also increase the immuno- infected targets. Although these effectors killed UR2AV-infected tar genicity of the TAs involved in host recognition and regression of gets (data not shown), this cytotoxicity was mostly specific for .yrr-induced tumors. 1702.Ã‡re-induceddeterminants. Thus, rASV1702-induced tumor pro In this study, we show that 157src- 1702irc-induced tumors are tection correlates with the induction of CTLs specific for ire-induced inherently tumorigenic in nude mice, i.e., they grow progressively in determinants. the absence of T-cell-mediated immunity. Thus, their regression in Progressive Growth of 1702src-induced Tumors in Nude Mice. immunocompetent hosts seems directly linked to their potent immu- The data from Fig. 6 suggest a requirement for active CTLs in the nogenicity. Moreover, 1702.crc seems to induce TRAs in the absence regression of rASV1702-induced tumors. To determine whether in the of viral replication or viremia, as assayed by regression of rd( 1702)- 918

60 n

Fig. 6. In virm CTL activity of rASV1702- and SR-RSV-induced splenic lymphocytes. Splenic lymphocytes effectors from mock (Ml), rASV-1702- (7702 ), and SR-RSV-A-infected (SR-A ) 7- or 58-day-old SC chickens were activated and proliferated in vitro against UV-irradiated. SR-RSV-B-infected SC-CEF (described in Fig. 5). Effectors were co- incuhatedat a 1:100 ratio with target SC-CEF either mock-infected (Mi), or infected with id 107 helper, rASVl57, rASVI702. or SR-RSV-B (SR- B). Target cells had been preincubated with MCr for 1 h. After 6 h of coincubalion, the cells were pelleted by centrifugaron, and the superna tant aspirated and counted for released S|Cr (i.e., level of cytoloxicity) as described in "Materials and Methods." These results are from a single experiment and represent the mean value of assays done in triplicate.

gg,5!g, ,Â»Â£5,5 S, ,Ã®g5,igg, 58d 7d 58d 7d

Table 2 Transforming and tumorigenic potential of various src genes in mouse not transfer with humoral immunity, we have not ruled out a possible ftbroblasts or in nude Â¡nice ancillary role such as described with antibody-dependent cytotoxic of foci/ug in Colony Tumor softc++++ agar size" production'1 P val uÃ¨ cells. CelllineN'v-jrt-lM ofDNA1.5 Both 1702.Ã‡/-0andv-.v/r-transformed cells induce transferable cel xIO41.8x 2-4++ +10 lular immunity and CTL activity against v-.w-transformed cells, al I7021N[I57]N|l702/c-.s/r|N|l57/c-Â«r]N'c-ifr]NIH3T3B[v-.m-|B[IO1''2.0 0.5++ +8 xIO-1''9.0 0.5+ +5 though the latter is much less capable in this regard. For rASV 1702- xIO2''9.0 <0.5+ +5 preinfected chickens to induce a protective immunity against RSV, the xIO2''2.5 <0.500++ +

919

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Irwin H. Gelman and Hidesaburo Hanafusa

Cancer Res 1993;53:915-920.

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