Proc. NatL Acad. Sci. USA Vol. 79, pp. 5384-5387, September 1982 Immunology

Protection against syngeneic by a long-lived cytotoxic T-cell clone (immunotherapy/ migration/interleuldn 2/Abelson virus) MoRRIs 0. DAILEY, ERIc PILLEMER, AND IRVING L. WEISSMAN Department of Pathology, Stanford University School of Medicine, Stanford, California 94305 Communicated by Ray D. Owen, June 7, 1982 ABSTRACT The effect of a cloned T-cell line on the in vivo cloning immune and expanding them in vitro, one growth of syngeneic lymphoma cells was studied. 1E4 is an H-2- should be able to generate large numbers of T cells that are restricted cytotoxic T-cell clone that efficiently kills Abelson virus- antigen specific and have well-defined functional activities. We induced lymphoma target cells (L1-2) at low effector/target ra- have examined the ability of one Tc clone (1E4) to inhibit the tios, as measured by in vitro cytotoxicity assays. In addition, it is in vivo growth of syngeneic Abelson virus-induced lymphoma long lived in vitro in the absence of stimulation and survives for cells. We chose this particular clone because, unlike other re- more than 1 wk in vivo in the absence of exogenous antigen or ported Tc clones, 1E4 lives for long periods of time in the ab- growth factors. Mice injected intraperitoneally with lethal doses sence of antigen or growth factors, both in vitro and in vivo of L1-2 and then treated with 1E4 survived longer than animals (unpublished results). The results reported here show that a treated with saline or with a control T-cell clone. Multiple weekly homogenous population ofcytotoxic T lymphocytes can protect injections of effector cells, or a single injection in animals given animals from the lethal outgrowth oftumorcells but suggest that a low dose oftumor cells, resulted in 50-80% long-term survivors. The observation that intravenous injection of killer cells was less such activity may be limited by the inability ofthe clone to gain effective than intraperitoneal treatment, coupled with the pre- access to the sites of tumor deposition, analogous to the very vious demonstration of markedly abnormal circulatory patterns abnormal migratory properties previously described for several for T-cell clones, suggests that those animals succumbing to pro- T-cell clones (12). gressively growing die because the effector cells are unable to home into peripheral sites of tumor deposition. Thus, MATERUILS AND METHODS although this cytotoxic T-cell clone does have useful invivo activity, Mice and Cell Lines. C57L, (C57L X A/J) F1, and B10.S its function may be partially limited by a generalized defect in mice were bred in our colony or obtained from Jackson Labo- migration. ratory. L1-2 is an Abelson virus-induced lymphoma cell line of The observation that carcinogen-induced tumors can cause spe- C57L origin. EL-4 is a chemically induced lymphoma from cific immunity in inbred mice has led to the beliefthat the im- C57BL mice. Both of these were maintained by serial in vitro mune response is important in determining an animal's resis- passage. tance to tumor growth (1, 2). While humoral immunity has Derivation of T-Cell Clones. 1E4 is a cytotoxic T-cell clone occasionally shown antitumor activity, it can also serve to block from a C57L mouse that was obtained by limiting-dilution clon- what might otherwise be an effective cell-mediated response ing from a secondary mixed lymphocyte tumor cell culture with to an immunogenic tumor (for review, see ref. 3). Cytotoxic T L1-2 stimulator cells. It is passed weekly in RPMI 1640/50 ,uM (Tc) lymphocytes have been considered to mediate the direct 2-mercaptoethanol containing penicillin, streptomycin, gluta- destruction oftumor cells in vivo, corresponding to their activity mine, 10% fetal calf serum, and 10% Lewis rat concanavalin A in killing assays in vitro. The exact role ofdifferentT-cell subsets (Con A) supernatant as a source ofinterleukin 2 (IL 2). Further in the antitumor response, however, is not known. The recent characterization of this clone will be published elsewhere. A demonstration of the selective increase in specific Tc cells in- control cell line, aB10. S, was generated by cloning at limiting filtrating murine sarcoma virus-induced tumors suggests an in dilution responder cells from a C57L anti-B1O.S mixed lym- vivo role for these cells (4). Other studies using heterogenous phocyte culture. It is maintained by weekly restimulation of 4 lymphocyte populations have shown that T cells of the helper X 105 cells with 2 X 107 irradiated B10.S spleen cells in 5 ml phenotype may also be active in tumor rejection (5, 6). Some of medium containing 10% Con A supernatant. types of T cells may suppress the immune response to a neo- Assay for Cell-Mediated Cytotoxicity. The cytolytic capacity plasm (7, 8), and others may even stimulate its growth (9, 10). ofthe two clones was measured with the standard 51Cr release The general importance of tumor-specific T cells in the re- assay. Serial dilutions ofeffector cells were incubated with 51Cr- sponse to naturally occurring tumors must also be viewed in x in 200 ,ul ofmedium in V-bottom relationship to the observation that many spontaneous murine labeled target cells (1 104) are poorly immunogeneic (11). Regardless ofthe role microtiterwells. After4 hr. the plates were centrifuged and 100- of T cells in the natural antitumor response, however, poten- ,ul aliquots of supernatant were assayed in a gamma counter. tiation ofan endogenous immune response or effective adoptive The percent specific lysis was calculated with reference to spon- immunotherapy would be of certain practical importance. taneous and maximal releases by the usual methods (13). Target Treatment with adoptively transferred immune lymphocytes cells for testing the specificity of the aB10.S clone were pre- can be effective, even in eliminating established tumors (5). By pared by incubating 1.2 x 107 normal spleen cells from various mouse strains in 8 ml of medium containing Con A at 2 ,g/ The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- Abbreviations: Tc, cytotoxic ; Con A, concanavalin A; IL 2, inter- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. leukin 2; i.v., intravenously; i.p., intraperitoneally. 5384 Downloaded by guest on September 29, 2021 Immunology: Dailey et aL Proc. Natl. Acad. Sci. USA 79 (1982) 5385 ml (Sigma). These cells were collected on day 3 and washed with there is no lysis ofL1-2 tumor cells. This T-cell clone therefore 50 mM a-methylmannoside before labeling with 51Cr. is specific for H-2S alloantigens. Tumor Protection Experiments. (C57L X A/J) F1 mice were Effect of 1E4 on in Vivo Growth ofTumor Cells. The effect injected intraperitoneally with 3-50 x 104 L1-2 cells in 0.5 ml of the specific antitumor T-cell clone on in vivo growth of ofsaline. The LD50 determined in preliminary experiments was L1-2 tumor cells was examined in several experiments. Fig. 2 found to be less than 1 x 103 cells. One hour after injection of and Table 1 (experiment 1) show the results of an experiment tumor, the animals were injected with 1-1.5 x 107 clone 1E4 in which (C57L x A/J) F1 mice were injected i.p. with a high cells, either intravenously (i.v.), intraperitoneally (i.p.), orboth. dose (5 x 105) of L1-2 and subsequently treated with 1 x 107 Within each experiment, animals were matched for age and sex, 1E4, either i.p. or i.v., orwith saline alone. A 50% prolongation randomized, and kept in cages in subgroups oftwo or three from of survival time was found in animals treated i.p. with clone each treatment group. No variation in survival was found be- 1E4. A smaller, but statistically significant, increase in survival tween subgroups of animals in different cages. Survival times time was observed for animals given 1E4 iv. (t) in Table 1 are reciprocals ofthe means ofthe reciprocal sur- In animals dying of progressive tumor growth, there was vival times and were calculated as follows: wide-spread involvement of the internal organs. Marked splenomegaly with diffuse involvement of the red pulp was a (Eff\ universal finding. In addition, leukemia infiltrates were present in the liver, lungs, lymph nodes, and bone marrow. This is sim- ilar to the pathological findings in animals dying from leukemia induced by injection ofthe Abelson virus (15). Localized tumor where si is the survival time for animal i and n is the number growth at the site of injection-that is, in the peritoneal cav- of animals in the group. This takes into account the long-term ity-varied from animal to animal. LL-2 has a striking predi- survivors more than does median or mean survival time. To test lection for growing subcutaneously, spreading out from the whether the survival times ofany two groups were significantly needle track at the site ofinjection. In many animals, especially different, the data were analyzed by the nonparametric in those treated with 1E4, there was a massive subcutaneous Mann-Whitney test (14). tumor without any apparent intraperitoneal growth. To rule out nonspecific protective effects that might occur RESULTS with the injection of T cells, experiments were carried out in Cytotoxic Cell Lines. 1E4 is a cytotoxic T-cell clone that spe- which animals were injected with aB10. S. a T, clone that does cifically lyses lymphoma cells induced by Abelson leukemia not recognize LL-2 tumor cells. The survival period of animals virus. Fig. 1 shows that this clone is highly efficient in killing injected with L1-2 and given 1E4, both i.v. and i.p., was more LI-2, a C57L Abelson lymphoma cell line, with 50% of maxi- than doubled, and one animal (of eight) survived indefinitely. mum killing at an effector/target cell ratio of less than 1:1. In contrast, aBlO.S did not increase survival beyond that of There is no significant killing ofEL-4, a chemically transformed animals treated with saline only (Fig. 3; Table 1, experiment H-2' cell line. Further experiments have shown that killing of 2). There was only a marginal increase in survival in animals Abelson targets is H-2 restricted to the H-2b-haplotype and that treated with 1E4 i.v. only, even when a lower challenge dose the clone has the cell surface phenotype Lyt-1-2+, Thy-l+, typ- of tumor cells was used (experiment 3). ical of cytotoxic T cells (unpublished results). These experiments show that injection of clone 1E4 has a A negative control syngeneic T-cell line of irrelevant speci- specific protective effect against the growth of L1-2. Experi- ficity, aB10.S, was obtained by limiting-dilution cloning from ments were done to test whether multiple doses of the anti- a C57L anti-B10. S mixed lymphocyte culture. Immunofluo- Abelson clone would have an even greater effect on animal sur- rescence analysis showed that it has a phenotype similar to that vival. When mice were i.v. and i.p. injected with 1.5 X 107 1E4 of 1E4 (Lyt-12+, Thy-1+, H-2Kb) (data not shown). The killing 1 hr after injection of L1-2 and, 3 days later, with 5 X 106 cells, specificity of this cell line was determined by using Con A-in- there was no increase in survival beyond that seen in a single duced blast cells as targets in chromium release assays. As injection of 1E4 (data not shown). In experiment 4, mice were shown in Fig. 1, aB10.S is highly cytolytic against B10.S blast treated at weekly intervals, beginning 1 hr after injection of cells but does not kill A/J, BALB/c, or C57L blasts. In addition, 100

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co co :> J41 560 I.- 0. 10 aQa2 0~~~~~~~~~~ 20 1--- 0~~~~~~~~- 10 1 .2 10 1 Effector: Target Ratio 5 15 25 35 45 FIG. 1. In vitro cytotoxic activity of T-cell clones. Lysis of 51Cr-la- Time (days) beled target cells was tested in the standard 4-hr assay. (Left) 1E4 was tested against L1-2 (o) and EL-4 (o). (Right) aBlO.S was tested against FIG. 2. Effect of clone 1E4 on in vivo growth of L1-2 lymphoma. L1-2 (e) and against Con A-induced blasts from B1O.S (o), A/J (A), (C57L x A/J) F1 mice were injected i.p. with 5 x 105 L1-2 and 1 hr later BALB/c (i), and C57L (A) mice. Spontaneous release values were 19% with 1 x 107 1E4 i.p. (- - - -) or i.v. (---) or with saline (-). (See Table for L1-2, 17% for EL-4, and 16-18% for Con A-induced blast targets. 1, experiment 1.) Downloaded by guest on September 29, 2021 5386 Immunology: Dailey et aL Proc. Natd Acad. Sci. USA 79 (1982) Table 1. Protective effect of clone 1E4 in mice injected with lethal doses of L1-2 lymphoma cells Long-term Treatment regimen Survival, survivors, Exp. Tumor cells, no. Cells Doses, no. n days no. Significance 1 5 x 105 Saline 1 12 16.2 0 1E4 i.v. 1 13 20.4 0 P << 0.001 1E4 i.p. 1 12 25.9 0 P << 0.001 2 5 x 105 Saline 1 8 16.2 0 aBlO.S i.v., i.p. 1 8 15.9 0 NS 1E4 i.v., i.p. 1 8 36.0 1 P << 0.001 3 1x105 aB1OMSi.v. 1 6 15.7 0 1E4i.v. 1 6 20.0 0 P < 0.05* 4 5 x105 Saline 6t 8 20.8 0 1E4 i.v. 6 7 25.4 1 NS 1E4 i.v., i.p. 6 8 69.8 4 P << 0.001 5 3 x 104 Saline 1 6 34.4 1 aBlO.S i.v., i.p. 1 6 27.4 0 NS 1E4 i.v., i.p. 1 5 >100 4 P << 0.001 (C57L x A/J) F1 mice were injected i.p. on day 0 with the indicated number of tumor cells and 1 hr later with saline or cloned T cells, and survival times and P values [treatment vs. saline-treated (control) groups] were calculated. NS, not significant. * 1E4 vs. aBlO.S treatment. tAnimals were injected weekly with saline or 1E4 for a total of six injections. L1-2,.with 1-1.5 X 107 1E4, either i.v. or i.v./i.p. combined several reasons. It is highly active in vitro, killing the specific for a total of 5 wk. As was seen previously, there wasa marginal tumor targets at low effector/target ratios. In addition, unlike increase in survival time in animals treated with i.v. adminis- previously reported Tr clones, which die rapidly when washed tered 1E4, with one animal surviving more than 100 days (Fig. free ofIL 2 (16, 17), 1E4 is long lived in the absence ofantigen 4 and Table 1). In contrast, mice treated simultaneously with or IL 2 stimulation, both in vivo and in vitro. When 1E4 cells both i.p. and i.v. injected 1E4 had markedly prolonged survival are washed and cultured in medium free ofantigen or IL2, they times, and 50% ofthe animals survived for more than 100 days. survive, but do notproliferate, for several months. After intra- Thus, multiple injections ofclone 1E4 are more effective than venous injection, 1E4 lymphocytes can be recovered for at least single injections in preventing the growth of L1-2. 2 wk (unpublished observations). This in vivo survival is im- To test the ability of1E4 toprevent the growth oflowerdoses portant since eradication ofthe neoplasm may require the pro- of tumor cells, animals were given 3 X 104 L1-2 i.p. and then longed action of effector cells in the recipient (18). Thus, this 1E4 or aBlO.S i.v. and i.p. (Table 1, experiment 5). This dose clone provides an opportunity for us to determine the in vivo ofL1-2 is more than 5 times the LD50for L1-2. Ofthe 12 control role ofa homogenous population ofcytolytic T cells, as well as animals in the two groups, 1 survived more than 100 days. In to develop practical immunotherapeutic protocols. the group ofanimals treated with 1E4, 80% became long-term Although C57L mice are relatively resistant to adoptively survivors, demonstrating the-effectiveness of1E4 in preventing transferred Abelson , the (C57L x A/J) F1 mice outgrowth of a smaller tumor inoculum. used in our studies are very susceptible. Within 2 wk of intra- peritoneal innoculation, untreated animals have widely dissem- DISCUSSION inated lymphoma. Results of experiments reported here show that 1E4 is able to protect mice from a lethal dose of the L1-2 Clone 1E4 is a particularly useful cell line for studying the in lymphoma, essentially converting the sensitive F1 to the more vivo interaction between cytolytic T cells and tumor cells for resistant parental phenotype. This effect is specific in that a

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50 5 15 25 35' 100 Time (days) Time (days) FIG. 4. Survival of animals givenmultiple injections of clone 1E4. FIG. 3. Protective effect of 1E4 compared with that of the control Mice were injected i.p. withS5 x 10" L1-2 and, 1 hr later, with saline clone (aBlOS). Mice inoculated with 5 x 10' L1-2 i.p. were injected 1 (-), with 1E4 at 1 x 107 each i.v. and i.p. (- - - -), or with 1 x 107 1E4 hr later with saline (-) or i.p. and i.v. with 1.5 x 107 1E4 (---) or i.v. only (---). Animals were subsequently ijectedweeklywith saline aBlO.S (----). or 1-1.5 x 107 1E4, i.v. and i.p., for a total of six injections. Downloaded by guest on September 29, 2021 Immunology: Dailey et d Proc. Nati Acad. Sci. USA 79 (1982) 5387 cytotoxic clone with different antigenic specificity, aB1O.S, has unable to migrate to peripheral lymphoid organs. At least some no effect on tumor growth. Multiple weekly doses of 1E4 are effector cells must encounter the tumor to cause its destruction, more effective than a single dose. We do not yet know whether and a general defect in homing function would greatly reduce this is due to the larger cumulative dose ofTc cells or whether their in vivo activity. Further experiments will be necessary to the temporal spacing ofinjections is important. Combined i.p. determine the ability of 1E4 to enter sites of tumor growth. and i.v. treatment is more effective than i.v. treatment alone, The results of this study demonstrate the in vivo activity of although the latter also effects occasional long-term survival. cloned cytolytic T cells. It is possible that other types of T-cell Animals inoculated with a low dose of tumor cells are more ef- clones, such as helper cell lines, may also showin vivo antitumor fectively treated than those given a larger tumor dose. This sug- activity, as suggested by studies using heterogenous lympho- gests that the decreasing the tumor burden by other methods, cyte populations (5, 6). Long-lived cytotoxic T-cell clones spe- such as chemotherapy, followed by treatment with the specific cific for tumor antigens, perhaps in conjunction with chemo- T-cell clone might be most effective in the treatment of estab- therapy (21), may be of practical use in the treatment of neo- lished tumors. plastic disease. Previous studies using heterogenous cell populations have This work was supported by National Institutes ofHealth Fellowship shown that, in some cases, adoptively transferred immune lym- AI-06032 and Medical Scientist Training Program Grant GM-07365 and phocytes are able to inhibit the growth of tumors (3, 5, 19). In by Grant ASC-IM 56 to I. L.W. general, these studies have shown that large numbers of spe- cifically sensitized cells are required to effect long-term sur- 1. Foley, E. J. (1953) Cancer Res. 13, 835-837. vival. Combined therapy with cyclophosphamide and immune 2. Prehn, R. T. & Main, J. M. (1957) J. Nati Cancer Inst. 18, 769-778. lymphocytes is quite effective in the eradication ofsome murine 3. Rosenberg, S. A. & Terry, W. D. (1977) Adv. Cancer Res. 25, tumors, even after the neoplasms have disseminated (5). Large 323-388. numbers of immune lymphocytes that are active in combined 4. Brunner, K. T., MacDonald, H. R. & Cerottini, J.-C. (1981) J. chemoimmunotherapy can be obtained by in vitro expansion Exp. Med. 154, 362-373. of the cells in IL 2-containing media (20). Since many of the T 5. Greenberg, P. D., Cheever, M. A. & Fefer, A. (1981) J. Exp. lymphocytes in heterogenous populations are not specific for Med. 154, 952-963. 6. Fernandez-Cruz, E., Gilman, S. C. & Feldman, J. D. (1982)J. the tumor and some may even stimulate tumor growth (9, 10), Immunol 128, 1112-1117. cloning such T cells should make it possible to select cells that 7. Berendt, M. J. & North, R. J. (1980) J. Exp. Med. 151, 69-80. are antigen specific and most active in the inhibition of tumor 8. Dye, E. S. & North, R. J. (1981) J. Exp. Med. 154, 1033-1042. growth in vivo. 9. Prehn, R. & Lappe, M. (1971) Transplant Rev. 7, 26-54. The cytotoxic clone 1E4 has easily demonstrable antitumor 10. Mule, J. J., Forstrom, J. W., George, E., Hellstrom, I. activity in vivo. However, given its efficiency in lysing tumor & Hellstrom, K. E. (1981) Int. J. Cancer 28, 611-614. cells in vitro, we question why these cells are not even more 11. Hewitt, H. B., Blake, E. R. & Walder, E. S. (1976) Br.J. Cancer effective in preventing progressive fatal tumor growth. Pre- 33, 241-259. mature death of the cloned T cells in vivo is an unlikely expla- 12. Dailey, M. O., Fathman, C. G., Butcher, E. C., Pillemer, E. nation in view of our observation that there is no decrease in & Weissman, I. L. (1982)J. Immunol 128, 2134-2136. 13. Hollander, N., Pillemer, E. & Weissman, I. L. (1980) J. Exp. the number of 1E4 cells in the spleen 1-7 days after injection Med. 152, 674-687. (unpublished data). There are several other possible mecha- 14. Snedecor, G. W. & Cochran, W. G. (1971) Statistical Methods nisms by which tumor cells may escape destruction, such as the (Iowa State Univ. Press, Ames, IA), pp. 130-132. generation of suppressor T cells in the host (7, 8) and the pro- 15. Siegler, R., Zajdal, S. & Lane, I. (1972) J. NatL Cancer Inst. 48, duction ofvarious blocking factors (3). The explanation that we 189-218. favor was in part suggested by the observation that animals in- 16. Gillis, S., Ferm, M. M., Ou, W. & Smith, K. A. (1978) J. Im- oculated with tumor cells i.p. and treated with 1E4 often died munoL 120, 2027-2032. with massive subcutaneous tumor around the site of injection 17. Schreier, M. H., Iscove, N. N., Tees, R., Aarden, L. & Von but little or no intraperitoneal neoplasm. Clone 1E4 is therefore Boehmer, H. (1980) Immunol Rev. 51, 315-336. able to eradicate the intraperitoneal tumor but probably does 18. Greenberg, P. D., Cheever, M. A. & Fefer, A. (1980) Cancer Res. 40, 4428-4432. not gain adequate access to peripheral sites oftumor deposition, 19. Borberg, H., Oettgen, H. F., Choudry, K. & Beattie, E. A., Jr. such as subcutaneous tissues. The inability of 1E4 to home into (1972) Int. J. Cancer 10, 539-547. tumor masses would then be analogous to the markedly abnor- 20. Cheever, M. A., Greenberg, P. D. & Fefer, A. (1981) J. Immu- mal migration pattern previously demonstrated for 1E4 and nol 126, 1318-1322. other-T-cell clones (12). In that study, we showed that, in con- 21. Cheever, M. A., Greenberg, P. D., Fefer, A. & Gillis, S. (1982) trast to normal T cells, in vitro T-cell clones are completely J. Exp. Med. 155, 968-980. Downloaded by guest on September 29, 2021