• Abstract and Figures
• Public Full-text

Proc. NatL Acad. Sci. USA Vol. 79, pp. 5384-5387, September 1982 Immunology Protection against syngeneic lymphoma by a long-lived cytotoxic T-cell clone (immunotherapy/lymphocyte 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 lymphocytes 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of antigen or growth factors, both in vitro and in vivo of L1-2 and then treated with 1E4 survived longer than animals (unpublished results).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 neoplasm 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) neoplasms 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 T cell; 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.

Load more

  4

Copy Link