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Characterization of interleukin 2 (IL-2)-dependent cytotoxic T-cell clones
CELLULAR
IMMUNOLOGY
86, 299-307 (1984)
Characterization of lnterleukin 2 (IL-2)-Dependent Cytotoxic T-Cell Clones V. Transfer of Resistance to Allografts and Tumor Grafts Requires Exogenous IL-2’
MICHAEL A. PALLADINO,**~ KARL WELTE, ANN M. CARROLL, AND HERBERT F. OETTGEN Memorial Sloan-Kettering
Cancer Center, 1275 York Avenue, New York, New York 10021
Received December 29. 1983; accepted February 14, 1984 The adoptive transfer of resistance to tumor grafts with cloned interleukin 2 (IG2)depcndent cytotoxic T-cell lines was examined. Two clones were used: clone CTLL-A2 which recognizes H-2D” determinants and clone CTLGRS which recognizes a unique cell surface antigen of BALB/c leukemia RMI. Systemic transfer of resistance with these clones was accomplished only when exogenous (rat or human) IL-2 was administered at the same time. Intraperitoneal injection of CTLL-A2 cells accelerated rejection of sarcoma Meth A (H-2@), but not ascites sarcoma BPS (H-2’) or leukemia EL4 (H-2b) inoculated subcutaneously into C57BL/6 mice. CTLL-RS cells were examined in local (Winn tests) as well as systemic transfer experiments. When mixed with leukemia cells before subcutaneous injection, they suppressed the growth of leukemia m 1 without exogenous B-2. When injected intraperitoneally, CTLLRS cells inhibited the growth of subcutaneous grafts of leukemia RL8 1 only when exogenous IL-2 was administered at the same time. CTLL-RS did not inhibit the growth of other radiation-induced BALB/c leukemias.
INTRODUCTION While tumor-specific resistance to subsequent tumor grafts has been transferred successfully with lymphocytes from immunized donors (l-6), and regression of established tumors have been induced in some cases (7-9), the difficulty of obtaining the large numbers of sensitized effector cells required for systemic transfer has discouraged exploration of this approach. The discovery of interleukin 2 (IL-2)4 (10, 1 l), which made it possible to maintain T cells in continuous culture and to select ’ Supported by Grants IM3 10 and JFRA39 from the American Cancer Society. ’ To whom reprint requests should be addressed and Recipient of an American Cancer Society Junior Faculty Research Award. 3 Present address: Genentech, Inc., Immunology, Pharmacological Sciences, 460 Point San Bruno Boulevard, South San Francisco, Calif. 94080. 4 Abbreviations used: B6, C57BL/6; CB6F,, (BALB/c X C57BL/6)F, ; GM, growth medium; Con A, concanavalin A; PHA-M, phytohemagglutinin-M; IL2, interleukin 2; IL-ZR, rat B-2; IGZPR, purified rat IL2; ILZPH, purified human IL2; MEM, minimal essential medium; ip, intraperitoneally; iv, intravenously; id, intradennally; LN, lymph node. 299 0008-8749184 $3.00 Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.
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clones of desired reactivity for expansion in vitro, has given new impetus to the investigation of adoptive transfer of tumor immunity as an approach to immunotherapy. Cloned IL-Zdependent cytotoxic T cells have been shown to inhibit tumor growth when mixed with the tumor cell innoculum ( 12- 14) but have not been found effective in systemic transfer experiments (12). IG2dependent uncloned T-cell lines and T-cell clones, which proliferate autonomously in vitro in response to stimulation with alloantigens but in the absence of exogenous IL-2, have been reported to affect systemic transfer of allograft ( 15, 16), and tumor immunity ( 17). More recently, augmentation of in vivo activity of tumor-specific long-term cultured T lymphocytes has been demonstrated by in vivo administration of IL-2 (18). We have confirmed and extended these studies, and report here that resistance to tumor grafts can be transferred to allogeneic and syngeneic hosts with cloned IL-2-dependent cytotoxic T-cell lines specific for alloantigens or tumor-specific antigens on the transplanted tumor cells. One of the T-cell clones used in this study was specific for an H-2Dd determinant, the other for a unique tumor antigen expressed by the radiation-induced BALB/c leukemia Rm 1 ( 19). Systemic transfer of resistance with these cytotoxic T-cell clones depended on simultaneous injection of IL-2. MATERIALS
AND
METHODS
Animals. BALB/c, CsH/He, C57BL/6 (B6), and (BALB/c X C57BL/6)FI (CB6F,) mice were obtained from the Jackson Laboratories (Bar Harbor, Maine) and the breeding colony of the Sloan-Kettering Institute. CD rats (Sprague-Dawley) were obtained from the Charles River Breeding Laboratories (Willmington, Mass.). Tumors. The following tumors were used: BALB/c sarcoma Meth A induced with methylcholanthrene (20), C3H sarcoma BP8 induced with benzopyrene (2), BALB/c leukemias RL6 1, RLS8, RLQ3, and RLQ6 induced by radiation ( 19), leukemia BALBRVI induced by the radiation leukemia virus (21), and C57BL leukemia EL4 induced with dimethylbenzanthracene. Production of IL-2. Spleen cells from CD rats (106/ml) were cultured in Eagle’s minimal essential medium (MEM), supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 1% nonessential amino acids, 100 U/ml penicillin, 100 &ml of streptomycin (complete MEM), 5 X lo-’ M 2-mercaptoethanol, and concanavalin A, 5 &lo6 cells (Con A; Miles Laboratories, Inc., Elkhart, Ind.) for 48 hr. After centrifugation for 10 min at 1000 rpm, the supematant was sterilized by passage through a 0.20-pm filter and stored at 4°C. To prepare rat growth medium, the IL-2 (IG2R) preparation was added to complete MEM at a final concentration of 20 U/ml (22). DEAE-Sephadex-purified rat IL2 (IG2PR) was obtained from Collaborative Research (Waltham, Mass.). Preparation of purljied human IL-2. The protocol for the purification of human IL2 (IL-2PH) to apparent homogeneity has been described (23). Briefly, peripheral blood mononuclear cells (at 4 X 106/ml) were suspended in RPM1 1640 (GIBCO, Grand Island, N.Y.) with 0.25% bovine serum albumin (BSA) (Sigma, St. Louis, MO.) and 1% phytohemagglutinin (PHA-M, GIBCO) for 48 hr at 37°C and costimulated with irradiated Daudi B-lymphoblastoid cells. IL2 was purified from this crude lymphocyte-conditioned medium to apparent homogeneity using sequential ammonium sulfate precipitation, ion-exchange chromatography (DEAE-cellulose, DE-52, What-
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man, Chemical Separation Inc., Clifton, N.J.), gel filtration (AcA 54 Ultrogel; LKB, Rockland, Md.), chromatography on blue agarose (MRL, Gaithersburg, Md.), and chromatography on procion red agarose (MRL). The final product represented a 37,000-fold purification with a specific activity of lo6 U/mg protein. The purified IL2 lacked detectable interferon ((wor y), granulocytemacrophage colony stimulating factor, B-cell growth factor, B-cell differentiating factor, macrophage activating factor, or thymocyte differentiating activity, and was free of other contaminating proteins as judged by silver staining in sodium dodecyl sulfatepolyacrylamide gel electrophoresis; its molecular weight was 14,000. IL-2 assay. The IL-2 activity of supernatants was determined by a T-cell growth factor assay (22, 24). Briefly, 5 X lo3 CTLL-Al 1 cells ( 19), an H-2Dd-specific IL-2dependent cytotoxic T-cell line developed in our laboratory, were cultured in flatbottom microtiter plates (3042; Falcon, Pittsburgh, Pa.) containing 200 ~1 of IL-2containing supernatants, diluted serially from 1:4 to 15 12 in complete MEM. After 20 hr of culture at 37°C the plates were pulsed for 4-6 hr with 1 &i of tritiated thymidine (6.7 Ci/mmol, New England Nuclear, Boston, Mass.). Cultures were treated with 2% EDTA for 5 min and harvested (Skatron, Flow Laboratories, McLean, Va), and radioactivity was measured in a Beckman liquid scintillation counter. An IL-2 standard was produced by culturing CD rat spleen cells (lO’/ml) in complete MEM for 24 hr in Con A (20 pg/ml). With this preparation, 50% maximum incorporation of tritiated thymidine by CTLL-Al 1 cells was always obtained at a dilution of 1:256 to 1:5 12. In reference to other standard preparations (25), the activity of the material was 420 U/ml. Mixed-lymphocyte culture supematants, assayed for comparison on Day 2, usually contained lo-30 U/ml of IL-2. The concentration of IL-2 in the test supematants was expressed in units per milliliter, as determined by Probit analysis of thymidine incorporation. CTLL-Al 1 cells cultured with the IL-2 standard showed dose-dependent incorporation of tritiated thymidine; CTLL-A 11 cells cultured without IL-2 showed only background (100-500 cpm) incorporation. In general, maximum proliferation of CTLL-Al 1 was 20 to 30 times greater in IL-2 than in medium without IL-2. The standard error of triplicate cultures did not exceed 10%. T-cell lines. The IL-2dependent T-cell clones CTLGA2 and CTLL-Al 1 were derived from the peritoneal exudate cells of a B6 mouse immunized with BALB/c sarcoma Meth A as previously described; they are specific for H-2Dd determinants (19, 22, 26). T-Cell clone CTLL-R5 was derived from spleen cells of CB6Fi mice immunized with BALB/c radiation-induced leukemia RM 1; it is specific for a unique cell surface determinant on that leukemia (19). The Lyt phenotype of CTLL-A2, CTLL-Al 1, and CTLL-R5 has been shown to be Lyt-1+,23+ (27). Maintenance of T-cell lines. T-Cell lines were seeded at lo5 cells in T-75 tissue culture flasks (Falcon 3024) containing 10 ml of IG2R. The cells were fed every 3 days with 5 ml of fresh IL-2R and passaged when the cell concentration approximated 5 X 106/flask. All cells were fed with fresh IG2R 18 hr prior to cell transfer. Labeling of CTLL-R5 before transfer For analysis of their tissue distribution after transfer, CTLL-R5 cells ( lO’/ml) were labeled with Naz5’Cr04 (100 &i/ml, New England Nuclear) in RPM1 1640 medium supplemented with 10% FCS and 20% IL-2R for 30 min at 37°C in a shaking water bath. Transfer experiments. The viability of the CTLL-R5 cells was assessed by trypan blue exclusion, and only cell suspensions with greater than 90% viability were used. Labeled CTLL-R5 cells (2 X 106) and IL-2PH (500 U) in serum-free medium were
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injected iv or ip into CB6Fi mice which had been injected id 10 days earlier with 2 X lo6 RL8 1 cells. Aliquots of the labeled suspensions were retained as indications of the total radioactivity injected. Assessment of distribution of transferred cells. Recipients (CB6Fi mice) were sacrificed 24 hr after ip or iv injection of labeled T cells. Lymph nodes (LN) (mesenteric, inguinal, and thoracic), liver, spleen, lungs, blood, and tumor were examined. These specimens and the retained aliquots of the injected cell suspensions were counted in a Packard Gamma Scintillation Counter (Packard Instruments, Downers Grove, Ill.). Recovery from each tissue was calculated as percentage of total radioactivity injected. Results were presented as means f SEM. RESULTS Description of T-Cell Clones A summary of the T-cell characteristics of the CTLL specific for an H-2Dd determinant or an RM 1 specific determinant is shown in Table 1 (19). By indirect immunofluorescence and protein A-SRBC rosetting assays, all cytotoxic T-cell clones typed Lyt-2,3+. By quantitative absorption analyses, low levels of Lyt- 1 were detected on all tested cell lines (19, 27). Effector cell to target cell ratios at which 50% targetspecific lysis was obtained in a 4-hr “Cr-release assay varied from 0.1: 1 to 15: 1 for the three T-cell lines. Acceleration of Tumor AllograjI
Rejection by Cytotoxic T-Cell Clones
In a first series of experiments, the effects of T-cell clone CTLL-A2 on the rejection of BALB/c sarcoma Meth A by B6 mice were examined. In cytotoxicity tests in vitro, clone CTLGAZ was shown to be specific for an H-2Dd determinant (19) (Table 1). As shown in Fig. 1, ip injection of 5 X 1O6 CTLGA2 cells, combined with 200 units of IL-2PR, 2 hr after id inoculation of IO7 sarcoma Meth A cells (H-2Dd), delayed the initial growth and accelerated the subsequent rejection of the Meth A tumor graft (P < 0.001, Student’s t test on Days 7, 10, and 14). Injection of CTLL-A2 or IL2PR alone was ineffective. Combined injection of CTLL-A2 and IG2PR failed to effect the growth of leukemia EL4 (H-2b). Similar results were obtained with another H-2Dd specific T-cell clone CTLL-Al 1 (results not shown). TABLE
1
Characterization of Murine IL-ZDependent
Cytotoxic T-Cell Clones
Clone’
Cell surface phenotype
Antigenic specificity
Cytolytic activity b
CTLL-A2 CTLL-A 11 CTLL-RS
Lyt-l+, 23+ Lyt-1+, 23+ Lyt-l+, 23+
H-2Dd H-2@ RLd 1, unique
O.l:l lo:1 15:l
0 Murine T-cell clones were derived as described under Materials and Methods and as previously described (19). Additional studies characterizing these and other T-cell clones can be found in Ref. (28). b Effector cell to target cell ratio at which 50% specific lysis is obtained during a 4-hr 5’Cr-release assay (19).
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B
30
Days After Tumor In]ection
FIG. 1. Acceleration of tumor allograft rejection by CTLLA2. IO’ BALB/c Meth A sarcoma cells I3 (A) or 10’ C57BL EL4 leukemia cells 0 (B) were injected id into B6 mice. Two hours later, the mice (5 in each group) were injected ip with 5 X lo6 CTLLA2 (0), 5 X lo6 CTLL-A2 combined with 200 units of IL-2PR 0 or 200 units of ILZPR w alone as indicated. Results are presented as mean tumor size +- SE for each group of 5 mice.
In subsequent experiments IG2PH was used (23). As shown in Fig. 2, ip injection of 5 X 1O6CTLGA2 cells combined with 600 units of IL-2PH, 2 hr after id inoculation of 10’ Meth A or BP8 (H-2k) sarcoma cells delayed the initial growth, and accelerated rejection, of Meth A cells only (P < 0.01 on Day 7, < 0.05 on Day 10, and NS on Day 14). Injection of IL-2PH alone had a slight inhibitory effect (P = NS). Local Transfer of Tumor-Specljic
Immunity
with Cytotoxic T-Cell Clones
To investigate the transfer of tumor-specific immunity, the cytotoxic T-cell clone CTLL-RS was used. This clone has been shown to be specific for a unique cell surface
;:
7.5
E .f 50 fn b s F 25
FIG. 2. Acceleration of tumor allograft rejection by CTLLAZ. 10’ BALB/c Meth A sarcoma cells 0 (A) or lo7 C,H BP8 sarcoma cells 0 (B) were injected id into B6 mice. Two hours later, the mice (4 in each group) were injected ip with 5 X IO6 CTLL-A2 0, 5 X lo6 CTLGAZ combined with 600 units of IL-2PH 0 or 600 units of IL-2PH n alone as indicated. Results are presented as mean tumor size ? SE for each group of 4 mice.
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antigen restricted to BALB/c leukemia RU 1 (19). CTLL-RS cells and RU 1 cells were mixed and the mixture inoculated id into BALB/c mice. As shown in Fig. 3, the growth of 1O5RL$ 1 cells was inhibited by 1O6CTLL-R5 cells (P < 0.00 1 Student’s t test, on Days 7, 10, and 14). Of 24 mice 7 showed delayed tumor growth, and the remaining mice did not develop any tumors. By contrast, no inhibitory effect was noted when CTLL-R5 cells were mixed with BALB/c leukemia cells not expressing the unique RL$l antigen recognized by CTLL-RS in vitro-RL93, RLQ6, RIJ8, and RVl (13). These results led us to attempt systemic transfer of immunity to RUl with CTLL-R5 .
Distribution of Labeled CTLL-R5 Cells Labeled CTLL-R5 cells (2 X 106) combined with 500 units of IL2PH were injected ip or iv into CB6Fi mice that had been inoculated id with 2 X lo6 RL6 1 cells 10 days earlier. The mice were sacrificed after 24 hr and the tissue distribution of radioactivity was determined (Table 2). Radioactivity of the RL$ 1 tumor (which measured 0.5-0.75 cm) was 0.3 1% of the injected dose after iv injection, and 3.45% after ip injection. Radioactivity of the spleen was 2.94 and 0.23%, respectively. After iv injection, radioactivity found in the liver was much greater than after ip injection. Based on these results and similar results from two additional experiments, the ip route was used in systemic transfer experiments.
2.5
25
I
URLUI .+ RLd / + CTLL-R5
75
WRLd8 *aRLdB+CTLL-R5
rD
Days Aftw Tumor Injection
FIG. 3. Local transfer of tumor-specific resistance to leukemia RL6 1 with CTLGRS. BALB/c mice were inoculated id with 10’ leukemia RUl (A), RLPS (B), RL93 (C), or W6 (D) cells, alone (-) or mixed with lo6 CTLLRS (- - -). Results are presented as mean tumor size 2 SE for each group of 5 mice for Groups B-D and 24 mice in Group A.
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TABLE 2 Distribution of slCr-Labeled CTLL-RS Cells after iv or ip Injection Route of CTLL-R5 injectionb
ip iv
In viva tissue localization” Tumor
Spleen
Liver
Blood
LN’
Lung
3.45 + 0.43 0.31 f 0.11
0.23 f 0.06 2.94 + 0.87
2.19 f 0.34 21.33 f 4.8
0.26 +- 0.11 0.17 + 0.06
0.10 f 0.07 0.08 f 0.04
0.49 + 0.14 0.16 k 0.11
’ Results are expressed as mean percentage + SE of injected radioactivity from four mice. b CB6Fi mice were injected id with 2 X IO6 RLb 1 cells 10 days prior to injection of the CTLL-RS cells and IL-2 PH. ’ LN (mesenteric, inguinal, and thoracic).
Systemic Transfer of Tumor-Specijic
Immunity
with Cytotoxic T-Cell Clones
To determine whether resistance to BALB/c leukemia RUI could be transferred systemically with CTLL-R5, CB6Fi mice were injected id with 2 X IO6 BALB/c leukemia cells, and 2 hr later ip with CTLL-R5. The requirement for IL-2 and the specificity of in vivo activity was also examined. As shown in Fig. 4, the growth of the U 1 cells was signiticantly inhibited by ip injection of 3 X IO6 CTLL-R5 combined with 200 units of IL-2PR, on Days 2, 4, and 6 after leukemia cell inoculation (P < 0.01 by Student’s t test on Days 7, 10, and 14). Tumors were detectable on Day 5 in 1 out of 5 of the mice injected with CTLL-R5 and IL-2PR and in 3 out of 5 of the tumor controls. On Day 7, the proportion of mice showing tumor growth was 40% (2 out of 5) and 80% (4 out of 5), respectively. The growth of BALB/c leukemias not expressing the unique RUI antigen (RLa8, RLP3) was not affected by the combined injection of CTLL-R5 and IL-2PR. Increasing the frequency of CTLL-R5 injections did not affect tumor growth (unpublished observations). Combined results loorc
‘0 Or A
25
L
I
1
I
7
IO
14
25-
Days After Tumor Inpction
FIG. 4. Specificity of systemic transfer of tumor-specific resistance to leukemia RMl with CTLL-R5. CB6Fi mice were inoculated id with lo6 RMl (A), RL08 (B), or RM3 (C) q leukemia cells. On Days 2, 4, and 6 the mice (5 in each group) were injected ip with 3 X IO6 CTLL-R5 n , 3 X lo6 CTLGRS combined with 200 units of IGZPR 0, or 200 units of IL-2PR alone 0 as indicated. Results are presented as mean tumor size + SE for each group of 5 mice.
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from similar experiments showed that on Day 35, of 36 CTLL-RS- and -IG2PHtreated mice 23 survived as compared to 10 of 35 (43%) controls (P < 0.01). DISCUSSION We have presented evidence that resistance to tumor grafts can be transferred both systemically and locally with cloned cytotoxic T-cell lines recognizing H-2 antigens or tumor-specific antigens, and that the specificity of transferred resistance is identical with the specificity of the cytotoxic reaction in vitro. In our experiments, successful systemic but not local transfer of resistance with IL-Zdependent T-cell lines required simultaneous administration of exogenous IL-2. Our studies confirm and extend recent studies which have clearly demonstrated that although IL-2 had no detectable intrinsic antitumor activity, when used with long-term cultured T lymphocytes it significantly enhanced their in vivo efficacy (18). However, others have also recently reported in vivo transfer of helper activity with cloned T cells without additional administration of IL-2 or with T-cell lines which do not require exogenous IL2 to proliferate (29). Unlike the T-cell lines with helper activity used in these experiments, the cytotoxic clones CTLL-A2 and CTLGRS do not themselves produce IL2 after appropriate antigenic or mitogenic stimulation (15, 28). At least in part, the failure to effect in vivo transfer of reactivity with IL-Zdependent cloned cytotoxic T cells reported in some earlier studies may be explained by the lack of exogenous IL-2 (16-19). The role IL-2 plays in this context remains obscure. While in vivo production of IL-2 has not yet been demonstrated, it seems unlikely that the lymphokine is an artifact of in vitro experiments. It is perhaps more likely that the levels of circulating IL-2 are too low to meet the needs of rapidly proliferating T-cell lines which are known to express an increased number of IL-2 receptors. Alternatively, either IL-2 may not circulate at all, or is rapidly cleared from the circulation (30) so that the transferred cells may not reach local production sites before they exhaust the supply of IL-2 they carry on their surface. It has also been suggested that an inhibitor of IL-2 in the circulation blocks the IL-2 activity required to sustain the growth of circulating T-cell clones (3 1). As exogenous IL-2 has been effective in our experiments, however, an inhibitor does not appear to be the limiting factor unless we assume that its capacity can be easily exhausted. In vivo augmentation of immune responsiveness by partially purified IL-2 has been reported (18, 32), although the level of augmentation was low. However, IL-2 did not enhance in vitro cytotoxic activity of the T-cell clones (M. A. Palladino, unpublished observations). Another point to consider is that IL2 may not only be required to sustain proliferation of the transferred cells, but may also play a role in directing them to the site of effective deployment. While this possibility is entirely speculative in the context of the present study, we have shown that administration of IL-2 changes the pattern of distribution of IG2dependent helper-T-cell lines or cytotoxic T-cell lines after intravenous or intraperitoneal injection (33). REFERENCES 1. Klein, G., Annu. Rev. Microbial. 20, 223, 1966. 2. Old, L. J., and Boyse, E. A., Annu. Rev. Med. 15, 167, 1964. 3. Prehn, R. T., Fed. Proc. 24, 1018, 1965.
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4. Palladino, M. A., and Thorbecke, G. J., Cell Immunol. 38, 350, 1978. 5. Cheever, M. A., Kempf, R. A., and Fefer, A., J. Immunol. 119, 714, 1977. 6. Rosenberg, S. A., and Terry, W. D., In “Advances in Cancer Research” (G. Klein and S. Weinhouse, Eds.), Vol. 25, p. 323. Academic Press, New York, 1977. 7. Delorme, E. J., and Alexander, P., Lancet 2, 117, 1964. 8. Borberg, J., Oettgen, H. F., Choudry, K., and Beattie, E. J., Jr., Int. J. Cancer 10, 539, 1972. 9. Femandez-Cruz, E., Halliburton, B., and Feldman, J., J. Immunol. 123, 1772, 1979. 10. Morgan, D. A., Ruscetti, F. W., and Gallo, R., Science 193, 1007, 1976. 11. Gillis, S., and Smith, K. A., Nature (London) 268, 154, 1977. 12. Giorgi, J. V., and Warner, N. L., J. Immunol. 126, 322, 1981. 13. Palladino, M. A., Scheid, M. P., Stutman, O., von Wussow, P., and Oettgen, H. F., In “Third International Lymphokine Congress, 1982” (J. J. Oppenheim and S. Cohen, Eds.), p. 731. Academic Press, New York, 1983. 14. Smith, K. A., Gillis, S., and Baker, P. E., Proc. Amer. Assoc. Cancer Res. 20, 93, 1979. 15. Engers, H. D., Glasebrook, A. L., and Sorenson, G. D., J. Exp. Med. 156, 1280, 1982. 16. Rosenstein, M., Eberlein, T., Kemeny, M. M., Sugarbaker, P. H., and Rosenberg, S. A., J. Immunol. 127, 566, 1981. 17. Cheever, M. A., Greenberg, P. D., and Fefer, A., J. Immunol. 126, 13 18, 198 I. 18. Cheever, M. A., Greenberg, P. D., Fefer, A., and Gillis, S., J. Exp. Med. 155, 968, 1982. 19. Palladino, M. A., Obata, E., Stocker& E., and Oettgen, H. F., Cancer Res. 43, 572, 1983. 20. DeLeo, A. B., Shiku, J., Takahashi, T., John, M., and Old, L. J., J. Exp. Med. 146, 720, 1977. 21. Stockert, E., DeLeo, A. B., O’Donnell, P. V., Obata, L., and Old, L. J., J. Exp. Med. 149, 200, 1979. 22. Palladino, M. A., Ranges, G. E., Scheid, M. P., and Oettgen, H. F., J. Immunol. 130, 2200, 1983. 23. Welte, K., Wang, C. Y., Mertlesmann, R., Venuta, S., Feldman, S. P., and Moore, M. A. S., J. Exp. Med. 156,454, 1982. 24. Gillis, S., Ferm, M. M., Ou, W., and Smith, K. A., J. Immunol. 120, 2027, 1978. 25. Miller, R. A., and Stutman, O., Eur. J. Immunol. 11, 75 1, 1981. 26. Palladino, M. A., Lattime, E. C., Pecoraro, G. A., Stutman, O., and Oettgen, H. F., Cell Immunol. 76, 286, 1983. 27. Palladino, M. A., Carroll, A. M., De Sousa, M., Gillis, S., Scheid, M. P., and Oettgen, H. F., Cell. Immunol. 76, 276, 1983. 28. Palladino, M. A., von Wussow, P., Pearlstein, K. T., Welte, K., and Scheid, M. P., Cell Immunol. 81, 313, 1983. 29. Kaufmann, S. H. E., and Hahn, H., J. Exp. Med. 155, 1754, 1982. 30. Donohue, J. H., and Rosenberg, S. A., J. Immunol. 130, 2203, 1983. 31. Hardt, C., Rollinghoff, F., Pfizenmaier, K., Mosmann, H., and Wagner, H., J. Exp. Med. 154, 262, 1981. 32. Wagner, H., Hardt, C., Heeg, K., Pfizenmaier, K., Solbach, W., Bartlett, R., Stockinger, H., and Rollinghoff, M., Nature (London) 284, 278, 1980. 33. Carroll, A. M., Palladino, M. A., Oettgen, H. F., and de Sousa, M., Cell Immunol. 76, 69, 1983.
IMMUNOLOGY
86, 299-307 (1984)
Characterization of lnterleukin 2 (IL-2)-Dependent Cytotoxic T-Cell Clones V. Transfer of Resistance to Allografts and Tumor Grafts Requires Exogenous IL-2’
MICHAEL A. PALLADINO,**~ KARL WELTE, ANN M. CARROLL, AND HERBERT F. OETTGEN Memorial Sloan-Kettering
Cancer Center, 1275 York Avenue, New York, New York 10021
Received December 29. 1983; accepted February 14, 1984 The adoptive transfer of resistance to tumor grafts with cloned interleukin 2 (IG2)depcndent cytotoxic T-cell lines was examined. Two clones were used: clone CTLL-A2 which recognizes H-2D” determinants and clone CTLGRS which recognizes a unique cell surface antigen of BALB/c leukemia RMI. Systemic transfer of resistance with these clones was accomplished only when exogenous (rat or human) IL-2 was administered at the same time. Intraperitoneal injection of CTLL-A2 cells accelerated rejection of sarcoma Meth A (H-2@), but not ascites sarcoma BPS (H-2’) or leukemia EL4 (H-2b) inoculated subcutaneously into C57BL/6 mice. CTLL-RS cells were examined in local (Winn tests) as well as systemic transfer experiments. When mixed with leukemia cells before subcutaneous injection, they suppressed the growth of leukemia m 1 without exogenous B-2. When injected intraperitoneally, CTLLRS cells inhibited the growth of subcutaneous grafts of leukemia RL8 1 only when exogenous IL-2 was administered at the same time. CTLL-RS did not inhibit the growth of other radiation-induced BALB/c leukemias.
INTRODUCTION While tumor-specific resistance to subsequent tumor grafts has been transferred successfully with lymphocytes from immunized donors (l-6), and regression of established tumors have been induced in some cases (7-9), the difficulty of obtaining the large numbers of sensitized effector cells required for systemic transfer has discouraged exploration of this approach. The discovery of interleukin 2 (IL-2)4 (10, 1 l), which made it possible to maintain T cells in continuous culture and to select ’ Supported by Grants IM3 10 and JFRA39 from the American Cancer Society. ’ To whom reprint requests should be addressed and Recipient of an American Cancer Society Junior Faculty Research Award. 3 Present address: Genentech, Inc., Immunology, Pharmacological Sciences, 460 Point San Bruno Boulevard, South San Francisco, Calif. 94080. 4 Abbreviations used: B6, C57BL/6; CB6F,, (BALB/c X C57BL/6)F, ; GM, growth medium; Con A, concanavalin A; PHA-M, phytohemagglutinin-M; IL2, interleukin 2; IL-ZR, rat B-2; IGZPR, purified rat IL2; ILZPH, purified human IL2; MEM, minimal essential medium; ip, intraperitoneally; iv, intravenously; id, intradennally; LN, lymph node. 299 0008-8749184 $3.00 Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.
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clones of desired reactivity for expansion in vitro, has given new impetus to the investigation of adoptive transfer of tumor immunity as an approach to immunotherapy. Cloned IL-Zdependent cytotoxic T cells have been shown to inhibit tumor growth when mixed with the tumor cell innoculum ( 12- 14) but have not been found effective in systemic transfer experiments (12). IG2dependent uncloned T-cell lines and T-cell clones, which proliferate autonomously in vitro in response to stimulation with alloantigens but in the absence of exogenous IL-2, have been reported to affect systemic transfer of allograft ( 15, 16), and tumor immunity ( 17). More recently, augmentation of in vivo activity of tumor-specific long-term cultured T lymphocytes has been demonstrated by in vivo administration of IL-2 (18). We have confirmed and extended these studies, and report here that resistance to tumor grafts can be transferred to allogeneic and syngeneic hosts with cloned IL-2-dependent cytotoxic T-cell lines specific for alloantigens or tumor-specific antigens on the transplanted tumor cells. One of the T-cell clones used in this study was specific for an H-2Dd determinant, the other for a unique tumor antigen expressed by the radiation-induced BALB/c leukemia Rm 1 ( 19). Systemic transfer of resistance with these cytotoxic T-cell clones depended on simultaneous injection of IL-2. MATERIALS
AND
METHODS
Animals. BALB/c, CsH/He, C57BL/6 (B6), and (BALB/c X C57BL/6)FI (CB6F,) mice were obtained from the Jackson Laboratories (Bar Harbor, Maine) and the breeding colony of the Sloan-Kettering Institute. CD rats (Sprague-Dawley) were obtained from the Charles River Breeding Laboratories (Willmington, Mass.). Tumors. The following tumors were used: BALB/c sarcoma Meth A induced with methylcholanthrene (20), C3H sarcoma BP8 induced with benzopyrene (2), BALB/c leukemias RL6 1, RLS8, RLQ3, and RLQ6 induced by radiation ( 19), leukemia BALBRVI induced by the radiation leukemia virus (21), and C57BL leukemia EL4 induced with dimethylbenzanthracene. Production of IL-2. Spleen cells from CD rats (106/ml) were cultured in Eagle’s minimal essential medium (MEM), supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 1% nonessential amino acids, 100 U/ml penicillin, 100 &ml of streptomycin (complete MEM), 5 X lo-’ M 2-mercaptoethanol, and concanavalin A, 5 &lo6 cells (Con A; Miles Laboratories, Inc., Elkhart, Ind.) for 48 hr. After centrifugation for 10 min at 1000 rpm, the supematant was sterilized by passage through a 0.20-pm filter and stored at 4°C. To prepare rat growth medium, the IL-2 (IG2R) preparation was added to complete MEM at a final concentration of 20 U/ml (22). DEAE-Sephadex-purified rat IL2 (IG2PR) was obtained from Collaborative Research (Waltham, Mass.). Preparation of purljied human IL-2. The protocol for the purification of human IL2 (IL-2PH) to apparent homogeneity has been described (23). Briefly, peripheral blood mononuclear cells (at 4 X 106/ml) were suspended in RPM1 1640 (GIBCO, Grand Island, N.Y.) with 0.25% bovine serum albumin (BSA) (Sigma, St. Louis, MO.) and 1% phytohemagglutinin (PHA-M, GIBCO) for 48 hr at 37°C and costimulated with irradiated Daudi B-lymphoblastoid cells. IL2 was purified from this crude lymphocyte-conditioned medium to apparent homogeneity using sequential ammonium sulfate precipitation, ion-exchange chromatography (DEAE-cellulose, DE-52, What-
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man, Chemical Separation Inc., Clifton, N.J.), gel filtration (AcA 54 Ultrogel; LKB, Rockland, Md.), chromatography on blue agarose (MRL, Gaithersburg, Md.), and chromatography on procion red agarose (MRL). The final product represented a 37,000-fold purification with a specific activity of lo6 U/mg protein. The purified IL2 lacked detectable interferon ((wor y), granulocytemacrophage colony stimulating factor, B-cell growth factor, B-cell differentiating factor, macrophage activating factor, or thymocyte differentiating activity, and was free of other contaminating proteins as judged by silver staining in sodium dodecyl sulfatepolyacrylamide gel electrophoresis; its molecular weight was 14,000. IL-2 assay. The IL-2 activity of supernatants was determined by a T-cell growth factor assay (22, 24). Briefly, 5 X lo3 CTLL-Al 1 cells ( 19), an H-2Dd-specific IL-2dependent cytotoxic T-cell line developed in our laboratory, were cultured in flatbottom microtiter plates (3042; Falcon, Pittsburgh, Pa.) containing 200 ~1 of IL-2containing supernatants, diluted serially from 1:4 to 15 12 in complete MEM. After 20 hr of culture at 37°C the plates were pulsed for 4-6 hr with 1 &i of tritiated thymidine (6.7 Ci/mmol, New England Nuclear, Boston, Mass.). Cultures were treated with 2% EDTA for 5 min and harvested (Skatron, Flow Laboratories, McLean, Va), and radioactivity was measured in a Beckman liquid scintillation counter. An IL-2 standard was produced by culturing CD rat spleen cells (lO’/ml) in complete MEM for 24 hr in Con A (20 pg/ml). With this preparation, 50% maximum incorporation of tritiated thymidine by CTLL-Al 1 cells was always obtained at a dilution of 1:256 to 1:5 12. In reference to other standard preparations (25), the activity of the material was 420 U/ml. Mixed-lymphocyte culture supematants, assayed for comparison on Day 2, usually contained lo-30 U/ml of IL-2. The concentration of IL-2 in the test supematants was expressed in units per milliliter, as determined by Probit analysis of thymidine incorporation. CTLL-Al 1 cells cultured with the IL-2 standard showed dose-dependent incorporation of tritiated thymidine; CTLL-A 11 cells cultured without IL-2 showed only background (100-500 cpm) incorporation. In general, maximum proliferation of CTLL-Al 1 was 20 to 30 times greater in IL-2 than in medium without IL-2. The standard error of triplicate cultures did not exceed 10%. T-cell lines. The IL-2dependent T-cell clones CTLGA2 and CTLL-Al 1 were derived from the peritoneal exudate cells of a B6 mouse immunized with BALB/c sarcoma Meth A as previously described; they are specific for H-2Dd determinants (19, 22, 26). T-Cell clone CTLL-R5 was derived from spleen cells of CB6Fi mice immunized with BALB/c radiation-induced leukemia RM 1; it is specific for a unique cell surface determinant on that leukemia (19). The Lyt phenotype of CTLL-A2, CTLL-Al 1, and CTLL-R5 has been shown to be Lyt-1+,23+ (27). Maintenance of T-cell lines. T-Cell lines were seeded at lo5 cells in T-75 tissue culture flasks (Falcon 3024) containing 10 ml of IG2R. The cells were fed every 3 days with 5 ml of fresh IL-2R and passaged when the cell concentration approximated 5 X 106/flask. All cells were fed with fresh IG2R 18 hr prior to cell transfer. Labeling of CTLL-R5 before transfer For analysis of their tissue distribution after transfer, CTLL-R5 cells ( lO’/ml) were labeled with Naz5’Cr04 (100 &i/ml, New England Nuclear) in RPM1 1640 medium supplemented with 10% FCS and 20% IL-2R for 30 min at 37°C in a shaking water bath. Transfer experiments. The viability of the CTLL-R5 cells was assessed by trypan blue exclusion, and only cell suspensions with greater than 90% viability were used. Labeled CTLL-R5 cells (2 X 106) and IL-2PH (500 U) in serum-free medium were
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injected iv or ip into CB6Fi mice which had been injected id 10 days earlier with 2 X lo6 RL8 1 cells. Aliquots of the labeled suspensions were retained as indications of the total radioactivity injected. Assessment of distribution of transferred cells. Recipients (CB6Fi mice) were sacrificed 24 hr after ip or iv injection of labeled T cells. Lymph nodes (LN) (mesenteric, inguinal, and thoracic), liver, spleen, lungs, blood, and tumor were examined. These specimens and the retained aliquots of the injected cell suspensions were counted in a Packard Gamma Scintillation Counter (Packard Instruments, Downers Grove, Ill.). Recovery from each tissue was calculated as percentage of total radioactivity injected. Results were presented as means f SEM. RESULTS Description of T-Cell Clones A summary of the T-cell characteristics of the CTLL specific for an H-2Dd determinant or an RM 1 specific determinant is shown in Table 1 (19). By indirect immunofluorescence and protein A-SRBC rosetting assays, all cytotoxic T-cell clones typed Lyt-2,3+. By quantitative absorption analyses, low levels of Lyt- 1 were detected on all tested cell lines (19, 27). Effector cell to target cell ratios at which 50% targetspecific lysis was obtained in a 4-hr “Cr-release assay varied from 0.1: 1 to 15: 1 for the three T-cell lines. Acceleration of Tumor AllograjI
Rejection by Cytotoxic T-Cell Clones
In a first series of experiments, the effects of T-cell clone CTLL-A2 on the rejection of BALB/c sarcoma Meth A by B6 mice were examined. In cytotoxicity tests in vitro, clone CTLGAZ was shown to be specific for an H-2Dd determinant (19) (Table 1). As shown in Fig. 1, ip injection of 5 X 1O6 CTLGA2 cells, combined with 200 units of IL-2PR, 2 hr after id inoculation of IO7 sarcoma Meth A cells (H-2Dd), delayed the initial growth and accelerated the subsequent rejection of the Meth A tumor graft (P < 0.001, Student’s t test on Days 7, 10, and 14). Injection of CTLL-A2 or IL2PR alone was ineffective. Combined injection of CTLL-A2 and IG2PR failed to effect the growth of leukemia EL4 (H-2b). Similar results were obtained with another H-2Dd specific T-cell clone CTLL-Al 1 (results not shown). TABLE
1
Characterization of Murine IL-ZDependent
Cytotoxic T-Cell Clones
Clone’
Cell surface phenotype
Antigenic specificity
Cytolytic activity b
CTLL-A2 CTLL-A 11 CTLL-RS
Lyt-l+, 23+ Lyt-1+, 23+ Lyt-l+, 23+
H-2Dd H-2@ RLd 1, unique
O.l:l lo:1 15:l
0 Murine T-cell clones were derived as described under Materials and Methods and as previously described (19). Additional studies characterizing these and other T-cell clones can be found in Ref. (28). b Effector cell to target cell ratio at which 50% specific lysis is obtained during a 4-hr 5’Cr-release assay (19).
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B
30
Days After Tumor In]ection
FIG. 1. Acceleration of tumor allograft rejection by CTLLA2. IO’ BALB/c Meth A sarcoma cells I3 (A) or 10’ C57BL EL4 leukemia cells 0 (B) were injected id into B6 mice. Two hours later, the mice (5 in each group) were injected ip with 5 X lo6 CTLLA2 (0), 5 X lo6 CTLL-A2 combined with 200 units of IL-2PR 0 or 200 units of ILZPR w alone as indicated. Results are presented as mean tumor size +- SE for each group of 5 mice.
In subsequent experiments IG2PH was used (23). As shown in Fig. 2, ip injection of 5 X 1O6CTLGA2 cells combined with 600 units of IL-2PH, 2 hr after id inoculation of 10’ Meth A or BP8 (H-2k) sarcoma cells delayed the initial growth, and accelerated rejection, of Meth A cells only (P < 0.01 on Day 7, < 0.05 on Day 10, and NS on Day 14). Injection of IL-2PH alone had a slight inhibitory effect (P = NS). Local Transfer of Tumor-Specljic
Immunity
with Cytotoxic T-Cell Clones
To investigate the transfer of tumor-specific immunity, the cytotoxic T-cell clone CTLL-RS was used. This clone has been shown to be specific for a unique cell surface
;:
7.5
E .f 50 fn b s F 25
FIG. 2. Acceleration of tumor allograft rejection by CTLLAZ. 10’ BALB/c Meth A sarcoma cells 0 (A) or lo7 C,H BP8 sarcoma cells 0 (B) were injected id into B6 mice. Two hours later, the mice (4 in each group) were injected ip with 5 X IO6 CTLL-A2 0, 5 X lo6 CTLGAZ combined with 600 units of IL-2PH 0 or 600 units of IL-2PH n alone as indicated. Results are presented as mean tumor size ? SE for each group of 4 mice.
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antigen restricted to BALB/c leukemia RU 1 (19). CTLL-RS cells and RU 1 cells were mixed and the mixture inoculated id into BALB/c mice. As shown in Fig. 3, the growth of 1O5RL$ 1 cells was inhibited by 1O6CTLL-R5 cells (P < 0.00 1 Student’s t test, on Days 7, 10, and 14). Of 24 mice 7 showed delayed tumor growth, and the remaining mice did not develop any tumors. By contrast, no inhibitory effect was noted when CTLL-R5 cells were mixed with BALB/c leukemia cells not expressing the unique RL$l antigen recognized by CTLL-RS in vitro-RL93, RLQ6, RIJ8, and RVl (13). These results led us to attempt systemic transfer of immunity to RUl with CTLL-R5 .
Distribution of Labeled CTLL-R5 Cells Labeled CTLL-R5 cells (2 X 106) combined with 500 units of IL2PH were injected ip or iv into CB6Fi mice that had been inoculated id with 2 X lo6 RL6 1 cells 10 days earlier. The mice were sacrificed after 24 hr and the tissue distribution of radioactivity was determined (Table 2). Radioactivity of the RL$ 1 tumor (which measured 0.5-0.75 cm) was 0.3 1% of the injected dose after iv injection, and 3.45% after ip injection. Radioactivity of the spleen was 2.94 and 0.23%, respectively. After iv injection, radioactivity found in the liver was much greater than after ip injection. Based on these results and similar results from two additional experiments, the ip route was used in systemic transfer experiments.
2.5
25
I
URLUI .+ RLd / + CTLL-R5
75
WRLd8 *aRLdB+CTLL-R5
rD
Days Aftw Tumor Injection
FIG. 3. Local transfer of tumor-specific resistance to leukemia RL6 1 with CTLGRS. BALB/c mice were inoculated id with 10’ leukemia RUl (A), RLPS (B), RL93 (C), or W6 (D) cells, alone (-) or mixed with lo6 CTLLRS (- - -). Results are presented as mean tumor size 2 SE for each group of 5 mice for Groups B-D and 24 mice in Group A.
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TABLE 2 Distribution of slCr-Labeled CTLL-RS Cells after iv or ip Injection Route of CTLL-R5 injectionb
ip iv
In viva tissue localization” Tumor
Spleen
Liver
Blood
LN’
Lung
3.45 + 0.43 0.31 f 0.11
0.23 f 0.06 2.94 + 0.87
2.19 f 0.34 21.33 f 4.8
0.26 +- 0.11 0.17 + 0.06
0.10 f 0.07 0.08 f 0.04
0.49 + 0.14 0.16 k 0.11
’ Results are expressed as mean percentage + SE of injected radioactivity from four mice. b CB6Fi mice were injected id with 2 X IO6 RLb 1 cells 10 days prior to injection of the CTLL-RS cells and IL-2 PH. ’ LN (mesenteric, inguinal, and thoracic).
Systemic Transfer of Tumor-Specijic
Immunity
with Cytotoxic T-Cell Clones
To determine whether resistance to BALB/c leukemia RUI could be transferred systemically with CTLL-R5, CB6Fi mice were injected id with 2 X IO6 BALB/c leukemia cells, and 2 hr later ip with CTLL-R5. The requirement for IL-2 and the specificity of in vivo activity was also examined. As shown in Fig. 4, the growth of the U 1 cells was signiticantly inhibited by ip injection of 3 X IO6 CTLL-R5 combined with 200 units of IL-2PR, on Days 2, 4, and 6 after leukemia cell inoculation (P < 0.01 by Student’s t test on Days 7, 10, and 14). Tumors were detectable on Day 5 in 1 out of 5 of the mice injected with CTLL-R5 and IL-2PR and in 3 out of 5 of the tumor controls. On Day 7, the proportion of mice showing tumor growth was 40% (2 out of 5) and 80% (4 out of 5), respectively. The growth of BALB/c leukemias not expressing the unique RUI antigen (RLa8, RLP3) was not affected by the combined injection of CTLL-R5 and IL-2PR. Increasing the frequency of CTLL-R5 injections did not affect tumor growth (unpublished observations). Combined results loorc
‘0 Or A
25
L
I
1
I
7
IO
14
25-
Days After Tumor Inpction
FIG. 4. Specificity of systemic transfer of tumor-specific resistance to leukemia RMl with CTLL-R5. CB6Fi mice were inoculated id with lo6 RMl (A), RL08 (B), or RM3 (C) q leukemia cells. On Days 2, 4, and 6 the mice (5 in each group) were injected ip with 3 X IO6 CTLL-R5 n , 3 X lo6 CTLGRS combined with 200 units of IGZPR 0, or 200 units of IL-2PR alone 0 as indicated. Results are presented as mean tumor size + SE for each group of 5 mice.
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from similar experiments showed that on Day 35, of 36 CTLL-RS- and -IG2PHtreated mice 23 survived as compared to 10 of 35 (43%) controls (P < 0.01). DISCUSSION We have presented evidence that resistance to tumor grafts can be transferred both systemically and locally with cloned cytotoxic T-cell lines recognizing H-2 antigens or tumor-specific antigens, and that the specificity of transferred resistance is identical with the specificity of the cytotoxic reaction in vitro. In our experiments, successful systemic but not local transfer of resistance with IL-Zdependent T-cell lines required simultaneous administration of exogenous IL-2. Our studies confirm and extend recent studies which have clearly demonstrated that although IL-2 had no detectable intrinsic antitumor activity, when used with long-term cultured T lymphocytes it significantly enhanced their in vivo efficacy (18). However, others have also recently reported in vivo transfer of helper activity with cloned T cells without additional administration of IL-2 or with T-cell lines which do not require exogenous IL2 to proliferate (29). Unlike the T-cell lines with helper activity used in these experiments, the cytotoxic clones CTLL-A2 and CTLGRS do not themselves produce IL2 after appropriate antigenic or mitogenic stimulation (15, 28). At least in part, the failure to effect in vivo transfer of reactivity with IL-Zdependent cloned cytotoxic T cells reported in some earlier studies may be explained by the lack of exogenous IL-2 (16-19). The role IL-2 plays in this context remains obscure. While in vivo production of IL-2 has not yet been demonstrated, it seems unlikely that the lymphokine is an artifact of in vitro experiments. It is perhaps more likely that the levels of circulating IL-2 are too low to meet the needs of rapidly proliferating T-cell lines which are known to express an increased number of IL-2 receptors. Alternatively, either IL-2 may not circulate at all, or is rapidly cleared from the circulation (30) so that the transferred cells may not reach local production sites before they exhaust the supply of IL-2 they carry on their surface. It has also been suggested that an inhibitor of IL-2 in the circulation blocks the IL-2 activity required to sustain the growth of circulating T-cell clones (3 1). As exogenous IL-2 has been effective in our experiments, however, an inhibitor does not appear to be the limiting factor unless we assume that its capacity can be easily exhausted. In vivo augmentation of immune responsiveness by partially purified IL-2 has been reported (18, 32), although the level of augmentation was low. However, IL-2 did not enhance in vitro cytotoxic activity of the T-cell clones (M. A. Palladino, unpublished observations). Another point to consider is that IL2 may not only be required to sustain proliferation of the transferred cells, but may also play a role in directing them to the site of effective deployment. While this possibility is entirely speculative in the context of the present study, we have shown that administration of IL-2 changes the pattern of distribution of IG2dependent helper-T-cell lines or cytotoxic T-cell lines after intravenous or intraperitoneal injection (33). REFERENCES 1. Klein, G., Annu. Rev. Microbial. 20, 223, 1966. 2. Old, L. J., and Boyse, E. A., Annu. Rev. Med. 15, 167, 1964. 3. Prehn, R. T., Fed. Proc. 24, 1018, 1965.
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4. Palladino, M. A., and Thorbecke, G. J., Cell Immunol. 38, 350, 1978. 5. Cheever, M. A., Kempf, R. A., and Fefer, A., J. Immunol. 119, 714, 1977. 6. Rosenberg, S. A., and Terry, W. D., In “Advances in Cancer Research” (G. Klein and S. Weinhouse, Eds.), Vol. 25, p. 323. Academic Press, New York, 1977. 7. Delorme, E. J., and Alexander, P., Lancet 2, 117, 1964. 8. Borberg, J., Oettgen, H. F., Choudry, K., and Beattie, E. J., Jr., Int. J. Cancer 10, 539, 1972. 9. Femandez-Cruz, E., Halliburton, B., and Feldman, J., J. Immunol. 123, 1772, 1979. 10. Morgan, D. A., Ruscetti, F. W., and Gallo, R., Science 193, 1007, 1976. 11. Gillis, S., and Smith, K. A., Nature (London) 268, 154, 1977. 12. Giorgi, J. V., and Warner, N. L., J. Immunol. 126, 322, 1981. 13. Palladino, M. A., Scheid, M. P., Stutman, O., von Wussow, P., and Oettgen, H. F., In “Third International Lymphokine Congress, 1982” (J. J. Oppenheim and S. Cohen, Eds.), p. 731. Academic Press, New York, 1983. 14. Smith, K. A., Gillis, S., and Baker, P. E., Proc. Amer. Assoc. Cancer Res. 20, 93, 1979. 15. Engers, H. D., Glasebrook, A. L., and Sorenson, G. D., J. Exp. Med. 156, 1280, 1982. 16. Rosenstein, M., Eberlein, T., Kemeny, M. M., Sugarbaker, P. H., and Rosenberg, S. A., J. Immunol. 127, 566, 1981. 17. Cheever, M. A., Greenberg, P. D., and Fefer, A., J. Immunol. 126, 13 18, 198 I. 18. Cheever, M. A., Greenberg, P. D., Fefer, A., and Gillis, S., J. Exp. Med. 155, 968, 1982. 19. Palladino, M. A., Obata, E., Stocker& E., and Oettgen, H. F., Cancer Res. 43, 572, 1983. 20. DeLeo, A. B., Shiku, J., Takahashi, T., John, M., and Old, L. J., J. Exp. Med. 146, 720, 1977. 21. Stockert, E., DeLeo, A. B., O’Donnell, P. V., Obata, L., and Old, L. J., J. Exp. Med. 149, 200, 1979. 22. Palladino, M. A., Ranges, G. E., Scheid, M. P., and Oettgen, H. F., J. Immunol. 130, 2200, 1983. 23. Welte, K., Wang, C. Y., Mertlesmann, R., Venuta, S., Feldman, S. P., and Moore, M. A. S., J. Exp. Med. 156,454, 1982. 24. Gillis, S., Ferm, M. M., Ou, W., and Smith, K. A., J. Immunol. 120, 2027, 1978. 25. Miller, R. A., and Stutman, O., Eur. J. Immunol. 11, 75 1, 1981. 26. Palladino, M. A., Lattime, E. C., Pecoraro, G. A., Stutman, O., and Oettgen, H. F., Cell Immunol. 76, 286, 1983. 27. Palladino, M. A., Carroll, A. M., De Sousa, M., Gillis, S., Scheid, M. P., and Oettgen, H. F., Cell. Immunol. 76, 276, 1983. 28. Palladino, M. A., von Wussow, P., Pearlstein, K. T., Welte, K., and Scheid, M. P., Cell Immunol. 81, 313, 1983. 29. Kaufmann, S. H. E., and Hahn, H., J. Exp. Med. 155, 1754, 1982. 30. Donohue, J. H., and Rosenberg, S. A., J. Immunol. 130, 2203, 1983. 31. Hardt, C., Rollinghoff, F., Pfizenmaier, K., Mosmann, H., and Wagner, H., J. Exp. Med. 154, 262, 1981. 32. Wagner, H., Hardt, C., Heeg, K., Pfizenmaier, K., Solbach, W., Bartlett, R., Stockinger, H., and Rollinghoff, M., Nature (London) 284, 278, 1980. 33. Carroll, A. M., Palladino, M. A., Oettgen, H. F., and de Sousa, M., Cell Immunol. 76, 69, 1983.