Transfer of immunity by transfer of bone marrow cells: T-cell dependency.

Thymectomized, lethally irradiated mice reconstituted with normal bone marrow cells succumbed when challenged ip with rat Yoshida ascites sarcoma (YAS) cells 40 days after irradiation and reconstitution. In contrast, thymectomized irradiated mice reconstituted with bone marrow cells from YAS-immune donors rejected the subsequent tumor challenge. Pretreatment of the bone marrow cells from immune donors with anti-Thy 1.2 antiserum and complement completely abolished the transfer of anti-YAS resistance.Bone marrow cells from donors thymectomized 2 months before immunization enabled almost all recipients to reject YAS, but bone marrow cells from donors thymectomized 8 months before immunization protected only 50% of the recipients. Further analysis showed that mice thymectomized 8 months before immunization failed to generate anti-YAS antibody response, whereas the antibody response of mice thymectomized 2 months before immunization did not differ from that of non-thymectomized age-matched control mice. The data suggest that the immune reaction of mice against xenogeneic YAS requires long-lived T₂ lymphocytes.     

Cell cooperation in abolition of tolerance of xenogeneic tumor.

Thymectomized, lethally irradiated mice reconstituted with syngeneic bone marrow cells are tolerant to xenogeneic Yoshida ascites sarcoma (YAS). The tolerance was abolished by an injection of syngeneic normal spleen, thymus, or lymph node cells given simultaneously with YAS. Allogeneic and semiallogeneic spleen cells were ineffective. The YAS-rejecting mice produced specific anti-tumor antibodies. The serum of these mice transferred to tolerant T-cell-deficient mice protected the latter from inoculated YAS cells. These serum-protected mice were not able to resist the reinoculum of the tumor cells as the mice restored with lymphoid cells did. The latter mice rejected the YAS at the time when donor cells were practically absent in their lymphoid tissue. The low effective ratio of injected syngeneic lymphoid to tumor cells, efficiency of injected thymus cells, and other data led to the conclusion that transferred lymphoid cells did not act directly on tumor cells but through cooperation with host lymphoid cells. The cooperation of donor T- and host B-lymphocytes enabled the activation of the latter, and YAS cells were rejected.     

Efficacy of BCG presensitization in combination with cyclophosphamide (CY) on murine tumor growth and immunity

Summary The effect of BCG in combination with cyclophosphamide (CY) was studied in a solid tumor system in syngeneic mice. There was a marked effect of intradermal (ID) presensitization with BCG (10⁷ organisms) followed by a single intraperitoneal (IP) injection of CY (200 mg/kg) one day after the tumor inoculation on tumor suppression and tumor immunity, while there was no effect by either BCG or CY treatment alone. The optimal interval between BCG sensitization and tumor inoculation seemed to be about 3 weeks.Research supported in part by Grant-in-Aid Cancer Research from the Ministry of Health and Welfare, Japan     

The use of cytotoxic T cells in the regulation of tumour growth in syngeneic mice

Summary Normal C57BL/6 (B6) spleen cells were cultured with syngeneic EL4 tumour cells, expanded in IL2-containing medium, and tested for anti-tumour activity in vitro and in vivo. The activated cells were highly cytotoxic for EL4 and to a lesser degree killed syngeneic B6 blasts and allogeneic (D2) P815 tumour cells. B6 or BDF1 mice that received these cultured cells by IP injection cleared ¹²⁵IUdR-labelled EL4 cells faster than untreated mice. However, this enhanced clearance was evident only 7–12 days after injection. Since the injected cells had a short half-life (<10% remaining after 48 h) the effect of these cells in vivo was most probably due to the activation of the host's immune system. Mice that received cultured cells survived significantly longer than untreated mice following a lethal dose of EL4 cells. Cultured cells were much more effective in prolonging survival when used in conjunction with cyclophosphamide (CY). In animals receiving either cultured cells with or without CY or CY alone tumour clearance was markedly enhanced 7–12 days after injection.When challenged with a small dose of EL4 tumour cells (1×10⁴ SC per mouse) three of ten B6 mice treated with B6 anti-EL4 cultured cells were able to survive indefinitely. The frequency of CTL precursors to EL4 from the spleen cells of these surviving animals was about five-fold higher than that of normal spleen cells. Furthermore, CTL derived from primed spleen cells were more specific for EL4 than those derived from normal spleen cells.Abbreviations B6 C57BL/6J - BDF1 (C57BL/6J×DBA/2J) F1 - ConA SN concanavalin A supernatant - CTL cytotoxic T lymphocytes - CTL-P cytotoxic T-lymphocyte precursors - CY cyclophosphamide - E/T effector-to-target ratio - IL2 interleukin 2 - IP intraperitoneal - IUdR iododeoxyuridine - IV intravenous - LPS lipopolysaccharide - MST mean survival time     

The addition of interleukin-2 to cyclophosphamide therapy can facilitate tumor growth of B16 melanoma

The role of interleukin-2 (IL-2) on tumor growth of B16F10 melanoma cells was assessed in two sets of mice with different immune status: normal (immunocompetent) mice and immunodeficient mice. The two sets of animals were treated with cyclophosphamide (CY) or IL-2 alone or with a combined therapy of CY+IL-2. On days 6 and 10 after tumor cell injection, we evaluated the incidence of hepatic B16 melanoma metastases and the percentage of hepatic volume occupied by metastatic tissue. We observed that the CY alone (300 mg/kg, days 3 and 8 post-tumoral inoculation) significantly reduced tumor growth in all treated mice; however, CY proved more effective in normal recipients than in immunodeficient hosts. On the other hand, whereas administration of IL-2 alone (10⁵ IU daily, from day 3 to day 7) in immunocompetent mice significantly reduced tumor growth on days 6 and 10, in immunodeficient mice, no significant differences were observed in tumor growth either on the 6th or on the 10th day, in comparison to control groups. Finally, when the combined CY+IL-2 therapy was administered, an antisynergistic effect between these therapeutic agents was achieved both in normal and in immunodeficient mice. Thus, the addition of low-dose IL-2 (25×10³ IU daily, from day 4 to day 7) to high-dose CY (300 mg/kg, days 3 and 8) significantly increased tumor growth in both the early and later periods, compared to the effect of CY alone. It is concluded that exogenous IL-2 can facilitate tumor growth of B16 melanoma cells in vivo.     

Field bean protease inhibitor preparations,unlike methotrexate,can completely suppress Yoshida sarcoma tumor in rats

Protease inhibitor preparations (PIP) with antitryptic and antichymotryptic activities, isolated from field bean legume as well as doxorubicin and cyclophosphamide could effectively suppress the growth of Yoshida sarcoma ascites tumor cells transplanted in adult rats and prevent their death. As against this, methotrexate and heat-inactivated PIP were ineffective in such rats at varied doses of treatment tried. The percent survival of animals appeared to be related to the purity, treatment mode and the dose of PIP used. Zymographic analysis of the trypsin activated sarcoma cell homagenate revealed the presence of six protease bands in the molecular weight range of 51kD to 206kD. Prolonged interactions of such zymograms with protease inhibitors such as 20mM EDTA or 5mM diisopropyl fluorophosphate (DIFP) or 400 · μg/ml of PIP in reaction buffer indicated that these are not metalloproteases but serine proteases whose activities are inhibited by PIP and DIFP. Since proteases are involved in cell growth regulation and cell transformation, we hypothesize a positive relationship between the field bean protease inhibitor;s blocking action on tumor cell proteases and its tumor suppressing activity     

Immune mechanisms in leukemia: suppression of cellular immunity by drugs and x-irradiation.

The relative suppressive effects of x-irradiation (XR), cyclophosphamide (CY), prednisolone (PRD), and methotrexate (MTX) on the primary and secondary cellular immune response of C58/wm mice to syngeneic line Ib transplantable leukemia (Ib cells) were quantified. An LD10 dose of each agent was used for immunosuppression. XR, CY, and PRD were markedly suppressive for the primary immune response if given 24 hr before mice were immunized to Ib cells but less immunosuppressive if given 24 hr later. MTX was only slightly immunosuppressive XR, CY, and PRD also suppressed the secondary immune response if given before but not after antigen. The immunosuppressive effect of these agents was evaluated by defining their median immunosuppressive dose or the median time in days required for mice to recover from graded doses of each immunosuppressive agent. For example, the median recovery time from an LD10 of XR, CY, and PRD was 29.3, 19.7, and 3.7 days, respectively. Immunologic competence remaining after XR or drug treatment was quantified in terms of the LD50 dose of Ib cells required to kill recipient mice. For XR, CY, PRD, and MTX it was 10(6.16), 10(2.15), 10(6.90) and greater than 10(7.0) viable Ib cells, respectively. The overall results provided evidence that the primary and secondary cellular immune responses to a weak syngeneic tumor antigen were resistant to immunosuppression once they were initiated. There was a good correlation between the relative immunosuppressive effect of the test agents and the amount that they reduced the number of immune spleen cells. The agents also impaired the immunocompetence of individual spleen cells. Mechanisms by which XR or drugs might exert their immunosuppressive effects were discussed.     

Cooperation of nonsyngeneic tolerant lymphocytes: genetic restriction.

We have used a previously devised in vivo experimental model to investigate the ability of mouse thymus-dependent (T) and bone marrow-derived (B) lymphocytes to cooperate in immune (humoral) rejection of rat Yoshida ascites sarcoma (YAS). Because of conflicting reports in the literature concerning the effectiveness of T-B cooperation across major histocompatibility complex (MHC) barriers, we explored the interaction of T and B lymphocytes from mutually tolerant animals. Tolerance was achieved by establishing radiation chimeras of B6 → B6D2F₁ and D2 → B6D2F₁ constitutions. Chimeras' erythrocytes and spleen cells were shown by serological analysis to be the donor type. When the chimera was to serve as the tumor host or B-cell source, it was thymectomized prior to irradiation and reconstitution (TIR). Tolerance was evaluated by noting the inability of chimeric spleen cells to effect graft-versus-host damage upon injection into TIR host-type mice and the markedly reduced anti-host-type reactivity in short-term [³H]thymidine-uptake tests. Successful cooperation, manifested by YAS rejection, was seen whenever donor T and host B lymphocytes were syngeneic. Parental (P) T cells enabled F₁ TIR mice to reject YAS, but the reciprocal was not true: F₁ donor T cells did not cooperate with B cells in parental TIR mice. However, when the host B lymphocytes were tolerant P cells, i.e., in a P → F₁ TIR chimera, injected F₁ T lymphocytes did cooperate successfully. The final test of allogeneic T and B cells gave the clear-cut negative answer that, even when tolerant mice are used as sources of lymphocytes, cooperation does not occur. These results therefore confirm that T and B lymphocytes must at least share one MHC haplotype in order to cooperate.     

Amino acid uptake in plasma membrane vesicles isolated from proliferating tumor cells and tissues

Summary The transport of L-alanine, a natural substrate of system A, across plasma membrane vesicle preparations has been studied in the early stages of rat DENA-PH hepato-carcinogenesis and in a very undifferentiated rat ascites hepatoma cell line (Yoshida AH-130) in the exponential and stationary phase of growth.Kinetic analyses indicated an increase of the V_max value in DENA-PH-treated rats 30 h after partial hepatectomy as well as in exponential growing Yoshida ascites cells. In DENA-PH-treated rats the Km value was drastically reduced 7 and 60 days after surgery, when enzyme-altered hyperplastic and preneoplastic lesions were present in rat liver. Drastically reduced Km values were also found in Yoshida ascites cells.The results suggest that an altered alanine transporter might take place in liver plasma membranes from carcinogen-treated rats. This appears to occur also in an established tumor cell line, grown in vivo.Abbreviations AAF 2-acetylaminofluorene - DENA diethylnitrosamine - PH partial hepatectomy - PMSF phenylmethanesulfonyl fluoride     

Chemoimmunotherapeutic effect of cyclophosphamide on the highly metastatic MAT 13762 tumor

Summary We have observed that cyclophosphamide (CY) treatment of 13762 mammary adenocarcinoma tumor-bearing rats was found to cause tumor regression. Tumor-bearing animals cured with three low doses of CY were partially immune against IV and SC challenge with a high dose of 13762 cells. This immune protection mechanism in CY-cured animals appears to be a T (Ig^–) cell-mediated response. Irradiated rats reconstituted with CY-cured animal spleen cells were also partially protected against IV and SC challenge with 13762 cells, whereas irradiated rats reconstituted with CY-control animal spleen cells were not. In vitro primary and secondary cell-mediated cytotoxic activity of CY-cured spleen cells against target 13762 cells was low. The possible relevance of this tumor-model study is in the understanding of CY-induced tumor immune response and its role in preventing metastases or perhaps recurrent tumor growth.     

Cathepsins inhibit Growth of Yoshida Ascites Sarcoma in Rat

IT has been shown that rabbit anti-serum against rat serum (RARS) injected intravenously into rats produces fatal anaphylactic shock¹. This was interpreted as a reaction of RARS with γ-globulins adsorbed on the cell surface². It therefore seemed reasonable to investigate the same effect on enhancing antibodies by injecting RARS into rats bearing Yoshida ascites sarcoma (YAS). The result was as expected–delayed death. It has been suggested that the “de-enhanced” tumour cells become more susceptible to lymphocytes³. Although the presence of enhancing antibodies coating YAS cells has not actually been shown, if they are responsible for the observed phenomena, other agents acting on γ-globulins should result in both anaphylaxis and “de-enhancement”. We therefore used cathepsins isolated from white blood cells, which act specifically on β and γ-globulins⁴. illustration

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Importance of signaling via the IFN-α/β receptor on host cells for the realization of the therapeutic benefits of cyclophosphamide for mice bearing a large MOPC-315 tumor

Here we show that low-dose cyclophosphamide (CY), that depends for its therapeutic effectiveness on the immunopotentiating activity of the drug for T cell-mediated tumor-eradicating immunity, is curative for ~80% of wild-type (WT) mice bearing a large s.c. MOPC-315 tumor, but only for ~10% of IFN-α/βR^−/− mice bearing a large s.c. MOPC-315 tumor. Histopathological examination of the s.c. tumors of such mice on day 4 after the chemotherapy revealed that the low dose of CY led to accumulation of T lymphocytes in both the WT and the IFN-α/βR^−/− mice. However, in the CY treated tumor bearing WT mice the T lymphocytes were present throughout the tumor mass and in direct contact with tumor cells, but in the CY treated tumor bearing IFN-α/βR^−/− mice most of the T lymphocytes remained in blood vessels. In addition to being important for CY-induced transendothelial migration of T lymphocytes into the tumor mass, we show here that signaling via the IFN-α/βR is also important for CY-induced control of metastatic tumor progression in the spleen and liver of the tumor bearing mice. Finally, CY cured tumor bearing WT mice were resistant to a subsequent challenge with MOPC-315 tumor cells, but the few CY cured tumor bearing IFN-α/βR^−/− mice were not. Thus, signaling via the IFN-α/βR on host cells in MOPC-315 tumor bearers is important for CY-induced: (a) transendothelial migration of T lymphocytes into the tumor mass and the eradication of the primary tumor, (b) control of metastatic tumor progression, and (c) resistance to a subsequent tumor challenge. This work was supported by Research Grant 03-19 from the American Cancer Society-Illinois Division.     

Effect of preliminary administration of allogenic cells on transplantation immunity in mice receiving cyclophosphamide

It was shown that injection of 1 X 10(8) spleen cells from C57Bl/6 mice to CBA mice one day before the injection of cyclophosphamide (CY) helped the take of 2 X 10(7) allogeneic or semiallogeneic cells (injected for the second time 3 to 6 hours after C)). Criterion of survival is the ability of the donor cells to produce antibodies to sheep red blood cells in the recipients tolerant of this antigen. Injection of 1 X 10(8) allogeneic cells two days before CY produces no protective effect. Killer-cells proved to appear on the second day after the immunization with allogeneic cells; their peak was reached on the 5th day. The data obtained suggest that CY eliminated the recipients' lymphocytes, which responded to the transplantation antigens, whereas the killer-cells already formed were stable to the CY action.     

The negative systemic effect of BCGcw inoculated intraperitoneally

Summary The present studies describe a systemic effect of BCGcw inoculated intraperitoneally by observing its influence on the development of Morris hepatomas inoculated intramuscularly. In none of our studies did we observe an inhibition of tumor growth. Instead, an enhancement of tumor growth was seen with four antigenically distinct Morris hepatomas (3924a, 9098, 7777, and 5123tc) in two strains of inbred rats (Buffalo and ACI). Extensive studies with Morris hepatoma 3924a showed consistent enhancement of intramuscular tumor growth in eight of eight experiments with a tumor cell dose of 1×10⁵ and a BCGcw dose of 900 g. In BCGcw-inoculated animals, palpable tumors (1–2 g) were detected approximately 1 week earlier than in controls, and significantly larger tumor masses were noted on the day of sacrifice. With the threshold dose of cells, 1×10⁴, an increase in tumor incidence was observed. It was not necessary for the host to be immunized with BCGcw prior to tumor inoculation as enhancement of tumor growth occurred when the BCGcw were inoculated the same day or 7 days after tumor inoculation. Splenectomized animals also demonstrated BCGcw-mediated enhancement of tumor growth. BCGcw immunization blocks the induction of tumor-specific immunity. When 3924a ascites tumor cells were inoculated intradermally into normal animals, no tumor growth was observed and tumor-specific immunity resulted. When 3924a ascites tumor cells were inoculated intradermally into BCGcw-immunized animals, progressive intradermal tumors grew in all the animals, implying that tumor-specific immunity was not induced. The administration of BCGcw did not effect established tumor-specific immunity to line 3924a as assessed by tumor-specific rechallenge resistance. Studies with an allograft system showed that the ACI tumor 3924a would not grow in normal Buffalo rats, but transient tumor growth was observed when the Buffalo rats were immunized with BCGcw. The abbreviations used are: BCGcw, bacillus Calmette-Guérin cell walls attached to oil droplets; MLC, mixed lymphocyte culture; MER, methanol extract residue; IM, intramuscular; IP, intraperitoneal; ID, intradermal; Con-A, concanavalin-A     

Studies of allogeneic tumor transplants: induced rejection of advanced tumors by immune alteration of recipients

In the present experiments, a methylcholanthrene-induced sarcoma (S-702) of B10.D2 origin was found to grow rapidly in B6AF1 mice leading to the death of all recipients in 5 to 9 wk. Nevertheless, immunity to MHC antigens presented by the tumor was readily demonstrable in tumor-bearing mice by their responses to donor strain skin grafts until late in the course of tumor growth, when a nonspecific form of immune suppression developed. In addition, B6AF1 mice preimmunized by exposure to B10.D2 donor strain antigens did not permit tumor growth. Treatment of tumor-bearing B6AF1 mice with CY at 18 days, when the tumors measured over 12-mm in diameter, followed by the i.p. injection of B10.D2 lymphoid cells (at a dosage of from 1.2 to 2.5 X 10(8) cells) resulted in the complete regression of 100% of these large tumors. CY treatment combined with localized immune stimuli in the form of donor strain skin grafts or secondary tumor implants was incapable of producing a sufficiently heightened immune response to cause tumor rejection. A dose of CY temporarily retarded tumor growth in most mice, and in a minority of animals so treated (less than 25%) tumors regressed completely. In syngeneic (B10.D2) animals, CY also temporarily slowed tumor growth, but total regression was never observed. An effective B10.D2 cell inoculum could consist not only of living lymphoid cells but of irradiated (1000 rad) cells as well. Tumor cell suspensions (after irradiation, 10,000 rad) were also effective. These observations suggest local immune factors at the host-tumor interface may have been of importance in the survival of these allogeneic tumor transplants and that CY influenced this state, perhaps through an influence on suppressor cells, allowing subsequent administration of donor strain cellular antigens to induce an effective tumor rejection response.     

Prevention of transplantable tumors by adoptive transfer of spleen cells from immunized rats.

Fischer 344 rats were specifically hyperimmunized with allogeneic, nonvirus-producing [Kirsten murine sarcoma virus (KiMSV)] or syngeneic, virus-producing [KiMSV (Rasheed)] rat tumors. Spleen cells taken from these rats adoptively transferred protection against a 100 to 1,000 X rat tumor dose50 cell challenge with several different transplantable rat tumors. Protection was obtained with spleen cells after removal of adherent cells and macrophages but not peritoneal cells. The spleen cells were not directly cytotoxic but required more than 3 days residence in the recipient before protecting the recipient against challenge. No protection against tumor cell challenge was observed when spleen cells were lethally x-ray irradiated before injection into nontreated rats. Spleen cells taken from rats immunized with normal histocompatibility antigens did not protect in this test system.     

Uridine diphosphate reductase of Ehrlich ascites tumor is insensitive to hydroxyurea

Uridine diphosphate (UDP) reductase was isolated in the supernatant fraction obtained after the acidification of the cytosol of Ehrlich ascites tumor cells, and was found insensitive to 10 mM hydroxyurea. However, cytidine diphosphate (CDP) reductase, being separated concurrently in the precipitate fraction, was readily inhibited. In the cytosol fraction of either Ehrlich ascites tumor, Yoshida ascites sarcoma or regenerating rat liver after partial hepatectomy, UDP reduction activity, in contrast to CDP reduction activity, is not sensitive to hydroxyurea.     

Intraperitoneal transplantation of Yoshida ascites hepatoma cells after fine needle aspiration from tumor bearing rats organs

The authors tested the ability of scattered tumor cells to re-form a tumor in vivo. Disseminated tumor cells are morphologically visible stained with May-Grünwald Giemsa in the lung, liver, kidney, and spleen of Yoshida ascites tumor bearing rats. Free tumor cells can easily be fine needle aspirated from those organs and injected in syngeneic Wistar rats. All the host rats show ascites tumor take after intraperitoneal transplantation of each aspirated sample. This biological model might be useful to study in vivo a wide range of properties of neoplastic and non-neoplastic host cells.     

Chemotherapy-induced modulation of natural killer and lymphokine-activated killer cell activity in euthymic and athymic mice

Combinations of chemotherapy and interleukin-2 (IL-2) aimed at improving therapeutic efficacy in cancer patients have generally proved disappointing. Although chemotherapy blocks tumor growth and sometimes boosts immune functions, most drugs are immunosuppressive, at least transiently. Therefore, it is reasonable to assume that maximal exploitation of the immunostimulatory and antitumor activity of both modalities requires careful coordination of chemotherapy and IL-2 timing. We analyzed the temporal effect of 5-fluorouracil (5-FU, 100–120 mg/kg), cyclophosphamide (CY, 100 mg/kg), Adriamycin (8 mg/kg) and dacarbazine (100 mg/kg) on the activation of natural killer/lymphokine-activated killer (NK/LAK) cells by IL-2 in several strains of euthymic mice and in athymic nude mice. Following in vivo or in vitro exposure to IL-2 1–15 days after chemotherapy, the total lytic activity of the spleen and the number of LAK precursors (LAK-p) were measured. In euthymic mice injected with IL-2 (5×10⁴ Cetus units twice daily for 4–5 days), 5-FU augmented (up to 37-fold, days 1–9) and CY reduced (up to day 6) LAK activity, as compared with that in the IL-2 control. In bulk cultures containing IL-2 (1000 CU/ml, 3–4 days), both 5-FU and CY reduced LAK activity of euthymic mice splenocytes for up to 6 days after chemotherapy, which was followed on day 9 by full recovery. In splenocytes of nude mice, 5-FU increased and CY diminished LAK activation in bulk cultures, starting 3 days after chemotherapy. In athymic mice, 5-FU markedly augmented the total number of LAK-p/spleen (up to 30-fold, days 3–9), as determined by limiting-dilution cultures with IL-2 (for 7–8 days). In euthymic mice, in contrast, LAK-p levels decreased for up to 6–9 days after treatment with 5-FU, Adriamycin or dacarbazine, later recovering to pretreatment levels, whereas CY markedly increased LAK-p (up to 15-fold) when administered 6–12 days before limiting-dilution culture initiation. The effect of chemotherapy on LAK and NK activity was essentially similar. In other experiments, a subset of asialoGM1-LAK-p was found in the spleens of 5-FU-treated mice, but not in untreated mice. Our results suggest that the immunomodulatory effect of chemotherapy on NK/LAK activity in mice is variable and largely depends on the drug itself, the interval between chemotherapy and IL-2 administration, the strain of mice and the assay used.     

Improved therapeutic effects of interleukin 2 after the accumulation of lymphokine-activated killer cells in tumor tissue of mice previously treated with cyclophosphamide

Summary We investigated the combined effects of human recombinant interleukin 2 (IL-2) and cyclophosphamide (CY) on s.c. transplanted 3LL lung carcinoma in C57BL/6 mice. A total of 95% of the tumors were competely cured when CY (150 mg/kg, i.v.) was given on day 5 (5 days after tumor implantation) and IL-2 (5×10⁴ Jurkat Units/day, i.p.) was then combined with it between day 6 and day 15. CY alone brought about the complete regression of tumors, although 60% of the mice died of local recurrence and pulmonary metastasis; IL-2 alone had no therapeutic effect. Satisfactory effects from the combination of CY and IL-2 were also obtained by 5 days administration of IL-2 between days 11 and 15, initiated 6 days after CY treatment, but not by that given before CY (days 1–5) or 1 day after CY (days 6–10). No therapeutic effects from IL-2 were observed when it was combined with other types of chemotherapy that showed not therapeutic effects by themselves. Nor were we able to observe any transplantation resistance to the rechallenge of 3LL tumor in cured mice. We particularly examined the lymphokine-activated killer (LAK) cells as we suspected that these were responsible for the development of active effector cells in the treated mice. LAK cell activity in fresh spleen cells was detected in mice treated with IL-2 alone but not in untreated mice nor in those treated with CY alone or CY plus IL-2. The number of LAK precursor cells in the spleen had increased on day 8 and on day 13 in untreated mice with 3LL, as compared with the incidence in normal mice, while the number of cells had decreased by day 18. On the other hand LAK precursor cells were suppressed on day 8 and tended to recover thereafter in CY-treated mice. Adoptively transferred LAK cells were found to accumulate in CY-treated tumors 2.5 times more densely than in untreated tumors. The preferential accumulation of LAK cells that had been activated systemically by the appropriately timed administration of IL-2 in tumor tissue was followed by the improved effects obtained by combined treatment with CY and IL-2.Supported in part by Grants-in-Aid for Cancer Research from the Japanese Ministry of Education, Science and Culture and from the Japanese Ministry of Health and Welfare