The anti-tumor efficacy of lymphokine-activated killer cells and recombinant interleukin 2 in vivo

We showed previously that adoptive immunotherapy with the combination of LAK cells and recombinant IL 2 (RIL 2) can markedly reduce pulmonary micrometastases from multiple sarcomas established 3 days after the i.v. injection of syngeneic tumor cells in C57BL/6 mice. In this report, we analyzed the factors required for successful therapy. Titration analysis in vivo revealed an inverse relationship between the number of pulmonary metastases remaining after treatment and both the number of LAK cells and the amount of RIL 2 administered. Fresh or unstimulated splenocytes had no anti-tumor effect; a 2- to 3-day incubation of splenocytes in RIL 2 was required. LAK cells generated from allogeneic DBA (H-2d) splenocytes were as effective in vivo as syngeneic, C57BL/6 (H-2b) LAK cells. The anti-metastatic capacity of LAK cells was significantly reduced or eliminated when irradiated with 3000 rad before adoptive transfer. The combined therapy of LAK cells plus RIL 2 was shown to be highly effective in mice immunosuppressed by 500 rad total body irradiation and in treating macrometastases established in the lung 10 days after the i.v. injection of sarcoma cells. Further, reduction of both micrometastases and macrometastases could also be achieved by RIL 2 alone when administered at higher levels than were required with LAK cells. The value of LAK cell transfer and of IL 2 administration for the treatment of tumors established at other sites is currently under investigation.     

Lysis of fresh murine mammary tumor cells by syngeneic natural killer cells and lymphokine-activated killer cells

Summary We have compared the ability of natural killer (NK) cells from two substrains of C3H mice that differ with respect to their susceptibility to the development of mammary adenocarcinomas to lyse fresh syngeneic mammary tumor cells. Single cell suspensions of mammary tumors from retired breeder females were used as targets in 22-h ⁵¹Cr-release cytotoxicity assays with syngeneic NK cells. Tumor cell suspensions were prepared by enzymatic digestion of finely minced tissue followed by centrifugation through a discontinuous Percoll gradient. Effector cells were prepared by passing spleen cells over nylon wool followed by centrifugation through Percoll fraction 7. Syngeneic NK cells had significant levels of lysis against 5/8 tumors studied. NK cells from low risk animals (C3Heb/FeJ) consistently demonstrated greater cytotoxicity against tumor cell preparations than did effectors from the high tumor substrain (C3H/OuJ). Study of cytocentrifuge preparations stained with Wright-Giemsa revealed that the two substrains were identical with respect to the number of azurophilic granules present in the cytoplasm of their NK cells. We have also shown that lymphokine-activated killer (LAK) cells can be generated from splenocytes in C3H mice. While LAK cells from both substrains were capable of lysing fresh syngeneic mammary tumor cells in vitro, LAK cells from the animals at high risk for the formation of mammary adenocarcinomas had greater cytotoxicity against tumor cell suspensions than LAK cells from the low tumor substrain.     

Effect of interleukin 2 on fetal thymocytes in organ cultures: generation of lymphokine-activated killer cells

Cells with cytolytic activity can be detected in mouse fetal thymic lobes cultured in the presence of interleukin 2 for 6 days. The lymphokine-activated killer cells from 14-day fetal thymic lobes are relatively resistant to treatment with anti-Ly-2 antibody and complement (CD8-) but sensitive to anti-Thy-1 and complement treatment (Thy-1+). They display major histocompatibility complex-unrestricted killing, lysing both syngeneic and allogeneic tumor cells, but will not lyse human xenogenic target cells. Low levels of cytotoxic activity can be detected in thymic lobes from Day 12-13 embryos and this activity increases with embryonic age. While the events which lead to the inhibition of normal maturation of fetal thymocytes by inclusion of IL-2 in fetal thymus organ cultures are unknown, the appearance of cytotoxic cells raises the question of whether they are involved in the normal intrathymic cell death process.     

Recombinant interleukin 2 stimulates in vivo proliferation of adoptively transferred lymphokine-activated killer (LAK) cells

We previously reported that the adoptive transfer of lymphokine-activated killer (LAK) cells plus repetitive injections of recombinant interleukin 2 (IL 2) produced a marked reduction in established pulmonary metastases from a variety of murine sarcomas. The requirement for the exogenous administration of IL 2 prompted a subsequent examination of the role of IL 2 in the in vivo function of transferred LAK cells. The in vivo proliferation and migration patterns of lymphoid cells in C57BL/6 mice were examined after i.v. transfer of LAK cells alone, i.p. injection of IL 2 alone, or the combination of LAK cells and IL 2. A model for in vivo labeling of the DNA of dividing cells was used in which mice were injected with 5-[125I]-iodo-2'-deoxyuridine (125IUdR) and, 20 hr later, their tissues were removed and were counted in a gamma analyzer. A proliferation index (PI) was calculated by dividing the mean cpm of organs of experimentally treated mice by the mean cpm of organs of control mice. In animals given LAK cells alone, the lungs and liver demonstrated little if any uptake of 125IUdR above saline-treated controls (PI = 2.5 and 0.8, respectively, on day 5), whereas the same organs of mice receiving 6000 U of IL 2 alone displayed higher radiolabel incorporation (PI = 7.1 and 5.9, respectively). When mice were given LAK cells plus 6000 U of IL 2, their tissues showed an additional increase in 125IUdR uptake. In the spleen, kidneys, and mesenteric lymph nodes, IL 2 treatment alone (6000 U) produced elevated PI values that were not, however, additionally increased if LAK cells were also administered. To separate the stimulatory effects of IL 2 on host lymphocyte proliferation from similar IL 2 effects on injected LAK cells, these studies were repeated in mice immunosuppressed by 500 rad total body irradiation. Pre-irradiation of the host sufficiently reduced endogenous lymphoid expansion stimulated by IL 2 so as to allow the demonstration that IL 2 also induced the proliferation of the transferred LAK cells. A variety of studies confirmed that the injected LAK cells were actively proliferating in tissues in vivo under the influence of IL 2. Substitution of "normal" LAK cells with fresh and cultured (without IL 2) splenocytes, or irradiated LAK cells did not result in increased 125IUdR uptake in tissues. Histologic studies corroborated the findings of the 125IUdR incorporation assays and revealed extensive lymphoid proliferation in irradiated mice receiving LAK cells plus IL 2.(ABSTRACT TRUNCATED AT 400 WORDS)     

The anti-tumor efficacy of lymphokine-activated killer cells and recombinant interleukin 2 in vivo: direct correlation between reduction of established metastases and cytolytic activity of lymphokine-activated killer cells

Our previous studies demonstrated that the incubation of human peripheral blood lymphocytes or murine splenocytes in recombinant interleukin 2 (RIL 2) resulted in the generation of lymphokine-activated killer (LAK) cells capable of lysing a broad spectrum of fresh tumors in short-term chromium-release assays. Moreover, injections of LAK cells plus RIL 2 were highly effective in eliminating established 3 day metastases in the lung and liver (1-3). We have examined several parameters to define whether or not the cytolytic activity of LAK cells as measured in vitro correlated directly with the in vivo anti-tumor efficacy of adoptively transferred LAK cells. LAK cells plus RIL 2 could mediate marked reductions of established pulmonary metastases in mice rendered T cell deficient by adult thymectomy and lethal, total body irradiation followed by reconstitution with T cell-depleted bone marrow and spleen cells. Thus there was no requirement for additional T lymphocytes of host origin for successful therapy with adoptively transferred LAK cells plus RIL 2. Fresh splenocytes depleted of T cells by anti-Thy-1.2 monoclonal antibody plus complement generated LAK cells that were as highly lytic to fresh tumor in vitro and were as effective in reducing established pulmonary metastases as those generated from untreated or complement-treated splenocytes. Thus the precursor to LAK cells with anti-tumor activity in vitro and in vivo did not express the Thy-1 antigenic marker. In contrast, treatment of LAK effector cells (those generated from a 3-day incubation of fresh, normal splenocytes in RIL 2) with anti-Thy-1.2 antibody plus complement reduced or abolished their in vitro cytolytic activity. However, when combined with the systemic administration of RIL 2, these T cell-depleted, non-lytic LAK cells remained as effective in reducing the number of established pulmonary metastases upon adoptive transfer as untreated or complement-treated lytic LAK cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

A regimen of surgical adjuvant immunotherapy for cancer with interleukin 2 and lymphokine-activated killer cells

We designed a unique regimen of adoptive immunotherapy with lymphokine-activated killer (LAK) cells and recombinant interleukin 2 (rIL-2) for application with surgical adjuvant therapy of cancer. The regimen features the prolonged (6 consecutive days) s.c. administration of low-dose rIL-2 and the transfer ofex vivo generated LAK cells from regional lymph node lymphocytes, obtained at the time of surgical operation. According to this regimen, 5 patients with primary lung cancer received immunotherapy about 2 weeks after surgery (pulmonary lobectomy). Clinical toxicities included fever(5/5), fatigue(5/5), slight(< 5%) weight gain(5/5), increase of pleural effusion at the lobectomy site(2/5), and edema formation(1/5). All toxicities reversed within 4 days after the completion of therapy. Rebound lymphocytosis after therapy ranged from 2.4 to 5.5-fold (mean, 4.3-fold) over the baseline. Peripheral blood lymphocytes obtained during this lymphocytosis exhibitedin vitro LAK activity in 4 of 5 patients. Thus, the regimen is considered to be well-tolerable and immunologically active in regard to the postoperative state of the patients.     

Physiology of natural killer cells. In vivo regulation of progenitors by interleukin 3

Adoptive transfer of bone marrow cells to syngeneic lethally irradiated C57BL/6 mice was used to study the maturation of natural killer (NK) cells from their progenitors. The NK progenitor cell was found to be asialomonoganglioside-negative, (aGM1-) Thy-1-, NK-1-, Ly-1-, Ly-2-, and L3T4-. The NK cells emerging from the bone marrow grafts were aGM1+, NK-1+, Thy-1+/-, Ly-1-, Ly-2-, and L3T4- and to have a target specter similar to that of NK cells isolated from the spleen of normal mice. The regulatory role of interleukin 2 (IL-2) and interleukin 3 (IL-3) for the maturation of NK cells was examined by exposure of the bone marrow cells to the lymphokines in vitro before bone marrow grafting or by treatment of bone marrow-grafted mice with lymphokines through s.c. implanted miniosmotic pumps. IL-3 antagonized the IL-2-induced maturation of NK cells in vitro and strongly inhibited the generation of NK cells after adoptive transfer of bone marrow cells in vivo. The suppressive effect of IL-3 was evident throughout the treatment period (8 or 16 days) but was apparently reversible because NK activity returned to control levels within 8 days after cessation of treatment. The inhibition of cytotoxic activity was accompanied by a reduced appearance of cells with the NK phenotypic markers aGM1 or NK-1, indicating that not only the cytotoxic activity of NK cells but also their actual formation was inhibited. Concomitantly, a moderate increase in cells expressing the T cell marker L3T4 and an increased proliferative response to the T cell mitogen concanavalin A was observed. A direct estimate of the effect of IL-3 on the frequency of NK cell progenitors was obtained by limiting dilution analysis of bone marrow cells at day 8 after bone marrow transplantation. The estimated minimal frequency of NK cell progenitors was reduced from 1/11,800 in control to 1/41,900 in IL-3-exposed mice. IL-3 may take part in the homeostasis of NK cells by the down-regulation of their progenitors.     

In vivo administration of low-dose human interleukin-2 induces lymphokine-activated killer cells for enhanced cytolysis in vitro

We have examined the effect of the intradermal administration of IL-2 on the generation of natural killer (NK) cell and lymphokine-activated killer (LAK) cell activity. Peripheral blood mononuclear cells (PBMC) obtained from borderline lepromatous (BL) and lepromatous leprosy (LL) patients and normal volunteers prior to and after IL-2 injection were stimulated in vitro with IL-2 and their cytolytic activities compared against 51Cr labeled target K562 cells, Daudi cells, and monocytes. Before IL-2 administration, PBMC obtained from BL/LL patients and normal volunteers possessed similar levels of NK cell activity indicating that the NK cell activity of the BL/LL patients was intact. LAK cell activity was induced with IL-2 in vitro in both BL/LL patients and in normal volunteers. The level of LAK cell activity in BL/LL patients was, however, suboptimal. A single intradermal dose of 25 micrograms IL-2 had no effect on the phenotype of circulating mononuclear cells in either patients or normal volunteers. However, 6-12 days after IL-2 injection and subsequent restimulation of the PBMC with IL-2 in vitro, cytolytic activity of LAK cells obtained from the BL/LL patients was enhanced while cells from normal volunteers expressed the same high levels of activity as observed before IL-2 injection.     

In vitro sensitization and expansion with viable tumor cells and interleukin 2 in the generation of specific therapeutic effector cells

We have investigated the efficacy and immunologic characteristics of immune effector cells generated from cultures containing large numbers of viable tumor cells and interleukin 2 (IL 2) in the adoptive immunotherapy of experimentally induced pulmonary metastases from the newly developed, weakly immunogenic MCA 105 sarcoma in mice. The current culture conditions allowed increases of either normal or MCA 105 immune spleen cells up to 94-fold in 15 days. The in vitro expanded normal and MCA 105 immune cells displayed nonspecific in vitro cytotoxicity against several syngeneic tumor targets. However, therapeutically effective cells could only be obtained from cultures initiated with MCA 105 immune spleen cells. Immunotherapy with expanded immune effector cells could lead to the reduction of established 3 day pulmonary metastases, prolongation of survival, and cure of tumor in the majority of animals. The generation and proliferation of therapeutic effector cells in vitro depended on the presence in cultures of specific tumor stimulator cells as well as the presence of IL 2. Although immunotherapy with either fresh noncultured or secondarily in vitro-sensitized (IVS) MCA 105 immune spleen cells was immunologically specific, the efficacy of the adoptive cellular therapy with cultured but not fresh immune cells could be improved by the administration to tumor-bearing hosts of exogenous IL 2. In addition to numerical expansion, the IVS immune cells, on a per cell basis, afforded an eightfold to 10-fold increase in therapeutic efficacy when compared with fresh noncultured MCA 105 immune cells. Our results indicate that the current culture procedure induced in vitro antigenic stimulation and expansion of tumor-specific immune effector cells that was otherwise not possible by conventional mixed lymphocyte-tumor cultures.     

Lymphokine-activated killer cells in rats: generation of natural killer cells and lymphokine-activated killer cells from bone marrow progenitor cells

The coculture of rat bone marrow cells with recombinant interleukin-2 induced the generation of cells mediating natural killer (NK) activity and subsequent lymphokine-activated killer (LAK) activity depending upon the dose of IL-2 and time of culture. NK activity was detected as early as 4 to 5 days after the addition of IL-2 and could be evoked with as little as 5 to 50 U/ml. The induced NK cells had large granular lymphocyte (LGL) morphology and expressed 0X8 and asialo GM1 surface markers but did not express 0X19 or W3/25 markers. LAK activity was detected only after 5 days of culture, and required above 100 U/ml IL-2. Cells mediating LAK activity also expressed 0X8 and asialo GM1 but not 0X19. The generation of detectable NK and subsequent LAK activity was due to induction of early progenitor cells and not contaminating mature LGL/NK cells within the bone marrow population since of removal of such mature NK cells with L-leucine methyl ester (L-LME) did not affect the subsequent generation of either activity. Moreover, the removal of actively dividing cells as well as mature NK cells from the bone marrow by treatment with 5-fluorouracil (5-FU) in vivo enriched the remaining bone marrow population for both NK and LAK progenitor cells. The phenotype of the L-LME- and 5-FU-resistant NK and LAK progenitor cells within populations of bone marrow was determined by antibody plus complement depletion analysis. Although treatment of normal bone marrow with anti-asialo GM1 + C reduced the induction of NK and LAK activity in 5-day cultures, treatment of 5-FU marrow with anti-asialo GM1 + C did not affect either activity. Treatment with a pan-T cell antibody + C did not affect the development of NK or LAK activity under any conditions. Thus, the 5-FU-resistant NK/LAK progenitors were asialo GM1 negative but became asialo GM1+ after induction by IL-2. Finally, evidence that bone marrow-derived LAK cells were generated directly from the IL-2-induced NK cells was obtained by treating the IL-2-induced LGL/NK cells with L-LME.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interleukin-6 does not support interleukin-2 induced generation of human lymphokine-activated killer cells

Summary The activity of lymphokine-activated killer (LAK) cells is supported by various cytokines. The objective of this study was to see if recombinant interleukin-6 (IL-6) either alone or in combination with interleukin-2 (IL-2) has any effect on the generation of LAK cells. Peripheral blood mononuclear cells of healthy donors were cultured for 4 or 6 days with both cytokines either alone or in combination. LAK activity against K562 and natural killer-resistant Daudi cells was assessed by a 4-h and an 18-h⁵¹Cr-release assay at various effector to target ratios. IL-6 alone in increasing concentrations did not induce LAK cell activity. Neither additive nor synergistic effects of IL-6 with IL-2 were observed. Immunofluorescence analysis with phycoerythrin-conjugated anti-CD56 antibody demonstrated that IL-6 could not maintain or increase the number of CD56-positive cells over a 6-day culture period. These results suggest that IL-6 does not support LAK cell generation by itself or increase LAK cell activity in combination with IL-2.     

Studies of serum-free medium for the generation of lymphokine-activated killer cells

We examined a serum-free medium (designated as TYI 101) for the generation of lymphokine-activated killer (LAK) cells from human lymphocytes, regional lymph node lymphocytes (RLNL) and peripheral blood lymphocytes (PBL). TYI 101 medium consisted of, in addition to nutrient mixture, transferrin, insulin, fetuin, sodium selenite, 2-mercaptoethanol, o-phosphorylethanolamine, chick egg yolk and porcine kidney extract. These hormones were effective for supporting RLNL proliferation as assessed by (³H)-thymidine uptake. When human lymphocytes from two different sources were cultivated with recombinant interleukin 2 (rIL-2) in TYI 101 medium, LAK activity was generated. In cultures of PBL from a healthy donor, LAK cells were generated in TYI 101 medium as efficiently as in RPMI 1640 medium supplemented with 10% human AB-type serum (RPMI-AB). In cultures of RLNL from lung cancer patients, LAK activity obtained in TYI 101 medium was about sixty-five percent of that in RPMI-AB. However, the addition of a small amount of AB-type serum improved the generation of LAK activity, LAK cell expansion, and cell viability in TYI 101 medium. We conclude that TYI 101 medium can be used for the generation of LAK cells from human lymph node lymphocytes with supplementation of none or only a reduced amount of human serum.Abbreviations IMDM Iscove's Modification of Dulbecco's Medium - rIL-2 recombinant Interleukin - LAK Lymphokine-Activated Killer - RLNL Regional Lymph Node Lymphocytes - PBL Pheripheral Blood Lymphocytes - PBS Phosphate-Buffered Saline - RBC Red Blood Cells - RPMI-AB RPMI 1640 medium supplemented with 10% human AB-type serum Address for offprints: Tsukuba Research Laboratory, Japan Synthetic Rubber Co., Ltd., 25 Miyukigaoka, Tsukuba-shi, Ibaraki, 305 Japan     

Lysis of endothelial cells by autologous lymphokine-activated killer cells

The mechanisms of lysis of endothelial cells derived from human umbilical vein (HUVEC) by autologous lymphokine-activated killer (LAK) cells, generated from cord blood lymphocytes of the same donor, were investigated. Freshly isolated HUVEC as well as HUVEC cultured for several passages were efficiently lysed by autologous LAK cells, and their susceptibility to the LAK cells was almost the some as that of allogenic HUVEC. Complement-depletion experiments revealed that the lysis was mainly dependent on CD16-natural killer (NK) LAK cells. Pretreatment of HUVEC with recombinant interferon (rIFN) for 24 h made them resistant to lysis by autologous LAK cells, while pretreatment with either rIL-1. rTNF, or acidic or basic fibroblast growth factor did not alter the lytic sensitivity of HUVEC. The resistance of rIFN-treated HUVEC was specific to lysis by CD16⁺ NK LAK cells, and their lysis by CD3⁺ T-LAK cells was not significantly altered. Moreover, in comparison with control HUVEC or rIL-1-treated HUVEC, rIFN-treated HUVEC had a significantly less potent inhibitory effect on the lysis of untreated HUVEC, when used as an unlabeled target. This suggests that rIFN treatment may down-regulate the recognition of some molecules on HUVEC by rIL-2-activated NK cells. These data suggest that damage of the endothelium during LAK therapy is mainly dependent on LAK cells with a NK phenotype that can specifically recognize a certain molecule on autologous endothelial cells.     

Lysis of human monocytes by lymphokine-activated killer cells

Human peripheral blood leukocytes (PBL), stimulated in vitro with recombinant human interleukin 2 (IL-2) for 2-7 days, were seen to lyse autologous and allogeneic monocytes in a 4-hr 51Cr-release assay. The lymphokine-activated killer (LAK) cells against monocytic cells were selective in that polymorphonuclear leukocytes (PMN) and nonadherent PBLs were not lysed by these cells. Monocytes which had been cultured for 2-7 days served as better targets than uncultured cells. Also, kinetic studies demonstrated parallel activation of cytolytic activity against monocyte targets and FMEX, an natural killer cell-insensitive human melanoma target. Separation of PBLs by discontinuous density centrifugation identified the effector population in the fractions enriched for large granular lymphocytes (LGL). Precursor cells were seen to express CD2, CD11, and some CD16 markers, but not CD3, CD4, CD8, CD15, Leu M3, or Leu 7. The effector population after IL-2 activation retained the phenotype of the precursor cell. These studies indicate that IL-2 can generate LAK cells against monocytic cells, and this cytolytic activity, especially against autologous monocytes, must be taken into account when IL-2 or LAK cells are used for immunomodulation in cancer patients.     

In vivo generation of hapten-specific killer T cells without elimination of suppressor cells.

We have provided evidence to demonstrate that hapten-specific killer cells can be generated in vivo toward hapten-conjugated syngeneic splenic cells. The critical aspect is to provide an auxillary cellular antigenic stimulus in addition to the hapten-conjugated syngeneic cells. In our experiments this stimulus was CBA/J splenic cells that possess MIs disparate but H-2 compatible antigens with C3H/HeN hosts. The Mls antigen has been shown by others to activate helper T cells in vitro to synthesize KAF, a signal that prekiller cells require besides target antigen. Killer cells (shown to be T cells) as well as helper T cells were found to be derived from the C3H host. Induction in the host animal of partial tolerance to the auxiliary cells possessing Mls antigen abrogated the response. This system, besides providing a better understanding of the control mechanisms involved in the development of T killer cells in vivo, points to a way in which the latter may be generated-ng suppressor cells. The latter principle may prove highly useful in certain clinical situations.     

In vivo migration and tissue localization of highly purified lymphokine-activated killer cells (A-LAK cells) in tumor-bearing rats

Our laboratory has previously reported that the adoptive transfer of highly purified lymphokine-activated killer cells (adherent-LAK, A-LAK) into Fischer 344 (F344) rats bearing established lung or liver micrometastases effectively reduced the resultant tumor growth more than 90%, leading to significant increases in animal survival (Cancer Res. 49, 1441, 1989). To begin to investigate the mechanism(s) by which A-LAK cells mediate this anti-tumor effect, we studied their migration patterns in F344 rats bearing experimentally induced lung and liver metastases as well as subcutaneous tumors. A-LAK cells which were phenotypically 95 to 100% natural killer cells/large granular lymphocytes were labeled with either 51Chromium or fluorescein diacetate (so as to be visualized microscopically). Intravenous injection of such labeled A-LAK cells did not show significant differences in their tissue distribution patterns in tumor-bearing versus normal rats, even when high levels of exogenous recombinant interleukin-2 (rIL-2) was administered. A-LAK cells first migrated to the lungs and then subsequently migrated to the liver and spleen as early as 2 to 6 hr following iv injection. The kinetics of exit of A-LAK cells from the pulmonary capillary beds was not significantly different in rats bearing 3-day micrometastases or 14-day macrometastases compared to normal rats. Moreover, the presence of metastases in the liver did not alter the extent or kinetics of entry of A-LAK cells into the liver even in the presence of exogenously administered rIL-2. Finally, in rats bearing subcutaneous tumors, no evidence could be obtained that A-LAK cells were selectively localized to the tumor site. Tissue sections of livers from metastases-bearing animals injected with fluorescein diacetate labeled A-LAK cells did not demonstrate significant numbers of A-LAK cells infiltrating tumor nests with or without the administration of exogenous IL-2. These data suggest that A-LAK cells may mediate tumor regression in vivo by direct and indirect mechanisms, possibly through the secretion of cytokines and/or the recruitment of secondary effector cells.     

Immunotherapy of intraperitoneal cancer with interleukin 2 and lymphokine-activated killer cells reduces tumor load and prolongs survival in murine models

The control of malignancy disseminated within the peritoneal cavity is an important problem in the management of low-grade gastrointestinal and ovarian neoplasms. A model of peritoneal carcinomatosis in the mouse was used to investigate the potential of lymphokine-activated killer (LAK) cells and exogenous interleukin 2 (IL-2) to control intraperitoneal tumor. LAK cells are splenocytes activated in vitro by IL-2. C57BL/6 mice were injected intraperitoneally with a lethal inoculum of syngeneic MCA-105 tumor. Three days later, the established tumor was treated with adoptively transferred LAK cells and/or exogenous IL-2 administration. LAK cells alone were ineffective in reducing intraperitoneal tumor. Administration of IL-2 alone resulted in limited tumor reduction. Treatment with exogenous IL-2 in conjunction with LAK cells resulted in the greatest reduction of intraperitoneal tumor. The larger the number of LAK cells given, the greater the reduction in tumor. Frequent intraperitoneal bolus administration of IL-2 was more effective than a single daily intraperitoneal injection and intraperitoneal administration of IL-2 and LAK was more effective than systemic treatments. Marked prolongation of life was seen in mice treated with LAK cells plus exogenous IL-2. We conclude that intraperitoneal LAK cells plus exogenous IL-2 is an effective treatment regimen for reducing intraperitoneal tumor in this murine model.     

In vivo modulation of lymphokine-activated killer cell activity by cell wall components of Candida albicans.

We have previously reported that inoculating CD2F1 mice intraperitoneally with five doses of 2 x 10(7) inactivated Candida albicans (CA) cells was associated with the induction of lymphokine-activated killer (LAK)-like effectors. In this study we investigated the ability of some purified cell wall components of CA (CA-CW) to induce LAK-like cells in vivo. Multiple administrations of glucan ghost (GG), a mannoprotein mixture (MP) and a low-protein mannan fraction (M) at variance with whole CA did not induce LAK-like cells in the peritoneal cavity. However, the broad-spectrum antitumor cytotoxicity induced by CA could be recalled to a high level by a booster dose of MP and M, but not GG, given up to 70 days after the multiple CA-treatment. This induced cytotoxicity was maximum when the booster was given on Day +14 after CA-treatment and minimum on Day +70. In CA-treated mice, inoculated on Day +30 with CA or MP, LAK-like cytotoxicity was already significantly increased 4 hr after the booster, but the maximum value was reached at 24 hr. Anti-mannan antibodies did not interfere with LAK-like cells induction by CA because splenectomy before CA-treatment or passive administration of anti-mannan antibodies had no effect on the rapid activation of cytotoxicity by CA or a booster dose of MP. Administration of recombinant human interleukin-2 (rhIL-2) to CA-treated mice induced a higher level of NK activity than that induced by the same dose in untreated control mice, but did not activate LAK-like effectors. The results indicate that LAK-like effectors are easily generated in the peritoneal cavity by a booster with a defined antigenic constituent of CA cell wall for a long period in CA-sensitized mice.     

Activation of nonspecific killer cells by interleukin 2-containing supernatants

In the absence of specific antigen stimulation, nonspecific killer cells were induced by culturing C57BL/6 lymph node or spleen cells with interleukin 2-containing supernatants. These supernatants were obtained from stimulation of either rat spleen cells with concanavalin A or a variant of the T cell lymphoma, EL4 (H-2b) with phorbol myristic acetate. The ability of the EL4 supernatant to induce nonspecific killer cells was abrogated by absorption with an interleukin 2-dependent T cell line or by concanavalin A-stimulated spleen cell blasts, but not by lipopolysaccharide-stimulated spleen cell blasts or by a non-interleukin 2-producing EL4 line. Partially purified interleukin 2 from EL4 supernatants could also support nonspecific killer cell induction. The induction of cytolytic cells by interleukin 2 is sensitive to gamma-irradiation and has a D omicron of 120 rad. The nonspecific killer cells induced are likely cytotoxic T lymphocytes; the majority of the precursor and effector cells bear the Thy-1 alloantigen marker. These nonspecific killer cells killed a broad spectrum of target cells, including concanavalin A- and lipopolysaccharide-induced splenic blasts of syngeneic or allogeneic mice, a syngeneic tumor, and a cloned allogeneic cytotoxic T cell line. The frequency of precursors for nonspecific killer cells in C57BL/6 lymph node and spleen cells are 1/7000 and 1/12,000, respectively. Clonal analyses revealed that these nonspecific killers exhibit heterogeneity with respect to their target cell specificities. The induction of nonspecific killers by interleukin 2-containing supernatants is partially dependent on nylon wool-adherent cells; in antigen-stimulated cultures the most specific killer cells were obtained from cultures in which nylon wool-nonadherent lymph node responder cells were stimulated with nylon wool-nonadherent allogeneic splenic stimulator cells that were treated with anti-Thy-1 antibody and complement. The relevance of these findings with respect to the frequencies and fine specificities of cytotoxic T lymphocytes generated in interleukin 2-supplemented cultures is discussed.