Synergy of Nocardia rubra cell wall skeleton and interleukin 2 in the in vivo induction of murine lymphokine-activated killer cell activity

Combination of an ip injection of Nocardia rubra cell wall skeleton (N-CWS) and 3 daily sc injections of human recombinant interleukin 2 (rIL 2) into C3H/HeN mice resulted not only in a significant increase in the number of peritoneal cells (PC) but also in a potent induction of their lymphokine-activated killer (LAK) activity, compared with results obtained with N-CWS or rIL 2 alone. The augmented LAK activity of PC was mediated by nonadherent, nonphagocytic, Thy-1.2+(-)- and asialo GM1+ cells. Nonadherent PC induced by an ip injection of N-CWS bound more 125I-labeled rIL 2 than did normal, nonadherent PC, and generated high LAK activity when cultured overnight with rIL 2. In contrast, normal, nonadherent PC responded only weakly to the overnight stimulation with rIL 2. The phenotype of N-CWS-induced PC with an elevated IL 2 responsiveness was Thy-1.2+(-)-, Lyt-1.1-, Lyt-2.1- and asialo GM1+, suggesting that the N-CWS-stimulated LAK precursors were derived mainly from the NK cell lineage. However, mature T cells may also be involved in this mechanism, because N-CWS failed to augment the IL 2 responsiveness of nonadherent PC in BALB/c nu/nu mice. Treatment of C57BL/6N mice bearing solid Lewis lung carcinoma (3LL) tumors with an intratumoral injection of N-CWS followed by 6 daily sc injections of rIL 2 resulted in the apparent suppression of tumor growth, while N-CWS or rIL 2 alone produced no such suppression. These results suggest that N-CWS augments the antitumor effect of rIL 2 by accumulating LAK precursors and elevating their responsiveness to rIL 2 at the injection site.     

Augmentation of murine lymphokine-activated killer cell induction by a factor produced by Nocardia rubra cell wall skeleton-stimulated T cells

Four-hour exposure of C3H/HeN mouse spleen cells to Nocardia rubra cell wall skeleton (N-CWS) before 4-day culture with a suboptimal dose of human recombinant interleukin 2 (rIL 2) augmented the induction of lymphokine-activated killer (LAK) cell activity, whereas the treatment with N-CWS alone induced no cytotoxicity. In accordance with this, the IL 2 binding activity of spleen cells was augmented by combined stimulation with N-CWS and rIL 2. The augmented cytotoxicity was mediated by Thy-1.2+, Lyt-1.1-, Lyt-2.1- and asialo GM1+ cells. Cell cultures in diffusion chambers revealed that N-CWS-treated spleen cells produced a LAK cell induction-helper factor (LAK-helper factor, LHF) when cultured with rIL 2. The LHF production required Thy-1.2+, Lyt-1.1+, Lyt-2.1+ and asialo GM1- cells, and the coexistence of unstimulated accessory cells was also essential for the LHF production. Cells responding to both LHF and rIL 2 to generate LAK activity were Thy-1.2-, Lyt-1.1-, Lyt-2.1- and asialo GM1+. The culture fluid of spleen cells stimulated with both N-CWS and rIL 2 contained no tumor necrosis factor (TNF) activity, and the additional stimulation with N-CWS caused no production of either IL 2 or interferon (IFN). Murine recombinant interleukin 1 alpha (Mu-rIL 1 alpha) could not replace the augmentative effect of N-CWS on LAK cell induction. These results suggest that in the presence of rIL 2, N-CWS stimulates murine T cells to produce LHF that is probably distinct from IL 1, IL 2, TNF and IFN.     

Effects of Nocardia rubra cell wall skeleton on interleukin 2 production and lymphocyte proliferation in former poison gas factory workers

Phytohemagglutinin (PHA)-induced interleukin 2 (IL-2) production and lymphocyte proliferation were measured in former workers of the Okunojima Poison Gas Factory (poison gas workers), who have a high incidence of lung cancer, and the efficacy of administration of Nocardia rubra cell wall skeleton (N-CWS) was studied. In comparison with normal controls and poison gas workers receiving N-CWS, lymphocyte proliferation in poison gas workers not receiving N-CWS showed a significant decrease, while IL-2 production showed a slight though not statistically significant decrease. When N-CWS was administered to poison gas workers, IL-2 production and lymphocyte proliferation were significantly elevated, with a peak two weeks after administration. N-CWS, by elevating IL-2 production of lymphocytes, is considered to have improved the depression of lymphocyte proliferation.     

Synergy of human recombinant interleukin 1 with interleukin 2 in the generation of lymphokine-activated killer cells

Peripheral blood mononuclear cells cultured in vitro with interleukin 2 (IL-2) become cytolytic towards both autologous and allogeneic tumor cells. We report here that IL-1 synergizes with IL-2 in serum-free conditions to produce increased (1.3-286-fold) lymphokine-activated killer (LAK) activity. The most dramatic synergy is seen with low IL-2 concentrations (10 U/ml, 222 pM) and 50-250 U/ml IL-1 alpha or beta. Kinetics of addition experiments demonstrate a specific requirement for IL-1 at or before addition of IL-2 to the culture. We postulate that one of the mechanisms whereby IL-1 augments LAK activity is by rendering LAK-precursors more responsive to IL-2. Up-regulation of the IL-2 receptor beta chain (Tac) and increased [3H]thymidine incorporation in cultures containing IL-1 and IL-2 support this view. In some instances, IL-1 alone is capable of maintaining/generating a small degree of cytolytic activity. Collectively, our data demonstrate that IL-1 is capable of interacting with low dose IL-2 to significantly augment LAK activity, potentially playing an important role in the early stages of LAK activation and differentiation. Because synergy is observed with dramatically reduced IL-2 concentrations, this system may offer an alternative approach to high dose IL-2 therapy for the treatment of neoplastic disease.     

Antitumor effect of Nocardia rubra cell wall skeleton on syngeneically transplanted P388 tumors.

The antitumor activity of the immunomodulator, Nocardia rubra cell wall skeleton (N-CWS), was investigated using syngeneically transplanted P388 leukemia cells in a solid form. The s.c. growth of P388 tumors in DBA/2 mice was significantly suppressed by systemically administered N-CWS, and the effect was dose dependent. The antitumor effect of N-CWS was partially but significantly abrogated in splenectomized mice but not in T-cell or natural killer cell-deficient mice. Although spleen cells from mice treated with 1600 micrograms N-CWS contained no cytolytic activity, they exerted a significant cytostatic effect on P388 cell growth both in vitro and in vivo. Splenic cytostatic activity did not reside in T- or natural killer cells, but in plastic adherent cell population, macrophages. The response to N-CWS immunotherapy appeared to be associated with the number of macrophages infiltrating into the tumor lesions, and this was confirmed by histological analysis showing that P388 tumors from N-CWS-treated mice were intensively and dominantly infiltrated by macrophages. Furthermore, these were shown to be strongly tumor necrosis factor-positive by immunohistochemical analysis. These findings indicate that macrophages are the main effector cells playing a critical role in the suppression of P388 tumor growth in DBA/2 mice, and that tumor necrosis factor produced by these cells may be involved in the macrophage-mediated cytostatic effect induced by N-CWS. The fact that N-CWS suppressed the growth of weakly immunogenic P388 cells in syngeneic DBA/2 mice even when it was systemically injected would support the clinical potential of this agent.     

In vivo induction of the lymphokine-activated killer phenomenon: interleukin 2-dependent human non-major histocompatibility complex-restricted cytotoxicity generated in vivo during administration of human recombinant interleukin 2

The availability of purified human recombinant interleukin 2 (IL-2) has enabled clinical trials to test its in vivo effects. We report here the immunological effects of 7 consecutive days of IL-2 treatment given to 25 patients with cancer in a clinical Phase I study. Peripheral blood lymphocytes obtained from patients following therapy with IL-2 had enhanced proliferative responses to IL-2 and enhanced direct cytotoxic activity on K562 target cells. This lytic activity was further augmented by the addition of IL-2 during the 51Cr release assay. Fresh peripheral blood lymphocytes from some patients who had just completed treatment at the higher IL-2 dose levels were able to kill both the natural killer-resistant Daudi cell line and fresh tumor cells while pretreatment samples and peripheral blood lymphocytes from healthy controls were not. This lytic activity was best detected when IL-2 was present in the in vitro effector assay. These results demonstrate that the administration of IL-2 to patients with cancer induces a population of effector cells able to directly destroy natural killer-resistant target cells, when assayed in the presence of IL-2.     

Adoptive immunotherapy of human cancer using low-dose recombinant interleukin 2 and lymphokine-activated killer cells

The adoptive transfer of recombinant-methionyl human interleukin 2 (rIL-2)-activated autologous peripheral blood mononuclear lymphokine-activated killer (LAK) cells to cancer patients is being evaluated as an alternative to conventional cancer therapy. We have independently developed an alternative regimen to previously reported adoptive immunotherapy protocols using rIL-2 and LAK cells which features the prolonged administration of low-dose rIL-2 (30,000 units/kg) and an automated, entirely enclosed system of peripheral blood cell procurement, culture, harvest, and reinfusion of activated cells. The cell culture system was tested with a murine tumor model in which LAK cells generated in plastic culture bags were reinfused into tumor-bearing mice. Tumor regression was as effective with cells activated in the bags as in conventional culture flasks. Twenty-eight cancer patients were treated for 5 consecutive days with low-dose rIL-2, followed by leukapheresis, infusion of LAK cells, and prolonged IL-2 administration. At least 50% tumor regression was observed in 46% of all patients treated. These data imply that human peripheral blood mononuclear cells retain fully their capacity for rIL-2-induced activation and effector cell function under this alternative approach, and further, that a low-dose rIL-2 regimen with markedly reduced toxicities can be as effective as high-dose rIL-2 regimens if low-dose rIL-2 is given for a prolonged period of time following LAK cell infusion.     

胞必佳治疗恶性胸膜腔积液8例

目的探讨胞必佳治疗恶性胸膜腔积液的疗效。方法将病人胸水抽完后，用800μg胞必佳加入生理盐水30mL内注入胸膜腔，随即用盐酸山茛菪碱10mg、地塞米松10mg注入胸膜腔，后病人变换不同体位，以使药液与胸膜广泛接触．结果1wk后复查胸部B超，提示胸水消失或基本消失。2例术后发热，37．5～38．2℃对症治疗后好转，未见肝肾功能损害等不良反应。结论胞必佳治疗恶性胸膜腔积液具有较好的疗效。     

Successful immunotherapy of murine experimental hepatic metastases with lymphokine-activated killer cells and recombinant interleukin 2

Lymphokine-activated killer (LAK) cells are generated in vitro by the incubation of normal murine splenocytes in interleukin 2. We have shown previously that the systemic injection of LAK cells in conjunction with recombinant interleukin 2 can reduce the number of established pulmonary metastases in mice. In an attempt to study this approach in the treatment of hepatic metastases, we developed a technique for the induction of hepatic metastases in mice based on the intrasplenic injection of tumor cells and have tested the effects of LAK cells and recombinant interleukin 2 produced in Escherichia coli (RIL-2) therapy on these metastases. Treatment with LAK cells alone in 14 consecutive experiments rarely produced significant reduction in metastases over control (mean percentage reduction, 12%). Therapy with RIL-2 alone produced a dose-dependent reduction in the number of liver metastases. In 20 consecutive experiments when RIL-2 was administered i.p. three times a day at doses varying from 1,000 to 5,000, 10,000 to 15,000, and 25,000 units, a statistically significant (P less than 0.05) reduction in liver metastases was seen in 2 of 12, 2 of 4, and 8 of 12 determinations, respectively (percentage reduction, 0 to 97; mean, 42%). At doses greater than 25,000 units, the reduction in metastases was highly reproducible (percentage reduction, 66 to 95; mean, 83%) and was statistically significant in 14 of 14 determinations. When LAK cells were given i.v. in addition to RIL-2 administration in 16 consecutive experiments, the percentage reduction in liver metastases was markedly increased over that seen with RIL-2 alone (mean percentage reduction, 77% at doses of 5,000 to 25,000 units of RIL-2 and mean reduction, 97% for doses greater than 25,000 units of RIL-2). At doses of 5,000, 10,000, 25,000, and greater than 25,000 units of RIL-2 plus LAK cells, significant reduction of liver metastases (P less than 0.05) was achieved in 3 of 7, 2 of 2, 8 of 8, and 6 of 6 determinations, respectively. When animals were given fresh splenocytes or splenocytes cultured in complete medium without RIL-2 instead of LAK cells, no reduction in liver metastases was seen except for that attributable to the administration of RIL-2 alone. Sublethal total body irradiation of the mice prior to therapy abrogated the therapeutic effects of RIL-2, but the effects of treatment with LAK cells plus RIL-2 were maintained. Thus, treatment with RIL-2 alone or in combination with LAK cells is effective in reducing the number of established hepatic micrometastases in a murine model.(ABSTRACT TRUNCATED AT 400 WORDS)     

Induction of a tumor necrosis factor-like activity by Nocardia rubra cell wall skeleton

Nocardia rubra cell wall skeleton (N-CWS) stimulated adherent cells harvested from the peritoneal cavities of thioglycollate-treated mice to produce cytotoxic activity. Depletion of macrophages from the adherent cells by 2-chloroadenosine or silica abrogated the production of this cytotoxic activity, whereas treatment of the adherent cells with anti-Thy-1.2 antibody and complement did not. This suggested that macrophages were the producer cells of the activity. Cytotoxic activity became detectable as early as 2 h after N-CWS treatment and reached peak activity at 9 h, then declined to a lower level, indicating rapid onset without persistent effects. N-CWS-induced cytotoxic factors have a fairly narrow temperature range, pH optimum for storage, and are sensitive to pronase and trypsin. By using column chromatography, N-CWS-induced cytotoxic factors were compared in detail with tumor necrosis serum obtained from Bacillus Calmette-Guérin endotoxin-treated mice. Both toxins were found to be nearly identical with respect to their behavior in ion-exchange, gel filtration, and concanavalin A affinity columns. N-CWS also induced human peripheral blood lymphocytes to release cytotoxic activity. Monocytes predominantly participated in production of this activity as confirmed by treatment with monoclonal antibody and complement. The cytotoxic activity was completely neutralized by anti-human tumor necrosis factor antiserum, but not by anti-human lymphotoxin antiserum. The fact that human peripheral blood lymphocytes release tumor necrosis-like factors after stimulation with N-CWS might account for the antitumor effects of this agent.     

Circulating cytokines in patients with metastatic cancer treated with recombinant interleukin 2 and lymphokine-activated killer cells

Treatment with recombinant interleukin 2 and lymphokine-activated killer cells (rIL-2/LAK) has produced a clinical antitumor effect in preliminary human trials. The cytokines gamma-interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and tumor necrosis factor beta (TNF-beta, lymphotoxin) have potent in vitro antitumor activity and some clinical toxicities similar to interleukin 2 (IL-2)/LAK. This study sought to determine whether these cytokines were detectable in sera of IL-2/LAK-treated patients. Ten patients were treated with a protocol of 5-day i.v. rIL-2 bolus priming (10(5) units/kg, every 8 h), followed by 5 daily phereses with harvested lymphocytes cultured in vitro to generate LAK, and 5 days of rIL-2 bolus with infusion of LAK cells. Five patients were treated with a protocol modified to a 3-day rIL-2 prime and 6-day continuous infusion rIL-2 (3 x 10(6) units/m2/day) with infusion of LAK cells. Serum specimens were obtained prior to and 0.5, 2, 3, and 5 h after IL-2 or LAK cell administrations. IFN-gamma was detected by enzyme-linked immunosorbent assay, TNF-alpha by WEHI 164 bioassay or enzyme-linked immunosorbent assay, and TNF-beta by WEHI 164 cell bioassay. During the prime, few patients manifested in vivo detectable serum cytokines: IFN-gamma, three of ten, 5-day prime (1.03 +/- 0.46 ng/ml), and zero of five, 3-day prime; TNF-alpha, one of ten, 5-day prime, and one of three, 3-day prime; TNF-beta, one of ten, 5-day prime. The supernatants of in vitro LAK generation cultures had detectable levels of cytokines at 24 h which increased progressively until culture harvest at Day 4 (IFN-gamma, 2.56 +/- 0.34 ng/ml; TNF-alpha, 356 +/- 110 pg/ml; TNF-beta, 8.2 +/- 4.4 units/ml). The highest levels of in vivo serum cytokines occurred following LAK cell infusion and were more often elevated in patients receiving rIL-2 by bolus than by continuous infusion: IFN-gamma, four of six bolus, zero of three continuous infusion; TNF-alpha, six of six bolus (maximum 679 pg/ml) versus two of three continuous infusion (maximum, 106 pg/ml). LAK cells in vitro responded with cytokine release on stimulation by tumor cell lines (IFN-gamma, 0.88 +/- 0.06 ng/ml; TNF-alpha, 426 +/- 16 pg/ml; TNF-beta, 0.64 +/- 0.06 units/ml). In summary, this preliminary study has detected circulating cytokines in sera of patients receiving IL-2/LAK therapy. The greatest cytokine elevations followed LAK cell infusion.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunotherapy of murine hepatic metastases with lymphokine-activated killer cells expanded in serum-free media and recombinant interleukin 2

It has been shown that the systemic administration of lymphokine-activated killer (LAK) cells with recombinant interleukin 2 (RIL-2) is effective in reducing the number of established pulmonary and hepatic metastases in murine models. Similarly, this modality of therapy has been proven effective against certain selected human tumors as well. In view of the rising concern with transmission of virally related communicable diseases such as hepatitis and AIDS, we have undertaken the evaluation of a serum-free medium (AIM V) for the generation and expansion of murine LAK cells for use in in vivo tumor immunotherapy against murine hepatic metastases. Day 3 LAK cells generated in AIM V medium demonstrated a greater percentage of viable cells than cells generated in serum containing complete medium (CM) (mean percentage of yield, 59 versus 25%, AIM V medium versus CM, respectively, P less than 0.001, N = 6 consecutive experiments). When day 3 LAK cells were transferred to new medium (CM to CM and AIM V to AIM V), a highly reproducible expansion of these cells was demonstrated which was significantly better for cells expanded in AIM V medium versus cells expanded in CM (mean fold expansion on day 21 of culture; 201 versus 54, AIM V medium versus CM, respectively, P less than 0.005, N = 4 consecutive experiments). When day 3 LAK cells, day 5 expanded LAK cells, and day 13 expanded LAK cells grown in CM or in AIM V medium were given in vivo with RIL-2 to mice harboring hepatic metastases, cells grown in AIM V medium demonstrated an increased antitumor activity compared to cells grown in CM. As an example in experiment 1, the mean number of metastases with day 5 expanded LAK cells grown in CM and given with RIL-2 was 47 while the mean number of metastases with day 5 expanded LAK cells grown in AIM V medium and given with RIL-2 was 5 (P less than 0.002). These experiments demonstrate that AIM V medium can be utilized to generate greater numbers of murine LAK cells with enhanced in vivo antitumor activity compared to cells generated in CM. These findings could be applied to the expansion of cytotoxic cells for human antitumor therapy.     

Randomized controlled study of adjuvant immunotherapy with Nocardia rubra cell wall skeleton for inoperable lung cancer

In order to evaluate the clinical benefit of Nocardia rubra cell wall skeleton (N-CWS), a randomized controlled study was performed with inoperable lung cancer patients entered in 5 institutions from October 1978 to June 1981. Patients without pleural effusions were treated initially with conventional therapies such as chemotherapy and/or radiotherapy, according to common protocol, and then patients in performance statuses 0 to 3 were randomized into control and N-CWS groups with stratification into 16 categories according to 4 histological types and 4 clinical stages In the N-CWS group, 400 micrograms N-CWS were initially injected once or twice into the bronchial tumor using a fiberoptic bronchoscope, and subsequently 200 micrograms of N-CWS were injected at monthly intervals into the skin from the shoulders to upper arms. Of 309 patients, 118 patients in the N-CWS group and 108 patients in the control group were eligible for statistical analysis. There was statistically no significant difference in survival rate between the control and the N-CWS groups. According to histological type, significant prolongation of the survival period was observed in patients with small-cell carcinoma. The 97 patients with pleural effusions were initially randomized into control and N-CWS groups. In the control group, local chemotherapy with Adriamycin was performed and, in the N-CWS group, local administrations and monthly intracutaneous injections of N-CWS were given. Tube thoracostomy was performed in both groups. The local response rate was statistically greater in the N-CWS group than in the control group, and survival period was also prolonged significantly in the N-CWS group. The main adverse reactions to N-CWS were skin lesions in the injected sites and fever, but these were temporary and not serious.     

Association of macrophage activation with antitumor effect on rat syngeneic fibrosarcoma by Nocardia rubra cell wall skeleton.

The antitumor activities of the cell wall skeleton (CWS) of Nocardia rubra were demonstrated for syngeneic fibrosarcoma (AMC-60) in ACI/N rats in regard to macrophage activation. In the 24-hr cytolytic test, activated macrophages which were fractionated from peritoneal exudate cells induced by i.p. injection of Nocardia CWS showed significant cytolytic activity for [125I]iododeoxyuridine-labeled tumor cells. Activated macrophages also strongly inhibited [3H]thymidine incorporation into the tumor cells during the 24-hr cytostatic test. When tumor cells were inoculated s.c. with activated macrophages in the Winn-type transfer assay, subsequent tumor growth was significantly inhibited. Repeated i.p. injection of the CWS seemed to enhance these antitumor activities of macrophages. The therapeutic effect of Nocardia CWS was assessed with the ascites tumor and with the solid tumor inoculated i.m. into the hind leg. In the former treatment, repeated i.p. injections completely prevented the accumulation of ascites fluid and resulted in prolongation of the survival period. The peritoneal macrophages harvested from these survivors had a strong cytolytic activity for tumor cells in the cytolytic test. In the latter treatment, repeated intratumoral injections inhibited the growth of primary tumor and prevented metastasis. Furthermore, peritoneal resident macrophages from these tumor-bearing rats treated intratumorally with the CWS were found to be cytolytic for tumor cells in the cytolytic test.     

Local administration of autologous lymphokine-activated killer cells and recombinant interleukin 2 to patients with malignant brain tumors

Lymphokine-activated killer cells (LAK cells) were induced from lymphocytes from patients with malignant glioma by using interleukin 2 (IL-2), and their killing activity was examined. Their LAK activity against Daudi cells was 66.2 +/- 13.1% and 48.7 +/- 12.7% against self glioma cells, 54.4 +/- 10.1% against K562 cells, 43.1 +/- 7.9% against Raji cells, and 33.5 +/- 16.2% against allogeneic glioma cells. The phenotype of these LAK cells was Leu 1 (++), 2a (+/-), 3a (++), 7 (+), and 11 (++). The phenotype of precursor LAK cells, on the other hand, was Leu 1 (-), 2a (-), 3a (+), 7 (-), and 11 (++). Other activated killer cells, including LAK cells, phytohemagglutinin-activated killer cells, autoactivated killer cells, and their precursor LAK cells, were studied serologically in order to identify their phenotypic characteristics. From these data, the LAK cell populations were considered to be polyclonal. Using these LAK cells plus IL-2, local adoptive immunotherapy was undertaken in 23 patients with recurrent malignant glioma. We injected, that is, autologous LAK cells plus IL-2 directly into the cavities of the brain tumors; 1.2 to 324 x 10(8) LAK cells per ml and 0.8 to 5.4 x 10(3) units of IL-2 were directly injected into the brain tumor by using an Ommaya reservoir. Definite tumor regression, improvement of some clinical symptoms, and continuous remission over 6 mo or more were observed in six, nine, and three patients, respectively. There were no marked side effects, except for slight fever and chill, in eight and three patients, respectively. These results suggested the possibility of induction of a sufficient number of LAK cells from the lymphocytes of the patients with recurrent malignant glioma, indicating that local adoptive immunotherapy by direct injections of LAK cells and IL-2 into the brain tumor will prove to be an effective means of immunotherapy. Additional follow-up of the patients will be required before its therapeutic value can be established.     

Effects of Nocardia rubra cell wall skeleton on lymphocyte subsets in former poison gas factory workers

Peripheral blood lymphocyte subsets were determined in former workers of the Okunojima Poison Gas Factory (poison gas workers) having a high incidence of lung cancer, and the effect of administration of Nocardia rubra cell wall skeleton (N-CWS) was studied. In poison gas workers not receiving N-CWS, both the percentage and absolute number of Leu-2a+ cells were significantly increased as compared with normal controls, while both the percentage and absolute number of Leu-7+ cells as well as the Leu-3a/Leu-2a ratio were significantly decreased. In poison gas workers receiving N-CWS, the percentage of Leu-2a+ cells was significantly decreased when compared with poison gas workers not receiving N-CWS, while the percentage of Leu-1+ cells and both the percentage and absolute number of Leu-7+ cells as well as the Leu-3a/Leu-2a ratio were significantly increased. When N-CWS was administered to poison gas workers, the percentage of Leu-2a+ cells significantly decreased, with a minimum two weeks after administration, while the percentage of Leu-3a+ and the Leu-3a/Leu-2a ratio significantly increased and the percentages of Leu-1+ and Leu-7+ cells also increased. Furthermore, the absolute number of Leu-3a+ cells increased markedly with a peak two weeks after administration. The absolute numbers of both Leu-1+ cells and Leu-7+ cells as well as total lymphocytes also significantly increased, but that of Leu-2a+ cells showed hardly any change. It is considered that a single administration of N-CWS transiently corrects the abnormality of lymphocyte subsets, and that repeated administration of N-CWS once every three months may maintain the lymphocyte subsets of poison gas workers at normal levels.     

Exogenous interleukin 2 recruits in vitro lymphokine-activated killer activity by in vivo activated lymphocytes

Cryopreserved and thawed lymphocytes can be used instead of fresh lymphocytes to avoid test-to-test variability in studies of fluctuations of natural killer (NK) and lymphokine-activated killer (LAK) activities as a function of time. We investigated the effects of 1-h versus 18-h resting of lymphocytes on their lytic activities, because the process of cryopreservation and thawing decreases NK and LAK activities. Lymphocytes from renal cell cancer patients receiving adoptive immunotherapy were studied. An 18-h versus 1-h resting period led to a significant increase in NK activity but had no significant effect on LAK activity. The presence of 1200 IU/ml interleukin 2 (IL-2) in the medium 1 h prior to and during the cytotoxicity (CTX) assay increased in vivo and in vitro IL-2-induced LAK activities. This phenomenon has been interpreted as IL-2 dependency of effector lymphocytes (J.A. Hank, P.C. Kohler, G. Weil-Hillman, N. Rosenthal, K. H. Moore, B. Storer, D. Minkoff, J. Bradshaw, R. Bechhofer, and P. M. Sondel. Cancer Res., 48: 1965-1971, 1988). We performed kinetic studies to assess the role of effector lymphocyte recruitment in these experiments. LAK activity was tested in the presence or absence of IL-2 during preincubations and CTX assays varying between 0 and 120 min. These kinetic studies showed that effector lymphocyte recruitment indeed contributed to the increased level of LAK activity when IL-2 was added to the CTX assay. A minimal incubation period of 30 min was required to detect recruitment of lymphocytes. Effector lymphocytes could be recruited for periods varying between 90 and greater than 240 min, depending on the lymphocyte donor. We conclude that: (a) in vitro, IL-2-mediated recruitment of lymphocytes due to presence of IL-2 in the CTX assay may lead to an overestimate of the actual LAK activity; and (b) in vivo, prolonged IL-2 infusion after the administration of activated lymphocytes seems warranted in order to recruit maximal levels of effector lymphocytes with LAK activity.     

In vivo induction of lymphokine-activated killer cells by interleukin-2 splenic artery perfusion in advanced malignancy

In an effort to stimulate in vivo LAK cell activity at relatively nontoxic doses, 20 patients with advanced metastatic malignancy (13 renal cell carcinoma, 6 melanoma, 1 lymphoma) were treated with recombinant human interleukin-2 (IL-2) by continuous 5-day splenic artery perfusion using the femoral approach. Two treatment cycles were administered 3 weeks apart; IL-2 doses ranged from 1.5-4 x 10(4) Cetus units/kg/day. Peripheral blood lymphocyte cytotoxicity in a 4-h 51Cr release assay was measured using as tumor cell targets K562 for natural killer (NK) activity, Daudi for LAK, and Daudi plus in vitro IL-2 for inducible LAK (I-LAK). For the 20 patients, an increase in mean peak percent cytotoxicity from pretreatment levels was seen for NK (36% to 53%), LAK (8% to 37%) and I-LAK (20% to 53%) activity, all significant at P = 0.001. On day 43, 16 days after completing the second cycle of treatment, NK activity remained elevated at 47% and I-LAK at 40% (P = 0.008 and 0.01, respectively). Lymphocyte phenotype analysis by flow cytometry demonstrated increases from pretreatment levels in Leu 11+ (13 to 23%), Leu 19+ (10 to 21%), Leu 11+ 19+ (7 to 17%), IL-2r+ (4 to 17%), and HLA-DR+ (12 to 25%) subsets, all significant at P less than or equal to 0.01. Dose effect was studied at 3 dose levels: 1.5, 3, and 4 x 10(4) Cetus units/kg/day. At the higher doses mean peak NK (57%) and I-LAK (57%) activity were greater than at the low dose (42 and 31%, respectively), both significant at P less than 0.05. A trend to positive dose effect was seen in LAK activity (P = 0.08). Splenic artery perfusion with IL-2 can result in significant in vivo peripheral LAK cell generation as well as enhancement of I-LAK and NK activity that persists at least 16 days after the cessation of treatment. Such sustained activity would not be expected with conventional high dose i.v. therapy.     

Cytokine gene expression during the generation of human lymphokine-activated killer cells: early induction of interleukin 1 beta by interleukin 2

Culture of human peripheral blood leukocytes with interleukin 2 (IL-2) stimulates their differentiation into lymphokine-activated killer (LAK) cells, with a broad range of cytotoxicity against fresh tumor cells and tumor cell lines (Grimm et al., J. Exp. Med., 155: 1823-1841, 1982). We chose to utilize a molecular approach to determine whether IL-2 stimulates the expression of cytokine genes by the mixed cell population which may be involved in the generation or regulation of lytic activity. Northern blot analysis performed with total cellular RNA from LAK cells cultured for varying periods of time with IL-2 revealed that the genes which code for cytokines [interleukin 1 (IL-1)alpha and beta, gamma-interferon, tumor necrosis factor alpha, and lymphotoxin] were not spontaneously expressed. As soon as 2 h after IL-2 treatment, IL-1 alpha and IL-1 beta mRNAs were expressed. Both nonadherent and adherent populations of LAK cells express IL-1 beta mRNA; however, the adherent population produced more IL-1 beta mRNA and maintained its expression for a prolonged period of time. Other cytokine mRNAs (gamma-interferon, tumor necrosis factor alpha, and lymphotoxin) were expressed later than the IL-1 mRNAs with maximal levels between Days 2 through 7. Our results indicate that LAK cell populations can generate a variety of cytokines which may be involved in the generation of lytic activity.     

Combination treatment with irritant and recombinant interleukin 2 in the peritoneal cavity for evoking effective anti-tumor activity: generation of lymphokine-activated killer cells and tumor-specific killer cells

Meth A sarcoma, growing in the subcutaneous tissue of syngeneic BALB/c mice, regressed completely after an intraperitoneal (ip) injection of proteose peptone (PP) (on day 6) followed by 2 ip administrations (on days 7 and 8) of human recombinant interleukin-2 (IL-2, 25 micrograms/day), whereas one such treatment alone had little effect on the tumor growth. While this combination treatment was effective in anti-asialo GM1 antibody-treated mice, no such effect was noted in T cell-depleted ATXFL (thymectomized, irradiated and fetal liver cell-reconstituted) mice. These results show that T cells are mainly responsible for this antitumor effect. Treatment with a combination of PP and IL-2, but not with either PP or IL-2 alone, resulted in a marked increase in the T cell population in the peritoneal cavity after the treatment. At an early stage after the combination treatment, both peritoneal exudate cells (PEC) and spleen cells exhibited killing activity with a promiscuous specificity. However, at a later stage, 7 days after the treatment, Meth A-specific killer activity was observed in both PEC and the spleen. Meth A rechallenge was rejected by the mice in which the tumor had regressed, but the antigenically different Meth 1 was accepted by them. A similar result was obtained in Winn's neutralization test. These results suggest that this combination treatment, which is effective in the generation of lymphokine-activated killer cells in the peritoneal cavity, finally resulted in the induction of tumor-specific killer cells in the periphery. These results clearly show the anti-tumor efficacy of combination treatment with PP and rIL-2.