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.     

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-stimnlated 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 CS7BL/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.     

Identification of a novel CD56- lymphokine-activated killer cell precursor in cancer patients receiving recombinant interleukin 2.

Circulating lymphokine-activated killer (LAK) cell activity in cancer patients receiving recombinant interleukin 2 (rIL-2) therapy is confined to cells expressing the CD56- surface marker. However, CD56- cells from these patients but not normal individuals have been reported to exhibit LAK cytotoxicity only following in vitro activation with rIL-2. Studies were performed to document the existence of CD56- LAK precursor cells and to phenotypically characterize this population in patients receiving rIL-2 therapy using fluorescence-activated cell sorter-purified CD56- cell subsets. Initial studies confirmed that CD56- cells exhibit NK activity [20 +/- 7 (SE) LU/10(6) cells] but not LAK activity (0 +/- 0 LU/10(6) cells) when evaluated directly from peripheral blood of patients receiving rIL-2. CD56- cells from patients but not normal individuals developed significant LAK cytolytic activity against NK-resistant COLO 205 targets (16 +/- 3 LU/10(6) cells) when cultured for 3 days with 1500 units/ml rIL-2. The CD56- LAK precursor activity was confined to cells expressing a CD56-CD16+ phenotype and a large granular lymphocyte morphology; little or no NK or LAK precursor activity was detectable in CD56-CD5+ T-cells from patients. Phenotypic characterization of CD16+CD56- cells revealed that this population is uniformly CD11a+,CD18+, and CD38+ and is heterogeneous in its expression of CD11b, CD11c, and CD16/Leu 11c. These results indicate that rIL-2 administration induces enhanced LAK precursor activity in a novel population of CD5-CD16+CD56- cells.     

Combined therapy of mice bearing a lymphokine-activated killer-resistant tumor with recombinant interleukin 2 and an antitumor monoclonal antibody capable of inducing antibody-dependent cellular cytotoxicity

The ability of lymphokine-activated killer (LAK) cells to mediate antibody-dependent cellular cytotoxicity and its efficacy against a LAK-resistant tumor were investigated. Cells of the MH134 murine hepatoma line are scarcely lysed by LAK cells generated in vitro by incubation of C3H/HeN mouse spleen cells with human recombinant interleukin 2 (rIL 2). However, the splenic LAK cells potently lysed the LAK-resistant tumor cells in the presence of 11G2, a monoclonal antibody (MAb) of the IgG1 isotype reactive with a part of MM antigen. Peritoneal cells induced by daily i.p. injections of rIL 2 not only exhibited LAK activity but also mediated antibody-dependent cellular cytotoxicity against MH134 tumor cells in the presence of 11G2. The peritoneal cells exhibiting these cytotoxic activities were found to be nonadherent and nonphagocytic mononuclear cells possessing a similar cell surface phenotype as that of splenic LAK cells, that is Thy-1.2+ approximately -, Lyt-1.1-, Lyt-2.1-, and asialo GM1+. Treatment of spleen cells with antibodies and complement before culture with rIL 2 revealed that the phenotype of splenic LAK precursors is Thy-1.2- and asialo GM1+. The in vivo induction of peritoneal LAK cells in response to i.p. injections of rIL 2 was markedly depressed in C57BL/6 beige mice but was normally accomplished in BALB/c nude mice. Combined therapy of C3H/HeN mice bearing MH134 ascitic tumor with i.p. injection of rIL 2 and 11G2 brought about potent suppression of the tumor growth, resulting in the significant increase in the number of tumor-free mice, whereas neither rIL 2 nor the MAb could exhibit such a potent antitumor effect when used alone. Injection (i.v.) of anti-asialo GM1 antibody not only blocked the induction of peritoneal LAK cells by rIL 2 but also abrogated the development of the antitumor effect of the combined therapy. These results strongly suggest that combination of antitumor MAbs capable of inducing antibody-dependent cellular cytotoxicity with rIL 2 therapy could result in the generation of potent antitumor effects against LAK-resistant tumors and that asialo GM1-positive non-T-cell populations including cells of the natural killer cell lineage are essential, at least in part, for development of the antitumor effects of the combined therapy with rIL 2 and MAbs.     

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 la (Mu-rIL 1α) 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.     

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.     

Effects of Recombinant Interferon-Gamma and Interleukin-2 on the Generation of Lymphokine-Activated Killer Cells in Vitro

In order to determine if recombinant interferon-γ (rIFN-γ) can augment the effect of recombinant interleukin-2 (rIL-2) in generating lymphokine-activated killer (LAK) cells, we have incubated normal peripheral blood mononuclear cells (PBMC) with these lymphokines for 3 days and then tested their LAK and natural killer (NK) cell activity. We have found that LAK activity in PBMC from 13 out of 13 normal donors was increased by the combined lymphokines above that due to either lymphokine alone, provided that rIL-2 was present at suboptimal concentration: Optimal levels of rIFN-γ (100 U/ml) were able to enhance the LAK-inducing activity of suboptimal levels (5 U/ml) but not optimal levels (100 U/ml) of rIL-2. NK activity showed a similar response to these concentrations of lymphokines. Activation of LAK/NK cells was accompanied by increases in the percentages of Leu 19⁺ (CD56) cells and TAC⁺ (IL-2-receptor) cells, and in the intensity of TAC antigen expression. These results indicate that combination rIFN-γ and rIL-2 may be more effective in generating LAK/NK cells than rIL-2 alone, particularly with suboptimal concentrations of rIL-2 such as occur during continuous infusion therapy with this agent.     

Functional and phenotypic characteristics of recombinant interleukin-2 or T-cell growth factor-activated splenic lymphoid cells from patients with gastric or hepatocellular carcinoma

Fourteen days' culture of human spleen cells with recombinant interleukin-2 (rIL-2) or T-cell growth factor (TCGF) results in the generation of lymphokine-activated killer (LAK) effector cells that have the unique property of lysing natural killer (NK)-resistant human tumor cells, Daudi, and NK-sensitive K562 cells. LAK cells were generated from patients with advanced cancer or liver cirrhosis. The splenic LAK-effector cell types were analyzed by two-color flow cytometry. The rIL-2-induced LAK cells showed an increased proportion of CD8+CD11- and CD57+CD16- and a decreased proportion of CD4+Leu-8- cells. In contrast, TCGF-induced LAK cells revealed a significantly increased proportion of CD8+CD11- and CD4+Leu-8- cells and a decreased proportion of CD57+CD16- cells. Thus, splenic LAK cells with different surface phenotypes were induced by the cultivation with rIL-2 or TCGF. Furthermore, TCGF-induced LAK cell activities in patients with cancer were found to be lower than the rIL-2-induced LAK cell activities. It was noted that the TCGF-activated splenic lymphoid cells did not inhibit the effector process of tumor cell lysis by LAK cells that had been activated by rIL-2. Other mechanisms of lower LAK cell activities of TCGF-activated splenic lymphoid cells from patients with cancer were discussed. The findings suggest that spleens of examined patients with gastric or hepatocellular carcinoma do not seem to be responsible for suppression of cell-mediated antitumor immunity.     

Human recombinant interleukin-4 inhibits lymphokine-activated killer activity of sheep erythrocyte rosette-forming (E+) and -non-forming (E-) human lymphocytes

The effect of human recombinant interleukin-4 (rIL-4) on lymphokine-activated killer activity (LAK) of peripheral blood lymphocytes (PBL) as well as sheep erythrocyte rosette-forming (E+) and -non-forming (E-) lymphocytes stimulated by human recombinant interleukin-2 (rIL-2) has been investigated. rIL-4 drastically inhibited LAK activity of PBL cultured for 18 hr and for 5 days in the presence of rIL-2. Distribution of T, B and IgG Fc-receptor-bearing lymphocytes, as assessed by immunofluorescence and flow cytometry, was no different at the end of the culture in the presence of rIL-2 plus rIL-4 than with rIL-2 alone. LAK activity of E+ and E- lymphocytes was similarly inhibited. Finally, rIL-4 did not affect the natural killer (NK) activity of rIL-2-activated PBL as assessed by the capacity of these cells to kill the appropriate NK target.     

Lymphokine-activated killer cells in rats: analysis of tissue and strain distribution, ontogeny, and target specificity

The coculture of lymphoid cells from Fischer 344 rats with recombinant human interleukin 2 (rIL-2) resulted in the generation of lymphokine-activated killer (LAK) cells. Maximal LAK activity was obtained between 200 and 1000 units/ml rIL-2. Lymphoid cells from spleen, thymus, bone marrow, peripheral blood, and lymph nodes were able to generate LAK activity although the kinetics and magnitudes of the responses were appreciably different among these tissues. Thus, while spleen and blood lymphocytes responded quickly (by day 3) and gave the highest level of LAK activity in response to rIL-2, bone marrow and thymus cells responded only by 7 to 9 days in culture. LAK activity could be generated from a variety of rat strains regardless of whether there were high or low levels of endogenous splenic natural killer (NK) activity, but the early (day 3) response was lower in the strains with low levels of NK activity. Cells with LAK activity could lyse a variety of tumor targets including fresh ascites or fresh syngeneic solid tumor explants but could not lyse fresh normal cells including syngeneic fibroblasts, peripheral blood lymphocytes, bone marrow cells, thymocytes, or T,B blasts. The generation of LAK activity required a concomitant proliferative response and could be completely abrogated by mitomycin C, actinomycin D, or X-irradiation above 500 rads. These treatments, however, did not affect natural killer activity or short-term (4 h) IL-2-boosted NK activity. LAK activity could be generated from spleen cells obtained from rats as early as 10 days of age but could not be generated from unfractionated neonatal spleen, neonatal liver, or peritoneal macrophages. The ontogeny of the development of splenic LAK activity correlated closely to the development of concurrent natural killer activity. When mixed with an NK-resistant mammary adenocarcinoma (MADB106) and adoptively transferred to normal syngeneic recipients in standard Winn-type assays, LAK cells were effective at inducing complete tumor inhibition.     

Lymphokine-activated killer cell plus recombinant interleukin-2 therapy of erythroleukemia in mice

The antileukemic effects of lymphokine-activated killer (LAK) cells plus recombinant interleukin-2 (rIL-2) therapy were assessed in mice with Friend virus (FV)-induced erythroleukemia. LAK cells were generated by incubating normal spleen cells for 72 hr in the presence of rIL-2 (1000 units/ml). At the time of injection, the LAK cells were cytotoxic in vitro against FV-infected fibroblasts and NK-sensitive and -resistant tumor targets but not normal controls. To determine in vivo activity, fully leukemic mice (spleen weight greater than 0.75 g) were injected with either PBS or LAK cells (10(8) cells/mouse IV at 14 and 17 days post virus) and rIL-2 (10,000 units/mouse IP every 8 hr on days 14 through 18 post virus). More than 70% of the progressively leukemic mice experienced permanent leukemia regressions (disease-free for greater than 100 days) following LAK cell plus rIL-2 therapy. Regressions were characterized by return of spleen and liver weights to normal and elimination of virus-infected erythroid (CFU-E) and macrophage (CFU-C) progenitor cells from spleen and marrow. Leukemic animals treated with either LAK cells alone or IL-2 alone experienced only transient leukemia regressions. These results demonstrate that LAK cell plus rIL-2 treatment can induce permanent regressions in progressively leukemic mice and provide a responsive and manipulable model system to elucidate the mechanisms involved in this form of immunotherapy.     

Two populations of mouse lymphokine-activated killer cells separated by use of soybean agglutinin

We separated lymphokine-activated killer (LAK) cells induced from spleen cells of BALB/c mice by culturing with recombinant interleukin-2 (rIL-2) into soybean agglutinin-positive (SBA+) and soybean agglutinin-negative (SBA-) fractions with a cell sorter at the time when LAK activity reached a maximum (day 3). We found that the cells with LAK activity were enriched in the SBA+ fraction. Analysis of cell surface phenotypes revealed that the SBA+ cells are of non-T cell origin, while the SBA- fraction consists of T cells. We also found that the large granular lymphocyte (LGL) fraction of spleen cells obtained with a Percoll gradient became SBA+ after culture for 3 days with rIL-2, whereas cells of high density did not. The change in SBA binding sites was also examined on C57BL/6 mouse spleen cells and we found that NK1.1+ non-T cells selectively acquire SBA binding sites at an early stage of activation with rIL-2. On the other hand, sorted SBA- cells gradually acquired SBA binding sites on extended culturing with rIL-2. These results suggest that the expression of SBA binding sites is related to the stage of cell activation with rIL-2, though cells of the NK-lineage became SBA+ at an earlier period of a culture with rIL-2 than cells of the T-lineage. Utilizing these findings, we separated NK-derived LAK cells by means of SBA-binding, and used the separated cells for adoptive immunotherapy for experimental pulmonary metastasis. We found that SBA(+)-NK cell-derived LAK cells showed stronger activity for the inhibition of experimental pulmonary metastasis than SBA(-)-T cell-derived LAK cells.     

Enhanced antimetastatic activity of lymphokine-activated killer cells purified and expanded by their adherence to plastic

We have recently reported a simple and reproducible technique for the purification and rapid expansion of homogeneous populations of large granular lymphocytes expressing a natural killer cell phenotype and high levels of broad antitumor cytotoxic activity [lymphokine-activated killer (LAK) activity]. This technique exploits the observation that, in the presence of recombinant interleukin 2 (rIL-2), large granular lymphocytes/natural killer cells become adherent to plastic surfaces, actively proliferate, and acquire high levels of LAK activity. Because of their adherent properties these cells have been termed adherent LAK or A-LAK cells. The present studies investigate the antimetastatic effects of A-LAK cells in a syngeneic rat model of experimental pulmonary and hepatic metastases. For pulmonary metastases, F344 rats received i.v. injections with a natural killer-resistant mammary adenocarcinoma, MADB106, and, for hepatic metastases, animals received an intrasplenic injection of MADB106 tumor cells followed by surgical splenectomy. Three days later, the animals were treated with A-LAK cells alone, A-LAK cells plus rIL-2, or rIL-2 alone. These treatments were compared to immunotherapy using standard cultures of LAK cells (unfractionated spleen cells) and rIL-2. The results indicate that the administration of unfractionated LAK cells plus interleukin 2 (IL-2) was effective in reducing established lung or liver metastases in this rat model. However, the results also indicate that purified populations of A-LAK cells in combination with rIL-2 demonstrate dramatic and superior antimetastatic effects when compared to LAK cells cultured under standard conditions. The antimetastatic effects of standard LAK cells or A-LAK cells plus IL-2 translated into significant survival benefits compared to animals receiving no therapy or IL-2 therapy alone. Survival after therapy with A-LAK cells plus IL-2 was significantly prolonged compared to treatment with standard LAK cells. These data suggest that purified populations of LAK cells (derived from natural killer cells) may prove superior for adoptive immunotherapy in the clinical setting.     

Phase I trial of intraperitoneal recombinant interleukin-2/lymphokine-activated killer cells in patients with ovarian cancer

Ten patients with ovarian cancer refractory to conventional therapy were treated with intraperitoneal (i.p.) recombinant interleukin-2 (rIL-2) and lymphokine-activated killer cells (LAK). The 28-day protocol consisted of 6 priming i.p. rIL-2 infusions on days 0, 4, 6, 8, 10, and 12. Leukapheresis was performed for mononuclear cell collection on days 15, 16, 17, and 18 and lymphokine-activated killer cells were given i.p. with the rIL-2 on days 19 and 21. Three additional i.p. rIL-2 infusions were given on days 23, 25, and 27. Three dose levels of rIL-2 were tested: 5 X 10(5), 2 X 10(6), and 8 X 10(6) units/m2 body surface area. The dose-limiting toxicity was abdominal pain secondary to ascites accumulation with significant weight gain. Other toxic effects included decreased performance status, fever, nausea and vomiting, diarrhea, and anemia. Peripheral lymphocytosis and eosinophilia were seen at all dose levels. The maximum tolerated dose is 8 X 10(6) units/m2/dose. Peripheral and peritoneal IL-2 levels were measured with a bioassay using an IL-2-dependent cell line. At the highest dose level, serum IL-2 was greater than 10 units/ml for 18 h. After the first infusion, a 2-log dilution of the i.p. IL-2 was measured in the serum. In the postleukapheresis i.p. IL-2-dosing period less IL-2 was detected in the serum than in the earlier i.p. IL-2-priming period. The induction and persistence of LAK activity were studied. Peritoneal LAK activity was detected as early as 4 days after the first i.p. infusion, by day 11 in all evaluable patients, and persisted for the 6-day interval between priming IL-2 and LAK/IL-2 infusion. Peritoneal lytic activity persisted until day 28 in 5 tested patients. These peritoneal cells retained lytic activity 48 h in culture medium without rIL-2 present. Peritoneal LAK activity correlated with the percentage of mononuclear cells and the percentage of CD56-positive mononuclear cells in the peritoneum. The yield of peripheral lymphocytes after the six i.p. priming doses of rIL-2 correlated with the dose level of rIL-2 infused. Peripheral blood LAK activity showed a minimal, however progressive, increase during the treatment protocol. LAK activity could be enhanced if rIL-2 was present during the 4-h assay. These studies indicate that i.p. rIL-2 infusion induced durable regional LAK activity and primes peripheral blood cells for LAK activity if exposed briefly to additional IL-2.     

Postoperative adjuvant adoptive immunotherapy with lymph node-LAK cells and IL-2 for pathologic stage I non-small cell lung cancer

We conducted a clinical trial of adoptive immunotherapy with lymph node-lymphokine-activated killer (LN-LAK) cells and recombinant interleukin 2 (rIL-2) for a surgical adjuvant therapy of pathologic stage I non-small cell lung cancer. The regimen consisted of the subcutaneous administration of low-dose rIL-2 for 6 consecutive days and the transfer of ex vivo generated LAK cells from regional lymph node lymphocytes, obtained at the time of surgical operation. A group of 19 patients with primary lung cancer received the immunotherapy about 2 weeks after surgery (pulmonary lobectomy). The regimen was postoperatively well tolerated by the patients. In peripheral blood lymphocytes (PBL) obtained after the treatment, the proportion of CD3+ T cells predominantly increased with the increase of CD4+ T cell subsets. On the other hand, the proportion of CD20+ B cells decreased. Both NK and LAK activity of PBL significantly increased. However, the immunomodulatory effects did not result in a prolongation of the postoperative survival time in comparison to the postoperative survival of patients (n = 21) with surgery alone during the same period. These results suggested that the treatment with low-dose LN-LAK cells and concurrent low-dose IL-2 could, therefore, neither reduce nor eradicate minimal micrometastatic diseases.     

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.     

Antitumor Effects of 5'-Deoxy-S-fluorouridine in Combination with Recombinant Human Interleukin 2 on Murine Colon Carcinoma 26

The antitumor activity of rccombinant human interleukin 2 (rIL-2) in combination with 5'-deoxy-5-fluorouridine (doxifluridine; 5'-DFUR) against murine colon carcinoma 26 (Colon 26) was studied. BALB/c mice were treated daily for 15 days with S'-DFUR, rIL-2 or both, beginning on day 7 after subcutaneous transplantation of Colon 26. While mice treated with 5'-DFUR or rIL-2 alone died of tumor growth with pulmonary metastases within 9 weeks posttransplantation, the survival time was significantly prolonged in mice treated with both 5'-DFUR and rIL-2. Most of the combination-treated animals showed the regression of local tumors and the inhibition of pulmonary metastasis. Histopathologically, many tumor cells were degenerated and necrotized, with marked infiltration of mononuclear cells including large granular lymphocytes (LGLs) with periodic acid-Schiff-positive cytoplasmic granules. The cells were positive for CD3s, asialo GM1 and NK1.1. Spleen cells from the combination-treated mice showed high activities of natural killer (NK) cytotoxicity as well as growth inhibition of Colon 26 and Meth A fibrosarcoma in mice. The results suggest that the combination therapy of 5'-DFUR plus rIL-2 enhanced non-specific cytotoxicity of LGL/NK cells for Colon 26 in tumor-bearing mice and was effective in the inhibition of tumor growth.     

Two Populations of Mouse Lymphokine-activated Killer Cells Separated by Use of Soybean Agglutinin

We separated lymphokine-activated killer (LAK) cells induced from spleen cells of BALB/c mice by culturing with recombinant interleukin-2 (rIL-2) into soybean agglutinin-positive (SBA⁺) and soybean agglutinin-negative (SBA^-) fractions with a cell sorter at the time when LAK activity reached a maximum (day 3). We found that the cells with LAK activity were enriched in the SBA⁺ fraction. Analysis of cell surface phenotypes revealed that the SBA⁺ cells are of non-T cell origin, while the SBA^- fraction consists of T cells. We also found that the large granular lymphocyte (LGL) fraction of spleen cells obtained with a Percoll gradient became SBA⁺ after culture for 3 days with rIL-2, whereas cells of high density did not. The change in SBA binding sites was also examined on C57BL/6 mouse spleen cells and we found that NK1.1⁺ non-T cells selectively acquire SBA binding sites at an early stage of activation with rIL-2. On the other hand, sorted SBA^- cells gradually acquired SBA binding sites on extended culturing with rIL-2. These results suggest that the expression of SBA binding sites is related to the stage of cell activation with rIL-2, though cells of the NK-lineage became SBA⁺ at an earlier period of a culture with rIL-2 than cells of the T-lineage. Utilizing these findings, we separated NK-derived LAK cells by means of SBA-binding, and used the separated cells for adoptive immunotherapy for experimental pulmonary metastasis. We found that SBA⁺-NK cell-derived LAK cells showed stronger activity for the inhibition of experimental pulmonary metastasis than SBA -T cell-derived LAK cells.     

Interleukin-2-stimulated natural killer activity against malignant and benign endometrium

Natural cell-mediated immunity against autologous tumor cells, autologous endometrial epithelium, and allogeneic epidermoid carcinoma cell line HeLa was tested in 8 patients with endometrial carcinoma and one patient with endometrial stromal sarcoma. The average cytotoxicity of unstimulated peripheral blood lymphocytes against autologous tumor and HeLa cells was weak but significant. Pretreatment of effector cells for 3-5 days with 300 U/ml recombinant interleukin-2 (rIL-2) resulted in increased cytotoxicity against malignant target cells in 7 out of 9 cases. The 2 patients' effector cells which were refractory to rIL-2 could be stimulated to appreciable lytic activity against the malignant target cells with a recently described cytokine which induces morphological differentiation of natural killer cells. Benign endometrial cells were weakly sensitive to rIL-2-activated lysis in 2 cases. The precursors of the rIL-2-activated killer cells were mostly CD16-positive and CD3-negative, and co-sedimented with endogenous natural killer cells in discontinuous density gradient centrifugations. These results indicate the rIL-2-activated killer cells have a capacity to distinguish between normal and malignant endometrial cells, and that the precursors of the lytic cells in this system belong to the same subpopulation of lymphocytes as endogenous natural killer cells. In addition, rIL-2 alone may not in all cases be sufficient for optimal generation of cytotoxicity against malignant cells.