Interferon-gamma (IFN) and tumor necrosis factor-alpha (TNF) were examined for their ability to enhance major histocompatibility complex (MHC) expression on a variety of human tumor and normal tissue targets. Enhanced expression of MHC correlated with decreased target susceptibility to lysis by fresh peripheral blood mononuclear cells (PBMCs) and IL-2-augmented PBMCs (aPBL) but not as clearly with cells with lymphokine-activated killer (LAK) activity. These studies revealed maximal MHC enhancement after 48-72 h of incubation in IFN. Resistance to lysis by natural killer (NK) cells was best demonstrated after 72 h. Further, IFN and TNF were synergistic in their effects on MHC expression and induction of resistance of the cultured leukemias K562 and Molt-4 to aPBL effectors. Conversely, LAK susceptibility was usually unaltered after target IFN and TNF treatment. Incubation of fibroblasts and vascular endothelial cells with IFN also consistently resulted in MHC class I enhancement and resistance to NK lysis, whereas LAK susceptibility was variably affected. The brief incubation of fresh PBL in IL-2 (4-6 h) resulted in effectors highly lytic toward cultured cells, but with no activity against fresh tumor. Cultured cell lines treated with IFN and TNF were rendered relatively resistant to lysis by these activated cells. Fresh tumor MHC expression and LAK susceptibility was unchanged after IFN incubation. Additionally, there was no correlation between the level of MHC class I or class II expression and LAK susceptibility to any fresh, uncultured melanoma studied. These data suggest that LAK effectors possess different mechanisms of tumor recognition or lysis than cells with NK activity or cells briefly incubated (4-6 h) in IL-2. The ability of tumor-infiltrating lymphocytes to lyse the cultured autologous tumor target was markedly increased by preincubation of the targets with IFN and TNF. Finally, it appears that IL-2 treatment and the resultant endogenous production of IFN by T-lymphocytes should not adversely affect tumor susceptibility to current immunotherapy using IL-2.
Eighteen patients with advanced renal cell carcinoma were treated with human lymphoblastoid interferon (Wellferon) and continuous fusion vinblastine. All patients received vinblastine as a continuous infusion at a dose of 1.5 mg/m2/day on days 1 to 5. The interferon was given by daily intramuscular injections on days 1 to 10. Three patients were treated with a dose escalation scheme that reached a maximum daily dose by day 3 of 5 X 10(6) units/m2/day and that was then continued until day 10. Fifteen patients received 3 X 10(6) units/m2/day on day, 1, and 5 X 10(6) units/m2/day on days 3 to 10. Treatments were repeated every 28 days. Neutropenia (less than 1,500/mm3) occurred in 14 of 18 patients. Transient increases in serum glutamic-oxaloacetic transaminase levels to greater than four times baseline were noted in nine patients. Thrombocytopenia (less than 100,000 platelets/mm3) occurred in one patient. Fatigue, lethargy, and decline in performance status were marked in four of the patients. None of the patients in the low-dose interferon group and only 1 of the 15 patients in the high-dose interferon group had an objective response (7%, with a 95% confidence interval of 0 to 31%). Of the 12 patients completing at least two courses of therapy, 10 were in the high-dose group, which included the 1 objective (partial) response. This response noted at the start of the fourth course. Ten others developed progressive disease and one stopped treatment because of neurologic toxicity.(ABSTRACT TRUNCATED AT 250 WORDS)
OM-89, a proteinaceous extract from Escherichia coli with very low endotoxin content, was tested for its capacity to stimulate in vitro cells involved in the immune response. OM-89 induced a marked proliferation of mouse spleen cells; E. coli lipopolysaccharide (LPS) at the same concentration as present in OM-89 was totally ineffective. Passage through nylon wool strongly decreased the OM-89-induced effect, suggesting that the responding lymphocytes were of the B lineage. Exposure of bone marrow-derived macrophages to OM-89 promoted glucose oxidation through the hexose monophosphate shunt pathway and the capacity to generate superoxide upon phorbol myristate acetate (PMA) stimulation. These effects were not blocked by polymyxin B, whereas this compound completely prevented induction of similar metabolic activation by E. coli lipopolysaccharide. In addition, OM-89 treatment induced marked PMA-dependent superoxide and hydrogen peroxide release by macrophages from the LPS low responder mouse strain C3H/HeJ. Incubation with recombinant murine interferon-gamma and OM-89, but not with either compound alone, led to functional activation, as shown by the killing of tumor target cells, and by the destruction of the intracellular parasite Leishmania enrietti by macrophages of both LPS-responsive and unresponsive mouse strains. These experiments indicate that OM-89 can stimulate metabolic and functional activities of lymphocytes and macrophages that are important for host defense.
Interactions between the cellular and humoral compartments of the human immune system are well documented. Leukoregulin (LR) is a hormone with the ability to upregulate the natural killer (NK) phenomenon by increasing target cell sensitivity to NK lymphocyte cytotoxicity. In the present report, the interactions between LR and human cytotoxic T-lymphocytes (CTLs) are described. Two cultured T-cell populations are specifically cytotoxic for autologous human melanoma and fail to lyse allogeneic melanoma, K562, or autologous peripheral blood lymphocytes (PBLs) in 4 h chromium release assays. Pretreating allogeneic melanoma or K562 with LR resulted in 19-67% lysis at an effector:target (E:T) ratio of 5:1, while no more than 10% lysis was observed without LR. Autologous PBLs were also subject to lysis when pretreated with LR (50% at an E:T of 40:1 with LR, and 2% without LR). The observed effect was dose-related and was most effectively inhibited by autologous cold targets, but persisted in the presence of antibody to human lymphocyte antigen class I antigens (w6/32). Lysis of autologous melanoma was not increased by pretreatment with LR. LR appears to mediate lysis of melanoma, NK targets, and normal PBLs by tumor specific CTLs. The effect is dose-related and non-major histocompatibility complex-restricted. The data do not support a significant role for LR in the normal physiology of human CTLs, but the striking effects in vitro may prove to be experimentally or therapeutically useful.
Six groups of two cynomolgus monkeys were treated with escalating intravesicular doses of recombinant human tumor necrosis factor (rHuTNF) for a 6 week interval. The doses of rHuTNF ranged from 10 ng to 1 mg and were instilled weekly. Two monkeys had instillation of saline only and served as controls. The monkeys were weighed and temperatures determined before, immediately after, and 2 days following each treatment. Cystoscopic examination was performed 2 days after each treatment and blood samples were obtained. At the conclusion of the study, animals were killed and necropsy was performed. There was no observable toxicity from treatment with rHuTNF. There was no difference between treated and control monkeys with respect to temperature, weight, or blood measurements. No drug-induced alteration in bladder morphology was found by either cystoscopic or microscopic pathologic examination.
Clinical immunotherapy trials have been performed recently where ex vivo interleukin-2 (IL-2)-activated peripheral blood mononuclear cells (i.e., the "LAK" cells) have been transfused in addition to IL-2 infusions. In such protocols, patients have received highly heterogeneous cell suspensions and the nature of the effector cells that may have contributed to tumor regression has remained unclear. In certain animal models, it has appeared that natural killer lymphocytes were the effector cell type responsible for tumor regression. To test whether NK cells could eventually be relevant for the treatment of human tumors, we have performed a feasibility trial where purified lymphokine-activated natural killer (LANAK) cells have been prepared and transfused to a limited series of renal cell carcinoma patients receiving IL-2 (continuous infusions at 3 x 10(6) U/m2/day). Natural killer lymphocytes (1-2 x 10(6] were purified from peripheral blood mononuclear cells and expanded during 4-5 weeks in the presence of IL-2 on microtiter plates containing feeder layers cells. In vitro, the resulting LANAK cell suspensions were 100 times (range of 2 to 10(3] more efficient against Daudi target cells than their autologous LAK counterparts. Twelve patients were included; 9 received the two planned courses of treatment with LANAK cells and IL-2. Overall toxicity was relatively moderate. Besides occasional chills, there were no apparent secondary effects due to cell infusions. The mean number of LANAK cells transfused per patients was 45.1 x 10(9), ranging from 7 to 125 x 10(9). The biodistribution of LANAK cells was similar to that reported previously for LAK cells with no preferential localization to tumor sites. We conclude from this study that using well-defined populations of effector lymphocytes is a feasible cellular therapy approach that may lead to improved understanding and efficacy of the novel immunotherapy methods.
Phenotypic markers and cytotoxic function were monitored in cultures of normal human mononuclear cells obtained from peripheral blood or spleen and stimulated by recombinant interleukin-2 (IL-2; 1,500 U/ml). Fresh spleen cells contained less than 5% natural killer (NK) cells (CD3-NKH1+), which increased about threefold after activation with IL-2. In both spleen and peripheral blood cultures, T cells with the NKH1 marker showed the highest relative increment among all cell types studied. Lymphokine-activated killer (LAK) cells from peripheral blood and spleen displayed very similar cytotoxic activity against K562, Daudi, and COLO carcinoma cell lines. Killing of the three targets peaked at 7 days of culture. Antibody-dependent cell cytotoxicity against a B-cell line was mediated by both circulating and splenic LAK cells from 2 to 14 days of culture. Cell sorting experiments showed that K562 targets were killed by both CD5+NKH1+ and CD5-NKH1+ cells whereas Daudi targets are only killed by CD5-NKH1+ activated NK cells from both spleen and peripheral blood. In summary, human spleen LAK cells have similar phenotypic and functional properties to circulating LAK cells, and they may be used for adoptive immunotherapy of human cancer.
We have examined the responsiveness to in vitro stimulation with high-dose recombinant interleukin-2 (IL-2) of peripheral blood leukocytes (PBLs), collected from normal donors, or from successive daily cytaphereses of cancer patients with a range of advanced malignancies, following 5 days of continuous infusion with IL-2 in vivo. Normal donor PBLs showed a transient release of tumor necrosis factor (TNF) (up to 400 pg/ml) during the first day, while factors including interferon-gamma (IFN-gamma), soluble IL-2 receptor, and soluble CD-8 showed a gradual increase to modest levels (at best) during the 4 day incubation with IL-2. In contrast, the cancer patients' PBLs, after 5 days of IL-2 activation in vivo, responded with one of two patterns of production of cytokines. In pattern I, exposure to the IL-2 resulted in a transient release of TNF during the first 48 h. The level of TNF released showed a progressive increase from PBLs harvested from the first cytapheresis (up to 50 pg of TNF/ml) through the fourth cytapheresis (up to 2,000 pg of TNF/ml). Additionally, pattern I PBLs showed significant levels of production of IFN-gamma, soluble IL-2 receptor, and soluble CD8. In pattern II, the patients' PBLs from each cytapheresis released only low levels of TNF (less than 300 pg/ml) and minimal levels of IFN-gamma, IL-2 receptor, and CD8. A pattern I response is considered to be consistent with an immunostimulatory role for IL-2, which induces a cooperative interaction of lymphocytes and macrophages that is mediated by other cytokines, while pattern II may reflect an immunosuppression in these patients.(ABSTRACT TRUNCATED AT 250 WORDS)
Systemic administration of biologic response modifiers (BRMs) is often limited by serious adverse effects. Local delivery of a BRM may provide a means to avoid systemic side effects while enhancing host defense. To test this hypothesis, the effects of intratracheal administration of the interferon inducer polyinosinic-polycytidilic acid (Poly-I:C) were examined. We demonstrated that intratracheal administration of Poly-I:C in rats results in alterations in pulmonary lavage cell number, population distribution, and function, suggestive of cellular activation. Following intratracheal instillation of 15, 75, or 150 mg of Poly-I:C, we observed a dose-dependent increase in Fc receptor-mediated phagocytosis, a dose-independent tumoricidal activity directed against xenogeneic P815 murine mastocytoma target cells, and a dose-independent decrease in superoxide anion (O2-) generation. With the exception of O2- generation, these functions returned to normal control levels by 7 days after a single dose of Poly-I:C. Thus, localized administration of Poly-I:C enhanced the host defense capability of pulmonary lavage cells, providing a rationale for compartmentalized immunoprophylaxis in the lung.
The purpose of this study was to characterize the antiproliferative activity of a recombinant interferon-alpha (IFN-alpha) against a newly established human adenocarcinoma cell line (DMS-4C) and to determine whether IFN-alpha entrapped in multilamellar liposomes had superior antitumor effects compared with free IFN-alpha. Treatment of DMS-4C cells with 100 U/ml of free IFN-alpha resulted in 34% cytostasis. IFN-alpha encapsulated in phosphatidylcholine/phosphatidylserine multilamellar vesicles produced growth inhibition of 67%, which was significantly greater than that produced by free IFN-alpha or by control liposomes containing only medium combined with free IFN-alpha. Moreover, kinetic analysis revealed that to produce significant cytolysis, free IFN-alpha had to be incubated with target cells for at least 24 h, whereas IFN-alpha encapsulated into liposomes required only 30 min of exposure.
Tumor-infiltrating lymphocytes (TILs) have been grown from a variety of human tumors. TILs from some patients with melanoma demonstrate lytic activity specific for autologous tumor, and can mediate tumor regression when adoptively transferred to select cancer patients. In this study, we have compared the in vitro properties of lymphocytes from peripheral blood (PBLs), from draining lymph nodes (DLNs), and from tumors (TILs) grown simultaneously from 10 patients: 2 with melanoma, 4 with breast cancer, 1 with gastric cancer, 1 with renal cancer, 1 with sarcoma and 1 with lung cancer. PBLs, TILs, and DLNs were cultured in RPMI 1640 + 10% human AB serum, 20% LAK cell culture supernatant, and 1,000 u/ml of recombinant interleukin-2. Half of each culture was restimulated with irradiated autologous tumor every 14 days. In all groups, tumor feeding enhanced lymphocyte proliferation, although TILs and DLNs consistently proliferated longer and more rapidly than PBLs. Eight of 10 early cultures of TILs and DLNs contained greater or equal proportions of CD8+ cells compared with CD4+ cells, but in long-term cultures an inversion of that ratio was seen (CD4+ greater than CD8+). In short-term chromium release assays, specific lysis of autologous tumor was seen in tumor-fed TILs and DLNs from one patient with melanoma, DLNs from one patient with breast cancer, and TILs from one patient with lung cancer. Other cultures had nonspecific lytic activity. Specific cytotoxicity against autologous tumor sometimes became apparent only after prolonged culture and repeated restimulation with autologous tumor. DLNs have in vitro properties similar to TILs and may be a useful immune reagent for cancer therapy.
Twenty patients with metastatic renal cell cancer were treated with a combination of recombinant interferon-alpha 2a (Roferon-A), 18 MU intramuscularly three times weekly and vinblastine 0.1 mg/kg intravenously once every 3 weeks. Three patients experienced a complete response (CR) (15%) and three a partial response (PR) (15%). The response duration was 3, 13, and 15 months in the CR group, and PRs lasted 11, 13, and 14 months. Constitutional symptoms like fever, anorexia, and fatigue were the most common side effects. One patient had reversible hepatitis, which was probably unrelated to antineoplastic therapy. Dose modifications had to be made in seven patients due to leukopenia or thrombocytopenia. No very serious side effects were noticed. In view of what has been reported previously, the overall response rate (30%) of this regimen is good and tolerance of the treatment is acceptable.
The antitumor activity of recombinant human interleukin-1 alpha (rHIL-1 alpha), recombinant human tumor necrosis factor, and recombinant murine interferon-beta (rIFN-beta) was augmented by concomitant administration of recombinant human interleukin-2 (rHIL-2) in the treatment of adenocarcinoma 755. Especially when a divided dose (two doses/day) of rHIL-1 alpha or rIFN-beta was combined with a divided dose of rHIL-2, the antitumor effect was markedly potentiated. However, only a marginal effect was seen by combination of rHIL-1 alpha and/or rIFN-beta and rHIL-2 against Lewis lung carcinoma, a tumor that is resistant to cytokines.
Prior studies with the XMMME-001-RTA immunoconjugate composed of an antimelanoma monoclonal antibody and ricin A chain demonstrated some antitumor activity. However, almost all patients studied developed human antimurine antibodies and antiricin antibodies. In an effort to abrogate these host anti-immunotoxin immune responses and thus enhance antitumor activity, we treated 20 patients with the immunoconjugate plus a single dose of intravenous cyclophosphamide. An overall response rate of 20% was observed-predominantly in pulmonary and soft tissue nodules. There was no diminution in antibody responses against either the murine antibody or the ricin moiety. Further studies to elucidate the role of cyclophosphamide in monoclonal antibody therapy are planned.
The in vivo administration of recombinant interleukin-2 (rIL-2) in humans has led to anemia, thrombocytopenia, and eosinophilia. In an attempt to evaluate the effects of the in vivo administration of rIL-2 on murine bone marrow, we administered rIL-2 to C57BL/6 female mice i.p. three times a day at doses ranging from 10,000 to 100,000 U for 10 days; we then harvested blood and bone marrow from these animals every other day and performed the following analyses: White blood cell count, red blood cell count, hemoglobin concentration, histogram analysis of nucleated cell volume, manual differential counts, and in vitro colony-forming assays for granulocytes and monocytes (CFU-GM). The administration of rIL-2 induced an overall increase in the total white blood cell count that was dose-dependent for its appearance and overall number. This increase was secondary to an increase in monocytes and granulocytes but not to a change in lymphocyte number. Myeloid proliferative activity measured by CFU-GM revealed a biphasic pattern of activity. An early proliferation at 2 days was not followed by lymphocytosis. However, a second peak of proliferation at 6 days was associated with peripheral blood granulocytosis and monocytosis. After rIL-2 was discontinued on day 10, the CFU-GM activity returned to normal by days 16-18. These results suggest that the in vivo administration of rIL-2 may play an important role in the regulation of hematopoiesis.
The binding of various lectins to human lymphokine-activated killer (LAK) cells was investigated. Human peripheral blood lymphocytes were cultured for 3 days with recombinant interleukin-2 (rIL-2), and then stained with 20 kinds of lectins. There were four types of lectin-binding patterns: (a) negative staining; (b) weakly positive staining; (c) strongly positive staining; and (d) two populations, one weakly and one strongly positive staining. Among the 20 kinds of lectins, both Lens culinaris agglutinin (LCA) and Pisum sativum agglutinin (PSA) showed two peaks in the staining patterns on LAK cells (type 4). We separated these two populations of LAK cells by using a cell sorter, and assayed them for cytotoxic activity against 51Cr-labeled Hela cells. The LAK cells that were strongly stained by LCA or PSA [LCA (or PSA)-bright LAK cells] showed stronger cytotoxic activity than the LAK cells that were weakly stained by LCA or PSA [LCA (or PSA)-dull LAK cells]. Furthermore, we separated lymphocytes with LCA or PSA before culturing them with rIL-2 (LAK precursor), and we found that both LCA-bright and PSA-bright lymphocytes can develop into LAK cells with higher cytolytic activity. LCA-bright lymphocytes kept their ability to be stained strongly with LCA even after culture with rIL-2 for 3 days. Similarly, LCA-dull lymphocytes were still stained weakly after culture with rIL-2. Two-color assay of LAK cells revealed that 78% of NK (CD16+)-LAK cells and 46% of T (CD3+)-LAK cells were LCA-bright cells.(ABSTRACT TRUNCATED AT 250 WORDS)
SKCO 1 human colon carcinoma cells have been shown to be synergistically inhibited in their growth by the combinations of alpha-interferon (IFN-alpha) or beta-interferon (IFN-beta ser) and gamma-interferon (IFN-gamma). To determine if a correlation could be established between this synergistic antiproliferative effect and a synergistic induction in IFN-inducible proteins, or a unique perturbation in the cell cycle, we studied the effects of IFN-beta ser and IFN-gamma, alone and in combination, on 2',5'-oligoadenylate (2-5A) synthetase, indoleamine-2,3-dioxygenase (IDO), a human analogue of the murine Mx protein (p78), and the phases of cell cycle. 2-5A synthetase was maximally induced after a 24-h exposure to both IFN-beta and IFN-gamma. A synergistic enhancement of 2-5A synthetase activity was observed only with low concentrations of each IFN (0.05 ng/ml). IDO activity was induced by IFN-gamma and the combination of IFN-beta ser and IFN-gamma, but not IFN-beta ser alone. The differences in IDO activity between IFN-gamma and the combination, however, were not statistically significant. The p78 protein was induced in a dose-dependent manner by IFN-alpha and IFN-beta ser. IFN-gamma enhanced the expression of p78 induction by IFN-alpha or IFN-beta ser, even at concentrations of IFN-gamma that did not induce the protein when administered as a single agent. The combination of IFN-alpha and IFN-beta ser, which results in an antagonistic antiproliferative effect, also resulted in an antagonistic induction of p78. No changes in the cell cycle were observed following exposure to IFN-beta ser, IFN-gamma, or the combination, and treatment with IFN-gamma did not inhibit the accumulation of cells in G2M caused by colchicine. Thus, the synergistic antiproliferative effect produced by IFN-beta ser and IFN-gamma in SKCO 1 cells could not be correlated with a synergistic enhancement in 2-5A synthetase or IDO activity, or with a perturbation in the cell cycle. In contrast, the combination of IFN-gamma and IFN-alpha or IFN-beta ser synergistically enhanced the expression of p78 protein in these cells.
The human Mx, an interferon (IFN)-alpha- and IFN-beta-induced 76-kd protein, is a homolog (Mx-homolog) to the murine Mx protein, which is necessary and sufficient to provide adequate resistance against influenza virus in murine cells and in mice. Leukocytes from 36 patients with tumors (chronic myelogenic leukemia, hairy cell leukemia, and malignant melanoma) were monitored for their Mx-homolog content before, during, and after rIFN-alpha-2b therapy. Before therapy, only one patient was slightly positive for Mx-homolog. All 36 patients showed a significant increase of Mx-homolog in their mononuclear cells within the first day of IFN therapy. During therapy, the Mx-homolog levels remained elevated. After cessation of treatment, the Mx-homolog content in the mononuclear cells decreased slowly; within 2 weeks, it was about 20-30% of its value during therapy. However, even after 3 weeks, the Mx-homolog was still detectable. The maximally induced Mx-homolog concentration showed a significant correlation to the IFN dose given in vivo. These data indicate that the Mx-homolog is an excellent marker for monitoring the activity of IFN during IFN therapy. In addition, the in vivo endogenous activation of the IFN system might be detectable by the determination of the Mx-homolog despite the lack of circulating IFN.
We examined the effect of levamisole (LMS) on the proliferative response and interleukin-2 (IL-2) concentration in OKT3-, phytohemagglutinin-, and concanavalin-A-stimulated lymphocyte cultures. Although proliferative response was enhanced in lymphocyte cultures stimulated in the presence of LMS, similar levels of IL-2 were observed in stimulated and unstimulated cultures. The mechanism of the enhancement effect of LMS on proliferative response was further characterized by studying its effects on the growth of IL-2-dependent CTLL-2 cells in culture. Since this cell line has been shown to require 2-mercaptoethanol (2-ME) for normal growth in recombinant IL-2, the effect of LMS on several parameters of its growth was compared with that of 2-ME. Unlike 2-ME, LMS did not enhance 35S-cystine uptake. Both compounds increased thiol concentration in the cell culture, but (oxidized) 2-ME induced a greater increase. Generally, the effects of LMS on CTLL-2 growth were quite similar to those of structurally unrelated compounds known to have antioxidant properties, and the demonstrated thiol requirement of this cell line for growth in recombinant IL-2 was met by substituting LMS for 2-ME. When the effect of LMS on IL-2 receptor (IL-2R) expression in CTLL-2 cells was examined by a receptor-ligand binding assay involving low levels (10-80 pM) of 125IL-2, a modest increase in the level of IL-2R expression was observed. The biologically active high-affinity IL-2R complex is believed to be preferentially bound at the low levels of 125IL-2 used here, suggesting a functional relevance for this effect of LMS. These observations should be useful in minimizing the cost and duration of in vitro expansion of lymphocytes for use in adoptive immunotherapy and should be applicable in improving the response of immunologically impaired patients to immunotherapy.
We have previously demonstrated that the successful transfer of rat chloroleukemia (Mia C51) cells to newborn rats is related to the host's inability to generate adequate levels of differentiation factor (DF). Thus, when the appropriate amount of DF was injected into rats bearing MIA C51 cells, the development of chloroleukemia was aborted. In the present study, we provide evidence that stimulation of endogenous differentiation activity (DA) production by the administration of a biologic response modifier (Imuvert) will like-wise abort the development of chloroleukemia. Imuvert at 50 micrograms/ml had no direct effect on growth, viability, or differentiation of MIA C51 cells. However, when monocytes from young rats or adult rats were stimulated with Imuvert in vivo or in vitro, there was significant increase in DA production. Treatment of young rats with Imuvert aborted the development of chloroleukemia from transplanted MIA C51 cells. It is concluded that stimulation of endogenous DA production may provide a potentially useful approach in the treatment of leukemia.
