In vivo antitumor effects of murine interferon- γ -inducing factor/interleukin-18 in mice bearing syngeneic Meth A sarcoma malignant ascites

Interferon-γ-inducing factor/interleukin-18 is a novel cytokine that reportedly augments natural killer (NK) activity in human and mouse peripheral blood mononuclear cell cultures in vitro and has recently been designated IL-18. In this study, IL-18 exhibited significant antitumor effects in BALB/c mice challenged intraperitoneally (i.p.) with syngeneic Meth A sarcoma when administered i.p. on days 1, 2 and 3 after challenge. Intravenous (i.v.) administration also induced antitumor effects in the tumor-bearing mice; however, subcutaneous (s.c.) administration did not. When mice were twice pretreated with 1 μg IL-18 3 days and 6 h before tumor challenge, all mice survived whereas control mice died within 3 weeks of challenge. Inhibitory effects on Meth A cell growth in vitro were not observed with either IL-18 or interferon γ. The effects of IL-18 pretreatment were abrogated by abolition of NK activity after mice had been injected with anti-asialo GM1 antibody 48 h before and, 24 h and 72 h after tumor challenge. Mice pretreated with IL-18 and surviving tumor challenge resisted rechallenge with Meth A cells but could not reject Ehrlich ascites carcinoma, and spleen cells from the resistant mice, but not control mice, exhibited cytotoxic activity against Meth A cells in vitro after restimulation with mitomycin C-treated Meth A cells for 5 days. The effector cells in the spleen cell preparations from resistant mice appear to be CD4⁺ cells because cytolytic activity was significantly inhibited after depletion of this subset by monoclonal antibodies and complement. In conclusion, IL-18 exhibits in vivo immunologically (primarily NK) mediated antitumor effects in mice challenged with syngeneic Meth A sarcoma and induces immunological memory and the generation of cytotoxic CD4⁺ cells. Received: 17 September 1996 / Accepted: 8 November 1996     

Contrasting effects of T cell growth factors on T cell responses to melanoma in vitro

 Determinants of T cell responses to tumor cells remain largely unknown. In the present study we have used long-term cultures of human melanoma cells and autologous peripheral blood lymphocytes to examine the influence of cytokines with T cell growth activity on the phenotype and cytotoxic and proliferative response of T cells to melanoma. It was found that addition of interleukin-4 (IL-4) inhibited the response of CD8⁺ T cells and promoted the response of the CD4 subset. IL-2 or IL-7 was effective in increasing melanoma-specific cytotoxic T lymphocyte (CTL) activity in cultures where CD8 T cells were predominant, whereas IL-4 followed by IL-2 was most effective in cultures where CD4 T cells predominated. IL-10 or IL-12 inhibited proliferation and CTL activity against melanoma in long-term cultures. The effects of IL-12 were reproduced in long-term cultures of T cells stimulated with mAb against CD3 and were shown to depend on prior exposure of T cells to IL-12 before IL-2. As yet unidentified factors, such as co-factor expression on melanoma, appear to be as important as exogenous cytokines in determining the nature of T cell responses to melanoma. These results suggest that analysis of responses in long-term culture may assist in defining the role of key cytokines and other determinants of immune responses to melanoma. Received: 4 June 1996 / Accepted: 12 November 1996     

Diabetes mellitus induced by low-dose interleukin-2

 Interleukin-2 (IL-2) is a potent immunomodulator that has been associated with the clinical development of autoimmune disorders. However, diabetes mellitus has not been reported in patients treated with single-agent IL-2. We conducted a clinical trial of a protracted daily schedule of subcutaneously administered low-dose IL-2. A patient with advanced colorectal cancer, treated with 1.5×10⁶ international units of IL-2 daily, developed insulin-requiring diabetes during therapy. Hyperglycemia improved during treatment interruption and recurred with reinstitution of IL-2. The diabetes in this patient developed in the context of T cell and natural killer cell expansion, and the presence of islet cell autoantibodies was documented. We postulate that, in this patient, IL-2 reversed the anergy of autoreactive T cells that had escaped clonal deletion. It is possible that prolonged daily exposure to immunomodulatory doses of IL-2 will result in the development of autoimmune phenomena not observed with other schedules of administration. Received: 15 April 1996 / Accepted: 1 August 1996     

Interleukin-1α synergistic in vivo enhancement of cyclophosphamide- and carboplatin-mediated antitumor activity

 Interleukin-1α (IL-1α) has potent acute antitumor activity in vivo and can enhance the efficacy of chemotherapeutic drug-mediated antitumor responses. Studies were undertaken to examine the ability of IL-1α to enhance the activity of cyclophosphamide (CTX) administered in combination with carboplatin. To determine the in vivo effect of IL-1α, CTX and/or carboplatin, mice bearing 14-day RIF-1 tumors were treated on day 0 with a concurrent i.p. injection of varying doses of CTX (5–150 mg/kg), human IL-1α (125 μg/kg), and carboplatin (50 mg/kg) and examined 24 h later for the surviving fraction by the in vivo excision clonogenic-tumor-cell assay. Even at the lowest doses of CTX, IL-1α significantly enhanced the clonogenic tumor cell kill when compared to treatment with CTX alone. When carboplatin was added to the treatment schema, significantly greater clonogenic cell killing and tumor regrowth delay were observed as compared to any agent alone or a two-drug combination (CTX/IL-1α or CTX/carboplatin). Significant enhancement was observed even at low doses of CTX in combination with carboplatin and IL-1α. The interaction between the three-drug combination was found to be synergistic as determined by the median dose effect with significant dose reduction apparent for IL-1α and CTX when used in this combination. These results demonstrate that IL-1α can synergistically enhance the antitumor efficacy of CTX and the combination of CTX and carboplatin. Received: 11 September 1996 / Accepted: 20 May 1997     

Effects of granulocyte-colony-stimulating factor and interleukin-2 on ascites formation and the survival time of nude mice bearing human ovarian cancer cells

 The aim of this study was to elucidate the effect of intraperitoneal (i.p.) instillations of granulocyte-colony-stimulating factor (G-CSF) and/or interleukin-2 (IL-2) on ascites formation and the survival time of nude mice with malignant ascites, produced by i.p. inoculation of human ovarian cancer cells. When the nude mice were treated with medium alone, ascites was observed in all mice 28 days after tumor inoculation. When the mice were treated with cis-diamminedichloroplatinum(II) (cisplatin) alone, G-CSF alone or IL-2 alone, it took 35 days for the ascites to form in all mice. When cisplatin was combined with G-CSF or IL-2, one of ten mice did not form ascites during the observation period. Surprisingly, when G-CSF and IL-2 were simultaneously administered, ascites formation was not observed in any mice. Although i.p. treatment with cisplatin alone significantly prolonged the survival time, compared to medium alone, the lytic activity of spleen cells against HRA cells was significantly suppressed. When G-CSF or IL-2 was combined with cisplatin, the suppression by cisplatin was eliminated and subsequently resulted in a prolongation of the survival time. When G-CSF was combined with IL-2, both the peritoneal and splenic macrophages/monocytes were stimulated and the splenic lytic activity was about double that following treatment with G-CSF alone on IL-2 alone, suggesting that complete inhibition of ascites formation results not only from a significant increase of the peritoneal macrophages but also from enhancement of the lytic activity. Two mice, died from dissemination of tumor in the abdominal cavity, but eight mice survived without tumor for more than 90 days. As confirmed by monitoring body weight and hematocrit, G-CSF and IL-2 seemed to have no adverse effect. From these results, we conclude that a combination therapy with G-CSF and IL-2 might be of clinical use for inhibiting large amounts of ascites, which may inhibit therapeutic effects for ovarian cancer patients. Received: 20 May 1996 / Accepted: 19 September 1996     

Induction of accessory cell function of human alveolar macrophages by inhalation of human natural interleukin-2

 Accessory function allows antigen-presenting cells to produce sufficient secondary signals for optimum T cell proliferation and interleukin-2 (IL-2) production. Alveolar macrophages are inferior accessory cells compared to monocytes (PBM). We report here that the accessory index (AI) of alveolar macrophages and PBM of patients with lung metastases of solid tumors treated with inhalations of human natural IL-2 (hnIL-2) increased following its administration (P<0.005). The accessory index was significantly elevated from baseline values after 2 weeks of inhalation of 300 000 IU hnIL-2/day (8.2±10.2 compared to 1.1±1; P<0.001). The inhalation of 150 000 IU also induced increases in the index (AI = 2.3±1.9), however, without reaching statistical significance. In addition at 300 000 IU IL-2/day a significant increase in the accessory index was observed for PBM (4±2.5; P<0.05). The indices of PBM and alveolar macrophages prior to inhalation showed a significant negative correlation with the age of the patients (r _s =  – 0.5; r _s =  – 0.8, respectively; P<0.03 for all comparisons). Our data demonstrate that the inhalational application of hnIL-2 enhances the accessory function of alveolar macrophages and, to lesser extent, the accessory index of PBM, indicating the occurrence of pharmacological immunostimulation. Received: 16 August 1995 / Accepted: 4 January 1996     

Cellular immune responses to autologous chronic myelogenous leukaemia cells in vitro

 Using a modification of the autologous mixed lymphocyte/tumour cell culture (MLTC), it is demonstrated here that lymphocytes from chronic-phase myelogenous leukaemia (CML) patients (n = 58), but not from their HLA-identical siblings, proliferated upon coculture with autologous tumour cells. However, in most cases, the level of proliferation measured was low (stimulation index <3, n = 37). This was most likely related to the amount of interleukin-10 (IL-10) released into the culture medium by the CML cells, because addition of neutralizing anti-IL-10 serum to MLTC markedly enhanced proliferative responses. In addition, supplementation of media with IL-1α further enhanced proliferative responses and a combination of anti-IL-10 serum and IL-1α was more effective than either agent alone. Only HLA-DR-matched CML cells, but not HLA-DR-mismatched CML cells or matched or mismatched PBMC restimulated proliferation of IL-2-dependent T cell lines derived from MLTC supplemented with IL-1α and anti-IL-10 serum. The responding cells under these conditions were predominantly CD4⁺ and secreted IL-2, and interferon γ; some secreted IL-4, but none secreted IL-10. These data therefore suggest the existence of an HLA-DR-restricted DTH/Th1-type of tumour-specific immunity in CML patients, which may be down-regulated in vitro by excessive secretion of IL-10 together with depressed secretion of IL-1. Received: 9 November 1995 / Accepted: 8 February 1996     

Effects of N G-methyl-L-arginine,an inhibitor of nitric oxide synthesis,on interleukin-2-induced capillary leakage and antitumor responses in healthy and tumor-bearing mice

 We tested whether treatment with an inhibitor of nitric oxide synthesis (N ^G-methyl-L-arginine, MeArg) can ameliorate interleukin-2(IL-2)-therapy-induced capillary leak syndrome in healthy or tumor-bearing mice without compromising the antitumor effects of IL-2 therapy. Healthy or C3-L5-mammary-adenocarcinoma-bearing C3H/HeJ mice were treated with one or two rounds of various doses of IL-2 (ten injections, i. p., every 8 h) or MeArg (ten injections s. c., every 8 h) or their combination. In an additional experiment, MeArg was given chronically in the drinking water, rather than s. c. to healthy mice subjected to one round of therapy as above. Mice were killed 1 h after their last IL-2 injection to measure the water content of the lungs and pleural cavities (markers of capillary leakage), NO production (given by NO₂ ^– and NO₃ ^– levels in the serum and pleural effusion), as well as the effect of therapies on the primary tumor size and number of spontaneous lung metastatic nodules. Results revealed that all doses of IL-2 (7500 – 35 000 Cetus U/injection), as well as both rounds of IL-2 therapy, caused capillary leakage. However, no pleural effusion was seen after the second round in any of the IL-2-treated groups. MeArg therapy, given subcutaneously (5 – 20 mg kg^–1 injection^–1 in healthy and 20 mg kg^–1 injection^–1 in tumor-bearing mice), did not ameliorate IL-2-induced capillary leakage in either group of mice, and did not compromise antitumor effects of IL-2. However, subcutaneous MeArg therapy alone reduced the growth of the primary tumors, the occurrence of spontaneous lung metastases and the amount of tumor-induced pulmonary edema. When MeArg therapy was given orally (1 mg/ml drinking water), a substantial drop in NO production, as well as reduction in capillary leakage was noted in IL-2-treated healthy mice. These findings suggest that NO inhibitors could be a valuable adjunct to IL-2 therapy of cancer and infectious diseases. Received: 23 October 1995 / Accepted: 22 November 1995     

Effects of interleukin-12 on in vitro culture with interleukin-2 of regional lymph node lymphocytes from lung cancer patients

 In the present study, we carried out a functional analysis of regional lymph node lymphocytes (RLNL) from patients with lung cancer after in vitro activation by interleukin-2 (IL-2) and interleukin-12 (IL-12). IL-12 (100 U/ml) enhanced both the proliferation and cytotoxic activity of RLNL in a culture with low doses of IL-2 (5 – 10 JRU/ml). After comparing an RLNL culture with a low dose of IL-2 alone, a higher proportion of CD8⁺ cells and CD56⁺ cells and a lower proportion of CD4⁺ cells were found in the culture with both IL-12 and a low dose of IL-2. Such a combination of the cytokines effectively activated RLNL in terms of the expression of IL-2 receptors. In the culture condition of IL-12 and a low dose of IL-2, a synergistic effect was observed in the production of such cytokines as interferon γ, tumor necrosis factor α (TNFα), and TNFβ, as well as in tumor cytotoxicity. However, the addition of IL-12 inhibited the cytotoxicity of RLNL in the culture with a high dose of IL-2 (100 JRU/ml). This inhibition is considered to be partially due to the endogenous production of TNFα by lymphocytes, because the neutralization of TNFα bioactivity partially restored the cytotoxic activities of RLNL. Furthermore, in the presence of hydrocortisone, IL-12 synergistically enhanced the cytotoxic activity of RLNL cultured with a high dose of IL-2. These results provide useful information about the improvement of adoptive immunotherapy against cancer using RLNL. Received: 2 February 1996 / Accepted: 30 July 1996     

Interleukin-4 is effective in restoring cytotoxic T cell activity that declines during in vivo progression of a murine B lymphoma

 We previously reported [Chakrabarti et al. (1992) Cell Immunol 142:54; 144:455] that, in a murine B lymphoma model 2C3, idiotype (Id)-specific CD8⁺ cytotoxic T lymphocytes (CTL) are generated in mice following hyperimmunization with irradiated tumor cells, and that they are effective in tumor rejection. The present study reveals that 2C3-specific CTL are also induced in spleens during tumor progression, but are not sustained. At the early stage of tumor growth, the splenic T cells following a 5-day incubation in vitro with killed 2C3 tumor targets, produce high levels of cytokines, namely interleukin-4 (IL-4), IL-10 and interferon γ (IFNγ). Their cytotoxic T lymphocyte (CTL) activity and cytokine levels, except IL-2, sharply decline at the late stage when the mice are increasingly moribund. Although the decline in cytokine level is also evident with CD4⁺ T cells, a precipitous and concurrent decrease occurs primarily in the IL-4 level with both CD4⁺ and CD8⁺ T cells of late-tumor-bearing animals (TBA). Study with the unseparated splenocytes also reveals that sevenfold less IL-4 is produced at the late stage. Furthermore, the cytotoxicity of CTL from late TBA can be effectively restored by addition of supernatants from the splenocyte culture of early TBA, or by IL-4, but not by IFNγ and IL-10. In addition, only IL-4-activated CD8⁺ T cells from the late TBA are found, by Winn assay, to be protective in vivo. Thus it appears that IL-4, required to sustain antitumor CTL activity, is consumed by T and possibly other cells at the late stage of tumor growth, thereby compromising host immunity against the tumor. We contend that induction or maintenance of protective immunity depends not only on the tumor antigen but also on the specific cytokine milieu in a tumor-bearing host. Received: 8 February 1997 / Accepted: 24 April 1997     

The tumor-bearing state induces augmented responses of organ-associated lymphocytes to high-dose interleukin-2 therapy in mice

 A high-dose bolus regimen for interleukin(IL)-2 administration to cancer patients frequently causes serious side-effects in which various organs are involved. In order to reveal the mechanism of toxicities associated with this regimen, we compared the augmenting effect of high-dose IL-2 on murine organ-associated lymphocytes between neoplastic and non-neoplastic states. Intraperitoneal administration of IL-2 at a dose of 10⁵ JRU (Japanese Reference Units) twice daily for 3 days led to the death of all the syngeneic MH134-hepatoma- or X5563-myeloma-bearing mice, whereas it had no lethal effect on non-tumor-bearing mice. Histological and morphometric analyses demonstrated that tumor-bearing mice displayed more extensive infiltration of large granular lymphocytes and agranular lymphocytes in the liver and lungs than did the non-tumor-bearing mice. Large granular lymphocytes had the ultrastructural characteristics of lymphokine-activated killer cells. Lymphocytes often underwent extravasation into the interstitial space and exhibited local proliferation without causing any direct injury to apposed parenchymal cells. Flow-cytometric analysis of hepatic mononuclear cells demonstrated that IL-2-receptor-β(IL-2Rβ)-bearing lymphocytes, i.e., natural killer cells and intermediate CD3 cells, were increased in number in the neoplastic state before the IL-2 injection. The present study indicates that the tumor-bearing state increases the number of organ-associated IL-2Rβ⁺ lymphocytes, which are then greatly amplified by the challenge of high-dose IL-2, leading to the functional disturbance of organs. We have further demonstrated here that an intermittent low-dose IL-2 regimen has a potential therapeutic effect on tumor regression without causing lethal side-effects. Received: 15 July 1996 / Accepted: 23 July 1997     

Immune modulation by interleukin-12 in tumor-bearing mice receiving vitamin D3 treatments to block induction of immunosuppressive granulocyte/macrophage progenitor cells

 Lewis lung carcinoma (LLC-LN7) tumors stimulate myelopoiesis and increase the presence of granulocyte/macrophage (GM) progenitor cells having natural suppressor activity. Treatment of these tumor-bearing mice with interleukin-12 (IL-12) resulted in minimal immune modulation. The objective of this study was to determine whether eliminating natural suppressor activity would allow for immune stimulation by IL-12. Treatment of LLC-LN7 tumor-bearing mice with vitamin D₃ eliminated natural suppressor activity. In mice that were first treated with vitamin D₃ and then also with IL-12, there was stimulation of splenic T cell proliferation in response to immobilized anti-CD3 plus IL-2. In addition, spleen and lymph node cells from vitamin-D₃/IL-12-treated tumor-bearing mice became stimulated in response to autologous tumor to produce interferon γ (IFNγ), although IL-2 production was not stimulated. A prominent effect of the combined vitamin-D₃/IL-12 treatment regimen was the synergistic augmentation of autologous tumor-specific cytolytic activity within the regional lymph nodes. The generation of these tumor-specific effector cells required the presence of the tumor mass since such activity was not elicited in the lymph nodes of mice from which the tumors had been surgically excised. The results of this study show that, after treatment of tumor bearers with vitamin D₃ to eliminate GM-suppressor cells, IL-12 can induce select regional antitumor immune responses, particularly IFNγ production and cytolysis by regional lymph node cells of autologous tumor. Received: 15 December 1995 / Accepted: 22 March 1996     

Bacillus Calmette-Guérin plus interleukin-2 and/or granulocyte/macrophage-colony-stimulating factor enhances immunocompetent cell production of interferon-γ, which inhibits B16F10 melanoma cell growth in vitro

 Although immunotherapy with bacillus Calmette Guérin (BCG) is an established adjuvant treatment for malignant melanoma, the mechanism of its role in this process is unclear. To investigate the possible contribution of tumor-inhibitory cytokines induced by BCG, B16F10 melanoma cell growth in culture was assessed in response to purified cytokines and conditioned media of BCG-stimulated splenocytes. Interferon-γ (IFNγ) was the most potent single agent (IC₅₀≈50 pg/ml). Tumor necrosis factor α was substantially weaker (IC50>10 ng/ml) but provided synergy with IFNγ. None of the other cytokines such as interleukin-2 (IL-2), IL-4, IL-6, IL-10, IL-12, or granulocyte/macrophage-colony-stimulating factor had direct antitumor activity against B16F10 melanoma cells. However, when IL-2 and/or GM-CSF were combined with BCG either by exogenous addition or through endogenous production by novel cytokine-secreting recombinant BCG (rBCG), a substantial increase in INFγ production by splenocytes was observed. Antitumor activity of this conditioned medium directly correlated with IFNγ concentration and was completely blocked by neutralizing antibody to IFNγ. These results suggest that BCG may exert part of its antitumor action on melanoma through the induction of IFNγ, which can be greatly enhanced through the concomitant addition of IL-2 and/or GM-CSF. Furthermore, by utilizing rBCG that secrete these cytokines, it may be possible to potentiate the antitumor effect of BCG directly at the site of BCG inoculation. Received: 29 January 1996 / Accepted: 9 April 1996     

Human T lymphocyte activation in the presence of acute myelogenous leukaemia blasts; studies of normal polyclonal T cells and T lymphocyte clones derived early after allogeneic bone marrow transplantation

 T cell clones (CD4⁺CD8^–TCRαβ⁺γδ^–) derived from bone marrow transplant recipients were stimulated with phytohaemagglutinin (PHA) +interleukin-2 (IL-2) in the presence of irradiated (50 Gy) peripheral blood mononuclear cells (PBMC) derived from acute leukaemia patients(leukaemic PBMC containing more than 95% blast cells). Leukaemic PBMC could function as accessory cells during mitogenic T cell activation resulting in both T cell proliferation and a broad T cell cytokine response [IL-3, IL-4, IL-10, granulocyte/macrophage-colony-stimulating factor (GM-CSF) tumour necrosis factor α (TNFα) and interferon γ (IFNγ) secretion]. Blockade of IL-1 effects by adding IL-1 receptor antagonist together with PHA+IL-2+leukaemia blasts increased T cell proliferation, whereas IL-6-neutralizing antibodies did not alter T cell proliferation. A qualitatively similar T cell cytokine response and a similar cytokine profile (highest levels detected for GM-CSF and IFNγ) were detected when normal polyclonal T cell lines were stimulated with PHA in the presence of non-irradiated leukaemic PBMC. When leukaemic PBMC derived from 18 acute myelogenous leukaemia patients were cultured with PHA and cells from a polyclonal T cell line, increased concentrations of the T cell cytokines IFNγ and IL-4 were detected for all patients. We conclude that T cell activation resulting in proliferation and a broad cytokine response can take place in the presence of excess acute myelogenous leukaemia blasts. Received: 30 November 1995 / Accepted: 9 January 1996     

Suppressive effects of Lactobacillus casei cells,a bacterial immunostimulant,on the incidence of spontaneous thymic lymphoma in AKR mice

 The mean survival age of female AKR/J mice was significantly prolonged, the enlargement of thymus was markedly suppressed, and the proliferation of ecotropic and recombinant murine leukemia viruses (MuLV) was markedly inhibited when 8-week-old female AKR/J mice were injected intraperitoneally (i. p.) with heat-killed Lactobacillus casei cells twice weekly for 8 weeks. In contrast, such actions of heat-killed L. casei cells were not seen in 20-week-old female AKR/J mice. The leukemogenic activity of the cell-free extract of thymus from adult female AKR/J mice in newborn female AKR/J mice was drastically reduced by i. p. treatment with heat-killed L. casei cells. The difference in adjuvant effectiveness of heat-killed L. casei cells on 8- and 20-week-old animals may be dependent on the difference in the enhancing activity of the cell-mediated immune systems between the groups induced by heat-killed L. casei cells, and, as a result, on the difference in the degree of proliferation of ecotropic and recombinant MuLV in thymus, which consequently causes thymic lymphoma. Received: 13 February 1996 / Accepted: 18 April 1996     

Human ex vivo carcinoma cells produce transforming growth factor β and thereby can inhibit lymphocyte functions in vitro

 We tested 20 human carcinoma samples for the production of transforming growth factor β (TGFβ) in vitro. Tumour cell suspensions without obvious contamination with non-malignant cells were kept in culture conditions for 16 h and their supernatants were added to CCL-64 cells. The proliferation of these cells is inhibited by TGFβ. According to this assay, the supernatants contained both active and latent TGFβ. In addition, the supernatants were found to suppress the spontaneous cytotoxic function and activation of T-cell-enriched lymphocyte populations. A specific monoclonal antibody (mAb) counteracted these effects and therefore we concluded that they were mediated to a large extent by TGFβ. In line with the results obtained with the supernatants, activation of lymphocytes could also be inhibited by tumour cells and their inhibitory effect was weaker in the presence of the TGFβ-specific mAb. It is important to note that, when TGFβ-specific mAb was added to autologous mixed lymphocyte/tumour cell cultures, lymphocyte activation occurred more often. These results thus substantiate the assumption that production of TGFβ may help the survival of potentially immunogenic tumour cells in immunocompetent patients. Received: 21 August 1996 / Accepted: 12 November 1996     

Human T lymphocyte activation in the presence of acute myelogenous leukaemia blasts: studies of allostimulated interferon-γ secretion

 Normal peripheral blood mononuclear cells (PBMC responders) were cultured together with non-irradiated allogeneic PBMC (more than 95% leukaemia blasts) derived from patients with acute leukaemia (referred to as leukaemic PBMC stimulators). Cytokine secretion was determined as cytokine concentrations in supernatants. Both normal PBMC and enriched CD4⁺ and CD8⁺ T cells responded to allostimulation with interferon (IFNγ) secretion. Interleukin-1 (IL-1) receptor antagonist and IL-2-neutralizing antibodies decreased IFNγ secretion. Exogenous IL-1β, IL-2 and IL-7 increased allostimulated IFNγ secretion, whereas decreased levels were seen in the presence of IL-6, IL-10 and granulocyte-colony-stimulating factor (G-CSF). During allorecognition IFNγ -neutralizing antibodies decreased acute myelogenous leukaemia (AML) blast secretion of G-CSF. We conclude that (i) both CD4⁺ and CD8⁺ T cells show allostimulated cytokine secretion in response to allogeneic stimulator cells containing a dominating population of native, cytokine-secreting leukaemia blasts, and (ii) IFNγ released during this response can modulate the function of allogeneic AML blasts. Received: 4 June 1996 / Accepted: 15 October 1996     

Interleukin-2 secretion by KHT sarcoma cells leads to loss of their vaccine potential

 We have investigated the effect of interleukin-2 (IL-2) secretion by KHT sarcoma cells upon their vaccine potential in syngeneic C3Hf/He mice. Parental KHT tumor cells were transfected with the plasmid pBCMG-neo-mIL-2 to obtain a transfectant KHT-2-3-7 that secreted 20 units IL-2. KHT-2-3-7 cells elicited protective immunity in only 10% of the immunized mice, compared with 40% of mice immunized with irradiated parental KHT tumor colls. To minimize the contribution of potential antigenic differences between the KHT-2-3-7 transfectant and parental KHT cells, a clone of KHT cells (KHT-C21) was isolated and used in subsequent experiments. A number of transfectants secreting various amounts of IL-2, ranging from 2 units to 200 units, were obtained following transfection of KHT-C21 cells with plasmid pBCMG-neo-mIL-2. Two of the transfectants, C21-13-4 and C21-1, each secreting 200 units IL-2, elicited protective immunity in a significantly lower fraction of mice than did irradiated KHT-C21 parental tumor cells (P<0.0l). Two other transfectants C21-10 and C21-11, secreting 2 and 23 units IL-2 respectively, also showed lower vaccine potential compared with the parental KHT-C21 clone (P<0.05). To minimize further any role for potential antigenic or other molecular differences between the individual transfectants and the clonal KHT-C21 parental cells in lowering their vaccine efficacy, mice were immunized with a mixture of five transfectants, and the results again showed significantly lower vaccine efficacy of the mixture compared with the irradiated parental C21 cells (P<0.0l). In view of published studies showing enhanced or unchanged efficacy of IL-2-secreting tumor cell vaccines, our observation of the lower vaccine potential of IL-2-transduced tumor cells indicates that the vaccine efficacy of IL-2-secreting tumor cells depends on the individual tumor. Such variability/unpredictability would hamper the clinical use of IL-2-secreting tumor cells as vaccines. Received: 23 April 1996 / Accepted: 7 February 1997     

Levamisole regulates the proliferation of murine liver T cells through Kupffer-cell-derived cytokines

 We have previously shown that levamisole increases the cytotoxic, cytostatic, and proliferative activity of murine nonparenchymal liver cells (NPC) in vitro. We have also shown that the nonadherent subpopulation of NPC, which are composed predominantly of T lymphocytes, is very responsive to this agent when administered to mice. Kupffer cells or immigrant macrophages are also responsive to levamisole but to a lesser extent. These findings prompted us to investigate changes in cytokine production by NPC following-treatment of mice with levamisole (25 mg/kg, i.p.), which may help explain the observed alterations in the immune functions of these cells. We found that levamisole treatment of mice causes a threefold increase in production of interferon (IFN) α/β by adherent NPC (more than 80% – 90% Kupffer cells) in vitro. When IFN α/β was added to cultured cells, it decreased the proliferative capacity of liver T cells in a dose-dependent manner. In contrast, the addition of anti-IFNα/β was shown to augment levamisole-induced proliferation of unfractionated NPC and Kupffer cells. NPC production of interleukin 1 (IL-1) and interleukin-6 (IL-6) in vitro was also increased threefold following treatment of mice with levamisole. IL-6 added in vitro to cells significantly augmented levamisole-induced proliferation of liver T cells while anti-IL-6 reduced proliferative activity to control levels. These findings suggested that IFNα/β, IL-6, and IL-1 play important regulatory roles in controlling the proliferative response of murine liver-associated T lymphocytes to levamisole. Finally, the proliferation of bone marrow cells was increased in mice given 5-fluorouracil (5FU). On the other hand, the proliferation of NPC was dramatically suppressed when 5FU was administered. However, the proliferation of these cells was restored when levamisole was given after 5FU. Received: 27 November 1995 / Accepted: 16 October 1996     

Antigen-presenting function of human peritoneum mesothelial cells isolated from a pancreatic carcinoma patient after mutant Ras peptide vaccination

 Mesothelial cells obtained from ascites fluid of a Ras-peptide vaccinated pancreatic adenocarcinoma patient were cultured in vitro. Fresh isolated cells expressed HLA class II molecules, which were lost upon culture, but could be up-regulated after coculture with human recombinant interferon-γ. The antigen-presenting capacity of these mesothelial cells was tested with allogeneic peripheral blood mononuclear cells (PBMC) in a mixed lymphocyte mesothelial cell culture and by stimulating autologous PBMC with purified protein derivative of Mycobacterium tuberculosis. Cloned T cells from the same patient allowed us to test the ability of mesothelial cells to present a mutant Ras-derived peptide to specific T cells in a DLA-DR-restricted manner. Mesothelial cells effectively stimulated allogeneic resting T lymphocytes to proliferate and presented soluble protein antigen or a mutant Ras-derived peptide to specific T cells, indicating that they display processing and presenting capabilities. Since mesothelial cells are in close anatomical relationship with intraabdominal malignancies, they may contribute to stimulation of specific T cells by endocytosing tumour-specific antigens and presenting them to T lymphocytes. This could be a possible mechanism in which mesothelial cells participate in maintaining local specific immunity in patients already primed. Received: 17 July 1996 / Accepted: 15 October 1996