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     

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     

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     

Dendritic cells modified with 6Ckine/IFNγ fusion gene induce specific cytotoxic T lymphocytes in vitro

Backgroud and objective Dendritic cells play an important role in initiation and regulation of immune responses. Previous studies demonstrated that intratumoral administration of 6Ckine-modified DCs enhanced local and systemic antitumor effects. Herein we report the investigation of the specific CTL responses elicited by adenoviral 6Ckine/IFNγ fusion gene-modified DCs in vitro. Methods Human monocyte-derived DCs were modified with an adenoviral vector encoding 6Ckine/IFNγ fusion protein (Ad-6Ckine/IFNγ), and then investigated the effect of 6Ckine/IFNγ fusion protein on the maturation, cytokine and chemokine secretion of DCs, and their activities of recruiting and activating T cells in vitro were investigated. Results 6Ckine/IFNγ fusion protein induced DC maturation characterized with the upregulation of CD83 and CCR7. And it up-regulated the expression of RANTES and IL-12p70, down-regulated that of IL-10 in DCs. Additionally, 6Ckine/IFNγ markedly increased DC’s recruiting ability for naive T cells, benefiting from the enhanced expression of chemokines 6Ckine and RANTES in DCs. Fusion gene-modified DCs significantly promoted the proliferation of autologous T cells, induced Th1 differentiation by upregulating the expression of IL-2 and T-bet in T cells, and increased specific cytotoxicity of CTLs against specific tumor cells, HepG2 or LoVo cells, respectively. Conclusion Combining the effects of 6Ckine and IFNγ, Ad-6Ckine/IFNγ modified DCs induced enhanced CTL responses in vitro, which indicated that Ad-6Ckine/IFNγ modified DCs might be used as an adjuvant to trigger an effective antitumor immune response. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Gang Xue, Ran-yi Liu, Yan Li contributed equally to this work.     

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     

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     

Interferon γ and antisense transforming growth factor β transgenes synergize to enhance the immunogenicity of a murine mammary carcinoma

Transforming growth factor β (TGFβ) is an immunosuppressive cytokine that contributes to the immunological escape of tumor cells. In a previous study we demonstrated that inhibition of TGFβ production by EMT6 murine mammary tumor cells expressing an antisense TGF-β transgene reduces their tumorigenicity. On the basis of this observation we hypothesized that down-regulation of TGFβ production coupled with interferon γ (IFNγ) stimulation would induce an immune response superior to that generated by either strategy alone. In this study, EMT6 tumor cells expressing antisense TGFβ were transduced with the murine IFNγ gene. Tumor cells expressing either or both transgenes grew more slowly than mock-transduced tumors. Dual-transgene-expressing tumor cells were more immunogenic than tumor cells expressing either transgene alone. Studies in mice depleted of T cell subsets indicated that CD8⁺ T cells are the primary effectors of the antitumor activity observed. These results suggest that down-regulation of immunosuppression combined with cytokine-mediated immune augmentation is a useful strategy to improve antitumor immunity. Received: 6 October 1998 / Accepted: 15 January 1999     

T cells remaining after intensive chemotherapy for acute myelogenous leukemia show a broad cytokine release profile including high levels of interferon-γ that can be further increased by a novel protein kinase C agonist PEP005

Cytokines are released during T cell activation, including the potentially anti-leukemic interferon-γ (IFNγ), but also the hematopoietic growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF) that enhance proliferation and inhibit apoptosis of acute myelogenous leukemia (AML) cells. In the present study we investigated the release of IFNγ and GM-CSF by circulating T cells in AML patients with chemotherapy-induced cytopenia. T cells were activated with anti-CD3 plus anti-CD28 in a whole-blood assay in the presence of their natural cytokine network. We examined 63 samples derived from 16 AML patients during 28 chemotherapy cycles. Activated T cells showed a broad cytokine release profile, but IFNγ and GM-CSF levels showed a significant correlation and were generally higher than the other cytokine levels. Higher IFNγ and GM-CSF responses were associated with a low CD4:CD8 ratio, older patient age and no ongoing chemotherapy indicating potential utility of T cell activation regimes for the older AML patient. The cytokine levels could be further increased by the novel protein kinase C agonist PEP005, which also induced significant production of IL2 and TNFα which could contribute to anti-tumor effects in AML patients. We conclude that remaining T cells after intensive AML therapy show a broad cytokine release profile including high and significantly correlated levels of potentially anti-leukemic IFNγ and the AML growth factor GM-CSF. The final outcome of an AML-initiated T cell cytokine response will thus depend on the functional characteristics of the AML cells, in particular the relative expression of IFNγ and GM-CSF receptors which differs between AML patients.     

Disregulation in TH1 and TH2 subsets of CD4+ T cells in peripheral blood of colorectal cancer patients and involvement in cancer establishment and progression

 Recent theories have established that, during an ongoing immune response, the lymphokines produced by TH1 and TH2 subsets of CD4⁺ T cells are critical to the effectiveness of that response. In vivo and in vitro studies have demonstrated that the type of environmental cytokines plays a determinant role in directing the development of naive T cells into TH1 or TH2 effector cells. Disregulated expansion of one or other subset may contribute to the development of certain diseases. To establish whether a similar situation might exist in the cells of the peripheral blood (PBMC) of colorectal cancer patients, we have performed immunological studies on a group of patients and a group of healthy subjects. We examined the interleukin-2 (IL-2), interferon γ (IFNγ), IL-4, IL-6 and tumour necrosis factor α levels in serum; the production of IL-4 and IL-2, with and without activating agents, by PBMC, tumour-draining lymph node lymphocytes and tumour cells; and the proliferative response of PBMC to IL-2, IL-4 and anti-CD3 monoclonal antibody (anti-CD3), which were variously combined. The data of the present study lead us to hypothesize that, because of suppressive effects probably due to environmental IL-4, in the peripheral blood of patients there seems to be a disregulation in the functionality of TH1 and TH2 subsets of CD4⁺ T cells, with an expansion in TH2 and a malfunction in TH1 cells. Moreover it seems that this disregulation increases with as the disease progresses through the stages, suggesting that it can be directly implicated in the mechanisms that allow the tumour to locate and progress in the host. Received: 27 June 1995 / Accepted: 13 November 1995     

Differential modulation by interferon γ of the sensitivity of human melanoma cells to cytolytic T cell clones that recognize differentiation or progression antigens

 Human melanoma is a highly immunogenic tumor capable of inducing a specific immune response. A number of melanoma-associated antigens have been characterized during the past several years and can be classified into two groups: differentiation antigens  –  present also in normal melanocytes  –  and tumor-specific antigens, which, with the exception of testis, are present only in tumor cells. In a previous publication [Kirkin A. F., Petersen T. R., Olsen A. C., Li L., thor Straten P., Zeuthen J. (1995) Cancer Immunol Immunother 41:71] we have described the production of clones of cytotoxic T lymphocytes (CTL) against the highly immunogenic human melanoma cell line FM3. Using these clones we have defined four previously unknown melanoma-associated antigens, which could be subdivided into differentiation and progression antigens. In the experiments reported in this paper, we have further compared CTL clones from different groups and shown that the sensitivity of melanoma cells to CTL that recognize differentiation or progression antigens is differentially modulated during tumor progression as well as by the lymphokines interferon γ (IFNγ) and interleukin-10 (IL-10). The interaction of CTL clones recognizing progression antigens was strongly increased after treatment of melanoma cells with IFNγ, while the recognition by CTL clones specific for differentiation antigens either was unchanged or significantly decreased. IL-10 treatment of melanoma cells induced up-regulation with respect to recognition by CTL clones specific for differentiation antigens without affecting the recognition of melanoma cells by CTL clones specific for progression antigens. Using cellular systems at different stages of tumor progression, we demonstrated that the progressed state of melanoma cells is associated with increased sensitivity to recognition by CTL clones detecting progression antigens, and with decreased sensitivity to CTL clones recognizing differentiation antigens. Mimicking tumor progression, treatment with IFN-γ induced apparent down-regulation of differentiation antigens. A hypothesis is suggested in which IFN-γ plays different roles in the immune response against poorly immunogenic and highly immunogenic melanoma cells, increasing the progression of poorly immunogenic tumor cells or promoting a strong immune response and regression of highly immunogenic melanoma cells. Received: 23 November 1995 / Accepted: 7 March 1996     

Roles of cytokines in the pathogenesis and therapy of type 1 diabetes

Type 1 diabetes (T1D) results from autoimmune destruction of the insulin-producing β-cells in the pancreatic islets of Langerhans by autoreactive T helper 1 (Th1) cells characterized by their cytokine secretory products, interleukin-2 (IL-2) and interferon γ (IFNγ). Th1-type cytokines (IL-2 and IFNγ) correlate with T1D, whereas Th2 (IL-4 and IL-10), Th3 (transforming growth factor beta [TGFβ]), and T regulatory cell-type cytokines (IL-10 and TGFβ) correlate with protection from T1D. Paradoxically, however, administrations of Th1-type cytokines (IL-2 and IFNγ) and immunotherapies that induce Th1-type cytokine responses actually prevent T1D, at least in animal models. Therefore, immunotherapies that inhibit IL-2 production/action will block Th1 cell/cytokine-driven effector mechanisms of pancreatic islet β-cell destruction; however, anti-IL-2 therapy will not allow immune tolerance to be established. In contrast, immunotherapies that increase IL-2 production/action may correct an immunodeficiency in IL-2 production that appears to underlie the autoimmunity of T1D, thereby restoring immune tolerance to islet β-cells and prevention of T1D.     

Antitumor Activity of T Cells Generated from Lymph Nodes Draining the SEA-expressing Murine B16 Melanoma and Secondarily Activated with Dendritic Cells

The successful use of tumor-draining lymph nodes (TDLN) as a source of effector cells for cancer immunotherapy depends largely on the immunogenicity of the tumor drained by the lymph nodes as well as the methods for secondary in vitro T cell activation and expansion. We transferred the bacterial superantigen staphylococcal enterotoxin A (SEA) gene into B16 murine melanoma tumor cells, and used them to induce TDLN (SEA TDLN) in syngeneic hosts. Wild-type (wt) TDLN induced by parental B16 tumor was used as a control. In vitro, SEA TDLN cells proliferated more vigorously, produced more IFNγ and demonstrated higher CTL activity than wt TDLN cells when activated with anti-CD3/anti-CD28/IL-2. In vivo, SEA TDLN cells mediated tumor eradication more effectively than similarly activated wt TDLN cells (p<0.01). Furthermore, use of dendritic cells (DC) plus tumor antigen in vitro in addition to anti-CD3/anti-CD28/IL-2 stimulation further amplified the immune function and therapeutic efficacy of SEA TDLN cells. DC-stimulated SEA TDLN cells eliminated nearly 90% of the pulmonary metastasis in mice bearing established B16 melanoma micrometastases. These results indicate that enforced expression of superantigen SEA in poorly immunogenic tumor cells can enhance their immunogenicity as a vaccine in vivo. The combined use of genetically modified tumor cells as vaccine to induce TDLN followed by secondary stimulation using antigen-presenting cells and tumor antigen in a sequential immunization/activation procedure may represent a unique method to generate more potent effector T cells for adoptive immunotherapy of cancer.     

Cellular immune responses in acute leukaemia patients with severe chemotherapy-induced leucopenia; characterization of the cytokine repertoire of clonogenic T cells

 T lymphocytes are important both for the host defence against infections and probably also as antileukaemic effector cells in patients with acute leukaemia. To investigate the T lymphocyte cytokine repertoire of clonogenic T lymphocytes, CD4⁺ and CD8⁺ T lymphocyte clones were prepared from acute leukaemia patients with chemotherapy-induced cytopenia (leucocytes <0.5×10⁹/l). A majority of both CD4⁺ and CD8⁺ clones secreted detectable interleukin-2 (IL-2), IL-10, IL-13, granulocyte/macrophage-colony-stimulating factor and interferon γ (IFNγ) in response to phytohaemagglutinin + accessory cells (Epstein-Barr-virus-transformed B cell line, 80-Gy-irradiated). The CD4⁺ clones showed significantly higher levels of IL-10 secretion than the CD8⁺ clones. Decreased levels of IL-2, IL-13 and IFNγ were observed when acute myelogenous leukaemia (AML) blasts were used instead of cells from the B cell line as accessory cells during phytohaemagglutinin activation, but the differences in IL-13 and IFNγ levels were reversed by addition of exogenous IL-2. On the basis of these results we conclude: (i) the remaining clonogenic T lymphocytes derived from acute leukaemia patients with therapy-induced leucopenia can respond to activation with a broad cytokine response, and T-cell-derived cytokines may then contribute to cytokine responses during complicating infections in these patients; (ii) although T cells can modulate AML blast functions and mediate antileukaemic effects, the leukaemia blasts will also modulate T cell functions and alter the cytokine profile of activated T lymphocytes. Received: 6 November 1997 / Accepted: 5 March 1998     

Recognition by human Vγ9/Vδ2 T cells of melanoma cells upon fusion with Daudi cells

 Daudi Burkitt’s lymphoma cells, unlike other tumor cell lines, stimulate human T cells coexpressing the variable (V) region genes TCRG-V9 and V TCRD-V2 to proliferate and secrete lymphokines. Hybrids, derived by the fusion of Daudi cells with the human melanoma cell line MZ2-MEL 2.2, retain the morphology of melanoma cells. Unlike the parental melanoma cell line, these Daudi × MZ2-MEL 2.2 hybrids stimulate secretion of tumor necrosis factor (TNF) and granulocyte/macrophage colony stimulating factor (GM-CSF) by CD4-positive Vγ9/Vδ2 T-cell clones. Whereas the stimulator phenotype of Daudi cells behaves as a dominant trait in Daudi × melanoma hybrids, the expression of B-cell differentiation markers is suppressed. Thus, the γ/δ T-cell ligand expressed by Daudi cells behaves as a dominant tumor antigen in Daudi × melanoma hybrids and is unrelated to the differentiated B-cell phenotype. Dominant expression of the Daudi ligand for human Vγ9/Vδ2 T cells in these hybrids may provide a basis for defining the stimulatory principle at the molecular level. Received: 2 May 1996 / Revised: 15 July 1996     

Regulation of proliferation of UM-SCV-1A and UM-SCV-6 vulvar carcinoma cells by cytokines

 The biology and pathogenesis of vulvar carcinoma are poorly understood at present. In order to understand this disease better, we have used recently developed squamous cell carcinoma lines of the vulva as models. Two cell lines originating from two individuals (UM-SCV-1A and UM-SCV-6) were cultured in vitro in 10% fetal calf serum. The effects of interleukins 10 and 13, interferons α and γ, granulocyte/macrophage-growth-stimulating factor (GM-CSF), tumor necrosis factor α (TNFα), and transforming growth factor β (TGFβ) on the proliferation of the cells was investigated by using radioactively labelled uridine as tracer. In addition, an investigation on the molecular structure of extracted cellular DNA was carried out to investigate whether programmed cell death (apoptosis) would be inducible by any of the factors. In UM-SCV-1A cells, interleukin-10 (IL-10) and interleukin-13 (IL-13) caused an approximately 12-fold decrease in DNA synthesis in cells cultured for 72 h (P<0.001), while GM-CSF had no significant effect. TGFβ showed a significant inhibitory effect on deoxyuridine incorporation (P<0.001), which was 2.0- and 4.2-fold at 48 h and 72 h, respectively. TFGα showed a 1.2-fold inhibitory effect on DNA synthesis at 48 h (P<0.01) and a 1.5-fold inhibition at 72 h (P<0.05). Interferon γ (IFNγ) showed an inhibitory effect on DNA synthesis (1.3-fold; P<0.01). In UM-SCV-6 cells, both IL-10 and IL-13 showed inhibitory effects on deoxyuridine incorporation (1.3- and 1.4-fold at 48 h, respectively; P<0.001) that were even more pronounced at 72 h (2.4- and 2.5-fold respectively; P<0.001). IFNγ caused a 3.6-fold inhibition of DNA synthesis by UM-SCV-6 cells at 72 h (P<0.001). Both TFGβ and TNFα inhibited uridine incorporation (3.0- and 1.6-fold at 48 h, respectively; 2.7-fold at 72 h for both factors). GM-CSF inihibited DNA synthesis by UM-SCV-6 cells 1.3- 2.0-fold at 48 h and 72 h, respectively. In dose/response analyses, the effect of INFα on DNA synthesis was inhibitory in both cell lines at 48 h, while stimulatory effects were observed at 72 h. Electrophoretic analyses of DNA isolated from cells cultured in the presence or absence of different factors did not reveal DNA fragmentation. All cytokines, with the exception of IFNα, showed inhibitory effects on DNA synthesis by vulvar carcinoma cells. Of the factors studied, the recently described interleukins 10 and 13 showed potent inhibition of cell growth, encouraging further investigation on the molecular mechanisms of the observed inhibition. Apoptosis does not seem to be induced in the two vulvar carcinoma cell lines by any of the cytokines studied. Received: 26 March 1996 / Accepted: 5 December 1996     

Antitumor response of regional lymph node lymphocytes in human lung cancer

 Monoclonal antibodies (mAb) are promising substances for the treatment of colorectal carcinoma, but the efficiency of this therapy still needs further improvement. We used a flow-cytometric cytotoxicity test to determine the efficacy of the cytokines interferon α (IFNα) and γ (IFNγ), interleukin-2 (IL-2), macrophage-colony-stimulating factor (M-CSF), granulocyte/macrophage-CSF (GM-CSF) and tumor necrosis factor α (TNFα) in enhancing the antibody-dependent cellular cytoxicity (ADCC) of the mAb 17-1A and the mAb BR55-2 against the colorectal carcinoma cell line HT29. In experiments performed at an effector to target ratio of 9:1, with peripheral blood mononuclear cells from five healthy volunteers as effector cells, we found that IFNα, IFNγ and IL-2 significantly augmented the ADCC of both mAb at concentrations between 3 ng/ml and 30 ng/ml. The other three cytokines were not effective. In further experiments we examined combinations of the three effective cytokines in different concentrations. The combination of IFNα and IL-2 proved to be optimal in enhancing ADCC of both mAb. Thus, the examination of ADCC by flow cytometry may reveal potentially useful combinations of cytokines and mAb for the treatment of colorectal carcinoma. Received: 11 September 1997 / Accepted: 19 February 1998     

CD4+ T cells kill HLA-class-II-antigen-positive melanoma cells presenting peptide in vitro

Purpose: Most melanoma cell lines express HLA class II antigens constitutively or can be induced to do so with interferon γ (IFNγ). We have previously demonstrated that peptide-specific CD4⁺ T cells proliferate in response to HLA-class-II-antigen-mediated peptide presentation by melanoma cells in vitro and produce interleukin-10 (IL-10) and (IFNγ). We asked whether the responding T cells kill the tumor cells and, if so, whether direct cell contact was required. Methods: Two HLA class II⁺ melanoma cell lines derived from metastases were co-cultured with a human CD4⁺ T cell clone specific for influenza hemagglutinin peptide (HA). T cells, melanoma, and HA were co-cultured for 48 h. Melanoma cells with and without HA and/or T cells served as controls. After 36 h, the medium was removed for cytokine analysis by enzyme-linked immunosorbent assay (ELISA). Twelve hours later non-adherent cells were washed away and the adherent melanoma cells were trypsinized and counted. Dual-chamber culture plates were used to determine whether cell contact and/or exposure to cytokine were required for tumor cell death. Results: Melanoma cell counts were over 80% lower in wells containing T cells than in wells with melanoma and peptide alone (P < 0.05). ELISA of supernatants revealed production of IFNγ and IL-10 by the responding T cells. Direct T cell contact with tumor cells was not required for tumor cell death, as melanoma cells were killed when they shared medium but had no contact with T cells responding to peptide presentation by HLA-class-II-antigen-positive melanoma cells in a separate chamber. Blocking antibody to IFNγ but not IL-10 prevented melanoma cell death at levels of cytokine similar to that present in co-culture assays. Conclusions: Peptide-specific CD4⁺ T cells kill melanoma cells in vitro when they recognize peptide presented by the tumor cell in the context of HLA class II antigen. Direct cell contact is not required, suggesting that it is a cytokine-mediated event. Immunotherapy, using primed CD4⁺ T cells and peptide, may be beneficial in patients whose tumors express HLA class II antigens or can be induced to do so with IFNγ. Received: 1 July 1999 / Accepted: 17 September 1999     

Modulation of Antigenic Phenotype in Cultured Human Osteoblast-like Cells by FGFb, TGFβ1, PDGF-BB, IL-2, IL-1β, LPS and IFNγ

Background/Aims Recent reports demonstrated that osteoblast-like cells can also exert activities directly associated with the immune system (cytokine synthesis, antigen presentation, phagocytosis and stimulation of T lymphocytes). The present study aimed to analyze the effect of Transforming growth factorβ1 (TGFβ1), Fibroblast growth factor basic (FGFb), Platelet-derived growth factor-BB (PDGF-BB), Interleukin-1β (IL-1β), Interleukin-2 (IL-2), Lipopolysaccharide (LPS) and Interferon-γ (IFNγ) on the expression on osteoblast-like cells of antigens involved in antigen presentation.Methods Flow cytometry was used to investigate whether the growth factors FGFb, TGFβ1, PDGF-BB, IL-2, IL-1β, LPS and IFNγ modulate the expression on cultured human osteoblast-like cells of different antigens involved in antigen-presentation and T cell activation.Results TGFβ1 treatment significantly reduced the expression of CD54 and CD86. IL-1β treatment significantly enhanced the expression of CD54, CD86 and HLA-DR. LPS and IFNγ treatments produced a major increase in CD54, CD80, CD86 and HLA-DR expression. Expression of these antigen-presenting molecules was not significantly modified by FGFb, PDGF-BB or IL-2 treatment.     

Cellular characteristics of peripheral blood lymphocytes and tumour-infiltrating lymphocytes in patients with gynaecological tumours

 Immunotherapy of gynaecological cancer with tumour-infiltrating lymphocytes (TIL) or peripheral blood lymphocytes (PBL) has become a valid treatment modality with varying degrees of success in obtaining an antitumour response. TIL consist of lymphocytes, mainly T cells and minor populations of natural killer cells or B cells. Conventional cytogenetic studies of tumour cells from patients with breast and ovarian cancer have shown multiple chromosomal abnormalities including chromosomes 7 and 12. This study was designed to analyse the surface further, as well as investigate the intracellular, characteristics of TIL by multicolour flow cytometry and the cytogenetic features by fluorescence in situ hybridization. Tumour cell, peripheral blood and TIL samples from 25 patients (15 ovarian tumours, 8 breast cancers, 1 uterine sarcoma, 1 cervical carcinoma) were analysed for their phenotype, the expression of major cytokines [interleukin-2 (IL-2), IL-4 and interferon γ (IFNγ)], their proliferation rate, their cytotoxic ability and for the presence of numerical aberrations of chromosomes 7 and 12. All the tumour cells showed a high frequency of numerical aberration in chromosomes 7 and 12, especially trisomies or tetrasomies and combined aberrations. Trisomies of both chromosomes also occured at a low percentage in TIL and PBL. Received: 20 June 1996 / Accepted: 4 January 1997     

Analysis of the immune reactivity of infiltrating and peripheral lymphocytes from patients with renal cell carcinoma by measuring cytokine secretion

 The immunological properties of tumor-infiltrating (TIL) and peripheral blood lymphocytes (PBL) from 29 patients with renal cell carcinomas were characterized with respect to their phenotypic expression and cytokine production. TIL were isolated from mechanically disaggregated tumor material and PBL from peripheral blood by gradient centrifugation. To eliminate all non-lymphoid cells, CD3-positive cells were specifically separated from these cell fractions with anti-CD3 magnetic beads. These pure CD3-positive PBL (CD3⁺PBL) and TIL (CD3⁺TIL) were cultured with pokeweed mitogen and the levels of the cytokines interleukin-1α (IL-1α), IL-1β, IL-2, interferon γ (IFNγ), and tumor necrosis factor α (TNFα) measured in the 4-day post-inductional cell culture supernatants. In all cell cultures a wide range of cytokine values was found, indicating a large variation in the immunological activity of the lymphocytes of each individual. When the cell cultures of the CD3⁺TIL and CD3⁺PBL were compared in each patient similar values for IL-1α, IL-1β, IFNγ and TNFα were found. However CD3⁺TIL produced significantly lower levels of IL-2 than CD3⁺PBL upon mitogenic stimulation. This may be due to a lower CD4/CD8 ratio in the CD3⁺TIL as compared to the CD3⁺PBL. These results suggest that there are no fundamental qualitative and quantitative differences in the lymphokine-producing capacity of CD3⁺TIL and CD3⁺PBL derived from patients with renal cell carcinomas. Received: 8 August 1995 / Accepted: 23 January 1996