TRAIL-induced cytotoxicity in a melphalan-resistant rhabdomyosarcoma cell line via activation of caspase-2.

The prognosis for patients with chemo-refractory rhabdomyosarcoma remains poor. The tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a hopeful candidate for new strategies in chemotherapy. The effects of TRAIL and melphalan (Mel) in the rhabdomyosarcoma cell line TE-671 were investigated by colorimetric caspase assays and flow cytometry. TRAIL induced the activation of caspases-2, -3 and -8, but not the activation of caspase-9, in the Mel-resistant TE-671 cells. Inhibition of caspase-2 with the caspase-2 inhibitor z-VDVAD-fmk significantly down-regulated the TRAIL-induced caspase-3 activation, as well as the TRAIL-induced cytotoxicity. When TE-671 cells were treated with a combination of Mel and TRAIL, a significant synergism of drug-induced cytotoxicity was obtained. The inhibition of caspase-2 could completely abolish caspase-3 activation, suggesting that TRAIL sensitises TE-671 cells for Mel-induced cytotoxicity via a caspase-2- and -3-dependent mechanism. In conclusion, it was shown, for the first time, that TRAIL could sensitise Mel-resistant tumour cells to melphalan.

Prognostic value of T-1 cell numbers prior to allogeneic stem cell transplantation in patients with severe graft-versus-host disease.

Assessment of risk factors for acute graft-versus-host disease (aGvHD) might help in tailoring the intensity of prophylactic immunosuppression after allogeneic stem cell transplantation (SCT), thereby decreasing the relapse rate in leukaemia patients. In this study, we analysed whether the number of recipient blood T cells and plasma levels of different cytokines were correlated with the risk of aGvHD after allogeneic SCT. Analyses were performed in 23 patients receiving pSCT immediately before or during the first 2 days of the conditioning regimen. In all, 40 or more Tc-1 cells/microl pretransplant were associated with a significantly increased risk of aGvHD (10/10 patients with GvHD>/=II; 4/13 patients without aGvHD with a Tc-1 number >40/microl, P<0.002, Fisher's exact test). In addition, 40 or more Th-1 cells/microl pretransplant were also associated with a significantly increased risk of aGvHD (P<0.04, Fisher's exact test). Furthermore, the number of Th-2 cells was significantly higher in patients with severe aGvHD even though the median absolute cell counts were very low. However, all other investigated parameters did not reveal predictive value. In conclusion, determination of T-1 cells prior to SCT might determine patients with high/low risk of aGvHD and could thus be used to control immunosuppression after SCT.

In vivo cytokine responses to interleukin-2 immunotherapy after autologous stem cell transplantation in children with solid tumors.

The potent immunostimulatory cytokine interleukin-2 (IL-2) has been extensively investigated for its potential to induce anti-tumor immunity in a number of tumor models. Only recently the complex interplay of mutually suppressive or supportive cytokines of the IL-2-induced network of cytokines has been better characterized. The aim of this study was to assess which of these in vitro findings are reproducible in vivo in recipients of stem cell transplants (SCT), since in these patients long- lasting impairments in cytokine inducibility have been described. We have therefore studied the kinetics of putative modulators and mediators of IL-2-induced immune activation, namely IL-1beta, IL-4, IL-5, IL-10, IL-12, soluble Fas ligand (sFasL), and GM-CSF during IL-2 therapy. All patients were children or adolescents suffering from solid tumors with poor prognosis who received three 5-day courses of high-dose intravenous IL-2 as an adjuvant to their radio-chemotherapy and autologous SCT. While IL-1beta, IL-4 and IL-12 were not, and sFasL was only mildly affected by the IL-2 therapy, we observed a consistent and early rise of IL-10, IL-5, and GM-CSF. These increases were rapidly reversible after discontinuation of IL-2 therapy. The inducibility of IL-10, IL-5 and GM-CSF was more pronounced with increasing time from the SCT, and in the third cycle reached an order of magnitude as in high-dose IL-2 patients without SCT. Together with the abundant in vitro data, these findings may help devise a combination immunotherapy permitting stronger anti-tumor effects, but lesser adverse effects.

Molecular mechanisms of hyperthermia- and cisplatin-induced cytotoxicity in T cell leukemia.

BACKGROUND: The prognosis of early and very early relapse in acute lymphoblastic leukemia of childhood is still very poor unless a hematopoietic stem cell transplant is performed if a second remission can be achieved by induction chemotherapy. Therefore an intensification of chemotherapy is required. MATERIALS AND METHODS: In the present study the molecular mechanisms of cisplatin- and/or hyperthermia-mediated cytotoxicity in CEM cells, a human T leukemia cell line, were investigated. RESULTS: Both hyperthermia and cisplatin induced the activation of the effector caspases-3 and -6. However, caspase activation followed different time kinetics. While hyperthermia exerted maximum caspase activation immediately after application, cisplatin activated caspase-3 and -6 after 24 hours. At both time-points significant caspase-3 and -6 activation was observed when the cells were stimulated by a combination of heat and cisplatin. The application of z-VAD-fmk, a general caspase inhibitor, showed that hyperthermia mediated cytotoxicity mainly via caspase-dependent mechanisms, while cisplatin induced both caspase-dependent and -independent cytotoxicity. Time kinetic experiments revealed that hyperthermia induced cell death immediately after the heating pulse. In contrast, cisplatin-induced cell death had its maximum between 6 hours and 12 hours after the heating pulse. The combined application of heat and cisplatin induced two peaks of cytotoxicity, one immediately after the heating pulse and the other between 6 hours and 12 hours. CONCLUSION: Hyperthermia and cisplatin induced cell death in T leukemic cells by different molecular mechanisms, which might explain the enhanced cisplatin-induced cytotoxicity by hyperthermia.

Mechanisms of hyperthermia- and 4-hydroperoxy-ifosfamide-induced cytotoxicity in T cell leukemia.

The prognosis of patients with early ALL (acute lymphoblastic leukaemia) relapse is poor with conventional chemotherapy alone. Thus, intensified chemotherapy strategies are required. The application of hyperthermia enhances the efficacy of certain antineoplastic drugs such as ifosfamide. In this study, the effects and molecular mechanisms of ifosfamide (4hydroperoxy-ifosfamide = 4OOH-IFA)- and/or hyperthermia-induced cell death are investigated in CEM cells. Hyperthermia enhanced the efficacy of 4OOH-IFA in a subaddictive manner. Analysis of caspase activation revealed an early hyperthermia-induced stimulation of caspase-3 and -6 directly after the heating pulse, while maximum activation following stimulation with 4OOH-IFA was obtained after 24 hours of culture. The combination of 4OOH-IFA and hyperthermia mediated an overaddictive caspase stimulation directly following the heating phase. At this time also an overaddictive cytotoxic effect was noticed, being mainly responsible for the enhancing effects of hyperthermia on 4OOH-IFA cytotoxicity. In conclusion, hyperthermia enhanced the cytotoxic effect of 4OOH-IFA on CEM cells by stimulation of an early 4OOH-IFA effect. Thus, thermochemotherapy might be considered as an intensifying treatment option in relapsed T cell leukemias.

Regulation of interleukin-2 induced soluble Fas ligand release from human peripheral blood mononuclear cells.

Adjuvant interleukin (IL)-2 immunotherapy has been used in the treatment of different malignant dieseases. However, clinical results have been rather disappointing. Therefore, further investigations on IL-2-induced mediators of cytotoxicity seem to be necessary in order to possibly create cytokine cocktails which could enhance the IL-2-induced cytotoxicity. We therefore investigated the regulation of IL-2-induced release of soluble Fas Ligand (sFasL), since this factor is known to possess anti-tumor activities. In CD3-stimulated peripheral blood mononuclear cells IL-2 induced sFasL in a dose-dependent fashion. Maximum sFasL concentrations were obtained after stimulation of MNC for 120 hrs. Inhibition of endogenous IL-12 production significantly reduced IL-2-mediated sFasL release by about 25%. In contrast, addition of IL-12 enhanced the IL-2-induced sFasL about 1,5-fold. IL-10 and IL-4 reduced the IL-2-stimulated sFasL by about 30%. Interestingly, these suppressive effects could be antagonized by the addition of IL-12. Not only exogenous IL-10 but also endogenously produced IL-10 decreased the sFasL release to that extent which had been stimulated by IL-12. Since IL-12 and IL-10 only marginally influenced the IL-2-mediated cell proliferation as well as the IL-2-induced cell death, the IL-12- and IL-10-controlled sFasL release seems to be based on an enhanced production per cell. However, the increase in cell numbers as well as the decrease of viability during cell culture might additionally contribute to the IL-2-induced increase of sFasL release. This secondary effect might explain why IL-2-mediated sFasL production is only partially controlled by regulatory cytokines such as IL-4, IL-10 or IL-12. In conclusion, addition of IL-12 might increase the efficacy of IL-2 immunotherapy by inhibition of the IL-10-mediated negative feed-back loop on IL-2-mediated sFasL release.

IGFs, basic FGF, and glucose modulate proliferation and apoptosis induced by IFNgamma but not by IL-1beta in rat INS-1E beta-cells.

We investigated the effects of glucose and beta-cell growth factors (IGF-I, IGF-II, bFGF) on growth and apoptosis in the presence and absence of apoptosis inducing cytokines (IFNgamma, Il-1beta, TNFalpha). Rat INS-1E beta-cell viability was measured by WST-1 viability assay and cell counting, apoptosis by FACS analysis of annexin-V-FITC and fluorescein-dUTP (TUNEL-staining)-positive cells. Glucose alone maintained INS-1E beta-cell viability at high physiological concentrations (6.2-12.5 mmol/l), addition of IGF-II alone or in combination with bFGF further increased these glucose effects. The cytokines IFNg and IL-1beta, but not TNFalpha strongly induced INS-1E beta-cell apoptosis. Interestingly, glucose alone induced apoptosis at extremely low or very high concentrations. In combination with IFNg, low glucose (1.6 mmol/l) increased apoptosis by 25.6% (1SD 5.0%) and high glucose (50 mmol/l) by 22.8% (1SD 2.8%) compared to 12.5 mmol/l glucose. In contrast, glucose failed to modulate IL-1beta-induced apoptosis. Most importantly, IGF-II and bFGF inhibited apoptosis induced by IFNg, but not by IL-1beta. Therefore, IGF signaling, supported by bFGF and optimal glucose levels, maintains beta-cell viability in vitro. Cytokines IFNg and IL-1beta differentially interfere with intracellular signaling cascades stimulated by IGFs and bFGF or glucose, respectively.

Heat- and 4-hydroperoxy-ifosfamide-induced apoptosis in B cell precursor leukaemias.

In the group of high risk childhood acute lymphoblastic leukaemia (ALL), very early and early relapses have a very poor prognosis with conventional chemotherapy alone. Remission induction in these patients is often hindered by drug resistance. Thus, intensifying chemotherapy strategies are required. Application of hyperthermia enhances efficacy of certain anti-neoplastic drugs such as ifosfamide. In this study, effects and molecular mechanisms of ifosfamide - and hyperthermia-induced apoptosis are investigated in a B cell precursor leukaemia cell line (REH) and in primary patient-derived B cell progenitor leukaemic blasts. Both 4OOH-IFA and hyperthermia are able to induce cell death in leukaemic cells, mainly by induction of caspase-dependent apoptosis. However, completely different kinetics of caspase-3, -8 and -9 activation are found for both stimuli. In addition, activation of caspase-1 is only observed following stimulation with hyperthermia. Combined application of ifosfamide and hyperthermia reveals increased cytotoxicity in both the leukaemia cell line and in 5/8 of the patient-derived leukaemic blast samples. In conclusion, hyperthermia and ifosfamide mediate cytotoxicity in B precursor leukaemic blasts by different kinetics of caspase activation. This might explain the additive effects of 4OOH-IFA and heat on leukaemic cell death. Therefore, whole body thermochemotherapy could be considered as a treatment option in relapsed leukaemic patients.

Co-operation of IL-1 and IL-2 on T-cell activation in mononuclear cell cultures.

In search of an optimized anti-cancer immunotherapy, the combination of IL-2 and IL-1 has been tried. In an in-vitro LAK model, this cytokine cocktail seemed to be quite promising. In our in-vitro model of IL-2 induced T-cell activation we have therefore investigated the co-operation of these two potent immunostimulators. Mononuclear cells were stimulated with CD3 activating antibody in the presence of different cytokines and blocking or neutralizing antibodies. Cytokine concentrations were detected in the supernatants with ELISA. Intracellular IFN-gamma and IL-4 in the different T-cell subsets was measured by flow cytometry. IL-1 and IL-1 receptor antagonist (IL-1Ra) were up-regulated by IL-2, this was achieved independently of IL-12 or CD40/CD40L interaction. As a negative feedback mechanism, IL-1beta induced its natural antagonist, IL-1Ra. Both endogenous and exogenous IL-10 suppressed IL-1beta and induced IL-1Ra, thus markedly decreased the amount of functional IL-1. The combination of IL-2 and IL-1beta lead to a mildly increased Interferon-gamma (IFN-gamma) secretion (+20%, p < 0.05), however, this appeared to be the result of an increased IFN-gamma production per secreting cell, rather than of an increased recruitment of non-secreting cells. Similarly, IL-6 was also induced in an additive fashion (+30%, p < 0.05). For both cytokines, this effect could be significantly augmented by neutralizing IL-1Ra. Concentrations of IL-2 induced IL-10 and soluble Fas ligand (sFasL) were not affected by IL-1beta. We were thus able to demonstrate that IL-1 relays its activity through different pathways than IL-2. Furthermore, we could show that the potentially synergistic action of IL-2 and IL-1 was hindered by the simultaneous induction of signficant amounts of IL-1Ra. From the latter findings we conclude that the combination of IL-2 and IL-1 for cytokine-induced anti-tumor activity may not, but a combination of IL-2 and anti-IL-1Ra might prove beneficial.

Release of the soluble interleukin-6 receptor from human T-cells.

The soluble Interleukin-6 receptor (sIL-6R) is capable of conferring the Interleukin-6 (IL-6) signal onto cells lacking the gp80 ligand binding protein. Here we investigate the release of sIL-6R from T-cells. After 2 h stimulation with PMA, a release of sIL-6R from peripheral human T-cells was observed which was insensitive to the protein synthesis inhibitor cycloheximide. This release was accompanied by a decrease of membrane-bound (mb) IL-6R. After 24 h, however, the observed sIL6-R release did prove to be sensitive to cycloheximide. These results suggest that both shedding and denovo-synthesis may be responsible for the PMA-induced sIL-6R release. In contrast to PMA, neither anti-CD3, a positive, nor IL-10, a negative regulator of IL-6 release from T-cells affected the production of the sIL-6R. The differential regulation of sIL-6R and IL-6 production by T-cells might be relevant for the immunomodulatory potential of the sIL-6R with respect to the interaction of T- and non-T-cells.

Interleukin 10 inhibits TNF-alpha production in human monocytes independently of interleukin 12 and interleukin 1 beta.

Previously we demonstrated that endogenously produced Interleukin (IL-)10 suppressed the production of tumor necrosis factor-alpha (TNF-alpha) in CD3 activated T-cells via down-regulation of paracrine IL-12 secretion from APC. Here we investigated the effect of endogenous IL-10 on TNF-alpha production in purified lipopolysaccharide (LPS) stimulated monocytes and its mechanism. Similarly to its effects on T-cells, IL-10 inhibited monocyte TNF-alpha production by about half. Unlike in T-cells, however, this effect was not mediated via IL-12. While blockade of endogenous IL-10 binding to the IL-10 receptor enhanced the autocrine production of TNF-alpha, IL-12 and IL-1 beta, the neutralization of IL-12 or IL-1 beta did not affect the IL-10 effects on TNF-alpha production. This suggests that despite its inhibitory effects on IL-12 and IL-1 beta, which is quite similarly observed in T-cells, in purified monocytes IL-10 does not effect its TNF-alpha suppression by this mechanism. These findings indicate that IL-10 regulates production of pro-inflammatory cytokines by distinct mechanisms in different cells and tissues. Our study thus adds to the appreciation of the complex cytokine regulation of the immune system.

Additive effects of infection and neutropenia on the induction of granulocytopoietic activity in vivo.

BACKGROUND: Granulocyte-colony stimulating factor (G-CSF) is a potent stimulator of granulocytopoiesis and granulocyte function. It has been used in the treatment of children with neutropenic infection; in this context, it was expected to shorten aplasia and limit the severity of infection. Clinical trials, however, have demonstrated conflicting results as to whether these aims can be met. Recently, the use of other, less lineage specific growth factors, such as interleukin (IL)-11 and stem cell factor (SCF), has also been discussed. The dynamics of growth factors and growth factor-regulating proteins during neutropenic infection, particularly in youngsters, are not well understood. METHODS: Serial blood samples from children and adolescents with infection during chemotherapy-induced neutropenia were assayed for C-reactive protein, white blood cell count, IL-11, SCF, G-CSF, IL-10, IL-4, IL-lalpha, and IL-1beta. RESULTS: Although no correlation could be demonstrated between endogenous IL-11 or SCF levels, infection, and leukocyte counts, endogenous G-CSF levels were increased during both aplasia and infection. However, only the additive effects of infection and neutropenia led to maximally stimulated endogenous G-CSF levels. CONCLUSIONS: The elevated G-CSF levels in a majority of patients during severe neutropenic infection may explain why a therapeutic benefit of G-CSF treatment cannot be demonstrated in all cases. There remains, however, a subgroup of patients in whom infection and cytopenia do not yield a good G-CSF response. This latter group should be identified, because they might derive some benefit from adjuvant growth factor therapy. The authors predict that the efficacy of G-CSF in the treatment of patients with neutropenic fever might depend on each individual's ability to initiate the necessary cytokine production.

Cytokine-mediated regulation of granulocyte colony-stimulating factor production.

Granulocyte colony-stimulating factor (G-CSF) is an important regulator of granulopoiesis and an inducer of T helper 2 (Th2)-related cytokines. In this study we investigated the mechanism of cytokine-modulated G-CSF production in lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMC) and bone marrow stromal cells (BMSC). In PBMC, LPS significantly induced G-CSF and interleukin (IL)-1, an inducer of G-CSF. Both were partly inhibited by IL-4, IL-6 and IL-10. None of these effects were the result of altered monocyte activation or proliferation. The effects of IL-4 and IL-10 appeared to be independent of IL-1 suppression or IL-1 receptor antagonist (IL-1ra) induction, but rather seemed to involve a blockade of IL-1 function, in addition to a blockade of IL-1-independent stimulatory effects on G-CSF production. The effect of the IL-6-induced suppression of G-CSF production differed from that of IL-4 and IL-10 in that it was much less pronounced and could be partially overridden by addition of functional IL-1, yet it also appeared to involve the interference with IL-1 function and the suppression of IL-1-independent mechanisms. In BMSC, G-CSF synthesis was regulated differently. Here, IL-1 was the main stimulator of G-CSF release, and the effect of IL-1 was neither affected by IL-10 nor IL-6, while IL-4 had a stimulatory effect. Thus, G-CSF production was found to be differently regulated in distinct cellular compartments, and a cross-regulation between these might be facilitated by IL-4-, IL-6- and IL-10-induced inhibition of IL-1. These results could be important for the understanding of G-CSF production in neutropenic patients during severe infection.

Regulation of interleukin-2 induced interleukin-5 and interleukin-13 production in human peripheral blood mononuclear cells.

Adjuvant interleukin (IL)-2 immunotherapy has been used for many years for a variety of malignant and nonmalignant entities. In many cases, a dose escalation might have seemed desirable, but was prevented by the rather severe adverse effects of systemic IL-2 application. Only recently has the regulation of IL-2 induced cytotoxicity been understood better, so that now efforts can be aimed at the design of cytokine cocktails that would selectively induce cytotoxicity but result in as few adverse effects as possible. Previously, induction of IL-5 under systemic IL-2 therapy has been described, and a number of the side-effects have been attributed to this event. We therefore investigated the regulation of IL-2 induced production of IL-5 and IL-13 (which, similarly to IL-5, is a mediator of allergy-like symptoms). At the same time, the effects of regulatory cytokines, such as IL-4, IL-10 and IL-12, on interferon-gamma (IFN-gamma), the major cytotoxic mediator of IL-2 therapy, were studied. All three have been discussed as antitumour immunotherapeutics, either alone or in combination with IL-2. In anti-CD3-treated peripheral blood mononuclear cells, IL-2 induced IL-5 and IL-13 alongside IFN-gamma, IL-10 and IL-12. In the presence of IL-2, inhibition of endogenous IL-12 production further enhanced the IL-5 and IL-13 responses, while IFN-gamma and IL-10 were markedly suppressed. Co-incubation with IL-2 and IL-12 suppressed IL-5/IL-13 below, but enhanced IFN-gamma and IL-10 above, levels induced by IL-2 alone. IL-10 was suppressive on all the investigated cytokines, while IL-4 interfered with IL-2 induced IFN-gamma and IL-12 production, but was additive to IL-2 in its effect on IL-5 and IL-13. These data suggest that the combination of IL-12 with IL-2 would enhance the cytotoxic activity of this regimen, but might reduce its adverse effects.

Transforming growth factor-beta1 suppresses interleukin-15-mediated interferon-gamma production in human T lymphocytes.

One of the most remarkable means by which tumour cells manage to evade recognition and elimination by the immune system is the release of immunosuppressive mediators, such as interleukin (IL)-10 or transforming growth factor-beta (TGF-beta). For antitumour immunotherapies to reach their full potential, cytokine cocktails will have to be custom-tailored to the tumour's individual cytokine microenvironment. One of the components of such a cytokine cocktail may be interleukin (IL)-15, which has demonstrated an excellent stimulatory potential of antitumour immunity. In an in vitro model, we have previously been able to show that the negative effects of IL-10 on IL-15-mediated cytotoxic T-cell activation can be outweighed by the addition of interleukin (IL)-12. The mechanism by which TGF-beta may influence the effect of IL-15 remains poorly understood, however. We have therefore taken our T-cell model further and have studied the effect of TGF-beta on IL-15-mediated interferon-gamma (IFN-gamma) production. In activated, IL-15-stimulated peripheral blood T lymphocytes, TGF-beta suppressed IFN-gamma mRNA and protein levels by approximately 75%. This effect was likewise observed on both CD4+ and CD8+ T cells and, in contrast to the effect of IL-10 in this system, could not be neutralized by the addition of IL-12. Thus, immunotherapy for TGF-beta-producing tumours may benefit from the addition of TGF-neutralizing activity rather than IL-12.

Systemic activation of the immune system during ganciclovir treatment following intratumoral herpes simplex virus type 1 thymidine kinase gene transfer in an adolescent ependymoma patient.

During ganciclovir treatment of an adolescent ependymoma patient two weeks after intracranial implantation of HSVtk retroviral vector producer cells, increasing numbers of peripheral T- and B-cells were found as well as enhanced T-cell activation and elevated serum levels of interleukin 12 and soluble Fas ligand. These findings suggest the systemic activation of the immune system during ganciclovir treatment in our patient. The induction of an immune response by HSVtk/ganciclovir supports the concept of an anti-tumor vaccination effect by prodrug activating gene therapy systems and may open new promising perspectives for enhancing therapeutic efficiency by combined prodrug activating and immunological gene therapy strategies.

Endogenous pro-inflammatory cytokines in children and adolescents during chemotherapy-induced neutropenia.

Monocyte-derived pro-inflammatory cytokines such as GM-CSF, IL-12, and IP-10 might protect patients with chemotherapy-induced neutropenia against infections. In settings with abundant neutrophils, G-CSF has been described as a suppressor of IL-12, but also as an inducer of GM-CSF. In 25 pediatric patients with chemotherapy-induced neutropenia the authors measured plasma levels of these four cytokines. GM-CSF was detectable in only a minority of patients (6/25). It was, however, positively correlated with high plasma levels of IL-12 and IP-10. G-CSF levels, however, were in no way correlated with the levels of any of these three cytokines.

The role of interleukin-10 (IL-10) in IL-15-mediated T-cell responses.

Interleukin-15 (IL-15) is a potent T-cell stimulating factor, which has recently been used for pre-clinical in vivo immunotherapy. Here, the IL-15 effect on CD3-stimulated peripheral human T cells was investigated. IL-15 induced a significant T-cell proliferation and upregulated CD25 expression. IL-15 significantly enhanced T-cell production of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and IL-10. Between 10- and 100-fold greater concentrations of IL-15 were necessary to reach a biological effect equivalent to that of IL-2. Blockade of IL-2 binding to the high-affinity IL-2 receptor did not affect the IL-15 effects, suggesting that IL-15 did not act by inducing endogenous IL-2. Exogenously administered IL-10 significantly reduced the IL-15 and IL-2-mediated IFN-gamma and TNF-alpha production, whereas T-cell proliferation and CD25 expression were not affected. The inhibitory effects of exogenously administered IL-10 on T-cell cytokine production appeared indirect, and are likely secondary to decreased IL-12 production by accessory cells. Inhibition of endogenous IL-10 binding to the IL-10 receptor significantly increased IFN-gamma and TNF-alpha release from T cells. These data suggest that endogenous IL-10 can regulate activated T-cell production of IFN-gamma and TNF-alpha via a paracrine negative feedback loop. The observations of this study could be of relevance for the therapeutic use of IL-15 in vivo.

Immune response induced by retrovirus-mediated HSV-tk/GCV pharmacogene therapy in patients with glioblastoma multiforme.

This study was conducted to investigate immunological components of somatic gene therapy for primary glioblastoma multiforme (GBM) in adults. It involved 13 patients treated by surgical resection of tumor with subsequent radiation therapy. Seven of them received additional herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) gene therapy by direct intracerebral injection of retrovirus (RV) vector producing cells (VPC) during tumor surgery and subsequent systemic administration of GCV. Peripheral blood for FACS immunophenotyping, isolation of peripheral mononuclear cells (PMNC), and serum ELISA assays for IL-12 and soluble Fas ligand (sFasL) was collected daily during the first 4 post-operative weeks. Tumor specimens were obtained at primary or recurrent surgery and at autopsy. Tumors from gene therapy patients showed varying degrees of peritumoral necrosis around the former tumor resection cavity. Numbers of tumor-infiltrating lymphocytes found weeks after gene therapy were not significantly increased compared with primary tumors. Mitotic tumor cells were sparse close to the VPC injection sites, but abundant in brain areas somewhat distant from these sites. Serum ELISA revealed significantly increased sFasL and IL-12 levels in the gene therapy group compared with controls. Immunophenotyping of PMNC did not show a significant activation of T cells or NK cells during gene therapy. Interferon gamma secretion was evaluated by ELISPOT assays employing PMNC cocultivated with autologous tumor cells. It demonstrated an antitumor immune response in the gene therapy group, but not in the control group. These findings support the concept of in vivo induction of a systemic immune response by local intracerebral HSV-tk/GCV pharmacogene therapy for primary human GBM.