Generation and functional characterisation of dendritic cells from patients with pancreatic carcinoma with special regard to clinical applicability

Background: We studied dendritic cell (DC) function in patients affected by pancreatic carcinoma, and the possibility of obtaining DC adequate for immunological treatment modalities. Methods: Leucocytes were isolated from buffy coats obtained by autotransfusion of six patients undergoing pancreatico-duodenectomy. The leucocytes were cryopreserved and, after thawing, were purified by density gradient and/or plastic adhesion. They were then cultured in vitro in cytokine-enriched medium (granulocyte/macrophage-colony-stimulating factor + interleukin-4) with different sources of serum: 10% fetal calf serum (FCS), 2% autologous human serum or 2% pooled human AB serum. Results: The DC obtained were identical to those from healthy donors in terms of phenotype, antigen uptake capacity, capacity for antigen presentation and their capacity to mature after exposure to stimuli like CD40L. DC differentiated in human serum demonstrated more mature behaviour than did DC cultured in FCS but, after exposure to CD40L, this difference disappeared. In one patient soluble factors in serum were able to inhibit the capacity of DC to stimulate T cells. Conclusion: It's possible to obtain DC from autotransfusion of patients with pancreatic carcinoma: these cells do not show evident quantitative or qualitative alterations, are able to present soluble antigen even when cultured in the presence of human serum and may be used in immunological tumour treatments. Received: 18 May 2000 / Accepted: 27 July 2000     

Potentiated autologous tumor cell and peripheral blood lymphocyte lysate vaccination

Immune stimulation is a promising prospect in cancer therapy. Immunotherapy may be local or systemic, aspecific or targeted and may use monoclonal antibodies or vaccines. The aim of using vaccines is to stimulate the body to produce its own antibodies. Autologous tumor-cell vaccination has no contraindications or side-effects, since the patients own materials (lymphocytes, tumor cells) are used. We describe a method for producing an autologous cancer vaccine. The material to be injected as a vaccine derives from a mixed culture of autologous lymphocytes cocultured with autologous cancer cells. Peripheral blood lymphocytes are obtained by lymphocytapheresis. Cancer cells may be obtained from tissue biopsies or biological fluids, or from long-term cultures from the patient who is to be vaccinated. The culture medium (RPMI 1640) is free of fetal calf serum (FCS). The coculture is mixed with autologous plasma in a 1:1 ratio with the addition of 200 IU of recombinant human interleukin-2/mL, and is incubated at 37 degrees C in a humidified 5% CO2-enriched atmosphere for 48 h. The cocultured material is frozen, thawed to lyse cells, aliquoted and stored at -20 degrees C.     

Immunogenicity of tumor peptides: importance of peptide length and stability of peptide/MHC class II complex

Nonameric P815AB, a cytotoxic-T-lymphocyte-defined minimal core peptide encoded by the murine mastocytoma gene P1A, fails to initiate CD4⁺ cell-dependent reactivity in vivo to class-I-restricted epitopes when mice are administered peptide-pulsed dendritic cells. Effective immunization requires T helper effects, such as those mediated by coimmunization with class-II-restricted (helper) peptides or by the use of recombinant interleukin-12 (rIL-12). Although P815AB does possess class-II-restricted epitopes, they are likely suboptimal, resulting in poor affinity and/or stability of MHC/P815AB complexes and inadequate activation of the antigen-presenting cell function of dendritic cells. The present study has examined a series of longer, P815AB-centered peptides (11–14 amino acids in length, all P1A-encoded) for their ability to initiate CD4⁺ and CD8⁺ cell-mediated responses to the nonamer in vivo, their ability to bind class II MHC in vitro, and their ability to assemble class II molecules stably. By means of a class-I-restricted skin test assay in mice receiving peptide-pulsed dendritic cells, we found that a 12-mer and a 13-mer effectively immunized against the core P815AB peptide, and that this correlated with IL-2 production in vitro by CD4⁺ cells in response to the nonamer. In vitro studies, involving affinity-purified class II molecules, showed that the capacity to assemble class II molecules stably, more than the affinity for class II MHC, correlated with the ability of the different P815AB peptides to prime the host to the core peptide seen by the T cells. Received: 25 February 1999 / Accepted: 14 April 1999     

Messenger RNA electroporation of human monocytes,followed by rapid in vitro differentiation,leads to highly stimulatory antigen-loaded mature dendritic cells

Dendritic cells (DC) are professional Ag-capturing and -presenting cells of the immune system. Because of their exceptional capability of activating tumor-specific T cells, cancer vaccination research is now shifting toward the formulation of a clinical human DC vaccine. We developed a short term and serum-free culture protocol for rapid generation of fully mature, viable, and highly stimulatory CD83(+) DC. Human monocytes were cultured for 24 h in serum-free AIM-V medium, followed by 24-h maturation by polyriboinosinic polyribocytidylic acid (polyI:C). Short term cultured, polyI:C-maturated DC, far more than immature DC, showed typical mature DC markers and high allogeneic stimulatory capacity and had high autologous stimulatory capacity in an influenza model system using peptide-pulsed DC. Electroporation of mRNA as an Ag-loading strategy in these cells was optimized using mRNA encoding the enhanced green fluorescent protein (EGFP). Monocytes electroporated with EGFP mRNA, followed by short term, serum-free differentiation to mature DC, had a phenotype of DC, and all showed positive EGFP fluorescence. Influenza matrix protein mRNA-electroporated monocytes cultured serum-free and maturated with polyI:C showed high stimulatory capacity in autologous T cell activation experiments. In conclusion, the present short term and serum-free ex vivo DC culture protocol in combination with mRNA electroporation at the monocyte stage imply an important reduction in time and consumables for preparation of Ag-loaded mature DC compared with classical DC culture protocols and might find application in clinical immunotherapy settings.     

A low-serum medium with BSA and ferrous sulfate for the production of tissue plasminogen activator by human embryo lung cells

A low-serum medium containing bovine serum albumin (BSA) was investigated with respect to the growth of and tissue plasminogen activator (TPA) production by human embryo lung (HEL) cells on microcarrier beads and in collagen gel. BSA and ferrous sulfate were chosen as substitutes for fetal calf serum (FCS) through a simple screening test involving many substances. The growth promoting effects of BSA and ferrous sulfate were independent of each other and from the FCS concentration. Though BSA inhibited initial cell attachment to the carrier surface, it did promote the growth of cells attached to microcarrier beads. Cells grown on microcarrier beads in the low-serum medium containing BSA, ferrous sulfate and 3% FCS produced an amount of TPA similar to that produced by ones grown in the 10% FCS medium. Although cells on the dish surface did not grow at all on serum-free media containing BSA and ferrous sulfate, cells in the collagen gel were able to grow slightly on the serum-free medium. Cells grown on the low-serum medium in collagen gel produced more TPA over a long period than those in the microcarrier beads using the low-serum medium. The optimum concentration of proteose peptone in the TPA production medium for the collagen gel culture was similar to that for the dish surface culture.     

Ablation of eosinophil and IgE responses with anti-IL-5 or anti-IL-4 antibodies fails to affect immunity against Schistosoma mansoni in the mouse

To investigate the role of anaphylactic immune responses in protective immunity against schistosomiasis, mice vaccinated with irradiated cercariae of Schistosoma mansoni were treated with neutralizing mAb antibodies against either IL-5 or IL-4 before and during challenge infection. Anti-IL-5-treated vaccinated mice showed a complete ablation of circulating as well as tissue eosinophils present in inflammatory reactions to migrating schistosomula in the skin and lungs but nevertheless eliminated challenge infections as effectively as vaccinated animals treated with a control mAb. Similarly, treatment of vaccinated mice with an anti-IL-4 mAb markedly reduced serum IgE although failing to diminish immunity. The effect of anti-IL-5 mediated eosinophil depletion was also assessed in a second model in which resistance is induced by concomitant chronic infection. Again, normal, unaltered protection was observed in the absence of circulating and tissue eosinophils. In contrast to the above findings, treatment with anti-IFN-gamma was found to cause a partial depletion of immunity in vaccinated mice whereas, paradoxically, increasing the numbers of inflammatory reactions against invading schistosomula in the lungs. These observations argue against a requirement for either eosinophils or IgE in the anti-schistosome immunity induced by vaccination with irradiated cercariae or for eosinophils in the resistance resulting from previous infection in mice and support previous data suggesting a role for an IFN-gamma dependent cell-mediated effector mechanism in vaccine-induced resistance.     

Differential recognition of tumor-derived and in vitro Epstein-Barr virus-transformed B-cell lines by fetal calf serum-specific T4-positive cytotoxic T-lymphocyte clones

Two interleukin-2 (IL-2)-dependent cytotoxic T-cell clones were obtained by limiting dilution from a lymphocyte culture stimulated in vitro with the autologous Epstein-Barr virus-transformed lymphoblastoid cell line (LCL) in the presence of fetal calf serum (FCS). Both clones uniformly had a T3+, T4+, Dr+ phenotype and lysed autologous B blasts, the autologous LCL, and allogeneic B cell lines sharing major histocompatibility complex (MHC) class II antigens. The cytotoxic function was triggered by FCS-derived components. There was no killing if the sensitive targets were cultured in serum-free medium or in medium supplemented with human serum. Sensitivity to lysis could be restored by exposing the targets to FCS for at least 6 hr at 37 degrees C. Monoclonal antibodies directed to T-cell-specific surface antigens and MHC class II antigens inhibited lysis with different efficiencies depending on the target cell origin. Killing of Burkitt's lymphoma (BL)-derived cell lines was blocked more easily than killing of LCLs. LCLs but not BL lines induced proliferation of the T-cell clones in the absence of exogenous IL-2. The differences were not related to quantitative variations in the expression of MHC class II antigens, indicating that BL lines differ from LCLs in other cell membrane properties that may influence antigen presentation. The results suggest that the affinity of effector/target binding, which is probably influenced by the concentration of antigenic determinants expressed on the target cell membrane, determines whether proliferative responses or cytotoxicity are induced in the antigen-recognizing T cells.     

Autoantibodies to thyroid gland antigens in chronic relapsing urticaria

The content of total IgE, antibodies to thyroglobulin (TG-Ab), antibodies to thyreoid peroxidase (TPO-Ab) in the blood serum and skin reaction to autologous serum were detected in patients with chronic relapsing urticaria (CRU). The skin test to autologous serum yielded positive results in 42% of the patients. The elevated levels of TG-Ab and TPO-Ab were detected in 30.7% and 35.4% of the patients, the elevated level of total IgE was detected in 60% of the patients. At the same time the detection rates of antithyreoid antibodies and the elevated level of IgE were not linked with skin reaction to autologous serum. Apparently, in addition to autoantibodies to IgE or its receptor (the positive skin test to autologous serum), thyroid gland antibodies may take part in the mechanism of the CRU formation.     

Differential patterns of blastulation in bovine morulae cultured in synthetic oviduct fluid medium containing FCS or BSA

This study examined the effects of fetal calf serum (FCS) supplementation of culture medium on blastulation and hatching of bovine morulae cultured in vitro. The presumptive zygotes derived from in vitro maturation and fertilization (IVM/IVF) were cultured in the modified synthetic oviduct fluid medium containing 3 mg/ml BSA (mSOF-BSA). At 120 h post insemination, morulae were randomly assigned to culture with mSOF-BSA (control) or mSOF containing 5% FCS (mSOF-FCS) instead of BSA. The replacement of BSA with FCS in mSOF significantly increased the percentage of blastocyst formation from Day 6 to Day 10 (Day 0 = the day of in vitro insemination) and the hatching rate of embryos on Days 8 and 9. The total number of cells in morulae and blastocysts on Day 6, in blastocysts on Day 7, and in blastocysts and hatched blastocysts on Day 8 were similar among the treatments. However, the replacement of BSA with FCS in mSOF significantly increased the total number of cells in hatched blastocysts on Day 10. Although the time of blastulation of embryos was significantly accelerated by the replacement of BSA with FCS in mSOF, the total number of cells in embryos at blastulation was lowered. The total number of cells in embryos at blastulation showed a time-dependent decrease when the embryos were cultured in mSOF-BSA. In contrast, the total number of cells in embryos that were cultured in mSOF-FCS depended little on the time after in vitro insemination. The results indicate that FCS supplementation of culture medium increased the percentage of embryos developing to the blastocyst stage without an increase in the total number of cells. However, an acceleration in the hatching rate and an increase in the total number of cells in hatched blastocysts were observed, compared with that in BSA-supplemented medium. It is suggested that FCS in the culture medium initiates earlier blastulation with fewer total numbers of cells in the morulae than BSA during in vitro culture of bovine embryos.     

Fetal calf serum enhances in vitro production of Bos taurus indicus embryos

The objective of this study was to determine the effect of fetal calf serum (FCS) on the quality of in vitro produced bovine embryos. Cumulus oocyte-complexes (COCs, n = 2 449) recovered by ovum pick-up from Bos taurus indicus donors were randomly assigned to experimental groups. Sperm selected by Percoll gradient was used for in vitro fertilization (insemination = Day 0). In Experiment 1 (n = 1 745 COCs), zygotes were cultured in vitro in Synthetic Oviduct Fluid + 4 mg/mL of bovine serum albumin (BSA), or BSA + 2% FCS (BSA+FCS). In Experiment 2 (n = 704 COCs), the COCs were cultured in SOF + BSA, BSA + 2% FCS, or BSA + 2% FCS on D4 (BSA + FCSD4). In Experiment 1, blastocyst yield (51%) and Quality I blastocysts (41%) at Day 7 were higher (P < 0.05) in the BSA + FCS treatment than in BSA (42 and 30%, respectively). In Experiment 2, blastocyst yield was higher (P < 0.05) in the BSA+FCS (47%) treatment. Quality I blastocyst yield was higher (P < 0.05) for BSA + FCS (34%) and BSA+FCSD4 (32%) compared to the BSA treatment (20%). A total of 820 embryos were transferred, with no significant differences among groups in pregnancy rates. In conclusion, in vitro culture in SOFaaci + BSA + FCS enhanced blastocyst yield and Quality I blastocysts; adding FCS to the culture medium increased the efficiency of IVP of bovine embryos.     

Cellular control of IgE induction by a polyphenol-rich compound. Preferential activation of Th2 cells

The polyphenol group rutin (R) appears to influence isotype expression, because R-BSA conjugates induce anti-BSA responses in mice that show a significant decrease in hemagglutinating antibodies (HA) to BSA, as compared to mice immunized with BSA. However, the level of IgE antibodies to BSA is unaltered. To determine if suppressor cells for isotypes other than IgE are induced by R-BSA, cell transfers were performed. The results were consistent with the view that the decrease in HA titer to BSA in R-BSA immunized mice is not due to the activation of suppressor cells for isotypes other than IgE. Inasmuch as the IgE response in mice is associated with the production of IL-4 by Th2 cells, we analyzed the factors produced by spleen cells cultured with R-BSA. We found that supernatant from spleen cells cultured with R-BSA contained IL-4 as determined by the enhanced expression of Fc epsilon R (CD23) on B cells. This enhancement was inhibited by 11B11, the anti-IL-4 mAb. IL-2, a product of Th1 cells, was not detected in these supernatants. Moreover, IL-4 mRNA, but not IL-2 mRNA, was detected by Northern blot analysis of RNA from spleen cells cultured with R-BSA. Taken together the data suggest that the polyphenol containing compounds preferentially activate Th2 cells, thereby favoring IgE production.     

Mechanisms of protective immunity against Schistosoma mansoni infection in mice vaccinated with irradiated cercariae. IV. Analysis of the role of IgE antibodies and mast cells

Mice resistant to challenge infection with Schistosoma mansoni by vaccination with highly irradiated cercariae were examined for the presence of circulating IgE antibodies and peritoneal mast cells sensitized against schistosome antigens. Significant levels of SWAP- or CAP-specific IgE antibodies could not be detected by solid phase radioimmunoassay in the sera of C57BL/6 mice during the first 6 wk after vaccination. Similarly, heatlabile antibodies capable of passively sensitizing normal mast cells for degranulation in response to SWAP could not be identified in the same sera. In contrast, peritoneal mast cells harvested from C57BL/6 mice 2 wk or later after vaccination gave strong degranulation responses when challenged with SWAP or CAP. Thus, vaccination with irradiated cercariae induces an unusual form of immediate-type hypersensitivity in which mast cells become sensitized in the absence of detectable circulating IgE antibodies. Mice deficient in mast cells (W/Wv mutant strain) were observed to develop the same resistance to challenge infection after vaccination with irradiated cercariae as nondeficient littermates. Similarly, vaccinated SJL/J mice were found to mount an extremely weak IgE response as measured by mast cell degranulation yet displayed the same level of resistance to challenge infection as other inbred mice developing potent mast cell responses. These findings argue that IgE antibodies and mast cells are not essential components in the effector mechanism of irradiated vaccine-induced immunity against schistosome infection.     

Human glioma cell culture: two FCS-free media could be recommended for clinical use in immunotherapy

Immunotherapy, particularly active vaccination, may be developed as an effective and safe treatment modality for malignant gliomas, which continue to have a poor prognosis, despite advances in surgical techniques and adjuvant chemotherapy and radiotherapy. Since no glioma-specific tumor-associated antigens (TAAs) have been discovered, autologous tumor cells or well-established glioma cell lines could be used in future vaccination protocols to induce antitumour immunity against unknown TAAs. One obstacle for clinical use of these tumour cell vaccines is related to foetal calf serum (FCS). Efforts are currently being directed toward developing FCS-free media and serum-free alternatives to culture these cell vaccines. In this study, a medium containing human serum and one serum-free medium (UltraCulture), supplemented or not with epidermal growth factor, were tested on morphology, survival, DNA content and TAA expression of human glioma cell lines and glioma biopsy primary cultures. Their effects were compared on FCS-containing medium. Results show that, whatever the medium used, no significant variations in morphology and survival were observed. Furthermore, human serum-containing medium or UltraCulture preserved at early passage cultures the cell population of interest present in the biopsies before culture. In addition, the expression profile of eight TAAs was similar between these media. These data indicate that human serum-containing medium and UltraCulture serum-free medium could be promising candidates to produce tumour-cell vaccines.     

Spontaneous and vaccine induced AFP-specific T cell phenotypes in subjects with AFP-positive hepatocellular cancer

We are investigating the use of Alpha Fetoprotein (AFP) as a tumor rejection antigen for hepatocellular carcinoma (HCC). We recently completed vaccination of 10 AFP+/HLA-A2.1+ HCC subjects with AFP peptide-pulsed autologous dendritic cells (DC). There were increased frequencies of circulating AFP-specific T cells and of IFNγ-producing AFP-specific T cells after vaccination. In order to better understand the lack of association between immune response and clinical response, we have examined additional aspects of the AFP immune response in patients. Here, we have characterized the cell surface phenotype of circulating AFP tetramer-positive CD8 T cells and assessed AFP-specific CD4 function. Before vaccination, HCC subjects had increased frequencies of circulating AFP-specific CD8 T cells with a range of naïve, effector, central and effector memory phenotypes. Several patients had up-regulated activation markers. A subset of patients was assessed for phenotypic changes pre- and post-vaccination, and evidence for complete differentiation to effector or memory phenotype was lacking. CD8 phenotypic and cytokine responses did not correlate with level of patient serum AFP antigen (between 74 and 463,040 ng/ml). Assessment of CD4+ T cell responses by ELISPOT and multi-cytokine assay did not identify any spontaneous CD4 T cell responses to this secreted protein. These data indicate that there is an expanded pool of partially differentiated AFP-specific CD8 T cells in many of these HCC subjects, but that these cells are largely non-functional, and that a detectable CD4 T cell response to this secreted oncofetal antigen is lacking.     

Human autologous serum obtained using a completely closed bag system as a substitute for foetal calf serum in human mesenchymal stem cell cultures

The major problem in cell therapy is the possibility of viral or bacterial infection and immune reactions. Therefore, it is expected of culture cells which are intended to be re-implanted with autologous serum rather than conventional bovine serum. Cell therapy with human mesenchymal stem cells (hMSC), differentiating to various cells, is thought to be curative. To culture hMSC with human autologous serum (HAS) and re-implant them for cell therapy, we developed a completely closed bag system separating serum, comparing proliferation and multipotency of hMSC cultured in HAS with those in foetal calf serum (FCS). HAS was simply, safely and efficiently obtained with the developed closed bag system. Cell proliferation of hMSC cultured in HAS was greater than that in FCS. hMSC, exposed to the defined induction medium containing HAS as well as FCS, differentiated into osteoblasts and adipocytes. These findings suggest that HAS obtained with the developed closed bag system is advantageous in a point of decrease in risk of virus or bacterial infection and foreign protein contamination and enhancement of proliferation of hMSC.     

血清及BSA对牛体外受精胚胎发育过程超微结构影响的研究

本研究将牛IVF胚胎分别在SOF FCS、SOF BSA和SOF PVA三种培养系统内进行培养,然后分别取三个系统中发育到原核期、2细胞、4细胞、8细胞、桑椹胚和囊胚阶段的胚胎进行透射电镜的观察,了解培养系统中血清和BSA的添加与否对胚胎发育过程中细胞内脂滴、细胞连接、细胞凋亡和微绒毛发育的影响。观察结果表明：各培养系统胚胎的细胞质中均存在大量的脂滴,表明外培养系统是造成脂滴积累的主要原因;血清的添加不会进一步促进脂滴的大量积累,反而可以避免多个脂滴聚合成更大的脂滴。三种培养系统条件下胚胎细胞连接无显著差异。培养系统中添加FCS或BSA时,桑椹胚期以后的胚胎细胞中存在凋亡小体,表明血清成分是引起细胞凋亡的重要原因。培养系统中血清成分的缺乏会影响胚胎表面微绒毛的发育。     

Dendritic cell–tumor cell hybrid vaccination for metastatic cancer

Dendritic cells are the most potent antigen-presenting cells, and the possibility of their use for cancer vaccination has renewed the interest in this therapeutic modality. Nevertheless, the ideal immunization protocol with these cells has not been described yet. In this paper we describe the preliminary results of a protocol using autologous tumor and allogeneic dendritic hybrid cell vaccination every 6 weeks, for metastatic melanoma and renal cell carcinoma (RCC) patients. Thirty-five patients were enrolled between March 2001 and March 2003. Though all patients included presented with large tumor burdens and progressive diseases, 71% of them experienced stability after vaccination, with durations up to 19 months. Among RCC patients 3/22 (14%) presented objective responses. The median time to progression was 4 months for melanoma and 5.7 months for RCC patients; no significant untoward effects were noted. Furthermore, immune function, as evaluated by cutaneous delayed-type hypersensitivity reactions to recall antigens and by peripheral blood proliferative responses to tumor-specific and nonspecific stimuli, presented a clear tendency to recover in vaccinated patients. These data indicate that dendritic cell–tumor cell hybrid vaccination affects the natural history of advanced cancer and provide support for its study in less advanced patients, who should, more likely, benefit even more from this approach.     

Effects of fetuin on zona pellucida hardening,fertilization and embryo development in cattle

Mammalian oocytes can undergo spontaneous meiotic maturation when they are liberated from their follicles and cultured in vitro; however, the zona pellucida (ZP) becomes resistant to chymotrypsin digestion, or hardens, when spontaneous maturation occurs in serum-free medium. Schroeder et al. [Biol. Reprod. 43 (1990) 891] described that fetuin, a component of fetal calf serum (FCS), inhibits ZP hardening during oocyte maturation. The aim of this experiment was to study the effect of the presence of cumulus cells and addition of hormones to maturation media on bovine zona hardening and embryo development in medium with and without fetuin. In Experiment I, different concentrations of fetuin were added to the maturation medium. The time necessary for digestion of 50% of the ZP (d50) was not different when oocytes were matured in presence of 10% FCS, 1mg/ml polyvinyl alcohol (PVA), or 4, 1 and 0.25mg/ml of fetuin; cleavage rates were also similar. However, significantly more blastocysts (P<0.05) were formed when FCS was used compared to PVA and 0.25mg/ml of fetuin. In Experiment II, we examined the influence of the presence of cumulus cells and hormones during the maturation of oocytes in media with PVA, BSA, FCS and fetuin. The d50 was significantly higher (P<0.05) when oocytes were matured in presence of cumulus cells. The cleavage rate of cumulus-intact oocytes was similar for all groups. However, when oocytes were partially stripped before maturation, the cleavage rate was significantly higher (P<0.05) when FCS or fetuin was used. In both stripped and non-stripped groups, significantly more blastocysts (P<0.05) were formed when oocytes were matured with FCS compared to BSA and PVA. These results indicate that zona hardening, as described for mouse and human oocytes, does not have a large effect on bovine cumulus-intact oocytes. Apparently fetuin can be used as a substitute for FCS during bovine oocyte maturation, since it leads to similar developmental rates as FCS in intact and partially stripped oocytes.