Rationale for the Combination of Dendritic Cell-Based Vaccination Approaches With Chemotherapy Agents.

Owing to their central role in the initiation and regulation of antitumor immunity, dendritic cells (DCs) have been widely tested for use in cancer immunotherapy. Despite several encouraging clinical applications, existing DC-based immunotherapy efforts have yielded inconsistent results. Recent work has identified strategies that may allow for more potent DC-based vaccines, such as the combination with antitumor agents that have the potential to synergistically enhance DC functions. Selected cytotoxic agents may stimulate DCs either by directly promoting their maturation or through the induction of immunogenic tumor cell death. Moreover, they may support DC-induced adaptive immune responses by disrupting tumor-induced immunosuppressive mechanisms via selective depletion or inhibition of regulatory subsets, such as myeloid-derived suppressor cells and/or regulatory T cells (Tregs). Here, we summarize our current knowledge on the capacity of anticancer chemotherapeutics to modulate DC phenotype and functions and the results of ongoing clinical trials evaluating the use of DC-based immunotherapy in combination with chemotherapy in cancer patients.

[The Concept of Immunogenic Cell Death in Antitumor Immunity]. / Význam imunogenní bunecné smrti v protinádorové imunite.

Cancer cell death can be immunogenic or nonimmunogenic depending on the initiating stimulus. The immunogenic characteristics of immunogenic cell death are mainly mediated by damage-associated molecular patterns represented by preapoptotic exposure of calreticulin and heat shock proteins (HSP70 and HSP90) from endoplasmic reticulum at the cell surface and active secretion of adenosintriphospate. Other damage-associated molecular patterns are produced in late stage apoptosis as high mobility group box 1 protein (HMGB1) into the extracellular milieu. Such signals operate on various receptors expressed by antigen presenting cells, mainly by population of dendritic cells, to stimulate the activation of antigen specific T-cell response. In this review, we describe the current known immunogenic cell death inducers and their potential to activate antitumor immune response.

[Immune System in Patients with Head and Neck Carcinoma]. / Imunitní systém u pacientu s nádory hlavy a krku.

The insight into the biological nature of head and neck squamous cell carcinoma has evolved significantly in the last few years. Tobacco use and alcohol consumption are proven risk factors of head and neck squamous cell carcinoma. Cancer patients possessing such a tumor are generally elderly, mostly in fifth or sixth decade of life. In addition, significant association of head and neck squamous cell carcinoma with infection by human papillomavirus (HPV) was proven. HPV is now considered to be one of the most important risk factors, particularly for oropharyngeal carcinoma. HPV  positive tumors of oropharynx are associated with significantly better prognosis. Experimental and clinical data indicate that HPV positive and HPV negative tumors can be considered as two different entities and it has not been clarified which factors are crucial for better prognosis of HPV  positive tumors. The character of immune reaction, which contributes to distinct prognosis, may be one of the important factors. This review summarizes current knowledge concerning various aspects of antitumor immune responses in HPV  positive and HPV  negative tumors. Recent studies have shown that a broad repertoire of tumor infiltrating HPV specific T-cells is detectable in almost all patients with HPV positive tumors. Despite this, there is a development of tumor, which may be facilitated by abnormalities in antigen processing, T-cell dysfunction or prevalence of immunosuppressive cells. Nonetheless, the immunologic profile of HPV positive vs. HPV negative head and neck squamous cell carcinoma is associated with better outcome, and HPV  specific immune response is suggested to play an essential role in the better response to conventional therapy of HPV  positive patients. We also discuss HPV specific antitumor immunotherapy approaches, which are now tested in clinical trials.

[K-ras mutational status and tumour-infiltrating lymphocytes in human colon cancer: state of the art and future perspectives]. / K-ras mutace a nádory infiltrující lymfocyty u karcinomu kolon, soucasnost a výhledy.

INTRODUCTION: Nowadays, the prognosis of newly diagnosed colorectal cancer patients relies mostly on the tumour-node-metastasis (TNM) classification which is also a determining criterion for the indication of adjuvant oncological treatment. Currently, new prognostic and predictive biomarkers are sought after in order to more precisely define prognosis and better predict the benefits of adjuvant treatment in colorectal cancer. Besides several molecular biomarkers, such as mutations in the proto-oncogene K-ras, analyses of tumour-infiltrating lymphocytes have shown promising prognostic value. The aim of the study is to examine the correlations between K-ras mutational status and tumour-infiltrating immune cells in colon cancer patients with respect to colon cancer recurrence. MATERIAL AND METHODS: Formalin-fixed paraffin-embedded specimens were obtained from 44 patients with surgically resected colon cancer (R0 resection) treated between 2004 and 2009. K-ras mutational status was detected using PCR amplification of exon 1 followed by direct sequencing and K-ras StripAssay. Tumour-infiltrating immune cells were detected by immunofluorescence staining using monoclonal antibodies against CD3, CD8, FoxP3, CD1a and DC-LAMP. RESULTS: All 44 patients in our cohort underwent radical resection of colon cancer. In 16 patients the tumour relapsed (36.4%). K-ras mutations were found in 45.5% (n=20) of the primary carcinomas: 65% in codon 12 and 35% in codon 13. Although codon 13 K-ras mutations were associated with disease relapse, they were present in both disease-free and relapsed patients. However, disease-free and relapsed patients differed markedly in their patterns of tumour-infiltrating immune cells. There was a trend towards decreased density of tumour-infiltrating lymphocytes within the group of relapsed patients. In addition, relapsed patients with codon 13 mutations had markedly lower levels of tumour-infiltrating mature DC-LAMP+ dendritic cells and higher frequency of CD1a+ cells compared to disease-free patients. CONCLUSION: Colon cancer patients with low levels of tumour-infiltrating lymphocytes, a high CD1a+/DC-LAMP+ tumour-infiltrating DC ratio and a K-ras mutation in codon 13 are at a high risk of disease recurrence.

[Active cellular immunotherapy of ovarian cancer using dendritic cells]. / Aktivní bunecná imunoterapie karcinomu ovaria pomocí dendritických bunek.

OBJECTIVE: Overview and comparison of current results of studies dealing with the development and application of anti-cancer vaccines based on dendritic cells in ovarian cancer. DESIGN: Review. SETTING: Department of Obstetrics and Gynaecology Charles University, Prague, 2nd Faculty of Medicine and University Hospital Motol, Department of Immunology 2nd Faculty of Medicine and University Hospital Motol. SUMMARY: Ovarian carcinoma (OVCA) is highly sensitive to chemotherapy; however despite this results from treatment are fairly unsatisfactory. Bearing this in mind, it is important to look for new ways to better understand the immunological mechanisms which could affect reactivation of the disease. It is likely that new knowledge in the field of the immunology of ovarian carcinoma could improve monitoring of the disease and help to ameliorate prognosis of the disease. One strategy in development is creation of anti-OVCA vaccines. Theese vaccines are made by the fusion of dendritic cell (DC) and tumor cells or its parts (NA, peptides). DC are bone-marrow derived leukocytes that are critical in the initiation of T cell mediated immunity. DC are fused to patient-derived ovarian carcinoma cells. The fusion cells induces cytotoxic T cell against autologous OVCA cells.

Selective increase in blood dendritic cell antigen-3-positive dendritic cells in bronchoalveolar lavage fluid in allergic patients.

Dendritic cells (DCs) are specific antigen-presenting cells that play critical roles in the initiation and polarization of immune responses. DCs residing in the lungs might be detected in the bronchoalveolar lavage fluid (BALF). We analysed DC compartment in the peripheral blood and BALF of patients with allergy and in controls. Plasmacytoid and four distinct subsets of myeloid DCs [characterized by the expression of blood dendritic cell antigen (BDCA)-1+ and -3+ and CD16 positivity or negativity] were detected in both tested compartments. We further evaluated the expression of C-type lectins [mannose receptor (MR), dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) and dendritic and epithelial cells (DEC)-205] relevant to the pathogenesis of asthma. Interestingly, we found a selective increase in the frequency of myeloid DC-expressing BDCA-3 and MR particularly in BALF from allergic patients. Specific and highly statistically significant increase in BDCA-3+ and/or MR+ DCs brings a novel characteristic to BAL analysis in allergic patients.

Expansion of T helper type 17 lymphocytes in patients with chronic granulomatous disease.

Hyper-immunoglobulin (Ig)E syndrome (HIES) is a primary immunodeficiency associated with mutations in STAT3 resulting in impaired development of T helper type 17 (Th17) lymphocytes. HIES patients with a reduced frequency of Th17 cells present with infections caused by Staphylococcus aureus and/or Candida strains. The same spectrum of pathogens is present in patients with chronic granulomatous disease (CGD).We analysed the characteristics of the Th17 compartment in HIES and CGD. HIES patients showed very low numbers of Th17 cells. By contrast, the frequency of Th17 cells and production of Th17-derived cytokines was significantly higher among CGD patients when compared to both control samples and HIES. Naive CD4(+) cells in CGD patients had a normal capacity to differentiate into IL-17-producing cells and the numbers of Th17 cells in the CGD patients normalized following successful bone marrow transplantation. Our findings complement recent data on the importance of Th17 cells for elimination of infections with C. albicans and S. aureus.

Effects of 5-azacytidine and trichostatin A on dendritic cell maturation.

Maturation of dendritic cells (DC) towards functional antigen-presenting cells is a complex process, the regulation of which may also involve epigenetic mechanisms. Thus, it is of interest to investigate how gene expression changes during DC maturation can be influenced with epigenetic agents, such as DNA methyltransferase or histone deacetylase inhibitors. Here, we document the effects of DNA methyltransferase inhibitor 5-azacytidine (5AC) and histone deacetylase inhibitor trichostatin A (TSA) on the murine bone marrow-derived, as well as on the human monocyte-derived DC maturation. The major impact of 5AC and TSA on the DC maturation process consisted in the inhibition of unmethylated CpG oligodeoxynucleotide (CpG ODN) 1826 or LPS-induced activation of pro- and anti-inflammatory cytokine gene expression activation. In the in vitro studies, TSA but not 5AC significantly reduced the capacity of the peptide-pulsed DC to induce total spleen as well as CD8(+) or CD4(+) cell proliferation. IFNγ production by the specific CD4(+) spleen cells co-cultured with TSA- but not with 5AC-treated DC was lower, as compared to the cytokine production after co-cultivation with untreated mature DC. Collectively, these results demonstrate the potential of epigenetic agents, which are under intensive investigation as promising anti-tumour agents, to hamper the immune response induction through their inhibitory effects on DC.

[Experimental tumor therapy using intratumoral injection of dendritic cells]. / Experimentální terapie nádoru pomocí intratumorózní aplikace dendritických bunek.

AIM OF STUDY: After the treatment of tumors, we often encounter a minimal residual sickness. However, the elimination of these leftover tumor cells is crucial for the patient. In the past years one of the most discussed options for this treatment is Imunotherapy, mainly by Dendritic cells. Dendritic cells are the most efficient cells out of the antigen presenting cell group. METHODS AND RESULTS: In the first part of the project, we perfected a technique of inducting a tumor on an experimental model. We inducted the tumor by the use of Carcinogenic substances or with the help of the Sarkom line imortalized fibroblasts. Another important part of the project was perfecting the method for the preparation of undeveloped dendritic cells from periphery blood monocytes. After these significant procedures were developed and perfected we moved onto the main part of the study. The Induction of a tumor by the carcenogenic substances Ethylennitrosamin and Phenobarbital was successful only in 20 % of the cases and therefore, was unusable for our experiment. We inducted the tumors with the Sarkom line method. After the application of dendritic cells into the tumor, a decrease in the development of the growth of the tumor was achieved. CONCLUSION: Imunotherapy using dendritic cells as a basis for treatment is a perspective method for treatment of tumors.

Dendritic cell-based immunotherapy induces transient clinical response in advanced rat fibrosarcoma - comparison with preventive anti-tumour vaccination.

In this study we present the models of preventive and therapeutic vaccination of sarcoma-bearing rats with dendritic cells that present tumour antigens from killed tumour cells. We present the characteristics of dendritic cell-based vaccine and its capacity to induce anti-tumour immune response both in vitro and in vivo. We show that preventive vaccination efficiently prevents tumour growth. On the other hand, vaccination of rats with established tumours did not lead to eradication of the tumours. Despite the induction of a vigorous immune response after administration of dendritic cell-based vaccine and transient decrease in tumour progression, tumours eventually resumed their growth and animals vaccinated with dendritic cells succumbed to cancer. In both settings, preventive and therapeutic, dendritic cell-based vaccination induced a vigorous tumour-specific T-cell response. These results argue for the timing of cancer immunotherapy to the stages of low tumour load. Immunotherapy initiated at the stage of minimal residual disease, after reduction of tumour load by other modalities, will have much better chance to offer a clinical benefit to cancer patients than the immunotherapy at the stage of metastatic disease.

Assessment of lymphocyte proliferation: CFSE kills dividing cells and modulates expression of activation markers.

The measurement of cell proliferation after mitogenic stimulation is an important parameter used in diagnosis of immunodeficiencies in clinical laboratory as well as in various fields of lymphocyte research. Recent methods try to overcome the radioactive assay using tritiated thymidine ((3)H) by flow-cytometric methods using different fluorochromes such as CFSE or even to substitute these direct methods by tracing the expression of cell-membrane activation markers associated with various steps of proliferation cycle. In our study we compared the (3)H assay with CFSE-staining method and expression of activation markers (CD69, HLA-DR, CD25, CD27, CD71, CD152, CD134 and CD195) on a sample of 128 consecutive patients and healthy controls evaluated in clinical laboratory. We also tested various concentrations of CFSE and its impact on proliferation activity and expression of activation markers. We found that CFSE in concentration from 37nM to 10microM decreases the proliferative capacity (expressed in cpm (3)H assay) due to the decreased viability of proliferating cells (measured as 7-AAD+) in concentration-dependent manner. Moreover, CFSE substantially modulates the expression of activation molecules (decreasing CD69, HLA-DR, CD25 the majority of examinated molecules). We found a good correlation between CFSE-staining method with (3)H assay, if CFSE low population is gated on CD3+ population (correlation coefficient 0.801), but only in samples with stimulation index (SI) higher then 25. In poorly proliferating samples (SI25) no correlation was found due to several false positive results in CFSE test. Statistically significant correlation between proliferation assessed as (3)H-thymidine incorporation and expression of activation markers was found in the case of CD25, CD27, CD38, CD152, CD71, still only in samples with higher proliferation activity (SI>25). No correlation was found with CD134, CD195, HLA-DR and CD69. We conclude that standard assay with (3)H-thymidine incorporation is unreplaceable assay in diagnosis of severe cellular immunodeficiencies as CFSE assay have high proportion of false positive results. Researchers tracing cell-membrane bound molecules on dividing cells stained by CFSE must take into account that CFSE may substantially modulate the expression of these markers and decrease the viability of stained cells.

[Familial haemophagocytic lymphohistiocytosis caused by perforin deficit can be successfully treated by haematopoietic stem cell transplantation--the first diagnosed case in the Czech Republic]. / Familiární hemofagocytující lymfohistiocytóza na podklade deficitu perforinu uspesne lécená transplantací hematopoetických kmenových bunek--první diagnostikovaný prípad v Ceské republice.

Familial haemophagocytic lymphohistiocytosis (FHL) is an inherited disorder characterized by an impaired cytotoxicity of T lymphocytes and NK cells typically manifesting within first few months after birth. If not treated adequately, it is inevitably fatal within several months. The incidence in Caucasians has been estimated to 1: 50 000 births. Haematopoietic stem cell transplantation represents the only curative treatment for FHL. Recently, several genetic defects underlying molecular defects in FHL have been identified. In approximately 30% of patients FHL is caused by mutations in PRF1 gene coding for perforin. Further 30% of patients were found to have mutations in UNC13D coding for hMunc13-4 protein. Very recent report has identified another cause of FHL, mutations in STX11 gene on chromosome 6, coding for syntaxin 11. Absence of any of those proteins severely impairs the process of exocytosis of cytotoxic granules. We describe patient with clinical symptoms of FHL. Immunological and molecular biology methods led to the identification of perforin mutation as a cause of the disease. Patient received an allogeneic SCT from HLA-matched unrelated donor. SCT was followed by rapid normalization of clinical symptoms and laboratory findings. In patient described in this study, FHL manifested with typical clinical and laboratory symptoms. Adequate immunosuppressive treatment and subsequent SCT led to the sustained remission of FHL and correction of molecular defect. This is the first case of FHL in Czech Republic where perforin mutation was identified as a molecular cause both at cellular and molecular level.

Polyomavirus EGFP-pseudocapsids: analysis of model particles for introduction of proteins and peptides into mammalian cells.

A vector for preparation of mouse polyomavirus capsid-like particles for transfer of foreign peptides or proteins into cells was constructed. Model pseudocapsids carrying EGFP fused with the C-terminal part of the VP3 minor protein (EGFP-VLPs) have been prepared and analysed for their ability to be internalised and processed by mouse cells and to activate mouse and human dendritic cells (DC) in vitro. EGFP-VLPs entered mouse epithelial cells, fibroblasts and human and mouse DC efficiently and were processed by both, lysosomes and proteasomes. Surprisingly, they did not induce upregulation of DC co-stimulation molecules or maturation markers in vitro; however, they did induce interleukin 12 secretion.

[Glucocorticoids and their effect on dendritic cell function]. / Glukokortikoidy a jejich vliv na funkci dendritických bunek.

BACKGROUND: Dendritic cells represent the most effective antigen presenting cells and they are the only cell type capable of initiating the primary immune response. They use several sets of germ-line encoded receptors to differentiate between self and non-self and to detect the presence of danger signals. Danger signals are mainly represented by microbial pathogens but it can be also a necrotic or malignant cell. At various stages of their lifecycle dendritic cells play a key role in maintaining the peripheral tolerance towards self-antigens and in the initiation of an effective immune response. Glucocorticoids have been widely used in the treatment of autoimmune or inflammatory disorders and their immunosuppressive effect has been mainly attributed to the inhibition of lymphocytes functions. METHODS AND RESULTS: In this study, we discuss the effects of glucocorticoids on in vitro generated myeloid dendritic cells and on peripheral blood myeloid and plasmacytoid dendritic cells subsets. CONCLUSIONS: Experimental results point to the profound suppressive effect of glucocorticoids on the antigen presenting functions of dendritic cells and to contribute to better understanding of glucocorticoids-mediated immunosuppressive effect.

[Immunotherapy--perspectives in therapy of ovarian carcinomas]. / Imunoterapie--perspektivy vyuzití v lécbe ovariálních karcinomu.

OBJECTIVE: To summarise recent knowledge and clinical studies of immunotherapy in the treatment of malignant ovarian epithelial tumors. DESIGN: A literature review. SETTING: Department of Gynecology and Obstetrics, Charles University Prague, 2nd Medical Faculty, University Hospital Motol. Department of Immunology Charles University Prague, 2nd Medical Faculty, University Hospital Motol. ABSTRACT: Combination of surgery and chemotherapy has been the usual standard of therapeutic protocols in ovarian cancer patients. However, this therapy is still not sufficient to eliminate all of the tumour cells. Immunotherapy seems to be an effective approach in combination with surgery and chemotherapy. Immunotherapy includes three types of strategies: cytokine therapy, monoclonal antibody therapy and vaccine therapy, especially vaccines with dendritic cells. All of them are shortly reviewed in this article. IFNalpha, IFNgamma, IL-2, GM-CSF are examples of cytokine therapy. Representatives of monoclonal antibody therapy include trastuzumab (monoclonal antibody against HER-2/neu peptide, MAb B.43.13 (antibody against CA 125), or radiolabeled antibody--pemtumomab (90Yttrium-CC49). Cancer vaccination is used in experiments because it should be effective in presenting tumour cells as foreign cells to effector cells of the immune system. Otherwise, tumour cells are not usually recognised by the immune system as dangerous cells. The efficiency of immunotherapy depends on tumor size and previous therapy. It seems to be effective in potentiation of primary chemotherapy or as a consolidation treatment of minimal residual disease. Immunotherapy is still at the experimental level, but in the future it could be a useful part of protocols for the treatment of ovarian cancer.

Anti-cancer therapy using dendritic cells and apoptotic tumour cells: pre-clinical data in human mesothelioma and acute myeloid leukaemia.

We have recently reported in an experimental model, that treatments based on the injections of dendritic cells which had phagocytosed apoptotic bodies derived from tumour cells were particularly effective in the cure of tumour-bearing animals. We proposed that systems using processing and presentation of antigenic molecules from antigen-presenting cells primed with apoptotic bodies can offer new opportunities in anti-cancer treatment. We first established the technical conditions for purification, characterisation and production of tumour cells isolated from fresh pleural liquid or blood. Then we compared efficacy of different apoptotic inducers agents on the cancer cells in culture. The apoptotic tumour cells were purified, characterised and maintained in coculture with monocytes-derived immature dendritic cells. We subsequently investigated the effect of the maturation process on phagocytosis of apoptotic bodies. We have shown that whatever the nature of the apoptotic cells they are phagocytosed by the dendritic cells which were efficiently matured using the combination of TNFalpha+Poly I:C. Furthermore, we demonstrated that the generation of the mature dendritic cells pulsed with apoptotic tumour cells, successfully generated CD4(+) (Th1) and CD8(+) (CTL) cells. All the experimental procedures that we have used were developed with clinical use in mind, using Good Manufacturing Products. We are presently investigating the feasibility of such a "vaccine" for the treatment of asbestos mesothelioma or acute myeloid leukaemia.

Standardized generation of fully mature p70 IL-12 secreting monocyte-derived dendritic cells for clinical use.

Dendritic cells (DC) have been shown to be efficient antigen-presenting cells (APC) and, as such, could be considered ideal candidates for cancer immunotherapy. Immature DC (iDC) efficiently capture surrounding antigens; however, only mature DC (mDC) prime naive T lymphocytes. Clinical trials using DC-based tumor vaccines have achieved encouraging, but limited, success, possibly due to the use of immature or incompletely mature DC. Thus, it was apparent that a method capable of generating large numbers of fully functional iDC, their pulsing with desired form of tumor antigens and the subsequent complete and reproducible maturation of iDC is needed. Therefore, we compared two different methods of producing large numbers of iDC. Both protocols yielded comparable numbers of cells with an iDC phenotype with phagocytic function. We next determined which of the clinically applicable activators could induce the complete and reproducible maturation of DC, in order to define the most suitable combination for future clinical trials. Only a combination of TNFalpha + Poly (I:C), or a previously described cytokine cocktail of TNFalpha + IL-1beta + IL-6 + prostaglandin E2, induced the complete activation of the whole DC population, as assessed by the cell surface expression of CD83 and costimulatory molecules. The matured DC were functionally superior to iDC in their ability to stimulate the proliferation of allogeneic lymphocytes and autologous keyhole limpet hemocyanin (KLH)-specific T lymphocytes. Furthermore, only the combination of TNFalpha + Poly (L:C) activated DC to produce large amounts of biologically active p70 IL-12. Thus DC maturation by TNFalpha + Poly (I:C) could efficiently bias T cell response towards Th1 response. Implementation of our results into clinical protocols used for DC generation could be beneficial for future immunotherapy trials.

[Dendritic cells and their use in the therapy of neoplastic diseases]. / Dendritické bunky a jejich vyuzití v terapii nádorových chorob.

Dendritic cells (DC) constitute a heterogeneous leukocyte population. Their main function is to capture and process antigens (Ag) and present them to immunocompetent cells. Heterogeneity of DC is reflected at several levels. Myeloid and lymphoid lineage of the DC can be distinguished according to the precursor cell they originate from. The functional differentiation is of the great importance. DC can induce either specific immune reaction or tolerance to certain Ag. It depends upon the microenvironment where the processing of Ag takes place. Phenotypic and functional differences between the subtypes of DC are being extensively investigated for the purpose of their use in the immunotherapy of various diseases, tumors in particular. It appears that one of the causes of the specific anti-tumor immunity failure is the insufficient function of DC in vivo in patients with malignant diseases. Recent technology advances has enabled to generate and cultivate DC in sufficient amounts in vitro from their precursors. Coculturing of DC with the tumor Ag in the presence of cytokine mixture leads to the efficient Ag presentation and to the generation of specific cytotoxic lymphocytes capable of killing tumor cells. Subsequent application of these tumor Ag pulsed DC to the laboratory animals, and to the patients in first clinical studies, can induce regression of malignant disease. On the other side the ability of DC to induce tolerance to certain Ag is the subject of investigation in the field of immunotherapy of hypersensitivity states induced either by outer Ag (allergy) or inner Ag (autoimmune diseases). In this review we summarize source and ontogeny of DC, their morphology, phenotype, function and different ways of their generation in vitro. We emphasize the use of DC in the clinical practice aimed at the immunotherapy of tumor diseases.