Jiřina Bartůňková

Paricalcitol (19-nor-1,25-dihydroxyvitamin D2) and calcitriol (1,25-dihydroxyvitamin D3) exert potent immunomodulatory effects on dendritic cells and inhibit induction of antigen-specific T cells

Paricalcitol (19-nor-1,25/OH(2)/D(2)), a second generation vitamin D receptor (VDR) activator, is a synthetic analogue of vitamin D3. In contrast to calcitriol, paricalcitol has a reduced effect on intestinal calcium resorption thus avoiding undesirable hypercalcemia. Information about immunomodulatory activity of paricalcitol is scarce. In this study we show that, in all investigated aspects, paricalcitol retains significant immunomodulatory activity, comparable to calcitriol. Both VDR agonists impaired differentiation of immature dendritic cells (DCs) from monocytes. The presence of VDR agonists during DC differentiation abolished their capacity to be activated and, despite potent Toll-like receptor mediated stimulation, VDR agonist-treated DCs remained in the immature state. In accordance with these findings, VDR-treated DCs produced no bioactive IL-12 and had a significantly decreased capacity to induce antigen-specific T cells while the capacity to induce functional Tregs remained unchanged when compared to control DCs. As DCs and T cells play an important role in the pathogenesis of atherosclerosis, in end-stage renal disease patients, paricalcitol should be a VDR agonist of choice for the reduction of the risk of atherosclerosis due to its immunomodulatory effect proven in this study and known limited hypercalcemic effect. The immunomodulatory potency of paricalcitol makes it a drug of interest in the therapy of chronic immune-mediated inflammatory diseases.

Dynamics of T-cell infiltration during the course of ovarian cancer: the gradual shift from a Th17 effector cell response to a predominant infiltration by regulatory T-cells.

The type of immune cells that are present within the tumor microenvironment can play a crucial role in the survival of patients. However, little is known about the dynamics of the tumor‐infiltrating immune cells during disease progression. We studied the immune cells that infiltrated the tumor tissues of ovarian cancer patients at different stages of disease. The early stages of development of ovarian carcinomas were characterized by a strong Th17 immune response, whereas in stage II patients, recruitment of high numbers of Th1 cells was observed. In disseminated tumors (Stages III–IV), we detected a dominant population of Helios+ activated regulatory T cells (Tregs) along with high numbers of monocytes/macrophages and myeloid dendritic cells (mDCs). Tumor‐infiltrating Tregs had markedly lower expression of CCR4 than circulating Tregs, and the numbers of tumor‐infiltrating Tregs significantly correlated with the levels of CCL22 in ovarian tumor cell culture supernatants, suggesting their recruitment via a CCR4/CCL22 interaction. CCL22 was mainly produced by tumor cells, monocytes/macrophages and mDCs in the primary ovarian tumors, and its expression markedly increased in response to IFNγ. Taken together, the specific recruitment of Tregs, probably triggered by inflammatory stimuli, leads to a significant immune suppression in the advanced stages of ovarian cancer.

Toll-like receptors on B-CLL cells: expression and functional consequences of their stimulation

Toll‐like receptor (TLR) stimulation plays a crucial role in the homeostasis of human B cells. We investigated the expression of TLRs 1–9 on the cells of B‐cell chronic lymphocytic leukemia (B‐CLL) and analyzed the functional consequences of TLR stimulation on leukemic cells. We showed that B‐CLL cells express similar set of TLRs as memory B cells of healthy donors, i.e. TLR‐1, TLR‐2, TLR‐6, TLR‐7 and TLR‐9. However, in contrast to memory B cells, B‐CLL cells lack TLR‐4 expression. Expression of TLRs correlates with their capacity to respond to specific TLR agonists. At the level of phenotype, ODN2006 (TLR‐9 agonist) is the most potent stimulus. B‐CLL cells also respond to the stimulation with loxoribine, Pam3CSK4 and MALP‐2 (TLR‐7, TLR1/TLR2 and TLR2/TLR6 agonists, respectively). TLR‐7 and TLR‐9 stimulation induces production of IL‐6 and TNFα. In 47% of tested patients, treatment with ODN2006, MALP‐2 and Pam3CSK4 reduced leukemic cells survival. Stimulation of B‐CLL cells with TLR‐9 agonists, loxoribine, MALP‐2 and Pam3CSK4 induces significant proliferation. We report that TLR stimulation induces expression of CD38, a negative prognostic marker, on B‐CLL cells. Expression of CD38 is induced by direct stimulation of B‐CLL cells through TLR‐7 and TLR‐9 or CD38 can be induced on B‐CLL cells indirectly by a soluble factor induced in non‐B‐CLL cells after stimulation with TLR‐2, TLR‐3 or TLR‐5 agonists; the nature of this factor remains unknown. Our results argue for cautious evaluation of immunointervention strategies based on the administration of TLR agonists in the treatment of B‐CLL as their effects on B‐CLL cells may be tumor promoting as well as tumor suppressing.

Coexpression of binding sites for A(B) histo-blood group trisaccharides with galectin-3 and Lag antigen in human Langerhans cells

Galectin‐3 is an immunomodulatory protein with binding capacity for various glycoconjugates including IgE. It has been shown to be produced by epidermal keratinocytes and is present on the surfaces of skin Langerhans cells (LC). Therefore, it may have a role in the pathogenesis of various skin diseases, such as atopic dermatitis. To study the expression of galectin‐3 in LC, we used, in addition to specific antibodies, a panel of synthetic, carrier‐immobilized, specific oligosaccharides of the A‐ and B‐histo‐blood group, which are recognized by this lectin. In the mean time, Birbeck granules were visualized with an anti‐Lag antibody. The double labeling experiments showed a remarkable colocalization of signals for Lag antigen (Birbeck granules) and galectin‐3, as well as the binding sites for A‐ and B‐histo‐blood group trisaccharides. The specificity of the oligosaccharide binding was demonstrated by the lack of binding by Lec, Led (H blood group antigen), and sLex, which are not recognized by galectin‐3. These results suggest that galectin‐3 is present in Birbeck granules, where it retains reactivity for its glycoligands. J. Leukoc. Biol. 66: 644–649; 1999.

Effect of chemical structure of hydrogels on the adhesion and phenotypic characteristics of human monocytes such as expression of galectins and other carbohydrate-binding sites

The reactivity of diverse immune aspects to the presence of synthetic polymers represents one of the most important aspects of implantable device biocompatibility. In this report, we show the effect of the chemical structure of a synthetic polymer support on monocyte adhesion and selected phenotypic characteristics in vitro as a model for the initial steps of non-self-recognition of an implant. The extent of monocyte adhesion was significantly influenced by the support chemistry. The highest level of monocyte adhesion was observed on a surface copolymer of 2-hydroxyethyl methacrylate with dimethyl aminoethyl methacrylate relative to results of experiments in which poly(2-hydroxyethyl methacrylate) or the copolymer of 2-hydroxyethyl methacrylate with the sodium salt of methacrylic acid was used. Cell adhesion to the polymers tested and to glass was accompanied by enhanced expression of the carbohydrate-binding sites tested for asialoglycoprotein beta-galactosides such as galectins, beta-N-acetylgalactosamine, alpha-mannoside, specific lectin for heparin as well as the lymphokine-macrophage migration inhibitory factor in the monocytes tested. These results suggest the importance of monocyte adhesion to the biomaterial surface for their development into macrophages and further non-self-recognition of the implanted device.

FOCUS on FOCIS: combined chemo-immunotherapy for the treatment of hormone-refractory metastatic prostate cancer.

Immunotherapy has emerged as another treatment modality in cancer. The goal of immunotherapy in advanced cancer patients does not have to be the complete eradication of tumor cells but rather the restoration of a dynamic balance between tumor cells and the immune response. Appropriate combination of tumor mass reduction (by surgery and/or chemotherapy) and neutralization of tumor-induced immunosuppression might set the right conditions for the induction of anti-tumor immune response by active immunotherapy. We review experimental basis and key concepts of combined chemo-immunotherapy and document its principles in the case report of patient with hormone refractory metastatic prostate cancer with sinister prognosis. More than four hundred prostate cancer patients have been treated with DC-based immunotherapy and tumor-specific immune responses have been reported in two-thirds of them. In half of these patients, DC immunotherapy resulted in transient clinical responses. Tregs, among other factors, potently inhibit tumor-specific T cells. Prostate cancer patients have elevated numbers of circulating and tumor infiltrating Tregs and there is evidence that Tregs increase tumor growth in vivo. Because of the high frequency of circulating Tregs in our patients, we first administered metronomic cyclophosphamide. After obtaining IRB approval, we started regular vaccinations with dendritic cells (DCs) loaded with killed prostate cancer cells. In accordance with the principles of combined immunotherapy, we continued palliative chemotherapy with docetaxel to reduce the tumor cell burden. DC-based vaccination induced prostate cancer cell-specific immune response. Combined chemo-immunotherapy consisting of alternate courses of chemotherapy and vaccination with mature DCs pulsed with LNCap prostate cancer cell line led to the marked improvement in the clinical and laboratory presentation and to the decrease of PSA levels by more than 90%.

Generation of functional dendritic cells for potential use in the treatment of acute lymphoblastic leukemia

Abstract. Immunotherapy of malignant diseases mediated by dendritic cells (DC) pulsed with tumor antigens ex vivo is a promising new tool in the individual treatment of malignant diseases. The present study focuses on the problem of how to optimize in vitro culture conditions and induce the maturation of DC with the capacity to induce antitumor immunity toward leukemic cells. DC were generated from peripheral mononuclear cells by co-cultivation with granulocyte/macrophage–colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). Tumor antigens were added for 2 h after 7 days in culture. Irradiated leukemic blasts, blast lysate, apoptotic cells from the Jurkat cell line (T ALL) and their lysate were used in various concentrations for antigen pulsing. Harvested DC were phenotyped by flow cytometry, and viability was assessed using trypan blue exclusion (Annexin test). After the cells had been pulsed with tumor antigens and co-cultured with autologous lymphocytes, the production of interferon-gamma (IFN-γ) and IL-12 was analyzed, and lymphocyte proliferative response and cytotoxicity against the target tumor cell line were assessed. The cultivation of monocytes under the described conditions led to the expression of surface markers typical of DC (i.e. CD83, CD86, HLA-DR, CD11c and CD40). Pulsation by antigens from leukemic cells further increased the cell populations expressing these markers. Antigen pulsation decreased the viability of generated DC depending on the increase in concentration of tumor antigens. Pulsed DC–lymphocyte interaction increased the proliferative response of lymphocytes and IFN-γ production depending on the type of tumor antigens used for pulsation. The highest proliferative response was detected with DC pulsed with Jurkat cell-line lysate. Similarly to the proliferation assay, cytotoxic testing showed the highest efficiency of DC pulsed with Jurkat cell-line lysate in killing the target malignant cells. Our results show that an appropriate antigen concentration used for DC pulsing is one of the crucial factors in an effective treatment strategy, as high concentrations of tumor antigens induce apoptosis of DC, thereby rendering them non-functional. Under optimal conditions, pulsation by lysate from leukemic blasts induced the maturation of DC and led to an increase in the proliferation of autologous lymphocytes, to the production of Th1-cytokines and to the induction of cytotoxicity toward the leukemic cell line. These results are encouraging for the possible application of pulsed DC in the therapy of acute lymphoblastic leukemia.

Kinetics of dendritic cells reconstitution and costimulatory molecules expression after myeloablative allogeneic haematopoetic stem cell transplantation: implications for the development of acute graft-versus host disease.

Allogeneic hematopoetic stem cell transplantation (HSCT) represents a unique opportunity to monitor the kinetics of reconstitution of dendritic cells (DCs) and their dynamics in distinct pathologies. We analyzed DCs reconstitution after myeloablative HSCT. We separately analyzed patients with acute GVHD. DCs were monitored from the earliest phase of hematopoetic reconstitution until day +365. Both myeloid DCs and plasmacytoid DCs appeared at earliest stages after engraftment and relative numbers within white blood cells compartment peaked between days 19-25 after HSCT. Their proportion then gradually declined and absolute numbers of both DC subsets remained lower than in controls during the whole follow-up. Patients with acute GVHD had significantly lower numbers of circulating DCs. Decrease in DC counts preceded onset of clinical symptoms by at least 24 h and was independent of corticosteroids administration. This study reveals quantification of plasmacytoid and myeloid DCs as a potential biomarker for the prediction of acute GVHD development.

Poly I: C-activated dendritic cells that were generated in CellGro for use in cancer immunotherapy trials.

BackgroundFor clinical applications, dendritic cells (DCs) need to be generated using GMP-approved reagents. In this study, we tested the characteristics of DCs generated in two clinical grade culture media and activated by three maturation stimuli, Poly I: C, LPS and the mixture of proinflammatory cytokines in order to identify the optimal combination of culture media and activation stimulus for the clinical use.MethodWe tested DCs generation using two GMP-certified culture media, CellGro and RPMI+5% human AB serum and evaluated DCs morphology, viability and capapability to mature. We tested three maturation stimuli, PolyI:C, LPS and the mixture of proinflammatory cytokines consisting of IL-1, IL-6, TNF and prostaglandin E2. We evaluated the capacity of activated DCs to induce antigen-specific T cells and regulatory T lymphocytes.ResultsCell culture in CellGro resulted in a higher yield of immature DCs resulting from increased number of adherent monocytes. DCs that were generated in CellGro and activated using Poly I:C were the most efficient in expanding antigen-specific T cells compared to the DCs that were generated in other media and activated using LPS or the cocktail of proinflammatory cytokines. A comparison of all tested combinations revealed that DCs that were generated in CellGro and activated using Poly I:C induced low numbers of regulatory T cells.ConclusionIn this study, we identified monocyte-derived DCs that were generated in CellGro and activated using Poly I:C as the most potent clinical-grade DCs for the induction of antigen-specific T cells.

Intensive physical activity increases peripheral blood dendritic cells.

We analyzed the frequency and absolute numbers of circulating myeloid and plasmacytoid DCs in peripheral blood and evaluated their maturation status to test the hypothesis that significant physical stress to the body might induce measurable changes in DCs subsets, phenotype and function, which would complete existing knowledge about the response of the cellular immune system to an acute exercise in top sportsmen. We evaluated the heart rate and draw blood samples before and after the physical load in 18 profesional ice-hockey players. We observed an increase in leukocytes numbers with a predominant increase in the population of DCs and lymphocytes after exercise. Both myeloid and plasmacytoid DCs increased significantly. We found a correlation between the increase of peripheral blood DCs and serum epinephrine and norepinephrine levels. Increase in peripheral blood DCs also correlates with the extent of heart rate elevation during exercise.