Maturation of monocyte-derived dendritic cells with Toll-like receptor 3 and 7/8 ligands combined with prostaglandin E2 results in high interleukin-12 production and cell migration

Dendritic cells (DC) are professional antigen-presenting cells of the immune system that play a key role in regulating T cell-based immunity. In vivo, the capacity of DC to activate T cells depends on their ability to migrate to the T cell areas of lymph nodes as well as on their maturation state. Depending on their cytokine-secreting profile, DC are able to skew the immune response in a specific direction. In particular, IL-12p70 producing DC drive T cells towards a T helper 1 type response. A serious disadvantage of current clinical grade ex vivo generated monocyte-derived DC is the poor IL-12p70 production. We have investigated the effects of Toll-like receptor (TLR)-mediated maturation on ex vivo generated human monocyte-derived DC. We demonstrate that in contrast to cytokine-matured DC, DC matured with poly(I:C) (TLR3 ligand) and/or R848 (TLR7/8 ligand) are able to produce vast amounts of IL-12p70, but exhibit a reduced migratory capacity. The addition of prostaglandin E2 (PGE2) improved the migratory capacity of TLR-ligand matured DC while maintaining their IL-12p70 production upon T cell encounter. We propose a novel clinical grade maturation protocol in which TLR ligands poly(I:C) and R848 are combined with PGE2 to generate DC with both high migratory capacity and IL-12p70 production upon T cell encounter

Blood compatibility of surfaces with immobilized albumin-heparin conjugate and effect of endothelial cell seeding on platelet adhesion

Endothelial cell (EC) seeding significantly improves the blood compatibility of artificial surfaces. Although a coating consisting of albumin and heparin (alb-hep) is a suitable substrate for seeded ECs, binding of ECs to the substrate further improves when small amounts of fibronectin are present in the alb-hep coating. Alb-hep conjugate was immobilized on carbon dioxide gas plasma-treated polystyrene (PS-CO2), thereby significantly increasing the recalcification time of blood plasma exposed to this surface. Furthermore, surface-immobilized alb-hep conjugate inhibited exogenous thrombin. Heparin activity was reduced by adding fibronectin on top of a monolayer of alb-hep conjugate, but not by simultaneous coating of fibronectin and alb-hep conjugate. Coating of PS-CO2 with alb-hep conjugate significantly decreased contact activation (FXII activation). The number of platelets deposited from blood plasma on PS-CO2 coated with alb-hep conjugate was twice as high as on PS-CO2 coated with albumin. Addition of fibronectin to alb-hep conjugate-coated PS-CO2 had no significant effect on the number of adhered platelets. Seeding of the substrates with ECs significantly reduced the number of adhered platelets under stationary conditions. Platelets deposited onto endothelialized surfaces were primarily found on endothelial cell edges, and sparingly on areas between ECs. In conclusion, alb-hep conjugate-coated surfaces display anticoagulant activity. ECs adhering to and proliferating on this coating significantly decrease the number of platelets which adhere to the surface. Therefore, alb-hep conjugate-coated surfaces form a suitable substrate for seeding of ECs in low density. Although application of fibronectin on top of the coating decreases the anticoagulant activity to some extent, it might be useful in view of the improved adherence of ECs to the coating

Endothelialization of crosslinked albumin-heparin gels

Crosslinked gels of albumin as well as heparinized albumin gels, potential sealants of prosthetic vascular grafts, were studied with regard to in vitro stability, binding of basic fibroblast growth factor (bFGF) and cellular interactions. A small percentage of the heparin present in these gels, was released during storage in SDS solution. During storage in cell culture medium at 37° C, heparin release was 21-25 percent. Release of albumin did not occur. Human umbilical vein endothelial cells (HUVECs) rapidly adhered and subsequently spread on (heparinized) albumin gels, but proliferation was only observed if heparin was present in the gel. Binding of 125I-bFGF to heparinized albumin gel was 35 percent higher than to non-heparinized albumin gel. Growth of HUVECs occurred only on heparinized albumin gel loaded with bFGF and not on bFGF-loaded albumin gel. The number of platelets deposited under stationary conditions onto heparinized albumin gel was about twice the number found on nonheparinized albumin gel. Seeding of HUVECs on heparinized albumin gel, significantly reduced the number of platelets adhering to this surface. Moreover, no spreading of platelets was observed on substrates seeded with HUVECs. It can be concluded that crosslinked gels of albumin to which heparin is immobilized, are candidate sealants for prosthetic vascular grafts and suitable substrates for endothelial cell seeding

Proliferation of endothelial cells on surface-immobilized albumin-heparin conjugate loaded with basic fibroblast growth factor

Seeding of endothelial cells (ECs) on the luminal surface of small-diameter vascular grafts is a promising method to avoid occlusion of these prostheses. Immobilization of basic fibroblast growth factor (bFGF) to substrates used to coat or fill porous prostheses may enhance the formation of a confluent monolayer of ECs. Human umbilical vein endothelial cells (HUVECs) were grown on bFGF-loaded albumin-heparin conjugate bound to CO2 gas-plasma-treated polystyrene. In the order of 2-3 ng/cm2 bFGF had to be immobilized to form a confluent monolayer of HUVECs. The most prominent effect of surface-immobilized bFGF was stimulation of the proliferation shortly after seeding, resulting within 3 days in confluent cell monolayers with high density. In contrast, in cultures with 0.3 ng/mL bFGF in the medium instead of bFGF bound to the surface, it took almost a week before the cell layers reached confluency. Binding of bFGF to heparin and the biological activity of bFGF towards ECs were not influenced by the (radio-)labeling of bFGF with iodine. However, only a minor part of the bFGF used in this study displayed heparin affinity. Furthermore, degradation and multimerization of labeled bFGF in time occurred when the growth factor was stored at 20°-37°C. This limits the use of labeled bFGF to short-term (hours) experiments. In conclusion, bFGF loading of vascular graft surfaces through complexation of bFGF with a heparin-containing matrix probably will lead to more rapid formation of a confluent monolayer of ECs on graft surfaces upon seeding of the cells

Adherence and Proliferation of Endothelial Cells on Surface-Immobilized Albumin-Heparin Conjugate

Small-diameter vascular grafts rapidly fail after implantation, due to occlusion caused by thrombosis. This problem cannot be overcome using medication. A promising improvement of graft patency is the seeding of endothelial cells (EC) on the luminal surface of the vascular graft. Conjugates of albumin and heparin, which were developed to obtain nonthrombogenic coatings, could form an ideal coating for vascular grafts. Besides presenting anticoagulant function, heparin will bind proteins with cell adhesive properties, thus facilitating adherence of EC to the graft surface. EC were able to grow to confluency on CO2 gas plasma–treated polystyrene (PS-CO2) coated with albumin-heparin conjugate. CO2 gas plasma treatment resulted in the introduction of functional groups at the surface (e.g., hydroxyl, aldehyde, carboxylic acid, and epoxide groups). Addition of albumin-heparin conjugate to the functionalized surface in an aqueous solution with pH 8.2 yielded a stable monolayer of covalently bound conjugate. The number of cells adhering and proliferating on this surface was comparable to the number of cells on fibronectin-coated PS-CO2. However, the structure and size of EC proliferating on surface-immobilized albumin-heparin was more irregular. Long-term adherence might be improved by adding fibronectin to the albumin-heparin surface, either as a mixture with albumin-heparin or in a separate incubation step

Maturation of dendritic cells is a prerequisite for inducing immune responses in advanced melanoma patients

We have investigated the capacity of immature and mature monocyte-derived DCs pulsed with melanoma-associated peptides (gp100 and tyrosinase) to induce a primary cytotoxic T-lymphocyte response in vivo. Advanced HLA-A2.1(+) melanoma patients were vaccinated with peptide- and keyhole limpet hemocyanin (KLH)-pulsed DCs, either immature (9 patients) or matured by monocyte-conditioned medium/tumor necrosis factor alpha/prostaglandin E(2) (10 patients). All patients vaccinated with mature DCs showed a pronounced proliferative T-cell and humoral response against KLH. By contrast, KLH responses were absent in most of the patients vaccinated with immature DCs. Delayed-type hypersensitivity (DTH) reactions against antigen-pulsed DCs were only observed in patients vaccinated with mature DCs and not in patients vaccinated with immature DCs. MHC-peptide tetramer staining of DTH-derived T cells revealed the presence of specific T cells recognizing the melanoma-associated peptides in 1 patient. In a second patient, DTH-derived T cells showed specific lysis of tumor cells expressing the antigens used for DC pulsing. Only patients vaccinated with mature DCs showed objective clinical responses. Interestingly, both patients with long-term progression-free survival (22 and >40 months) were both vaccinated with mature DCs and demonstrated antigen-specific T-cell reactivity of DTH-derived T cells. We conclude that mature DC are superior to immature DC in the induction of immunological responses in melanoma patients, which may translate into improved clinical result

Immunogenicity of dendritic cells pulsed with CEA peptide or transfected with CEA mRNA for vaccination of colorectal cancer patients

Dendritic cells (DCs) are the professional antigen-presenting cells of the immune system. We have demonstrated that vaccination of autologous ex vivo cultured DCs results in the induction of tumor-specific immune responses in cancer patients, which correlates with clinical response. Optimization of antigen loading is one of the possibilities for further improving the efficacy of DC vaccination. Theoretically, transfection of DCs with RNA encoding a tumor-specific antigen may induce a broader immune response as compared to the most widely used technique of peptide pulsing. In this clinical study, RNA transfection was compared with peptide pulsing as an antigen loading strategy for DC vaccination. Patients with resectable liver metastases of colorectal cancer were vaccinated intravenously and intradermally 3 times weekly with either carcinoembryogenic antigen (CEA)-derived HLA-A2 binding peptide-loaded or CEA mRNA electroporated DCs prior to surgical resection of the metastases. All DCs were loaded with keyhole limpet hemocyanin (KLH) as a control protein. Evaluation of vaccine-induced immune reactivity consisted of T-cell proliferative responses and B-cell antibody responses against KLH in peripheral blood. CEA reactivity was determined in T-cell cultures of biopsies of post-treatment delayed type hypersensitivity skin tests. Sixteen patients were included. All patients showed T-cell responses against KLH upon vaccination. CEA peptide-specific T-cells were detected in 8 out of 11 patients in the peptide group, but in none of the 5 patients in the RNA group. In our study, DC CEA mRNA transfection was not superior to DC CEA peptide pulsing in the induction of a tumor-specific immune response in colorectal cancer patient

Effective migration of antigen-pulsed dendritic cells to lymph nodes in melanoma patients is determined by their maturation state

Dendritic cells are the professional antigen-presenting cells of the immune system. To induce an effective immune response, these cells should not only express high levels of MHC and costimulatory molecules but also migrate into the lymph nodes to interact with naïve T cells. Here, we demonstrate that in vitro-generated mature, but not immature dendritic cells, efficiently migrate into the T-cell areas of lymph nodes of melanoma patients. This difference is confirmed by in vitro studies, in which immature dendritic cells are strongly adherent, whereas mature dendritic cells remain highly motile. Our present findings demonstrate that the ability of dendritic cells to mount a proper immune response correlates with their ability to migrate both in vitro and in viv