http://repub.eur.nl/res/aut/7844/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/31714/ 2012-03-01T00:00:00Z Antigen-specific immunity is crucially important for containing viral replication in human immunodeficiency virus (HIV)-1-infected hosts. Several epitopes have been predicted for the early expressed HIV-1 proteins Tat and Rev, but few have been studied in detail. We characterized the human leukocyte antigen (HLA)-B44-restricted Rev epitope EELLKTVRL (EL9) in an HIV-1-infected subject treated with antiretroviral therapy. Interestingly, a high sequence similarity was found between the EL9 epitope and the human nucleolar protein 6 (NOL6). However, this similarity does not seem to impede immunogenicity as CD8+T-cells, previously stimulated with EL9-pulsed dendritic cells, were able to specifically recognize the HIV-1 Rev epitope without cross-recognizing the human self-antigen NOL6. After the subject interrupted antiretroviral therapy and virus rebounded, mutations within the EL9 epitope were identified. Although the emerging mutations resulted in decreased or abolished T-cell recognition, they did not impair Rev protein function. Mutations leading to escape from T-cell recognition persisted for up to 124 weeks following treatment interruption. This study shows that the HLA-B44-restricted Rev CD8+T-cell epitope EL9 is immunogenic notwithstanding its close resemblance to a human peptide. The epitope mutates as a consequence of dynamic interaction between T-cells and HIV-1. Clinical status, CD4+T-cell count and viral load remained stable despite escape from T-cell recognition. http://repub.eur.nl/res/pub/31846/ 2012-03-01T00:00:00Z In a phase I/IIa clinical trial, 17 HIV-1 infected patients, stable on cART, received 4 vaccinations with autologous dendritic cells electroporated with mRNA encoding Tat, Rev and Nef, after which cART was interrupted. Vaccination was safe and feasible. During the analytical treatment interruption (ATI), no serious adverse events were observed. Ninety-six weeks following ATI, 6/17 patients remained off therapy. Although induced and/or enhanced CD4+and CD8+T-cell responses specific for the immunogens were observed in most of the patients, we found no correlation with the number of weeks off cART. Moreover, CD4+T-cell counts, plasma viral load and the time remaining off cART following ATI did not differ from historical control data. To conclude, the vaccine was safe, well tolerated and resulted in vaccine-specific immune responses. Since no correlation with clinical parameters could be found, these results warrant further research in order to optimize the efficacy of vaccine-induced T-cell responses. http://repub.eur.nl/res/pub/28724/ 2010-12-01T00:00:00Z Conventional assays for quantification of allo-reactive T-cell precursor frequencies (PF) are relatively insensitive. We present a robust assay for quantification of PF of T-cells with direct donor-specificity, and establish the kinetics of circulating donorspecific T cells after liver transplantation (LTx). B cells from donor splenocytes were differentiated into professional antigenpresenting cells by CD40-engagement (CD40-B cells). CFSE-labelled PBMC from LTx-recipients obtained before and at several time points after LTx, were stimulated with donor-derived or 3rdparty CD40-B cells. PF of donor-specific T cells were calculated from CFSE-dilution patterns, and intracellular IFN-γ was determined after re-stimulation with CD40-B cells. Compared to splenocytes, stimulations with CD40-B cells resulted in 3 to 5-fold higher responding T-cell PF. Memory and nai{dotless}̈ve T-cell subsets responded equally to allogeneic CD40-B cell stimulation. Donor-specific CD4+and CD8+T-cell PF ranged from 0.5 to 19% (median: 5.2%). One week after LTx, PF of circulating donor-specific CD4+and CD8+T cells increased significantly, while only a minor increase in numbers of T cells reacting to 3rdparty allo-antigens was observed. One year after LTx numbers of CD4+and CD8+T cells reacting to donor antigens, as well as those reacting to 3rdparty allo-antigens, were slightly lower compared to pre-transplant values. Moreover, CD4+and CD8+T cells responding to donor-derived, as well as those reacting to 3rdparty CD40-B cells, produced less IFN-γ. In conclusion, our alternative approach enables detection of allo-reactive human T cells at high frequencies, and after application we conclude that donor-specific T-cell PF increase immediately after LTx. However, no evidence for a specific loss of circulating T-cells recognizing donor allo-antigens via the direct pathway up to 1 year after LTx was obtained, underscoring the relative insensitiveness of previous assays. http://repub.eur.nl/res/pub/27245/ 2009-10-22T00:00:00Z http://repub.eur.nl/res/pub/28937/ 2008-07-07T00:00:00Z Although dendritic cells (DCs) play an important role in mediating protection against influenza virus, the precise role of lung DC subsets, such as CD11b-and CD11b+conventional DCs or plasmacytoid DCs (pDCs), in different lung compartments is currently unknown. Early after intranasal infection, tracheal CD11b-CD11chiDCs migrated to the mediastinal lymph nodes (MLNs), acquiring co-stimulatory molecules in the process. This emigration from the lung was followed by an accumulation of CD11b+CD11chiDCs in the trachea and lung interstitium. In the MLNs, the CD11b+DCs contained abundant viral nucleoprotein (NP), but these cells failed to present antigen to CD4 or CD8 T cells, whereas resident CD11b-CD8α+DCs presented to CD8 cells, and migratory CD11b-CD8α-DCs presented to CD4 and CD8 T cells. When lung CD11chiDCs and macrophages or langerin+CD11b-CD11chiDCs were depleted using either CD11c - diphtheria toxin receptor (DTR) or langerin-DTR mice, the development of virus-specif c CD8+T cells was severely delayed, which correlated with increased clinical severity and a delayed viral clearance. 120G8+CD11cintpDCs also accumulated in the lung and LNs carrying viral NP, but in their absence, there was no effect on viral clearance or clinical severity. Rather, in pDC-depleted mice, there was a reduction in antiviral antibody production after lung clearance of the virus. This suggests that multiple DCs are endowed with different tasks in mediating protection against influenza virus. http://repub.eur.nl/res/pub/29560/ 2008-07-04T00:00:00Z The limitations of highly active anti-retroviral therapy (HAART) have necessitated the development of alternative therapeutic strategies. One of the approaches that has gained prominence in recent years is therapeutic vaccination. We decided to assess the capacity of mature dendritic cells, derived from blood monocytes of HIV-1 infected patients, to generate functional T-cell responses. For this purpose, we constructed a chimeric mRNA encoding the proteins Tat, Rev and Nef. The TaReNef encoding information was linked to the HLA class II-targeting sequence of DC-LAMP. Broadly directed HIV-specific CD4+and CD8+cytotoxic T cells exhibiting a poly-functional cytokine secretion pattern were generated by co-culturing with autologous chimeric mRNA electroporated dendritic cells. Thus, administration of ex vivo generated dendritic cells expressing the early proteins Tat, Rev and Nef might offer a promising approach for therapeutic vaccination in HIV-1 infection. http://repub.eur.nl/res/pub/8183/ 2002-01-01T00:00:00Z Airway dendritic cells (DCs) are held responsible for inducing sensitization to inhaled antigen, leading to eosinophilic airway inflammation, typical of asthma. However, less information is available about the role of these cells in ongoing inflammation. In a mouse model of asthma, sensitization to ovalbumin (OVA) was induced by intratracheal injection of myeloid OVA-pulsed DCs. Upon OVA aerosol challenge and induction of eosinophilic airway inflammation in sensitized mice, there was a time-dependent and almost 100-fold increase in the number of MHCII(+) CD11b(+) CD11c(+) endogenous airway DCs as well as CD11b(+) blood DCs. The mechanism of this increase was studied. Adoptive transfer experiments demonstrated that accumulation of airway DCs was not due to reduced migration to the mediastinal lymph nodes. Rather, the massive increase in airway and lymph node DCs was supported by an almost 3-fold expansion of myeloid CD31(hi)Ly-6C(neg) hematopoietic precursor cells in the bone marrow (BM). There was no change in any of the other 5 populations revealed by CD31/Ly-6C staining. When these CD31(hi)Ly-6C(neg) BM precursors were sorted and grown in granulocyte macrophage-colony-stimulating factor, they differentiated into MHCII(+) CD11c(+) DCs. The same CD31(hi)Ly-6C(neg) precursors also expressed the eotaxin receptor CCR3 and differentiated into eosinophils when grown in interleukin 5. Serum levels of eotaxin were doubled in mice with inflammation. These findings in an animal model of asthma suggest that the BM increases its output of myeloid precursors to meet the enhanced demand for DCs and eosinophils in inflamed airways. http://repub.eur.nl/res/pub/9442/ 2000-01-01T00:00:00Z The aim of this study was to investigate whether dendritic cells (DCs) can induce sensitization to aeroallergen in a mouse model of allergic asthma. Ovalbumin-pulsed (OVA-pulsed) or unpulsed myeloid DCs that were injected into the airways of naive mice migrated into the mediastinal lymph nodes. When challenged 2 weeks later with an aerosol of OVA, activated CD4 and CD8 lymphocytes, eosinophils, and neutrophils were recruited to the lungs of actively immunized mice. These CD4(+) lymphocytes produced predominantly IL-4 and IL-5 but also IFN-gamma, whereas CD8(+) lymphocytes produced predominantly IFN-gamma. Histological analysis revealed perivascular and peribronchial eosinophilic infiltrates and goblet cell hyperplasia. Studies in IL-4(-/-) and CD28(-/-) mice revealed that production of IL-4 by host cells and provision of costimulation to T cells by DCs were critical for inducing the response. Lung CD4(+) T cells strongly expressed the Th2 marker T1/ST2, and signaling through this molecule via a ligand expressed on DCs was essential for the establishment of airway eosinophilia. These data demonstrate that DCs in the airways induce sensitization to inhaled antigen and that molecules expressed on the surface of these cells are critical for the development of Th2-dependent airway eosinophilia.