Biomedical subjects

K Thielemans

Publications and source records attributed to K Thielemans.

At least 19 recordsLinked to original sources

PubMed2006

Induction of antigen-specific CD8+ cytotoxic T cells by dendritic cells co-electroporated with a dsRNA analogue and tumor antigen mRNA.

The maturation state of dendritic cells (DCs) is an important determinant for the initiation and regulation of adaptive immune responses. In this study, we wanted to assess whether functional activation of human monocyte-derived DCs can be achieved by electroporation of an activation signal in the form of double-stranded (ds) RNA and whether simultaneous electroporation of the dsRNA with tumor antigen encoding mRNA can lead to the induction of a cytotoxic T-lymphocyte (CTL) response. Electroporation of immature DCs with poly(I:C(12)U), a dsRNA analogue, resulted in phenotypic as well as functional changes, indicative of DC maturation. Co-electroporation of DCs with both poly(I:C(12)U) and Melan-A/MART-1 encoding mRNA induced strong anti-Melan-A/MART-1 CD8(+) T-cell responses in vitro. Higher numbers of Melan-A/MART-1-specific CTLs were consistently obtained with poly(I:C(12)U)-activated DCs compared to DCs matured in the presence of an inflammatory cytokine cocktail. These results indicate that DC co-electroporation with both dsRNA and tumor antigen encoding mRNA induces fully activated and antigen-loaded DCs that promote antigen-specific CTL responses and may provide the basis for future immunotherapeutic strategies.

Antigen Presentation↗

PubMed2006

Induction of effective therapeutic antitumor immunity by direct in vivo administration of lentiviral vectors.

Ex vivo lentivirally transduced dendritic cells (DC) have been described to induce CD8+ and CD4+ T-cell responses against various tumor-associated antigens (TAAs) in vitro and in vivo. We report here that direct administration of ovalbumin (OVA) encoding lentiviral vectors caused in vivo transduction of cells that were found in draining lymph nodes (LNs) and induced potent anti-OVA cytotoxic T cells similar to those elicited by ex vivo transduced DC. The cytotoxic T-lymphocyte (CTL) response following direct injection of lentiviral vectors was highly effective in eliminating target cells in vivo up to 30 days after immunization and was efficiently recalled after a boost immunization. Injection of lentiviral vectors furthermore activated OVA-specific CD4+ T cells and this CD4 help was shown to be necessary for an adequate primary and memory CTL response. When tested in therapeutic tumor experiments with OVA+ melanoma cells, direct administration of lentiviral vectors slowed down tumor growth to a comparable extent with the highest dose of ex vivo transduced DC. Taken together, these data indicate that direct in vivo administration of lentiviral vectors encoding TAAs has strong potential for anticancer vaccination.

Animals↗

PubMed2005

Electroporation of immature and mature dendritic cells: implications for dendritic cell-based vaccines.

Until now, studies utilizing mRNA electroporation as a tool for the delivery of tumor antigens to human monocyte-derived dendritic cells (DC) have focused on DC electroporated in an immature state. Immature DC are considered to be specialized in antigen capture and processing, whereas mature DC present antigen and have an increased T-cell stimulatory capacity. Therefore, the consensus has been to electroporate DC before maturation. We show that the transfection efficiency of DC electroporated either before or after maturation was similarly high. Both immature and mature electroporated DC, matured in the presence of an inflammatory cytokine cocktail, expressed mature DC surface markers and preserved their capacity to secrete cytokines and chemokines upon CD40 ligation. In addition, both immature and mature DC can be efficiently cryopreserved before or after electroporation without deleterious effects on viability, phenotype or T-cell stimulatory capacity including in vitro antigen-specific T-cell activation. However, DC electroporated after maturation are more efficient in in vitro migration assays and at least as effective in antigen presentation as DC electroporated before maturation. These results are important for vaccination strategies where an optimal antigen presentation by DC after migration to the lymphoid organs is crucial.

Antigen Presentation↗

PubMed2004

Human pancreatic duct cells can produce tumour necrosis factor-alpha that damages neighbouring beta cells and activates dendritic cells.

AIMS/HYPOTHESIS: In the human pancreas, a close topographic relationship exists between duct cells and beta cells. This explains the high proportion of duct cells in isolated human islet preparations. We investigated whether human duct cells are a source of TNFalpha-mediated interactions with beta cells and immune cells. This cytokine has been implicated in the development of autoimmune diabetes in mice. METHODS: Human duct cells were isolated from donor pancreases and examined for their ability to produce TNFalpha following a stress-signalling pathway. Duct-cell-released TNFalpha was tested for its in vitro effects on survival of human beta cells and on activation of human dendritic cells. RESULTS: Exposure of human pancreatic duct cells to interleukin-1beta (IL-1beta) induces TNFalpha gene expression, synthesis of the 26,000 M(r) TNFalpha precursor and conversion to the 17,000 M(r) mature form, which is rapidly released. This effect is NO-independent and involves p38 MAPK and NF-kappaB signalling. Duct-cell-released TNFalpha contributed to cytokine-induced apoptosis of isolated human beta cells. It also induced activation of human dendritic cells. CONCLUSIONS/INTERPRETATION: Human pancreatic duct cells are a potential source of TNFalpha that can cause apoptosis of neighbouring beta cells and initiate an immune response through activation of dendritic cells. They may thus actively participate in inflammatory and immune processes that threaten beta cells during development of diabetes or after human islet cell grafts have been implanted.

Apoptosis↗

PubMed2004

Unexpected effects of viral interleukin-10-secreting dendritic cells in vivo: preferential inhibition of TH2 responses.

Viral interleukin (IL)-10 (vIL-10) has been widely described as an immunoregulatory cytokine that does not possess the T-cell costimulatory activities of cellular IL-10; it was therefore believed to be a more potent tolerogenic mediator. The immunosuppressive properties of this cytokine are partly attributed to its capacity to render dendritic cells (DCs) unable to undergo full maturation and to activate T cells. We reported here that myeloid DCs retrovirally transduced with vIL-10 had an impaired production of IL-12 and a decreased expression of MHC class II molecules but had minor defects in costimulatory molecule expression and no alteration on CCR5 and CCR7 expression. In mixed leukocyte reaction, vIL-10-transduced C57BL/6 bm12 (MHC class II mismatch) DCs had a reduced capacity to stimulate C57BL/6 wild-type CD4+ T-cell proliferation. We show that bm12 vIL-10-transduced DC administration in CD8-/- C57BL/6 mice promoted IFN-gamma production, down-regulated TH2-type cytokine production, and did not induce skin graft tolerance. These findings suggest that vIL-10-transduced DC may surprisingly facilitate Th1-type inflammatory responses in vivo.

Animals↗

PubMed2002

A MAGE-3 peptide presented by HLA-B44 is also recognized by cytolytic T lymphocytes on HLA-B18.

Antigens encoded by MAGE genes are of particular interest for cancer immunotherapy because of their tumoral specificity and because they are shared by many tumors. Antigenic peptide MEVDPIGHLY, which is encoded by MAGE-3 and is known to be presented by human leukocyte antigen (HLA)-B44, is currently being used in therapeutic vaccination trials. We report here that a cytolytic T lymphocyte (CTL) clone, which is restricted by HLA-B*1801, recognizes the same peptide and, importantly, lyzes HLA-B18 tumor cells expressing MAGE-3. These results imply that the use of peptide MEVDPIGHLY can now be extended to HLA-B18 patients. We also provide evidence that, under limiting amounts of protein MAGE-3, HLA B*1801 and B*4403 compete for binding to the peptide.

Antigen Presentation↗

PubMed2001

Implementation and evaluation of a 3D one-step late reconstruction algorithm for 3D positron emission tomography brain studies using median root prior.

A fully three-dimensional (3D) one-step late (OSL), maximum a posteriori (MAP) reconstruction algorithm based on the median root prior (MRP) was implemented and evaluated for the reconstruction of 3D positron emission tomography (PET) studies. The algorithm uses the ordered subsets (OS) scheme for convergence acceleration and data update during iterations. The algorithm was implemented using the software package developed within the EU project PARAPET (www.brunel.ac.uk/~masrppet). The MRP algorithm was evaluated using experimental phantom and real 3D PET brain studies. Various experimental set-ups in terms of activity distribution and counting statistics were considered. The performance of the algorithm was assessed by calculating figures of merit such as: contrast, coefficient of variation, activity ratio between two regions and full width at half of maximum for resolution measurements. The performance of MRP was compared with that of 3D ordered subsets-expectation maximisation (OSEM) and 3D re-projection (3DRP) algorithms. In all the experimental situations considered, MRP showed: (1) convergence to a stable solution, (2) effectiveness in noise reduction, particularly for low statistics data, (3) good preservation of spatial details. Compared with the OSEM and 3DRP algorithms, MRP provides comparable or better results depending on the parameters used for the reconstruction of the images.

Algorithms↗

PubMed2001

Myeloma isotype-switch variants in the murine 5T myeloma model: evidence that myeloma IgM and IgA expressing subclones can originate from the IgG expressing tumour.

Isotype-switch variants can easily be detected in a significant proportion of multiple myeloma (MM) patients. The biological significance of these isotype-switch variants remains obscure. Therefore, we studied the appearance of these isotype-switch variants in two murine MM models, 5T2MM and 5T33MM, both of IgG isotype. With a MM-specific PCR assay we could detect isotype-switch variants in the bone marrow of both the 5T2MM and the 5T33MM bearing mice, reflecting again the close resemblance of this mouse model to the human MM. These isotype-switch variants were not found in an in vitro stroma-independent variant of the 5T33MM line. However, when this 5T33MMvitro line was injected into young syngeneic mice, isotype-switch variants appeared thereafter in the isolated tumour cells. These isotype-switch variants could only originate from the MM-IgG expressing cell since IgG subclones from the 5T33MMvitro line again gave rise to isotype-switch variants. The appearance of IgA cells can be explained by down-stream switching of IgG to IgA, while the emergence of IgM cells have to occur via trans-switching to the sister chromatid as the Cmu region is deleted from the CIS-chromosome. This study demonstrates that isotype-switch variants originate from the major tumour clone suggesting no role for the MM-IgM expressing cell as a pre-switch precursor MM cell. The appearance of isotype-switch variants should be considered as a rare but normal event now becoming visible due to the high number of clonal cells present in MM.

Amino Acid Sequence↗

PubMed2001

CD40L-induced IL-12 production is further enhanced by the Th2 cytokines IL-4 and IL-13.

Interaction of the CD40L (CD154) molecule on activated T cells with its receptor, CD40, on macrophages and dendritic cells (DC) provides a strong signal for interleukin (IL)-12 production. As IL-12 is the most important factor in driving Th precursor (Thp) cells into T(h)elper 1 cells, CD40-CD40L interactions strongly promote Th1 differentiation. Th2 cytokines (IL-4, IL-13, IL-10) on the other hand, are known to inhibit Th1 differentiation, and to promote either directly or indirectly, Th2 differentiation. Inhibition of lipopolysaccharide (LPS)-induced IL-12 production by IL-4, IL-13 and IL-10 is supposed to be one such mechanism. However, we here report that IL-4 and IL-13 enhance p70 IL-12 production and p40 mRNA transcription by human monocytes when the latter are stimulated trough triggering of CD40. This effect on IL-12 induction is most clear in the presence of interferon (IFN)-gamma, which upregulates CD40 expression. IL-10 potently inhibits IL-12 production. The increased IL-12 production in the presence of IL-4 and IL-13 is however, not the indirect result of a reduction in IL-10 production, but is most likely owing to a direct effect of IL-4 and IL-13. We conclude that IL-4 and IL-13 enhance rather than decrease the IL-12 production by human monocytes during interaction with T cells. This effect can potentially contribute in vivo to switching of an ongoing Th2 response towards a Th1 response and the findings also support the dominant effect of CD40/CD40L interaction on Th1 development, even in the presence of Th2 cytokines.

Autocrine Communication↗

PubMed2001

Autoimmune lymphoproliferative syndrome type III: an indefinite disorder.

Autoimmune Lymphoproliferative Syndrome (ALPS) is a childhood disorder characterized by chronic nonmalignant lymphoproliferation and autoimmunity. Although the pathogenesis is not fully understood, deficient Fas mediated apoptosis appears to be an important factor. This deficiency can be caused by a mutation of the APT1 gene (ALPS type Ia), of the FasL gene (ALPS type Ib), or of the Caspase-10 gene (ALPS type II). In one sub population of patients, no mutations have been identified as yet (ALPS type III). According to published data, the latter group is much smaller than the group of patients with ALPS type Ia. However, because of the variability of the clinical presentation and the absence of a known genetic defect, this disease is difficult to diagnose, the more so as few data have been reported on these patients. Thus, ALPS type III could be more common than believed until now. In this review we provide evidence for this hypothesis.

Animals↗

PubMed2001

Autoimmune lymphoproliferative syndrome type III, an indefinite disorder.

Autoimmune Lymphoproliferative Syndrome (ALPS) is a childhood disorder characterized by chronic nonmalignant lymphoproliferation and autoimmunity. Although the pathogenesis is not fully understood, deficient Fas mediated apoptosis appears to be an important factor. This deficiency can be caused by a mutation of the APTI gene (ALPS type Ia), of the FasL gene (ALPS type Ib), or of the Caspase-10 gene (ALPS type II). In one sub population of patients, no mutations have been identified as yet (ALPS type III). According to published data, the latter group is much smaller than the group of patients with ALPS type Ia. However, because of the variability of the clinical presentation and the absence of a known genetic defect, this disease is difficult to diagnose, the more so as few data have been reported on these patients. Thus, ALPS type III could be more common than believed until now. In this review we provide evidence for this hypothesis.

Animals↗

PubMed2000

A MAGE-A3 peptide presented by HLA-DP4 is recognized on tumor cells by CD4+ cytolytic T lymphocytes.

Antigens encoded by MAGE-A3 and recognized by T cells are interesting targets for tumor immunotherapy because they are strictly tumor specific and shared by many tumors of various histological types. A number of MAGE-A3 antigenic peptides presented by HLA class I molecules have been used in clinical trials, and regressions of melanoma metastasis have been observed. We report here the identification of a MAGE-A3 epitope, TQHFVQENYLEY, presented to CD4+ T lymphocytes by HLA-DP4 molecules, which are expressed in approximately 76% of Caucasians. This new epitope may be useful both for therapeutic vaccination and for the evaluation of the immune response in cancer patients. Interest ingly, the CD4+ T cells lysed HLA-DP4 tumor cells expressing MAGE-A3, indicating that this epitope, in contrast to other class-II MAGE-A3 epitopes, is presented at the surface of tumor cells. The study of this disparity in the presentation of two epitopes from the same protein may lead to a better understanding of the endogenous class II presentation pathway.

Animals↗

PubMed2000

B lymphocytes regulate dendritic cell (DC) function in vivo: increased interleukin 12 production by DCs from B cell-deficient mice results in T helper cell type 1 deviation.

Increasing evidence indicates that dendritic cells (DCs) are the antigen-presenting cells of the primary immune response. However, several reports suggest that B lymphocytes could be required for optimal T cell sensitization. We compared the immune responses of wild-type and B cell-deficient (muMT) mice, induced by antigen emulsified in adjuvant or pulsed on splenic dendritic cells. Our data show that lymph node cells from both control and muMT animals were primed, but each released distinct cytokine profiles. Lymph node T cells from control animals secreted interferon (IFN)-gamma, interleukin (IL)-2, and IL-4, whereas those from muMT mice produced IFN-gamma and IL-2 but no IL-4. To test whether B cells may influence the T helper cell type 1 (Th1)/Th2 balance by affecting the function of DCs, we immunized mice by transferring antigen-pulsed DCs from wild-type or mutant mice. Injection of control DCs induced the secretion of IL-4, IFN-gamma, and IL-2, whereas administration of DCs from muMT animals failed to sensitize cells to produce IL-4. Analysis of IL-12 production revealed that DCs from muMT mice produce higher levels of IL-12p70 than do DCs from wild-type animals. These data suggest that B lymphocytes regulate the capacity of DCs to promote IL-4 secretion, possibly by downregulating their secretion of IL-12, thereby favoring the induction of a nonpolarized immune response.

Adjuvants, Immunologic↗

PubMed2000

B7-1, IFN gamma and anti-CTLA-4 co-operate to prevent T-cell tolerization during immunotherapy against a murine T-lymphoma.

We previously reported on a murine T lymphoma cell line, BW-Sp3, with inherent immunogenicity. BW-Sp3 tumors can elicit an anti-tumor CD8(+) CTL response capable of mediating a regression of subcutaneous tumors. However, this immune response is inadequate to eliminate cancer cells completely in a significant percentage of the recipients, resulting in progressing tumors. In this tumor model, tumor progression correlated with a tolerization of tumor-reactive T cells and cellular immunotherapy of tumor bearing animals, with or without B7-mediated costimulation, even increased tumor progression (Raes et al, 1998). In the present study, we investigated whether the co-expression of IFN gamma, together with B7-1, could have beneficial effects on immunotherapy. Although immunotherapy with IFN gamma and B7-1 single transfectants tended to tolerize anti-tumor T-cells and consequently increased tumor growth, the B7-1/IFN gamma double transfectants resulted in a more beneficial outcome. This phenomenon correlated with an increased CTL-inducing potential of the double transfectants. Secondly, we wondered whether CTLA-4 signalling was involved in the down-regulation of the anti-tumor response. Indeed, when immunotherapy was provided along with anti-CTLA-4, the protection by B71/IFN gamma double transfectants was further improved and the tumor-promoting effect of BW-Sp3(B7-1) was compensated for. Our results indicate that B7-1, IFN gamma and the blockade of CTLA-4 cooperate to tilt the balance in favour of tumor elimination, while either factor alone fails to do so or even promotes tumor growth.

Abatacept↗

PubMed2000

A human antibody directed to the factor VIII C1 domain inhibits factor VIII cofactor activity and binding to von Willebrand factor.

The occurrence of factor VIII (fVIII) inhibitory antibodies is a rare complication of fVIII substitution therapy in mild/moderate hemophilia A patients. fVIII mutations in certain regions such as the C1 domain are, however, more frequently associated with inhibitor, for reasons which remain unclear. To determine whether inhibitors could map to the mutation site, we analyzed at the clonal level the immune response of such a patient with an inhibitor to wild-type but not self-fVIII and an Arg2150His substitution in the C1 domain. Immortalization of the patient B lymphocytes provided a cell line producing an anti-fVIII IgG4kappa antibody, LE2E9, that inhibited fVIII cofactor activity, following type 2 kinetics and prevented fVIII binding to von Willebrand factor. Epitope mapping with recombinant fVIII fragments indicated that LE2E9 recognized the fVIII C1 domain, but not the Arg2150His-substituted C1 domain. Accordingly, LE2E9 did not inhibit Arg2150His fVIII activity. These observations identify C1 as a novel target for fVIII inhibitors and demonstrate that Arg2150His substitution alters a B-cell epitope in the C1 domain, which may contribute to the higher inhibitor incidence in patients carrying such substitution. (Blood. 2000; 95:156-163)

Amino Acid Sequence↗

PubMed2000

Clonally related IgA- and IgE-secreting plasma cells in a myeloma patient.

OBJECTIVES: The purpose of this work was to study the clonal relationship between the cells that secrete monoclonal proteins in an IgA/ IgE double multiple myeloma patient. Double monoclonal gammopathy is a rare condition in which two types of monoclonal proteins can be found in the serum and/or urine of patients with multiple myeloma or gammopathy of undetermined significance. The study of the relationship between the cells expressing the different monoclonal proteins may provide insight in the pathogenesis of these disorders. METHODS: The clonal relationship of the two tumoral plasma cell populations was examined by immunophenotyping and sequence analysis of the variable regions of the immunoglobulin heavy chain genes. Both immunoglobulin sequences were isolated from the bone marrow using a polymerase chain reaction (PCR)-based cloning strategy. Rare isotype-switch variants were detected by a myeloma-specific PCR in combination with different isotype-specific primers. An in vitro culture system, based on the activation of the CD40 molecule on the B cell, was used in order to isolate and expand myeloma-related B cells from peripheral blood that could possibly be regarded as myeloma precursor cells. RESULTS: The variable parts of the immunoglobulin heavy chains linked to either Calpha or Cepsilon were exactly the same, including the same somatic mutations. From the in vitro CD40 cultures B cells could be isolated that either expressed IgA or IgE with exactly the same variable immunoglobulin part as the myeloma clone. No pre-switched IgM myeloma-related B cells could be found. CONCLUSION: Both cell populations in this IgA/IgE myeloma patient shared a common clonal origin. No evidence for a pre-switched IgM precursor myeloma cell was found in this patient.

Aged↗

PubMed2000

Enhanced 3D PET OSEM reconstruction using inter-update Metz filtering.

We present an enhancement of the OSEM (ordered set expectation maximization) algorithm for 3D PET reconstruction, which we call the inter-update Metz filtered OSEM (IMF-OSEM). The IMF-OSEM algorithm incorporates filtering action into the image updating process in order to improve the quality of the reconstruction. With this technique, the multiplicative correction image--ordinarily used to update image estimates in plain OSEM--is applied to a Metz-filtered version of the image estimate at certain intervals. In addition, we present a software implementation that employs several high-speed features to accelerate reconstruction. These features include, firstly, forward and back projection functions which make full use of symmetry as well as a fast incremental computation technique. Secondly, the software has the capability of running in parallel mode on several processors. The parallelization approach employed yields a significant speed-up, which is nearly independent of the amount of data. Together, these features lead to reasonable reconstruction times even when using large image arrays and non-axially compressed projection data. The performance of IMF-OSEM was tested on phantom data acquired on the GE Advance scanner. Our results demonstrate that an appropriate choice of Metz filter parameters can improve the contrast-noise balance of certain regions of interest relative to both plain and post-filtered OSEM, and to the GE commercial reprojection algorithm software.

Algorithms↗

PubMed2000

Myeloid dendritic cells induce Th2 responses to inhaled antigen, leading to eosinophilic airway inflammation.

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.

Administration, Inhalation↗