Biomedical subjects

Kris Thielemans

Publications and source records attributed to Kris Thielemans.

At least 19 recordsLinked to original sources

PubMed2006

Melan-A/MART-1-specific CD4 T cells in melanoma patients: identification of new epitopes and ex vivo visualization of specific T cells by MHC class II tetramers.

Over the past decade, many efforts have been made to identify MHC class II-restricted epitopes from different tumor-associated Ags. Melan-A/MART-1(26-35) parental or Melan-A/MART-1(26-35(A27L)) analog epitopes have been widely used in melanoma immunotherapy to induce and boost CTL responses, but only one Th epitope is currently known (Melan-A51-73, DRB1*0401 restricted). In this study, we describe two novel Melan-A/MART-1-derived sequences recognized by CD4 T cells from melanoma patients. These epitopes can be mimicked by peptides Melan-A27-40 presented by HLA-DRB1*0101 and HLA-DRB1*0102 and Melan-A25-36 presented by HLA-DQB1*0602 and HLA-DRB1*0301. CD4 T cell clones specific for these epitopes recognize Melan-A/MART-1+ tumor cells and Melan-A/MART-1-transduced EBV-B cells and recognition is reduced by inhibitors of the MHC class II presentation pathway. This suggests that the epitopes are naturally processed and presented by EBV-B cells and melanoma cells. Moreover, Melan-A-specific Abs could be detected in the serum of patients with measurable CD4 T cell responses specific for Melan-A/MART-1. Interestingly, even the short Melan-A/MART-1(26-35(A27L)) peptide was recognized by CD4 T cells from HLA-DQ6+ and HLA-DR3+ melanoma patients. Using Melan-A/MART-1(25-36)/DQ6 tetramers, we could detect Ag-specific CD4 T cells directly ex vivo in circulating lymphocytes of a melanoma patient. Together, these results provide the basis for monitoring of naturally occurring and vaccine-induced Melan-A/MART-1-specific CD4 T cell responses, allowing precise and ex vivo characterization of responding T cells.

Amino Acid Sequence↗

PubMed2006

A new tumor-specific antigen encoded by MAGE-C2 and presented to cytolytic T lymphocytes by HLA-B44.

A panel of cytolytic T lymphocyte (CTL) clones was isolated from metastases and blood samples of a melanoma patient vaccinated with MAGE-3.A1-pulsed autologous dendritic cells. We report here the identification of a new antigen encoded by the MAGE-C2 cancer-germline gene. This antigen is recognized by some of these CTL on HLA-B*4403. The sequence of the peptide is SESIKKKVL. It is processed in various melanoma cell lines expressing MAGE-C2 and HLA-B*4403. Because of the expression pattern of gene MAGE-C2, this new antigen is strictly tumor-specific and could therefore be used for peptide-based antitumoral vaccination.

Amino Acid Sequence↗

PubMed2006

From pathogen to medicine: HIV-1-derived lentiviral vectors as vehicles for dendritic cell based cancer immunotherapy.

Over the years, the unique capacity of dendritic cells (DC) for efficient activation of naive T cells has led to their extensive use in cancer immunotherapy protocols. In order to be able to fulfil their role as antigen-presenting cells, the antigen of interest needs to be efficiently introduced and subsequently correctly processed and presented by the DC. For this purpose, a variety of both viral and non-viral antigen-delivery systems have been evaluated. Amongst those, HIV-1-derived lentiviral vectors have been used successfully to transduce DC. This review considers the use of HIV-1-derived lentiviral vectors to transduce human and murine DC for cancer immunotherapy. Lentivirally transduced DC have been shown to present antigenic peptides, prime transgene-specific T cells in vitro and elicit a protective cytotoxic T-lymphocyte (CTL) response in animal models. Different parameters determining the efficacy of transduction are considered. The influence of lentiviral transduction on the DC phenotype and function is described and the induction of immune responses by lentivirally transduced DC in vitro and in vivo is discussed in detail. In addition, direct in vivo administration of lentiviral vectors aiming at the induction of antigen-specific immunity is reviewed. This strategy might overcome the need for ex vivo generation and antigen loading of DC. Finally, future perspectives towards the use of lentiviral vectors in cancer immunotherapy are presented.

Animals↗

PubMed2006

Correction of head movement on PET studies: comparison of methods.

UNLABELLED: Head movement presents a continuing problem in PET studies. Head restraint minimizes movement but is unreliable, resulting in the need to develop alternative strategies. These include frame-by-frame (FBF) realignment or use of motion tracking (MT) during the scan to realign PET acquisition data. Here we present a comparative analysis of these 2 methods of motion correction. METHODS: Eight volunteers were examined at rest using (11)C-raclopride PET with the radioligand administered as a bolus followed by constant infusion to achieve steady state. Binding potential (BP) was estimated using the ratio method during 2 periods of the scan at steady state. Head movement was compensated by using coregistration between frames (FBF) and 3 methods using MT measurements of head position acquired with a commercially available optical tracking system. RESULTS: All methods of realignment improved test-retest reliability and noise characteristics of the raw data, with important consequences for the power to detect small changes in radiotracer binding, and the potential to reduce false-positive and false-negative results. MT methods were superior to FBF realignment using coregistration on some indices. CONCLUSION: Such methods have considerable potential to improve the reliability of PET data with important implications for the numbers of volunteers required to test hypotheses.

Algorithms↗

PubMed2005

Dendritic cells differentiated in the presence of IFN-{beta} and IL-3 are potent inducers of an antigen-specific CD8+ T cell response.

Dendritic cells (DC) are professional antigen-presenting cells that are used in vaccine approaches to cancer. Classically, mature monocyte-derived DC are generated in vitro in the presence of interleukin (IL)-4, granulocyte macrophage-colony stimulating factor, and inflammatory cytokines (G4-DC). Recently, it has been described that DC can also be generated in the presence of IL-3 and interferon (IFN)-beta and that these DC are efficiently matured using polyriboinosinic polyribocytidylic acid (I3-DC). In this study, a series of in vitro experiments was performed to compare side-by-side I3-DC and G4-DC as vaccine adjuvants. Phenotypic characterization of the DC revealed differences in the expression of the monocyte marker CD14 and the maturation marker CD83. Low expression of CD14 and high expression of CD83 characterized G4-DC, whereas I3-DC displayed intermediate expression of CD14 and CD83. Both types of DC were as potent in the induction of allogeneic T cell proliferation. Upon CD40 ligation, G4-DC produced lower amounts of IFN-alpha and pulmonary and activation-regulated chemokine, similar amounts of IL-6, macrophage-inflammatory protein (MIP)-1alpha, and MIP-1beta, and higher amounts of IL-12 p70, tumor necrosis factor alpha, and MIP-3beta than I3-DC. We further evaluated whether the DC could be frozen/thawed without loss of cell number, viability, phenotype, and function. After freezing/thawing, 56.0% +/- 9.0% of I3-DC and 77.0% +/- 3.0% of G4-DC (n=9) were recovered as viable cells, displaying the same phenotype as their fresh counterparts. Finally, in vitro stimulations showed that fresh and frozen peptide-loaded I3-DC are more potent inducers of Melan-A-specific CD8(+) T cell responses than G4-DC. The antigen-specific T cells were functional as shown in cytotoxicity and IFN-gamma secretion assay.

CD8-Positive T-Lymphocytes↗

PubMed2005

Monitoring of anti-vaccine CD4 T cell frequencies in melanoma patients vaccinated with a MAGE-3 protein.

Quantitative evaluation of T cell responses of patients receiving antitumoral vaccination with a protein is difficult because of the large number of possible HLA-peptide combinations that could be targeted by the response. To evaluate the responses of patients vaccinated with protein MAGE-3, we have developed an approach that involves overnight stimulation of blood T cells with autologous dendritic cells loaded with the protein, sorting by flow cytometry of the T cells that produce IFN-gamma, cloning of these cells, and evaluation of the number of T cell clones that secrete IFN-gamma upon stimulation with the Ag. An important criterion is that T cell clones must recognize not only stimulator cells loaded with the protein, but also stimulator cells transduced with the MAGE-3 gene, so as to exclude the T cells that recognize contaminants generated by the protein production system. Using this approach it is possible to measure T cell frequencies as low as 10(-6). We analyzed the frequencies of anti-vaccine CD4 T cells in five metastatic melanoma patients who had been injected with a MAGE-3 protein without adjuvant and showed evidence of tumor regression. Anti-MAGE-3 CD4 T cells were detected in one of the five patients. The frequency of the anti-MAGE-3 CD4 T cells was estimated at 1/60,000 of the CD4 T cells in postvaccination blood samples, representing at least an 80-fold increase in the frequency found before immunization. The frequencies of one anti-MAGE-3 CD4 T cell clonotype were confirmed by PCR analysis on blood lymphocytes. The 13 anti-MAGE-3 clones, which corresponded to five different TCR clonotypes, recognized the same peptide presented by HLA-DR1.

Amino Acid Sequence↗

PubMed2005

Genetic approaches for the induction of a CD4+ T cell response in cancer immunotherapy.

Recently, it has become more and more obvious that not only CD8+ cytotoxic T lymphocytes, but also CD4+ T helper cells are required for the induction of an optimal, long-lasting anti-tumor immune response. CD4+ T helper cells, and in particular IFN-gamma-secreting type 1 T helper cells, have been shown to fulfill a critical function in the mounting of a cancer-specific response. Consequently, targeting antigens into MHC class II molecules would greatly enhance the efficacy of an anti-cancer vaccine. The dissection of the MHC class II presentation pathway has paved the way for rational approaches to achieve this goal: novel systems have been developed to genetically manipulate the MHC class II presentation pathway. First, different genetic approaches have been used for the delivery of known epitopes into the MHC class II processing pathway or directly onto the peptide-binding groove of the MHC molecules. Second, several strategies exist for the targeting of whole tumor antigens, containing both MHC class I and class II restricted epitopes, to the MHC class II processing pathway. We review these data and describe how this knowledge is currently applied in vaccine development.

Animals↗

PubMed2005

In vivo depletion of CD4+CD25+ regulatory T cells enhances the antigen-specific primary and memory CTL response elicited by mature mRNA-electroporated dendritic cells.

We previously described mRNA electroporation as an efficient gene delivery method to introduce tumor-antigens (Ag) into murine immature dendritic cells (DC). Here, we further optimize the protocol and evaluate the capacity of mRNA-electroporated DC as a vaccine for immunotherapy. First, the early DC maturation kinetics and the effect of different lipopolysaccharide incubation periods on the phenotypic maturation profile of DC are determined. Next, we show that either immature or mature DC are equally well electroporated and express and present the transgene at a comparable level after electroporation. We point out that the mRNA electroporation results in a negative effect on the interleukin (IL)-12p70, IL-6, and tumor necrosis factor-alpha secretion after maturation. Nevertheless, mRNA-electroporated DC induce an effective cytotoxic T lymphocyte (CTL) response in vivo. Mature electroporated DC are significantly more potent in eliciting an Ag-specific CD8+ CTL response compared to their immature electroporated counterparts. In addition, a significant improvement in CTL response is obtained both in the primary and in the memory effector phases when CD4+CD25+ regulatory T cells (Treg) are depleted in vivo prior to immunization. These findings are further substantiated in tumor protection experiments and hold convincing evidence for the merit of Treg cell depletion prior to immunization with mRNA-electroporated DC.

Animals↗

PubMed2004

Correlation between tumor regression and T cell responses in melanoma patients vaccinated with a MAGE antigen.

The cancer-germline gene MAGE-3 codes for tumor-specific antigens recognized on many tumors by T lymphocytes. A MAGE-3 antigen presented by HLA-A1 has been used in several vaccination trials on metastatic melanoma patients. Only a small minority of patients have shown evidence of tumor regression. Attempts to correlate the tumor rejections with the cytotoxic T lymphocyte (CTL) response against the vaccine have been hampered by the low level of these responses. In noncancerous individuals, the frequency of the T cell precursors against antigen MAGE-3.A1 is approximately 4 x 10(-7) CD8 T cells. The diversity of the T cell receptor repertoire of these anti-MAGE-3.A1 precursors was analyzed in one individual. The results indicate that it is very likely that the repertoire comprises >100 clonotypes. On this basis, it is possible to use not only the frequency of CTL precursors in the blood but also the presence of dominant clonotypes to ascertain in patients the existence of anti-MAGE-3.A1 responses as low as 10(-6) of CD8. With this approach, we observed a correlation between tumor regression and anti-MAGE-3.A1 CTL responses in patients vaccinated with a recombinant virus encoding the antigen and also in patients vaccinated with peptide-pulsed dendritic cells. In contrast, for patients showing tumor regression after vaccination with peptide alone, CTL responses were almost never observed. It is possible that even those CTL responses that are below our present detection level can trigger a sequence of events that leads to tumor regression.

Amino Acid Sequence↗

PubMed2004

Dendritic cells retrovirally overexpressing IL-12 induce strong Th1 responses to inhaled antigen in the lung but fail to revert established Th2 sensitization.

It has been postulated that low-level interleukin (IL)-12 production of antigen-presenting cells is associated with the risk of developing atopic asthma. To study the relationship between IL-12 production capacity of dendritic cells (DCs) and development of T helper type 2 (Th2) responses in the lung, we genetically engineered DCs to constutively overexpress bioactive IL-12. Retrovirally mediated overexpression of IL-12 in DCs strongly polarized naive ovalbumin (OVA)-specific CD4+ T cells toward Th1 effector cells in vitro. After intratracheal injection, OVA-pulsed IL-12-overexpressing DCs failed to induce Th2 responses in vivo and no longer primed mice for Th2-dependent eosinophilic airway inflammation upon OVA aerosol challenge, readily observed in mice immunized with sham-transfected, OVA-pulsed DCs. Analysis of a panel of cytokines and chemokines in the lung demonstrated that the lack of Th2 sensitization was accompanied by increased production of the Th1 cytokine interferon-gamma (IFN-gamma), chemokines induced by IFN-gamma, and the immunoregulatory cytokine IL-10. When Th2 priming was induced using OVA/alum prior to intratracheal DC administration, DCs constitutively expressing IL-12 were no longer capable of preventing eosinophilic airway inflammation and even enhanced it. These data show directly that high-level expression of IL-12 in DCs prevents the development of Th2 sensitization. Enhancing IL-12 production in DCs should be seen as a primary prevention strategy for atopic disorders. Enhancing IL-12 production in DCs is less likely to be of benefit in already Th2-sensitized individuals.

Animals↗

PubMed2004

Messenger RNA-electroporated dendritic cells presenting MAGE-A3 simultaneously in HLA class I and class II molecules.

An optimal anticancer vaccine probably requires the cooperation of both CD4(+) Th cells and CD8(+) CTLs. A promising tool in cancer immunotherapy is, therefore, the genetic modification of dendritic cells (DCs) by introducing the coding region of a tumor Ag, of which the antigenic peptides will be presented in both HLA class I and class II molecules. This can be achieved by linking the tumor Ag to the HLA class II-targeting sequence of an endosomal or lysosomal protein. In this study we compared the efficiency of the targeting signals of invariant chain, lysosome-associated membrane protein-1 (LAMP1) and DC-LAMP. Human DCs were electroporated before or after maturation with mRNA encoding unmodified enhanced green fluorescent protein (eGFP) or eGFP linked to various targeting signals. The lysosomal degradation inhibitor chloroquine was added, and eGFP expression was evaluated at different time points after electroporation. DCs were also electroporated with unmodified MAGE-A3 or MAGE-A3 linked to the targeting signals, and the presentation of MAGE-A3-derived epitopes in the context of HLA class I and class II molecules was investigated. Our data suggest that proteins linked to the different targeting signals are targeted to the lysosomes and are indeed presented in the context of HLA class I and class II molecules, but with different efficiencies. Proteins linked to the LAMP1 or DC-LAMP signal are more efficiently presented than proteins linked to the invariant chain-targeting signal. Furthermore, DCs electroporated after maturation are more efficient in Ag presentation than DCs electroporated before maturation.

Antigen Presentation↗

PubMed2004

Identification of new antigenic peptide presented by HLA-Cw7 and encoded by several MAGE genes using dendritic cells transduced with lentiviruses.

Antigens encoded by MAGE genes are of particular interest for cancer immunotherapy because they are tumor specific and shared by tumors of different histological types. Several clinical trials are in progress with MAGE peptides, proteins, recombinant poxviruses, and dendritic cells (DC) pulsed with peptides or proteins. The use of gene-modified DC would offer the major advantage of a long-lasting expression of the transgene and a large array of antigenic peptides that fit into the different HLA molecules of the patient. In this study, we tested the ability of gene-modified DC to prime rare Ag-specific T cells, and we identified a new antigenic peptide of clinical interest. CD8(+) T lymphocytes from an individual without cancer were stimulated with monocyte-derived DC, which were infected with a second-generation lentiviral vector encoding MAGE-3. A CTL clone was isolated that recognized peptide EGDCAPEEK presented by HLA-Cw7 molecules, which are expressed by >40% of Caucasians. Interestingly, this new tumor-specific antigenic peptide corresponds to position 212-220 of MAGE-2, -3, -6, and -12. HLA-Cw7 tumor cell lines expressing one of these MAGE genes were lysed by the CTL, indicating that the peptide is efficiently processed in tumor cells and can therefore be used as target for antitumoral vaccination. The risk of tumor escape due to appearance of Ag-loss variants should be reduced by the fact that the peptide is encoded by several MAGE genes.

Amino Acid Sequence↗

PubMed2004

Depending on their maturation state, splenic dendritic cells induce the differentiation of CD4(+) T lymphocytes into memory and/or effector cells in vivo.

There is increasing evidence that dendritic cells (DC) display opposite functions in the immune system, as they may induce immunity or tolerance depending on intrinsic and environmental factors. In mice, adoptive transfer of mature DC pulsed extracorporeally with antigen induces the development of antigen-specific Th1- and Th2-type CD4(+) cells. In this work, we compared the adjuvant properties of immature (freshly isolated) and mature (cultured) splenic DC in vivo. Our data show that injection of either cell population induces the clonal expansion of CD4(+) T cells but that only mature DC trigger their differentiation into effector cells producing IFN-gamma. In contrast, transfer of immature DC provokes the development of intermediates in the differentiation process, similar to the central memory cells. These observations, together with data in the literature, suggest that DC may induce tolerance, memory, or immunity depending on their maturation state.

Adoptive Transfer↗

PubMed2004

Exploiting dendritic cells for cancer immunotherapy: genetic modification of dendritic cells.

Dendritic cells (DCs) are pivotal regulators of immune reactivity and immune tolerance. The observation that DCs can recruit naive T cells has invigorated cancer immunology and led to the proposal of DCs as the basis for vaccines designed for the treatment of cancer. Designing effective strategies to load DCs with antigens is a challenging field of research. The successful realization of gene transfer to DCs will be highly dependent on the employed vector system. Here, we review various viral and non-viral gene transfer systems, and discuss their distinct characteristics and possible advantages and disadvantages in respect to their use in DC-based immunotherapy.

Cancer Vaccines↗

PubMed2004

Side-by-side comparison of lentivirally transduced and mRNA-electroporated dendritic cells: implications for cancer immunotherapy protocols.

The use of tumor antigen-loaded dendritic cells (DC) is one of the most promising approaches to inducing a tumor-specific immune response. We compared electroporation of mRNA to lentiviral transduction for the delivery of tumor antigens to human monocyte-derived and murine bone marrow-derived DC. Both lentiviral transduction and mRNA electroporation induced eGFP expression in on average 81% of human DC. For murine DC, eGFP mRNA electroporation (62%) proved to be more efficient than lentiviral transduction (47%). When we used tNGFR as a transgene we observed lentiviral pseudotransduction that overestimated lentiviral efficiency. Neither gene transfer method had an adverse effect on viability, phenotype, or allostimulatory capacity of either human or murine DC. Yet, the mRNA-electroporated DC showed a reduced production of IL-12p70 compared to their lentivirally transduced and unmodified counterparts. Human Ii80MAGE-A3-modified DC and murine Ii80tOVA-modified DC were able to present antigenic epitopes in the context of MHC class I and class II. Both types of modified murine DC were able to induce OVA-specific cytotoxic T cells in vivo; however, the mRNA-electroporated DC were less potent. Our data indicate that this may be related to their impaired IL-12 production.

Animals↗

PubMed2004

Activation of monocytes via the CD14 receptor leads to the enhanced lentiviral transduction of immature dendritic cells.

In this study, we compared dendritic cells (DCs) differentiated from positively selected monocytes (CD14-DCs) to DCs differentiated from adherence-selected monocytes (adh-DCs) with emphasis on lentiviral transduction. Using a second-generation, triple-helix containing, self-inactivating lentiviral vector at a multiplicity of infection (MOI) of 15, we observed enhanced transduction of CD14-DCs (72.8 +/- 5.3%, mean fluorescence intensity [MFI] = 166 +/- 76) compared to adh-DCs (32.3 +/- 13.1%, MFI = 119 +/- 76, n = 5). More importantly, the efficiency to transduce adh-DCs was significantly increased when monocytes were incubated with antiCD14 antibody coupled beads, anti-CD14 antibodies, or lipopolysaccharide (LPS), reaching transduction efficiencies up to 86.6%, 53.3%, and 80.9%, respectively. We showed that this enhanced transduction was correlated to an activation of the monocytes, characterized by the up regulation of the cytokines interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha and the de novo synthesis of IL-6 and IL-10. However, the enhanced transduction of immature CD14-DCs was not correlated with a progression in the cell cycle from G(0) to G(1). We further showed that CD14-DCs were phenotypically comparable to adh-DCs. Functional analysis revealed that there were no differences in allostimulatory capacity, production of IL-12 p70 on CD40 ligation or expression of IL-1beta, IL-6, IL-8, IL-10, IL-12, and TNF-alpha as evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). Finally, we showed that lentivirally transduced CD14-DCs were equally capable as adh-DCs in stimulating MAGE-A3 antigen-specific CD4(+) and CD8(+) T cells in vitro.

Antibodies, Monoclonal↗

PubMed2004

Object dependency of resolution in reconstruction algorithms with interiteration filtering applied to PET data.

In this paper, we study the resolution properties of those algorithms where a filtering step is applied after every iteration. As concrete examples we take filtered preconditioned gradient descent algorithms for the Poisson log likelihood for PET emission data. For nonlinear estimators, resolution can be characterized in terms of the linearized local impulse response (LLIR). We provide analytic approximations for the LLIR for the class of algorithms mentioned above. Our expressions clearly show that when interiteration filtering (with linear filters) is used, the resolution properties are, in most cases, spatially varying, object dependent and asymmetric. These nonuniformities are solely due to the interaction between the filtering step and the Poisson noise model. This situation is similar to penalized likelihood reconstructions as studied previously in the literature. In contrast, nonregularized and postfiltered maximum-likelihood expectation maximization (MLEM) produce images with nearly "perfect" uniform resolution when convergence is reached. We use the analytic expressions for the LLIR to propose three different approaches to obtain nearly object independent and uniform resolution. Two of them are based on calculating filter coefficients on a pixel basis, whereas the third one chooses an appropriate preconditioner. These three approaches are tested on simulated data for the filtered MLEM algorithm or the filtered separable paraboloidal surrogates algorithm. The evaluation confirms that images obtained using our proposed regularization methods have nearly object independent and uniform resolution.

Algorithms↗

PubMed2004

Post-transplantation tumour load in bone marrow, as assessed by quantitative ASO-PCR, is a prognostic parameter in multiple myeloma.

High-dose therapy (HDT) and autologous transplantation prolongs remission duration and survival in multiple myeloma (MM), but relapse still occurs at a median of 2 years post-HDT. In order to investigate whether the number of residual tumour cells in the bone marrow (BM) after transplantation can predict the duration of response, a quantitative allele-specific oligonucleotide polymerase chain reaction (qASO-PCR) assay was used to measure tumour load in BM at 3-6 months post-HDT in 67 patients. The method of maximally selected log-rank statistics was used to test for the existence of a cut-off value in the BM tumour load data set. A cut-off value with respect to progression-free survival (PFS) was identified (P = 0.001). The estimated threshold for placing patients into a "good" or "bad" prognostic group was 0.015% (n = 22 and 38 respectively) with a median PFS of 64 months vs. 16. Multivariate analysis showed grouping by PCR result to be an independent prognostic factor for PFS (estimated hazard ratio after shrinkage, 3.91). This study identifies for the first time a threshold of the post-HDT tumour load with prognostic significance for PFS in MM. Quantitative molecular assessment thus may help to identify those patients who are in need of further treatment early after autologous transplantation.

Adult↗