1,721,050 research outputs found
Advanced Magnetic Resonance Imaging in the Workup of Gliomas
No full textGliomas are the most common primary brain tumours in adults. They cover a broad range of lesions with distinct differences in malignancy. Glioblastoma are the most aggressive and the most frequent of gliomas. With the current standard of care consisting of surgery and combined radiochemotherapy, the average life expectancy for patients with glioblastoma is 14 months. As such, there is an obvious need for more effective therapies. Among several innovative treatment strategies, dendritic cell immune therapy has emerged as a promising, safe and feasible treatment option for inducing long-term survival in at least a subpopulation of patients with high grade gliomas. The diagnosis and follow-up of patients with gliomas relies mainly on medical imaging and in specific magnetic resonance imaging (MRI). However, routinely used MRI techniques still present many difficulties in clinical practice. Current conventional MRI techniques are insufficient to accurately define glioma grade. However, adequate determination of the aggressiveness of gliomas is of capital importance because treatment regimens and prognosis depend on malignancy grade. Moreover, in the follow-up of patients with glioblastoma, the differentiation between therapy-induced inflammatory reactions and early tumour relapse remains challenging, as the radiological characteristics of both entities are similar. This is a frequently encountered problem in patients treated with the standard of care as well as in patients treated with more innovative therapies such as immune therapy.Currently used conventional MRI techniques do not enable adequate documentation of the nature of the tumoural pathophysiology of gliomas. Advanced MR methods, however, address parameters like diffusion, tissue organisation, blood flow or metabolism, induced by pathological changes. Therefore, advanced MR techniques potentially provide more insights in the problems encountered with the currently used conventional MR techniques as various aspects of tumoural pathophysiology can be demonstrated. Diffusion MRI allows non-invasive mapping of the diffusion process of water molecules in a biological environment and is therefore able to provide information on the organization of biological tissue. In this thesis work, we use diffusion weighted imaging (DWI) and diffusion kurtosis imaging (DKI). DKI is the most recent technique in diffusion MRI and provides information on diffusional heterogeneity and microstructural complexity. Furthermore, we used dynamic susceptibility-weighted imaging, a perfusion technique that measures brain perfusion and provides markers of blood flow and blood volume in healthy and diseased tissue. Finally, chemical shift imaging (CSI) was used to study the metabolism of gliomas. For example, CSI provides markers of neuronal density, cell turn over and energy metabolism. In this thesis work, we explore the potential of these advanced MRI techniques to improve the workup of patients with gliomas in five experiments. In a first experiment, we validated the reproducibility of a 2D - CSI acquisition protocol accelerated with parallel imaging, using automated repositioning software. CSI provides measures relating to the metabolism of the tissue under investigation. The reproducibility was tested on ten healthy volunteers, each imaged three times on two different 3T MRI scanners, receiving anatomical imaging and two identical CSI measurements. The automated repositioning software was tested for consistent repositioning of the CSI grid. This proved to be very robust which is of utmost importance in conducting longitudinal studies. Comparison of CSI data acquisition on the same volunteers using different MR scanners indicated acceptable reproducibility, However, great care should be taken for the interpretation of the results of metabolite signals with an inherent low SNR as the error estimates were still substantial. Moreover, the effect of the MRI scanner on the variability of the results was negligible, which implies that scanners could be interchanged, a pre-requisite for inter-institutional studies. As such, in this first experiment, we proposed a CSI protocol with good reproducibility, applicable in clinical routine. The proposed protocol was afterwards implemented in two clinical studies in this thesis work. In a second experiment of this thesis, we assessed the diagnostic accuracy of a novel diffusion imaging technique, diffusion kurtosis imaging (DKI), in determining glioma grade. We found that kurtosis parameters better separated between low and high grade gliomas, compared to conventional diffusion metrics. Although the outcome of this study was promising, results were demonstrated on a group level. Nonetheless, to get acceptance in clinical setting, diagnosis should be made for the individual patient. Therefore, in a third study, in order to increase diagnostic performance, we combined DKI with dynamic susceptibility-weighted MRI (DSC-MRI) and CSI, as established in the first experiment, in a multiparametric approach to assess glioma grade. By integrating these three advanced MRI techniques in a decision tree algorithm, diagnostic accuracy could be increased up to individual patient level. The best performing modality, when considering the techniques separately, was DSC-MRI. These results are considerably beneficial for patients with gliomas because using this algorithm, an accurate diagnosis can be made before any invasive procedure is performed. An early diagnosis allows optimizing the therapeutic strategy in view of the patient's individual needs. The last part of this thesis work focused on the follow-up of glioblastoma patients treated with dendritic cell immune therapy. The two experiments presented in this thesis work are, to our knowledge, the first studies to explore immune therapy-induced inflammatory responses with MRI. In a first small, retrospective study, we reviewed the MR imaging follow-up in patients with recurrent GBM, treated with immune therapy as single treatment approach. We focused on the available DSC-MRIand DWI exams obtained in routine clinical setting, and found that mainly perfusion measures and to a lesser extent diffusion measures, were potential markers to differentiate between immune therapy inflammatory responses and tumour relapse.These findings were further explored in a more elaborate prospective, longitudinal imaging follow-up study. Patients with relapsed glioblastoma treated with immune therapy as the single approach as well as patients with primary diagnosis of GBM receiving the standard treatment strategy combined with immune therapy were imaged with an extensive imaging protocol, consisting of DSC-MRI, DKI and CSI. The aims of this study were threefold: (i) documentation of the time course of immune therapy-induced inflammatory changes versus tumour relapse on conventional imaging, (ii) characterization of the vaccine-induced inflammatory immune responses and tumour relapse using advanced MRI techniques and (iii) defining prognostic biomarkers. The first results of this ongoing study indicate that patients with relapsed glioblastoma display distinct imaging patterns compared to patients with the primary diagnosis of glioblastoma receiving immune therapy. Furthermore, we were able to confirm the results of the retrospective study. Perfusion related measures were able to differentiate inflammatory reactions from tumour relapse and possibly provide clues for the patients prognosis. Also kurtosis related parameters seemed promising. As such, these results bring further insights in the working mechanism of immune therapy and can help guide clinical trials. Furthermore, treatment strategy can be improved and the patient can be informed more accurately on his or her disease status. Although the results of this thesis contribute to establish the role of advanced MRI techniques in the characterization of the pathophysiology of gliomas, future research and further standardization is warranted to gain acceptance for these advanced MRI techniques as clinically valid and applicable techniques.status: Publishe
Mechanismen van tolerantie inductie door middel van costimulatieblokkade in een muismodel van graft-versus-host ziekte
Get PDFTargeting costimulatory interactions between T cells and APC can potenti ally be useful to treat auto-immune diseases and to prevent transplant r ejection. Blocking of these interactions has already been shown to lead to tolerance in several autoimmunity and transplantation models, but the underlying mechanisms are incompletely understood. Here, we targeted tw o particular costimulatory pathways (the CD28 CD80/CD86 and the CD40 - CD40L pathway) and tried to elucidate the mechanisms responsible for pr omoting tolerance when both pathways are blocked. More specifically, we investigated the involvement of regulatory T cells and of deletion of al lo-reactive cells in the induction and maintenance of tolerance after co stimulation blockade. To study the mechanisms responsible for promoting tolerance after costim ulation blockade, a MHC-mismatched parent-in-F1 mouse model of GVHD was developed. Injection of splenocytes from the C57BL/6 parent strain into a sublethally irradiated F1 offspring (C57BL/6xC3H) induced a GVHD chara cterized by severe pancytopenia. Treatment with anti-CD40L mAb and CTLA4 -Ig every three days during three weeks after splenocyte injection preve nted disease development and induced a long lasting state of stable mixe d chimerism (> 120 days). In parallel, host-specific tolerance was achie ved as demonstrated by lack of host-directed allo-reactivity of donor-ty pe T cells both in vitro and in vivo. Chimerism and tolerance were also obtained after transfer of CD25+ cell depleted splenocytes, showing that CD25+ natural Treg cells are not essential for tolerance induction. We further showed that costimulation blockade results in enhanced Treg cell activity at early timepoints (day 6 day 30) after splenocyte transfer . This was demonstrated by the presence of a high percentage of Foxp3+ c ells among donor CD4+ cells in the spleen of treated animals, and the fi nding that isolated donor T cells at an early timepoint (day 30) after s plenocyte transfer displayed suppressive capacity in vitro. When sorted Foxp3- splenocytes were used as donor cells, there was no de novo induct ion of Foxp3+ cells, indicating that the high percentage of Foxp3+ cells among donor cells originated from Foxp3+ cells present in the donor cel l inoculum. At later timepoints (> 30 days after splenocyte transfer), d eletion of host-reactive T cells was found to be a major mechanism respo nsible for tolerance. Finally, we provided evidence that IL-10 is involv ed in the induction of tolerance by costimulation blockade. In the absen ce of IL-10 producing donor cells, costimulation blockade could not prev ent lethal GVHD in most of the recipient animals.status: Publishe
Costimulatory signals and activation of regulatory Tcells
No full textNormal 0 21 false false false DE X-NONE X-NONE mso-ansi-language:EN-GB" lang="EN-GB">Since costimulation is crucial for T cell-activation,attempts to block those pathways have been effective in preventing unwantedimmune reactions. In particular, blocking the CD28/B7 (using CTLA-4Ig) and theCD40/CD40L (using anti-CD40L antibodies) interaction is highly effective inpreventing T cell-mediated autoimmune diseases, transplant rejection and graft versus host disease (GvHD). Despite itssuccess, the mechanisms by which costimulation blockade induces tolerance arestill not completely understood. In this study we therefore aimed to elucidatethe effects of CD28/B7 and CD40/CD40L blockade on effector T (Teff) cells andon regulatory T (Treg) cells. mso-ansi-language:EN-GB" lang="EN-GB">To this end we used a fully MHC-mismatch parent intoF1 mouse-model of GvHD as well as murine mixed lymphocyte reactions (MLRs), inwhich CD4+ responder cells were stimulated with allogeneicsplenocytes. We showed that costimulatory signals arerequired for efficientpriming and activation of naïve T cells, as CTLA-4Ig and MR1 (an anti-CD40Lantibody) could prevent allogeneic T cell-responses normal">in vitro and in vivo .Further characterization of the effect of CTLA-4Ig and MR1 in a mouse-model of GvHDshowed that MR1 targets only CD4+ T cells,while CTLA-4Ig exerts amild suppressive effect on CD4+ and CD8+ T cells.Consequently, both molecules are necessary to induce tolerance to allo-antigensand completely prevent Teff cell-expansion and activation. In addition, weshowed that the suppressive effect of CTLA-4Igand MR1 is accompanied by anincrease in Treg cell-frequency in vitro and in vivo . We further demonstrated in vitro that Treg cells are lessdependent on CD28/B7 and CD40/CD40L signalling concerning activation, survivaland proliferation compared to Teff cells. However, high concentration of CTLA-4Ig(and MR1) prevented the increase in Treg cell-frequency normal">in vivo and in vitro suggesting that Treg cells are not completely independent of costimulation. Mostlikely this dose dependent effect on Treg cells is linked to the availabilityof IL-2 produced by Teff cells. mso-ansi-language:EN-GB" lang="EN-GB">As a next step we explored the effect of CTLA-4Ig andMR1 in a setting of autoimmunity. To this end, we used a mouse model of experimentalautoimmune encephalomyelitis (EAE), which resembles the human autoimmunedisease multiple sclerosis (MS). In order to be relevant for MS therapy, thetreatment should be able to interfere with an ongoing inflammatory reaction.Consequently, the mice received CTLA-4Ig and MR1 on day 7 and 9 after disease inductionwhen T cells were already primed. While MR1 was also effective in this setting,treatment with CTLA-4Ig surprisingly resulted in exacerbation of the disease. Asimilar exacerbation of the disease could be observed if the CTLA-4/B7interaction was blocked with monoclonal antibodies against CTLA-4 or B7. Basedon this we hypothesize that at this stage during T cell-activation, CTLA-4Igpreferentially blocks the CTLA-4/B7 interaction rather than the CD28/B7interaction. This could equally affect cell-intrinsic regulation of Teff cellsor cell-extrinsic regulation mediated by Treg cells. Although the frequency andthe suppressive function of Treg cells at the peak of the disease was notaltered in animals treated with CTLA-4Ig, it remains to be examined if Tregcells are affected at the time of treatment. A transient inhibition of Tregcells might indeed result in an enhanced expansion of Teff cells. mso-ansi-language:EN-GB" lang="EN-GB">Together these results show that Teff cells and Tregcells have different requirements regarding costimulation. Blockadeof costimulation therefore alters the balance between Teff and Treg cell-activationin favor of Treg activity. line-height:150%;font-family:"Times New Roman","serif";mso-ansi-language:EN-GB" lang="EN-GB">However,the dose of costimulatory blocking reagents as well as the timing of treatmentis crucial for the outcome of the therapy. mso-ansi-language:EN-GB" lang="EN-GB">These findings stressthe importance of detecting themechanisms underlying costimulation blockade in order to modify this promisingtreatment strategy to match different disease settings. 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Wilms' tumor gen 1 (WT1) als een doel voor immunotherapie in uteriene tumoren
No full textHooggradige uteriene sarcomen en recidiverende uteriene carcinomen zijn aggressieve vormen van baarmoederkanker. De standaard behandelingsopties zijn beperkt, hetgeen resulteert in een zeer slechte prognose. Er is dan ook een grote nood aan nieuwe en meer gerichte behandelingen. In dit doctoraatsonderzoek hebben we ons in de eerste plaats gefocust op het aantonen van een sterk immunogeen kanker-antigen, Wilms tumor gen 1 (WT1), in baarmoederkanker. We ontdekten dat het in hoge mate aanwezig was in de tumorcellen van hooggradige sarcomen en carcinosarcomen. Maar er was meer. Patiënten met een dergelijke WT1 postieve tumor hadden een significant slechtere prognose dan deze met een WT1 negatieve tumor. Het WT1 verhaal voor uteriene carcinomen was complexer. We vonden WT1 expressie in een beperkt deel van de tumorcellen maar in het overgrote deel van de bloedvaten in de tumor. Ook kleine bloedvaten rond de tumor (in het normale omgevende weefsel), bleken WT1 positief, hetgeen ons deed vermoeden dat WT1 in carcinomen wel eens een rol in angiogenese zou kunnen hebben. WT1 werd niet of nauwelijks geproduceerd in de normale baarmoeder en de goedaardig tumorale baarmoeder (myomen en poliepen). Het volgende luik in dit onderzoek was de ontwikkeling van een doelgerichte behandeling tegen dit WT1 in baarmoederkanker. We kozen voor immunotherapie op basis van dendritische cellen. Verrassend genoeg konden we in monocyten en in vitro gekweekte onbeladen dendritische cellen reeds WT1 aantreffen. Echter, de electroporatie van dendritische cellen met WT1-RNA zorgde voor meer WT1 expressie. WT1-mRNA beladen dendritische cellen werden gebruikt voor de in vivo stimulatie van T cellen, hetgeen resulteerde in de aanreiking van WT1-specifieke T cellen en een tijdelijke afname van de tumormarker, CA125, in een patiente met een uitbehandeld endometriumcarcinoom. Er waren geen nevenwerkingen. In een recent opgestarte fase I studie (WT1-2009; EudraCT 2009-016868-37) in ons centrum zal moeten duidelijk worden of deze nieuwe behandeling in staat is een immunologische en mogelijks klinische respons te induceren in 20 patienten met uitbehandelde, WT1 positieve baarmoederkanker.status: Publishe
Aktieve Immuuntherapie tegen Maligne Glioma op basis van RNA-beladen Dendritische Cellen
Get PDFDe prognose voor patiënten met maligne glioma blijft bijzonder slecht ondanks aanzienlijke vooruitgang in chirurgische technieken, chemo- en radiotherapie. Er is bijgevolg een absolute nood aan nieuwe therapeutische invalshoeken. In deze studie hebben wij in een experimenteel muismodel het concept onderzocht van aktieve immunisatie tegen maligne glioma op basis van dendritische cellen (DC) die beladen worden met tumorantigenen. De resultaten uit dit experimenteel werk tonen aan dat muis DC op een efficiënte manier kunnen opgeladen worden met totaal RNA van tumorale oorsprong en dat deze DC specifieke antitumorale cytotoxische T cellen kunnen induceren in vitro. Het preventief toedienen van deze cellen in vivo in een experimenteel muis gliomamodel is bovendien voldoende om de helft van de dieren te beschermen tegen intracraniële tumorgroei. Implementatie van in vivo bioluminescentie (BLI) in ons model maakte het mogelijk om verschillende responstypes ten opzichte van DC vaccinatie te onderscheiden. Deze immuunrespons is tumorspecifiek en op zijn minst deels gemedieerd door CD8+ T cellen. Om het effect van immuunsuppressieve CD25+ regulatoire T cellen (Treg) die het opwekken van antitumorale immuniteit kunnen belemmeren tegen te gaan werden de muizen geïnjecteerd met een anti-CD25 depleterend antilichaam. Alle anti-CD25 behandelde dieren bleken beschermd tegen daaropvolgende challenge met glioma tumorcellen. Voor protectie op lange termijn was anti-CD25 behandeling alleen niet voldoende en was immunisatie met DC noodzakelijk. Beide behandelingen resulteerden in een aanzienlijke infiltratie van lymfocyten in de hersenen van dieren met glioma implantatie. Zowel CD4+ als CD8+ effector en geheugencellen waren aanwezig in dit infiltraat en anti-CD25 behandeling verhoogde bovendien de cytotoxische aktiviteit van de effector T cellen. Naast effectorcellen werden ook Treg geëxpandeerd door vaccinatie met DC. Onverwachts resulteerde anti-CD25 behandeling maar niet immunisatie met DC in de lokale aantrekking van myeloïde cellen naar de hersenen van dieren met intracranieel glioma. Deze cellen presenteerden zich als macrofagen en granulocyten en konden niet als Gr-1+ myeloïde suppressor cellen beschouwd worden. Wij menen dat deze gerecruteerde cellen evenmin als tumorgeassocieerde macrofagen kunnen gezien worden maar eerder antitumorale M1 macrofagen zijn. Verder onderzoek naar de functionaliteit van deze cellen en hun verband met Treg is daarom absoluut noodzakelijk. Samengevat hebben wij in deze studie immunotherapie met RNA-beladen DC gevalideerd in een experimenteel glioma model en enkele belangrijke mechanismen ontrafeld inzake antitumorale immuniteit en immuunsuppressie. In het bijzonder willen we het translationeel aspect van dit onderzoek benadrukken met als finaal doel het verbeteren van de prognose van GBM patiënten.status: Publishe
Modulation of the natural and adaptive immune system by MultiStem®
No full textBecause of their long-term self-renewal ability and functional multi-lineage differentiation capacity, stem cells are highly attractive c andidates for many applications. Both embryon ic and adult stem cells have been explored as ther apeutic strategies in the context of tissue regene ration. Over the years, it has become clear that a dult stem cells also possess a wide rang e of immune and inflammation modulating properties . Many research groups have studied the immunologi cal behavior of mesenchymal stem cells (MSCs) in v itro and their role as an adoptive immune reg ulatory cell population in several immune-related disorders. Nowadays, other adult adheren t stem cell populations have been characterized an d are being considered to exhibit more potenc y compared to MSCs. Recently, human multipote nt adult progenitor cells (hMAPCs) have thoroughly been studied and have been shown to be clear ly distinct from hMSCs. In contrast to hMSCs, hMAP Cs can be expanded for a significantly longer time ¨and – besides mesenchymal cell types & ndash; also differentiate into functional end othelium in vitro and in vivo. Clinical-grade prod uction of MAPCs has been achieved and, curren tly, this proprietary large-scale expanded stem ce ll product (MultiStem®) is being evaluated as¨ an allogeneic ‘off-the-shelf’ stem cel l product in the clinic in the fields of regenerat ive medicine for cardiovascular (acute myocardial¨ infarction; AMI) and neurological diseases (i schemic stroke) and as immunotherapy in immun e-associated disorders [graft-versus-host disease¨ (GvHD) after allogeneic hematopoietic stem cell tr ansplantation (HSCT), solid organ transplantation¨ and autoimmunity).
Even though administ ration of clinical-grade hMAPCs appears to be ¨safe and well-tolerated, and leads to benefi cial (preliminary) clinical results, data regardin g their immunological behavior in vitro are s carce. A better understanding of the exact immune¨ regulatory mechanism would be very valuable t o optimize future clinical studies on the immunotherapeutic function of hMAPCs and of adult stem cel ls in general. In the presented manuscript, in con tinuation of a former project in this specific res earch line, we addressed the issue by investi gating the reciprocal interaction between (clinica l-grade) hMAPCs and immune cells of a functional h uman immune system in vitro. Given the fact t hat cytotoxic immune effector cells play a cr ucial role in immune homeostasis and in the p athogenesis of some autoimmune diseases, ¨we analyzed the influence of (clinical-grade ) hMAPCs on the phenotype and functionality of nat ural killer (NK) cells and CD8+ cytotoxic T cells¨ (CTLs).
The first specific objective of this ¨research was to explore the immunogenic and immun e modulatory properties of (clinical-grade) hMAPCs , with regard to functional cytotoxic responses of ¨NK cells and T cells. More specific, it was quest ioned whether these stem cells of alloge neic origin could act as stimulators, as fore ign target cells or as modulators of NK cells and CTLs. We showed that hMAPCs are not able to induce an antigen-specific ‒ a nd only a minimal anti-CD3-redirected ‒ CTL response in vitro. Furthermore, allogene ic hMAPCs were insensitive to antigen-sp ecific CTL-mediated lysis and to killing by r esting KIR-mismatched NK cells, despite expression ¨of some ligands (PVR and ULBP-2/5/6) of activatin g NK cell receptors (respectively DNAM-1 and NKG2D). On the other hand, IL-2-preactiv ated NK cells efficiently lysed allogeneic hMAPCs. ¨However, increased MHC class I expression af ter IFN-γ-pretreatment of hMAPCs did not res ult in higher MHC-I-dependent allorecognition by C TLs and reduced hMAPC susceptibility to preac tivated NK cell-mediated killing, by shifting the¨ balance towards MHC-I-associated inhibitory N K cell signalling. These results indicate low ¨immunogenicity of (clinical-grade) hMAPCs in vitr o, and suggest that survival of hMAPCs in viv o will primarily be dictated by the inflammat ory status and the cytokine balance in the local microenvironment.
Secondly, we proved that hMA PCs exert strong immune suppressive effects, as th ey are able to suppress alloreactive CTL prol iferation and IL-2-induced expansion of allogeneic ¨NK cells. The suppressive effect on NK cell proliferation is dose- and IDO-dependent, and is m ore pronounced for hMAPCs than for hMSCs ¨from the same donor. In addition, we observed a reduced cytotoxic function of restin g NK cells and activated CTLs (in an ant igen-specific and anti-CD3-redirected cytotoxicity ¨system) in the presence of allogeneic third-party ¨hMAPCs during the lytic effector phase. hMAP Cs also directly and dose-dependently impaired ind uction of CTL cytotoxicity during their priming phase (alloantigen-induced or polyclonal a ctivation) by interfering with their perforin expression. On the other hand, in contrast t o their inhibitory effect on NK cell prolifer ation, hMAPCs had no influence on the intrins ic cytotoxic properties of NK cells during th e IL-2-mediated activation phase. Because of compe tition between hMAPCs and K562 cells as targe t cells for IL-2-activated NK cell-mediated k illing, we could not conclude if hMAPCs influ ence the killer function of IL-2-activated NK cells during their effector phase. Taken together , our observations imply that (clinical-grade) hMA PCs impair both proliferative and cytotoxic NK and T cell responses, resulting in a lower immun e response in vitro.
The second specific goa l of this thesis was to further elaborate the immune modulatory mechanism of (clinical-gra de) hMAPCs and to identify responsible immune ¨regulatory factors and pathways. We demonstrated¨ that the mode of action is multifactorial and ¨depends on the studied immune effector response.¨ In case of proliferation suppression, we coul d ascribe a partial role to IDO for hMAPC-med iated impairment of NK cell proliferation, in line ¨with previously obtained results for T cell¨ expansion inhibition. In contrast, we could n ot identify a single responsible soluble factor (IDO, PGE2, IL-10 and TGF-β) in NK ce ll cytotoxicity suppression. Transwell experiments showed contact-dependency of hMAPCs to¨ exert their suppressive function on T cell cytotox icity. Until now, as is the case for NK cells, we did not detect a single responsible ¨contact-dependent mechanism in hMAPC-mediated T c ell cytotoxicity suppression. Though, we desc ribed for the first time hMAPC expressio n of galectin-1 and ligands of the immun e inhibitory PD-1 pathway (PD-L1/2) and¨ of the Fas-mediated apoptosis pathway (FasL). Functional blocking studies however did not confirm the latter two pathways as suppressiv e candidates in CTL function inhibition by hMAPCs. With regard to the suppressed T cell phenoty pe, we observed that the presence of hMAPCs does n ot result in T cell apoptosis or anergy and t hat T cells retain their memory response duri ng a secondary immune reaction after removal¨ of hMAPCs. However, hMAPCs largely disturb th e activation marker expression of T cells. In ¨hMAPC-modulated T cells, CD69 expression was ¨clearly and persistently increased, in both CD8-¨ and CD8+ T cell populations. Moreover, HLA-DR ¨and CD25 (IL-2Rα) upregulation were signifi cantly decreased in the presence of hMAPCs. hMAPCs ¨downregulated CD25 expression to a lower ext ent on CD8 T cells and shifted those cells p ossibly towards a regulatory phenotype.
In conclusion, we have provided evidence that (cl inical-grade) hMAPCs are low immunogenic ¨and have potent immunosuppressive effects in ¨vitro on NK cells and CTLs. In addition, we have¨ contributed to a better understanding of the¨ complex immunomodulatory mechanism. Based on these findings, we can state that the rationa le for therapeutic use of hMAPCs or its clinical-g rade counterpart MultiStem as an off-the-shel f adoptive cell population in the context of¨ immune-related disorders is legitimate. These data may represent an extensive co ntribution to the current knowledge and, in combination with the results of future phase II/III ¨trials using MultiStem, will lead to an intriguing continuation of stem cell-based resear ch for immunotherapy.status: Publishe
Galectine-1 moduleert glioma immuniteit en is een potentiële biomerker in de monitoring van kwaadaardige gliomen
No full textDespite major advances in the management of malignant gliomas GBM remains incurable. The aggressive nature of GBM is believed to be due to the extensive spread of tumor cells into surrounding regions of the brain and to the development of treatment resistance. A large amount of experimental evidence has highlighted a role for galectin-1, a hypoxia-sensitive, glycan-binding protein, in the aggressiveness of astrocytic tumors. Within astrocytic tumors, a positive correlation has been described between galectin-1 expression levels and the grade of malignancy. Moreover, high-grade astrocytic tumors with high levels of galectin-1 expression are associated with dismal prognosis as compared to high-grade astrocytic tumors with low expression levels of galectin-1. Galectin-1 regulation of glioma aggressiveness is multimodal. Galectin-1 has been shown to modulate tumor astrocyte migration. In addition, intratumoral galectin-1 expression was demonstrated to protect glioma cells against cytotoxic insults such as chemotherapy and radiotherapy. Moreover, a pro-angiogenic role for galectin-1 has been described in several other malignancies as well as in gliomas. In a variety of cancer models, the presence of galectin-1 within the tumor microenvironment has been shown to contribute significantly to the establishment of local immune resistance. Although the abundance of galectin-1 in malignant glioma is well-known, it remains to be determined whether this lectin also plays an important role in glioma-mediated immune evasion. Hence, we investigated whether glioma-derived galectin-1 is capable of modulating innate or adaptive anti-glioma immune responses in an experimental murine glioma model. This syngeneic mouse model comprises the intracranial challenge of immune competent mice with GL261 HGG cells. To investigate whether glioma-derived galectin-1 exerts an immune modulating function, we developed a GL261 tumor cell line in which the galectin-1 expression was permanently and stably silenced. Among the different galectin-1 knockdown clones that were generated we selected one monoclonal cell line (GL261-KD) in which a strong decrease in galectin-1 protein and gene expression level was observed. In a first set of experiments, we analyzed the impact of intratumoral galectin-1 downregulation on in vivo glioma progression. These experiments revealed that the silencing of glioma-derived galectin-1 significantly improves the outcome of glioma-bearing mice. This observation could not be explained by a direct role of galectin-1 on tumor cell proliferation, as wild-type GL261-wt tumor cells as well as GL261-KD tumor cells exhibited a similar in vitro proliferation rate. Interestingly, absence of host-derived galectin-1 had no effect on in vivo glioma progression. In a next step, we analyzed whether the silencing of glioma-derived galectin-1 induced proportional or phenotypic changes in brain-infiltrating myeloid cells. Both flow cytometric and pathological analysis revealed a significant decrease in the percentage of brain-infiltrating macrophages and MDSC amongst the total population of infiltrating CD45high immune cells upon intratumoral silencing of the galectin-1 expression. To further explore the link between tumor-derived galectin-1 and myeloid cell recruitment towards the tumor microenvironment, we analyzed CCL2 and VEGF mRNA expression levels in in vitro cultured GL261-WT and GL261-KD tumor cells. This experiment revealed that the downregulation of intratumoral galectin-1 reduced the mRNA expression levels of both CCL2 and VEGF in in vitro cultivated GL261 tumor cells. Moreover, CCL2 expression was also significantly downregulated in brain-infiltrating myeloid cells that were isolated from GL261-KD tumor-bearing mice, suggesting the presence of a negative, autocrine feedback loop. Interestingly, we observed a similar negative correlation between intratumoral galectin-1 expression levels and the magnitude of macrophage / microglia influx in biopsies of patients with newly diagnosed GBM. Whether glioma-derived galectin-1 also regulates macrophage function in the GL261 glioma model remains elusive. From our experiments, it is clear that myeloid cells that are recruited to a galectin-1-depleted glioma microenvironment have a phenotype that is considerably different from the phenotype of myeloid cells that were recruited to a galectin-1 enriched glioma microenvironment. However, further ex vivo functional studies are required to address the role of glioma-derived galectin-1 in the education of TAM. To investigate the involvement of the adaptive immune response in the prolonged survival of GL261-KD tumor-bearing mice, tumor cells were injected into immune compromised NSG mice which lack mature T cells, B cells and functional NK cells. These experiments demonstrated that the prolonged survival upon an intracranial challenge with GL261-KD tumor cells requires the presence of an intact adaptive immune system. Whereas compelling evidence has accumulated regarding the modulating effects of tumor-derived galectin-1 on T cell viability, we found no correlation between the amounts of tumor-derived galectin-1 and the apoptosis of brain-infiltrating lymphocytes. However, we did observe a modulating effect of galectin-1 on T cell function, as in the absence of galectin-1 the brain-infiltrating T cells produced significantly higher amounts of IFN-ɣ. Interestingly, quantification of the number of blood vessels in GL261-WT or GL261-KD tumor-bearing mice revealed that the silencing of glioma-derived galectin-1 also impaired angiogenesis, thereby underscoring the multimodal role of this lectin in the glioma microenvironment. Finally, we explored whether the silencing of glioma-derived galectin-1 could improve the survival of vaccinated tumor-bearing mice. These experiments revealed that prophylactic DC vaccination of either GL261-WT or GL261-KD tumor-bearing mice significantly prolongs the survival of tumor-bearing mice. In addition, prophylactic DC vaccination protected a small subset of mice against primary intracranial tumor challenge. Moreover, comparison of the outcome of vaccinated GL261-WT tumor-challenged mice and vaccinated GL261-KD tumor-challenged mice revealed the presence of a significant survival benefit in vaccinated GL261-KD tumor-challenged mice. Moreover, we demonstrated that this survival benefit was only observed upon the silencing of tumor-derived but not of host-derived galectin-1. Collectively, these data demonstrate that tumor-derived galectin-1 is a modulator of anti-glioma immune responses and that the silencing of intratumoral galectin-1 expression improves the outcome of tumor-bearing mice both in the presence or absence of DC vaccination. Hence, the targeting of galectin-1 might offer an adjuvant modality in the treatment of patients with malignant gliomas.Thus far, it is unknown whether increased galectin-1 expression levels are exclusively found at the site of the tumor or whether altered galectin-1 levels can also be found in the serum of patients with HGG as a consequence of a leaky brain tumor vasculature. Hence, we evaluated the galectin-1 serum levels in a prospective dataset of 43 healthy controls and 125 patients with newly diagnosed or recurrent HGG. Patient blood samples were collected at the moment of neurosurgical intervention and/or at the moment of leukapheresis (approximately 2-3 weeks after surgery) in case of postoperative tumor vaccination therapy. Galectin-1 serum levels were measured by means of ELISA. The comparison of the galectin-1 serum levels in healthy controls revealed that the galectin-1 serum levels depended significantly on age and sex. Therefore, a multivariable model for galectin-1 with age and sex as predictors has been fitted in the control patients. Age- and sex-adjusted galectin-1 serum levels were significantly higher in all patient subgroups compared to healthy controls with a high discriminative ability that increased with age. Collectively, the data presented in this study may represent a first step to establish galectin-1 as a biomarker in glioma disease monitoring. Further longitudinal evaluation is required and ongoing to investigate the value of galectin-1 serum levels in HGG patients as an additional diagnostic marker and as a predictor of treatment response and prognosis. Furthermore, galectin-1 serum levels could also provide an important tool for the identification of HGG patients that could benefit from galectin-1 directed therapies that are currently under development.status: Publishe
DC vaccinatietherapie voor hoog-gradige gliomen
No full textIn spite of full, state-of-the-art oncological therapy, including maximal safe surgical resection, external beam radiotherapy and chemotherapy, the prognosis of glioblastoma multiforme (GBM) remains poor with a median survival of 14.6 months. Notwithstanding this maximal treatment, relapse is universal. At time of recurrence, prognosis is even worse and virtually all patients die within 18 months after relapse. There is a clear need for well-tolerated long-term treatments that are tumor-specific and able to kill all (residual) tumor cells that infiltrate in the adjacent areas of the brain. The immune system provides us with some promising tools in that regard, and therefore we have investigated the integration of dendritic cell (DC)-based immunotherapy in the current therapeutic concepts, both in newly diagnosed and relapsed GBM patients.First, we have implemented immunotherapy for the treatment of patients with relapsed high-grade glioma (HGG), as a stepwise process using a cohort-comparison study concept (HGG-IMMUNO-2003). We slightly modified the original RTOG RPA classification, a known model to define prognostic classes based on treatment and pre-treatment prognostic variables, and made it applicable to patients treated in this study. In this heterogeneous group of patients, we could define simple, clinically relevant, prognostic classes using this modified RTOG RPA model. Also, we found long-term survivors after relapse in classes III and IV, which is a remarkable clinical finding, comparing favorably to almost all other trials in (multi-)relapsed patients with HGG. Based on the already published importance of age in DC vaccination in the patients with relapsed HGG, we analyzed the results in 45 children (18 years and younger) with a recurrent malignant brain tumor, who were treated with autologous DC-based immunotherapy (HGG-IMMUNO-2003). We could demonstrate the clinical feasibility of this approach without major adverse events, even in young children. HGG seem to respond more favorably to vaccination than ependymoma and medulloblastoma/primitive neuro-ectodermal tumor, and for atypical teratoid-rhabdoid tumor the addition of immunotherapy to radio- and chemotherapy might be beneficial. Although preliminary, and derived from a heterogeneous patient group, our results on DC-based vaccination support testing of the integration of this innovative immunotherapy approach in new treatment protocols for HGG and atypical teratoid-rhabdoid tumor.Next, we integrated DC-based immunotherapy in the standard primary treatment of GBM, consisting of maximal safe surgical resection, radiotherapy and temozolomide (TMZ) chemotherapy. In 8 pilot, adult newly-diagnosed GBM patients, tumor vaccination proved to be feasible and well tolerated. The survival data were used to power the phase I/II HGG-2006 trial for patients with newly-diagnosed GBM, in which 77 patients were included. Median overall survival (OS) based on intent-to-treat analysis was 18.3 months, which compares favorably to the survival data reportedby Stupp et al. with a median OS of 14.6 months. Survival seems to be improved especially in the patients belonging to EORTC RPA classes III and IV. The integration of immunotherapy within the standard postoperative therapy for patients with a newly diagnosed GBM is based on the presumed mutually beneficial effect of the conventional treatment strategies and immunotherapy. It is hypothesized that the combination of radiotherapy, chemotherapy and immunotherapy could potentiate the cumulative antitumoral activity, when applied in a well designed strategy. For this radio-chemo-immunotherapy, we provide feasibility data in terms of clinical responses, as well as an acceptable QOL.Finally, we addressed the issue of immune monitoring in vaccinated, newly-diagnosed GBM patients. Both in the pilot and HGG-2006 trial, we applied flow cytometry on peripheral blood as immune monitoring tool to assess the induction and/or maintenance of vaccine-induced antitumoral immunity in the close temporal relationship with radiochemotherapy. For this, we first validated IL-7 receptor alpha subunit (CD127) dim expression as a marker for human Treg cells, since these cells have claimed a very prominent role in tumor immunology as potential suppressors of immune responses. Thus, monitoring of Treg cells is essential in patients treated with immunotherapy. Immunological profiles based on flow cytometry, were compared to clinical outcome, but no clear correlation between immunological and clinical responses was found. A possible explanation for this lack in correlation might be that the peripheral immune status does not mirror the immune responses that occur in the tumor itself. Therefore, we established a baseline phenotypical characterization of brain tumor-infiltrating cells, which we compared to the immune status of the patient as measured in the peripheral blood. Preliminary results seem to indicate in fact, that the peripheral immune status does not mimic the intratumoral immune reaction. How these findings relate to immune monitoring in vaccinated patients has to be further elucidated, but one might argue that peripheral immune monitoring tools are not well suited for the full monitoring of the immune response against these tumors.status: Publishe
Immunogene op oxidatietherapie-gebaseerde tweede generatie dendritische celvaccins voor de behandeling van hooggradige gliomen
No full textHigh-grade glioma (HGG) represents a highly aggressive central nervous system (CNS) malignancy with mortality rates matching incidence rates. Dendritic cell vaccines aiming to stimulate the endogenous antiglioma immune response constitute a safe and promising treatment modality. Thus far, the efficacy of DC vaccines is considered suboptimal, in part owing to a lack of attention towards the immunogenicity of the tumor lysate used as an antigen source for loading the DC vaccines. Given the recent evidence that oxidation and the resultant oxidation-associated molecular patterns (OAMPs) can impact tumor cell immunogenicity, we applied two physico-chemical oxidizing therapies to obtain a more immunogenic tumor cell cargo: x-ray irradiation and hypericin-based photodynamic therapy (Hyp-PDT).
DC vaccine immunogenicity was investigated in the orthotopic, syngeneic GL261 HGG model. Mice prophylactically immunized with DC vaccines loaded with x-ray irradiated freeze-thawed lysates survived significantly longer as compared to mice vaccinated with DCs loaded with freeze-thaw lysate only. This was associated, both in prophylactic and curative vaccination setups, with increased intrabrain infiltration of T helper-1 cells (Th1)/Cytotoxic T lymphocytes (CTLs) and reduced infiltration of regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs). Through further application of anti-oxidants and hydrogenperoxide, we found a striking correlation between the amount of lysate-associated protein carbonylation/OAMPs and the capacity of the DC vaccines to mediate glioma rejection. This result fostered the preclinical investigation of other strong oxidizers like Hyp-PDT, a potent type II inducer of ICD, in our HGG model. Hyp-PDT induced prototypical ICD in the GL261 cell line. In prophylactic as well as curative vaccination settings, both biologically and clinically-relevant versions of Hyp-PDT-based ICD-based DC vaccines provided strong anti-HGG survival benefit. The ability of DC vaccines to elicit HGG rejection was significantly blunted if cancer cell-associated reactive oxygen species (ROS) and emanating danger signals were blocked (either singly or concomitantly; showing hierarchical effect on immunogenicity i.e. extracellular-HMGB1>extracellular-ATP>ecto-calreticulin) or if DCs, the DCs-associated MyD88 signal or the adaptive immune system (especially CD8+ T cells) were depleted. In a curative setting, ICD-based DC vaccines synergized with standard-of-care chemotherapy (temozolomide) to increase survival of HGG-bearing mice by ~300% resulting in ~50% long-term survivors. Additionally, DC vaccines also induced an immunostimulatory shift in the brain immune-contexture from Tregs to Th1/CTLs/Th17 cells. Analysis of the TCGA glioblastoma-cohort confirmed that increased intra-tumor prevalence of Th1/CTLs/Th17 cells-linked genetic signatures associated with good patient prognosis.
Our research demonstrates that combining DC vaccination with oxidizing treatment modalities can induce Th1-driven antitumor immune responses, enabling CTL-mediated glioma rejection. ICD-based DC vaccines exploiting ER-directed oxidative stress might be considered next generation DC vaccines.status: Publishe
Exploratie en ontwikkeling van combinatie immuuntherapie als behandeling voor uteriene tumoren
No full textAlthough current treatments are often curative for uterine cancer when it is diagnosed in an early stage, with a 5-year survival rate of around 80 %, these treatments are often no longer effective in case of far-advanced disease. The mean survival rate for stage III uterine tumors is 57.8 %, while for stage IV, this number decreases even further to a mere 22.8 %. Consequently, additional treatments are needed.The main focus of the current project was to further develop an autologous dendritic cell-based vaccine for uterine tumors. The initial vaccine using WT1 mRNA loaded monocyte-derived DC, showed an immunological response in 5/8 patients and a transient clinical response in 2/8 patients. In the current study, the vaccine was further improved and extended.The first objective was to identify additional antigenic targets to include in the vaccine. We selected 6 different antigens, MAGE-A3, BORIS, MUC1, Sp17, hTERT and survivin, for validation using immunohistochemistry as well as qRT-PCR. In addition, to verify the immunogenicity of these antigens, spontaneous ex vivo T cell responses to these antigens were analyzed. From these analyses, we found that survivin was the best suitable target to proceed with.In addition to adding a new target antigen to the DC vaccine, we aimed to further improve the functional characteristics of the DC used in the vaccine. To this end, an optimal protocol for the generation of monocyte-derived DC was established using an IL-4/GM-CSF/TriMix maturation cocktail. As shown in previously published data this improves the maturity profile of the DC and improves the T cell stimulatory capacity.Tumor-induced immunosuppression is an important factor hampering the efficacy of immunotherapy. More and more awareness is being raised for this issue. We aimed to map some of the immunosuppressive mechanisms in uterine tumors. To this end, 3 members of the B7 family, B7-H1 (PD-L1), B7-DC (PD-L2) and B7-H4 were analyzed as well as IDO, galectin-1 and galectin-3 as well as the presence and activity of myeloid-derived suppressor cells in uterine tumors. IHC analysis showed that the PD-1:PD-L1 axis and B7-H4 are promising targets in the vast majority of tumors and IDO in a minority of cases. Flow cytometric analysis showed that MDSC do infiltrate uterine tumors and are likely functionally active, thus presenting another route at which tumor immune escape may be interrupted.Beside using active immunotherapeutic regimens or direct intervention of immunosuppression, substances that are used for current anti-cancer treatment may also be averted in a different dosage to modulate the immune system. Low-dose cyclophosphamide, for example, has been shown to have immunomodulatory effects. We verified the effect of oral administration of low-dose cyclophosphamide on the Treg population in order to determine whether or not this modality could be applied for uterine tumors. However, we could not confirm the suppressive effect of cyclophosphamide on Treg. Consequently, this method is according to our results, not applicable in uterine tumors.Taken together, the current research is the next step in the identification of the role the immune system plays in uterine tumors and provides novel indications for its use and its modulation in the treatment of uterine cancer.status: Publishe
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