Hematopoietic stem cell transplantation (HSCT) is used to treat patient with hematological diseases such as leukemia and genetic conditions such as sickle cell anemia. After HSCT the patients are supervised for signs of relapse of disease or rejection of transplanted cells. This is done by using chimerism analysis. At the department of clinical genetics at Akademiska sjukhuset fragment analysis of short tandem repeats is used for chimerism analysis, which is to be replaced by a Next generation sequencing (NGS) based method called Devyser chimerism, which includes an IVDR labelled kit. The aim of this project was to validate the new method for chimerism analysis. DNA samples from twelve HSCT patients and their donors were analyzed with Devyser chimerism and the results were compared to the results from the current method. The sensitivity of the new method was tested by analysis of artificial chimerism samples from blood donors. The results from the comparison showed a good correlation between methods (R2 = 0,9864) and the sensitivity of the method was confirmed to be 0,1% mixed chimerism. There was some difficulty in identifying enough informative markers for re-transplanted patients two had separate donors. This is a known problem for chimerism analysis in general and not a specific problem to the new method and will not be a hindrance for the implementation of Devyser chimerism at the clinical laboratory.

Osteoblasts (OST) provide strong intrinsic growth modulatory activities on hematopoietic stem and progenitor cells via different mechanisms that include secretion of growth factors, and cellular interaction. Previously we showed that medium conditioned by mesenchymal stromal cell (MSC)-derived osteoblasts (M-OST) improve the expansion of cord blood (CB) CD34+ cells. I hypothesize that the hematopoietic supporting activity of M-OST would vary as a function of their maturation. This was tested by producing osteoblast conditioned media (OCM) from M-OST at distinct stages of maturation, and testing their growth regulatory activities in CB CD34+ cell cultures. My results showed that some of the growth promoting activity of OCM on CB cells are not dependent on the maturation status, while others are and those are largely independent of Notch signalling. In conclusion, these results provide further evidence that osteoblasts release factors that can promote the growth of immature CB progenitors in a Notch-independent way.

Background: Multiple sclerosis (MS) is a neurological, autoimmune disease which mainly affects people in the age of 20 to 40. The disease course is unpredictable affecting each patient differently, leading to progressiveand irreversible degradation of the central nervous system. There is no treatment that cures this disease, however, there are treatments that either slows down the disease course or prevents progressive disabilities. A treatment called autologous hematopoietic stem cell transplantation (AHSCT) is thought to reset the immune system and induce a new, more tolerant one, thus haltering the disease course. However, the knowledge about the effects causing the improvement seen in patients treated with AHSCT is limited. Methods: To investigate the effect of AHSCT in MS patients, serum lipidomics data from 16 patients was collected at ten timepoints. The lipidomics data was collected for both positively and negatively charged molecules separately as well as within a single experiment called polarity switching, using mass spectrometry. Since the standard method requires two separate experiments to analyze both positively and negatively charged lipids it requires twice the time and resources compared to polarity switching. Results: Comparing the two mass spectrometry protocols showed that the coefficient of variation (CV) was slightly higher for polarity switching compared to the standard method. Nevertheless, the difference was not significant and both methods had in general a good CV, indicating low technical variation. In addition, this thesis showed that polarity switching has a slightly higher percentage of lipids with zero carryover compared to the standard method. The results also indicated that the expression levels of differentially expressed lipids follow two distinct patterns throughout the AHSCT treatment. The largest intensity variation arises after stem cellreinfusion and the lipid intensities are back to nearly initial levels atthe three month follow-up. Finally, many lipids were found to be associated with the change in c-protein levels as well as erythrocyte, leukocyte, and thrombocyte levels that occurred during treatment. Conclusions: This master thesis showed that polarity switching is a good alternative to the standard method, saving both time and resources without losing too much in specificity. In addition, this thesis has shown that differentially expressed lipids follow two distinct expression patterns through the treatment. The lipids levels for both differentially expressed lipids and lipids associated with clinical data were nearly back to baseline levels three months after AHSCT. Hence, AHSCT has a major but short-lasting impact on the lipid levels in peripheral blood.

The discovery of novel genes involved in hematopoietic development and lymphoid function is necessary for the understanding of these systems. To this end, we utilized transmembrane protein-specific gene trapping in embryonic stem (ES) cells, a method of forward genetics, to identify a novel, complex locus from which several splice variants arise. The trapped locus identified in the KST30 ES cell clone encodes several genes including outer membrane protein 25 (OMP25) and activin receptor interacting protein (ARIP2) and two novel genes, AK74 and AK88. AK74 is highly conserved between human and mouse with 85% identity at the amino acid level. The human homolog was cloned from CD34+ cord blood hematopoietic stem cell progenitors (HSCPs) implying that it may have a role in the hematopoietic system. We generated mice from the gene trapped ES cells, called KST30 mice, to analyze the expression pattern of transcripts from the trapped locus in the hematopoietic system. Utilizing the gene trap LacZ reporter and RT-PCR, we found that AK88 and AK74 are expressed in hematopoietic stem cells and thymocytes and that AK88 and ARIP2 are dramatically up-regulated in activated Band T lymphocytes. In addition, we found restricted expression of the gene trap in most non-lymphoid tissues. Interestingly, the expression pattern of the gene trap coincides with the expression of activin signaling components in many cell types including thymocytes, activated B cells, hematopoietic stem cells and the ductal cells of the pancreas. AK74, AK88 and ARIP2 share two exons that encode a 44 amino acid region. ARIP2 negatively regulates activin signaling through endocytosis of Activin type II receptors. The N-terminal PDZ domain associates with ActRII and mediates endocytosis via association with RalBP1. The region of ARIP2 that associates with RalBP1 encompasses the 44 amino acid region also found in AK74 and AK88, suggesting that these proteins may also associate with RalBP1, perhaps sequestering it from ARIP2. This possibility combined with the similarities between gene trap expression and expression of the components of activin signaling indicates a role of the trapped genes in activin signaling. AK74 and AK88 have a signal sequence and transmembrane domain that are predicted to direct them to mitochondria. To confirm this prediction, we examined the subcellular localization of AK74 and found that it localizes to a punctate, perinuclear structure identified as mitochondria using a mitochondria specific dye. AK74 was not seen in the cytoplasm, nucleus or at the plasma membrane of cells. To determine the function of these novel genes, AK74 was retrovirally over-expressed in a double positive thymoma cell line and examined the global expression profile using Affymetrix gene chip. AK74 changed the expression levels of 36 genes greater than 3-fold compared to vector alone. Of these genes, several are involved in cytoskeletal rearrangement, apoptosis or are regulated by calcium signaling. Using yeast two-hybrid, several candidate binding partners for AK74 were identified, one of which is the receptor for activated protein kinase C (RACK1). RACK1 was also identified as a potential binding partner for AK88. RACK1 is a WD40 domain-containing scaffolding protein that has been implicated in many pathways but most prominently in the protein kinase C signaling pathway. Association with RACK1 by either AK74 or AK88 suggests that they may be involved in RACK1 function. Both RACK1 and PKC are involved with Ca2+ signaling through different mechanisms. This, combined with global gene expression changes in AK74 over-expressing cells suggests a role for AK74, AK88 or ARIP2 in Ca2+ signaling. When we examined the expression of the trapped genes in mice homozygous for the gene-trapped allele (KST30tr/tr) we found that insertion of the gene trap caused a severe decrease in AK88 and ARIP2 but not AK74 transcripts. Analysis of KST30tr/tr mice showed no abnormalities in conventional lymphoid populations and precursors, however, intraepithelial lymphocyte (IEL) populations were altered by the loss of AK88 and/or ARIP2. There was an approximate 2-fold decrease CD8αα+ T cells in the small intestine while CD8αβ+ T cells were largely unaltered. Using gene trap technology, we have identified two novel, mitochondria-localized proteins. The cumulative findings described in this thesis, including the homology between AK74, AK88 and ARIP2, their expression pattern and the phenotype of KST30tr/tr mice, suggest possible roles of AK74 and AK88 in diverse pathways.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Multiple myeloma (MM) is an incurable malignancy characterized by the proliferation of neoplastic plasma cells in the bone marrow (BM) and by multiple osteolytic lesions throughout the skeleton. We previously reported that CD166 is a functional molecule on normal hematopoietic stem cells (HSC) that plays a critical role in HSC homing and engraftment, suggesting that CD166 is involved in HSC trafficking and lodgment. CD166, a member of the immunoglobulin superfamily capable of mediating homophilic interactions, has been shown to enhance metastasis and invasion in several tumors. However, whether CD166 is involved in MM and plays a role in MM progression has not been addressed. We demonstrated that a fraction of all human MM cell lines tested and MM patients’ BM CD138+ cells express CD166. Additionally, CD166+ cells preferentially home to the BM of NSG mice. Knocking-down (KD) CD166 expression on MM cells with shRNA reduced their homing to the BM. Furthermore, in a long-term xenograft model, NSG mice inoculated with CD166KD cells showed delayed disease progression and prolonged survival compared to mice receiving mock transduced cells. To examine the potential role of CD166 in osteolytic lesions, we first used a novel Ex Vivo Organ Culture Assay (EVOCA) which creates an in vitro 3D system for the interaction of MM cells with the bone microenvironment. EVOCA data from MM cells lines as well as from primary MM patients’ CD138+ BM cells demonstrated that bone osteolytic resorption was significantly reduced when CD166 was absent on MM cells or calvarial cells. We then confirmed our ex vivo findings with intra-tibial inoculation of MM cells in vivo. Mice inoculated with CD166KD cells had significantly less osteolytic lesions. Further analysis demonstrated that CD166 expression on MM cells alters bone remodeling by inhibiting RUNX2 gene expression in osteoblast precursors and increasing RANKL to OPG ratio in osteoclast precursors. We also identified that CD166 is indispensable for osteoclastogenesis via the activation of TRAF6-dependent signaling pathways. These results suggest that CD166 directs MM cell homing to the BM and promotes MM disease progression and osteolytic disease. CD166 may serve as a therapeutic target in the treatment of MM.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20077号 / 医博第4170号 / 新制||医||1018(附属図書館) / 33193 / 京都大学大学院医学研究科医学専攻 / (主査)教授 江藤 浩之, 教授 斎藤 通紀, 教授 山田 泰広 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21018号 / 医博第4364号 / 新制||医||1028(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 小川 誠司, 教授 柳田 素子, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Hämatopoetische Stammzellen besitzen die Fähigkeit, die dauerhafte Erhaltung ihrer eigenen Population im Knochenmark zu gewährleisten und gleichzeitig zur Neubildung der verschiedenen Zelltypen des peripheren Blutes beizutragen. Die Sequenz von Entscheidungsprozessen, die den Übergang einer undifferenzierten Stammzelle in eine funktionale ausgereifte Zelle beschreibt, wird als Linienspezifikation bezeichnet. Obwohl viele Details zu den molekularen Mechanismen dieser Entscheidungsprozesse mittlerweile erforscht sind, bestehen noch immer große Unklarheiten, wie die komplexen phänotypischen Veränderungen hervorgerufen und reguliert werden. Im Rahmen dieser Dissertation wird ein geeignetes mathematisches Modell der Linienspezifikation hämatopoetischer Stammzellen entwickelt, welches dann in ein bestehendes Modell der hämatopoetischen Stammzellorganisation auf Gewebsebene integriert wird. Zur Verifizierung des theoretischen Modells werden Simulationsergebnisse mit verschiedenen experimentellen Daten verglichen. Der zweite Teil dieser Arbeit konzentriert sich auf die Beschreibung und Analyse der Entwick- lungsprozesse von Einzelzellen, die aus diesem integrierten Modell hervorgehen. Aufbauend auf den entsprechenden Modellsimulationen wird dazu eine topologische Charakterisierung der resultierenden zellulären Genealogien etabliert, welche durch verschiedener Maße für deren Quantifizierung ergänzt wird. Das vorgestellte mathematische Modell stellt eine neuartige Verknüpfung der intrazellulären Linienspezifikation mit der Beschreibung der hämatopoetischen Stammzellorganisation auf Populationsebene her. Dadurch wird das Stammzellm- odell von Röder und Löffler um die wichtige Dimension der Linienspezifikation ergänzt und damit in seinem Anwendungsbereich deutlich ausgedehnt. Durch die Analyse von Einzelzellverläufen trägt das Modell zu einem grundlegenden Verständnis der inhärenten Heterogenität hämatopoetischer Stammzellen bei.

Le transfert adoptif de lymphocytes T virus-spécifiques ou ‘’Chimeric Antigen Receptors’’ (CAR) s’est avéré efficace pour le traitement de plusieurs types d’infections virales et certains cancers à la suite d’une greffe de cellules souches hématopoïétiques. Cependant, l’immunosuppression administrée pour la prévention du rejet de greffe et de la maladie du greffon contre l’hôte limite l’efficacité et la persistance à long terme des réponses médiées par ces lymphocytes. L’agent immunosuppresseur Tacrolimus (FK506) est parmi les plus utilisés, et fonctionne en liant la protéine FK506-Binding protein (FKBP12) afin d’exercer ses effets immunosuppresseurs sur les lymphocytes T. Dans le but de fournir une protection anti-virale, mais également anti-lymphoprolifératif des lymphocytes B et permettre la poursuite de cette immunosuppression préventive, nous avons pour objectif de générer des lymphocytes CAR-T et virus-spécifiques résistants à l’action du FK506 par l’invalidation du FKBP12. En utilisant la méthode d’édition génique basée sur les CRISPR ciblés par la nucléase Cas9, nous avons pu invalider le gène du FKBP12 sur des lymphocytes T stimulés par CD3-CD28. L’efficacité du knockout a été validée par Western Blot et TIDE sequencing. Le knock-out du gène du FKBP12 a conféré un maintien de la croissance cellulaire et des fonctions effectrices telles que la synthèse de cytokines IL-2, TNFα et IFN γ en présence de Tacrolimus comparativement aux cellules contrôles. Par la même méthode, nous avons pu invalider le gène du FKBP12 sur des lymphocytes T multivirus-spécifiques dont l’efficacité a été validée par cytométrie en flux. Les fonctions effectrices ont également été maintenues en présence de tacrolimus et ont été évaluées par ELISpot. Enfin, des lignées de lymphocytes T multivirus spécifiques dont le gène du FKBP12 a été invalidé ont été transduites avec un vecteur lentiviral dans le but d’exprimer un CAR CD19 dont l’expression a été validée par cytométrie en flux et la réactivité maintenue en présence de tacrolimus. En conclusion, ces résultats nous ont permis de démontrer la faisabilité de génération de lymphocytes T « triple fonction » anti-tumorales, anti-virales et résistantes au tacrolimus. L’application de cette approche semble prometteuse dans un contexte d’une immunothérapie adoptive anti-virale et anti-tumorale post-transplantation de moelle osseuse. / Adoptive transfer of virus-specific T lymphocytes or CAR-T cells has been shown to be effective for the treatment of several types of viral infections and certain cancers following hematopoietic cell transplantation. However, immunosuppression administered for the prevention of transplant rejection and graft-versus-host disease limits the efficacy and long-term persistence of responses mediated by these lymphocytes. The widely used immunosuppressive agent Tacrolimus (FK506) requires FK506-Binding protein (FKBP12) to exert its immunosuppressive effects on T cells. We undertook to engineer a multifunctional T-cell therapy to both optimally prevent viral reactivation and relapse of B-cell malignancies post-transplant in the context of immunosuppression. The objective of our work is to generate tacrolimus resistant, multivirus-specific T-cell lines expressing an anti-CD19 CAR. Using the gene editing method based on Clustered Regular interspaced short palidromic repeats (CRISPR) targeted by the CRISPR-associated protein 9 (Cas9) nuclease, we were able to invalidate the FKBP12 gene on activated T cells (confirmed by TIDE sequencing and western blotting). Invalidation of FKBP12 conferred maintenance of cell growth and effector functions such as the synthesis of cytokines IL-2, TNFα and IFNγ in the presence of Tacrolimus. Using the same method, we were able to delete the FKBP12 gene in virus-specific T lymphocytes. Effector functions were also maintained in the presence of tacrolimus. Finally, we integrated an anti-CD19 CAR by lentiviral transduction into FKBP12-edited multi-virus T-cell lines, and the efficiency of transduction was determined by flow cytometry. The cells maintained their viral reactivity in the presence of tacrolimus. In conclusion, we were able to confirm the feasibility of generation of ‘’triple function’’ T cells (anti-viral, anti-tumoral and tacrolimus resistant). Multifunctional T-cell product manufacturing is a promising approach to optimize post-transplant T-cell immunity against opportunistic pathogens and underlying malignancies.