Dissertationen zum Thema „Killer cells. Lymphocytic leukemia“

Clinical application of natural killer (NK) cells against leukemia is an area of intense investigation. In human leukocyte antigen-mismatched allogeneic hematopoietic stem cell transplantations (HSCT), alloreactive NK cells exert powerful anti-leukemic activity in preventing relapse in the absence of graft-versus-host disease, particularly in acute myeloid leukemia patients. Adoptive transfer of donor NK cells post-HSCT or in non-transplant scenarios may be superior to the currently widely used unmanipulated donor lymphocyte infusion. This concept could be further improved through transfusion of activated NK cells. Significant progress has been made in good manufacturing practice (GMP)-compliant large-scale production of stimulated effectors. However, inherent limitations remain. These include differing yields and compositions of the end-product due to donor variability and inefficient means for cryopreservation. Moreover, the impact of the various novel activation strategies on NK cell biology and in vivo behavior are barely understood. In contrast, reproduction of the thirdparty NK-92 drug from a cryostored GMP-compliant master cell bank is straightforward and efficient. Safety for the application of this highly cytotoxic cell line was demonstrated in first clinical trials. This novel ‘off-theshelf’ product could become a treatment option for a broad patient population. For specific tumor targeting chimeric-antigen-receptor-engineered NK-92 cells have been designed.

T-cells appear to have multiple conflicting roles in CLL. On the one hand tumour-specific T-cells could be used to deliver effective immunotherapy; on the other hand, certain T-cell populations may enhance CLL survival and disease progression. The aim of this thesis was to address these contradictory aspects and to provide a deeper understanding of the role of T-cells in CLL. Firstly, candidate peptides from the pro-apoptotic protein Bax were used to activate potential CLL specific T-cells from HLA-A2+ patients. A CD8+ T-cell clone (6C5) was isolated and it’s specificity was initially mapped to (Bax161-170; LLSYFGTPT) and(Bax160–170; GLLSYFGTPT). However, 6C5 failed to recognise HLA-A2+ CLL cells in vitro, and failed to recognise highly purified forms of the peptides. Further characterisation, involving mass spectrometry and HPLC, mapped T-cell specificity to a modified peptide (LLSY(3-tBu)FGTPT). A second strand of this project involved detailed phenotypic analysis of T-cells from CLL patients (n=97) in order to investigate the basis for immune dysfunction. This analysis indicated that patients with an inverted CD4:CD8 ratio (CLLIR), displayed a skewing towards a highly differentiated T-cell phenotype, as well as expression of markers associated with replicative senescence (CD57+, CD27-) within CD4+ and CD8+ T-cell compartments. In addition, CD4+ T-cells expressing markers associated with immunosuppression (PD-1+, TIM-3+) were also increased in CLLIR. Importantly, the inversion of the CD4:CD8 ratio was associated with shorter progression-free survival. Furthermore, the frequencies of distinct T-cell populations were also shown to haveprognostic impact in both univariate analysis (CD4+PD-1+, CD4+CD57+, CD8+CD57+ and CD8+CD27-) and multivariate analysis (CD4+CD27-PD-1+LAG-3+ and CD8+CD27- CD57+PD-1+). To further evaluate the differences between CLLIR and CLLNR patients, preliminary transcriptional analysis was performed, focusing on genes associated with T-cell function. By contrast, transcriptional analysis suggested that genes associated with activation rather than suppression were enriched in CLLIR.

B cell Chronic lymphocytic leukemia (B-CLL) is a neoplastic disorder characterized by accumulation of B lymphocytes due to uncontrolled growth and resistance to apoptosis. Src family kinases (SFKs) are non receptor tyrosine kinases present in the cytosol, which couple with downstream B cell receptor signaling and thus mediate growth, survival, proliferation and antiapoptosis. In CLL cells SFKs are remarkably overexpressed, especially Lyn kinase. This gives the rational to use SFKs inhibitor to treat CLL. Addition of the specific pharmacological inhibitors of SFKs, bosutinib and saracatinib, inhibited the global tyrosine phosphorylation as well as the basal auto-phosphorylation of SFKs. Mechanistically, inhibition of SFKs is coupled to apoptosis induction via decreased protein levels of the anti-apoptotic proteins Bcl-2, Mcl-1 and survivin, which were demonstrated by Western blotting. To assess apoptosis induction, annexin V binding to freshly isolated CLL cells with or without treatment with kinase inhibitors was measured flow cytometrically. Using the inhibitors at a concentration of 10 μM the average percentages of annexin V-positive, apoptotic cells in 11 CLL samples increased from 24 % in untreated controls to 55 %, 45 % and 37 % after treatment with bosutinib, saracatinib and dasatinib, respectively. The response to each of the inhibitors showed a high but comparable degree of variation among the investigated CLL samples. On the average bosutinib induced apoptosis with significantly higher efficiency than dasatinib, which calls for further investigation of its pre-clinical potential for treatment of CLL.

Mestrado em Biomedicina Farmacêutica
Cellular immunotherapies, or Advanced Therapy Medicinal Products (ATMPs), are emerging as novel and specific therapeutic approaches to treat diseases, such as certain types of leukemias, which are difficult or impossible to treat with today’s biopharmaceutical products. Breakthroughs in basic, preclinical, and clinical science spanning cellular immunology, and cellprocessing technologies has allowed clinical applications of chimeric antigen receptor–based therapies. A recent example is CTL019, a lentivirus-based gene therapy for autologous T cells, acquired by Novartis in 2012 through a global alliance with the University of Pennsylvania. Although this technology is still in its infancy, clinical trials have already shown clinically significant antitumor activity in chronic lymphocytic leukemia and acute lymphocytic leukemia. Trials targeting a variety of other adult and pediatric malignancies are under way. The potential to target essentially any tumor-associated cell-surface antigen for which a monoclonal antibody can be made opens up an entirely new arena for targeted therapy of cancer. The regulatory environment for these Advanced Therapies Medicinal Products is complex and in constant evolution. Many challenges lie ahead in terms of manufacturing process, non-conventional supply chain logistics, business models, intellectual property, funding and patient access.

To supplement and modify the diagnosis and clinical research of B-cell Chronic Lymphocytic Leukemia (B-CLL), a new method based on cell imaging and image processing was developed and applied to the B-CLL patient samples. The fluorophore-labelled leukemia cells were clearly visualized, reflecting the positive/negative expression of the corresponding surface markers and their distribution. Computer algorithms were devised and used to analyze a large number of images. The fluorescence intensity of the labelled antibodies on a given cell directly reflects the expression of the corresponding surface markers. The morphology and size of leukemia cells were not identical even in the same patient’s sample and the size variation does not correlate with the number of surface markers. The amount of each surface marker was approximately fixed for each patient, but there were some relationships, for instance, the number of CD19 and CD38 markers were correlated to each other. The heterogeneous expression of surface markers confirmed an assumption that surface markers have their preferred membrane positions. One of the most important results is that the cell imaging and our image processing method has provided an alternative and reliable way to diagnose B-CLL and new insights in the prognosis of subtype of B-CLL.

Les cellules Natural Killer (NK) représentent une population de cellules innées lymphoïdes aux fonctions anti-infectieuses et antitumorales. Les leucémies aiguës lymphoblastiques pré-B (LAL pré-B) constituent le cancer de l’enfant le plus fréquent et ont été décrites comme résistantes à la cytotoxicité médiée par les NK bien que les bases moléculaires demeurent inconnues.L’objectif de ces travaux a été de caractériser cette résistance. En développant un essai de cytotoxicité par cytométrie en flux et en utilisant des cellules effectrices activées in vitro, nous avons établi la sensibilité retardée des LAL pré-B à la cytotoxicité NK : initialement résistantes après 4h d’incubation, elles sont fortement tuées après 25h.Cette cytotoxicité est contact-dépendante mais ni la voie de l’exocytose des granules cytotoxiques ni celle des récepteurs de mort n’y contribuent. La mort cellulaire des cibles est de profil apoptotique mais indépendante des caspases ; la signalisation mitochondriale l’amplifie partiellement. Interférer avec les dérivés de l’oxygène par un antioxydant diminue la cytotoxicité. Nous montrons que les cellules NK de patients atteints de granulomatose septique chronique liée à l’X présentent un défaut de cette nouvelle cytotoxicité. Nous démontrons l’expression par les NK des composants clefs d’une NADPH oxydase distincte du complexe utilisé par les phagocytes. Nos travaux établissent l’existence d’une voie de cytotoxicité non conventionnelle et en définissent les principaux prérequis moléculaires
Natural Killer (NK) cells are innate lymphoid cells with anti-infectious and anti-tumoral activities. Among neoplasia, pre-B acute lymphoblastic leukemias (pre-B ALL) represent the most common form of cancer in childhood and were shown to be resistant to NK cell mediated cytotoxicity although the mechanisms explaining this phenomenon are incompletely understood.In the present work, we investigated the relative immune resistance of pediatric pre-B ALL targets to activated NK cells. We developed a flow cytometry based cytotoxicity assay to assess the NK activity and the involvement of long term cytotoxic pathways. Although pre-B ALL blasts were strongly resistant at 4h, we found a considerable delayed NK killing at 25h.Further investigations revealed that cell contact was mandatory for efficient killing but also that neither the granule exocytosis nor the death receptor pathway were involved. Target cell death was caspase independent but mitochondria signaling amplified it. We then showed that NK cells from patients with X-linked chronic granulomatous disease could not kill efficiently ALL blasts and that NK cells expressed key components of a NADPH oxidase complex that was distinct from the phagocyte type. Our work reveals an uncharacterized effector pathway among cytotoxic lymphocytes and establishes key molecular requirements for this unconventional pathway

Natural killer (NK) cells are lymphocytes that comprise part of the innate immune system and play a key role in the early defence against pathogenic organisms and cancer. CpG oligodeoxynucelotides (ODNs) are short synthetic ODN containing unmethylated CpG dinucleotide motifs that have immune-enhancing effects. NK cell-derived IFN-γ is essential for the effects of CpG ODNs, but how NK cells become activated by CpG ODNs remains unclear. We found that CpG ODN-mediated stimulation of NK cells requires IL-12 or IL-18. CpG ODNs did not stimulate IL-12-deficient mouse spleen cells and IL-12 neutralization almost completely inhibited IFN-γ production. Although IL-18 was undetectable in cultures, neutralization significantly dampened the IFN-γ response and addition of exogenous IL-18 greatly enhanced CpG ODN-mediated NK cell stimulation. IL-12 is mainly produced by Gr-1⁺ monocytes and neutrophils, while what cells produce IL-18 remains unknown. We then tested the anti-leukemia effects of CpG ODN-stimulated NK cells. Studies with human acute myeloid leukemia (AML) patients have shown that haploidentical NK cells effectively kill AML blasts, but their ability to lyse leukemia initiating cells (LICs) has not been studied. Therefore, we tested NK cells from haploidentical F1 mice against the mouse AML cell line MN1. F1 mouse NK cells expanded in cultures in the presence of IL-15 and stimulated by CpG ODNs plus IL-18, effectively killed bulk MN1 cells in vitro and reduced the numbers of in vitro colony forming cells. NK cell-treated MN1 cells were also injected into irradiated B6 mice to test whether AML LICs were also killed. F1 mouse NK cells seemed to kill some AML initiating cells since mice receiving NK-treated MN1 cells survived significantly longer than those given untreated MN1 cells, but the frequency of LICs did not significantly differ between MN1 cells incubated with or without NK cells. For NK cells to be used as a treatment for AML, we must find a way to induce a higher cytotoxicity in NK cells or to target them specifically towards LIC.

The telomeres are specialized structures at the end of the chromosomes composed of the repeated DNA sequence (TTAGGG)n and specific proteins bound to the DNA. The telomeres protect the chromosomes from degradation and end to end fusions. Due to the end-replication problem, the telomeric DNA shortens every cell division, forcing the cells into senescence at a critical telomere length. This process can be counteracted by activating a specialized enzyme, telomerase, which adds telomeric repeats to the chromosome ends leading to an extended or infinite cellular life span. Telomerase activity is absent in most somatic tissues but is found in germ cells, stem cells, activated lymphocytes and the vast majority of tumor cells and permanent cell lines. Hence, telomerase has been suggested as a target for cancer treatment as malignant cells almost exclusively express the enzyme and in that context telomere length measurements will be of great importance.

Telomere length is traditionally measured with a Southern blot based technique. A new method for telomere analysis of cells in suspension, called flow-FISH, was developed based on fluorescence in situ hybridization using a telomeric peptide nucleic acid (PNA) probe,

DNA staining with propidium iodide and quantification by flow cytometry. Flow-FISH had high reproducibility and the telomere length measurements showed good correlation with Southern blotting results. The flow-FISH technique also allows studies of cells in specific phases of the cell cycle and the replication timing of telomeric, centromeric and other repetitive sequences were analyzed in a number of cells. Like previous studies, centromeres were shown to replicate late in S phase while the telomere repeats were found to replicate early in S phase or concomitant with the bulk DNA, which is opposite to the patterns described in yeast.

In benign immunopurified lymphocytes from tonsils, high telomerase activity was found in germinal center (GC) B cells. This population also had high hTERT mRNA levels and displayed a telomere elongation as shown by flow-FISH and Southern blotting. Combined immunophenotyping and flow-FISH on unpurified tonsil cells confirmed the results.

Chronic lymphocytic leukemia (CLL), the most common leukemia in adults, can be divided into pre-GC CLL, characterized by unmutated immunoglobulin VH genes and worse prognosis, and post-GC CLL, with mutated VH genes and better prognosis. In 61 cases of CLL, telomere length was measured with Southern blotting and VH gene mutation status was analyzed. A new association was found between VH mutation status and telomere length, where cases with longer telomeres and mutated VH genes (post-GC CLL) had better prognosis

than CLL with short telomeres and unmutated VH genes (pre-GC CLL). A larger study of 112 CLL cases was performed using flow-FISH. The same correlation between telomere length and VH mutation status was found but gender seemed to be of importance as telomere length was a significant prognostic factor for the male CLL patients but not in the female group. Age of the patients and spread of disease seemed to affect the prognostic value of VH gene mutation status.

Les dernières avancées dans les traitements des hémopathies aboutissent à un meilleurs taux de rémission complète ainsi qu' à de meilleurs taux de survie après traitement. Cependant les risques de rechutes restent élevés. Notre projet s'inscrit dans la compréhension du rôle des cellules NK dans l'évolution de ce type de pathologies. Dans une première partie nous nous sommes intéressés à la polyglobulie de Vaquez. Cette pathologie présente une évolution lente et progressive, et elle est caractérisée par une mutation de JAK2 présente dans la lignée myéloïde chez plus de 95% des patients. Nous avons cherché à détecter la mutation dans les cellules NK de patients, puis, pour savoir si la mutation avait un effet sur les NK, nous avons exploré leurs fonctions in vitro. Nos résultats ont montré que, bien que la mutation soit présente dans les cellules NK, elle ne semble pas avoir d'impact sur les fonctions des cellules NK que nous avons pu tester. Nous en avons conclu que l'évolution de la polyglobulie de Vaquez en leucémie n'était peut-être pas due à une perte de fonction des NK mais plutôt à leur inhibition par l'environnement cellulaire.Dans une deuxième partie nous avons étudié la régulation des natural cytotoxicity receptors dans la leucémie aiguë myéloïde. D'apres des travaux antérieurs nous avons émis l'hypothèse que l'expression des trois NCR aurait une régulation commune s'effectuant au niveau de transcription de leurs gènes. Nos recherches bio-informatiques ainsi que notre expérimentation d'immunoprécipitation de la chromatine (Chip) montrent que le facteur de transcription ETS-1 semble être impliqué dans la régulation commune aux trois NCR
The latest advances in blood disorders treatments lead to a better complete remission rate and a better survival rate after treatment. However, the risk of relapse remains high. Our project is included in the understanding of NK cells role in the development of these diseases.In a first part, we focused on polycythemia Vera for several reasons: the pathology has a slowly progressive disease, and it is characterized by the presence of JAK2 mutation for > 95% patients. We wanted to know if this mutation was found in NK cells from PV patients and what effects the mutation had on NK cells functions. Our results have shown that although the mutation was found in NK cells, it appears to have no impact on NK cells functions. We conclude that the evolution of PV to leukemia is not due to a loss of NK cell functions but to their inhibition by cellular environment.In a second part, we investigated the regulation of natural cytotoxicity receptors in acute myeloid leukemia because previous works have shown that NCR are weakly expressed in AML patients, that this down-regulation is acquired during evolution of AML and reversible after complete remission, ant that NCR weak expression is related to poor prognosis. We supposed that the expression of the three NCR has a common regulation at genes transcription level. Our bioinformatic researches and our experiment of chromatin immunoprecipitation show that ETS-1 transcription factor is a good candidate involved in the common regulation of the three NCR

La leucémie lymphoïde chronique (LLC) est la plus fréquente des leucémies de l'adulte, caractérisée par l'accumulation de lymphocytes B CD5+ anormaux dans le sang, la moelle osseuse, et les organes lymphoïdes secondaires. Les événements oncogéniques à l'origine du développement de la LLC sont peu connus. L'identification de nouveaux gènes dérégulés est importante afin d'améliorer notre compréhension du développement et de l'évolution de la LLC, et de proposer de nouvelles stratégies ciblées. Tout d'abord, nous avons montré que la LLC se développe à partir de progéniteurs hématopoïétiques multipotentes pré-leucémiques portant des mutations somatiques. J'ai également analysé la délétion 14q dans une large série de patients, et nous avons conclu que la del(14q) est associé à la trisomie 12 et à des facteurs pronostiques péjoratifs : IGHV non mutée, mutations NOTCH1, et une survie sans traitement plus courte. Enfin, la partie principale de ma thèse portrait sur la caractérisation du gain du bras court du chromosome 2 (2p). J'ai identifié une région minimale de gain chez les patients LLC/2p+ et démontré que CRM1/XPO1 (Exportin-1) se trouve dans cette région, et muté de façon récurrente dans la LLC. J'ai démontré que le gain 2p provoque la résistance aux traitements RFC, Ibrutinib, R-Idélalisib, et au Selinexor. Nos résultats révèlent également que le Selinexor est inefficace pour induire l'apoptose dans les cellules LLC-B muté pour XPO1. Au total, mon travail préconise l'évaluation du gain 2p et des mutations de XPO1 avant de décider d'un traitement de la LLC
Chronic lymphocytic leukemia (CLL), the most common adulthood leukemia, is characterized by an accumulation of abnormal CD5+ B-lymphocytes in the peripheral blood, bone marrow, and secondary lymphoid organs. The origin and pathogenic mechanisms of CLL are not fully understood. Identifying the deregulation of new genes is important to improve our understanding about CLL development and evolution, and to propose new targeted strategies. First, we have shown that CLL develops from pre-leukemic multipotent hematopoietic progenitors carrying somatic mutations. I have also analyzed the deletion 14q in a large series of patients, and we have concluded that del(14q) was associated with trisomy 12 and with pejorative prognostic factors: unmutated IGHV, NOTCH1 mutations, and a short treatment-free survival. Finally, the main part of my thesis was about the characterization of the short arm of chromosome 2 (2p). I identified a minimal region gained in 2p+/CLL patients and demonstrated that CRM1/XPO1 (Exportin-1) is located in this region, and recurrently mutated in CLL. I demonstrated that 2p gain provokes FCR, Ibrutinib, R-Idelalisib, and Selinexor drug resistance. Our results also reveal that Selinexor is inefficient in inducing apoptosis in CLL B-cells with mutations in XPO1. Altogether, my work advocates for the assessment of the 2p+ and XPO1 mutations before deciding a CLL therapy

La leucémie lymphoïde chronique (LLC) est particulièrement associée aux cytopénies auto-immunes (AIC). L’expression de ZAP-70 dans la LLC est un facteur pronostique,due à une forte signalisation par le BCR. Nous décrivons une nouvelle population B normale (LyBn) qui exprime ZAP-70. Ces LyBn ZAP-70+ ne semble pas appartenir à une sous-population LyB particulière, et elles n’ont pas un phénotype activé. L’expression de ZAP-70 dans les LyB naïves suggère que ce phénomène est précoce. Il y a une bonne corrélation dans le niveau de ZAP-70 entre les LyBn et les LyB de LLC. Les LyBn ZAP-70+ ont été retrouvé dans tous les cas de AIC associée à la LLC.L’analyse des anticorps monoclonaux prévenant de LyB ZAP-70+ et des souris ZAP-70KI, réalisés pendant ce travail, définiront les conséquences de la surexpression de ZAP-70 dans les LyB dans la pathogénèse de l’auto-immunité et de la lymphoprolifération
Chronic lymphocytic leukemia (CLL) is particularly associated with autoimmune cytopenia (AIC). The expression of ZAP-70 in CLL cells is a prognostic factor, through increased BCR signaling. We described a novel normal B cell population (LyBn) that expresses ZAP-70. ZAP-70+ LyBn do not seem to belong to a certain subset of B cells, nor seem to have an activated phenotype. The presence of ZAP-70 in the naïve B cell subset suggests that this is an early process, which probably occurs before malignant transformation. There is a good correlation in the level of ZAP-70 expression, between normal B cells and CLL B cells. We found a significant percentage of ZAP-70+ LyBn in all AIC-associated CLLs. Analysis of monoclonal antibodies and of conditional ZAP-70KI mouse model synthesized in this work will clarify the consequences of aberrant ZAP-70 expression in B cells on autoimmunity and lymphoproliferation

Chronic Lymphocytic Leukemia is the primary lymphoid neoplasm in adults and and it is especially manifested in the elderly. Because it is a heterogeneous disease it awakens great interest regarding its prognosis. Rai and Binet developed staging systems to predict the evolution of the disease and currently, the analysis of expression of CD38 and Zap-70 has been investigated as a prognostic factor for indicating presence or absence of the mutation in the gene IgVH, so, the objective of this study was to analyze the clinical and laboratory profiles of patients with Chronic Lymphocytic Leukemia taking as reference the clinical staging of Rai and Binet and quantification of CD38 and Zap-70 expression as prognosis factors. We searched the medical records of 64 patients treated at University Hospital of Santa Maria and the variables considered were swollen lymph nodes, presence or absence of hepatomegaly and / or splenomegaly, hematological evaluation of peripheral blood and immunophenotype. The data obtained were correlated with the staging of Rai (1975) and Binet (1981), the expression of CD38 and Zap-70 and clinical stage. The results showed no association between ataging Rai and Binet and the expression of CD38, Zap-70 and Binet clinical staging.
A Leucemia Linfocítica Crônica é a principal neoplasia linfóide em adultos e se manifeta principalmente em indivíduos idosos. Por ser uma doença heterogênea, desperta grande interesse quanto ao seu prognóstico. Rai e Binet desenvolveram sistemas de estadiamento capazes de prever a evolução da doença e atualmente, a análise da expressão de CD38 e Zap- 70 tem sido investigada como fator prognóstico por indicar presença ou ausência da mutação no gene IgVH, assim, o objetivo deste estudo foi analisar o perfil clínico-laboratorial dos pacientes com Leucema Linfocítica Crônica, tomando como referência os estadiamentos clínicos de Rai e Binet e a quantificação da expressão de CD38 e Zap-70 como fatores prognóstico. Foram pesquisados 64 prontuários médicos de pacientes atendidos no Hospital Universitário de Santa Maria e as variáveis consideradas foram aumento de linfonodos, presença ou ausência de hepatomegalia e/ou esplenomegalia, avaliação hematológica de sangue periférico e imunofenótipo. Os dados obtidos foram correlacionados com o estadiamento de Rai (1975) e Binet (1981), a expressão de CD38 e Zap-70 com o estádio clínico de Binet. Os resultados demonstraram que não há associação entre o estadiamento de Raí e Binet e a expressão de CD38, Zap-70 com o estadiamento clínico de Binet.

Les cellules Natural Killer (NK) sont jouent un rôle essentiel dans la surveillance des hémopathies malignes. Cependant, les cellules tumorales développent des mécanismes immunosuppresseurs pour échapper à l'immunité cellulaire NK. Ainsi, le maintien ou l'amélioration des performances des cellules NK sont considérés comme des défis majeurs. Les objectifs de cette partie étaient d'évaluer l'impact de la perfusion de cellules NK activées sur la récupération et la biologie des cellules NK circulantes après l'allo-SCT. Des doses croissantes de cellules NK activées par IL-2 ex-vivo ont été perfusées chez des patients atteints de tumeurs malignes hématologiques 3 mois après allo-SCT. Nos résultats ont montré une fréquence plus élevée des cellules NK dans la périphérie des patients traités. Bien que le phénotype immature soit remarquable peu après le traitement, les cellules NK circulantes, présentaient un état d'activation avec un profil de maturation amélioré après 6 mois de traitement. Nous avons également constaté que l'expression des récepteurs NK activateurs (NKG2D, NKp30 et NKp46) augmentait sur les cellules NK circulantes des patients. De plus, ces cellules ont montré une augmentation significative de la capacité de dégranulation ainsi que de la sécrétion de cytokines (IFN-Ƴ et TNF-α) tout au long de l'étude. Ces différences ont notamment été observées chez les patients ayant reçu des doses plus importantes de cellules NK activées. En conclusion, nous supposons que la perfusion de fortes doses de cellules NK activées ex-vivo pourrait être associée à l'amélioration du phénotype et des fonctions des cellules NK au cours de la reconstitution immunitaire après allo-SCT
Natural killer (NK) cells are effector lymphocytes of the innate immune system that have the ability to kill transformed cells. They play a critical role in hematological malignancies surveillance, however, tumor cells develop immunosuppressive mechanisms to escape NK cell-mediated killing. So, maintaining or improving NK cell performance is considered a major challenge. Our goals are to evaluate the impact of activated NK cells infusion on the recovery and biology of circulating NK cells post allo-SCT.Three different doses (dose 1: 106 NK cell/Kg, n=3; dose 2: 5x106 NK cell/Kg, n=7; dose 3: >5x106 NK cell/Kg, n=6) of ex-vivo IL-2 activated NK cells were infused into patients with hematological malignancies after all-SCT. Our results showed higher frequency of NK cells in the periphery of patients treated with larger doses of activated NK cells. Although the notable immature phenotype shortly after treatment, the circulating NK cells in patients receiving larger doses of activated NK cells displayed more activation status with improved maturation profile after 6 months of treatment. We also found that the expression of activating NK receptors (NKG2D, NKp30, and NKp46) augmented on circulating CD56dim NK cells of patients receiving larger doses of activated NK cells. Moreover, these cells showed a significant increase in degranulation capacity as well as cytokine secretion (IFN-Ƴ and TNF-α) throughout study period. In conclusion, we hypothesize that infusion of high-dose of ex-vivo activated NK cells could be associated with improvements of NK cell phenotype and function during immune reconstitution after allo-SCT

CAR-T-Zell-Therapie ist eine vielversprechende neuartige Behandlungsform für Patienten mit aggressiven B-Zell Non-Hodgkin-Lymphomen (B-NHL). In dieser Arbeit wurde die anti-CXCR5 CAR-T-Zell-Therapie als Alternative zur anti-CD19 CAR-T-Zell-Therapie für die Behandlung von reifen B-NHLs untersucht. CXCR5 ist ein B-Zell-homing Rezeptor, der von reifen B Zellen und follikulären T-Helferzellen (TFH Zellen) exprimiert wird. TFH Zellen wurden als tumor-unterstützend in chronisch lymphatischer Leukämie (CLL) und im follikulären Lymphom (FL) beschrieben. Dieses Expressionsmuster erlaubt es, auf einzigartige Weise zeitgleich die malignen Zellen und die tumorunterstützende Mikroumgebung mithilfe von CAR-T-Zell-Therapie gerichtet gegen einen Chemokinrezeptor anzugreifen. Die wichtigsten Ergebnisse dieser Arbeit waren, dass (1) die anti-CXCR5 CAR T-Zellen zielgerichtet CXCR5 positive reife B-NHL Zelllinien und Patientenproben in vitro eliminierten und eine starke anti-Tumor Reaktivität in einem immundefizienten Xenotransplantationsmausmodell zeigten, (2) die anti-CXCR5 CAR T-Zellen zielgerichtet die tumorunterstützenden TFH Zellen in CLL und FL Patientenproben in vitro erkannten und dass (3) CXCR5 ein sicheres Expressionsprofil zeigte. CXCR5 war stark und häufig auf B-NHL exprimiert und die Expression auf gesundem Gewebe war auf lymphoide Zellen beschränkt. Zusammenfassend lässt sich sagen, dass die anti-CXCR5 CAR-T-Zell-Therapie eine neue Behandlungsmöglichkeit für Patienten mit reifen B-NHL darstellt, indem durch die anti-CXCR5 CAR-T Zellen sowohl der Tumor als auch ein Anteil der tumorunterstützende Mikroumgebung eliminiert werden. Im zweiten Teil der Arbeit wurde das Eμ-Tcl1 murine CLL Lymphommodell genutzt um die Auswirkung der Lymphomentwicklung auf die CXCR5+ T Zellen zu untersuchen. Mittels RNA-Einzelzell-Sequenzierung konnte ein profunder Einfluss des Lymphomwachstums auf das T Zell-Kompartiment der Mäuse, denen Eμ-Tcl1 Zellen gespritzt wurden, gezeigt werden.
CAR T cell therapy is a promising new treatment option for patients suffering from aggressive B non-Hodgkin lymphomas (NHLs). In CAR T cell therapy, patient-derived T cells are genetically modified to express a chimeric receptor commonly directed towards a surface antigen expressed by neoplastic cells. In this thesis, anti-CXCR5 CAR T cell therapy was investigated as an alternative to anti-CD19 CAR T cell therapy for the treatment of mature B-NHLs. CXCR5 is a B cell homing receptor expressed by mature B cells and follicular helper T (TFH) cells. TFH cells were described to support the tumor cells in chronic lymphocytic leukemia (CLL) and follicular lymphoma (FL). This expression pattern allows simultaneous targeting of the malignant cells and the tumor-supporting microenvironment by CAR T cell therapy against a chemokine receptor in an unprecedented manner. Main findings included that (1) anti-CXCR5 CAR T cells targeted specifically CXCR5 expressing mature B-NHL cell lines and patient samples in vitro and showed strong in vivo anti-tumor reactivity in an immunodeficient xenograft mouse model, (2) anti-CXCR5 CAR T cells targeted tumor-supportive TFH cells derived from CLL and FL patient samples in vitro and (3) CXCR5 showed a safe expression profile. CXCR5 was strongly and frequently expressed by B-NHLs and its expression on healthy tissue was restricted to lymphoid cells. In summary, anti-CXCR5 CAR T cell therapy presents a novel treatment option for patients suffering from mature B-NHLs by eliminating the tumor and part of the tumor-supportive microenvironment. The second part of the project, the Eμ-Tcl1 murine lymphoma model, which mimics human CLL, was used to study the impact of lymphomagenesis on CXCR5+ T cells. Using single cell RNA sequencing, a profound influence of lymphoma growth on the T cell compartment in Eμ-Tcl1 tumor-challenged mice could be shown.

Dissertação de mestrado em Bioquímica, apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra.
As células Natural Killer (NK) são linfócitos granulares com capacidade de matar células infectadas ou transformadas sem sinalização prévia, para além de produzirem citocinas que permitem modular a resposta imune inata e adaptativa. Representam cerca de 5-20% dos linfócitos no sangue periférico e, com base na sua função, são divididas em duas subpopulações CD56brightCD16- (produção de citocinas) e CD56dimCD16+ (citotoxicidade). A Leucemia Mielóide Crónica (CML) é uma doença genética mieloproliferativa e estudos anteriores indicam que células NK de doentes com CML são disfuncionais, mas os mecanismos que estão por base destas disfunções não estão ainda bem definidos. Desta forma, o principal objectivo deste trabalho é contribuir para o entendimento do controlo insuficiente das células NK sobre as células leucémicas. 62 Amostras de sangue de doentes com CML e de 4 dadores saudáveis foram analisadas. Todas as amostras foram marcadas com anticorpos e, por citometria de fluxo, analisamos a expressão de alguns marcadores, receptores e a produção de citocinas nas células NK. Neste estudo demonstramos que a frequência das células NK de doentes com CML está diminuída e a expressão de marcadores de superfície, receptores e a produção de citocinas está alterada. Para além disso, reportamos também que diferentes tipos de terapia ou a dosagem do fármaco têm diferentes efeitos nestas células. Concluímos então que os doentes com CML têm disfuncionalidades ao nível das células NK, o que indica uma disfuncionalidade na resposta imune do organismo contra a doença. Assim, entender como estas células estão a ser afectadas pode trazer novos desenvolvimentos no tratamento destes doentes, nomeadamente por modulação das células NK, estratégias que permitam potenciar a sua actividade.
Natural Killer (NK) cells are lymphocytes of the innate immune system killing skills against infected or transformed cells, without prior signaling. NK cells represent 5-20% of peripheral blood lymphocytes and based on their functions NK cells are subdivided into two subsets CD56brightCD16- (cytokine production) and CD56dimCD16+ (cytotoxicity). Chronic myeloid leukemia (CML) is a genetic myeloproliferative disease and previous studies indicate that NK cells are deficient in CML patients, although the mechanisms behind the dysfunction are not completely understood. The main goal of this work was to contribute to the cellular understanding of the insufficient control of NK cells over malignant CML cells. For this propose, we analyzed 62 blood samples from CML patients and 4 from healthy donors. All samples are labelled with extra or intracellular antibodies and through flow cytometry, we analyzed the expression of some surface markers, receptors and cytokine production NK cells from CML patients. In this study we demonstrate that NK cells from CML patients are reduced in number and the expression of cell surface markers and receptors, and cytokine production are altered. Besides that we reported that different biologic and TKI therapies affect in different ways the NK cells as well as imatinib dosage. We conclude that CML patients actually have dysfunctional NK cells, which reveal a dysfunction in the immune response against the disease. Thus, understand how these cells are affected can bring new developments in the treatment of these patients, particularly through modulation of NK cells, strategies to boost its activity.

B cell chronic lymphocytic leukemia (B-CLL) is a hematologic neoplasm characterised by the proliferation and accumulation of sIgM+/D+ B cells that fail to progress to the final stages of B cell development. The malignant cells in B-CLL also express the pan-T cell antigen CD5, suggesting that CLL is a malignancy of the CD5+ subset of B cells. Additional characteristics of the malignant clone include a low proliferative index, enhanced in vivo survival and constitutive expression of the anti-apoptosis oncoprotein bcl-2. The behaviour of leukemic CD5 B cells in vitro contrasts their arrested in vivo state. That is, despite the majority of cells being arrested in the G0 phase of the cell cycle, the leukemic B cells are not irreversibly frozen as they can be induced to differentiate to Ig-secreting cells under appropriate in vitro conditions. Furthermore, leukemic CD5 B cells rapidly undergo death by apoptosis following in vitro culture. This thesis describes the requirements for in vitro activation of leukemic CD5+ B cells, the characterisation of the events involved in apoptosis of these cells as well as the identification of various growth factors capable of modulating these events. Stimulation of unfractionated peripheral blood lymphocytes (PBLs) from three patients with B-CLL with the phorbol ester PMA and the mitogens PHA and PWM resulted in significant increases in cell proliferation, RNA synthesis and 1gM secretion when compared to unstimulated cell populations. PMA was the most potent inducer of 1gM secretion and this occurred irrespective of the presence of residual T cells. PMA-induced proliferation and RNA synthesis were also independent of T cells. However, in the presence of T cells, these parameters of cellular activation were enhanced during in vitro culture. Thus, the inductive ability of PMA on leukemic CD5 B cells was independent of T cells. In contrast, activation and differentiation of the leukemic CD5 B cells into 1gM-secreting cells following culture with mitogens did not occur in the absence of T cells. Interestingly, co-stimulation of leukemic CD5+ B cells with PMA and anti-Ig induced cellular responses that exceeded those induced by either activator alone. Thus, leukemic CD5+ B cells from patients with B-CLL can be activated in vitro and differentiate in response to stimulation via both T cell-dependent and T cell-independent mechanisms. Apoptotic cell death was characterised in purified leukemic CD5 B cells obtained from six B-CLL patients. All leukemic CD5 B cell populations entered an apoptotic pathway in vitro as evidenced by a reduction in cell size, loss of cell viability and fragmentation of DNA into multimers of -180 base pairs. Following 24 hours of in vitro culture 24.0±16% of DNA was fragmented. After 8 days, the majority of DNA was fragmented, and fewer than 10% of cultured cells were viable. Examination of bcl-2 expression in the malignant B cells by flow cytometry revealed a unimodal pattern of expression in greater than 85% of cells from each B-CLL patient prior to culture. During in vitro culture, bcl-2 expression became bimodal such that the B cells displayed a bcl-2hjgh and bcl-2iow phenotype. The level of expression by the bCl2hjgh cells was similar to that observed prior to in vitro culture, indicating that bcl-2 is down-regulated in apoptosing cells. Interestingly, despite this downregulation, the overall number of cells positive for bcl-2 remained constant. This suggests that the enhanced survival of leukemic CD5+ B cells in vivo is mediated by the sustained expression of bcl-2 and that additional mechanisms exist capable of overriding the protective effect of bcl-2 when bcl-2 is present at reduced levels. Leukemic B cell apoptosis has previously been reported to be delayed or prevented by IL-4, IFN-y and IFN-a. These results were confirmed in this study where it was found that culture of leukemic CD5 B cells with IL-4 or IFN-y enhanced cell viability and delayed apoptosis in 6/6 and 5/6 populations of leukemic B cells, respectively. This function was also found to be shared by IL-2, IL-6, IL-13 and TNF-a as these cytokines enhanced cell viability and delayed apoptosis in some of the cell populations examined at a level similar to that observed for IL-4 and IFN-y. These cytokines may mediate their effect via the expression of bcl2 as culture in the presence of IL-2, IL-4, IL-6, IL-13, IFN-y or TNF-a resulted in a higher percentage of cells displaying the bcl-2high phenotype, compared to unstimulated cells. Taken together, these results suggest that autocrine and/or paracrine growth loops may play a role in the pathogenesis of B-CLL and that cytokines that prevent apoptosis in vitro may be targets for treatment of this B cell malignancy.

Adequate cell production for adoptive cell transfer therapies such as Chimeric Antigen Receptor (CAR)-T cell therapy remains a critical barrier to treatment for indications that fail to achieve clinical success. One such disease is Chronic Lymphocytic Leukemia (CLL), a B-cell lymphoma with their characteristically exhausted T cells, marked by a progressive loss of the ability to secrete cytokines and proliferate, as well as an increase in the expression of checkpoint inhibitor molecules such as PD-1. The goal of this thesis is to characterize the functional differences or specific biomarkers within the CLL patient population that is indicative of the proliferation outcomes. Conventional clinical markers such as Rai stage or PD-1 expression alone were inadequate to describe the complex variability among patients. In order to better characterize exhaustion using microscopy-based cell function assays, we developed a sample sparing microscopy chamber that requires as little as 1000 cells per sample. The microscopy chambers were mass produced via injection molding, and made compatible with the antibody microcontact printing technique developed in the Kam lab. The chambers typically reduced cell usage per experiment by 20-fold. This reduction allowed us to measure IL-2 secretion, T cell arrest response to activating antibody patterns (pattern alignment), and motility of scarce human samples simultaneously from a single experiment. Results from these functional readouts along with other clinical markers were used as inputs for a multifactor exploratory analysis to cluster patients according to their functional similarities from the combination of responses in an unbiased manner. The resulting clusters based on the combination of the top 3 parameters IL-2, pattern alignment, and PD-1 resulted in better separation of patient groups and provided a basis for predicting max doubling outcomes from these inputs. We further used motility measurements as a way to understand initial T cell response to activation before the stop response, which was measured as pattern alignment previously. The time it takes for cells to come to a stop at the signal was most informative for translating T cell activation response to a stop response, and eventually to downstream effector functions of cytokine secretion and proliferation. The results of this work provide a powerful framework to describe different donors, and can be applied to cells from additional donors to guide future cell expansion studies.

Dissertação de Mestrado em Bioquímica apresentada à Faculdade de Ciências e Tecnologia
A leucemia mieloide crónica (LMC) é uma doença mieloproliferativa maligna que se desenvolve quando as células estaminais hematopoiéticas adquirem o cromossoma Philadelphia, sendo que este resulta da translocação entre os cromossomas 9 e 22. O resultado molecular desta translocação é uma proteína de fusão BCR-ABL,que se encontra constitutivamente ativada com ação de tirosina cinase . O uso de inibidores de tirosina cinase (TKIs) representam a terapia de referência em LMC, no entanto o único tratamento curativo é o transplante alogénico de células estaminais hematopoiéticas. O sistema imunitário, em particular as células Natural Killer e as CD8+ T, têm vindo a demonstrar ter um papel importante no controlo da LMC. No entanto as células tumorais desenvolvem diferentes mecanismos imunosupressores para escapar ao controlo do sistema imune. Um destes mecanismos é o aumento da expressão de immune checkpoints nas células imunes. O mais destacado nos últimos tempos é o recetor de morte programada-1 (PD-1); os anticorpos monoclonais para este recetor e para os seus ligandos tem vindo a ser descritos como uma arma promissora na imunoterapia. O objetivo principal deste trabalho foi estudar o papel do PD-1 nas células NK na LMC. Em primeiro lugar, a expressão do PD-1 e dos seus ligandos foi analisada por citometria de fluxo em 37 doentes com diferentes características clínicas. Os doentes em diagnóstico tiveram a expressão mais elevada de PD-1, demonstrando que a terapia tem a capacidade de modular o papel do PD-1. Além disso, quando os resultados foram analisados de acordo com a resposta molecular, a terapêutica também demonstrou a sua capacidade de diminuir a expressão do PD-1. A partir do momento em que os doentes atingiram uma pre resposta molecular major verificou-se uma tendência para a percentagem de células que expressam PD-1 diminuir com uma resposta molecular mais profunda . Foi medida uma maior concentração do PD-L1 solúvel comparativamente com o PD-1 solúvel, e para além disto o sPD-L1 teve a tendência para aumentar com uma resposta molecular mais profunda. Desta forma, 4 doentes em tratamento com uma resposta molecular 5.0 logs (MR5.0) foram selecionados para proceder as experiências com os anticorpos bloqueadores. O efeito destes anticorpos foi avaliado pela medição das funções citotóxicas e citolíticas das células NK. Desta forma, a desgranulação e a produção de IFN- e Granzima B foram medidos por citometria de fluxo. Adicionalmente, também se mediu a citotoxicidade direta mediada pelas células NK através do ensaio NK-TVA. Os resultados desta experiência com doentes de LMC, demonstraram que os anticorpos bloqueadores não aumentaram o potencial citotóxico das células NK, excluindo a possibilidade dos doentes com estas características clínicas poderem vir a beneficiar de imunoterapia com anticorpos bloqueadores.
Chronic myeloid leukemia (CML) is a malignant myeloproliferative disease developed when hematopoietic stem cells (HSCs) acquire the Philadelphia (Ph) chromosome, which is caused by the translocation between chromosomes 9 and 22. The molecular result of this translocation is a fusion protein BCR-ABL, which is a constitutively active tyrosine kinase. Tyrosine kinase inhibitors (TKIs) targeting the BCR-ABL kinase represent the standard care in CML, however the only curative treatment is the allogeneic HSCs transplantation. The immune system, in particular Natural Killer cells and CD8+ T cells, have been demonstrate to have an important role in CML control. Still, different immunosuppressive mechanisms are developed by tumour cells in order to escape immune surveillance. One of the immunosuppressive mechanisms is the expression of checkpoint inhibitors by the immune cells. Programmed death receptor 1 (PD-1) is the most remarkable of recent times, likewise monoclonal antibodies against this receptor and its ligands have been described as the new weapon of immunotherapy. The main goal of this work was to understand the role of PD-1 on NK cells in CML disease. In the first place, the expression of PD-1 and its ligands were analysed by flow cytometry in 37 CML patients with different clinical characteristics. Naïve patients showed the highest expression of PD-1, demonstrating that TKIs have the capacity to modulate PD-1 role. From the moment that CML patients achieved a pre major molecular response (Pre-MMR), the percentage of cells expressing PD-1 tends to increase with a deep molecular response. Higher concentrations of sPD-L1 were measured compared to sPD-1 and it was also shown a tendency to increase the sPD-L1 with a deepness MR. In this way, 4 patients undergoing Imatinib with a molecular response of 5.0logs (MR5.0) were chosen to proceed blocking experiences. The effect of blockade was assessed by the measurement of NK cytotoxicity functions and cytokine production. For this purpose, degranulation, IFN- and the Granzyme B production were measured by flow cytometry. Additionally, a NK-TVA assay was also done to measure the direct NK-mediated cytotoxicity. The results of blocking experiences in CML patients did not show the improvement of NK cells functions, excluding the possibility that CML patients, with these clinical characteristics, could benefit with the blocking antibodies to improve NK cells functions.
Outro - Funded by the FEDER Funds through COMPETE 2020 and by FCT within the framework of the Strategic Project with reference assigned by COMPETE: POCI-01-0145-FEDER-007440.

碩士
國立陽明大學
醫學生物技術暨檢驗學系
103
Natural killer (NK) cells, one of the important innate immune cells, have potential to kill virus-infected cells, transformed cells and tumor cells without further stimulation. The cytotoxic functions of the NK cells were regulated by the signals from a large set of activating receptors and inhibitory receptors. Moreover, either the directly interaction with other immune cells, including dendritic cells, macrophages and T cells, or the cytokines secreted from these immune cells indirectly enhance the killing activity of NK cells. Acute myeloid leukemia is characterized by the accumulation of immature cells in bone marrow and peripheral blood then further affects the hematopoiesis and cause disease. By the standard chemo- and radiation therapy, leukemia could be controlled on the remission status. However, the leukemia stem cells are resistant to conventional treatments and cause the relapse leading to the death of AML patients. According to previous study, NK cells seem to have the potential to kill LSCs and lower the disease relapse. In this study, we tend to develop a NK cell model with the ability to eliminate both leukemia cells and LSCs. Here, we demonstrated that the 2B4- and NKp30-mediated singling involved the NK-92MI-mediated cytotoxicity in killing Raji cells. We also have result showed that the stimulators IL-12+15+18+ODN2216/2336 dramatically increased the killing activity of NK-92MI cells. The killing mechanism needs further investigation. To test the cytotoxicity of the established NK-92MI cells in vivo, we successfully developed a THP-1-induced leukemia mouse model. Next, the killing of the stimulated NK model will be applied on the AML treatment of the mouse model. We believe the more understanding of the mechanism for NK-mediated cytotoxicity and the regulation of killing function would improve the outcome of current NK-based therapy in leukemia treatment.

Tese de doutoramento em Biociências, no ramo de Biologia Celular e Molecular, apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra
Hoje o conhecimento dos mecanismos envolvidos na patogenia das doenças linfoproliferativas crónicas de célula-B (B-CLPD) assume uma importância crescente. De forma geral, a sobrevivência e/ou proliferação da célula tumoral depende tanto das anomalias genéticas das células neoplásicas como do microambiente tumoral. Neste sentido, o desenvolvimento generalizado de técnicas moleculares para a caracterização quer das alterações genéticas presentes nas células tumorais, quer das características do recetor das células B (BCR), mostrou-se fundamental. Como consequência, a leucemia linfocítica crónica (CLL) é hoje considerada como o protótipo para várias doenças de células B em que as interações com o microambiente, mais que a presença de uma anomalia genética específica, são cruciais no surgimento, expansão ou mesmo na progressão da doença, em pelo menos uma fração dos casos. Neste sentido, a existência de um repertório de genes da região variável da cadeia pesada da imunoglobulina (IGHV) tendencioso juntamente com um estado mutacional particular e a recente identificação em casos não relacionados de locais de ligação ao antigénio (Ag) praticamente homólogos (BCR “estereotipados") é, regra geral, indicativo do envolvimento de um conjunto limitado de Ags, superantigénios ou ambos, no desenvolvimento da doença, fomentando a investigação das fases iniciais da mesma, p.e., através do estudo da linfocitose monoclonal de células B (MBL). Neste sentido, a citometria de fluxo veio facilitar a identificação de casos de MBL com (MBLhigh) ou sem (MBLlow) linfocitose B absoluta, a qual precede a maioria dos casos de CLL, permitindo assim a investigação de potenciais mecanismos envolvidos na transição de tais estados precursores tipo MBL, para CLL. Uma vez que a tumorigénese consiste num processo em várias etapas, os primeiros eventos transformantes podem ainda ocorrer em etapas mais precoces, quer diretamente na contrapartida normal da célula de CLL ou talvez, mesmo no compartimento de células estaminais hematopoiéticas de doentes com CLL. Para resolver esta questão, na presente tese de doutoramento investigámos múltiplas características fenotípicas e do BCR de células B clonais assim como do seu microambiente, numa série relativamente ampla de clones MBL, CLL/B-CLPD, tanto de casos monoclonais como multiclonais. De forma a explorar se determinados Ags poderão estar envolvidos em vias citogenéticas específicas durante as fases inicias do processo oncogénico, na primeira parte do estudo, focámos o nosso interesse na potencial associação entre determinados perfis citogenéticos e repertórios IGHV específicos. Num segundo passo, foram comparadas as características do BCR e as alterações citogenéticas dos clones de células B de casos monoclonais vs. casos multiclonais para determinar neste último grupo de doentes, a possível existência de uma maior homologia nos BCR que fosse potencialmente indicadora da ocorrência de respostas imunes mediadas por células B. Por fim, comparámos as características dos casos com clones MBL e CLL estereotipados vs. não estereotipados. De forma geral, foram detetados três grupos principais de clones com padrões distintos, mas parcialmente sobrepostos, relativamente ao uso dos genes IGHV, ao estado mutacional desses genes e às alterações citogenéticas: 1) um grupo enriquecido em clones MBLlow expressando genes IGHV específicos (p.e. VH3-23) sem alterações citogenéticas ou com alterações isoladas de bom prognóstico; 2) um grupo principalmente constituído por clones MBLhigh e estágios avançados de CLL com um repertório IGHV restrito, mas diferente (p.e., VH1-69), muitas vezes associado com cariótipos complexos e alterações citogenéticas de mau prognóstico, e; 3) um grupo com características intermédias, com prevalência de genes IGHV mutados e com números mais elevados de células clonais B del(13q)+. Estes resultados sugerem que as características do BCR de clones de células B com fenótipo de CLL podem modular o tipo de alterações citogenéticas adquiridas pela célula transformada, a sua taxa de aquisição, e eventualmente também, as suas consequências clínicas. Tal como referido anteriormente, os resultados recentes apoiam a existência em doentes com CLL, de uma estimulação crónica subjacente das células B por um conjunto restrito de epítopos. Neste sentido, expansões de ≥ 2 clones de células B têm sido frequentemente relatadas em B-CLPD, principalmente na MBL, a qual parece constituir um epifenómeno de estimulação antigénica crónica e persistente. Assim, foi colocada a hipótese de a multiclonalidade se encontrar associada com características particulares do BCR indicando uma maior probabilidade de interação com determinantes imunológicos partilhados. A análise comparativa de clones de células B com fenótipo de CLL e com fenótipo não-CLL de casos de MBL, CLL/B-CLPD multiclonais vs. monoclonais mostrou que, nos casos multiclonais o BCR clonotípico apresenta um grau ligeiramente maior de homologia de HCDR3, juntamente com características hematológicas e citogenéticas únicas, que estão tipicamente associadas com os estágios iniciais da doença. De entre estes casos foi ainda identificado um subgrupo de clones de células B (coexistentes) filogeneticamente relacionados que exibiam características moleculares e citogenéticas únicas. No seu conjunto, esses resultados apoiariam a natureza de tais expansões de células B multiclonais associada ao Ag e o potencial envolvimento de múltiplos epítopos em promover o desenvolvimento da MBL e favorecer a sua progressão para doença (p.e., LLC). No entanto, o cenário no qual podem ocorrer esses eventos permanece desconhecido. De forma a ganhar um maior conhecimento acerca deste cenário, na última parte do nosso trabalho, investigamos ainda a potencial relação entre uma hematopoiese alterada/ clonal e o estímulo antigénico durante a expansão dos clones de CLL e MBL estereotipados vs. não estereotipados. No geral, os casos estereotipados exibiam mais frequentemente genes IGHV1 em vez de IGHV3, juntamente com sequências HCDR3 mais longas e genes IGHV não mutados. O tamanho dos clones de células B estereotipados no sangue periférico (PB) não mostrou estar relacionado com o seu perfil citogenético, mas sim com a presença de imunofenótipos associados com mielodisplasia em células mielóides do PB. Tal associação particular sugere que o surgimento e/ou expansão de clones de células B de CLL nestes casos estereotipados pode ser favorecido por uma hematopoiese alterada subjacente. Em conclusão, os nossos resultados destacam o potencial envolvimento de diferentes vias induzidas pelo Ag nos estágios iniciais de desenvolvimento da MBL e de transformação para CLL, onde o reconhecimento de múltiplos epítopos pelo BCR, juntamente com a coexistência ou não de uma hematopoiese alterada subjacente, poderão modular os padrões de aquisição de alterações citogenéticas na patogénese da CLL, através de diferentes vias de transição desde os estágios de MBL multiclonal até aos clones de CLL monoclonal com perfis citogenéticos mais complexos.
Increasing knowledge exists about the mechanisms involved in the pathogenesis of B-cell chronic lymphoproliferative disorders (B-CLPD). Generally, tumor cell survival and/or proliferation depend both on the genetic abnormalities of neoplastic cells and the tumor microenvironment. Therefore, the development and widespread of molecular techniques for the characterization of both tumor cell genetic alterations and B-cell receptor (BCR) features, have been pivotal in the understanding of B-CLPD. As a consequence, chronic lymphocytic leukemia (CLL) is now considered as the prototype for several B-cell diseases where microenvironmental interactions, rather than a specific genetic abnormality, are critical in the onset, expansion and even progression of the disease, in at least a fraction of cases. Thus, a biased repertoire of the immunoglobulin heavy chain variable region (IGHV) genes with a particular mutational status, or even closely homologous antigen (Ag) binding sites among otherwise unrelated cases (“stereotyped” BCR), is generally considered as evidence for the involvement of a limited set of Ags, superantigens or both, in the development of CLL, fostering research about the early phases of the disease, e.g. monoclonal B-cell lymphocytosis (MBL). In this regard, flow cytometry has facilitated the identification of MBL cases with (MBLhigh) or without (MBLlow) absolute B-lymphocytosis which precedes most CLL cases, allowing the investigation of potential mechanisms involved in the transition from such MBL precursor states to overt CLL. Since tumorigenesis is a multi-step process, the first transforming events may occur at earlier stages, either directly in the normal counterpart of a CLL cell or perhaps, even in the hematopoietic stem cell compartment of CLL patients. In order to address this issue, in the present doctoral thesis we investigated multiple phenotypic and BCR features of clonal B-cells and their microenvironment in a relatively large series of MBL, CLL/B-CLPD clones, from both monoclonal and multiclonal cases. In order to explore whether particular Ag could be involved in specific cytogenetic pathways during early oncogenesis, we first investigated the potential association between unique cytogenetic profiles and specific IGHV repertoires. In a second step, we compared the BCR and cytogenetic features of B-cell clones from monoclonal vs. multiclonal cases to determine whether or not the latter were associated with a higher BCR homology, potentially reflecting occurrence of B-cell mediated immune responses. Finally, we compared the features of stereotyped vs. non-stereotyped MBL and CLL cases. Overall, we detected three major groups of clones with distinct but partially overlapping patterns of IGHV gene usage, mutational status and cytogenetic alterations: 1) a group enriched in MBLlow clones expressing specific IGHV genes (e.g. VH3-23) with no or isolated good-prognosis cytogenetic alterations; 2) a group which mainly consisted of MBLhigh and advanced stage CLL with a skewed, but different, IGHV gene repertoire (e.g. VH1-69), often associated with complex karyotypes and poor-prognosis cytogenetic alterations, and; 3) a group with intermediate features, prevalence of mutated IGHV genes and higher numbers of del(13q)+ clonal B-cells. Altogether, these results suggest that BCR features of CLL-like B-cell clones may modulate the type of cytogenetic alterations acquired by the transformed cell, their rate of acquisition, and potentially also, their clinical consequences. As referred above, recent findings support the existence of underlying chronic B-cell stimulation by a restricted set of epitopes in CLL. In line with this, expansion of ≥2 B-cell clones has been frequently reported in B-CLPD, mainly in MBL, which could be an epiphenomenon of a chronic and persistent antigenic stimulation. Thus, we hypothesized that multiclonality could be associated with particular BCR features indicating a greater probability of interaction with shared immunological determinants. Comparative analysis of CLL-like and non-CLL-like B-cell clones from multiclonal vs. monoclonal MBL, CLL/B-CLPD cases showed clonotypic BCR of multiclonal cases have a slightly higher degree of HCDR3 homology, together with unique hematological and cytogenetic features, which are typically associated with earlier disease stages. Among these cases a subgroup of phylogenetically related (coexisting) B-cell clones which displayed unique molecular and cytogenetic features, was identified. Altogether, these results would support the Ag-driven nature of such multiclonal B-cell expansions and the potential involvement of multiple epitopes in promoting the development of MBL and favor their progression into full disease (e.g. CLL). However, the scenario in which these events occur, remains unknown. In order to gain insight into the above scenario in the last part of our work we further investigated the potential relationship between an altered/clonal hematopoiesis and antigenic driving forces, during the expansion of stereotyped vs. non-stereotyped CLL and CLL-like MBL clones. Overall, former cases more frequently used IGHV1 rather than IGHV3 genes, together with longer HCDR3 and unmutated IGHV sequences. The overall size of the stereotyped B-cell clones in peripheral blood (PB) did not appear to be associated with their cytogenetic profile but it was more closely related to presence of myelodysplasia-associated immunophenotypes on PB myeloid cells. Such unique association suggests that the emergence and/or expansion of CLL-like B-cell clones in these stereotyped cases could be favored by an underlying altered hematopoiesis. In conclusion, our results highlight the potential involvement of different Ag-driven pathways in the early stages of development of MBL and transformation to CLL, where BCR recognition of multiple epitopes together with the co-existence or not of an underlying altered hematopoiesis, would modulate further patterns of acquisition of cytogenetic alterations in the pathway to CLL, through different transitional stages from multiclonal MBL to monoclonal CLL clones carrying more complex cytogenetic profiles.
FCT - SFRH/BD/31609/2006

Tumor-induced immunosuppression can occur by multiple mechanisms, each posing a significant obstacle to immunotherapy. Evidence presented in this dissertation suggests that aberrant cytokine signaling, as a result of altered metabolism of Chronic Lymphocytic Leukemia (CLL) cells, confers a selective advantage for tumor survival and growth. Cells from CLL patients with aggressive disease (as indicated by high-risk cytogenetics) were found to exhibit prolongation in Interferon (IFN)-induced STAT3 phosphorylation, and increased levels of reactive oxygen species (ROS) in these cells reflected these signaling processes. Changes in the relative balance of phospho-STAT3 and phospho-STAT1 levels, in response to combinations of IL-2 + Toll-like receptor (TLR)-7 agonist + phorbol esters, as well as IFN, were associated with the immunosuppressive and immunogenic states of CLL cells. In addition, immunosuppressive leukemic cells were found to express high levels of proteins with O-linked N-acetylglucosamine (O-GlcNAc) modifications, due to increased metabolic activity through the Hexosamine Biosynthetic Pathway (HBP), which caused impaired intracellular signaling responses and affected disease progression. A conclusion of the studies presented here is that the intrinsic immunosuppressive properties of leukemic cells may be overcome by agents such as Resveratrol that target metabolic pathways of these cells.

L’ostéonécrose (ON) et les fractures (FR) sont des complications qui prennent de plus en plus place dans le traitement pédiatrique de la leucémie aiguë lymphoblastique (LAL). L’ON peut être causée par différents facteurs, dont principalement l’utilisation de glucocorticoïdes. Les glucocorticoïdes sont administrés lors du traitement de la leucémie dans le but d’initier l’apoptose des cellules malignes tout en ayant un effet anti-inflammatoire. Cependant, l’utilisation de ces corticostéroïdes comprend des effets secondaires sérieux, notamment le développement d’ostéonécrose. Des variantes génétiques peuvent mettre certains patients plus à risque que d’autres. Plusieurs gènes ont déjà été signalés comme régulés par les actions glucocorticoïdes (GC). Les variations génétiques présentes dans les régions régulatrices de ces gènes peuvent affecter leur fonctionnement normal et, en fin de compte, de déterminer un risque accru de développer l’ON associé au traitement contre la leucémie. Pour cette raison, plusieurs polymorphismes ont été identifiés et étudiés dans la cohorte QcALL de Ste-Justine, concernant les gènes suivants : ABCB1, ACP1, BCL2L11, NFKB1, PARP1, et SHMT1. Ces gènes jouent majoritairement un rôle dans les mécanismes d’action des glucocorticoïdes, mais quelques-uns ont plutôt un effet direct sur le développement d’ostéonécrose. Nos recherches ont démontré une corrélation entre ces polymorphismes et l’apparition d’ostéonécrose chez les patients de la cohorte QcALL, traités aux glucocorticoïdes. L'incidence cumulative de l'ostéonécrose a été évaluée rétrospectivement chez 305 enfants atteints de la leucémie qui ont subi un traitement à l’hôpital Ste-Justine selon les protocoles DFCI de Boston (87-01, 91-01, 95-01 et 2000-01). Parmi les huit polymorphismes de BCL2L11 étudiés, les 891T> G (rs2241843) et 29201C> T (rs724710) ont été significativement associés à ON (p = 0.01 et p = 0.03, respectivement). L'association du polymorphisme 891T> G a été modulée par le type de corticostéroïde (CS), l’âge, le sexe et le groupe à risque (p ≤ 0,05). Le polymorphisme 29201C> T était particulièrement apparent chez les patients à haut risque (p = 0,003). La même étude était conduite en parallèle sur des patients de la cohorte DFCI de Boston (N = 192), et montrait des résultats significatifs pour les polymorphismes étudiés. En conclusion, les résultats de cette étude permettront de confirmer l’association de ces polymorphismes au développement d’ON chez les patients de LLA traités aux GC.
Osteonecrosis (ON) and fractures (FR) are complications that take place in the treatment of children acute lymphoblastic leukemia (cALL). They can be caused by various factors, mainly using glucocorticoids. The corticosteroids, dexamethasone (DXM) and prednisone (PDN) are administered during the treatment of leukemia to initiate apoptosis of malignant cells; while having an anti-inflammatory effect. However, the use of these corticosteroids has severe side effects, including the development of osteonecrosis. Moreover, some patients develop resistance to treatment, and are at risk of developing side effects. The genetic variants predispose some patients at higher risk than others. Several genes have been previously reported as up- or down regulated by the GCs actions. The genetic variations present in gene coding or regulatory regions can affect their function and ultimately determine an increased risk of developing ON associated to ALL therapy. Therefore, we investigated the association between several single nucleotide polymorphisms (SNPs) in six candidate genes: BCL2L11, NFKB1, PARP1, ABCB1, ACP1, and SHMT1. These genes play a role in the mechanisms of action of glucocorticoids, but some have more of a direct effect on the development of osteonecrosis. Our research has shown a correlation between these polymorphisms and the occurrence of osteonecrosis in patients in the QCALL cohort, treated with glucocorticoids. Cumulative incidence of osteonecrosis was assessed retrospectively in 305 children with ALL who underwent treatment with DFCI protocols (87-01, 91-01, 95-01 and 2000-01) in childhood ALL cohort from Quebec (QcALL). Among the eight tag BCL2L11 polymorphisms studied the 891T>G (rs2241843) and 29201C>T (rs724710) were significantly associated with ON (p = 0.01 and p = 0.03, respectively). Association of 891T>G polymorphism was modulated by type of corticosteroid (CS), age, sex and risk group (p ≤ 0.05 and that of 29201C>T was particularly apparent among high risk (p = 0.003) patients. These polymorphisms have shown significant ON association in several QcALL risk groups, mainly in corticosteroid groups, age < 10 years, and high risk (HR) group. Furthermore, the same study was conducted in parallel with patients in the replication (DFCI) cohort (N = 192), and we showed significant genetic association results for all studied polymorphisms. In conclusion, this study identifies that some ALL children have a high incidence of ON during the treatment that is highly associated with polymorphisms in different genes regulated by corticosteroids and ALL prognostic factors.

La leucémie aigüe lymphoblastique de précurseurs des cellules B (pré-B LAL) est le cancer le plus fréquent chez l’enfant. La transplantation de cellules souches hématopoïétiques (TCSH) est nécessaire dans environ 20 à 30 % des enfants ayant une pré-B LAL. Les rechutes après TCSH sont habituellement réfractaires aux thérapies actuelles, et par conséquent, il est important de développer et d’optimiser de nouvelles stratégies thérapeutiques. Dans cette étude, nous nous sommes intéressés aux cellules « cytokine-induced killer » (CIK). En effet, ces cellules ont été montrées comme hautement cytotoxique contre beaucoup de types de cancers. Cependant, leur activité cytotoxique contre les pré-B LAL n’est pas vraiment efficace. Par conséquent, nous avons étudié la possibilité de combiner l’immunothérapie des cellules CIK avec l’interféron alpha (IFN-α) afin d’optimiser l’activité lytique de ces cellules contre les cellules pré-B LAL. De plus, vu qu’il a été démontré que l’activité cytotoxique des cellules CIK provient de la fraction CD56+, plus particulièrement les cellules CD3+CD56+, nous avons décidé d’utiliser la fraction CD56+ (cellules CD56+) dans l’ensemble de nos expériences. Nous avons observé in vitro que les cellules CD56+ lysent mieux les lignées cellulaires pré-B LAL comparativement aux cellules CIK non purifiées. Aussi, leur activité cytotoxique peut être augmentée par le traitement avec l’IFN-α. Par ailleurs, nous avons démontré l’efficacité des cellules CD56+ traitées par l’IFN-α contre les lignées cellulaires pré- B LAL in vivo, dans le modèle de souris NOD/SCID/gamma c- (NSG). La survie des souris est significativement prolongée lorsqu’elles reçoivent les cellules pré-B LAL avec les cellules CD56+ traitées par l’IFN-α. Nous avons par la suite étudié le mécanisme d’action des cellules CD56+ contre les lignées cellulaires pré-B LAL. Nous avons observé que les cellules CD56+ provenant de sang de cordon sont plus efficaces que les cellules CD56+ provenant de sang I périphérique pour tuer les lignées cellulaires pré-B LAL. Nous avons également montré que les cellules CD56+ utilisent seulement la voie NKG2D ou bien les voies NKG2D et TRAIL selon la lignée cellulaire pré-B LAL cible et selon la provenance de la source des cellules CD56+. Par ailleurs, nous avons remarqué que les cellules CIK sont sensibles à l’apoptose par Fas, et que cette sensibilité influence leur activité cytotoxique contre les cellules tumorales. En conclusion, les cellules CD56+ sont cytotoxiques contre les lignées cellulaires pré-B LAL, et leur effet lytique est augmenté par l’IFN-α aussi bien in vitro qu’in vivo dans le modèle de souris NSG. Ces données précliniques sont encourageantes pour tester cette nouvelle approche d’immunothérapie dans le traitement contre la pré-B LAL.
Precursor B-cell acute lymphoblastic leukemia (B-ALL) is the most common form of leukemia in children. Hematopoietic stem cell transplantation (HSCT) is required in around 20 to 30% of children with a B-ALL. The relapses occuring post-HSCT are usually insensitive to current therapy. Therefore, it is important to develop and optimize a new therapeutic strategy. In this study, we were interested to study « cytokine-induced killer » (CIK) cells. These cells have been shown to be very cytotoxic against many types of tumor. However, their cytotoxic activity against B-ALL cells is not very efficient. Consequently, we have studied the effect of combining adoptive immunotherapy of CIK cells with the interferon alpha (IFN-α) to increase their lytic activity against B-ALL cells. In addition, in the literature, the cytotoxic activity of CIK cells has been shown to come from the CD56+ fraction (CD56+ CIK), in particular CD3+CD56+ cells. Therefore, we used the CD56+ fraction in all the experiments. We have observed in vitro that CD56+ CIK cells killed more efficiently B-ALL cell lines than did non-purified CIK cells. Also, their cytotoxic activity could be enhanced with IFN-α. Moreover, we have demonstrated the efficacy of IFN-α-treated-CD56+ CIK cells against B-ALL cell lines in vivo in the model of NOD/SCID/gamma c- (NSG) mice by showing that the survival of mice injected with B-ALL cell lines was significantly increased when they were injected with IFN-α-treated-CD56+ CIK cells. Subsequently, we have studied the lytic mechanism of CD56+ CIK cells against B-ALL cell lines. We have observed that CD56+ CIK cells from cord blood were more efficient than CD56+ CIK cells from peripheral blood to kill B-ALL cell lines. CD56+ CIK cells used only the NKG2D pathway or the both NKG2D and TRAIL pathways depending on the B-ALL cell line and the source of CIK cells. In addition, we showed that CIK cells were sensitive to Fas apoptosis. This sensitivity III influenced the cytotoxic activity of CIK cells against tumor cells. In conclusion, CD56+ CIK cells are cytotoxic against B-ALL cell lines, and their effect can be increased with IFN-α in vitro and in vivo. Taken together, our pre-clinical data are very interesting for testing the potential clinical utility of purified CD56+ CIK cells as an immunotherapeutic strategy for B- ALL patients.