Immunotherapy for cancer is a young research field progressing at high speed. The first chimera of an antibody and a signaling chain was designed by Zelig Eshhar and was later further developed to enhance existing T cell therapy by combining a single-chain fragment of an antibody with the CD3 zeta chain of the TCR complex. T cells expressing these chimeric antigen receptors (CARs) could recognize and specifically kill tumor cells. However the T cells, lacked in persistence and tumor rejection did not occur. Thus, the CAR constructs have been improved by providing the T cell with costimulatory signals promoting activation. The focus of this thesis has been to evaluate second and third generation αCD19-CAR T cells for the treatment of B cell leukemia and lymphoma. B cell tumors commonly upregulate anti-apoptotic proteins such as Bcl-2, which generates therapy resistance. In the first paper a second generation (2G) αCD19-CD28-CAR T cell was combined with the Bcl-2 family inhibitor ABT-737. ABT-737 sensitized tumor cells to CAR T cell therapy and may be an interesting clinical combination treatment. In paper II, the phenotype and function of a third generation (3G) αCD19-CD28-4-1BB-CAR T cell were evaluated. B cell-stimulated CAR T cells showed increased proliferation and an antigen-driven accumulation of CAR+ T cells. 3G CAR T cells had equal cytotoxic capacity, similar lineage, memory and exhaustion profile phenotype compared to 2G CARs. However, 3G CAR T cells proliferated better and had increased activation of intracellular signaling pathways compared to 2G CAR T cells. In paper III, αCD19-CD28-4-1BB-CAR T cells were used to stimulate immature dendritic cells leading to an upregulation of maturation markers on co-cultured dendritic cells. Hence, CAR T cells may not only directly kill the tumor cells, but may induce bystander immunity that indirectly aids tumor control. This thesis also include supplementary information about the development and implementation of protocols for GMP production of CAR T cell batches for a phase I/IIa clinical trial currently ongoing for patients with refractory B cell leukemia and lymphoma. So far, two patients have safely been treated on the lowest dose.

The studies presented in this thesis focus on human and canine models for autoimmune disease, with the main aim to gain new knowledge about disease mechanisms and to further evaluate the dog as a model for autoimmune disease. Autoimmune Polyendocrine Syndrome type 1 (APS-1) is a hereditary human multiorgan disease caused by mutations in the autoimmune regulator (AIRE) gene. Hallmarks of APS-1 are chronic mucocutaneous candidiasis caused by Candida albicans, together with the autoimmune endocrine disorders hypoparathyroidism and adrenocortical failure. Many human diseases have an equivalent disease in dogs. Because humans share environment, and in part life style with the dogs they provide an interesting model for further genetic studies. Immune responses to Candida albicans in APS-1 patients displayed an increased secretion of the proinflammatory cytokine IL-17A and similar results were also found in AIRE deficient mice. Anticytokine autoantibodies to IL-17A, IL-17F and IL-22 were detected in APS-1 patients, and a radioligand binding assay for measuring these autoantibodies was developed and evaluated. In the canine studies we investigated whether canine diabetes mellitus could serve as a model for human autoimmune diabetes mellitus. Furthermore, we investigated type I IFN responses in Nova Scotia duck tolling retriever dogs with a systemic autoimmune disease resembling human SLE. Four assays were used in search for signs of humoral autoimmunity in diabetic dogs. However, no evidence for a type 1 diabetes-like phenotype in dogs was found. Sera from Nova Scotia duck tolling retrievers suffering from steroid-responsive meningitis arteritis elicited an increased expression of IFN-inducible genes in the canine MDCK cell line. This suggests that these dogs have an IFN signature, as seen in human SLE.

[Truncated abstract] Human complement receptor 2 (CR2/CD21) is a cell surface glycoprotein detected on specific cells involved in immunity, which binds complement C3 cleavage fragments, cellular ligands IFN-? and CD23 as well as the EBV coat protein, gp350/220. During the early stages of B-cell development CR2/CD21 is silenced. Expression is initiated on immature B-cells escaping negative selection. During peripheral maturation CR2/CD21 is up-regulated with B-cell sub-populations showing distinctive surface levels (comparatively low, intermediate or high). CR2/CD21 is silenced upon terminal plasmacytic differentiation. Appropriate timing and expression level of CR2/CD21 is important for the development of a healthy B-cell repertoire. Previous studies have identified sequences within the proximal promoter and first intron of CR2/CD21 that cooperate within native chromatin to control cell-specific silencing. Further, analysis of cultured human cells has revealed chromatin structural variation causing DNase I hypersensitivity at these regulatory sites in a CR2/CD21-expressing mature B-cell line (Raji) which are absent in a non-lymphoid cell type (K562). The primary focus of the present study involved characterising chromatin structural variation over previously recognized DNase I hypersensitive regions at the CR2/CD21 locus in human cells to understand how chromatin structure might regulate developmental expression of CR2/CD21. ... These studies provide evidence that notch signaling influences CR2/CD21 expression in human cell lines. First, in vivo binding of CBF1 to CR2/CD21 sequences in the proximal promoter and CRS implies that CR2/CD21 is a direct target of notch activation. Second, the effect of exogenous notch signalling molecules on CR2/CD21 proximal promoter activity was modulated by factors binding tandem E-boxes near the transcriptional start site suggesting that the notch pathway may also influence CR2/CD21 expression via control of HLH molecules. Third, initiation of CR2/CD21 expression was observed in a nonexpressing pre-B cell line (Reh) by co-culture with stromal cells expressing a notch ligand (OP9-DL) but not control stroma (OP9-GFP). Together, these findings support a role for notch regulation of B-cell maturation and invite speculation that initiation of CR2/CD21 expression following negative selection of immature B-cells involves crosstalk between HLH transcriptional regulators and the notch pathway. Furthermore, the Reh/OP9-DL co-culture system may provide a model to directly study the relationship between cell signalling molecules, transcription factor regulation, chromatin structural variation and differentiation of B-cells.

RANKL (ligand du récepteur activateur de NF-KB) est un membre de la famille des TNF dont la signalisation passe par RANK et qui joue un rôle important dans la régulation immunitaire. Chez l'adulte, RANKL est exprimé constitutivement par des cellules réticulaires marginales (MRC) des ganglions lymphatiques. Comme les MRCs sont physiquement proches des lymphocytes B (LB) et ont été proposé d’être des précurseurs de cellules dendritiques folliculaires (FDC), RANKL pourrait jouer un rôle dans la différenciation du stroma associé aux LB et dans la réponse humorale. Afin de mieux comprendre la fonction de RANKL exprimé par les MRC, nous avons généré des souris déficitaires pour RANKL dans les cellules stromales. Nous avons constaté que la formation du follicule B était perturbée ainsi que le réseau FDC. Bien que RANKL ne soit pas requis pour la formation des MRC, il est nécessaire pour l'expression de la chimiokine CXCL13 par ces mêmes cellules. Parmi les TNFRSF dont la signalisation est requise pour l’expression de CXCL13 et la différenciation des FDC, le TNFR1 était significativement réduit dans les cellules stromales des souris dépourvues de RANKL stromal. Ainsi, RANKL pourrait constituer une nouvelle cible thérapeutique contre les immunopathologies des LB en agissant sur son stroma. / RANKL (receptor activator of NF-κB ligand), a member of the TNF family that signals via RANK, plays an important role for immune regulation. In the adult, RANKL is constitutively expressed by marginal reticular cells (MRCs) of the lymph nodes. Because MRCs are positioned in close vicinity to B cells and may be precursors of follicular dendritic cells (FDCs), RANKL could play a role in the differentiation of B cell-associated stroma and the humoral immune response. In order to better understand the role of RANKL expressed by the MRCs, we generated mice with conditional RANKL deficiency in the stromal compartment. We found that the B cell follicle structure was disrupted and FDC network formation was reduced. Although RANKL was not required for MRC formation, it was necessary for the expression of B cell attracting chemokine CXCL13. Among the TNFRSF members known to control CXCL13 expression and FDC formation, we found that TNFR1 was significantly reduced in the RANKL cKO mice. Thus, RANKL may present a novel therapeutic strategy against B cell-mediated immunopathologies by acting on its stroma.

The most widely transfused blood component is red blood cells (RBCs), and voluntary donation is the main resource for RBC transfusion. In the UK, 7,000 units of RBCs are transfused daily but this life-saving cell therapy is completely dependent on donors and there are persistent problems associated with transfusion transmitted infections and in blood group compatibility. Furthermore, the quality, safety and efficiency of donated RBCs gradually decrease with storage time. A number of novel sources of RBCs are being explored including the production of RBCs from adult haematopoietic progenitor cells, erythroid progenitor cell lines and induced pluripotent stem cells (iPSCs). The iPSC source could essentially provide a limitless supply and a route to producing cells that are matched to the recipient. A number of protocols have been described to produce mature RBCs from human pluripotent stem cells but they are relatively inefficient and would be difficult to scale up to the levels required for clinical translation. We tested and evaluated a defined feeder- and serum-free differentiation protocol for deriving erythroid cells from hiPSCs. RBC production was not efficient, the cells that were produced did not enucleate efficiently and they expressed embryonic rather than adult globin. We hypothesised that the production of RBCs from iPSCs could be enhanced by enforced expression of erythroid-specific transcription factors (TFs). Previous studies had demonstrated that Krüppel-like factor 1 (KLF1) plays an important role in RBC development and maturation so we generated iPSC lines expressing a tamoxifen-inducible KLF1-ERT2 fusion protein. Using zinc finger nuclease technology, we targeted the expression cassette to the AAVS1 locus to ensure consistent expression levels and to avoid integration site specific effects and/or silencing. These iKLF1 iPSCs were applied to our defined RBC differentiation protocol and the activity of KLF1 was induced by adding tamoxifen. Activation of KLF1 from day 10 accelerated erythroid differentiation and maturation with an increase in the proportion of erythroblasts, a higher level of expression of erythroid genes associated with maturation and an apparently more robust morphology. However, KLF1 activation had an anti-proliferation effect resulting in significantly less cell generated overall and HPLC analysis demonstrated that KLF1-activated cells expressed higher levels of embryonic globin compared to control iPSCs-derived cells. Many of the effects that were observed when KLF1 was activated from day 10 were not observed when activated from day 18. We therefore concluded that activation of exogenous KLF1 is able to promote erythroid cell production and maturation in progenitors (day 10) but not at the later stage of erythropoiesis (day 18). We hypothesised that KLF1 might require a co-factor to regulate RBC maturation and adult globin expression at the later stage of erythropoiesis. The TF, B-cell lymphoma/leukaemia 11a (BCL11A), plays a key role in the suppression of foetal globin expression, thereby completing globin switching to adult globin. Preliminary data showed that iPSC-derived erythroid cells were able to express adult globin when transduced with a BCL11A-expressing lentiviral-vector. Based on that finding we then generated an iPSC line expressing tamoxifen-inducible BCL11AERT2 and KLF1-ERT2 fusion proteins, applied this iBK iPSC line to our differentiation protocol. Activation of both TFs from day 18 slightly increased the expression of genes associated with RBC maturation and the inclusion of BCL11A appeared to eliminate the anti-proliferation effect of KLF1. Most importantly, activation of both BCL11A and KLF1 from day 18 of the differentiation protocol increased the production of α- globin (foetal / adult globin) indicating that some definitive-like erythroid cells might be generated by activation of both TFs at the later stage of erythroid differentiation. Collectively, these findings demonstrate that enforced expression of erythroid TFs could be a useful strategy to enhance RBC maturation from iPSCs.

Leucemia Linfóide Crônica (LLC) é a leucemia mais comum em países ocidentais, tem apresentação clínica e evolução heterogênea. Especula-se que todos os casos sejam precedidos por Linfocitose B Monoclonal (LBM). Não são bem conhecidos os mecanismos moleculares responsáveis por esta evolução. Alterações na expressão de miRNAs e em comprimento telomérico podem contribuir para desencadear esta neoplasia. O objetivo deste estudo foi identificar diferenças em comprimento telomérico e expressão de miRNAs entre pacientes com LLC, portadores de LBM clínica e populacional e controles saudáveis. Estudamos 21 pacientes com LLC, 11 portadores de LBM clínica, 6 de LBM populacional e 10 voluntários saudáveis. Para o controle de comprimento telomérico, utilizamos dados de estudo anterior do nosso serviço com grupo de 261 voluntários saudáveis de 0 a 86 anos. Realizamos separação de células CD19+CD5+ por citometria de fluxo nos grupos de estudo e de linfócitos B no grupo controle. Analisamos expressão dos miRNAs 15a, 16-1, 29b, 34a, 155, 181a e 181b por RT-qPCR e comprimento telomérico por qPCR. O miR- 155 foi o único que demonstrou expressão diferente entre os grupos LLC e LBM, sendo maior nos pacientes com LLC. Este miRNA e o miR-34a têm aumento de expressão nas células com fenótipo anormal, apesar desta diferença não ter tido significância estatística quando considerada a expressão do miR-155 na LBM. Os miRNAs 15a, 16-1, 181a e 181b são hipoexpressos nas células com fenótipo anormal. O miR-29b teve expressão semelhante nos grupos estudados. O comprimento telomérico foi semelhante nos 3 grupos de estudo e menor quando comparados ao grupo controle. O miR-155 tem diferente expressão em LBM e LLC, podendo ser um dos responsáveis por esta evolução. Alterações nos miRNAs 34a, 15a, 16-1, 181a e 181b contribuem para expansão clonal de linfócitos B CD5+. O papel do miR-29b na fisiopatogênese e evolução da LLC ainda não está bem definido. O comprimento telomérico diminuído em LLC e LBM pode fazer parte dos eventos iniciais da fisiopatogênese desta leucemia. / Chronic Lymphocytic Leukemia (CLL) is the most common leukemia on Western countries, it has an heterogeneous clinical presentation and outcome. Monoclonal BCell Lymphocytosis (MBL) may precede all CLL cases. The molecular mechanisms responsible for this evolution are not known. Aberrant miRNA expression and telomere shortening may contribute for the pathophysiology of this disease. The objective of this study was to identify differences on telomere length and miRNA expression between CLL patients, subjects with clinical and population-screening MBL and healthy volunteers. 21 CLL patients, 11 subjects with clinical MBL, 6 with population-screening MBL and 10 healthy volunteers were enrolled on this study. As control for telomere length, we used a group of 261 healthy volunteers aged 0 to 86 years old that had been enrolled on a previous study from our group. After diagnosis confirmation, it has been done a flow citometry CD19+CD5+ cell sorting for the study groups and CD19+ cell sorting for the control group. The expression of the miRNAs 15a, 16-1, 29b, 34a, 155, 181a and 181b was determined by RT-qPCR. The telomere length was determined by qPCR. miR-155 was the only one with different expression between the CLL and MBL groups, presenting higher expression on the CLL group. This miRNA and the miR-34a are overexpressed on the study groups when compared to the control group, although this difference did not reach statistical significance when the miR-155 expression in MBL is considered. miRNAs 15a, 16-1, 181a and 181b are underexpressed on the study groups. The miR-29b was the only one with similar expression on all groups. The telomere length was similar on the 3 study groups and shorter on these groups when compared to normal subjects. The expression of miR-155 is different in CLL and MBL, it may contribute for this evolution. Aberrant expression of miR 34a, 15a, 16-1, 181a and 181b may contribute for the clonal expansion of CD5+ B lymphocytes. The role of miR-29b on the CLL pathogenesis and evolution is still not understood. The reduced telomere length on CLL and MBL may be part of the initial events of this leukemia pathogenesis.

A família paraoxonase (PON1, PON2 e PON3) tem sido objeto de grande interesse por prevenir o estresse oxidativo e o processo inflamatório, condições importantes na carcinogênese. O Linfoma Difuso de Grandes Células B (LDGCB) consiste no subtipo histológico mais comum dentre os linfomas, doenças que se originam a partir das células do tecido linfoide e exibem distintos comportamentos clínicos, fatores patológicos e características epidemiológicas. Há escassez de dados sobre a atuação das paraoxonases na susceptibilidade diferencial ao risco de linfomas. Deste modo, o objetivo do presente estudo foi investigar a frequência alélica e genotípica dos polimorfismos 192QR e 55LM, no gene da PON1, e 148AG e 311SC, no gene da PON2 e o efeito desses polimorfismos sobre as atividades da enzima PON e perfil lipídico em 182 indivíduos (78 pacientes com LDGCB e 104 indivíduos saudáveis). O sangue foi coletado, em 4 momentos, para a determinação do perfil lipídico e das atividades arilesterase e paraoxonase da PON. O DNA foi extraído de leucócitos do sangue periférico pelo método de extração salina. A análise dos polimorfismos foi realizada por PCR/RFLP. Não houve diferença estatística na distribuição de genótipos e frequência de alelos dos polimorfismos nos genes da PON1 e PON2. A atividade sérica da arilesterase apresentou valores significativamente maiores apenas entre os indivíduos saudáveis (p=0,001). As variantes 55MM e 192QQ, do gene da PON1, influenciaram as atividades arilesterase (p=0,011) e paraoxonase (0,001). O polimorfismo PON2 311SS associou-se a atividade arilesterase (p=0,021). A concentração de autoanticorpos oxLDL foi alterada, pela presença do genótipo 55LM (p=0,037) nos indivíduos com LDGCB / The paraoxonase family (PON1, PON2 and PON3) have been the subject of great interest, since they are responsible for preventing oxidative stress and inflammation, conditions important in carcinogenesis. The Diffuse Large B Cell Lymphoma (DLBCL) is the most common histological subtype among lymphomas, diseases that originate from cells of the lymphoid tissue and exhibit clinically distinct behaviors and pathological and epidemiological factors. There are paucity of data on the activity of paraoxonase in the differential susceptibility to the risk of lymphoma. Thus, the objective of this study was to investigate the genotypic and allelic frequency of polymorphisms 192QR and 55LM, in the PON1 gene and 148AG and 311SC, in the PON2 gene and the effect of these polymorphisms on PON enzyme activities and lipid profile in 182 subjects (78 patients with DLBCL and 104 healthy subjects). Blood was collected in four moments for the determination of lipid profile and paraoxonase and arylesterase activities of PON. The DNA was extracted from peripheral blood leukocytes by salt extraction method. The analysis of polymorphisms was performed by PCR/RFLP. There was no statistical difference in the distribution of genotypes and allele frequencies of polymorphisms in the PON1 and PON2 genes. The serum arylesterase activity was significantly higher only among healthy subjects (p=0.001). 192QQ and 55MM variants of the PON1 gene, influenced arylesterase (p=0.011) and paraoxonase (0.001) activities. The PON2 polymorphism was associated with 311SS arylesterase activity (p = 0.021). The concentration of oxLDL autoantibodies was altered by the presence of 55LM genotype (p = 0.037) in patients with DLBCL

The most common human leukemia is B-cell chronic lymphocytic leukemia (B-CLL), which is characterized by a progressive accumulation of abnormal B-lymphocytes in blood, bone marrow and secondary lymphoid organs. Typically disease progression is slow, but as the number of leukemic cells increases, they interfere with the production of other important blood cells, causing the patients to be in an immunosuppressive state. To study the basis of this immunoregulation, we used cells from the transgenic Eμ-TCL1 mouse, which spontaneously develop B-CLL due to a B-cell specific expression of the oncogene, TCL1. Previously we showed that Eμ-TCL1 CLL cells constitutively produce an anti-inflammatory cytokine, IL-10. Here we studied the role of IL-10 in CLL cell survival in vitro and the development of CLL in vivo. We found that neutralization of IL-10 using anti-IL-10 antibodies or blocking the IL-10 receptor (IL-10R) using anti-IL-10R antibodies did not affect the survival of CLL cells in vitro. On the other hand, adoptively transferred Eμ-TCL1 cells grew at a slower rate in IL-10R KO mice vs. wild type (WT) mice. There was a significant reduction in CLL cell engraftment in the spleen, bone marrow, peritoneal cavity and liver of the IL-10R KO compared to WT mice. Further studies revealed that IL-10 could be playing a role in the tumor microenvironment possibly by affecting anti-tumor immunity. This was seen by a reduction in the activation of CD8+ T cells as well as a significantly lower production of IFN-γ by CD4+ T cells purified from CLL-injected WT mice compared to those purified from CLL-injected IL-10R KO mice. Also CLL-primed IL-10R null T cells were more effective than those from similarly CLL-primed wild type mice in controlling CLL growth in immunodeficient recipient mice. These studies demonstrate that CLL cells suppress host anti-tumor immunity via IL-10 production. This led us to investigate possible mechanisms by which IL-10 is produced. We found a novel role of B-cell receptor (BCR) signaling pathway in constitutive IL-10 secretion. Inhibition of Src or Syk family kinases reduces the constitutive IL-10 production by Eμ-TCL1 cells in a dose dependent manner. We identified the transcription factor Sp1 as a novel regulator of IL-10 production by CLL cells and that it is regulated by BCR signaling via the Syk/MAPK pathway.

The immune system will maintain the integrity of the organism from harmful non-malicious recognizes and protects the body against exo- and endogenous toxic substances and together with the nervous and endocrine system are among regulatory systems of the organism. Recently the evidence has supported the emerging concept of different B cell subpopulations to play a direct or indirect role in a pathogenesis of spectrum of disorders. However, so far the knowledge has been limited mainly in the way of how the specific differentiation stages of B lymphocytes are involved in pathogenesis of diseases and how course of disease, stage, and eventually different treatment can affect B cell homeostasis. This work is focused on the study of peripheral CD27+ B lymphocytes (one of the least explored human B lymphocytes) in healthy controls and patients with various immunopathologies, in this case we present representative results for patients with inflammatory bowel disease. Using polychromatic flow cytometry we examined 31 of peripheral blood samples, including 14 controls, 7 patients with Crohn's disease (CD) and 5 with ulcerative colitis (UC). In 6 patients with CD, we were able to perform immunophenotyping also 2 hours after intravenous administration of infliximab, and in one patient 14 days after drug...

La réponse humorale est altérée lors de l’infection par le virus de l’immunodéficience humaine (VIH). Les lymphocytes T CD4+ folliculaires helper (Tfh) sont impliqués dans la maturation des lymphocytes B (LB) dans les organes lymphoïdes secondaires. Mon travail de thèse s’est articulé autour de deux axes complémentaires visant à étudier les Tfh et les réponses B mémoires lors de l’infection par le VIH. J’ai d’abord étudié les Tfh spléniques lors de la phase chronique de l’infection par le VIH. J’ai mis en évidence une augmentation des populations de Tfh dans les rates de patients VIH+. D’autre part l’infection par le VIH a un impact sur le profil transcriptionnel des Tfh de la rate et la production de cytokines impliquées dans la différenciation des LB, suggérant un défaut fonctionnel des Tfh. Parallèlement, la maturation des LB est altérée dans les rates VIH+. Dans le second axe de ma thèse, j’ai étudié les réponses B mémoires anti-VIH dans différentes cohortes de patients VIH+ : Elite controller (EC) contrôlant l’infection sans traitements et des patients VIH+ traités. J’ai mis en évidence que les EC préservent naturellement leurs compartiments B mémoires et que les réponses B mémoires spécifiques du VIH sont maintenues dans le sang de ces patients. Les réponses B mémoires IgG1+ anti-VIH sont majoritaires chez les EC, tandis que les réponses IgG2+ et IgG3+ sont plus rares. Ces travaux permettent une meilleure compréhension de la physiopathologie de l’infection par le VIH en apportant de nouveaux éléments sur la fonctionnalité des Tfh et les réponses B mémoires anti-VIH. / HIV infection is associated with a defect of humoral response. T follicular helper cells (Tfh) support multiple steps of B cell maturation and antibody production. My work was divided in two complementary axes aiming to characterize Tfh and memory B cell responses in HIV-infected patients.I identified several Tfh populations in HIV+ and HIV- spleens by FACS. These three populations were increased in HIV+ spleen. I also evidenced an impact of HIV infection on transcriptional profile and a compromised production of B cell differentiation-related cytokines by splenocytes from HIV+ donors. These results suggest Tfh functions impairment during HIV-infection. In parallel, we noticed an altered maturation of B cells in HIV+ spleens. In a cohort study, we compared memory B cell responses in the blood of Elite controllers (EC) who naturally control HIV and treated HIV+ patients. I evidenced that EC naturally preserve their memory B cell compartments. In contrast to anti-HIV IgG2 and IgG3 secreting B cells, most EC exhibit a high frequency of anti-HIV IgG1 secreting B cells. My work highlights a defective Tfh differentiation, which might explain why B cell maturation is severely affected in HIV-progressors. The status of HIV-controller seems associated with the presence of an IgG1 B cell memory response. Further work will highlight whether Tfh functions are preserved in EC.