Chronic lymphocytic leukemia (CLL) is a heterogeneous disease where a significant proportion of patients will develop an aggressive disease. Today, the mutational status of the immunoglobulin heavy variable (IGHV) genes is one of the strongest prognostic markers in CLL, where unmutated IGHV genes correlate with poor outcome. In addition, IGHV3-21 gene usage is associated with poor prognosis independent of mutational status. Recently, several genes were shown to be differently expressed between IGHV mutated and unmutated CLL and were suggested as prognostic markers. The aim of this thesis was to examine the applicability of these RNA-based prognostic markers in CLL. In papers I and II, the prognostic significance of LPL and TCL1A mRNA expression in CLL was investigated in 140 and 144 patients, respectively. High expression was found to be associated with inferior clinical outcome for both markers. However, CLL cases with mutated IGHV3-21 genes displayed low levels of LPL expression, indicating that LPL cannot identify this poor-risk patient group. In contrast, high TCL1A expression was detected in all IGHV3-21 cases. To elucidate the functionality of LPL in CLL, LPL lipase activity was measured in 33 cases. The lipase activity was found to be invariably low, implying an alternative function for LPL in CLL. In paper III, a comprehensive analysis of five RNA-based markers (LPL, TCL1A, ZAP70, CLLU1 and MCL1) was performed in 252 CLL patients. All RNA-based markers except MCL1 predicted clinical outcome, with LPL being the strongest. Moreover, LPL expression independently predicted overall survival when adjusted for established markers. All of the RNA-based markers added additional prognostic information to established markers, e.g. high LPL expression predicted an inferior outcome in patients with mutated IGHV genes or good-risk cytogenetics. For clinical application, over time stability of prognostic markers is crucial. In paper IV, the expression of LPL, TCL1A, ZAP70 and MCL1 was investigated in samples taken at diagnosis and at a follow-up of seven years in 104 CLL patients. LPL was found to be the most stable marker, displaying high correlation between the sequential samples, whereas ZAP70 and MCL1 varied significantly. TCL1A expression increased at follow-up, which may indicate disease progression as TCL1A promotes cell survival. In summary, this thesis highlights the applicability of RNA-based markers in CLL prognostication, both as single markers or in combination with established markers. In particular, LPL was shown to be the strongest RNA-based marker in terms of prognostic strength and stability.

B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia among the adult population in western countries and accounts for 30-40% of all leukemias. With survival time ranging from months to decades, the clinical course of individual CLL patients is highly variable. This heterogeneity and in the end the need for means to identify the patients with less favorable disease has encouraged the search for biomarkers that can predict the prognosis. Telomeres are repetitive structures protecting the chromosomal endings and shorten at each cell division. Telomere length (TL) has been indicated as a prognostic factor both in hematological malignancies and solid tumors. In B-CLL, TL is associated with mutation status of the immunoglobulin heavy chain variable (IGHV) gene and with clinical course. In the present thesis the main aim was to evaluate TL as a biomarker in B-CLL using a quantitative PCR-based method for TL determination. In paper I, TL was shown to be a prognostic factor for stage A and stage B/C patients, whereas IGHV mutation status predicted outcome only in stage A patients. Moreover, IGHV mutated CLL cases were subdivided by TL into two groups with different prognosis, a subdivision not seen for unmutated cases. Interestingly, the IGHV-mutated group with short telomeres had en overall survival close to that of the unmutated cases. Thus, a combination of IGHV mutation status and telomere length gave an improved subclassification of CLL identifying previously unrecognized patient groups with different outcomes. TL correlates with cellular origin of B-cell malignancies in relation to the germinal center (GC). In paper II different B-cell lymphoma/leukemia subtypes were analyzed. Shortest telomeres were found in IGHV unmutated CLLs, differing significantly from IGHV mutated cases. Contrary to this, mantle cell lymphomas (MCL) demonstrated similar TL regardless of IGHV mutation status. TL differed significantly between GC-like and non-GC-like diffuse large B-cell lymphomas (DLBCL) and follicular lymphomas (FL) had shorter telomeres than GC-like DLBCL. Hairy cell leukemias, which display Ig gene intraclonal heterogeneity, had longer telomeres than FLs and non-GC-DLBCL, but shorter than GC-DLBCL. In conclusion, TL seemed not to simply correlate with GC origin. Paper III presents a B-CLL cohort assessed for TL, genomic aberrations, IGHV mutation status, CD38 and ZAP-70 expression. An inverse correlation existed between TL and IGHV homology, CD38 and ZAP-70 expression. The presence of genomic aberrations was similar among patients regardless of TL. In contrast, 13q deletion, a favorable biomarker, was more frequent in patients with long telomeres, while 11q and 17p deletions (markers of less favorable outcome) were more frequent in the subgroup with short telomeres. In paper IV a large group of mainly indolent CLL cases from a population based cohort was studied again showing an association between TL and prognosis, especially in “good” prognosis cases as defined by other biomarkers. Multivariate analysis indicated a strong connection between IGHV mutation status, lipoprotein lipase (LPL) expression and TL. A comparison of TL in diagnostic and follow up samples demonstrated a significant correlation, and also in the follow samples TL constituted a significant biomarker for survival.

La leucemia linfática crónica (LLC) es la leucemia más frecuente en los países occidentales. En esta enfermedad es frecuente hallar complicaciones autoinmunes. A pesar de que la relación entre LLC y trastornos autoinmunes se conoce desde hace prácticamente cinco décadas, los mecanismos responsables de la autoinmunidad en la LLC y sus repercusiones clínicas todavía no se conocen con precisión. Mientras la asociación entre citopenias autoinmunes y LLC está perfectamente demostrada, la relación entre trastornos inmunes no hemáticos y la LLC es controvertida. En esta tesis doctoral se analizó la relación entre citopenias autoinmunes con la LLC y sus características biológicas, así como implicaciones clínicas, y el papel de las moléculas BAFF y APRIL como vínculo de unión entre la autoinmunidad y la LLC. Los principales hallazgos de esta tesis se pueden resumir de la siguiente forma. En el primer lugar, la incidencia de citopenia autoinmune en una serie de 960 pacientes con LLC por nosotros seguidos fue del 7%, en concordancia con otras series. Los pacientes con LLC y citopenias autoinmunes presentaban datos de mal pronóstico, como un recuento linfocitario elevado, un tiempo de duplicación linfocitario rápido, y B2M sérica, ZAP-70 y CD38 elevadas. De forma sumamente importante, los enfermos con estadio avanzado atribuible a un origen inmune tenían un pronóstico mejor que aquellos en los que la fase avanzada de la enfermedad reflejaba una alta carga tumoral. Ello invita, de acuerdo con otro estudio de la Clínica Mayo, y de la Editorial en Blood que acompañó a nuestra publicación, a diferenciar dentro de los pacientes con LLC en estadio avanzado aquellos en los que la citopenia tiene un origen autoinmune (C “inmune”) y los que la anemia se debe al fallo de la medula ósea a causa de la infiltración linfocitaria (C “infiltrativo”). BAFF y APRIL dos miembros de la familia de proteínas TNF claves en la regulación del desarrollo y supervivencia de los linfocitos B, están implicadas en la patogénesis de la LLC y en la autoinmunidad por lo que pueden constituir un vínculo fisiopatológico entre los componentes neoplásico e inmune de la LLC. En esta tesis observamos que los pacientes con LLC presentaban niveles altos de APRIL y bajos de BAFF en comparación con sujetos sanos. Además, los niveles de BAFF se correlacionaban con el recuento linfocitario en sangre, el estadio avanzado de la enfermedad y la presencia de la anemia hemolítica autoinmune. Por último, la valoración conjunta de BAFF y APRIL ofreció mayor información pronóstica que ambas moléculas por separado. / Chronic lymphocytic leukemia (CLL) is the most common leukemia in Western countries. Although it is well documented that autoimmune complications are common in these patients, the clinical relevance and the biological basis of autoimmune phenomena in CLL have not yet been clearly established. Autoimmune cytopenias are clearly related to LLC, but no causal link between CLL and autoimmune diseases not targeting blood cells has been established. The results of this doctoral thesis can be summarized as follows. First, the incidence of autoimmune cytopenia in 960 patients with CLL from our institution was 7%, which is consistent with other reports. Although patients with autoimmune cytopenias presented poor prognostic factors, such as a high lymphocyte count, a rapid lymphocyte doubling time, and high levels of serum B2M, ZAP-70 and CD38, their prognosis was similar to that of other patients. In fact, the cohort of patients classified as in advanced due to autoimmune cytopenia had a better prognosis than those classified in advanced disease because of bone marrow failure related to the infiltration by the disease. Our results and similar findings from another study suggest that patients in advanced stage of disease should be separated according to the origin of the cytopenia ("autoimmunity" or "infiltration"). BAFF and APRIL, two members of the TNF family proteins, are key regulators of the development and survival of B lymphocytes. These molecules are involved in CLL pathogenesis and autoimmunity and can constitute a link between the neoplastic and the immune components of CLL. We found that patients with CLL had higher levels of APRIL and lower levels of BAFF as compared to healthy subjects. In addition, BAFF levels correlated with blood lymphocyte count, advanced clinical stage and autoimmune hemolytic anemia. Finally, the combination of serum levels of both BAFF and APRIL provided better prognostic information on disease progression than any of these molecules independently considered.

Les dérivés de la cytosine sont d’importantes modifications épigénétiques dont le rôle dans l’évolution de la leucémie lymphoïde chronique (LLC) n’est pas totalement exploré. Dans ce contexte, notre première étude vise à examiner le niveau global de la 5-methylcytosine (5-mCyt), 5-hydroxymethylcytosine (5-hmCyt), 5-carboxylcytosine (5-CaCyt) et 5-hydroxymethyluridine (5-hmU) dans des lymphocytes B purifiés de patients LLC (n=56) et d’individus sains (n=17). Les principaux acteurs de la régulation épigénétique (DNMT1/3A/3B, MBD2/4, TET1/2/3, SAT1) ont été évalués par PCR quantitative en temps réel. L’analyse a permis de mettre en exergue trois groupes de patients. En premier lieu, un groupe de patients stables (délai médian de progression [PFS] et délai au premier traitement [TFT] >120 mois), avec un profil épigénétique similaire au groupe contrôle. Deuxièmement, un groupe intermédiaire (PFS=84; TFT=120 mois) qui présente une augmentation de la déméthylation de l’ADN expliquée par l'induction SAT1 / TET2 pendant la progression de la maladie. Troisièmement, un groupe de patients avec une forme active de la maladie (PFS=52; TFT=112 mois) qui présentent une hyperlymphocytose, une réduction du temps de doublement des lymphocytes et des modifications épigénétiques majeures. Au sein de ce groupe, une réduction est observée pour la 5-mCyt, 5-hmCyt, 5-CaCyt et serait associée à une diminution des DNMTs, TETs et MBDs au cours de la progression de la maladie. Les profils épigénétiques mis en évidence sont indépendants du statut mutationnel IGHV mais sont associés avec les anomalies cytogénétiques. Nous nous sommes également intéressés à cette association et nous avons montré dans la deuxième étude que les modifications des dérivées de la cytosine peuvent affiner le pouvoir pronostic des anomalies cytogénétiques. En conclusion, nos résultats suggèrent que les variations de la méthylation ainsi que des intermédiaires de la déméthylation de l’ADN sont impliqués dans la progression de la LLC. / Cytosine derivatives are important epigenetic modifications whose role in the pathogenesis and evolution of chronic lymphocytic leukemia (CLL) is not fully explored. In this context, our first study aims to examine the global DNA level of 5-methylcytosine (5-mCyt), 5-hydroxymethylcytosine (5-hmCyt), 5-carboxylcytosine (5-CaCyt) and 5-hydroxymethyluridine (5-hmU) in purified B lymphocytes of CLL patients (n = 56) and healthy individuals (n = 17). The main actors in epigenetic regulation (DNMT1 / 3A / 3B, MBD2 / 4, TET1 / 2/3, SAT1) were evaluated by quantitative real time PCR. The analysis highlighted three groups of patients. First, a group of patients with stable disease (median time to progression [PFS] and time to first treatment [TFT]> 120 months), with an epigenetic profile similar to the control group. Secondly, an intermediate group (PFS = 84, TFT = 120 months) which shows an increase in DNA demethylation explained by SAT1 / TET2 induction during disease progression. Third, a group of patients with an active form of the disease (PFS = 52, TFT = 112 months) who have hyperlymphocytosis, a short lymphocyte doubling time, and major epigenetic changes. Within this group, a reduction is observed for 5-mCyt, 5-hmCyt, 5-CaCyt which is associated with a decrease in DNMTs, TETs and MBDs during disease progression. The identified epigenetic profiles are independent of IGHV mutational status but are associated with cytogenetic abnormalities. We were also interested in this association and we showed in the second study that modifications of cytosine derivatives levels can refine the prognostic power of cytogenetic abnormalities.In conclusion, our results suggest that methylation variations as well as DNA demethylation intermediates are involved in the progression of CLL.

Submitted by Maike Costa (maiksebas@gmail.com) on 2017-03-16T14:10:50Z No. of bitstreams: 1 arquivototal.pdf: 1122971 bytes, checksum: 96c05223167558b53c43c710feee4238 (MD5) / Made available in DSpace on 2017-03-16T14:10:50Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1122971 bytes, checksum: 96c05223167558b53c43c710feee4238 (MD5) Previous issue date: 2015-12-14 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Introduction: Acute lymphocytic leukemia (ALL) is the most common malignancy in childreen accounting for 75% of all diagnosed leukemias and being 25% of all malignancies in childhood. There are few studies that deal directly with the understanding of the clinical profile and other variables associated with oral Candida colonization in pediatric patients with ALL, especially in developing countries. Objective: The aim of this study was to evaluate antifungal resistance and Candida colonization in oral cavity of infant juvenile ALL patients. Material and Methods: This was a cross sectioned design, controlled, with dual observational and descriptive characteristics. To carry out this study, two groups were formed: a ALL group with 40 patients diagnosed with ALL and a control group, formed by 40 healthy individuals matched by age and gender. All these individuals were clinically evaluated and submitted to salivary collection with sterile swab. The saliva was seeded onto CHROMagar CandidaTM, incubed for 48hs at 37oC, and the obtained colony formation unities (CFU) were counted and presumptively identified. Variable data of the participants were collected and stored in individual files. The found species was submitted to microdilution for Nystatin and Amphotericin B, widely used treating candidosis, to establish their susceptibility/resistance. Results: Of 40 patients evaluated in case group, 13 (32.5%) were positive to Candida and only 1 (2.5%) was positive in control group (p<0.001). Candida albicans was the most prevalent strain (87.5%). All patients who had 10³ CFU/mL counts were on induction phase of chemotherapy. Of analysed variables, only mucositis was directly associated with Candida positiveness (p=0.017) on ALL group. Five out 14 strains of C. albicans (35.7%) were resistant to Nystatin and all species were not susceptible to Ampothericin B. Conclusion: Candida colonization was associated with ALL probably vinculated to mucositis events being the higher counts found on induction phase of chemotherapy. C. albicans was the prevalent strain and resistance and lack of susceptibility to Nystatin and Amphotericin B was found. / Introdução: Leucemia linfocítica aguda (LLA) é a neoplasia maligna mais comum em crianças, representando 75% de todas as leucemias diagnosticados e sendo 25% de todas as neoplasias malignas na infância. Existem poucos estudos que lidam diretamente com o entendimento do perfil clínico e outras variáveis associadas com a colonização por Candida em pacientes oncológicos infanto-juvenis, especialmente nos países em desenvolvimento. Objetivo: O objetivo deste estudo foi avaliar a colonização e resistência antifúngica de Candida spp. na cavidade bucal de pacientes infanto-juvenis com LLA. Material e Métodos: Este foi um estudo transversal, controlado, com características observacionais e descritivas. Para realizar este estudo, foram formados dois grupos: um grupo LLA com 40 pacientes diagnosticados com LLA e um grupo controle, formado por 40 indivíduos saudáveis pareados por idade e sexo. Todos esses indivíduos foram avaliados clinicamente e submetidos à coleta de saliva com cotonete estéril. A saliva foi semeada em CHROMagar CandidaTM, incubada por 48 hs a 37ºC, e as unidades formadoras de colônias (UFC) obtidas foram contadas e identificadas presuntivamente. Dados variáveis dos participantes foram coletados e armazenados em arquivos individuais. As espécies encontradas foram submetidas a teste de susceptibilidade antifúngica por ensaio de microdiluição para nistatina e anfotericina B, amplamente utilizadas no tratamento de candidose, para estabelecer a sua susceptibilidade/resistência. Resultados: Dos 40 pacientes avaliados no grupo LLA, 13 (32,5%) foram positivos para Candida e apenas 1 (2,5%) foi positivo no grupo controle (p <0,001). Candida albicans foi a espécie mais prevalente (87,5% ). Todos os pacientes que tiveram 10³ UFC/ml de quantificação estavam em fase de indução da quimioterapia. Das variáveis analisadas, apenas a presença de mucosite esteve diretamente associada com a Candida (p = 0,017) no grupo LLA. Das 14 cepas de C.albicans, 5 cepas (35,7%) eram resistentes à Nistatina e todas as espécies não foram suscetíveis ao Anfotericina B. Conclusão: A colonização por Candida foi associada a LLA e vinculadas a mucosite sendo as contagens mais elevadas encontradas em fase de indução da quimioterapia. C. albicans foi a cepa predominante e a resistência e falta de susceptibilidade à Nistatina e Anfotericina B foram observadas.

Made available in DSpace on 2014-12-17T14:16:26Z (GMT). No. of bitstreams: 1 MariaCAL_DISSERT.pdf: 1577232 bytes, checksum: 21520a348f865554e71416a87d0342f6 (MD5) Previous issue date: 2010-02-24 / Chronic lymphocytic leukemia (B-CLL) is a clonal proliferation of mature B lymphocytes characterized by indolent clinical course. Biologically this clonallity is characterized by low expression of surface immunoglobulin (sIg) with restriction to a single immunoglobulin light chain associated with high expression of CD5 antigen and positivity to B cell antigens lymphocytes such as CD19, CD20 and CD23 and negativity to FMC7. The immunological profile and morphological analysis of lymphoid cells are the main means for the differential diagnosis of B-CLL from other chronic lymphoproliferative diseases. The aim of this study was to evaluate the expression pattern of a variety of membrane antigens in leukemic cells originating from patients with B-CLL. In this study, peripheral blood samples from 80 patients with B-CLL were analyzed by multiparametric flow cytometry in addition to routine hematologic exams, using a panel of monoclonal antibodies (MoAb): CD45/CD14, CD3/CD19/CD45, CD4/CD8 / CD3, CD20/CD5/CD3, CD3/CD16-56/CD45, CD2/CD7, FMC7/CD23, CD103/CD22/CD20, HLADR/CD38, CD10/CD19, CD1a, CD11b and also IgM/gD, kappa and lambda immunoglobulin light chains for the detection of surface immunoglobulin and clonal restriction for immunoglobulin light chain. The Hematological data were obtained from the hematological analyzer and cytomorphological analysis in blood film stained by Leishmann. The study samples consisted of 45 men and 35 women, ages ranging from 55 to 84 years (mean 65 years). Complete white blood count showed count ranging from 10.0 to 42.0 x 109/l. (mean 50.0 x 109/l) and lymphocytes count greater than 5.0 x 109/l in all cases. The neoplastic cells displayed B-CLL phenotype (CD5+/CD19+/CD20+/HLADR+/CD23+) in the vast majority of the cases, associated to failed to stain for T cell markers (CD1a, CD2, CD4, CD3, CD7, CD8), CD103, CD14 and FMC7. Leukemic cells of most patients also expressed low intensity of IgM and IgD with restricted kappa light chain, in most cases (59,7%). This observation highlights the importance of immunophenotyping for correct diagnosis of chronic lymphoproliferative syndromes and the panel of MoAb used was sufficient for diagnostic confirmation of B-CLL / A leucemia linfoc?tica cr?nica (LLC-B) ? uma prolifera??o clonal de linf?citos B maduros caracterizada por curso cl?nico indolente. Biologicamente esta clonalidade ? caracterizada pela baixa express?o de imunoglobulina de superf?cie (sIg) com restri??o a uma ?nica cadeia leve de imunoglobulina, associada a alta express?o do ant?geno CD5 e positividade a ant?genos relacionados a linf?citos B tais como: CD19, CD20 e CD23 e negatividade ao FMC7. O perfil imunol?gico e a an?lise morfol?gica das c?lulas linf?ides s?o os principais meios para o diagn?stico diferencial LLC-B de outras doen?as linfoproliferativas cr?nicas. O objetivo deste estudo foi avaliar o padr?o de express?o de uma variedade de ant?genos de membrana em c?lulas leuc?micas procedentes de pacientes com LLC-B. No presente estudo, amostras de sangue perif?rico de 80 pacientes com LLC-B foram analisados por citometria de fluxo multiparam?trica juntamente com an?lises hematol?gicas de rotina, com um painel de anticorpos monoclonais (AcMo): CD45/CD14, CD3/CD19/CD45, CD4/CD8/CD3, CD20/CD5/CD3, CD3/CD16-56/CD45, CD2/CD7, FMC7/CD23, CD103/CD22/CD20, HLADR/CD38, CD10/CD19, CD1a, CD11b al?m de IgM/gD, e cadeias leves das imunoglobulinas kappa e lambda visando a detec??o sIg e do perfil de restri??o clonal das cadeia leves das imunoglobulinas. Os dados hematol?gicos foram obtidos a partir do analisador hematol?gico e as an?lises citomorfologicas em distens?o sangu?nea coradas pelo Leishmann. As amostras deste estudo foram procedentes de 45 homens e 35 mulheres, com idade variando entre 55 e 84 anos (m?dia de 65 anos). O hemograma revelou contagem total de c?lulas branca variando de 10,0-42,0 x 109/l. (m?dia de 50,0 x 109/l) e contagem de linf?citos superior a 5,0 x 109/l em todos os casos. As c?lulas neopl?sicas demonstraram um fen?tipo caracter?stico para LLC-B (CD5+/CD19+/CD20+/HLADR+/CD23+) na maioria dos casos, associados ? aus?ncia de express?o para marcadores de c?lulas T (CD1a, CD2, CD4, CD3, CD7, CD8), CD103, CD14 e FMC7. As c?lulas leuc?micas da maioria dos pacientes expressaram tamb?m IgM e IgD de baixa intensidade com predom?nio da restri??o da cadeia leve kappa, na maioria dos casos (59,7%). A presente observa??o destaca a import?ncia da imunofenotipagem para o correto diagn?stico das s?ndromes linfoproliferativas cr?nicas e sendo o painel de AcMo utilizado capaz de estabelecer a confirma??o diagn?stica da B-CLL

La leucémie lymphoïde chronique (LLC) constitue l'hémopathie maligne la plus fréquente dans les pays occidentaux et résulte d’une accumulation de lymphocytes B (LB) monoclonaux matures porteurs de la glycoprotéine CD5. Les LB de LLC sont également caractérisées par une altération de l'homéostasie du calcium avec, d'une part, la mise en évidence de facteurs de survie contrôlés par le calcium tels que phospho-ERK, NFAT-2 et IL-10, et d'autre part par une progression de la maladie qui est associée à la réponse au calcium. Tout d'abord, afin de mieux comprendre l'impact de la molécule CD5 sur les dérégulations du calcium, il a été montré que l'introduction d'un plasmide d'expression pour CD5 dans les lignées de cellules B humaines s'accompagnait d'une entrée calcique à l’état basale. Ensuite, cette entrée, appelée entrée constitutive, a été recherchée dans les LB de LLC pour montrer qu'elle caractérisait les patients non traités en phase d'évolution. Comme pour les lignées de cellules B CD5, cette nouvelle voie d'entrée du calcium est autonome et indépendante de la voie classique de signalisation du LB de LLC : BCR-IP3R. L'étude des protéines responsables de cet influx a permis de mettre en évidence, premièrement trois partenaires STIM1, Orai1 et TRPC1, et deuxièmement l'importance de la fraction membranaire de STIM1 (STIMPM) puisque l'utilisation d'un anticorps monoclonal anti-STIM1 (Acm) est capable d’inhiber l'entrée constitutive du calcium qui à son tour agit sur la survie des LB, pour les patients STIMPM positifs, lorsque l'Acm anti-STIM1 est utilisé en association avec le rituximab, un Acm thérapeutique anti-CD20. Enfin, la modélisation de la partie Cterminale de STIM1 permet d'envisager plusieurs cibles potentielles pour le développement de nouveaux Acm anti-STIM1. En conclusion, la mise en place, par le clone malin de LLC, d'une entrée constitutive du calcium favorise son agressivité et constitue donc une nouvelle voie thérapeutique contrôlable par l'utilisation d’Acm anti-STIM1 ce qui ouvre de nouvelles perspectives comme outils diagnostiques et thérapeutiques. / Chronic lymphocytic leukemia (CLL) is the most common hematological malignancy in Western countries and is a result of the accumulation of mature monoclonal B lymphocytes (B-CLL) carrying the CD5 glycoprotein. The B-CLL are also characterized by an alteration of calcium homeostasis with, on the one hand, the demonstration of calcium-controlled survival factors such as phospho-ERK, NFAT- 2 and IL-10, and on the other hand by a progression of the disease which is associated with the response to calcium. Initially, in order to better understand the impact of the CD5 molecule on calcium deregulations, it has been shown that the introduction of an expression plasmid for CD5 into human B cell lines was accompanied by a calcium entry in the basal state. Then, this entry, called constitutive entry, was sought in the B-CLL to show that it characterized untreated patients in the evolution phase.As with CD5 B cell lines, this new calcium entry pathway is autonomous and independent of the classical B-CLL signaling pathway: BCR-IP3R. The study of the proteins responsible for this influx made it possible to highlight, firstly three partners (STIM1, Orai1 and TRPC1), and secondly the importance of the membrane fraction of STIM1 (STIMPM) since the use of a human monoclonal antibody (mAb) anti-STIM1 is able to inhibit the constitutive entry of calcium which in turn acts on the survival of B-CLL, for STIMPM positive patients, when the anti-STIM1 mAb was used in combination with rituximab, a therapeutic anti-CD20 mAb. Finally, the modeling of the C-terminal part of STIM1 makes it possible to envisage several potential targets for the development of new anti-STIM1 mAbs. In conclusion, the introduction by the CLL malignant clone of a constitutive entry of calcium favors its aggressiveness and thus constitutes a new therapeutic pathway controllable by the use of anti-STIM1 mAb, which opens new perspectives like diagnostic and therapeutic tools.

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.

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.

During virus infections, activation of CD8 T cells takes place in secondary lymphoid organs including spleen and lymph nodes. The kinetics of the T cell response in lymphoid tissues has been clearly studied. However, a large number of virus-specific T cells disseminate into various nonlymphoid tissues. As reservoirs for effector and memory cells, nonlymphoid organs play an important role for defending against infections. T cell responses in nonlymphoid organs may differ from lymphoid organs. T cell repertoire usage in lymphoid and nonlymphoid tissues was studied in an acute lymphocytic choriomeningitis virus (LCMV)-infected murine model. The hierarchy of CD8 T cell specificities was examined with cytotoxic T lymphocyte (CTL) sodium 51 chromate (51Cr) release assays and intracellular interferon (IFN)γ assays. T cell receptor (TCR) repertoire usage was determined by complementarity determining region (CDR)3 length spectratyping analysis. Both T cell specificity and TCR repertoire usage revealed some similarities and differences between several organs. Within an epitope-specific CD8 T cell population, the TCR repertoire usage was similar in different organs of the same mouse, but highly heterogeneous between individual mice with genetically identical backgrounds. A very restricted CD4 TCR repertoire was observed in BALB/c mice after secondary respiratory syncytial virus (RSV) infection. Most of the CD4 T cells of BALB/c mice pre-immunized with RSV glycoprotein (GP) predominantly express Vβ14 TCR with discrete oligoclonal CDR3 regions. Depletion of Vβ14 CD4 T cells dramatically reduced immunopathology. The apoptotic phenotype of LCMV-specific CD8 T cells was studied in various lymphoid and nonlymphoid tissues during acute and memory stages of infections. Peripheral tissues (peritoneal cavity (PEC), fat pad, and lung) reacted with a much lower frequency with the early apoptotic marker Annexin V than those in spleen and lymph nodes. This was not due to a TCR-based selection because similar TCR spectratypes were seen in different organs. Activated lymphoid and nonlymphoid T cells from LCMV GP33 transgenic mice, which have identical TCR α and β chains on all T cells, had differential Annexin V binding. When incubated shortly in vitro, most Annexin V+ T cells rapidly fragmented their DNA and became terminal transferase-mediated dUTP nick end-labeling positive (TUNEL+), while much fewer Annexin V- cells became TUNEL+. Therefore, those Annexin-V+ cells were truly in a pre-apoptotic stage. The differential spontaneous apoptosis in different tissues is independent of several death/survival-related molecules, including Fas/Fas ligand (FasL), turner necrosis factor (TNF)α, interleukin (IL-15), perforin, B cell lymphoma (Bcl)-2 and independent of virus tropism. I further investigated the significance of the high Annexin V reactivity of lymphoid T cells. Pre-apoptotic cells were prevented from fragmenting their DNA by anti-CD3 or IL-2 stimulation in vitro. However, this pre-apoptotic phenotype precluded generation of memory. Annexin V reactive cells did not give rise to long-lived memory after being transferred into naïve hosts. The pre-apoptotic phenotype is also an intrinsic property of the epitope. Different proportions of apoptotic cells were found in LCMV effector and, memory T cells specific to two different epitopes, nucleoprotein (NP)396 and GP33. Higher Annexin V reactivity of NP396-specific CD8 T cells was independent of virus tropism and duration of encounter with antigen. Higher expression of IL-7R was found in peripheral, Annexin V- and GP33-specific CD8 T cells, indicating that IL-7-dependent signals may inhibit apoptosis. Nonlymphoid T cells were more resistant than lymphoid T cells to activation-induced cell death (AICD). When stimulated with anti-CD3 in vitro for 40 hours (hr), a significantly reduced number of splenic transgenic T cells were recovered with much higher frequency of Annexin V reactivity and TUNEL staining than transgenic T cells from PEC. Consistent with the finding that Fas and FasL regulates AICD, a much lower expression of Fas and FasL was observed in PEC and lung transgenic T cells than spleen and lymph nodes after short time stimulation. FasL blockage largely increased cell-number recovery and reduced Annexin V and TUNEL staining of spleen transgenic T cells. Interestingly, the leukocyte environment played an important role of deciding the fate of transgenic T cells. When placing activated spleen transgenic T cells with excess infected PEC cells, spleen transgenic cells rapidly reduced their Annexin V staining and TUNEL staining and were recovered with greater number after stimulation. Vice versa, PEC transgenic T cells became Annexin V and TUNEL positive with lower numbers of cells recovered when placed with excess splenocytes. Less detection of Annexin V+ cells in peripheral tissues was not due to rapid phagocytosis by macrophages, because Cytochalasin D, which can inhibit phagocytosis, did not induce equal amount of pre-apoptotic cells in spleen and PEC. This reduced death in the periphery may contribute to the long-term maintenance of nondividing nonlymphoid memory T cells, enabling them to efficiently function without being driven into apoptosis. Overall, this study characterizes in detail the different T cell repertoire usage and apoptosis of virus-specific T cells based on their organ localization and specificities and helps to better understand T cell immunity after infections and vaccine design.