Orientadora : Profª. Drª. Karin Braun Prado / Coorientador : Prof. Dr. Danillo G. Augusto / Dissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Genética. Defesa: Curitiba, 28/03/2017 / Inclui referências : f. 92-102 / Resumo: A familia de genes KIR (do ingles, killer cell immunoglobulin-like receptors) desempenha um papel central na imunidade inata e adaptativa e seu polimorfismo tem sido associado a suscetibilidade diferencial a diversas doencas. Esses genes exibem extensa variabilidade, tanto em termos de ausencia e presenca de genes, quanto em nivel alelico. Os receptores codificados por esses genes sao expressos na superficie das celulas NK e de alguns subtipos de celulas T e podem transduzir sinais ativadores ou inibidores. Pouco se conhece a respeito dos seus niveis de expressao genica diferencial, tampouco dos mecanismos de regulacao. O gene KIR3DL2 codifica um receptor inibidor e e o segundo gene KIR mais polimorfico e polialelico do complexo, com mais de 80 alelos descritos. O objetivo desse trabalho foi analisar se o polimorfismo alelico de KIR3DL2 impacta na sua expressao genica diferencial. Para isso, caracterizamos a diversidade alelica de KIR3DL2 em uma populacao de euro-descendentes de Curitiba e regiao metropolitana (n = 235), atraves de sequenciamento de DNA. As frequencias genotipicas estavam de acordo com as esperadas pelo equilibrio de Hardy-Weinberg, sendo os genotipos mais comuns: 3DL2*001/007 (11,5%), 3DL2*001/002 (6,8%) e 3DL2*002/007 (6,8%). Os alelos mais frequentes encontrados nessa populacao foram 3DL2*007 (21,7%), 3DL2*001 (21,3%) e 3DL2*002 (20%). Um total de 40 individuos com genotipos especificos e comuns na populacao desse estudo foram selecionados para a analise de expressao diferencial por citometria de fluxo. A analise de expressao diferencial foi realizada com os individuos portadores dos alelos mais frequentes encontrados na populacao, sendo eles: 3DL2*001, 3DL2*002, 3DL2*003, 3DL2*005, 3DL2*007, 3DL2*009, 3DL2*010 e 3DL2*011. Verificamos que o polimorfismo alelico de KIR3DL2 esta associado nao somente com niveis de expressao diferencial, mas tambem com diferentes quantidades de celulas NK que exibem KIR3DL2 em sua superficie. O alelo KIR3DL2*002 foi associado ao maior nivel de expressao de KIR3DL2 e ao maior numero de celulas NK 3DL2 positivas. Ja 3DL2*010 foi associado ao menor nivel de expressao e a menor quantidade de celulas NK com KIR3DL2 presente na superficie celular. Alem disso, demonstramos que um polimorfismo na regiao 3' UTR, na posicao 16545A>G (rs1865095) marca os niveis de expressao de KIR3DL2. Sugerimos que esta expressao diferencial possa estar relacionada a ligacao de miRNAs nesta regiao, especificamente o miR-2114-3p. A presenca de ligantes especificos (HLA-A3, -A11 e -B27) nao esta associada a diferentes niveis de expressao de KIR3DL2 (p = 0,5739) nem a diferentes quantidades de celulas NK KIR3DL2 positivas (p = 0,7772). Alem disso, nenhuma correlacao foi encontrada entre a expressao diferencial dos alelos de KIR3DL2 e a expressao diferencial dos alelos de HLA-A. Como perspectivas futuras pretendemos analisar, atraves de co-cultivo celular, a atividade citotoxica das celulas NK de individuos com diferentes genotipos homozigotos, como KIR3DL2*001/KIR3DL2*001, KIR3DL2*002/KIR3DL2*002, KIR3DL2*007/KIR3DL2*007 e KIR3DL2*010/KIR3DL2*010 a fim de corroborar as hipoteses geradas. Palavras-chave: Celulas NK, KIR3DL2, variabilidade alelica, HLA-A3, HLA-A11, HLA-B27, expressao diferencial. / Abstract: The KIR (killer cell immunoglobulin-like receptors) gene family plays a central role in innate and adaptive immunity and has been associated with differential susceptibility to diseases. Besides the uncommon presence and absence polymorphism that occurs in KIR, these genes also exhibit an extensive allelic variation. The receptors encoded by these genes are expressed on the surface of NK cells and on some subset of T cells. They can transduce either activating or inhibiting signals. The differential expression levels and the mechanisms of genetic regulation of these receptors are poorly known. The KIR3DL2 gene encodes an inhibitory receptor and it is one of the most polymorphic and polyallelic KIR, with more than 80 alleles described so far. This study aimed to analyze if there are a profound impact of KIR3DL2 allelic polymorphism on its differential gene expression. Allelic diversity of KIR3DL2 was characterized in an euro-descendant population from Curitiba and metropolitan region, state of PR (n = 235), by sequencing-based typing. Genotype frequencies were in accordance with Hardy-Weinberg equilibrium and the most frequent genotype was 3DL2*001/007 (11.5%), followed by 3DL2*001/002 (6.8%) and 3DL2*002/007 (6.8%). The most frequent alleles in the population were 3DL2*007 (21.7%), 3DL2*001 (21.3%) and 3DL2*002 (20%). A total of 40 individuals with specific and commom genotypes were selected for differential expression analysis by flow citometry. The differential expression analysis was made for the most common alleles found in the population: 3DL2*001, 3DL2*002, 3DL2*003, 3DL2*005, 3DL2*007, 3DL2*009, 3DL2*010 and 3DL2*011. We verified that the allelic polymorphism of KIR3DL2 was associated not only with the differential expression but also with the different amount of NK cells that display KIR3DL2 on their surface. KIR3DL2*002 allele was associated with higher expression of KIR3DL2 and higher number of KIR3DL2 positive NK cells, while KIR3DL2*010 was related to the lowest expression and lowest level of KIR3DL2 positive NK cells. It has also been found that a polymorphism in the 3' UTR, 16545A>G (rs1865095), was associated with KIR3DL2 differential expression level. We suggest that it may be related to miRNAs binding, specifically miR-2114-3p. The presence of specific HLA class I ligands (HLA-A3, -A11 and -B27) was not associated with KIR3DL2 differential expression levels (p = 0.5739) nor with different amount of NK KIR3DL2+ cells (p = 0.7772). In addition, no correlation was found between the differential expression of the KIR3DL2 alleles and the differential expression of the HLA-A alleles. As future perspectives, it is intended to analyze the citotoxic activity of NK cells from individuals with different genotypes like KIR3DL2*001/KIR3DL2*001, KIR3DL2*002/KIR3DL2*002, KIR3DL2*007/KIR3DL2*007 and KIR3DL2*010/KIR3DL2*010 through co-culture to corroborate the hypotheses generated. Key-words: NK cells, KIR3DL2, allelic variability, HLA-A3, HLA-A11, HLA-B27, differential expression.

Les lymphomes T périphériques (PTCLs) sont des entités hétérogènes dont la classification a récemment été révisée. Plus de 25 entités de lymphomes T ou NK matures sont ainsi classées selon leur présentation clinico-biologique et leur origine cellulaire présumée, justifiant l’étude approfondie de leur phénotype. Des récepteurs NK (NKRs) ont été mis en évidence dans certains lymphomes cutanés. En outre, un lymphome intra-épithélial intestinal, appelé maladie coeliaque réfractaire de type II (MCRII), dérive d’un lymphocyte caractérisé par des marqueurs T et NK. Ces constatations nous ont amenés à évaluer l’expression de NKRs sur un panel représentatif de PTCLs, constitué en particulier de PTCLs primitifs intestinaux et de leucémies/lymphomes T de l’adulte associés à l’HTLV-1 (ATL). Dans l’ATL, nous montrons que KIR3DL2 est exprimé par les cellules tumorales des formes aigües. Le virus HTLV-1 et la méthylation de son promoteur jouent un rôle dans l’expression de KIR3DL2. Enfin, un anticorps monoclonal dirigé contre KIR3DL2, IPH4102, est capable de tuer spécifiquement les cellules primaires d’ATL KIR3DL2 ex vivo par un mécanisme dépendant d’effecteurs NK autologues. Dans les lymphoproliférations T intestinales primitives, nous montrons que NKp46 est un nouveau biomarqueur pour leur diagnostic et leur stratification thérapeutique. En effet, NKp46 est exprimé sur les cellules anormales de la MCRII, et sur les lymphomes T primitifs intestinaux agressifs, mais pas sur les indolents et les MC non compliquées. Enfin, les cellules primaires exprimant NKp46 sont sensibles ex vivo à la cytotoxicité induite par un anticorps monoclonal dirigé contre NKp46 et couplé à une toxine.Ces résultats nous ont permis de discuter l’origine cellulaire de ces lymphomes et le rôle de ces NKRs dans la lymphomagénèse. Dans une optique translationnelle, l’expression des NKRs peut aider au diagnostic de ces entités parfois difficiles à individualiser, et enfin constituer une cible thérapeutique intéressante. / Peripheral T-cell lymphoma (PTCLs) are heterogeneous entities whose classification has recently been revised. More than 25 entities are thus classified according to their clinico-biological presentation and their presumed cellular origin, justifying the in-depth study of their phenotype. NK receptors (NKRs) have been demonstrated in some cutaneous lymphomas. In addition, a less known intestinal lymphoma, called type II refractory celiac disease (RCDII), arises from an intraepithelial lymphocyte characterized by T and NK markers. These findings led us to evaluate the expression of NKRs on a representative panel of PTCLs, focusing on primary gastrointestinal (GI) T-cell lymphoproliferative diseases (T-LPD) and HTLV-1 associated adult T leukemia/lymphoma (ATL).In ATL, we show that KIR3DL2 expression is mainly associated with acute-type ATL. HTLV-1 has a preferential tropism for KIR3DL2+ lymphocytes and may play a role in KIR3DL2 expression induction, combined with the hypomethylation status of KIR3DL2 promoter. The benefit of targeting KIR3DL2 by IPH4102 should be further investigating in acute ATL patients.In GI T-LPD, we show that NKp46 is a novel biomarker useful for diagnosis and therapeutic stratification, as NKp46 is a hallmark of RCDII tumor cells, shared by EATL. Strong preclinical rationale identifies anti-NKp46-PBD as a promising therapy for RCDII, EATL and MEITL.These results allowed us to discuss the cellular origin of these lymphomas and the role of NKRs in lymphomagenesis. From a translational point of view, NKRs could represent useful biomarkers in these entities that are sometimes difficult to individualize, and finally constitute an interesting therapeutic target.

Reproduction de : Thèse d'exercice : Médecine. Biologie médicale : Paris 12 : 2008. / Titre provenant de l'écran-titre. Bibliogr. f. 44-48.