NKT cells are a population of T cells that have a broad range of regulatory effects on the immune system. Somewhat paradoxically, they can both suppress and potentiate cell-mediated immune responses; for example, while they can suppress some autoimmune diseases, they can also promote potent tumour rejection. There is accumulating evidence to suggest that this functional dichotomy can be explained by the existence of functionally distinct NKT cell subsets, which can differentially regulate the behaviour of other immune cells and drive remarkably different outcomes in disease settings. / Studies in vivo and in vitro have demonstrated remarkable functional diversity between NKT cell subsets of different phenotypes, and deriving from different tissues. This thesis examined functional differences between NKT cell subsets in the context of type 1 diabetes in NOD mice, identified a phenotypically and developmentally distinct IL-17 producing NKT cell subset, and investigated the functional effects of thymic NKT cells on the development and maintenance of conventional T cells and thymic stromal cells. The data presented in this thesis adds to the accumulating evidence that NKT cells are a functionally heterogeneous population, and reiterates the important point that subsets of NKT cells should be studied separately in order to properly understand the biological function of this important regulatory T cell population, and to maximise their clinical potential.

Le développement de nouvelles stratégies d’immunothérapie représente de nos jours un enjeu majeur de santé publique. Dans ce contexte, les lymphocytes T Natural Killer invariant (iNKT) exercent de puissantes activités immuno-modulatrices. Ces cellules ont la particularité de reconnaitre par l’intermédiaire de leur récepteur T (TCR), des (glyco) lipides, présentés par la molécule CD1d exprimée par les cellules présentatrices d’antigènes (APC), notamment les cellules dendritiques (DC). Initialement découvert à partir d’une éponge marine pour ses activités anti-métastatiques, l’alpha-galactosylcéramide (a-GalCer ou KRN) induit rapidement une sécrétion massive, par les cellules iNKT, de cytokines immunomodulatrices telles l’IFN-g, conduisant à la transactivation de nombreuses cellules immunitaires, notamment les cellules Natural Killer (NK), les DC ou encore les lymphocytes Tgd. Cette propriété unique permet aux cellules iNKT de contrôler, chez la souris, le développement des réponses immunes, notamment la réponse anti-tumorale. Face à ces résultats encourageants, des essais cliniques chez l’homme ont été réalisés mais les résultats se sont avérés décevants. Actuellement, il existe deux stratégies pour cibler un antigène à une population cellulaire particulière : 1) le ciblage passif, basé sur la taille des particules ou leur composition et 2) le ciblage actif, basé sur les fortes interactions anticorps/antigène ou ligand/récepteur. Nous avons testé les deux types de ciblages et avons utilisé le même polymère pour constituer nos vecteurs : le PLGA ou poly(lactic coglycolic acid). Dans une première étude (ciblage passif), nous avons comparé l’efficacité de l’encapsulation de l’a-GalCer dans des particules de tailles différentes: des nano (NP) et microparticules (MP). L’a-GalCer vectorisé dans les NP et les MP est endocyté par les DC (voie des clathrines) et active les cellules iNKT in vitro et in vivo mais ne peuvent empêcher leur anergie. Ces résultats décevants nous ont conduits à opter pour le ciblage actif des DC. Dans un premier temps, étant donné les études controversées sur le rôle des DC dans l’anergie des cellules iNKT, nous avons revisité l’implication de ces dernières dans la primo-activation/anergie des cellules iNKT. Nous confirmons ainsi le rôle primordial de DC dans la primo-activation des cellules iNKT mais surtout, nous montrons qu’elles n’induisent pas leur anergie. Représentant une population hétérogène, nous montrons que parmi les DC, les DC CD8a+ sont de puissantes activatrices des cellules iNKT. Nos résultats nous ont ainsi menés à délivrer spécifiquement l’a-GalCer aux DC CD8a+. Pour cela, nous avons greffé sur les NP de PLGA un anticorps anti-DEC205, récepteur lectinique fortement exprimé par les DC CD8a+. In vitro et in vivo, les NP/DEC205/a-GalCer induisent une plus forte activation des cellules iNKT comparativement à l’a-GalCer libre. De même, la co-délivrance d’a-GalCer et d’ovalbumine (OVA) au sein des DC CD8a+ améliore les propriétés adjuvantes de l’a-GalCer en induisant des réponses humorale et cellulaire (lymphocytes T CD8+) spécifiques de l’OVA plus importantes comparativement à la délivrance des deux composés sous leur forme libre. Finalement, de façon intéressante, nous montrons que suite à une primo-activation par les NP/DEC205/a-GalCer, les cellules iNKT sont capables de répondre de nouveau à une seconde stimulation, traduisant l’absence d’anergie des cellules iNKT. En conclusion, nos résultats indiquent que la délivrance spécifique de l’a-GalCer aux DC CD8a+ amplifie la primo-activation des cellules iNKT tout en évitant la mise en place du phénomène d’anergie et ouvrent de nombreuses perspectives dans le cadre de thérapies anti-tumorales et anti-infectieuses. / Nowdays, the development of new immunotherapy strategies represent a major issuein public health. In this context, the invariant Natural Killer T lymphocytes (iNKT) have strongimmunomodulatory properties. This cell population recognizes (glycol)lipid presented by theCD1d molecule expressed by antigen presenting cell (APC) as dendritic cells (DC). Initiallyfound in a marine sponge for its anti-metastatic activities, alpha-galactosylceramide (-GalCer or KRN) induces a massive cytokine production (IFN-, IL-4, IL-17) by iNKT cells.This cytokine burst lead to downstream activation of numerous immune cells like naturalkiller cell (NK), DC or CD8+ T cells. Through this property, iNKT cells regulate numerousimmune responses, including anti-tumoral response. Based on encouraging results in themouse model, -GalCer has been used in anti-tumour therapy in human. Although the drugwas well tolerated, no or moderate clinical responses were observed in patients repeatedlyinoculated with -GalCer. As observed in the mouse system, one potential explanation forthis disappointing observation may lie in the induction of a long-term anergy of human iNKTcells, thus preventing cytokine release upon a recall stimulation. Although controversial,various studies suggest that this phenomonen should be due to a lack of delivery of -GalCer into dendritic cells (DC) and so its presentation by non adequate antigen presentingcell (APC) as B cells. The objective of our work was to optimize -GalCer activity by avoidingiNKT anergy using vectorisation approach. Actually, there are 2 strategies using nanotechnologies to target an antigen to a specific cell population: 1) passive targeting based on the size of the particles, their composition and their surface charge and 2) active targeting based on the strong interactions between an antibody and its antigen or a ligand and its receptor. We have tested the two strategies and therefore we use the same composition of our particles: PLGA or poly(lactic co glycolic acid). This biodegradable and biocompatible molecule is already used in therapy.In a first study (passive targeting), we compared the efficiency of -GalCer encapsulation inparticles with different size: nano (NP) and microparticle (MP). Vectorised -GalCer in NPand MP rapidly activates iNKT cells in vitro. Both type of particles are uptake by DC via aclathrin dependent mechanism. In in vivo approaches, NP/-GalCer and MP/-GalCeractivate iNKT cells but unfortunately could not prevent iNKT cell anergy. These disappointingresults led us to use an active targeting. In first time, because of controversial role of DC iniNKT anergy, we have revisited the role of DC in iNKT primo-activation and anergy. Weconfirm the primordial role of DC in iNKT primo-activation but especially we show that DC donot induce iNKT anergy. DC are heterogeneous and we show that among DC, CD8a+ DCsubpopulation are potent iNKT cells activation. Our results led us to deliver specifically a-GalCer to CD8+ DC. For this, anti-DEC205 antibodies were covalently linked to the surfaceof PLGA NP, DEC205 being highly expressed by CD8+ DC. In vitro and in vivo,NP/DEC205/-GalCer induce a stronger iNKT cell activation relative to free -GalCer (or NPIgG/-GalCer). Moreover, -GalCer and ovalbumin co-delivery in CD8+ DC improve -GalCer adjuvanticity leading to more important humoral and cellular responses. Interestingly,after a primo-activation by NP/DEC205/-GalCer, iNKT cells are able to respond to secondstimulation thus avoiding iNKT cell anergy.In conclusion, our results indicate that specific -GalCer delivery to CD8+ DC improve iNKT cells primo-activation and avoid anergy phenomenon. These findings open several perspectives in anti-tumoral and anti-infectious therapies.

Semi-invariant natural killer T (iNKT) cells are critical components of the host immune response in peripheral tissues such as the lung, liver, and gut, and they play important roles in cancer, bacterial infections, autoimmunity, wound repair, and atherosclerosis. Tissue-resident iNKT cells exert their effects early in the developing immune response by rapidly producing a wide variety of cytokines and chemokines, and it was recently discovered that different tissues possess iNKT cell subsets that preferentially produce IFN-γ (NKT1), IL-4 (NKT2), or IL-17 (NKT17). Despite their critical role in the immune response, the mechanisms that regulate iNKT cell function in the periphery remain unclear. Signaling lymphocyte activation marker (SLAM) proteins are cell surface-expressed molecular switches that are expressed on all hematopoietic cells. The nine SLAM family receptors serve a variety of functions including promotion of cell-cell adhesion, regulation of cytokine production, co-stimulation, and inhibition. Importantly, SLAM family receptors are critical for the development of iNKT cells. Yet, numerous efforts to ascribe discrete roles of SLAM family receptors in iNKT cell function has proven difficult. We conducted a comprehensive analysis of SLAM family receptor co-expression on iNKT cell subsets in the lung, spleen, liver, and thymus and identified co-expression profiles that varied in a tissue and strain-dependent manner. Interestingly, we found that SLAM family receptor expression profiles varied among different iNKT cell subsets. In particular, we noted a close association of SLAMf6 expression with the NKT2 and NKT17 subsets in both the periphery and in the thymus. Further investigation using SLAMf6-deficient mice revealed a critical role for SLAMf6 in NKT2 and NKT17 subset development, and in iNKT IL-4 and IL-17 cytokine production in the periphery. This investigation also revealed that the SLAMf6high NKT2 and NKT17 subsets exhibited significantly higher proliferative capacity than the NKT1 subset and the NKT2 and NKT17 proliferation was dependent, in part, on SLAMf6 expression. Since Slam family genes are highly polymorphic, we next investigated whether these polymorphisms regulated iNKT function. We employed a B6.129 congenic mouse exhibiting impaired NKT cell function, in which a 6.6 Mbp 129/SvJ locus encompassing Slam genes was introgressed onto the C57BL/6 background. To test the hypothesis that Slam gene polymorphisms regulate iNKT cell function, we refined this genetic interval by generating B6.129 subcongenic lines and assessing iNKT cell function. Unexpectedly, we found that while Slam gene polymorphisms in this model do regulate iNKT cell function, the dominant regulator was in a 0.14 Mbp interval centromeric to the Slam genes. Further experimentation revealed that impaired iNKT cell development and function was associated with changes in the expression of Fcgr3 (Fc gamma receptor III) on iNKT cells, suggesting it as a novel candidate gene regulating iNKT cell function. Taken together, these data reveal for the first time a specific role for SLAMf6 on NKT2 and NKT17 subset development and function. In addition, these data identify Fcgr3 as a novel candidate gene that regulates iNKT cell subset development and cytokine production. Cumulatively, these data reveal the presence of discrete regulatory mechanisms at work in different iNKT subsets, a finding that has broad implications for our understanding of iNKT-cell mediated immunity.

Submitted by Pós Imunologia (ppgimicsufba@gmail.com) on 2017-07-31T19:06:05Z No. of bitstreams: 1 Dissertação Cayo Amaral Abreu (1).pdf: 920431 bytes, checksum: ab2e71045643d125d189674441bd23b6 (MD5) / Approved for entry into archive by Edvaldo Souza (edvaldosouza@ufba.br) on 2017-08-01T20:27:18Z (GMT) No. of bitstreams: 1 Dissertação Cayo Amaral Abreu (1).pdf: 920431 bytes, checksum: ab2e71045643d125d189674441bd23b6 (MD5) / Made available in DSpace on 2017-08-01T20:27:18Z (GMT). No. of bitstreams: 1 Dissertação Cayo Amaral Abreu (1).pdf: 920431 bytes, checksum: ab2e71045643d125d189674441bd23b6 (MD5) / PAPES/CNPq, CAPES / O melanoma é um tipo de tumor de pele com altas taxas de incidências por todo mundo. Buscando formas de terapia auxiliar, destaca-se o Imiquimode (IMIQ), um creme de uso tópico que atua no receptor tipo toll-7 (TLR-7) estimulando o sistema imune inato a promover atividades tumoricidas. No presente estudo o tumor de linhagem B16F0 foi inoculado na orelha dos camundongos, seguidos do tratamento com o creme IMIQ. A mortalidade e o desenvolvimento tumoral foram acompanhados, nos diferentes grupos experimentais. Após 12 dias de tratamento, baços e linfonodos cervicais foram removidos em grupos de animais tratados e não tratados. As células foram colocadas em cultivo, plaqueadas e marcadas para análise em citômetro de fluxo. Grupos tratados com IMIQ apresentaram considerável eficácia no aumento da sobrevida e na contenção do tumor. Além disso, foi demonstrado que a atividade antitumoral é dependente da molécula de CD1, comprovando a importância das células NKT no combate ao melanoma. Ademais, nos grupos tratados, houve maior produção de IFN-γ por linfócitos TCD8 esplênicos, e TNF-α por células TCD4 e TCD8 de linfonodos drenantes. Pôde-se ainda notar o aumento de granzima B (GZB) produzida por células dendríticas (DCs) no baço de animais com melanoma. Já na estimulação de esplenócitos com o mitógeno Concanavalina A, foi evidenciada inibição na produção de GZB por células dendríticas. Conclui-se que NKT pode ser a principal responsável pela migração dessa população celular e que a produção de GZB por DCs pode ser inibida mediante a presença de antígenos tumorais. / Melanoma is a type of skin tumor with high incidence rates worldwide. Aiming for forms of auxiliary therapy, Imiquimode (IMIQ), a topical cream that acts on the toll-7 receptor (TLR-7), stimulates the innate immune system to promote tumoricidal activities. In the present study the B16F0 lineage tumor was inoculated into the ears of the mice, followed by treatment with the IMIQ cream. Mortality and tumor development were monitored in the different experimental groups. After 12 days of treatment, spleens and cervical lymph nodes were removed in groups of treated and untreated animals. Cells were plated, plated and labeled for flow cytometer analysis. Groups treated with IMIQ showed considerable efficacy in increasing survival and tumor containment. In addition, it has been demonstrated that the antitumor activity is dependent on the CD1 molecule, proving the importance of NKT cells in the fight against melanoma. In addition, in the treated groups, there was a greater production of IFN-γ by splenic TCD8 lymphocytes, and TNF-α by TCD4 and TCD8 cells from draining lymph nodes. It was also possible to note the increase of granzyme B (GZB) produced by dendritic cells (DCs) in the spleen of animals with melanoma. Already in the stimulation of splenocytes with the mitogen Concanavalin A, inhibition was demonstrated in the production of GZB by dendritic cells. It is concluded that NKT may be the main responsible for the migration of this cell population and that the production of GZB by DCs can be inhibited by the presence of tumor antigens.

Natural killer T (NKT) cells are a subset of T cells that modify a variety of immune responses. NKT cells recognize glycolipid antigen presented by a molecule called CD1d, a nonclassical antigen-presenting molecule. The best known subset of CD1d-dependent NKT cells expresses an invariant T cell receptor Vα (TCR-α) chain. These are referred to as type I or invariant NKT (iNKT) cells. When stimulated by a glycolipid, NKT cells rapidly release large amounts of cytokines. Cytokines released by NKT cells can induce either Th1 or Th2 responses. Th1 cytokines are effective in regulating bacterial, parasitic, and viral infections. But Th1 responses are also involved in some autoimmune diseases, such as type 1 diabetes, lupus, rheumatoid arthritis, and allergen-induced asthma. Th2 cytokines can attenuate proinflammatory Th1 responses and therefore prevent some autoimmune diseases. Lysosomal processing and CD1d loading is very important in regulating the stimulatory properties of antigens. Analogs of KRN7000, with small molecules appended on C6” position of the galactose portion, do not significantly change stimulation of NKT cells. The question is if the substitution at this position would influence the lysosomal processing. Two sets of mono- and disaccharides with and without substitution at C6” position were prepared and evaluated the NKT cell stimulatory properties. The substitution at the C6” position of the sugar moiety of glycolipids do not significantly impact the stimulatory properties of glycolipids and their processing in lysosome. Small changes at C6” are well tolerated. A double bond in the acyl chain and modification of the C6” functional group helped the glycolipid loading into CD1d and NKT cells stimulation. PBS57 is 100 times more active than KRN 7000 in stimulation of NKT cells responses in vitro and in vivo. This improvement is probably due to increasing solubility and improving binding ability with the CD1d.

No description available.

A fibrose pulmonar é uma via comum de várias doenças agudas e crônicas do interstício pulmonar que pode resultar na cicatrização anormal do pulmão. Há acúmulo excessivo das proteínas da matriz extracelular levando a desestruturação das paredes alveolares, e consequente perda das trocas gasosas pelos pulmões. As células NKT são grande fonte de citocinas e podem ser cruciais na polarização de macrófagos para o fenótipo M2. O projeto tem a hipótese de que as células NKT podem influenciar o desenvolvimento da fibrose pulmonar via modulação de macrófagos. Para isso, animais selvagens e knockout para células NKT invariante (Ja18-/-) foram submetidos ao protocolo de indução de fibrose pulmonar pela bleomicina. Os resultados indicam que o grupo Ja18-/- assim como os grupos experimentais que receberam agonistas para células NKT apresentaram uma proteção contra a fibrose pulmonar uma vez que houve menor síntese de hidroxiprolina, deposição de colágeno, citocinas pró-fibróticas e a manutenção de macrófagos M1 no tecido pulmonar. / Pulmonary fibrosis is a common pathway of various acute and chronic interstitial lung diseases that may result in abnormal healing of the lung. There is excessive accumulation of extracellular matrix proteins, leading to disruption of the alveolar walls and the consequent loss of gas exchange through the lungs. NKT cells are a big source of cytokines and may be crucial in the polarization of macrophages to the M2 phenotype. This project has hypothesized that NKT cells can influence the development of pulmonary fibrosis through modulation of macrophages. For this, wild and knockout invariant NKT cells (Ja18-/-) mice were subjected to the protocol of bleomycin induced pulmonary fibrosis. The results indicate that the group Ja18-/- as well as the experimental groups receiving agonists for NKT cells showed protection against lung fibrosis since there was less synthesis of hydroxyproline, collagen deposition, pro-fibrotic cytokines and maintenance of macrophages M1 in lung tissue.

As células NKT compreendem um grupo distinto de linfócitos caracterizados pela reatividade a glicolipídios apresentados pela molécula CD1d. Em nosso trabalho, nós investigamos a participação das células NKT na homeostase intestinal utilizando animais nocautes para essas células e também caracterizamos os subtipos de NKT no intestino. Nos animais nocautes observamos que a ausência de células NKT influencia a microbiota e a homeostase intestinal. Os animais nocautes possuem diminuição no filo Firmicutes, diminuição dos níveis de IgA nas fezes e de TGF-β no intestino. Esses animais também são mais suscetíveis à colite induzida por DSS, apresentando menor sobrevida, menor tamanho do cólon e aumento no score. Nos animais selvagens, a indução do subtipo NKT10 diminuiu a inflamação intestinal, não causou intensa redução do cólon e reduziu a frequência de células NKT e do subtipo NKT17 no intestino. Nossos resultados indicam que as células NKT intestinais são peças importantes para a homeostase intestinal e para a composição da microbiota intestinal. / NKT cells comprise a distinct group of lymphocytes characterized by their reactivity to glycolipids presented by CD1d. In our work, we investigated the involvement of NKT cells in intestinal homeostasis using knockout (KO) mice and we also characterized NKT subtypes in the intestine. The absence of NKT cells in KO mice influenced the intestinal microbiota and homeostasis. KO mice had a decrease in the Firmicutes phyla, in the levels of fecal IgA and TGF-β in the intestine. These mice are also more susceptible to DSS-induced colitis, exhibiting worse survival, severe shortening of the colon and higher score. In wild type mice, the induction of the NKT10 subtype decreased intestinal inflammation, did not cause intense reduction of colon and reduced the frequency of NKT cells and the NKT17 subtype in the intestine. Our results indicate that intestinal NKT cells are important for intestinal homeostasis and microbiota composition.

A fibrose pulmonar é uma via comum de várias doenças agudas e crônicas do interstício pulmonar que pode resultar na cicatrização anormal do pulmão. Há acúmulo excessivo das proteínas da matriz extracelular levando a desestruturação das paredes alveolares, e consequente perda das trocas gasosas pelos pulmões. As células NKT são grande fonte de citocinas e podem ser cruciais na polarização de macrófagos para o fenótipo M2. O projeto tem a hipótese de que as células NKT podem influenciar o desenvolvimento da fibrose pulmonar via modulação de macrófagos. Para isso, animais selvagens e knockout para células NKT invariante (Ja18-/-) foram submetidos ao protocolo de indução de fibrose pulmonar pela bleomicina. Os resultados indicam que o grupo Ja18-/- assim como os grupos experimentais que receberam agonistas para células NKT apresentaram uma proteção contra a fibrose pulmonar uma vez que houve menor síntese de hidroxiprolina, deposição de colágeno, citocinas pró-fibróticas e a manutenção de macrófagos M1 no tecido pulmonar. / Pulmonary fibrosis is a common pathway of various acute and chronic interstitial lung diseases that may result in abnormal healing of the lung. There is excessive accumulation of extracellular matrix proteins, leading to disruption of the alveolar walls and the consequent loss of gas exchange through the lungs. NKT cells are a big source of cytokines and may be crucial in the polarization of macrophages to the M2 phenotype. This project has hypothesized that NKT cells can influence the development of pulmonary fibrosis through modulation of macrophages. For this, wild and knockout invariant NKT cells (Ja18-/-) mice were subjected to the protocol of bleomycin induced pulmonary fibrosis. The results indicate that the group Ja18-/- as well as the experimental groups receiving agonists for NKT cells showed protection against lung fibrosis since there was less synthesis of hydroxyproline, collagen deposition, pro-fibrotic cytokines and maintenance of macrophages M1 in lung tissue.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.