Influenza A virus (IAV) infection is a highly contagious respiratory disease, which can cause substantial morbidity and occasionally death. Invariant natural killer T (iNKT) cells, a subset of CD1d-restricted T lymphocytes, have been identified as important modulators of immunity, mediating both pro- and anti-inflammatory responses. We show that iNKTs play an important role for the generation of protective innate and adaptive immune responses to IAV, and enhance heterotypic immunity to influenza virus following vaccination with a novel pseudotyped virus, S-FLU, which lacks HA expression. iNKT-deficient mice (Jα18^-/-) showed increased susceptibility and lung pathology following IAV infection, which correlated with an exaggerated accumulation of inflammatory monocytes and neutrophils in the lung. Consistent with these findings, we demonstrated in IAV-infected CD1d^-/-:CD1d^{+/+} mixed bone marrow chimeric mice, that the lack of CD1d expression on myeloid cells purified from the lungs of IAV-infected mice significantly increased expression of genes linked to cell activation, survival and polarisation between pro- and antiinflammatory responses. We extended these results by examining the role of chemokine signalling during IAV infection, and identified a novel role for fractalkine (CX3CL1) and its receptor (CX3CR1) in innate immune cell recruitment. The use of CX3CR1-iNKT cell double knockout mice revealed that, although upregulated in Jα18^-/- mice, CX3CR1-CX3CL1 signalling is not required for cell migration during exacerbated IAV-responses. Finally, we showed that iNKT-deficient mice displayed reduced longevity of peripheral IAVspecific CD8^+ T cells following S-FLU vaccination, compared with wild-type mice. S-FLU vaccination protected mice following 5 day heterotypic challenge, however vaccinated mice exhibited reduced viral clearance, and importantly a significant reduction in IAV specific memory T cell response, suggesting a possible role of iNKT cells during T cell priming in modulating the lifespan of antigen-specific T cell responses. Although additional experiments are warranted, these results suggest that harnessing iNKT cells should be considered to modulate the innate and adaptive immune response to optimise heterotypic vaccine design and for therapeutic intervention against acute influenza infection.

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Durant l’infection par le virus Influenza A (IAV), les changements physiques et immunologiques du poumon prédisposent l’hôte aux surinfections bactériennes. Les cellules T Natural Killer invariantes (iNKT) sont des lymphocytes T innés pouvant avoir des rôles bénéfiques ou délétères durant l’infection. Nos objectifs ont visé à (i) étudier le rôle naturel des cellules iNKT et (ii) à rechercher l’effet d’une activation exogène des cellules iNKT dans la surinfection bactérienne post-influenza.Lors de mon arrivée, le laboratoire venait de décrire, pour la première fois en contexte infectieux, que les cellules iNKT étaient capables de produire de l’IL-22 au cours de l’infection grippale. Cette cytokine joue un rôle majeur dans les processus de maintien et de réparation des épithéliums. L’une des causes des surinfections bactériennes post-grippales étant l’altération et/ou la perte de l’intégrité de l’épithélium pulmonaire, nous nous sommes proposés d’étudier le rôle potentiel de cette cytokine dans un modèle expérimental de surinfection bactérienne à S. pneumoniae. Nous avons ainsi pu montrer que si cette cytokine ne joue pas un rôle majeur dans la réponse anti-virale de l’hôte, l’IL-22 participe au contrôle de l’inflammation au cours de l’infection grippale et joue un rôle protecteur dans la surinfection bactérienne.Par ailleurs, l’utilisation de souris dépourvues en cellules iNKT (Jα18-/-) a permis de montrer que les cellules iNKT limitent la susceptibilité aux surinfections et réduisent le synergisme létal de la coinfection virus/bactérie. Au moment de l’infection bactérienne, les cellules iNKT des souris grippées sont incapables de produire de l’IFN-γ, cytokine dont nous avons montré le rôle essentiel dans les mécanismes de défense antibactérienne. Le défaut d’activation des cellules iNKT chez les souris surinfectées est lié à l’interleukine-10 (IL-10), cytokine immunosuppressive induite par l’infection virale, plutôt qu’à un défaut intrinsèque des cellules iNKT. L’IL-10 inhibe l’activation des cellules iNKT en réponse au pneumocoque en inhibant la production d’IL-12 par les cellules dendritiques dérivées de monocytes (MoDCs). La neutralisation de l’IL-10 restaure l’activation des cellules iNKT et augmente la résistance à la surinfection. Ainsi, les cellules iNKT ont un rôle bénéfique (en amont de la colonisation bactérienne) dans le contrôle de la surinfection bactérienne de la grippe et représentent une cible de l’immunosuppression.Nous avons par la suite étudié la possibilité que le superagoniste des cellules iNKT, l’ α-galactosylceramide (α-GalCer) puisse limiter la surinfection bactérienne. Pour cela, les souris ont été traitées par voie intranasale avec de l’α-GalCer à différents temps post-influenza, juste avant l’infection par le pneumocoque. Le traitement à jour 3, au pic de la réplication virale, limite fortement la surinfection. Cependant, l’inoculation d’α-GalCer pendant la phase aiguë du virus (jour 7) ne permet pas d’activer les cellules iNKT pulmonaires et n’a pas d’effet sur la surinfection. L’absence d’activation des cellules iNKT n’est pas intrinsèque et est associée à une disparition complète des cellules dendritiques CD103+ respiratoires (cDCs), lesquelles sont cruciales dans l’activation des cellules iNKTs. À des temps plus tardifs (jour 14), les cDCs repeuplent le poumon et l’α-GalCer promeut l’activité antibactérienne des cellules iNKT.Pris dans son ensemble, cette étude souligne le rôle des cellules iNKT dans la surinfection bactérienne de la grippe et ouvre de nouvelles voies thérapeutiques afin de limiter les surinfections bactériennes post-influenza. / XDurant l’infection par le virus Influenza A (IAV), les changements physiques et immunologiques du poumon prédisposent l’hôte aux surinfections bactériennes. Les cellules T Natural Killer invariantes (iNKT) sont des lymphocytes T innés pouvant avoir des rôles bénéfiques ou délétères durant l’infection. Nos objectifs ont visé à (i) étudier le rôle naturel des cellules iNKT et (ii) à rechercher l’effet d’une activation exogène des cellules iNKT dans la surinfection bactérienne post-influenza.Lors de mon arrivée, le laboratoire venait de décrire, pour la première fois en contexte infectieux, que les cellules iNKT étaient capables de produire de l’IL-22 au cours de l’infection grippale. Cette cytokine joue un rôle majeur dans les processus de maintien et de réparation des épithéliums. L’une desDuring the infection by the virus Influenza A ( IAV), the physical and immunological changes of the lung predispose the host to the bacterial secondary infections. The invariant cells(units) T Natural Killer iNKT ) are lymphocytes T innate being able to have beneficial or noxious roles during the infection. Our objectives aimed at i) to study the natural role of cells(units) iNKT and ii) to look for the effect of an exogenous activation of cells(units) iNKT in the bacterial secondary infection post-influenza. During my arrival, the laboratory had just described, for the first time in infectious context, that cells(units) iNKT were capable of producing of IL-22 during the flu-like infection. This cytokine plays a major role in the processes of preservation and repair of epitheliums [...]

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第12957号 / 論医博第2099号 / 新制||医||1011(附属図書館) / 32356 / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 岩井 一宏, 教授 椛島 健治 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

The Lyme disease spirochete Borrelia burgdorferi is the only known human pathogen that directly activates invariant natural killer T (iNKT) cells. The number and activation kinetics of iNKT cells vary greatly among different strains of mice. Here, we report the role of the iNKT cell response in the pathogenesis of Lyme disease using C57BL/6 (B6) mice, a strain with optimal iNKT cell activation that is resistant to the development of spirochetal-induced inflammation. During experimental infection of B6 mice with B. burgdorferi , iNKT cells localize to the inflamed heart where they are activated by CD1d-expressing macrophages. Activation of iNKT cells in vivo results in the production of IFNγ, which we demonstrate controls the severity of murine Lyme carditis by at least two mechanisms. First, IFNγ greatly enhances the recognition of B. burgdorferi by macrophages, leading to increased phagocytosis of the spirochete. Secondly, IFNγ activation of macrophages increases the surface expression of CD1d, thereby facilitating further iNKT activation. Collectively, our data demonstrate that in the resistant background, B6, iNKT cells modulate acute murine Lyme carditis through the action of IFNγ, which appears to self-renew through a positive feedback loop during infection. Inflammation during infection with B. burgdorferi is dependent on the ability of the spirochete to evade local mechanisms of clearance. Even though macrophages are the main infiltrating cell during Lyme carditis, the identification of a receptor capable of mediating phagocytosis of B. burgdorferi has been elusive. Here, we demonstrate that the integrin CR3 is able to mediate binding to the spirochete and facilitate phagocytosis in a complement-dependent and independent manner. Expression of CR3, but not CR4, in CHO cells markedly enhanced their capacity to interact with B. burgdorferi , in the absence and presence of complement opsonization. Furthermore, the interaction between CR3 and B. burgdorferi is dependent on the metal-ion-dependent adhesion site (MIDAS) and could be blocked with EDTA. Inhibition of CR3 with blocking antibody was able to completely abrogate phagocytosis of B. burgdorferi by the macrophage-like RAW264.7 cells and partially block uptake by bone marrow-derived macrophages (BMMs), a finding that was recapitulated with CD11b-deficient BMMs. We further show that activation with recombinant IFNγ increases the transcription of CD11b and CD18, which correlates with increased surface expression of CR3, and that the effect of IFNγ on the phagocytosis of B. burgdorferi is circumscribed to CR3 activity, because inhibition of CR3 is able to completely diminish the effect of IFNγ on the phagocytosis of the B. burgdorferi . Lastly, our results demonstrate that CR3 is a negative regulator of proinflammatory cytokine induction in macrophages responding to B. burgdorferi . Overall, our data demonstrate roles for CR3 in the binding, phagocytosis and proinflammatory cytokine elicited by B. burgdorferi and shed light on the role of IFNγ in mediating the clearance of the spirochete during Lyme disease.

As células T Natural Killer (NKT) são linfócitos que expressam o rearranjo V24V11 com TCR invariante e reconhecem glicolipídeos como o alfagalactosilceramida (-GalCer) apresentado no contexto da molécula MHC-não clássica chamada CD1d. As NKT são divididas em subgrupos distintos: os fenótipos CD4+ e CD4- em camundongos e humanos, e CD8+ e CD4-CD8- em humanos. Após estímulo, as NKT secretam citocinas Th1 e Th2. A frequência das NKT representa menos de 1% da população de linfócitos T no sangue periférico humano. O objetivo deste trabalho é primeiro, identificar a frequência, o fenótipo e a função da população de células NKT nos grupos de indivíduos saudáveis de procedências distintas. Segundo, avaliar a influência da idade, gênero e país de moradia na frequência das NKT, incluindo suas subpopulações, no sangue periférico; Terceiro, verificar se há variabilidade ou não na apresentação da molécula CD1d. Quarto, verificar se há alteração na função das NKT utilizando o estímulo -GalCer e por fim, verificar o perfil das NKT quanto ao seu estado de ativação e migração celular quando submetidas a expansão. As amostras são provenientes de São Paulo, Brasil; São Francisco, Estado Unidos da América e Estocolmo, Suécia. Os parâmetros imunológicos foram analisados por citometria de fluxo. Demonstramos que o grupo de São Paulo, não apresentou maiores valores de linfócitos T CD3+, no entanto, apresentou valores significantes de células NKT V24+/V11+. Não estabelecemos a expressão dos marcadores de ativação e migração nas NKT. Estabelecemos, no grupo de São Paulo, a capacidade funcional das NKT que expressaram IFN- e TNF- assim como de expansão, sob estímulo de -GalCer. Os resultados sugerem que vários fatores podem alterar a quantidade das células NKT na periferia. Mais estudos devem ser endereçados para esclarecer melhor estes fatos / Natural Killer T cells (NKT cells) are lymphocytes that express the invariant TCR rearrangement V24V11 and recognize glycolipid as alpha-galactosylceramide (-GalCer) presented in conjunction with a non-MHC pathway molecule named CD1d. The NKT cells are divided into distinct subsets: the CD4+ and CD4- phenotype in mice and humans and the CD8+ and CD4-CD8- phenotype in humans. After stimulation, the NKT secrete Th1 and Th2 cytokines. The frequencies of NKT cells represent <1% of the population of T lymphocytes in human peripheral blood. The objective of this study is to 1) identify the frequency, phenotype and function of NKT cell population in groups of healthy individuals from different racial/ethnic origins, 2) evaluate the influence of age, gender and country of residence on the frequency of NKT cells/subpopulations, 3) investigate the variability of presentation of the CD1d molecule, 4) investigate the functional variability of NKT cells using -GalCer stimulation, and 5) study the activation and migration profile of NKT cells after expansion. Immunologic parameters of clinical samples from Sao Paulo, Brazil; San Francisco, California and Stockholm, Sweden were analyzed by flow cytometry. We demonstrate that the São Paulo group does not have an elevated number of CD3+ T lymphocytes, but does have significantly more NKT V24+/V11+ cells. We have not yet determined expression of NKT markers for activation and migration. Thus, we have shown that among sample obtained in São Paulo, there is increased functional capacity of IFN- and TNF--expressing NKT cells along with NKT cells expansion under stimulation with the -GalCer. These results suggest that there are several factors that can modulate NKT cell number and function in the peripheral blood and additional studies are needed to further clarify these findings

As células T Natural Killer (NKT) são linfócitos que expressam o rearranjo V24V11 com TCR invariante e reconhecem glicolipídeos como o alfagalactosilceramida (-GalCer) apresentado no contexto da molécula MHC-não clássica chamada CD1d. As NKT são divididas em subgrupos distintos: os fenótipos CD4+ e CD4- em camundongos e humanos, e CD8+ e CD4-CD8- em humanos. Após estímulo, as NKT secretam citocinas Th1 e Th2. A frequência das NKT representa menos de 1% da população de linfócitos T no sangue periférico humano. O objetivo deste trabalho é primeiro, identificar a frequência, o fenótipo e a função da população de células NKT nos grupos de indivíduos saudáveis de procedências distintas. Segundo, avaliar a influência da idade, gênero e país de moradia na frequência das NKT, incluindo suas subpopulações, no sangue periférico; Terceiro, verificar se há variabilidade ou não na apresentação da molécula CD1d. Quarto, verificar se há alteração na função das NKT utilizando o estímulo -GalCer e por fim, verificar o perfil das NKT quanto ao seu estado de ativação e migração celular quando submetidas a expansão. As amostras são provenientes de São Paulo, Brasil; São Francisco, Estado Unidos da América e Estocolmo, Suécia. Os parâmetros imunológicos foram analisados por citometria de fluxo. Demonstramos que o grupo de São Paulo, não apresentou maiores valores de linfócitos T CD3+, no entanto, apresentou valores significantes de células NKT V24+/V11+. Não estabelecemos a expressão dos marcadores de ativação e migração nas NKT. Estabelecemos, no grupo de São Paulo, a capacidade funcional das NKT que expressaram IFN- e TNF- assim como de expansão, sob estímulo de -GalCer. Os resultados sugerem que vários fatores podem alterar a quantidade das células NKT na periferia. Mais estudos devem ser endereçados para esclarecer melhor estes fatos / Natural Killer T cells (NKT cells) are lymphocytes that express the invariant TCR rearrangement V24V11 and recognize glycolipid as alpha-galactosylceramide (-GalCer) presented in conjunction with a non-MHC pathway molecule named CD1d. The NKT cells are divided into distinct subsets: the CD4+ and CD4- phenotype in mice and humans and the CD8+ and CD4-CD8- phenotype in humans. After stimulation, the NKT secrete Th1 and Th2 cytokines. The frequencies of NKT cells represent <1% of the population of T lymphocytes in human peripheral blood. The objective of this study is to 1) identify the frequency, phenotype and function of NKT cell population in groups of healthy individuals from different racial/ethnic origins, 2) evaluate the influence of age, gender and country of residence on the frequency of NKT cells/subpopulations, 3) investigate the variability of presentation of the CD1d molecule, 4) investigate the functional variability of NKT cells using -GalCer stimulation, and 5) study the activation and migration profile of NKT cells after expansion. Immunologic parameters of clinical samples from Sao Paulo, Brazil; San Francisco, California and Stockholm, Sweden were analyzed by flow cytometry. We demonstrate that the São Paulo group does not have an elevated number of CD3+ T lymphocytes, but does have significantly more NKT V24+/V11+ cells. We have not yet determined expression of NKT markers for activation and migration. Thus, we have shown that among sample obtained in São Paulo, there is increased functional capacity of IFN- and TNF--expressing NKT cells along with NKT cells expansion under stimulation with the -GalCer. These results suggest that there are several factors that can modulate NKT cell number and function in the peripheral blood and additional studies are needed to further clarify these findings

Introdução: A endometriose é uma doença com características inflamatórias que atinge as mulheres em idade reprodutiva. A patogênese da endometriose não está esclarecida. Tem sido demonstrada associação entre distúrbios imunológicos e endometriose, como alterações nos macrófagos, células NK, citocinas e nas repostas Th1, Th2 e Th17. As células iNKT, um tipo especial de linfócitos T, tem importante papel na resposta inflamatória como mediadores das repostas Th1, Th2 e Th17. Objetivos: o objetivo principal deste estudo foi comparar as frequências das células iNKT e seus subtipos entre pacientes com endometriose e pacientes sem endometriose. Procuramos também comparar as frequências destas células em ambos os grupos relacionando com alguns os aspectos clínicos e cirúrgicos da doença. Métodos: Realizamos estudo transversal, prospectivo entre fevereiro de 2013 a fevereiro de 2015 que avaliou a porcentagem de células iNKT e os subtipos iNKT CD4+, iNKT CD4+ CCR7+, iNKT CD4+ CD25+, iNKT DN, iNKT DN CCR7+, iNKT DN CD25+, iNKT CD4+ IL6+, iNKT CD4+ IL10+, iNKT CD4+ IL17+, iNKT CD8+ IL6+, iNKT CD8+ IL10+, iNKT CD8+ IL17+, iNKT DN IL6+, iNKT DN IL10+, iNKT DN IL17+ no sangue periférico, por citometria de fluxo, em pacientes com endometriose profunda (n = 47) e sem endometriose (n = 26). As frequências de células iNKT e seus subtipos foram comparadas entre os grupos de acordo com os sintomas, fase do ciclo, estádio da doença e classificação histológica. Resultados: Na avaliação da frequência das células iNKT, iNKT DN e iNKT DN IL17+ foi evidenciada diminuição significativa nas pacientes com endometriose (p=0,010, p=0,020, p=0,050; respectivamente). Além disso, foi observada diminuição significativa nas frequências das células iNKT CD4+ CCR7+ e aumento significativo das células iNKT CD4+ IL-17+ em pacientes com endometriose e dismenorréia severa em comparação a dismenorréia ausente/leve. Nas pacientes com endometriose e dor acíclica severa observou-se diminuição significativa da frequência das células iNKT CD4+ IL17+ em comparação a dor acíclica ausente/leve (p=0,048). Houve diminuição das células iNKT nas pacientes com endometriose em relação ao grupo controle na fase secretora do ciclo menstrual (p=0,030). Na avaliação da fase do ciclo menstrual foi observado na fase proliferativa aumento significativo na frequência das células iNKT CD4+ CD25+ (p=0,022) e diminuição significativa das células iNKT DN (p=0,011) nas pacientes com endometriose. Na fase secretora foi evidenciado diminuição significativa na frequência das células iNKT DN IL17+ (p=0,049) nas pacientes com endometriose. Foi identificado também nas pacientes com endometriose uma diminuição na frequência das células iNKT DN CD25+ na fase secretora em relação a fase proliferativa do ciclo menstrual. Conclusões: As células iNKT e os subtipos iNKT DN e iNKT DN IL17+ se mostraram alteradas nas pacientes com endometriose profunda. Subtipos específicos de células iNKT estão alteradas nas pacientes com endometriose profunda em pacientes com dismenorréia e dor acíclica severas. As fases do ciclo menstrual estão relacionadas a alteração nas frequências das células iNKT e dos subtipos iNKT CD4+ CD25+, iNKT DN, iNKT DN IL17+ e iNKT DN CD25+ nas pacientes com endometriose profunda. Estes resultados sugerem participação das células iNKT no desenvolvimento da endometriose / Introduction: Endometriosis is a disease with inflammatory characteristics that affects women of reproductive age. The pathogenesis of endometriosis is unclear. There has been an association between immune disorders and endometriosis, such as changes in macrophages, NK cells, cytokines, and Th1, Th2 and Th17 responses. iNKT cells, a special type of T lymphocytes, play an important role in the inflammatory response as mediators of the Th1, Th2 and Th17 responses. Objectives: The main objective of this study was to compare the frequencies of iNKT cells and their subtypes between patients with endometriosis and patients without endometriosis. We also compare the frequencies of these cells in both groups relating to some clinical and surgical aspects of the disease. Methods: We performed a prospective cross-sectional study between February 2013 and February 2015, which evaluated the percentage of iNKT cells and iNKT CD4+, iNKT CD4+ CCR7+, iNKT CD4+ CD25+, iNKT DN, iNKT DN CCR7+, iNKT DN CD25+, iNKT CD4+ IL6+ , iNKT CD8+ IL17+, iNKT DN IL6+, iNKT DN IL10+, iNKT DN IL17+ in the peripheral blood, by flow cytometry, in patients with deep endometriosis (n = 47), iNKT CD4+ IL10+, iNKT CD4+ IL17+, iNKT CD8+ ) and without endometriosis (n = 26). The frequencies of iNKT cells and their subtypes were compared between groups according to symptoms, stage of the cycle, stage of the disease and histological classification. Results: iNKT, iNKT DN and iNKT DN IL17+ cells showed significant decrease in patients with endometriosis (p = 0.010, p = 0.020, p = 0.050, respectively). In addition, a significant decrease in iNKT CD4+ CCR7+ cell numbers and a significant increase of iNKT CD4+ IL17+ cells were observed in patients with endometriosis and severe dysmenorrhea compared to absent / mild dysmenorrhea. In patients with endometriosis and severe acyclic pain, there was a significant decrease in the frequency of iNKT CD4+ IL17+ cells compared to absent / mild acyclic pain (p = 0.048). There was a decrease in iNKT cells in patients with endometriosis compared to the control group in the secretory phase of the menstrual cycle (p = 0.030). In the menstrual cycle, a significant increase in iNKT CD4+ CD25+ cells (p = 0.022) and a significant decrease in iNKT DN cells (p = 0.011) was observed in the proliferative phase in patients with endometriosis. In the secretory phase there was a significant decrease in the frequency of iNKT DN IL17 + cells (p = 0.049) in patients with endometriosis. It was also identified in patients with endometriosis a decrease in the frequency of iNKT DN CD25+ cells in the secretory phase in relation to the proliferative phase of the menstrual cycle. Conclusions: iNKT cells and subtypes iNKT DN e iNKT DN IL17+ have been altered in patients with deep endometriosis. Specific subtypes of iNKT cells are altered in patients with deep endometriosis in patients with severe dysmenorrhea and acyclic pain. The phases of the menstrual cycle are related to changes in the frequencies of iNKT cells and subtypes iNKT CD4+ CD25+, iNKT DN, iNKT DN IL17+ and iNKT DN CD25+ in patients with deep endometriosis. These results suggest the participation of iNKT cells in the development of endometriosis

Introdução: A endometriose é uma doença com características inflamatórias que atinge as mulheres em idade reprodutiva. A patogênese da endometriose não está esclarecida. Tem sido demonstrada associação entre distúrbios imunológicos e endometriose, como alterações nos macrófagos, células NK, citocinas e nas repostas Th1, Th2 e Th17. As células iNKT, um tipo especial de linfócitos T, tem importante papel na resposta inflamatória como mediadores das repostas Th1, Th2 e Th17. Objetivos: o objetivo principal deste estudo foi comparar as frequências das células iNKT e seus subtipos entre pacientes com endometriose e pacientes sem endometriose. Procuramos também comparar as frequências destas células em ambos os grupos relacionando com alguns os aspectos clínicos e cirúrgicos da doença. Métodos: Realizamos estudo transversal, prospectivo entre fevereiro de 2013 a fevereiro de 2015 que avaliou a porcentagem de células iNKT e os subtipos iNKT CD4+, iNKT CD4+ CCR7+, iNKT CD4+ CD25+, iNKT DN, iNKT DN CCR7+, iNKT DN CD25+, iNKT CD4+ IL6+, iNKT CD4+ IL10+, iNKT CD4+ IL17+, iNKT CD8+ IL6+, iNKT CD8+ IL10+, iNKT CD8+ IL17+, iNKT DN IL6+, iNKT DN IL10+, iNKT DN IL17+ no sangue periférico, por citometria de fluxo, em pacientes com endometriose profunda (n = 47) e sem endometriose (n = 26). As frequências de células iNKT e seus subtipos foram comparadas entre os grupos de acordo com os sintomas, fase do ciclo, estádio da doença e classificação histológica. Resultados: Na avaliação da frequência das células iNKT, iNKT DN e iNKT DN IL17+ foi evidenciada diminuição significativa nas pacientes com endometriose (p=0,010, p=0,020, p=0,050; respectivamente). Além disso, foi observada diminuição significativa nas frequências das células iNKT CD4+ CCR7+ e aumento significativo das células iNKT CD4+ IL-17+ em pacientes com endometriose e dismenorréia severa em comparação a dismenorréia ausente/leve. Nas pacientes com endometriose e dor acíclica severa observou-se diminuição significativa da frequência das células iNKT CD4+ IL17+ em comparação a dor acíclica ausente/leve (p=0,048). Houve diminuição das células iNKT nas pacientes com endometriose em relação ao grupo controle na fase secretora do ciclo menstrual (p=0,030). Na avaliação da fase do ciclo menstrual foi observado na fase proliferativa aumento significativo na frequência das células iNKT CD4+ CD25+ (p=0,022) e diminuição significativa das células iNKT DN (p=0,011) nas pacientes com endometriose. Na fase secretora foi evidenciado diminuição significativa na frequência das células iNKT DN IL17+ (p=0,049) nas pacientes com endometriose. Foi identificado também nas pacientes com endometriose uma diminuição na frequência das células iNKT DN CD25+ na fase secretora em relação a fase proliferativa do ciclo menstrual. Conclusões: As células iNKT e os subtipos iNKT DN e iNKT DN IL17+ se mostraram alteradas nas pacientes com endometriose profunda. Subtipos específicos de células iNKT estão alteradas nas pacientes com endometriose profunda em pacientes com dismenorréia e dor acíclica severas. As fases do ciclo menstrual estão relacionadas a alteração nas frequências das células iNKT e dos subtipos iNKT CD4+ CD25+, iNKT DN, iNKT DN IL17+ e iNKT DN CD25+ nas pacientes com endometriose profunda. Estes resultados sugerem participação das células iNKT no desenvolvimento da endometriose / Introduction: Endometriosis is a disease with inflammatory characteristics that affects women of reproductive age. The pathogenesis of endometriosis is unclear. There has been an association between immune disorders and endometriosis, such as changes in macrophages, NK cells, cytokines, and Th1, Th2 and Th17 responses. iNKT cells, a special type of T lymphocytes, play an important role in the inflammatory response as mediators of the Th1, Th2 and Th17 responses. Objectives: The main objective of this study was to compare the frequencies of iNKT cells and their subtypes between patients with endometriosis and patients without endometriosis. We also compare the frequencies of these cells in both groups relating to some clinical and surgical aspects of the disease. Methods: We performed a prospective cross-sectional study between February 2013 and February 2015, which evaluated the percentage of iNKT cells and iNKT CD4+, iNKT CD4+ CCR7+, iNKT CD4+ CD25+, iNKT DN, iNKT DN CCR7+, iNKT DN CD25+, iNKT CD4+ IL6+ , iNKT CD8+ IL17+, iNKT DN IL6+, iNKT DN IL10+, iNKT DN IL17+ in the peripheral blood, by flow cytometry, in patients with deep endometriosis (n = 47), iNKT CD4+ IL10+, iNKT CD4+ IL17+, iNKT CD8+ ) and without endometriosis (n = 26). The frequencies of iNKT cells and their subtypes were compared between groups according to symptoms, stage of the cycle, stage of the disease and histological classification. Results: iNKT, iNKT DN and iNKT DN IL17+ cells showed significant decrease in patients with endometriosis (p = 0.010, p = 0.020, p = 0.050, respectively). In addition, a significant decrease in iNKT CD4+ CCR7+ cell numbers and a significant increase of iNKT CD4+ IL17+ cells were observed in patients with endometriosis and severe dysmenorrhea compared to absent / mild dysmenorrhea. In patients with endometriosis and severe acyclic pain, there was a significant decrease in the frequency of iNKT CD4+ IL17+ cells compared to absent / mild acyclic pain (p = 0.048). There was a decrease in iNKT cells in patients with endometriosis compared to the control group in the secretory phase of the menstrual cycle (p = 0.030). In the menstrual cycle, a significant increase in iNKT CD4+ CD25+ cells (p = 0.022) and a significant decrease in iNKT DN cells (p = 0.011) was observed in the proliferative phase in patients with endometriosis. In the secretory phase there was a significant decrease in the frequency of iNKT DN IL17 + cells (p = 0.049) in patients with endometriosis. It was also identified in patients with endometriosis a decrease in the frequency of iNKT DN CD25+ cells in the secretory phase in relation to the proliferative phase of the menstrual cycle. Conclusions: iNKT cells and subtypes iNKT DN e iNKT DN IL17+ have been altered in patients with deep endometriosis. Specific subtypes of iNKT cells are altered in patients with deep endometriosis in patients with severe dysmenorrhea and acyclic pain. The phases of the menstrual cycle are related to changes in the frequencies of iNKT cells and subtypes iNKT CD4+ CD25+, iNKT DN, iNKT DN IL17+ and iNKT DN CD25+ in patients with deep endometriosis. These results suggest the participation of iNKT cells in the development of endometriosis

Les lymphomes T périphériques (PTCL) sont des néoplasmes rares et agressifs représentant environ 12% des lymphomes chez l’Homme. Nos travaux récents dans des souris p53-/- ont révélé une nouvelle entité de PTCL, émergeant de cellules Natural-Killer T-cell (NKT), un type particulier de lymphocyte T reconnaissant des antigènes lipidiques. Nous avons montré que ces lymphomes NKT (PTCL-NKT) présentent des caractéristiques de NKT stimulés chroniquement, et que la lymphomagenèse est initiée via l’activation chronique du TCR. Chez l’Homme, de nombreux PTCL sont suspectés pour être associés à des stimulations antigéniques chroniques, mais les mécanismes de transformation impliqués sont encore mal connus. Borrelia burgdorferi (Bb), l’agent responsable de la maladie de Lyme, provoque des infections chroniques dont l’implication dans certains lymphomes T cutanés (CTCL) a été suggérée. Cependant, cette observation manque de preuves cliniques et expérimentales. De manière intéressante, Bb est connue pour exprimer des glycolipides activateurs des NKT. Nous avons donc infecté des souris p53-/- avec des Bb vivantes, et montré que l’infection augmente significativement la fréquence des PTCL-NKT. Par traitement antibiotique précoce de souris infectées et par injections de Bb inactivées, nous avons également démontré que la chronicité de l’infection est nécessaire au développement de ces lymphomes. L’analyse phénotypique de ces PTCL-NKT a confirmé nos observations précédentes, montrant des caractéristiques de cellules NKT activées chroniquement, telles que l’expression de marqueurs d’activation et d’exhaustion (perte de NK1.1, surexpression de PD-1). Ces résultats suggèrent une implication de Borrelia dans la lymphomagenèse T. En se basant sur l’analyse de différents marqueurs phénotypiques et de leur production cytokinique, nous avons également montré que ces lymphomes présentent un profil dérégulé se rapprochant du sous-type NKT2. Une étude génomique par séquençage whole-exome sur 6 PTCL-NKT a révélé de larges pertes récurrentes du chromosome 13. Au sein de la zone minimale de délétion, nous avons identifié Jarid2, codant un facteur épigénétique impliqué dans le développement NKT par une activité histone-methytransférase. Ce gène est retrouvé altéré dans 20% des CTCL. De manière intéressante, les souris Jarid2-/- présentent une expansion périphérique de NKT au profil immature/NKT2, partageant donc des caractéristiques avec les PTCL-NKT. La perte de Jarid2 a été détectée dans presque tous les PTCL-NKT. Nous avons confirmé la perte de Jarid2 au niveau ARN et protéique. Nos résultats préliminaires montrent une hypométhylation de la lysine 9 de l’histone H3 (H3K9), la cible de Jarid2, soutenant un effet fonctionnel dans la physiopathologie des PTCL-NKT. Par conséquent, nous pensons que la perte de Jarid2 pourrait être un événement important de la lymphomagenèse NKT, puisque de plus en plus d’altérations de facteurs épigénétiques sont retrouvées dans les PTCL humains. Pour réponse à cette question, nous sommes notamment en train de générer des souris p53-/- x Jarid2-/-. En conclusion, nos données viennent renforcer le concept selon lequel certaines infections peuvent initier la transformation des cellules T par l’activation chronique du TCR. Nous avons également identifié un nouveau facteur épigénétique potentiellement impliqué dans la lymphomagenèse NKT / Peripheral T-cell lymphomas (PTCL) are aggressive and heterogeneous neoplasms that represent around 12% of Human lymphomas. Our recent work in p53-/- mice revealed a new PTCL entity, arising from Natural-Killer T-cell (NKT), a particular type of T cell recognizing lipidic antigens. We found that NKT lymphomas (NKTL) present features of chronically stimulated NKT-cells and that lymphomagenesis is driven through chronic TCR activation by microbial glycolipids. In human, many PTCL are suspected to be associated with chronic antigenic stimulation, but this transformation mechanism is still poorly understood.Borrelia burgdorferi (Bb), the causative agent of Lyme disease, induces chronic infection and has recently been suggested to be involved in cutaneous T-cell lymphomas (CTCL). However, this observation lacks clinical and experimental proofs. Interestingly, Bb is known to express NKT-activating glycolipids. We therefore infected p53-/- mice by live intradermal Bb injection and showed that Bb infection significantly increased NKTL rate. Phenotypic characterization of these NKTL confirmed our previously described features of chronically stimulated NKT-cells, with expression of activation and exhaustion markers (loss of NK1.1, upregulation of PD-1). Based on surface markers, transcription factors and cytokine production analysis, we also found that our lymphomas mostly present a NKT2 subtype profile, sometimes surprisingly mixed with NKT17 or NKT1. Genomic study by whole-exome sequencing on few of these lymphomas revealed recurrent large losses in the chromosome 13. Within the minimal deletion region, we identified Jarid2, a gene involved in NKT development by epigenetic regulation and which is found altered in 20% of CTCL. Jarid2 loss was detected in almost all NKTL. Interestingly, Jarid2-/- mice show increased NKT number in the periphery with an immature/NKT2 phenotype, sharing features with our NKTL.Thus, we believe that Jarid2 loss may be an important event in NKT lymphomagenesis, as more and more epigenetic factors are found mutated in several human PTCL. To answer this question we are currently breeding p53-/- x Jarid2-/- mice. In conclusion, our data reinforced the concept that chronic bacterial activation of T-cells through their TCR can effectively drive T-cell transformation. We also identified a new potential epigenetic factor that may be involved in lymphomagenesis