Both CD200 and its receptor(s), CD200R(s), are type I membrane glycoproteins belonging to the immunoglobulin (Ig) supergene family. CD200:CD200R(s) interaction manipulates host immunity in multiple models, including those exploring allograft rejection, autoimmune disease, tumor development, spontaneous fetal loss, infection/inflammation, and virus infection. The studies described in this thesis were focused on investigation possible mechanism(s) involved in CD200-mediated regulation, using transgenic mice over-expressing CD200, and exploring models of skin allograft rejection and LPS-induced abortion in mice. A Tet-on system was chosen to create CD200tg mice (rtTA CD200tg animal line), in which transgenic expression of CD200 is induced by the presence of doxycycline (Dox-treated mice). Splenocytes from Dox-treated transgenic mice, used as either responder cells or stimulator cells in mixed leukocyte cultures, showed antigen-specific suppressed lymphocyte proliferation and induction of CTL. Although enhanced survival of skin allografts was achieved in Dox-treated transplanted CD200tg mice (BALB/c to BL/6 Tg), all grafts were rejected by 28 days post transplantation (see chapter 2). A superior “second generation” Tet-on system was used to create a new CD200tg animal referred to as rtTA2s-M2 CD200tg mice. Transgenic overexpression of CD200 in this mouse was stably induced at much lower Dox concentrations, with less (or no) “leaky” expression of the transgene in the absence of Dox. Using these mice in an LPS-induced murine abortion model, transgenic expression of CD200 was found to reduce the LPS-induced abortion rate from ~49% to 6% (see chapter 3). Long term increased survival of grafted tissues (of both cardiac and skin allografts) was achieved using the rtTA2s-M2 CD200tg mice as recipients. To explore the potential molecular mechanism(s) involved in this allograft tolerance, a commercial microarray kit focusing on detecting altered expression of genes related with T-cell anergy/tolerance was used to investigate the gene expression profile in grafted tissue of mice with transgenic expression of CD200. Expression of genes associated with Foxp3+ regulatory T-populations (Foxp3, CTLA4 and GITR) and type 2 cytokine genes showed increased expression in CD200tg recipients. With particular note in regards to Foxp3+ regulatory T cells, expression of the gene encoding chemokine receptor CCR4, reported to play a key role in attracting Foxp3+ regulatory T cells to grafted tissues and DLNs, was found to be increased in Dox-treated CD200tg recipients, along with genes encoding chemokines CCL22/17, the ligands for CCR4. Immunochemistry staining also showed increased numbers of Foxp3+ cells in both grafted skin tissues and the DLNs of transplant at day 14 post transplantation. Using CCR4-shRNA lentivirus administered to Dox-treated CD200tg recipients to block expression of CCR4, the transgene-induced increased presence of regulatory T cell populations in grafted tissues and DLNs was attenuated, along with loss of enhanced skin graft survival and the histological appearance of graft acceptance (see chapter 4). These data provide support for a model suggesting that altered migration of Treg mediated through a CCR4:CCL17/22 pathway is an important mechanism underlying increased allograft acceptance following CD200tg expression.

Both CD200 and its receptor(s), CD200R(s), are type I membrane glycoproteins belonging to the immunoglobulin (Ig) supergene family. CD200:CD200R(s) interaction manipulates host immunity in multiple models, including those exploring allograft rejection, autoimmune disease, tumor development, spontaneous fetal loss, infection/inflammation, and virus infection. The studies described in this thesis were focused on investigation possible mechanism(s) involved in CD200-mediated regulation, using transgenic mice over-expressing CD200, and exploring models of skin allograft rejection and LPS-induced abortion in mice. A Tet-on system was chosen to create CD200tg mice (rtTA CD200tg animal line), in which transgenic expression of CD200 is induced by the presence of doxycycline (Dox-treated mice). Splenocytes from Dox-treated transgenic mice, used as either responder cells or stimulator cells in mixed leukocyte cultures, showed antigen-specific suppressed lymphocyte proliferation and induction of CTL. Although enhanced survival of skin allografts was achieved in Dox-treated transplanted CD200tg mice (BALB/c to BL/6 Tg), all grafts were rejected by 28 days post transplantation (see chapter 2). A superior “second generation” Tet-on system was used to create a new CD200tg animal referred to as rtTA2s-M2 CD200tg mice. Transgenic overexpression of CD200 in this mouse was stably induced at much lower Dox concentrations, with less (or no) “leaky” expression of the transgene in the absence of Dox. Using these mice in an LPS-induced murine abortion model, transgenic expression of CD200 was found to reduce the LPS-induced abortion rate from ~49% to 6% (see chapter 3). Long term increased survival of grafted tissues (of both cardiac and skin allografts) was achieved using the rtTA2s-M2 CD200tg mice as recipients. To explore the potential molecular mechanism(s) involved in this allograft tolerance, a commercial microarray kit focusing on detecting altered expression of genes related with T-cell anergy/tolerance was used to investigate the gene expression profile in grafted tissue of mice with transgenic expression of CD200. Expression of genes associated with Foxp3+ regulatory T-populations (Foxp3, CTLA4 and GITR) and type 2 cytokine genes showed increased expression in CD200tg recipients. With particular note in regards to Foxp3+ regulatory T cells, expression of the gene encoding chemokine receptor CCR4, reported to play a key role in attracting Foxp3+ regulatory T cells to grafted tissues and DLNs, was found to be increased in Dox-treated CD200tg recipients, along with genes encoding chemokines CCL22/17, the ligands for CCR4. Immunochemistry staining also showed increased numbers of Foxp3+ cells in both grafted skin tissues and the DLNs of transplant at day 14 post transplantation. Using CCR4-shRNA lentivirus administered to Dox-treated CD200tg recipients to block expression of CCR4, the transgene-induced increased presence of regulatory T cell populations in grafted tissues and DLNs was attenuated, along with loss of enhanced skin graft survival and the histological appearance of graft acceptance (see chapter 4). These data provide support for a model suggesting that altered migration of Treg mediated through a CCR4:CCL17/22 pathway is an important mechanism underlying increased allograft acceptance following CD200tg expression.

The ability of cancer cells to escape anti-tumor immune responses is acknowledged as one of the hallmarks of cancer. Overexpression of immunoregulatory molecules is one mechanism responsible for the immunsuppressive network that is characteristic of the tumor microenvironment. In this thesis, we investigated the role of CD200, a potent immunoregulatory molecule, in Chronic Lymphocytic Leukemia. We showed that functional blockade of CD200 on lymphoma cells or primary CLL cells, both of which express CD200 at high levels, augmented cytotoxic killing of these cells by effector CD8+ T cells in vitro. We also identified and characterized a previously unrecognized soluble form of CD200, sCD200, present in elevated levels in CLL plasma when compared to plasma from controls. The data reported show that patients with high sCD200 levels have more aggressive disease, inferring that sCD200 may be a novel prognostic marker for CLL. The in vivo function of sCD200 was investigated for its ability to support engraftment of CLL splenocytes in NOD.SCID mice. Infusion of sCD200hi CLL plasma, but not sCD200lo normal plasma, enhanced engraftment of CLL-splenocytes in vivo, an effect which was abrogated by depletion of sCD200 from CLL plasma. The prolonged engraftment of CLL cells seen in this model (>6 months) suggests these mice represent a useful pre-clinical model for drug screening. The effect of CD200 blockade was tested in this model, and was found to be as effective in eliminating engrafted CLL cells as rituximab. Investigation of the mechanisms leading to the release of sCD200 from CLL cells showed that sCD200 was produced following ectodomain shedding by ADAM proteases and MMPs. Results from studies reported in this thesis support the hypothesis that CD200 plays a major role in CLL biology, and suggests it may represent a novel therapeutic target for CLL.

The ability of cancer cells to escape anti-tumor immune responses is acknowledged as one of the hallmarks of cancer. Overexpression of immunoregulatory molecules is one mechanism responsible for the immunsuppressive network that is characteristic of the tumor microenvironment. In this thesis, we investigated the role of CD200, a potent immunoregulatory molecule, in Chronic Lymphocytic Leukemia. We showed that functional blockade of CD200 on lymphoma cells or primary CLL cells, both of which express CD200 at high levels, augmented cytotoxic killing of these cells by effector CD8+ T cells in vitro. We also identified and characterized a previously unrecognized soluble form of CD200, sCD200, present in elevated levels in CLL plasma when compared to plasma from controls. The data reported show that patients with high sCD200 levels have more aggressive disease, inferring that sCD200 may be a novel prognostic marker for CLL. The in vivo function of sCD200 was investigated for its ability to support engraftment of CLL splenocytes in NOD.SCID mice. Infusion of sCD200hi CLL plasma, but not sCD200lo normal plasma, enhanced engraftment of CLL-splenocytes in vivo, an effect which was abrogated by depletion of sCD200 from CLL plasma. The prolonged engraftment of CLL cells seen in this model (>6 months) suggests these mice represent a useful pre-clinical model for drug screening. The effect of CD200 blockade was tested in this model, and was found to be as effective in eliminating engrafted CLL cells as rituximab. Investigation of the mechanisms leading to the release of sCD200 from CLL cells showed that sCD200 was produced following ectodomain shedding by ADAM proteases and MMPs. Results from studies reported in this thesis support the hypothesis that CD200 plays a major role in CLL biology, and suggests it may represent a novel therapeutic target for CLL.

A L. (L.) amazonensis é capaz de evadir a resposta imune do macrófago hospedeiro induzindo a expressão de CD200 na célula. Porém, ainda não se sabe como ocorre este mecanismo. O objetivo deste trabalho foi avaliar a participação dos TLRs na indução de CD200 em macrófagos infectados por L. (L.) amazonensis. Os resultados mostraram que a indução de CD200 por L. (L.) amazonensis é dependente de TLR9 e das proteínas adaptadoras MyD88 e TRIF. Além disso, observamos que CD200 pode ser induzida pelo DNA do parasito, assim como por vesículas extracelulares (VEs) contendo DNA liberadas por ele. Os resultados in vivo mostraram que a ausência de TLR9 não altera o tamanho da lesão e nem a expressão de CD200 nos macrófagos presentes. Contudo, a carga parasitária foi maior nos camundongos selvagens. A partir dos resultados obtidos podemos concluir que a L. (L.) amazonensis induz CD200 de maneira dependente da via de TLRs e que esta indução pode ser estimulada pelo DNA do parasito. / L. (L.) amazonensis evades the immune response of host macrophage inducing the CD200 expression in the cell. However, it is not yet known how this mechanism occurs. The objective of this work was to evaluate the participation of TLRs in CD200 induction in infected macrophages by L. (L.) amazonensis. The results showed that the CD200 induction by the parasite is dependent on TLR9 and the adaptor proteins MyD88 and TRIF. In addition, we observed that the CD200 can be induced by the parasite DNA, as well as by extracellular vesicles (EVs) containing DNA released by it. In vivo results showed that the absence of TLR9 does not alter the lesion size nor the CD200 expression in macrophages present in the lesion. However, the parasite load was higher in wild type mice. Therefore, we can conclude that the CD200 induction by L. (L.) amazonensis amastigotes is TLR dependent and this can be stimulated by the parasite DNA.

No description available.

A leishmaniose é um termo global para doenças causadas por parasitos do gênero Leishmania, sendo a Leishmaniose Visceral (LV) a forma mais grave da doença. No Brasil é causada pelo parasita Leishmania (Leishmania) infantum chagasi. Para garantir a sua sobrevivência, alguns parasitas são capazes de manipular respostas de defesa das células do sistema imune. Recentes estudos demonstraram a participação da proteína imunomodulatória CD200 durante o processo de infecção de L. (L.) amazonenses. O presente estudo teve como objetivo investigar se os parasitos L. (L.) infantum chagasi são capazes de induzir a expressão da proteína CD200 durante o processo infeccioso. Em ensaios de infecção ex vivo, não foi observado proliferação de parasitas intracelulares. Apesar disso, L. (L.) infantum chagasi foi capaz de induzir a expressão do gene CD200. De maneira interessante, diferente de infecções por L. (L.) amazonenses, a indução de CD200 nessas células foi observada em tempos mais tardios de infecção. Ensaios de imunoprecipitação e Western blot indicaram a síntese da proteína, que atingiu os seus maiores níveis a 120 horas pós-infecção. A presença de CD200 sugere o envolvimento dessa molécula em tempos mais tardios de infecção por L. (L.) infantum chagasi. / Leishmaniasis is a global term for diseases caused by parasites of the genus Leishmania, and Visceral Leishmaniasis (VL) are the most severe form of the disease. In Brazil is caused by the parasite Leishmania (Leishmania) infantum chagasi. To ensure their survival, some parasites can handle defensive responses of the cells of the immune system. Recent studies have demonstrated the participation of immunomodulatory protein CD200 during the infection process of L. (L.) amazonenses. This study aimed to investigate whether the parasites L. (L.) infantum chagasi are capable of inducing the expression of CD200 protein during the infectious process. In trials of ex vivo infection, there was no proliferation of intracellular parasites. Nevertheless, L. (L.) infantum chagasi was able to induce the expression of CD200 gene. Interestingly, unlike infection by L. (L.) amazonenses, CD200 induction of these cells was observed at later times in infection. Immunoprecipitation assays and Western blot indicated protein synthesis, which reached their highest levels at 120 hours post-infection. The presence of CD200 suggests the involvement of this molecule at later times of infection with L. (L.) infantum chagasi.

Leishmania (Leishmania) amazonenses está associada à leishmaniose cutânea difusa (DCL) e localizada (LCL). LCL se apresenta como uma lesão única com cura espontânea e DCL apresenta nódulos não ulcerativos que se espalham pela pele. Até o momento não se conhecem os fatores que influenciam na apresentação destas manifestações. A virulência do parasito tem sido associada com a capacidade de sobreviver no macrófago ativado. CD200 é uma glicoproteína reguladora que ativa o CD200R, gerando a inibição da resposta próinflamatória do macrófago. Recentemente foi demonstrado que no processo de infecção por L. (L.) amazonenses, há indução de CD200 no começo da infecção, aumentando a supervivência do parasito. O objetivo principal foi determinar a associação da expressão de CD200 na infecção de macrófagos com isolados de L. (L.) amazonenses associados à LCL ou DCL. Observamos que o aumento da expressão da proteína nos tempos avançados de infecção está relacionado com um aumento na infectividade dos isolados e um maior número de parasitos por célula infectada. / Leishmania (Leishmania) amazonenses is one of the etiologic agents of two forms of leishmaniasis: diffuse (DCL) and localized (LCL) cutaneous leishmaniasis. LCL is presents a unique ulcerated lesion and DCL multiple non-ulcerative nodules. Until now, it is not well known which factors influence the development of LCL or DCL. The parasites virulence has been associated with the ability to survive inside the activated macrophage. CD200 is a glycoprotein that interacts with CD200R inhibiting the proinflammatory response of the cell. Recently it was revealed during the infection process in macrophages L. (L.) amazonenses enhances CD200 expression in the early stages of infection resulting in the intracellular survival of amastigotes. The aim of this study was to determine the association between the induction of CD200 during infection with L. (L.) amazonenses isolates associated with LCL or DCL. An increase in CD200 expression was noteworthy in the late stages of infection, which was also related to a higher infectivity rate of L. (L.) amazonenses isolates.

Leishmania (Leishmania) amazonenses está associada à leishmaniose cutânea difusa (DCL) e localizada (LCL). LCL se apresenta como uma lesão única com cura espontânea e DCL apresenta nódulos não ulcerativos que se espalham pela pele. Até o momento não se conhecem os fatores que influenciam na apresentação destas manifestações. A virulência do parasito tem sido associada com a capacidade de sobreviver no macrófago ativado. CD200 é uma glicoproteína reguladora que ativa o CD200R, gerando a inibição da resposta próinflamatória do macrófago. Recentemente foi demonstrado que no processo de infecção por L. (L.) amazonenses, há indução de CD200 no começo da infecção, aumentando a supervivência do parasito. O objetivo principal foi determinar a associação da expressão de CD200 na infecção de macrófagos com isolados de L. (L.) amazonenses associados à LCL ou DCL. Observamos que o aumento da expressão da proteína nos tempos avançados de infecção está relacionado com um aumento na infectividade dos isolados e um maior número de parasitos por célula infectada. / Leishmania (Leishmania) amazonenses is one of the etiologic agents of two forms of leishmaniasis: diffuse (DCL) and localized (LCL) cutaneous leishmaniasis. LCL is presents a unique ulcerated lesion and DCL multiple non-ulcerative nodules. Until now, it is not well known which factors influence the development of LCL or DCL. The parasites virulence has been associated with the ability to survive inside the activated macrophage. CD200 is a glycoprotein that interacts with CD200R inhibiting the proinflammatory response of the cell. Recently it was revealed during the infection process in macrophages L. (L.) amazonenses enhances CD200 expression in the early stages of infection resulting in the intracellular survival of amastigotes. The aim of this study was to determine the association between the induction of CD200 during infection with L. (L.) amazonenses isolates associated with LCL or DCL. An increase in CD200 expression was noteworthy in the late stages of infection, which was also related to a higher infectivity rate of L. (L.) amazonenses isolates.

A leishmaniose é um termo global para doenças causadas por parasitos do gênero Leishmania, sendo a Leishmaniose Visceral (LV) a forma mais grave da doença. No Brasil é causada pelo parasita Leishmania (Leishmania) infantum chagasi. Para garantir a sua sobrevivência, alguns parasitas são capazes de manipular respostas de defesa das células do sistema imune. Recentes estudos demonstraram a participação da proteína imunomodulatória CD200 durante o processo de infecção de L. (L.) amazonenses. O presente estudo teve como objetivo investigar se os parasitos L. (L.) infantum chagasi são capazes de induzir a expressão da proteína CD200 durante o processo infeccioso. Em ensaios de infecção ex vivo, não foi observado proliferação de parasitas intracelulares. Apesar disso, L. (L.) infantum chagasi foi capaz de induzir a expressão do gene CD200. De maneira interessante, diferente de infecções por L. (L.) amazonenses, a indução de CD200 nessas células foi observada em tempos mais tardios de infecção. Ensaios de imunoprecipitação e Western blot indicaram a síntese da proteína, que atingiu os seus maiores níveis a 120 horas pós-infecção. A presença de CD200 sugere o envolvimento dessa molécula em tempos mais tardios de infecção por L. (L.) infantum chagasi. / Leishmaniasis is a global term for diseases caused by parasites of the genus Leishmania, and Visceral Leishmaniasis (VL) are the most severe form of the disease. In Brazil is caused by the parasite Leishmania (Leishmania) infantum chagasi. To ensure their survival, some parasites can handle defensive responses of the cells of the immune system. Recent studies have demonstrated the participation of immunomodulatory protein CD200 during the infection process of L. (L.) amazonenses. This study aimed to investigate whether the parasites L. (L.) infantum chagasi are capable of inducing the expression of CD200 protein during the infectious process. In trials of ex vivo infection, there was no proliferation of intracellular parasites. Nevertheless, L. (L.) infantum chagasi was able to induce the expression of CD200 gene. Interestingly, unlike infection by L. (L.) amazonenses, CD200 induction of these cells was observed at later times in infection. Immunoprecipitation assays and Western blot indicated protein synthesis, which reached their highest levels at 120 hours post-infection. The presence of CD200 suggests the involvement of this molecule at later times of infection with L. (L.) infantum chagasi.