published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

Thesis (Ph. D.)--University of Nevada, Reno, 2008. / "May 2008." Includes bibliographical references. Online version available on the World Wide Web.

Regulatory T cells (Tregs), lymphocytes that suppress immunological reactions, are of great interest for our comprehension of basic immunology and as a therapeutic agent to treat immune-mediated pathologies. Understanding the physiology of these cells will help to inform clinical strategies targeting Tregs. In order to study the homing of human Tregs, we utilised genetic engineering to drive expression of fluorescent protein in human Tregs, permitting in vivo cell tracking. We optimised a protocol for lentivirus-mediated transduction of human Tregs during in vitro expansion, to generate high yields of stably-engineered cells. After infusing labelled cells into a humanised mouse model of skin allotransplantation, we detected human Tregs within a human skin graft by PCR and visualised Tregs moving in the graft, in a live mouse, by two-photon microscopy. Through reverse genetic analyses, we explored molecular mechanisms that allow Tregs to respond adaptively to environmental cues. Neuropilin-1 (NRP1), a transmembrane co-receptor, has been implicated in the function of mouse Tregs. Tregs transduced with shRNA to knock down NRP1 were severely impaired in their capacity to suppress cell proliferation in vitro and to prolong allograft survival in a humanised mouse model. qRT-PCR analysis revealed that transcription the gene encoding the anti-inflammatory cytokine IL-10, and the autophagy-associated genes BECN1, COPS4 and MAP1LC3B, was significantly diminished in NRP1-deficient Tregs. We concluded that in human Tregs, NRP1 is necessary for suppressive function, most likely via regulation of NRP1-dependent regulation of cytokine production and metabolism. Having identified a molecular target via which Treg function might be potentiated, we explored methods to target such molecules for cell therapy applications. Tregs engineered to over-express IL-10, but not NRP1, exerted significantly enhanced suppression of cell proliferation in vitro. Thus, relatively straightforward genetic engineering, compatible with generation of therapeutic cell yields, could be exploited to improve the efficacy of Treg cellular therapy.

Call number: LD2668 .T4 1985 C66 / Master of Science

published_or_final_version / abstract / Surgery / Doctoral / Doctor of Philosophy

A leptina é um mediador tanto de respostas neuroendócrinas como imunes, e tem sido associada a diversas autoimunidades. O nosso objetivo foi estudar a importância da leptina na modulação da resposta imunológica no transplante experimental, com ênfase em seu papel na plasticidade de linfócitos T e de células dendríticas (DC). Observamos que os animais deficientes em leptina têm uma sobrevida aumentada do enxerto de pele e uma menor frequência de células Th1 e maior T reguladoras (Treg), Th2 e Th17. Ademais, células T CD4+ naive diferenciam-se mais eficientemente em células Treg e Th17 tanto com DC como sem DC, na ausência de leptina. Nossos dados indicam que BMDC, imaturas e maduras, é comprometida na ausência de leptina, induzindo menor proliferação de linfócitos CD4+ e maior geração/expansão de células Treg, Th17 e Th2. Assim, a ausência de leptina resultou num predomínio de células Treg um padrão Th2 que, em conjunto com o perfil tolerogênico das DC, poderiam ser um dos mecanismos responsáveis pelo aumento da sobrevida do enxerto alogenêico de pele. / Leptin is a mediator of both neuroendocrine and immune responses, and has been associated with several autoimmune diseases. Our objective was to study the importance of leptin in modulating the immune response in experimental transplantation, with emphasis on its role in the plasticity of T lymphocytes and dendritic cells (DC). We observed that animals leptin deficient had an increased skin graft survival and lower frequency of Th1 and increased regulatory (Treg), Th2 and Th17 T cells. Furthermore, naive CD4+ T cells differentiate more efficiently in Treg and Th17 cells, with DC and without DC, in the absence of leptin. Our data indicate that BMDC, mature and immature, is compromised in the absence of leptin, leading to less proliferation of CD4+ and increased generation / expansion of Treg cells, Th2 and Th17. Thus, the lack of leptin resulted in a predominance of Th2 and Treg T cells, which together with the profile of tolerogenic DC could be one of the mechanisms responsible for increased survival of allograft skin.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 90-112).

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

A leptina é um mediador tanto de respostas neuroendócrinas como imunes, e tem sido associada a diversas autoimunidades. O nosso objetivo foi estudar a importância da leptina na modulação da resposta imunológica no transplante experimental, com ênfase em seu papel na plasticidade de linfócitos T e de células dendríticas (DC). Observamos que os animais deficientes em leptina têm uma sobrevida aumentada do enxerto de pele e uma menor frequência de células Th1 e maior T reguladoras (Treg), Th2 e Th17. Ademais, células T CD4+ naive diferenciam-se mais eficientemente em células Treg e Th17 tanto com DC como sem DC, na ausência de leptina. Nossos dados indicam que BMDC, imaturas e maduras, é comprometida na ausência de leptina, induzindo menor proliferação de linfócitos CD4+ e maior geração/expansão de células Treg, Th17 e Th2. Assim, a ausência de leptina resultou num predomínio de células Treg um padrão Th2 que, em conjunto com o perfil tolerogênico das DC, poderiam ser um dos mecanismos responsáveis pelo aumento da sobrevida do enxerto alogenêico de pele. / Leptin is a mediator of both neuroendocrine and immune responses, and has been associated with several autoimmune diseases. Our objective was to study the importance of leptin in modulating the immune response in experimental transplantation, with emphasis on its role in the plasticity of T lymphocytes and dendritic cells (DC). We observed that animals leptin deficient had an increased skin graft survival and lower frequency of Th1 and increased regulatory (Treg), Th2 and Th17 T cells. Furthermore, naive CD4+ T cells differentiate more efficiently in Treg and Th17 cells, with DC and without DC, in the absence of leptin. Our data indicate that BMDC, mature and immature, is compromised in the absence of leptin, leading to less proliferation of CD4+ and increased generation / expansion of Treg cells, Th2 and Th17. Thus, the lack of leptin resulted in a predominance of Th2 and Treg T cells, which together with the profile of tolerogenic DC could be one of the mechanisms responsible for increased survival of allograft skin.

A inflamação é um evento intrínseco ao transplante que é desencadeado após o dano causado pela cirurgia. No presente trabalho realizou-se a caracterização fenotípica e funcional das células inflamatórias presentes no enxerto, após o transplante alogênico ou singênico de pele em camundongos. Os resultados obtidos mostraram diferenças significativas na produção de citocinas pró e anti-inflamatórias entre o transplante alogênico e singênico, diferenças já detectáveis nas primeiras 24 horas pós-transplante. Mostrou-se que existe produção aumentada de IL-10 no transplante singênico em relação ao transplante alogênico indicando que a produção de IL-10 no enxerto possui um importante papel para o aceite de transplantes de pele. Além disso, na ausência de IL-10, a rejeição de enxertos alogênicos apresentou-se acelerada e surpreendentemente uma grande parcela dos enxertos singênicos não foi aceita e apresentava aspecto de fibrose com grande deposição de colágeno. O conjunto dos dados indica que a IL-10 possui um importante papel regulatório na inflamação local do enxerto. / Inflammation is an intrinsic event of transplantation that occurs due to to damage caused by surgery. In this study, phenotypic and functional characterization of inflammatory cells present in the graft was performed, after allogeneic or syngeneic skin transplantation in mice. Our results show significant differences in the production of pro and anti-inflammatory cytokines between the syngeneic and allogeneic grafts, detectable as early as 24 hours after transplantation. Higher production of IL-10 was shown in the syngeneic grafts in comparison to allogeneic grafts, indicating that production of IL-10 in the graft is important for acceptance. This is reinforced by data that shows that in the absence of IL-10, rejection of allografts is accelerated and that, surprisingly, a high percentage of the syngeneic grafts is not accepted as well, or shows fibrosis with high deposition of collagen. Taken together, this data indicates that IL-10 has an important regulatory function in the local inflammation of the graft.