Investigation of the cell biology of human regulatory T cells in the context of transplantation
<p>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...
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| Format: | Thesis |
| Language: | English |
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2016
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| _version_ | 1826315839281823744 |
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| author | Milward, K |
| author2 | Hester, J |
| author_facet | Hester, J Milward, K |
| author_sort | Milward, K |
| collection | OXFORD |
| description | <p>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.</p> <p>In order to study the homing of human Tregs, we utilised genetic engineering to drive expression of fluorescent protein in human Tregs, permitting <em>in vivo</em> cell tracking. We optimised a protocol for lentivirus-mediated transduction of human Tregs during <em>in vitro</em> 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.</p> <p>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 <em>NRP1</em> were severely impaired in their capacity to suppress cell proliferation <em>in vitro</em> 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 <em>BECN1</em>, <em>COPS4</em> and <em>MAP1LC3B</em>, 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.</p> <p>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 <em>in vitro</em>. Thus, relatively straightforward genetic engineering, compatible with generation of therapeutic cell yields, could be exploited to improve the efficacy of Treg cellular therapy.</p> |
| first_indexed | 2024-03-06T19:31:59Z |
| format | Thesis |
| id | oxford-uuid:1dc5105f-a74c-4451-a8dd-9b37daf3c01d |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-12-09T03:33:32Z |
| publishDate | 2016 |
| record_format | dspace |
| spelling | oxford-uuid:1dc5105f-a74c-4451-a8dd-9b37daf3c01d2024-12-01T16:43:10ZInvestigation of the cell biology of human regulatory T cells in the context of transplantationThesishttp://purl.org/coar/resource_type/c_db06uuid:1dc5105f-a74c-4451-a8dd-9b37daf3c01dTransplantation immunologyEnglishORA Deposit2016Milward, KHester, JWood, K<p>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.</p> <p>In order to study the homing of human Tregs, we utilised genetic engineering to drive expression of fluorescent protein in human Tregs, permitting <em>in vivo</em> cell tracking. We optimised a protocol for lentivirus-mediated transduction of human Tregs during <em>in vitro</em> 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.</p> <p>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 <em>NRP1</em> were severely impaired in their capacity to suppress cell proliferation <em>in vitro</em> 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 <em>BECN1</em>, <em>COPS4</em> and <em>MAP1LC3B</em>, 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.</p> <p>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 <em>in vitro</em>. Thus, relatively straightforward genetic engineering, compatible with generation of therapeutic cell yields, could be exploited to improve the efficacy of Treg cellular therapy.</p> |
| spellingShingle | Transplantation immunology Milward, K Investigation of the cell biology of human regulatory T cells in the context of transplantation |
| title | Investigation of the cell biology of human regulatory T cells in the context of transplantation |
| title_full | Investigation of the cell biology of human regulatory T cells in the context of transplantation |
| title_fullStr | Investigation of the cell biology of human regulatory T cells in the context of transplantation |
| title_full_unstemmed | Investigation of the cell biology of human regulatory T cells in the context of transplantation |
| title_short | Investigation of the cell biology of human regulatory T cells in the context of transplantation |
| title_sort | investigation of the cell biology of human regulatory t cells in the context of transplantation |
| topic | Transplantation immunology |
| work_keys_str_mv | AT milwardk investigationofthecellbiologyofhumanregulatorytcellsinthecontextoftransplantation |