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18 results on '"Milward, Kate"'

1. A pseudotyped adenovirus serotype 5 vector with serotype 49 fiber knob is an effective vector for vaccine and gene therapy applications

Author

Bliss, Carly M., Hulin-Curtis, Sarah L., Williams, Marta, Marušková, Mahulena, Davies, James A., Statkute, Evelina, Baker, Alexander T., Stack, Louise, Kerstetter, Lucas, Kerr-Jones, Lauren E., Milward, Kate F., Russell, Gabrielle, George, Sarah J., Badder, Luned M., Stanton, Richard J., Coughlan, Lynda, Humphreys, Ian R., and Parker, Alan L.

Published
2024
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2. Investigation of the cell biology of human regulatory T cells in the context of transplantation

Author

Milward, Kate, Hester, Joanna, and Wood, Kathryn

Subjects
617, Transplantation immunology, transplant, graft rejection, allograft, transplantation, immunology, immunoregulation, Tregs, cell therapy, human immunology, immune, regulatory T cells, humanised mouse model
Abstract

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.

Published
2016

3. Systemic silencing of Phd2 causes reversible immune regulatory dysfunction

Author

Yamamoto, Atsushi, Hester, Joanna, Macklin, Philip S., Kawai, Kento, Uchiyama, Masateru, Biggs, Daniel, Bishop, Tammie, Bull, Katherine, Cheng, Xiaotong, Cawthorne, Eleanor, Coleman, Mathew L., Crockford, Tanya L., Davies, Ben, Dow, Lukas E., Goldin, Rob, Kranc, Kamil, Kudo, Hiromi, Lawson, Hannah, McAuliffe, James, Milward, Kate, Scudamore, Cheryl L., Soilleux, Elizabeth, Issa, Fadi, Ratcliffe, Peter J., and Pugh, Chris W.

Subjects
Thermo Fisher Scientific Inc., Tetracyclines -- Physiological aspects, B cells -- Physiological aspects, Autoimmunity -- Physiological aspects, Enzymes -- Physiological aspects, RNA -- Physiological aspects, Scientific equipment industry -- Physiological aspects, T cells -- Physiological aspects, RNA interference, Alleles, Animal genetic engineering, Phenotypes, Health care industry, University of Oxford
Abstract

Physiological effects of cellular hypoxia are sensed by prolyl hydroxylase (PHD) enzymes, which regulate HIFs. Genetic interventions on HIF/PHD pathways have revealed multiple phenotypes that extend the known biology of hypoxia. Recent studies have unexpectedly implicated HIF in aspects of multiple immune and inflammatory pathways. However, such studies are often limited by systemic lethal effects and/or use tissue- specific recombination systems, which are inherently irreversible, unphysiologically restricted, and difficult to time. To study these processes better, we developed recombinant mice that expressed tetracycline-regulated shRNAs broadly targeting the main components of the HIF/ PHD pathway, permitting timed bidirectional intervention. We show that stabilization of HIF levels in adult mice through PHD2 enzyme silencing by RNA interference or inducible recombination of floxed alleles results in multilineage leukocytosis and features of autoimmunity. This phenotype was rapidly normalized on reestablishment of the hypoxia-sensing machinery when shRNA expression was discontinued. In both situations, these effects were mediated principally through the Hif2[alpha] isoform. Assessment of cells bearing Treg markers from these mice revealed defective function and proinflammatory effects in vivo. We believe our findings reveal a new role for the PHD2/Hif2[alpha] pathway in the reversible regulation of T cell and immune activity., Introduction HIFs are transcription factors composed of 1 of 3 a chains (HIF1[alpha], Hif2[alpha], and HIF3[alpha]) dimerized to a [beta] chain that mediates the transcriptional response to low oxygen tension [...]

Published
2019
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4. Matching or genetic engineering of HLA Class I and II facilitates successful allogeneic 'off-the-shelf' regulatory T cell therapy

Author

McCallion, Oliver, primary, Du, Weijie, additional, Glaser, Viktor, additional, Milward, Kate, additional, Franke, Clemens, additional, Kath, Jonas, additional, Valkov, Mikhail, additional, Yang, Mingxing, additional, Künkele, Annette, additional, Polansky, Julia K., additional, Schmueck-Henneresse, Michael, additional, Volk, Hans-Dieter, additional, Reinke, Petra, additional, Wagner, Dimitrios L., additional, Hester, Joanna, additional, and Issa, Fadi, additional

Published
2023
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13. Systemic silencing of PHD2 causes reversible immune regulatory dysfunction

Author

Yamamoto, Atsushi, Hester, Joanna, Macklin, Philip S, Kawai, Kento, Uchiyama, Masateru, Biggs, Daniel, Bishop, Tammie, Bull, Katherine, Cheng, Xiaotong, Cawthorne, Eleanor, Coleman, Mathew L, Crockford, Tanya L, Davies, Ben, Dow, Lukas E, Goldin, Rob, Kranc, Kamil, Kudo, Hiromi, Lawson, Hannah, McAuliffe, James, Milward, Kate, Scudamore, Cheryl L, Soilleux, Elizabeth, Issa, Fadi, Ratcliffe, Peter J, and Pugh, Chris W

Subjects
hypoxia, Autoimmune diseases, FOS: Clinical medicine, Immunology, Mice, Transgenic, Therapeutics, Mouse models, T-Lymphocytes, Regulatory, 3. Good health, Hypoxia-Inducible Factor-Proline Dioxygenases, Mice, Basic Helix-Loop-Helix Transcription Factors, Animals, RNA Interference, Signal Transduction
Abstract

Physiological effects of cellular hypoxia are sensed by prolyl hydroxylase (PHD) enzymes which regulate HIFs. Genetic interventions on HIF/PHD pathways reveal multiple phenotypes that extend the known biology of hypoxia. Recent studies unexpectedly implicate HIF in aspects of multiple immune and inflammatory pathways. However such studies are often limited by systemic lethal effects and/or use tissue-specific recombination systems, which are inherently irreversible, un-physiologically restricted and difficult to time. To study these processes better we developed recombinant mice which express tetracycline-regulated shRNAs broadly targeting the main components of the HIF/PHD pathway, permitting timed bi-directional intervention. We have shown that stabilization of HIF levels in adult mice through PHD2 enzyme silencing by RNA interference, or inducible recombination of floxed alleles, results in multi-lineage leukocytosis and features of autoimmunity. This phenotype was rapidly normalized on re-establishment of the hypoxia-sensing machinery when shRNA expression was discontinued. In both situations these effects were mediated principally through the Hif2a isoform. Assessment of cells bearing regulatory T cell markers from these mice revealed defective function and pro-inflammatory effects in vivo. We believe our findings have shown a new role for the PHD2/Hif2a couple in the reversible regulation of T cell and immune activity.

14. Systemic silencing of Phd2 causes reversible immune regulatory dysfunction

Author

Yamamoto, Atsushi, Hester, Joanna, Macklin, Philip S, Kawai, Kento, Masateru Uchiyama, Biggs, Daniel, Bishop, Tammie, Bull, Katherine, Xiaotong Cheng, Cawthorne, Eleanor, Coleman, Mathew L, Crockford, Tanya L, Davies, Ben, Dow, Lukas E, Goldin, Rob, Kranc, Kamil, Kudo, Hiromi, Lawson, Hannah, McAuliffe, James, Milward, Kate, Scudamore, Cheryl L, Soilleux, Elizabeth, Issa, Fadi, Ratcliffe, Peter J, and Pugh, Chris W

Subjects
Signalling & Oncogenes, Metabolism, Cell Biology, Biochemistry & Proteomics, 3. Good health
Abstract

Physiological effects of cellular hypoxia are sensed by prolyl hydroxylase (PHD) enzymes which regulate HIFs. Genetic interventions on HIF/PHD pathways reveal multiple phenotypes that extend the known biology of hypoxia. Recent studies unexpectedly implicate HIF in aspects of multiple immune and inflammatory pathways. However such studies are often limited by systemic lethal effects and/or use tissue-specific recombination systems, which are inherently irreversible, un-physiologically restricted and difficult to time. To study these processes better we developed recombinant mice which express tetracycline-regulated shRNAs broadly targeting the main components of the HIF/PHD pathway, permitting timed bi-directional intervention. We have shown that stabilization of HIF levels in adult mice through PHD2 enzyme silencing by RNA interference, or inducible recombination of floxed alleles, results in multi-lineage leukocytosis and features of autoimmunity. This phenotype was rapidly normalized on re-establishment of the hypoxia-sensing machinery when shRNA expression was discontinued. In both situations these effects were mediated principally through the Hif2a isoform. Assessment of cells bearing regulatory T cell markers from these mice revealed defective function and pro-inflammatory effects in vivo. We believe our findings have shown a new role for the PHD2/Hif2a couple in the reversible regulation of T cell and immune activity.

15. Systemic silencing of Phd2 causes reversible immune regulatory dysfunction

Author

Yamamoto, Atsushi, Hester, Joanna, Macklin, Philip S, Kawai, Kento, Masateru Uchiyama, Biggs, Daniel, Bishop, Tammie, Bull, Katherine, Xiaotong Cheng, Cawthorne, Eleanor, Coleman, Mathew L, Crockford, Tanya L, Davies, Ben, Dow, Lukas E, Goldin, Rob, Kranc, Kamil, Kudo, Hiromi, Lawson, Hannah, McAuliffe, James, Milward, Kate, Scudamore, Cheryl L, Soilleux, Elizabeth, Issa, Fadi, Ratcliffe, Peter J, and Pugh, Chris W

Subjects
Signalling & Oncogenes, Metabolism, Cell Biology, Biochemistry & Proteomics, 3. Good health
Abstract

Physiological effects of cellular hypoxia are sensed by prolyl hydroxylase (PHD) enzymes which regulate HIFs. Genetic interventions on HIF/PHD pathways reveal multiple phenotypes that extend the known biology of hypoxia. Recent studies unexpectedly implicate HIF in aspects of multiple immune and inflammatory pathways. However such studies are often limited by systemic lethal effects and/or use tissue-specific recombination systems, which are inherently irreversible, un-physiologically restricted and difficult to time. To study these processes better we developed recombinant mice which express tetracycline-regulated shRNAs broadly targeting the main components of the HIF/PHD pathway, permitting timed bi-directional intervention. We have shown that stabilization of HIF levels in adult mice through PHD2 enzyme silencing by RNA interference, or inducible recombination of floxed alleles, results in multi-lineage leukocytosis and features of autoimmunity. This phenotype was rapidly normalized on re-establishment of the hypoxia-sensing machinery when shRNA expression was discontinued. In both situations these effects were mediated principally through the Hif2a isoform. Assessment of cells bearing regulatory T cell markers from these mice revealed defective function and pro-inflammatory effects in vivo. We believe our findings have shown a new role for the PHD2/Hif2a couple in the reversible regulation of T cell and immune activity.

16. Investigation of the cell biology of human regulatory T cells in the context of transplantation

Author

Milward, Kate and Milward, Kate

Abstract

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 gene

17. Systemic silencing of PHD2 causes reversible immune regulatory dysfunction.

Author

Atsushi Yamamoto, Hester, Joanna, Macklin, Philip S., Kawai, Kento, Masateru Uchiyama, Biggs, Daniel, Bishop, Tammie, Bull, Katherine, Xiaotong Cheng, Cawthorne, Eleanor, Coleman, Mathew L., Crockford, Tanya L., Davies, Ben, Dow, Lukas E., Goldin, Rob, Kranc, Kamil, Hiromi Kudo, Lawson, Hannah, McAuliffe, James, and Milward, Kate

Subjects
*SUPPRESSOR cells, *GENETIC engineering, *RNA interference, *CELLULAR control mechanisms, *CATALYTIC RNA
Abstract

Physiological effects of cellular hypoxia are sensed by prolyl hydroxylase (PHD) enzymes which regulate HIFs. Genetic interventions on HIF/PHD pathways reveal multiple phenotypes that extend the known biology of hypoxia. Recent studies unexpectedly implicate HIF in aspects of multiple immune and inflammatory pathways. However such studies are often limited by systemic lethal effects and/or use tissue-specific recombination systems, which are inherently irreversible, un-physiologically restricted and difficult to time. To study these processes better we developed recombinant mice which express tetracycline-regulated shRNAs broadly targeting the main components of the HIF/PHD pathway, permitting timed bi-directional intervention. We have shown that stabilization of HIF levels in adult mice through PHD2 enzyme silencing by RNA interference, or inducible recombination of floxed alleles, results in multi-lineage leukocytosis and features of autoimmunity. This phenotype was rapidly normalized on re-establishment of the hypoxia-sensing machinery when shRNA expression was discontinued. In both situations these effects were mediated principally through the Hif2a isoform. Assessment of cells bearing regulatory T cell markers from these mice revealed defective function and pro-inflammatory effects in vivo. We believe our findings have shown a new role for the PHD2/Hif2a couple in the reversible regulation of T cell and immune activity. [ABSTRACT FROM AUTHOR]

Published
2019
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18. Isolation of Human Regulatory T Lymphocytes by Fluorescence-Activated Cell Sorting.

Author

Milward K, Hester J, and Wood KJ

Subjects
CD4 Antigens genetics, Humans, Interleukin-2 Receptor alpha Subunit genetics, Interleukin-7 Receptor alpha Subunit genetics, Leukocytes, Mononuclear cytology, Lymphocyte Count, T-Lymphocytes, Regulatory immunology, Cell Separation methods, Flow Cytometry methods, T-Lymphocytes, Regulatory cytology
Abstract

Regulatory T cells (Tregs) are a population of lymphocytes that exerts suppressive effects upon the immune system. In human peripheral blood, the major population of T lymphocytes with suppressive capacity are defined by expression of the T cell co-receptor CD4 and the interleukin-2 receptor α-chain (CD25), combined with minimal expression of the interleukin-7 receptor α subunit (CD127). We begin by outlining the method for isolating peripheral blood mononuclear cells (PBMCs) from human blood by centrifugation of whole blood overlayed on a hydrophilic polysaccharide, with an additional erythrocyte lysis step. The protocol that follows utilizes Fluorescence-Activated Cell Sorting (FACS) for the isolation of this CD4 + CD25 + CD127 lo population of regulatory T cells, with high yield and purity, from immunostained PBMCs. Prior to FACS isolation, this protocol exploits magnetic immunoselection for pre-enrichment of CD25 + PBMC, which reduces the duration of the subsequent FACS isolation.

Published
2019
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