{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/329027"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/329027","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Regulation of LFA-1 on T cells by phosphoinositide signalling","abstract":"Lymphocyte Function-Associated Antigen 1 (LFA-1) is the major integrin in T cells and binds Intercellular Adhesion Molecule 1 and 2 (ICAM-1 and ICAM-2) expressed on endothelial cells and antigen presenting cells. LFA-1 affinity for ICAM is increased following chemokine and T cell receptor (TCR) engagement by inside-out signalling. This process coordinates T cell migration, adhesion, and activation of T cells. LFA-1 activation is mediated by phosphoinositide 3-kinase (PI3K) and the downstream phosphoinositide PtdIns(3,4,5)P3 (PIP3). To investigate how PI3K regulates LFA-1, I optimised CRISPR/Cas9-mediated mutagenesis in T cells, and designed a retroviral library of CRISPR/single guide RNAs targeting all known and potential PIP3-binding proteins. Using this library, I screened the targeted genes for effects on ICAM-1-binding by a flow cytometry-based ICAM-1-binding assay, using Cas9-expressing primary mouse T cells. I identified multiple proteins regulating LFA-1-mediated adhesion to ICAM-1, including the RAP1/RAS GTPase-activating protein RASA3. I found that RASA3 is a critical negative regulator of LFA-1 activation and that RASA3 is inhibited by PI3K signalling. T cells without RASA3 have greatly increased ICAM-1-binding. Mice with conditional deletion of Rasa3 in T cells have altered T cell homeostasis including increased numbers of mature thymocytes in the thymus, but decreased T cells in lymph nodes, spleen, and especially in the blood. Further, Rasa3 deletion in T cells resulted in reduced germinal centre and antigen-specific antibody responses to immunisation, likely as a result of decreased T follicular helper (TFH) cell numbers. The presented results thus describe a novel genetic screen in T cells which has uncovered a critical role for RASA3 as a PI3K-regulated inhibitor of T cell adhesion and migration that is required for T cell homeostasis and function.","abstract_html":"Lymphocyte Function-Associated Antigen 1 (LFA-1) is the major integrin in T cells and binds Intercellular Adhesion Molecule 1 and 2 (ICAM-1 and ICAM-2) expressed on endothelial cells and antigen presenting cells. LFA-1 affinity for ICAM is increased following chemokine and T cell receptor (TCR) engagement by inside-out signalling. This process coordinates T cell migration, adhesion, and activation of T cells. LFA-1 activation is mediated by phosphoinositide 3-kinase (PI3K) and the downstream phosphoinositide PtdIns(3,4,5)P3 (PIP3). To investigate how PI3K regulates LFA-1, I optimised CRISPR/Cas9-mediated mutagenesis in T cells, and designed a retroviral library of CRISPR/single guide RNAs targeting all known and potential PIP3-binding proteins. Using this library, I screened the targeted genes for effects on ICAM-1-binding by a flow cytometry-based ICAM-1-binding assay, using Cas9-expressing primary mouse T cells. I identified multiple proteins regulating LFA-1-mediated adhesion to ICAM-1, including the RAP1/RAS GTPase-activating protein RASA3. I found that RASA3 is a critical negative regulator of LFA-1 activation and that RASA3 is inhibited by PI3K signalling. T cells without RASA3 have greatly increased ICAM-1-binding. Mice with conditional deletion of Rasa3 in T cells have altered T cell homeostasis including increased numbers of mature thymocytes in the thymus, but decreased T cells in lymph nodes, spleen, and especially in the blood. Further, Rasa3 deletion in T cells resulted in reduced germinal centre and antigen-specific antibody responses to immunisation, likely as a result of decreased T follicular helper (TFH) cell numbers. The presented results thus describe a novel genetic screen in T cells which has uncovered a critical role for RASA3 as a PI3K-regulated inhibitor of T cell adhesion and migration that is required for T cell homeostasis and function.","abstract_has_math":false,"creators":["Johansen, Kristoffer"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Okkenhaug, Klaus","Schwartzberg, Pamela L"],"committee_chairs":[],"committee_members":[],"year":2021,"date_issued":"2021-06-01","date_published":"2021-06-01","updated_at":"2026-07-22T22:24:10Z","subjects":["CRISPR/Cas9","T cells","LFA-1","Phosphoinositide","TCR signalling","PI3K","Integrin","Adhesion","T cell migration","T cell adhesion"],"languages":["eng"],"rights":[],"rights_urls":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/cb82729c-9fbb-4941-a97e-36674181bf45/download","https://creativecommons.org/licenses/by/4.0/"],"identifier_entries":[{"key":"dc:creator.authoridentifier","label":"Author Identifier","values":["0000000277119451","0000000294324051"],"render_values":[{"text":"0000-0002-7711-9451","href":"https://orcid.org/0000-0002-7711-9451","code":true},{"text":"0000-0002-9432-4051","href":"https://orcid.org/0000-0002-9432-4051","code":true}]}]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.76471","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Okkenhaug, Klaus","Schwartzberg, Pamela L"]},{"key":"dc:contributor.sponsor","label":"Sponsor","values":["Wellcome Trust (200925/Z/16/Z) NIH intramural funding"]},{"key":"dc:creator","label":"Author","values":["Johansen, Kristoffer"]},{"key":"dc:creator.authoridentifier","label":"Author Identifier","values":["0000000277119451","0000000294324051"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2021-06-01"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/329027"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["CRISPR/Cas9","T cells","LFA-1","Phosphoinositide","TCR signalling","PI3K","Integrin","Adhesion","T cell migration","T cell adhesion"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/cb82729c-9fbb-4941-a97e-36674181bf45/download","https://creativecommons.org/licenses/by/4.0/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["10.17863/CAM.76471"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/82a6fb85-5464-4669-8516-874b8c621cfe/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Lymphocyte Function-Associated Antigen 1 (LFA-1) is the major integrin in T cells and binds Intercellular Adhesion Molecule 1 and 2 (ICAM-1 and ICAM-2) expressed on endothelial cells and antigen presenting cells. LFA-1 affinity for ICAM is increased following chemokine and T cell receptor (TCR) engagement by inside-out signalling. This process coordinates T cell migration, adhesion, and activation of T cells. LFA-1 activation is mediated by phosphoinositide 3-kinase (PI3K) and the downstream phosphoinositide PtdIns(3,4,5)P3 (PIP3). To investigate how PI3K regulates LFA-1, I optimised CRISPR/Cas9-mediated mutagenesis in T cells, and designed a retroviral library of CRISPR/single guide RNAs targeting all known and potential PIP3-binding proteins. Using this library, I screened the targeted genes for effects on ICAM-1-binding by a flow cytometry-based ICAM-1-binding assay, using Cas9-expressing primary mouse T cells. I identified multiple proteins regulating LFA-1-mediated adhesion to ICAM-1, including the RAP1/RAS GTPase-activating protein RASA3. I found that RASA3 is a critical negative regulator of LFA-1 activation and that RASA3 is inhibited by PI3K signalling. T cells without RASA3 have greatly increased ICAM-1-binding. Mice with conditional deletion of Rasa3 in T cells have altered T cell homeostasis including increased numbers of mature thymocytes in the thymus, but decreased T cells in lymph nodes, spleen, and especially in the blood. Further, Rasa3 deletion in T cells resulted in reduced germinal centre and antigen-specific antibody responses to immunisation, likely as a result of decreased T follicular helper (TFH) cell numbers. The presented results thus describe a novel genetic screen in T cells which has uncovered a critical role for RASA3 as a PI3K-regulated inhibitor of T cell adhesion and migration that is required for T cell homeostasis and function."]},{"key":"dc:format.checksum.md5","label":"Dc Format Checksum Md5","values":["e45a24e02e48633f772c5ce86f3aa758","353adac0d1ebdfd65ab16480263c3c87"]},{"key":"dc:title","label":"Title","values":["Regulation of LFA-1 on T cells by phosphoinositide signalling"]}]}],"canonical_facts":{"dc:contributor.advisor":["Okkenhaug, Klaus","Schwartzberg, Pamela L"],"dc:contributor.sponsor":["Wellcome Trust (200925/Z/16/Z) NIH intramural funding"],"dc:creator":["Johansen, Kristoffer"],"dc:creator.authoridentifier":["0000000277119451","0000000294324051"],"dc:date.issued":["2021-06-01"],"dc:description.abstract":["Lymphocyte Function-Associated Antigen 1 (LFA-1) is the major integrin in T cells and binds Intercellular Adhesion Molecule 1 and 2 (ICAM-1 and ICAM-2) expressed on endothelial cells and antigen presenting cells. LFA-1 affinity for ICAM is increased following chemokine and T cell receptor (TCR) engagement by inside-out signalling. This process coordinates T cell migration, adhesion, and activation of T cells. LFA-1 activation is mediated by phosphoinositide 3-kinase (PI3K) and the downstream phosphoinositide PtdIns(3,4,5)P3 (PIP3). To investigate how PI3K regulates LFA-1, I optimised CRISPR/Cas9-mediated mutagenesis in T cells, and designed a retroviral library of CRISPR/single guide RNAs targeting all known and potential PIP3-binding proteins. Using this library, I screened the targeted genes for effects on ICAM-1-binding by a flow cytometry-based ICAM-1-binding assay, using Cas9-expressing primary mouse T cells. I identified multiple proteins regulating LFA-1-mediated adhesion to ICAM-1, including the RAP1/RAS GTPase-activating protein RASA3. I found that RASA3 is a critical negative regulator of LFA-1 activation and that RASA3 is inhibited by PI3K signalling. T cells without RASA3 have greatly increased ICAM-1-binding. Mice with conditional deletion of Rasa3 in T cells have altered T cell homeostasis including increased numbers of mature thymocytes in the thymus, but decreased T cells in lymph nodes, spleen, and especially in the blood. Further, Rasa3 deletion in T cells resulted in reduced germinal centre and antigen-specific antibody responses to immunisation, likely as a result of decreased T follicular helper (TFH) cell numbers. The presented results thus describe a novel genetic screen in T cells which has uncovered a critical role for RASA3 as a PI3K-regulated inhibitor of T cell adhesion and migration that is required for T cell homeostasis and function."],"dc:format.checksum.md5":["e45a24e02e48633f772c5ce86f3aa758","353adac0d1ebdfd65ab16480263c3c87"],"dc:identifier.doi":["10.17863/CAM.76471"],"dc:identifier.uri":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/82a6fb85-5464-4669-8516-874b8c621cfe/download"],"dc:language":["eng"],"dc:publisher.institution":["University of Cambridge"],"dc:relation.isreferencedby.uri":["https://www.repository.cam.ac.uk/handle/1810/329027"],"dc:rights":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/cb82729c-9fbb-4941-a97e-36674181bf45/download","https://creativecommons.org/licenses/by/4.0/"],"dc:subject":["CRISPR/Cas9","T cells","LFA-1","Phosphoinositide","TCR signalling","PI3K","Integrin","Adhesion","T cell migration","T cell adhesion"],"dc:title":["Regulation of LFA-1 on T cells by phosphoinositide signalling"],"dc:type":["Thesis"],"dc:type.qualificationlevel":["Doctoral"],"dc:type.qualificationname":["Doctor of Philosophy (PhD)"]},"updated_at":"2026-07-22T22:24:10Z"}