{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/386521"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/386521","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Autogramin-2, a synthetic sterol, inhibits T cell adhesion and effector function","abstract":"Plasma membrane resident cholesterol is a key regulator of T cell immunity as it stabilises immune synapses, thus supporting effector T cell function. Recent publications suggest that increasing plasma membrane cholesterol content boosts anti-viral and anti-cancer immunity in T cells. Inhibition of cholesterol transport from the plasma membrane to the endoplasmic reticulum in adoptively transferred T cells therefore constitutes a promising therapeutical avenue for improving cancer therapy. I hypothesised that inhibition of the cholesterol transport protein Aster A using the synthetic sterol autogramin-2 enhances T cell effector function by increasing plasma membrane cholesterol content. Treatment of effector T cells with autogramin-2 inhibited, rather than boosted, effector T cell function: proliferation and activation induced increase of activation marker CD69 were impaired. Production of pro-inflammatory cytokines (TNF, IFN-), degranulation, and cytotoxic capacity, as measured by a co-culture killing assay with Raji cells and bi-specific antibody blinatumomab, were decreased upon treatment. This was accompanied by a reduction in cell-to-cell contact. Furthermore, LFA-1/integrin dependent adhesion to ICAM-1 and extracellular matrix components was inhibited within 30min of treatment with 3.75M autogramin-2 in an Aster A independent manner. Autogramin-2 did not significantly inhibit LFA-1/TCR downstream signalling in PHA blasts, nor affect activation induced conformational change of LFA-1. Instead, plasma membrane architecture was altered upon treatment: membrane asymmetry decreased independently of phosphatidyl-serine, and membrane fluidity increased in a sphingomyelin-complexed cholesterol independent manner. Plasma membrane lipid raft enriched fractions isolated from autogramin-2 treated samples showed a near significant reduction in LFA-1 -chain (ITGAL) staining (p=0.0535) compared to vehicle control (ctrl.) treated fractions. Concomitantly, relative abundance of lipolysis substrates triacyl-glyceride and diacyl-glyceride decreased significantly in Jurkat T cells treated with autogramin-2, accompanied by a trend towards decrease in monoacyl-glycerides (p=0.0711). Furthermore, relative abundance of acyl-carnitines increased significantly, with a similar trend observed for free fatty acids (p=0.0511). Based on the data presented here, I propose the following working model: autogramin-2 inhibits T cell integrins, including LFA-1, by rapidly stimulating lipolysis. Fatty acids released during lipolysis react with carnitine to form acyl-carnitine. Both acyl-carnitines and free fatty acids cause plasma membrane remodelling resulting in the expulsion of LFA-1 from stabilising lipid rafts, thus inhibiting T cell adhesion and effector function. Further investigation is needed to refine this model and elucidate the underlying mechanism.","abstract_html":"Plasma membrane resident cholesterol is a key regulator of T cell immunity as it stabilises immune synapses, thus supporting effector T cell function. Recent publications suggest that increasing plasma membrane cholesterol content boosts anti-viral and anti-cancer immunity in T cells. Inhibition of cholesterol transport from the plasma membrane to the endoplasmic reticulum in adoptively transferred T cells therefore constitutes a promising therapeutical avenue for improving cancer therapy. I hypothesised that inhibition of the cholesterol transport protein Aster A using the synthetic sterol autogramin-2 enhances T cell effector function by increasing plasma membrane cholesterol content. Treatment of effector T cells with autogramin-2 inhibited, rather than boosted, effector T cell function: proliferation and activation induced increase of activation marker CD69 were impaired. Production of pro-inflammatory cytokines (TNF, IFN-), degranulation, and cytotoxic capacity, as measured by a co-culture killing assay with Raji cells and bi-specific antibody blinatumomab, were decreased upon treatment. This was accompanied by a reduction in cell-to-cell contact. Furthermore, LFA-1/integrin dependent adhesion to ICAM-1 and extracellular matrix components was inhibited within 30min of treatment with 3.75M autogramin-2 in an Aster A independent manner. Autogramin-2 did not significantly inhibit LFA-1/TCR downstream signalling in PHA blasts, nor affect activation induced conformational change of LFA-1. Instead, plasma membrane architecture was altered upon treatment: membrane asymmetry decreased independently of phosphatidyl-serine, and membrane fluidity increased in a sphingomyelin-complexed cholesterol independent manner. Plasma membrane lipid raft enriched fractions isolated from autogramin-2 treated samples showed a near significant reduction in LFA-1 -chain (ITGAL) staining (p=0.0535) compared to vehicle control (ctrl.) treated fractions. Concomitantly, relative abundance of lipolysis substrates triacyl-glyceride and diacyl-glyceride decreased significantly in Jurkat T cells treated with autogramin-2, accompanied by a trend towards decrease in monoacyl-glycerides (p=0.0711). Furthermore, relative abundance of acyl-carnitines increased significantly, with a similar trend observed for free fatty acids (p=0.0511). Based on the data presented here, I propose the following working model: autogramin-2 inhibits T cell integrins, including LFA-1, by rapidly stimulating lipolysis. Fatty acids released during lipolysis react with carnitine to form acyl-carnitine. Both acyl-carnitines and free fatty acids cause plasma membrane remodelling resulting in the expulsion of LFA-1 from stabilising lipid rafts, thus inhibiting T cell adhesion and effector function. Further investigation is needed to refine this model and elucidate the underlying mechanism.","abstract_has_math":false,"creators":["Patommel, Katharina Alice"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Hess, Christoph","Edwards-Hicks, Joy","Booty, Lee","Martí I Líndez, Adrià-Arnau"],"committee_chairs":[],"committee_members":[],"year":2024,"date_issued":"2024-12-20","date_published":"2024-12-20","updated_at":"2026-07-22T22:24:24Z","subjects":["Adhesion","Aster","Aster A","Autogramin-2","Effector Function","GRAMD1","GRAMD1A","LFA-1","Small Molecule Compound","Sterol","T-cell"],"languages":[],"rights":[],"rights_urls":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/8872f2d0-8434-4b7e-8147-58fbb8942ed2/download","http://purl.org/NET/rdflicense/allrightsreserved"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.119701","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Hess, Christoph","Edwards-Hicks, Joy","Booty, Lee","Martí I Líndez, Adrià-Arnau"]},{"key":"dc:contributor.sponsor","label":"Sponsor","values":["BBSRC-GSK I Case Studentship"]},{"key":"dc:creator","label":"Author","values":["Patommel, Katharina Alice"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2024-12-20"]},{"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/1810/386521"]},{"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":["Adhesion","Aster","Aster A","Autogramin-2","Effector Function","GRAMD1","GRAMD1A","LFA-1","Small Molecule Compound","Sterol","T-cell"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:rights","label":"Dc Rights","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/8872f2d0-8434-4b7e-8147-58fbb8942ed2/download","http://purl.org/NET/rdflicense/allrightsreserved"]},{"key":"dc:rights.embargodate","label":"Dc Rights Embargodate","values":["2026-07-08"]},{"key":"dc:rights.embargotype","label":"Dc Rights Embargotype","values":["embargo"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.17863/CAM.119701"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/3ae394ce-f980-4c86-b14c-7a2b53aeff4d/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Plasma membrane resident cholesterol is a key regulator of T cell immunity as it stabilises immune synapses, thus supporting effector T cell function. Recent publications suggest that increasing plasma membrane cholesterol content boosts anti-viral and anti-cancer immunity in T cells. Inhibition of cholesterol transport from the plasma membrane to the endoplasmic reticulum in adoptively transferred T cells therefore constitutes a promising therapeutical avenue for improving cancer therapy. I hypothesised that inhibition of the cholesterol transport protein Aster A using the synthetic sterol autogramin-2 enhances T cell effector function by increasing plasma membrane cholesterol content. Treatment of effector T cells with autogramin-2 inhibited, rather than boosted, effector T cell function: proliferation and activation induced increase of activation marker CD69 were impaired. Production of pro-inflammatory cytokines (TNF, IFN-), degranulation, and cytotoxic capacity, as measured by a co-culture killing assay with Raji cells and bi-specific antibody blinatumomab, were decreased upon treatment. This was accompanied by a reduction in cell-to-cell contact. Furthermore, LFA-1/integrin dependent adhesion to ICAM-1 and extracellular matrix components was inhibited within 30min of treatment with 3.75M autogramin-2 in an Aster A independent manner. Autogramin-2 did not significantly inhibit LFA-1/TCR downstream signalling in PHA blasts, nor affect activation induced conformational change of LFA-1. Instead, plasma membrane architecture was altered upon treatment: membrane asymmetry decreased independently of phosphatidyl-serine, and membrane fluidity increased in a sphingomyelin-complexed cholesterol independent manner. Plasma membrane lipid raft enriched fractions isolated from autogramin-2 treated samples showed a near significant reduction in LFA-1 -chain (ITGAL) staining (p=0.0535) compared to vehicle control (ctrl.) treated fractions. Concomitantly, relative abundance of lipolysis substrates triacyl-glyceride and diacyl-glyceride decreased significantly in Jurkat T cells treated with autogramin-2, accompanied by a trend towards decrease in monoacyl-glycerides (p=0.0711). Furthermore, relative abundance of acyl-carnitines increased significantly, with a similar trend observed for free fatty acids (p=0.0511). Based on the data presented here, I propose the following working model: autogramin-2 inhibits T cell integrins, including LFA-1, by rapidly stimulating lipolysis. Fatty acids released during lipolysis react with carnitine to form acyl-carnitine. Both acyl-carnitines and free fatty acids cause plasma membrane remodelling resulting in the expulsion of LFA-1 from stabilising lipid rafts, thus inhibiting T cell adhesion and effector function. 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Inhibition of cholesterol transport from the plasma membrane to the endoplasmic reticulum in adoptively transferred T cells therefore constitutes a promising therapeutical avenue for improving cancer therapy. I hypothesised that inhibition of the cholesterol transport protein Aster A using the synthetic sterol autogramin-2 enhances T cell effector function by increasing plasma membrane cholesterol content. Treatment of effector T cells with autogramin-2 inhibited, rather than boosted, effector T cell function: proliferation and activation induced increase of activation marker CD69 were impaired. Production of pro-inflammatory cytokines (TNF, IFN-), degranulation, and cytotoxic capacity, as measured by a co-culture killing assay with Raji cells and bi-specific antibody blinatumomab, were decreased upon treatment. This was accompanied by a reduction in cell-to-cell contact. Furthermore, LFA-1/integrin dependent adhesion to ICAM-1 and extracellular matrix components was inhibited within 30min of treatment with 3.75M autogramin-2 in an Aster A independent manner. Autogramin-2 did not significantly inhibit LFA-1/TCR downstream signalling in PHA blasts, nor affect activation induced conformational change of LFA-1. Instead, plasma membrane architecture was altered upon treatment: membrane asymmetry decreased independently of phosphatidyl-serine, and membrane fluidity increased in a sphingomyelin-complexed cholesterol independent manner. Plasma membrane lipid raft enriched fractions isolated from autogramin-2 treated samples showed a near significant reduction in LFA-1 -chain (ITGAL) staining (p=0.0535) compared to vehicle control (ctrl.) treated fractions. Concomitantly, relative abundance of lipolysis substrates triacyl-glyceride and diacyl-glyceride decreased significantly in Jurkat T cells treated with autogramin-2, accompanied by a trend towards decrease in monoacyl-glycerides (p=0.0711). Furthermore, relative abundance of acyl-carnitines increased significantly, with a similar trend observed for free fatty acids (p=0.0511). Based on the data presented here, I propose the following working model: autogramin-2 inhibits T cell integrins, including LFA-1, by rapidly stimulating lipolysis. Fatty acids released during lipolysis react with carnitine to form acyl-carnitine. Both acyl-carnitines and free fatty acids cause plasma membrane remodelling resulting in the expulsion of LFA-1 from stabilising lipid rafts, thus inhibiting T cell adhesion and effector function. 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