{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/297674"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/297674","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Study of the intracellular trafficking of the pre-T-cell receptor and its role in T-cell lineage commitment","abstract":"T-cell development is a complex multi-step process, driving the maturation of hematopoietic stem cells into fully functional mature T-cells that play a major role in the adaptive immune response against pathogens. This developmental process is regulated by a series of checkpoints in which the signalling capacity of the T-cell antigen receptor (TCR) is tested. One such checkpoint is the generation of the clonotypic chains of the two TCR variants (αβ-TCR or γδ-TCR) by V(D)J recombination. Importantly, the TCRα chain is rearranged only after correct TCRβ expression, which requires the latter chain to pair with a surrogate invariant pre-Tα chain to form the pre-TCR complex. While both TCR variants activate the same signalling pathway, they commit the T cells to distinct functional lineages. Insight into how thymocytes are nonetheless able to distinguish between these two signals is crucial to our fundamental understanding of T-cell development and how it might be manipulated therapeutically. Pertinently, the pre-TCR is undetectable at the cell surface, unlike the γδ TCR which is primarily located at the plasma membrane. In this thesis, I aim to investigate whether the distinct intracellular localisation of the pre-TCR provides a mean for this signal to be distinguished from that of the γδ-TCR. Through the reconstitution of pre-TCR expression in a non-immune cell, I show that although the pre-TCR can in fact translocate to the cell surface, it is rapidly removed via clathrin-mediated endocytosis and transported to lysosomes. The use of a non-immune cell permits the study of pre-TCR trafficking in the absence of other signalling components and demonstrates that this intracellular localisation of the pre-TCR is intrinsic to the receptor. Using a panel of truncated and engineered pre-TCR, I demonstrate that the absence of a second Ig domain in the extracellular domain of the pre-Tα chain promotes the rapid endocytosis of the receptor. In a thymocyte-OP9-DL1 co-culture system, I show that recombination-deficient thymocytes expressing a pre-TCR variant lacking the TCRβ variable domain is sufficient to drive cells through the TCRβ-selection checkpoint and give rise to double positive cells. Likewise, wild type thymocytes over-expressing the pre-TCR or a receptor with a fused TCRα variable domain may push thymocytes down the γδ-lineage. Finally, I employ a biotin proximity-labelling assay to screen for binding partners of the pre-TCR that might mediate its internalisation. Using this method, I identify TMEM131, a novel protein with an unknown function and characterise some of its biochemical and cell biological properties.","abstract_html":"T-cell development is a complex multi-step process, driving the maturation of hematopoietic stem cells into fully functional mature T-cells that play a major role in the adaptive immune response against pathogens. This developmental process is regulated by a series of checkpoints in which the signalling capacity of the T-cell antigen receptor (TCR) is tested. One such checkpoint is the generation of the clonotypic chains of the two TCR variants (αβ-TCR or γδ-TCR) by V(D)J recombination. Importantly, the TCRα chain is rearranged only after correct TCRβ expression, which requires the latter chain to pair with a surrogate invariant pre-Tα chain to form the pre-TCR complex. While both TCR variants activate the same signalling pathway, they commit the T cells to distinct functional lineages. Insight into how thymocytes are nonetheless able to distinguish between these two signals is crucial to our fundamental understanding of T-cell development and how it might be manipulated therapeutically. Pertinently, the pre-TCR is undetectable at the cell surface, unlike the γδ TCR which is primarily located at the plasma membrane. In this thesis, I aim to investigate whether the distinct intracellular localisation of the pre-TCR provides a mean for this signal to be distinguished from that of the γδ-TCR. Through the reconstitution of pre-TCR expression in a non-immune cell, I show that although the pre-TCR can in fact translocate to the cell surface, it is rapidly removed via clathrin-mediated endocytosis and transported to lysosomes. The use of a non-immune cell permits the study of pre-TCR trafficking in the absence of other signalling components and demonstrates that this intracellular localisation of the pre-TCR is intrinsic to the receptor. Using a panel of truncated and engineered pre-TCR, I demonstrate that the absence of a second Ig domain in the extracellular domain of the pre-Tα chain promotes the rapid endocytosis of the receptor. In a thymocyte-OP9-DL1 co-culture system, I show that recombination-deficient thymocytes expressing a pre-TCR variant lacking the TCRβ variable domain is sufficient to drive cells through the TCRβ-selection checkpoint and give rise to double positive cells. Likewise, wild type thymocytes over-expressing the pre-TCR or a receptor with a fused TCRα variable domain may push thymocytes down the γδ-lineage. Finally, I employ a biotin proximity-labelling assay to screen for binding partners of the pre-TCR that might mediate its internalisation. Using this method, I identify TMEM131, a novel protein with an unknown function and characterise some of its biochemical and cell biological properties.","abstract_has_math":false,"creators":["Smid, Andrei Iosif"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["James, John Robert"],"committee_chairs":[],"committee_members":[],"year":2019,"date_issued":"2019-11-30","date_published":"2019-11-30","updated_at":"2026-07-22T22:24:11Z","subjects":["T-cell","T-cell development","Pre-T-cell receptor","Trafficking","TCR signalling","γδ-TCR","OP9-DL1 co-culture","hematopoietic stem cells","BioID2"],"languages":["en"],"rights":[],"rights_urls":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/1556d7c0-8e21-4cb7-95b3-2efe8f7a3fe6/download","https://www.rioxx.net/licenses/all-rights-reserved/"],"identifier_entries":[{"key":"dc:creator.authoridentifier","label":"Author Identifier","values":["0000000197320059","0000000314527578"],"render_values":[{"text":"0000-0001-9732-0059","href":"https://orcid.org/0000-0001-9732-0059","code":true},{"text":"0000-0003-1452-7578","href":"https://orcid.org/0000-0003-1452-7578","code":true}]}]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.44728","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["James, John Robert"]},{"key":"dc:contributor.sponsor","label":"Sponsor","values":["This work was funded by the Wellcome Trust."]},{"key":"dc:creator","label":"Author","values":["Smid, Andrei Iosif"]},{"key":"dc:creator.authoridentifier","label":"Author Identifier","values":["0000000197320059","0000000314527578"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2019-11-30"]},{"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/1810/297674"]},{"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":["T-cell","T-cell development","Pre-T-cell receptor","Trafficking","TCR signalling","γδ-TCR","OP9-DL1 co-culture","hematopoietic stem cells","BioID2"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/1556d7c0-8e21-4cb7-95b3-2efe8f7a3fe6/download","https://www.rioxx.net/licenses/all-rights-reserved/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["10.17863/CAM.44728"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/72e83c16-cf9f-40b4-9d87-75728dcc95d6/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["T-cell development is a complex multi-step process, driving the maturation of hematopoietic stem cells into fully functional mature T-cells that play a major role in the adaptive immune response against pathogens. 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In this thesis, I aim to investigate whether the distinct intracellular localisation of the pre-TCR provides a mean for this signal to be distinguished from that of the γδ-TCR. Through the reconstitution of pre-TCR expression in a non-immune cell, I show that although the pre-TCR can in fact translocate to the cell surface, it is rapidly removed via clathrin-mediated endocytosis and transported to lysosomes. The use of a non-immune cell permits the study of pre-TCR trafficking in the absence of other signalling components and demonstrates that this intracellular localisation of the pre-TCR is intrinsic to the receptor. Using a panel of truncated and engineered pre-TCR, I demonstrate that the absence of a second Ig domain in the extracellular domain of the pre-Tα chain promotes the rapid endocytosis of the receptor. In a thymocyte-OP9-DL1 co-culture system, I show that recombination-deficient thymocytes expressing a pre-TCR variant lacking the TCRβ variable domain is sufficient to drive cells through the TCRβ-selection checkpoint and give rise to double positive cells. Likewise, wild type thymocytes over-expressing the pre-TCR or a receptor with a fused TCRα variable domain may push thymocytes down the γδ-lineage. Finally, I employ a biotin proximity-labelling assay to screen for binding partners of the pre-TCR that might mediate its internalisation. 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In this thesis, I aim to investigate whether the distinct intracellular localisation of the pre-TCR provides a mean for this signal to be distinguished from that of the γδ-TCR. Through the reconstitution of pre-TCR expression in a non-immune cell, I show that although the pre-TCR can in fact translocate to the cell surface, it is rapidly removed via clathrin-mediated endocytosis and transported to lysosomes. The use of a non-immune cell permits the study of pre-TCR trafficking in the absence of other signalling components and demonstrates that this intracellular localisation of the pre-TCR is intrinsic to the receptor. Using a panel of truncated and engineered pre-TCR, I demonstrate that the absence of a second Ig domain in the extracellular domain of the pre-Tα chain promotes the rapid endocytosis of the receptor. In a thymocyte-OP9-DL1 co-culture system, I show that recombination-deficient thymocytes expressing a pre-TCR variant lacking the TCRβ variable domain is sufficient to drive cells through the TCRβ-selection checkpoint and give rise to double positive cells. Likewise, wild type thymocytes over-expressing the pre-TCR or a receptor with a fused TCRα variable domain may push thymocytes down the γδ-lineage. Finally, I employ a biotin proximity-labelling assay to screen for binding partners of the pre-TCR that might mediate its internalisation. 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