{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/362957"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/362957","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Identifying regulators of natural killer cell development through CRISPR screening of common lymphoid progenitors","abstract":"Natural Killer (NK) cells are cytotoxic lymphocytes that perform a key role in the innate immune response, lysing abnormal host cells such as those that are cancerous or virally infected. They are the prototypic member of the innate lymphoid cell (ILC) family, which also comprises the functionally divergent ILC1s, ILC2s, ILC3s and lymphoid tissue inducer (LTi) cells. Most NK cells arise from progenitors in the bone marrow (BM), from which other ILCs are also derived. However, the precise signals that specify the commitment of ILC progenitors to the NK cell lineage are poorly characterised. The aim of this thesis was to study the transcriptional networks and BM niche factors that promote NK cell development from uncommitted BM progenitors, using the mouse as a model organism. Pooled CRISPR screens were performed by culturing CRISPR-edited primary common lymphoid progenitors in vitro to generate NK cells and ILC2s, so that the regulators unique to each lineage could be discovered. Reassuringly, this approach identified previously studied regulators of NK cell development such as *Nfil3*, *Runx3* and *Eomes*. Additional putative regulators, such as *Tcf3* (encoding E2A), *Rere*, *Bach1* and *Zfp652* were identified and validated using arrayed CRISPR screens. Due to the known role of *Tcf3*/E2A in the decision between the innate and adaptive lymphocyte lineages, this was an attractive target for further study. Using a novel *Tcf3<sup>FLAG</sup>* mouse strain, E2A protein was detectable in progenitors but not mature ILC subtypes. However, transcriptional analysis revealed that *Tcf3* was expressed by mature NK cells so a conditional knockout of *Tcf3* in NK cells/ILC1s was generated and characterised. Fewer NK cells in these mice produced effector molecules such as granzyme B, and mice challenged with B16F10 melanoma cells had more lung metastasis. Finally, the developmental niche of ILCs was studied using immunohistochemistry of bone cryosections, and the role of Notch signalling as a potential regulator of E2A was studied. Overall, the work presented in this thesis adds to our knowledge of innate lymphopoiesis, with potential implications for how this might be distorted in systemic diseases such as cancer or targeted therapeutically.","abstract_html":"Natural Killer (NK) cells are cytotoxic lymphocytes that perform a key role in the innate immune response, lysing abnormal host cells such as those that are cancerous or virally infected. They are the prototypic member of the innate lymphoid cell (ILC) family, which also comprises the functionally divergent ILC1s, ILC2s, ILC3s and lymphoid tissue inducer (LTi) cells. Most NK cells arise from progenitors in the bone marrow (BM), from which other ILCs are also derived. However, the precise signals that specify the commitment of ILC progenitors to the NK cell lineage are poorly characterised. The aim of this thesis was to study the transcriptional networks and BM niche factors that promote NK cell development from uncommitted BM progenitors, using the mouse as a model organism. Pooled CRISPR screens were performed by culturing CRISPR-edited primary common lymphoid progenitors in vitro to generate NK cells and ILC2s, so that the regulators unique to each lineage could be discovered. Reassuringly, this approach identified previously studied regulators of NK cell development such as *Nfil3*, *Runx3* and *Eomes*. Additional putative regulators, such as *Tcf3* (encoding E2A), *Rere*, *Bach1* and *Zfp652* were identified and validated using arrayed CRISPR screens. Due to the known role of *Tcf3*/E2A in the decision between the innate and adaptive lymphocyte lineages, this was an attractive target for further study. Using a novel *Tcf3&lt;sup&gt;FLAG&lt;/sup&gt;* mouse strain, E2A protein was detectable in progenitors but not mature ILC subtypes. However, transcriptional analysis revealed that *Tcf3* was expressed by mature NK cells so a conditional knockout of *Tcf3* in NK cells/ILC1s was generated and characterised. Fewer NK cells in these mice produced effector molecules such as granzyme B, and mice challenged with B16F10 melanoma cells had more lung metastasis. Finally, the developmental niche of ILCs was studied using immunohistochemistry of bone cryosections, and the role of Notch signalling as a potential regulator of E2A was studied. Overall, the work presented in this thesis adds to our knowledge of innate lymphopoiesis, with potential implications for how this might be distorted in systemic diseases such as cancer or targeted therapeutically.","abstract_has_math":false,"creators":["Murphy, Jane"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["McKenzie, Andrew"],"committee_chairs":[],"committee_members":[],"year":2023,"date_issued":"2023-08-02","date_published":"2023-08-02","updated_at":"2026-07-24T01:33:33Z","subjects":["CRISPR","NK cells"],"languages":["eng"],"rights":[],"rights_urls":["https://www.repository.cam.ac.uk/bitstreams/1d1a101d-66ea-4170-8f06-a49dbc3d9275/download","https://www.rioxx.net/licenses/all-rights-reserved/"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.104851","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["McKenzie, Andrew"]},{"key":"dc:creator","label":"Author","values":["Murphy, Jane"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2023-08-02"]},{"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/1810/362957"]},{"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","NK cells"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["https://www.repository.cam.ac.uk/bitstreams/1d1a101d-66ea-4170-8f06-a49dbc3d9275/download","https://www.rioxx.net/licenses/all-rights-reserved/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.17863/CAM.104851"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://www.repository.cam.ac.uk/bitstreams/e15a7158-b29d-461b-b493-a693be10c754/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Natural Killer (NK) cells are cytotoxic lymphocytes that perform a key role in the innate immune response, lysing abnormal host cells such as those that are cancerous or virally infected. 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Additional putative regulators, such as *Tcf3* (encoding E2A), *Rere*, *Bach1* and *Zfp652* were identified and validated using arrayed CRISPR screens. Due to the known role of *Tcf3*/E2A in the decision between the innate and adaptive lymphocyte lineages, this was an attractive target for further study. Using a novel *Tcf3<sup>FLAG</sup>* mouse strain, E2A protein was detectable in progenitors but not mature ILC subtypes. However, transcriptional analysis revealed that *Tcf3* was expressed by mature NK cells so a conditional knockout of *Tcf3* in NK cells/ILC1s was generated and characterised. Fewer NK cells in these mice produced effector molecules such as granzyme B, and mice challenged with B16F10 melanoma cells had more lung metastasis. Finally, the developmental niche of ILCs was studied using immunohistochemistry of bone cryosections, and the role of Notch signalling as a potential regulator of E2A was studied. 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