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Investigating lymphocyte development in the foetal thymus and modulation by the melanocortin 5 receptor https://doi.org/10.17863/CAM.96659 Heycock, Morgan McKenzie, Andrew Lymphocyte development and activation are critical for the generation of effective protective immunity. Innate lymphoid cells and T cells play central roles in orchestrating bespoke immune reactions by producing specialised repertoires of immuno-regulatory cytokines. ILC2 and natural killer (NK) cells are generated in the bone marrow and thymus, whilst T cell development is restricted to the thymus. Once in the tissues, these cells respond to microenvironmental cues that refine their responses, for example, via receptor-mediated cell-cell contacts, soluble cytokines and neuropeptides. The co-development of ILC2, NK cells and T cells within a 5-day window in the foetal thymus provides a tractable system for studying their genesis and differentiation. Using combinatorial multicolour transcription factor reporter mouse lines, flow cytometry and confocal imaging I investigated the temporal stages of thymic innate lymphoid cell development. I found that NK cell progenitors (NKp) were first visible at embryonic (E) day E13.5, followed by the appearance of ILC2 progenitors (ILC2p) and developing T cells. Notably, the NKp and ILC2p formed clusters in the vicinity of medullary thymic epithelial cells (mTEC). To provide greater molecular insight into this cellular niche, single-molecule RNA fluorescence in situ hybridisation (smFISH) was performed on E17.5 foetal thymus to visually identify gene expression of 100 genes with single-cell resolution. This highlighted potential foetal thymus niche factors that may support innate lymphoid cell development. In the periphery ILC2, NK cells and T cells can respond to niche-derived factors to help initiate or potentiate immunity. Recent reports have highlighted the importance of tissue-resident stromal cells and neurons in this process, especially for ILC2. By analysing bulk RNAseq gene expression data, I identified Mc5r, encoding the fifth melanocortin receptor (Mc5r), as expressed more highly by ILC2 than ILC3. Treatment of mice with the neuropeptide alpha-melanocortin-stimulating hormone (αMSH), an agonist for melanocortin receptors, induced increases in the proportions of lung NK cells and T cells. The generation of a mouse line in which Mc5r was deleted in all lymphocytes (Il7r^CreMc5r^fl/fl mice) confirmed that Mc5r is required for normal NK cell and T cell representation in the lung at basal state, after IL-25 or IL-33 alarmin challenge and after viral respiratory infection of mice by the Pneumonia Virus of Mice. University of Cambridge 2022-09-01 Thesis Doctoral Doctor of Philosophy (PhD) eng https://www.repository.cam.ac.uk/handle/1810/349905 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/fca705ed-af96-45b5-ac45-350e29f8606e/download 8e49ca124e74b000fed40975a37633cd https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/8d660b56-15f6-4eea-90e3-a7a4b87c8f41/download 87eda9de84448d1f82354d60eee3eb5f https://www.rioxx.net/licenses/all-rights-reserved/ immunology lung mouse thymus