In vivo imaging of regulatory T cell mediated transplant tolerance

    Student thesis: Doctoral ThesisDoctor of Philosophy


    Regulatory T cells (Tregs) were identified several years ago and are key in controlling autoimmune diseases and limiting immune responses to foreign antigens. These cells have been used successfully in animal models first and more recently in the clinic to prevent Graft vs Host disease and transplant rejections. However, their locations in vivo, their migratory abilities and their in vivo survival have not been extensively investigated. Imaging of the human sodium/iodide symporter via Single Photon Emission Computed Tomography (SPECT) has been used as a reporter gene to image various cell types in vivo. It has several advantages over other imaging techniques including high sensitivity, it allows non-invasive whole body studies of viable cell migration and localisation over time and lastly it may offer the possibility to be translated to the clinic. The study presented in this thesis addresses whether SPECT/CT imaging can be used to visualise the migratory pattern and survival of Tregs in vivo. At first, CD4+ T cells were directly radiolabelled and were subsequently imaged in vivo to demonstrate that T cells can be imaged using NanoSPECT/CT. Then Treg lines derived from CD4+CD25+FoxP3+ cells \ were/ retrovirally transduced with a construct encoding for the human Sodium Iodide Symporter (NIS) and the fluorescent protein mCherry. NIS expressing self-specific Tregs were specifically radiolabelled in vitro with Technetium-99m pertechnetate (9 mTcC)4~) and exposure of these cells to radioactivity was shown not to affect cell viability, phenotype and Treg function. In addition adoptively transferred Treg-NIS cells were imaged in vivo in C57BL/6 (BL/6) mice by SPECT/CT using 99mTcO4". After 24 hours NIS expressing Tregs were observed in the spleen and their localisation was further confirmed by organ biodistribution studies and flow cytometry analysis.
    Later, Treg lines with direct or indirect alloantigen specificity were imaged in skin transplant models using the same technique. It was observed that adoptively transferred Tregs migrate to the site of transplant at earlytime points and then migrated to various lymph nodes. The data presented here suggests that SPECT/CT imaging can be utilised in preclinical imaging studies of adoptively transferred Tregs without affecting Treg function and viability thereby allowing longitudinal studies within disease models. Moreover, new insight into pattern of migration of Tregs was identified.
    Date of Award2012
    Original languageEnglish
    Awarding Institution
    • King's College London
    SupervisorGiovanna Lombardi (Supervisor) & Gregory Mullen (Supervisor)

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