Generation of MHC class I allospecific regulatory T cells using chimeric antigen receptors, tools for eliciting targeted transplant tolerance

    Student thesis: Doctoral ThesisDoctor of Philosophy

    Abstract

    Regulatory T cells (Treg) therapy using autologous Tregs expanded ex vivo is currently being assessed clinically as a means of limiting graft rejection. However, pre-clinical data has demonstrated that graft-specific Tregs protect from graft rejection more effectively than polyclonal Tregs. Chimeric antigen receptor (CAR) technology is currently being investigated clinically as a means of conferring tumour antigen-specificity onto T cells in cancer research. CARs are synthetic fusion proteins which translate the engagement of extracellular target antigens into the activation of intracellular T cell signalling cascades. The hypothesis tested in this thesis was that the efficacy of polyclonal Treg therapy to inhibit transplant rejection could be enhanced by using CARs to confer specificity for donor MHC class I, alloantigens which are ubiquitously expressed in allografts.
    A human CAR was constructed to incorporate a patient-derived HLA-A2-targeting moiety and a CD28-CD3ζ signalling domain. Delivery of this CAR into human Tregs which were isolated using GMP-compatible protocols did not influence the phenotype or suppressive capacity of these cells. Compared to polyclonal Tregs, A2 CAR Tregs exhibited a greater suppressive function in the presence of HLA-A2+ antigen presenting cells, without eliciting cytotoxic activity. Furthermore, these cells preferentially transmigrated across HLA-A2-expressing endothelial cell monolayers, suggesting a favoured migration in to HLA-A2+ target tissues. In a human skin xenograft transplant model, A2 CAR Tregs alleviated alloimmune-mediated damage of HLA-A2+ skin more effectively than polyclonal Tregs.
    A second CAR was designed to redirect mouse Tregs towards BALB/c MHC class I (Kd) with the rationale of comparing the efficacy of CAR Tregs and Tregs with different allospecificities at reducing graft rejection. Murine CAR Tregs maintained their phenotype and suppressive ability and appeared to proliferate in the presence of Kd in vivo although conclusive evidence for the functionality of this CAR remained to be acquired.
    The results obtained demonstrated that CARs can be used to generate MHC class I-allospecific Tregs which are functionally superior to polyclonal Tregs at protecting from alloimmune-mediated transplant rejection, suggesting that CAR technology is a clinically applicable refinement of Treg therapy for organ transplantation.
    Date of Award2017
    Original languageEnglish
    Awarding Institution
    • King's College London
    SupervisorGiovanna Lombardi (Supervisor), Sandy FinesilverSmith (Supervisor) & John Maher (Supervisor)

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