Friday, May 10, 2024
5/10/2024
7. Project Summary/Abstract Complement may be activated in the peri-transplant period by insults such as ischemia reperfusion injury or binding of preformed host-anti-graft antibodies as well as post-transplant by binding of de novo donor specific antibody. In these settings, complement membrane attack complexes (MACs) are deposited on allograft endothelial cells (ECs) which increases the capacity of graft ECs to activate alloreactive host T and NK cells, providing a mechanistic link between such insults and an increased incidence and severity of T cell-mediated rejection (TCMR) without overt EC damage. We have shown that MACs are internalized and initiate IL-1¿ synthesis, processing and secretion followed by IL-1-mediated autocrine/paracrine activation of human ECs. A key component of this activation response is expression of IL-15/IL-15R¿ complexes on the EC surface that can be trans-presented to circulating lymphocytes. Blocking IL-15 trans-presentation reverses much of the MAC-induced augmentation of EC alloimmunogenicity. We hypothesize that gene editing of graft ECs ex vivo prior to transplant will reduce the severity of TCMR and prolong graft survival while still enabling the host immune system to respond to and control graft infection. Here we propose to develop approaches to do so, but preliminary data have revealed that the response of ECs to MAC and IL-1 is unexpectedly complex. Human ECs transcribe multiple different versions of IL-15 mRNA and increase these transcripts in response to IFN-¿ or MAC/IL-1, but only MAC/IL-1 leads to surface expression. The MAC/IL-1 response requires activation and nuclear uptake of NF-¿B, but NF-¿B appears to regulate IL-15 translation rather than transcription, possibly mediated by miRNA. Furthermore, co-induction of IL-15R¿, rather than pre-existing copies of this protein, is required for endogenous IL-15 surface expression. It is unknown if exogenous IL-15 can be trans-presented by ECs. We need to better understand these pathways in order to exploit them therapeutically. In this R21, we will identify and quantify the IL-15 mRNA species expressed by cultured human ECs that encode surface expressed protein isoforms and elucidate the mechanism of their translational control (aim 1). We then propose to optimize strategies for the elimination of MAC-induced IL-15 surface expression and trans- presentation using CRISPR/Cas9 gene editing and determine if and how this can be achieved using mRNA transfection while preserving EC homeostatic functions (aim 2). We will use the results of these studies to guide our future approach to gene edit human graft EC in ex vivo machine perfused organs.
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