Delivery of regulatory t-cell therapy via ex-situ machine perfusion

Hann, Angus ORCID: 0000-0003-4431-3642 (2024). Delivery of regulatory t-cell therapy via ex-situ machine perfusion. University of Birmingham. Ph.D.

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Abstract

Pharmacological immunosuppression following liver transplantation has improved considerably over the last half century. Despite an improved understanding, both acute and chronic rejection are frequently encountered by liver transplant clinicians and the latter is a significant cause of graft loss. In addition, the modern-day immunosuppressive medications such as calcineurin inhibitors, antimetabolites, anti-IL2 receptor antibodies and corticosteroids remain to be associated with sequalae such as diabetes, renal failure and post-transplant lymphoproliferative disorder. Regulatory T-cells (Tregs) represent a possible alternate therapy that may induce tolerance of the liver graft, rather than systemically supress the entire immune system. This research aimed to investigate the potential for Treg cell therapy to be administer directly to the liver graft through ex-situ normothermic machine perfusion (NMP). In order to do this, we firstly investigated the effect NMP has on the immune profile of the graft and immunological outcomes. Subsequently, we developed a liver wedge perfusion model to assess therapeutic Treg cell localisation after infusion, and the effect ex-situ administration has on the Treg phenotype. This was also then applied using a whole liver perfusion model consistent with that used in clinical practice (Liver Assist®). All experiments were done using human Tregs and liver. Post infusion Treg phenotype was assessed by re-isolating the infused cells from the liver tissue and perfusate, then performing flow cytometry to assess the expression of functional markers CD39, TIGIT and CTLA4. Clinical investigation comparing NMP preservation with cold storage demonstrated that early acute rejection occurred at a similar incidence in those undergoing primary transplant but was more frequent in those undergoing retransplant with an NMP preserved graft. The time of onset and severity were similar. After optimisation of our wedge perfusion model, expansion of human Tregs, and labelling with a fluorescent dye (Cell tracker red), these cells were infused via a portal vein branch. These cells were visible within the liver parenchyma and appeared to have migrated out of the hepatic sinusoids. This process appears to occur within 4 hours, as evidenced by the findings of the whole liver experiments using the Liver Assist®. The Treg suppressive phenotype, remained stable for the infused cells that were re-isolated from the liver, but was reduced in the cells within the perfusate. A proportion of Tregs administer via ex-situ machine perfusion engraft within the liver parenchyma, and therefore could exert a long lasting local immunosuppressive effect.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):