Cellular therapy for reconstitution of immunity and tolerance to transplantation

This is a CASE project funded through the UCL-Birkbeck MRC Doctoral Programme:

https://www.ucl.ac.uk/mrc-dtp/

This project aims to develop new ways to cryopreserve human thymus tissue slices and to purify human thymic epithelial cells (TEC) in order to develop cellular therapies to restore thymus function in athymic infants and to test if tolerance to organ transplants can be induced by donor TEC or thymus in heart transplant recipients.

Currently thymus, a key component of newborns’ immune systems, is transplanted into athymic infants by insertion of ~1mm slices of non-MHC matched thymus, after culturing to deplete thymocytes, which could cause graft-versus-host disease1. The procedure is life-saving, but recipients have low T-cell counts and develop autoimmunity because MHC-matching is not possible, as it would require a bank of cryopreserved tissue-typed thymus. Thymus is removed during cardiac surgery to allow access to the heart, so tissue is available if cryopreservation were feasible. In order both to allow MHC-matching and to investigate if TEC/thymus tissue could be used to induce tolerance, it is first necessary to be able to cryopreserve large quantities of human thymus tissue or TEC, while depleting thymocytes. We hypothesise that cryopreservation could be used both to establish a bank of viable frozen tissue for transplantation, and to selectively deplete thymocytes while retaining TEC.

Cryopreservation of thymus slices: We will develop protocols for the cryopreservation of thymus slices. Cryopreservation of tissue for regenerative medicine is challenging because of heat transfer issues, cryoprotectant toxicity, and the control of ice nucleation, complex events that impact on viability of thawed tissue2. We will use a GMP-compliant large-scale cryocooler-based control-rate freezer that can process large volumes of tissue required for transplantation, in a method compatible with ultimate clinical applications. In pilot experiments, histological analysis of thawed slices identified areas of tissue damage3, which may be reduced by introduction of cryobeads or ice nucleants to provide foci of cryogenesis, potentially lessening ice crystal formation and consequent damage. We will compare freezing protocols, with and without cryobeads, with different cycles and rates of cooling, different ice nucleation strategies, and different cryoprotectants. We will assess outcome by flow cytometry for cellular composition and viability, histology for tissue architecture, and functional ability to support T-cell development and induce tolerance in our mouse model3, 4. Currently, the relatively thick 1mm thymus slices for transplantation are cultured for two to three weeks on filters at the air interface to deplete thymocytes. This is inefficient: some TEC are also lost, and depletion of thymocytes is incomplete1. We will optimize protocols to maximize TEC viability but selectively deplete thymocytes. Thymocytes are susceptible to apoptosis, whereas epithelial cells are known to be more tolerant to cryopreservation, so freezing may be an alternative to the two-week culture, and we will also compare changes in incubator CO2 levels, or culturing submerged. We will carry out time-series analysis measuring apoptosis and viability to determine optimal freezing protocols to maximize TEC survival, while killing thymocytes.

References
1. Davies et al 2017 JACI 17, 30576.
2. Massie et al 2014 Tissue Eng Part C Methods 20, 693.
3. Ross et al 2018 EJI (in press).
4. Furmanski et al 2013 JID 133, 1221.