Background
Targeting the process of central (thymic) selection of developing T lymphocytes is the key mechanism for transplant tolerance induction mediated bybone marrow transplantation (BM-Tx)-based protocols. However, they are not amenable to most transplant recipients. Thymic Epithelial Cells (TEC), a population of stromal cells residing in the thymus, exert a major contribution to central selection. Unfortunately, donor TEC do not develop following BM-Tx protocols. Therefore, we propose a new strategy that voids the need for heavy pre-conditioning and is based on generating a donor-recipient “Hybrid Thymus,” through donor TEC engraftment, to re-engineer the thymic microenvironment and promote lasting central tolerance.
Methods
We developed an improved protocol for isolating mouse TEC via a combination of negative and positive selection. Allogeniec (Allo)-TEC purified from BALB/c were injected into the right thymic lobe of C57BL/6, Recipients were divided in groups based on treatment with CTLA4-Ig +/− anti-CD40L +/− T depletion. 14, 28, and 45 days post-injection Allo-TEC survival was assessed and the selection and reactivity of T cells was analyzed via flow cytometry and CFSE-based mixed lymphocyte reaction.
Results
Our optimized purification protocol yields an average 70% TEC purity. Unmanipulated recipients promptly rejected Allo-TEC, indicating the existence of effective immunity in the thymus. However, CTLA4-Ig and MR1 co-administration exerted a significant (but not lasting) protection. In this latter group, the percentage of peripheral Vβ11+ T cells (inversely correlated with central selection) on d24 was significantly lower, indicating functional activity of the engrafted donor TEC. Addition of transient T depletion enabled long-term engraftment of Allo-TEC that, on d45, were in a normal architecture within the thymus integrated with recipient thymocytes. However, anti-donor T cell reactivity remained unaltered, suggesting the need for engraftment in both thymic lobes for proper regulation.
Conclusion
Our preliminary data show that the thymic engraftment, survival, and function of allo-TEC can be promoted via costimulation blockade + T depletion. These exciting results indicate that engineering a donor-recipient Hybrid Thymus is feasible and has the potential to promote a dominant regulation of alloreactivity that could be conducive to transplant tolerance induction.