Low-intensity conditioning for the induction of allogeneic tolerance in aged recipients
thesisposted on 22.02.2017, 23:15 by Morison, Jessica Kate
Organ transplantation is the curative technique for end-stage organ failure. However, the success of this technique is opposed by the immunological rejection of the transplanted tissue. Traditionally strategies to combat allogeneic rejection have been based on life-long immunosuppression, which leads to high levels of morbidity stemming from opportunistic infections and malignancy. This can be overcome via the induction of allogeneic tolerance, where bone marrow transplant (BMT) induced allogeneic mixed chimerism can produce a permanent state of donor-specific tolerance, to both skin and solid organ grafts, across major histocompatibility complex (MHC) barriers. The clinical translation of such protocols has been hampered by the toxicities associated with recipient preconditioning, involving lethal or sub-lethal doses of irradiation and/or chemotherapy and a concern for the development of graft versus host disease (GvHD). To reduce conditioning-mediated toxicities, non-myeloablative conditioning regimes have been widely explored. However, despite the success of these protocols in small animal models and nonhuman primates translation to clinical trials has been limited. Age-related thymic involution is often overlooked when trying to induce allogeneic tolerance via BMT. This could pose a significant barrier, considering previous reports demonstrate that to maintain stable allogeneic tolerance in mixed chimeras, there is an absolute requirement for donor antigen-dependent, intrathymic deletion of alloreactive T cells and therefore a functioning thymus. Thymic involution begins at the onset of puberty/sex steroid production and is characterised by a marked disorganisation of the microenvironment, the replacement of lymphoid tissue by adipocytes and a significant decrease in T cell output, leaving the thymus functioning at only 5 % capacity by 10-12 months in the mouse and 40 years in humans. This may be particularly important in a clinical setting, as the majority of transplant recipients are well into adulthood. This thesis aimed to investigate the ability to induce allogeneic tolerance, via a low-intensity conditioning regime in aged mice. This was achieved by (1) investigating the effects of the chemotherapeutic drug busulfan on the bone marrow (BM), spleen and thymus of both young and aged mice, (2) assessing the level of engraftment achieved when busulfan-treated mice receive both low and high-dose HSCT or allogeneic BMT, (3) investigating the role of sex steroid ablation (SSA) in boosting donor-cell engraftment and hematopoietic chimerism in both young and aged mice and finally (4) designing a thymus-sparing, low-intensity conditioning protocol that allowed the induction of allogeneic tolerance via mixed chimerism. Consistent with previous reports, low-dose busulfan was myeloablative, allowing transplanted cells to engraft in the BM, but not immunosuppressive, having only mild effects on the thymus and peripheral lymphoid compartment. Additionally, SSA coupled to chemotherapy increased both thymic cellularity and the number of donor-derived thymocytes following both high and low-dose HSCT. Indicating that SSA could be coupled to non-myeloablative HSCT to boost T cell output. Importantly, low-dose busulfan could be used in conjunction with T cell depleting antibodies and short-term sirolimus immunosuppression to induce mixed chimerism and allogeneic tolerance following the transplantation of both whole BM (WBM) or purified progenitor cells. Surprisingly, the tolerance generated from BMT was not dependent on the presence of a thymus, but instead the induction of T regulatory cells. The BM of aged mice was more resistant to chemotherapy than young BM and this lead to a decrease in donor cell engraftment and chimerism following congeneic HSCT. Sensitivity to chemotherapy could be restored by activating BM-progenitor cells with granulocyte colony stimulating factor (G-CSF), however, G-CSF prior to low-dose busulfan and HSCT did not boost engraftment of congeneic progenitor cells. Because the engraftment of progenitor cells was poor and allogeneic T cells can promote donor cell engraftment, the ability for WBM to engraft, promote mixed chimerism and induce allogeneic tolerance in aged mice conditioned with T cell depleting antibodies, sirolimus immunosuppression and low-dose busulfan was investigated. Utilising this protocol robust mixed chimerism and allogeneic tolerance could be achieved in aged mice. To our knowledge, this is the first study to examine mixed chimerism induced allogenic tolerance in aged mice. In this model age-related thymic involution does not impede the ability to induce donor-specific tolerance. This is an important finding for the clinical application of protocols designed to induce tolerance, considering most transplant recipients are aged individuals, who have experienced significant thymic involution and other signs of immunosenescence.