Induction of Durable Mixed Hematopoietic Chimerism and Immune Tolerance in Monkeys

Mixed hematopoietic chimerism is a promising approach to achieving transplantation tolerance.  Transient mixed hematopoietic chimerism leads to renal allograft tolerance in about 70% of cynomolgus monkeys when the kidney is co-transplanted with MHC-mismatched bone marrow (BMT). This same approach failed to achieve tolerance with more immunogenic organs such as lungs and hearts. Murine studies have demonstrated that the addition of regulatory T cells (Tregs) to BMTs using a non-myeloablative protocol (that is insufficient to allow engraftment), permits lasting mixed chimerism, BM engraftment and skin graft tolerance. We aim to extend this approach into the pre-clinical cynomolgus monkey model.

We have developed a protocol for the expansion of (100 million-1 billion) host polyclonal cynomolgus macaque Tregs (pTregs) (CD4+, CD25hi, CD127-/lo, FoxP3+). Cryopreserved Tregs are thawed and washed prior to infusion on days 0, 2, 5, 7 and (+/-) 50 to recipients conditioned with total body irradiation (1.25-1.5cGy) on days -6 and -5, thymic irradiation (7Gy) on day -1, ATGAM on days -2, -1 and 0, anti-CD40L on days 0, 2, 5, 7, 9 and 12 and cyclosporine or rapamycin (monotherapy) for 30 days (from day -2 to day 28 after BMT). To assess for tolerance, a skin or kidney allograft from the same donor was grafted four months post-BMT, after discontinuation of all immunosuppression.

Control animals (which did not receive Tregs (n=4)) lost chimerism within 40 days. In vitro, anti-donor proliferative responses were observed,and donor kidneys were rejected. In contrast, animals that received Tregs together with BMT (n = 2) developed remarkably high and long-lasting chimerism, with a peak of >90% in myeloid lineages and range of 110-335 days. Unfortunately, experimental animals that reactivated CMV early after BMT either succumbed to CMV viremia or lost the BM graft when treated with myelotoxic antivirals. The substitution of rapamycin for CyA allowed control of early CMV reactivation (n=3 animals) and boosted the number of Tregs in the peripheral blood. T cell chimerism was observed only in Treg recipients, with high CD31 expression, suggesting new thymic emigrants. Tregs animals were donor-hyporesponsive 7 weeks post-BMT. One animal received a kidney graft on day +120 which was accepted without immunosuppression until euthanasia on day 335 (without evidence for histopathological rejection and with normal kidney function). The second Treg recipient received skin grafts on day 119. The third party graft is suspected to be rejected (histopathology to be confirmed) within 7 days and the donor skin is visually healthy three weeks post-transplant.

In summary, host pTregs given at the time of BMT across MHC barriers prolongs donor chimerism up to 335 days (extending it beyond 60 days for the first time in 20 years using this protocol) without GVHD and allows for the survival of donor organs transplanted four months post-BMT.