Makoto Tonsho

Uterine autotransplantation in cynomolgus macaques: the first case of pregnancy and delivery

BACKGROUND For women with congenital uterine infertility, or for those who have undergone hysterectomy, uterine transplantation is one of the potential treatments to regain fertility. In this study, we utilized a primate model of uterine transplantation, and evaluated the patency of our microsurgical anastomoses, and the perfusion of the transplanted uterus. METHODS Two female cynomolgus monkeys underwent surgery. We anastomosed two arteries and one vein in Case 1 and two arteries and two veins in Case 2. The arteries used were the uterine arteries and the anastomosis was done to the external iliac artery. We used one of the ovarian veins in both animals, but resected the ovary from the Fallopian tube. Uterine arterial blood flow and uterine size were determined by intraoperative indocyanine green (ICG) angiography and ultrasonography. The biopsy of the uterine cervix was performed after surgery. RESULTS ICG angiography showed that the unilateral uterine artery perfused the bilateral uterine bodies and cervix. In Case 1, ICG angiography showed the occlusion of one of the anastomosed arteries during the operation and the uterus appeared atrophied 2 months after operation. In Case 2, the transplanted uterus survived and normal menstruation occurred. The animal achieved a natural pregnancy and was delivered by the Caeserean section due to early separation of the placenta. The newborn suffered fetal distress. CONCLUSIONS These results show the anastomosis of at least the bilateral uterine arteries and the unilateral ovarian vein is required for uterus transplantation. This is the first report of a natural pregnancy in a primate following uterine autotransplantation.

Tolerance of Lung Allografts Achieved in Nonhuman Primates via Mixed Hematopoietic Chimerism

While the induction of transient mixed chimerism has tolerized MHC‐mismatched renal grafts in nonhuman primates and patients, this approach has not been successful for more immunogenic organs. Here, we describe a modified delayed‐tolerance‐induction protocol resulting in three out of four monkeys achieving long‐term lung allograft survival without ongoing immunosuppression. Two of the tolerant monkeys displayed stable mixed lymphoid chimerism, and the other showed transient chimerism. Serial biopsies and post‐mortem specimens from the tolerant monkeys revealed no signs of chronic rejection. The tolerant recipients also exhibited T cell unresponsiveness and a lack of alloantibody. This is the first report of durable mixed chimerism and successful tolerance induction of MHC‐mismatched lungs in primates.

Innate Immunity and Resistance to Tolerogenesis in Allotransplantation

The development of immunosuppressive drugs to control adaptive immune responses has led to the success of transplantation as a therapy for end-stage organ failure. However, these agents are largely ineffective in suppressing components of the innate immune system. This distinction has gained in clinical significance as mounting evidence now indicates that innate immune responses play important roles in the acute and chronic rejection of whole organ allografts. For instance, whereas clinical interest in natural killer (NK) cells was once largely confined to the field of bone marrow transplantation, recent findings suggest that these cells can also participate in the acute rejection of cardiac allografts and prevent tolerance induction. Stimulation of Toll-like receptors (TLRs), another important component of innate immunity, by endogenous ligands released in response to ischemia/reperfusion is now known to cause an inflammatory milieu favorable to graft rejection and abrogation of tolerance. Emerging data suggest that activation of complement is linked to acute rejection and interferes with tolerance. In summary, the conventional wisdom that the innate immune system is of little importance in whole organ transplantation is no longer tenable. The addition of strategies that target TLRs, NK cells, complement, and other components of the innate immune system will be necessary to eventually achieve long-term tolerance to human allograft recipients.

Heart Transplantation: Challenges Facing the Field

There has been significant progress in the field of heart transplantation over the last 45 years. The 1-yr survival rates following heart transplantation have improved from 30% in the 1970s to almost 90% in the 2000s. However, there has been little change in long-term outcomes. This is mainly due to chronic rejection, malignancy, and the detrimental side effects of chronic immunosuppression. In addition, over the last decade, new challenges have arisen such as increasingly complicated recipients and antibody-mediated rejection. Most, if not all, of these obstacles to long-term survival could be prevented or ameliorated by the induction of transplant tolerance wherein the recipients immune system is persuaded not to mount a damaging immune response against donor antigens, thus eliminating the need for chronic immunosuppression. However, the heart, as opposed to other allografts like kidneys, appears to be a tolerance-resistant organ. Understanding why organs like kidneys and livers are prone to tolerance induction, whereas others like hearts and lungs are tolerance-resistant, could aid in our attempts to achieve long-term, immunosuppression-free survival in human heart transplant recipients. It could also advance the field of pig-to-human xenotransplantation, which, if successful, would eliminate the organ shortage problem. Of course, there are alternative futures to the field of heart transplantation that may include the application of total mechanical support, stem cells, or bioengineered whole organs. Which modality will be the first to reach the ultimate goal of achieving unlimited, long-term, circulatory support with minimal risk to longevity or lifestyle is unknown, but significant progress in being made in each of these areas.

Alefacept promotes immunosuppression-free renal allograft survival in nonhuman primates via depletion of recipient memory T cells

Renal allograft tolerance has been achieved in MHC‐mismatched primates via nonmyeloablative conditioning beginning 6 days prior to planned kidney and donor bone marrow transplantation (DBMT). To extend the applicability of this approach to deceased donor transplantation, we recently developed a novel‐conditioning regimen, the “delayed protocol” in which donor bone marrow (DBM) is transplanted several months after kidney transplantation. However, activation/expansion of donor‐reactive CD8+ memory T cells (TMEM) occurring during the interval between kidney and DBM transplantation impaired tolerance induction using this strategy. In the current study, we tested whether, Alefacept, a fusion protein which targets LFA‐3/CD2 interactions and selectively depletes CD2highCD8+ effector memory T cells (TEM) could similarly induce long‐term immunosuppression‐free renal allograft survival but avoid the deleterious effects of anti‐CD8 mAb treatment. We found that Alefacept significantly delayed the expansion of CD2high cells including CD8+ TEM while sparing naïve CD8+ T and NK cells and achieved mixed chimerism and long‐term immunosuppression‐free renal allograft survival. In conclusion, elimination of CD2high T cells represents a promising approach to prevent electively the expansion/activation of donor‐reactive TEM and promotes tolerance induction via the delayed protocol mixed chimerism approach.

Treg depletion in non-human primates using a novel diphtheria toxin-based anti-human CCR4 immunotoxin.

Regulatory T cells (Treg) play an important role in modulating the immune response and has attracted increasing attention in diverse fields such as cancer treatment, transplantation and autoimmune diseases. CC chemokine receptor 4 (CCR4) is expressed on the majority of Tregs, especially on effector Tregs. Recently we have developed a diphtheria‐toxin based anti‐human CCR4 immunotoxin for depleting CCR4+ cells in vivo. In this study, we demonstrated that the anti‐human CCR4 immunotoxin bound and depleted monkey CCR4+ cells in vitro. We also demonstrated that the immunotoxin bound to the CCR4+Foxp3+ monkey Tregs in vitro. In vivo studies performed in two naive cynomolgus monkeys revealed 78–89% CCR4+Foxp3+ Treg depletion in peripheral blood lasting approximately 10 days. In lymph nodes, 89–96% CCR4+Foxp3+ Tregs were depleted. No effect was observed in other cell populations including CD8+ T cells, other CD4+ T cells, B cells and NK cells. To our knowledge, this is the first agent that effectively depleted non‐human primate (NHP) Tregs. This immunotoxin has potential to deplete effector Tregs for combined cancer treatment.

Long‐Term Lung Transplantation in Nonhuman Primates

Despite advances in surgical technique and clinical care, lung transplantation still remains a short‐term solution for the treatment of end‐stage lung disease. To date, there has been limited experience in experimental lung transplantation using nonhuman primate models. Therefore, we have endeavored to develop a long‐term, nonhuman primate model of orthotopic lung transplantation for the ultimate purpose of designing protocols to induce tolerance of lung grafts. Here, we report our initial results in developing this model and our observation that the nonhuman primate lung is particularly prone to rejection. This propensity toward rejection may be a consequence of 1) upregulated nonspecific inflammation, and 2) a larger number of pre‐existing alloreactive memory T cells, leading to augmented deleterious immune responses. Our data show that triple‐drug immunosuppression mimicking clinical practice is not sufficient to prevent acute rejection in nonhuman primate lung transplantation. The addition of horse‐derived anti‐thymocyte globulin and a monoclonal antibody to the IL‐6 receptor allowed six out of six lung recipients to be free of rejection for over 120 days.