A. Aoyama

Overcoming memory T-cell responses for induction of delayed tolerance in nonhuman primates.

The presence of alloreactive memory T cells is a major barrier for induction of tolerance in primates. In theory, delaying conditioning for tolerance induction until after organ transplantation could further decrease the efficacy of the regimen, since preexisting alloreactive memory T cells might be stimulated by the transplanted organ. Here, we show that such “delayed tolerance” can be induced in nonhuman primates through the mixed chimerism approach, if specific modifications to overcome/avoid donor‐specific memory T‐cell responses are provided. These modifications include adequate depletion of CD8+ memory T cells and timing of donor bone marrow administration to minimize levels of proinflammatory cytokines. Using this modified approach, mixed chimerism was induced successfully in 11 of 13 recipients of previously placed renal allografts and long‐term survival without immunosuppression could be achieved in at least 6 of these 11 animals.

Natural killer cells in rejection and tolerance of solid organ allografts.

Purpose of reviewA series of recent studies defy conventional wisdom by showing that natural killer (NK) cells exert a powerful and long-lasting influence on the immune response to whole organ allografts. The early activation of NK cells following transplantation is associated with killing of allogeneic target cells and release of immunomodulatory chemokines and cytokines, which can contribute to either rejection or tolerance. Here, we review findings describing NK cell receptors, potential mediators and mechanisms underlying the dual influence of NK cells in solid organ transplantation. Recent findingsNew studies show that NK cells can discriminate between self and foreign tissues and play a key role in the initiation and regulation of adaptive immune responses after solid organ transplantation. Depending upon the types of NK cell receptors engaged and the nature of cytokines released, early NK cell activation can promote either rejection or tolerance. SummarySolid organ transplantation is associated with the early activation of NK cells, which are then licensed to kill allogeneic target cells directly or via antibody-dependent cellular cytotoxicity and release various chemokines and immunomodulatory cytokines. Depending upon the nature of NK cell subsets activated and their ability to kill allogeneic target cells and release certain types of cytokines, NK cells can promote the activation/expansion of pro-inflammatory Th1 cells or regulatory Th2/Treg cells thus tilting the balance of alloimmunity towards rejection or tolerance. An in-depth understanding of these mechanisms will be necessary in order to design therapies targeting NK cells in human transplantation.

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.

Comparison of lung and kidney allografts in induction of tolerance by a mixed-chimerism approach in cynomolgus monkeys.

BACKGROUND We have previously reported the successful induction of renal allograft tolerance in non-human primates using a nonmyeloablative conditioning regimen to produce a mixed-chimeric state in the recipient. In the present study, we applied this same technique to lung allotransplantation in cynomolgus monkeys. METHODS Nine pairs of fully major histocompatibility complex (MHC)-mismatched cynomolgus monkeys were used. The conditioning regimen consisted of total body irradiation, thymic irradiation, and antithymocyte globulin. The recipients underwent lung and bone marrow transplantation, followed by anti-CD154 monoclonal antibody (mAb), and a 1-month course of cyclosporine. The regimen included anti-CD8 mAb in the last 5 recipients and alpha 1-antitripsin in the last 3 recipients. The results were compared with 8 recipients that received kidney allografts using the same regimen. RESULTS Transient chimerism developed in all lung recipients, as was previously seen in the kidney recipients. Nonetheless, the lung recipients rejected their allografts significant earlier than the kidney recipients (P < .01). CONCLUSIONS Despite the successful induction of mixed chimerism in recipients of fully MHC-mismatched lung allografts, we have not observed long-term graft survival, as has been seen in an analogous kidney model. Strategies to overcome this problem include organ-specific modifications of the transplant regimen.

Differential Effects of Denileukin Diftitox IL-2 Immunotoxin on NK and Regulatory T Cells in Nonhuman Primates

Denileukin diftitox (DD), a fusion protein comprising IL-2 and diphtheria toxin, was initially expected to enhance antitumor immunity by selectively eliminating regulatory T cells (Tregs) displaying the high-affinity IL-2R (α-β-γ trimers). Although DD was shown to deplete some Tregs in primates, its effects on NK cells (CD16+CD8+NKG2A+CD3−), which constitutively express the intermediate-affinity IL-2R (β-γ dimers) and play a critical role in antitumor immunity, are still unknown. To address this question, cynomolgus monkeys were injected i.v. with two doses of DD (8 or 18 μg/kg). This treatment resulted in a rapid, but short-term, reduction in detectable peripheral blood resting Tregs (CD4+CD45RA+Foxp3+) and a transient increase in the number of activated Tregs (CD4+CD45RA−Foxp3high), followed by their partial depletion (50–60%). In contrast, all NK cells were deleted immediately and durably after DD administration. This difference was not due to a higher binding or internalization of DD by NK cells compared with Tregs. Coadministration of DD with IL-15, which binds to IL-2Rβ-γ, abrogated DD-induced NK cell deletion in vitro and in vivo, whereas it did not affect Treg elimination. Taken together, these results show that DD exerts a potent cytotoxic effect on NK cells, a phenomenon that might impair its antitumoral properties. However, coadministration of IL-15 with DD could alleviate this problem by selectively protecting potentially oncolytic NK cells, while allowing the depletion of immunosuppressive Tregs in cancer patients.

Low‐Dose IL‐2 for In Vivo Expansion of CD4+ and CD8+ Regulatory T Cells in Nonhuman Primates

IL‐2 is a known potent T cell growth factor that amplifies lymphocyte responses in vivo. This capacity has led to the use of high‐dose IL‐2 to enhance T cell immunity in patients with AIDS or cancer. However, more recent studies have indicated that IL‐2 is also critical for the development and peripheral expansion of regulatory T cells (Tregs). In the current study, low‐dose IL‐2 (1 million IU/m2 BSA/day) was administered to expand Tregs in vivo in naïve nonhuman primates. Our study demonstrated that low‐dose IL‐2 therapy significantly expanded peripheral blood CD4+ and CD8+ Tregs in vivo with limited expansion of non‐Treg cells. These expanded Tregs are mainly CD45RA− Foxp3 high activated Tregs and demonstrated potent immunosuppressive function in vitro. The results of this preclinical study can serve as a basis to develop Treg immunotherapy, which has significant therapeutic potential in organ/cellular transplantation.

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.

Donor Brain Death Inhibits Tolerance Induction in Miniature Swine Recipients of Fully MHC‐Disparate Pulmonary Allografts

We have previously shown that a short course of high‐dose tacrolimus induces long‐term tolerance to fully mismatched lung allografts procured from healthy MHC‐inbred miniature swine. Here, we investigate whether donor brain death affects tolerance induction. Four recipient swine were transplanted with fully mismatched lung grafts from donors that were rendered brain dead and mechanically ventilated for 4 h before procurement (Group 1). These recipients were compared to two control groups (Group 2: 4 h of donor ventilation without brain death [n = 5]; and Group 3: no donor brain death with <1 h of ventilation [n = 6]). All recipients were treated with a 12‐day course of tacrolimus. In contrast to both groups of control animals, the swine transplanted with lung allografts from brain dead donors all rejected their grafts by postoperative day 45 and showed persistent responsiveness to donor antigen by MLR. Several additional swine underwent brain death induction and/or mechanical ventilation alone to determine the effects of these procedures on the expression of proinflammatory molecules. Significant increases in serum concentrations of IL‐1, TNF‐α and IL‐10 were seen after brain death. Upregulation of IL‐1 and IL‐6 gene expression was also observed.