The intragraft vascularized bone marrow component plays a critical role in tolerance induction after reconstructive transplantation.

The role of the vascularized bone marrow component as a continuous source of donor-derived hematopoietic stem cells that facilitate tolerance induction of vascularized composite allografts is not completely understood. In this study, vascularized composite tissue allograft transplantation outcomes between recipients receiving either conventional bone marrow transplantation (CBMT) or vascularized bone marrow (VBM) transplantation from Balb/c (H2d) to C57BL/6 (H2b) mice were compared. Either high- or low-dose CBMT (1.5 × 108 or 3 × 107 bone marrow cells, respectively) was applied. In addition, recipients were treated with costimulation blockade (1 mg anti-CD154 and 0.5 mg CTLA4Ig on postoperative days 0 and 2, respectively) and short-term rapamycin (3 mg/kg/day for the first posttransplant week and then every other day for another 3 weeks). Similar to high-dose conventional bone marrow transplantation, 5/6 animals in the vascularized bone marrow group demonstrated long-term allograft survival (>120 days). In contrast, significantly shorter median survival was noted in the low-dose CBMT group (~64 days). Consistently high chimerism levels were observed in the VBM transplantation group. Notably, low levels of circulating CD4+ and CD8+ T cells and a higher ratio of Treg to Teff cells were maintained in VBM transplantation and high-dose CBMT recipients (>30 days) but not in low-dose VBM transplant recipients. Donor-specific hyporesponsiveness was shown in tolerant recipients in vitro. Removal of the vascularized bone marrow component after secondary donor-specific skin transplantation did not affect either primary allograft or secondary skin graft survival.

Enabling allogeneic therapies: CIRM-funded strategies for immune tolerance and immune evasion.

A major goal for the field of regenerative medicine is to enable the safe and durable engraftment of allogeneic tissues and organs. In contrast to autologous therapies, allogeneic therapies can be produced for many patients, thus reducing costs and increasing availability. However, the need to overcome strong immune system barriers to engraftment poses a significant biological challenge to widespread adoption of allogeneic therapies. While the use of powerful immunosuppressant drugs has enabled the engraftment of lifesaving organ transplants, these drugs have serious side effects and often the organ is eventually rejected by the recipient immune system. Two conceptually different strategies have emerged to enable durable engraftment of allogeneic therapies in the absence of immune suppression. One strategy is to induce immune tolerance of the transplant, either by creating "mixed chimerism" in the hematopoietic system, or by retraining the immune system using modified thymic epithelial cells. The second strategy is to evade the immune system altogether, either by engineering the donor tissue to be "invisible" to the immune system, or by sequestering the donor tissue in an immune impermeable barrier. We give examples of research funded by the California Institute for Regenerative Medicine (CIRM) in each of these areas, ranging from early discovery-stage work through clinical trials. The advancements that are being made in this area hold promise that many more patients will be able to benefit from regenerative medicine therapies in the future.

ECDI-fixed donor splenocytes prolong skin allograft survival by promoting M2 macrophage polarization and inducing regulatory T cells.

Rejection is a common complication of allogeneic tissue transplantation. Fixation of splenocytes (SP) with 1-ethyl-3-(3'-dimethylaminopropyl)-carbodiimide (ECDI) induces immune tolerance in recipients post-transplantation; however, the mechanism underlying this effect remains unclear. Here, we determined the mechanisms of ECDI-fixed donor SP (ECDI-SP) in inducing tolerance in skin allograft transplantation. C57BL/6-recipient mice that received Balb/c full-thickness skin transplants with two infusions of donor-derived ECDI-SP, along with rapamycin showed superior skin allograft survival and lower inflammatory cell infiltration than mice that received rapamycin-only treatment. In ECDI-SP-treated mice, the levels of anti-inflammatory cytokines such as interleukin (IL)-10 in sera were markedly increased, whereas the expression of inflammatory cytokines was significantly suppressed. Splenic macrophages were significantly polarized to the alternative activated macrophage (M2) phenotype, with expansion of CD4+Foxp3+ regulatory T cells (Tregs) in the spleen and draining lymph nodes. Allostimulatory activity of ECDI-SP in vitro and donor-specific ex vivo hyporesponsiveness were observed. C57BL/6 macrophages engulfed allogeneic Balb/c-derived ECDI-SP, polarized to the M2 phenotype, with pronounced cAMP response element-binding (CREB) protein phosphorylation. By facilitating increased IL-10 expression, ECDI-SP induced M2 polarization and Treg production, inhibiting effector T-cell proliferation. Thus, ECDI-SP modulates macrophage M2 polarization by increasing CREB phosphorylation and promoting Treg production to suppress allogeneic skin graft rejection.

Changes in the Content of Mononuclear Liver Cells Expressing Immune Proteasomes after Transplantation of Ovarian Tissues Depending on Donor-Recipient Differences in Rats.

Implantation of ovarian tissue allografts in outbred Wistar rats and inbred August rats against the background of induction of donor-specific tolerance was accompanied by an increase in liver content of mononuclear cells expressing LMP2 immune of proteasome subunit by day 37 after transplantation in comparison with day 0. Graft rejection, on the contrary, was associated with a decrease in the number of LMP2+ cells in the liver of rats of both lines during this period. The difference in the content of these cells and the graft take rate were higher in Wistar rats. The number of mononuclear cells expressing LMP7 immune proteasome subunit in the liver did not change in rats of both lines by day 37 in comparison with day 0. Thus, the level of immune proteasomes with LMP2 subunit in mononuclear cells of the liver is related to fine mechanisms of regulation of immune responses and their shift toward graft take or rejection.

Changes in the Expression of Immune Proteasomes in the Liver after the Induction of Portal Tolerance Depending on Donor-Recipient Differences in Rats.

Induction of donor-specific tolerance in outbred Wistar rats (RT1u) and inbred August rats (RT1c) increased the expression of immune proteasome subunits in liver with a peak on day 7 after beginning of the induction. The increase in the level of immune subunits LMP2 and LMP7 was more pronounced in the liver of August rats in comparison with Wistar rats (by 2 and 6 times, respectively), which was associated with higher concentrations of monoamines in the CNS of August rats. After induction of donor-specific tolerance in August and Wistar rats, the immune subunits were in cells of sinusoidal lining and in cells located in sinusoid lumens. It can be suggested that immune proteasomes in these cells producing antigenic peptides for presentation to immunocompetent participate in the suppression of their activity and form the molecular basis for the development of donor-specific tolerance at very early stages of this process.

Immune monitoring of transplant patients in transient mixed chimerism tolerance trials.

This review focuses on mechanistic studies performed in recipients of non-myeloablative bone marrow transplant regimens developed at Massachusetts General Hospital in HLA-identical and HLA-mismatched haploidentical combinations, initially as a platform for treatment of hematologic malignancies with immunotherapy in the form of donor leukocyte infusions, and later in combination with donor kidney transplantation for the induction of allograft tolerance. In patients with permanent mixed chimerism, central deletion may be a major mechanism of long-term tolerance. In patients in whom donor chimerism is only transient, the kidney itself plays a significant role in maintaining long-term tolerance. A high throughput sequencing approach to identifying and tracking a significant portion of the alloreactive T cell receptor repertoire has demonstrated biological significance in transplant patients and has been useful in pointing to clonal deletion as a long-term tolerance mechanism in recipients of HLA-mismatched combined kidney and bone marrow transplants with only transient chimerism.

Change in the Content of Immunoproteasomes and Macrophages in Rat Liver At the Induction of Donor-Specific Tolerance.

Induction of donor specific tolerance (DST) by the introduction of donor cells into a recipient's portal vein is one of the approaches used to solve the problem of transplant engraftment. However, the mechanism of DST development remains unclear to this moment. In the present work, we first studied the change in the content of immunoproteasomes and macrophages of the liver at early stages of the development of allospecific portal tolerance in rats by Western blotting and flow cytofluorimetry. On the basis of the data obtained, we can conclude that the induction of DST is an active process characterized by two phases during which the level of the proteasome immune subunits LMP2 and LMP7 in liver mononuclear cells, including Kupffer cells, and the number of Kupffer cells change. The first phase lasts up to 5 days after the beginning of DST induction; the second phase - from 5 to 14 days. In both phases, the level of the subunits LMP2 and LMP7 in the total pool of mononuclear cells and Kupffer cells increases, with maximum values on days 1 and 7. In addition, the total number of Kupffer cells increases in both phases with a shift in several days. The most noticeable changes take place in the second phase. The third day is characterized by a lower content of mononuclear cells expressing immunoproteasomes compared to the control value in native animals. Presumably, at this time point a "window of opportunity" appears for subsequent filling of an empty niche with cells of different subpopulations and, depending on this fact, the development of tolerance or rejection. The results obtained raise the new tasks of finding ways to influence the cellular composition in the liver and the expression of immunoproteasomes on the third day after the beginning of DST induction to block the development of rejection.

Donor-specific tolerance induction in organ transplantation via mixed splenocytes chimerism.

We have shown previously that donor-derived splenocytes can replace recipients' bone marrow and induce donor-specific tolerance (DST). We have also shown the usefulness of the chimeric state for the induction of DST. Further analysis of mixed splenocytes chimera, especially the role of each T cells in mixed splenocytes chimera, is indispensable issue for its clinical use. A chimeric state has been shown to achieve long-term survival in major histocompatibility complex (MHC)-mismatched grafts. The donor-derived splenocytes can replace recipients' bone marrow and induce DST. The long-term survival of allogeneic skin grafts was achieved without immunosuppressants. In this study we show the role of each T cell type in a splenocyte mixed chimera. This review provides a short summary of our original work, adding some supplemental interpretations. Mixed chimerism is thus considered an attractive approach for the induction of DST without the use of immunosuppressants. In this paper, we summarize some of the findings on mixed splenocyte chimeras and review mixed chimerism in recent organ transplantation.

Induction of Donor-Specific Tolerance: Is This Achievable?

No abstract available.

Cryopreservation and the age of the allotransplant.

For centuries, reconstructive surgeons have restored form and function with autografts. These techniques are highly effective, but they are associated invariably with donor site morbidity. To avoid this, surgeons have long dreamed of using cadaveric sources for reconstructive material. However, allografts have two major limitations: rejection and limited donor tissue. In order to limit rejection, the allograft must be rendered more tolerable to the host or the host must be rendered more tolerant of the allograft. Both strategies have been used with considerable success in recent years. As understanding of the human immune response increases, clinical immunosuppressive regimens will undoubtedly become less morbid, and the indications for allotransplantation will broaden. This will place an even greater burden on the already small donor pool. One way to relieve this burden would be through the development of strategies for the long-term preservation of donated tissues and organs. Cryopreservation has been used clinically for decades, and recent advances in the field have allowed the preservation of an ever widening array of tissues and organs. As cold storage has been shown to reduce the antigenicity of parts, cryopreservation may actually serve to improve the survival rate of transplanted parts, as well as increase their availability. As the era of autotransplantation gives way to the age of allotransplantation, cryopreservation will play an increasingly important role.

The Differential Effects of Donor-Strain Immature Dendritic Cell Subtypes and the Blockade of CD40/CD154 Pathway on the Creation of Allograft Tolerance / 대한신장학회잡지

BACKGOUND: Dendritic cells (DC) are pivotal antigen presenting cells and serve a unique role in initiating immunity. To test the hypothesis that pre-immunization of recipient with certain DC subsets of donor origin can influence graft outcome. METHODS: We have studied the effects of immunization with allogeneic CD4+CD11c+(MDC) and CD8+CD11c+(LDC) DCs on the allograft response. RESULTS: Both immature MDC and LDC subsets from DBA/2 were able to prime naive allogeneic C57BL/6 (B6) T cells in MLR. In vitro allogeneic T cell responses were attenuated by the addition of anti-CD154 mAb into the culture. T cells from B6 mice that received DBA/2 MDC intravenously 4 weeks before testing mounted weaker MLR driven cell proliferation than T cells from LDC pretreated B6 mice. Consistent with the MLR results, combined pretreatment with MDC, but not LDC, plus anti-CD154 mAb produced donor-strain specific long-term graft survival and induced tolerance while treatment with LDC plus anti-CD154 mAb created minimal prolongation of allograft survival in a pancreas islet transplant model (DBA/2->B6). The beneficial effects exerted by MDC and anti-CD154 mAb pretreatment were correlated with TH1 to TH2 immune deviation and with the amplified donor-specific suppressive capacity by recipient CD4+CD25+T cells. CONCLUSION: These findings highlight the capacity of MDC to modulate alloimmune responses, and suggest therapeutic approaches for the induction of donor specific tolerance.