Donor-specific Transfusion Research Articles

Stem Cell Transplantation in Living Donor Renal Transplantation for Minimization of Immunosuppression

We present our experience of living-donor renal transplantation (LDRT) using pretransplant stem cell transplantation (SCT) where we have successfully achieved minimization of immunosuppression. Nine hundred sixteen patients underwent LDRT between 2007 and 2011: 606 under tolerance induction protocol (TIP) and 310 with our usual triple immunosuppression of calcineurin inhibitors (CNI), mycofenolate sodium (MMF), and prednisone (controls). The test group (TIP) was stratified into group 1, 1 haplomatch or greater (n=392), group 3, less than 1 haplomatch (n=214); controls were similarly stratified to group 2, 1 haplomatch or greater (n=179) and group 4, less than 1 haplomatch (n=131). The TIP consisted of donor-specific transfusion, adipose tissue-derived mesenchymal and hematopoietic stem cell transplantation, and nonmyeloablative conditioning with total lymphoid irradiation, cyclophosphamide, and rabbit-antithymocyte globulin. Posttransplant IS consisted of prednisone, CNI, or MMF, all in low doses. Four-year patient survival was 93.5%, 90.7%, 88.7%, and 82.7% in groups 1 through 4, respectively, and death-censored 4-year allograft survival was 94.8%, 95.4%, 94.5%, and 74.6%, respectively. Mean serum creatinine (mg/dL) for groups 1 through 4, respectively, at 4 years was 1.26, 1.57, 1.29, and 2.1. The number of rejection episodes was highest in group 4 and lowest in group 1. Minimization of IS was successfully achieved in 82.9% patients in group 1 and in 61.7% patients in group 3, whereas no minimization in groups 2 and 4. Stem cell transplantations were safe. Stem cell transplantation is effective in IS minimization in LDRT resulting in good graft function and patient and graft survival at 4 years.

Combined donor specific transfusion and anti-CD154 therapy achieves airway allograft tolerance

Objective To define the molecular mechanism of obliterative bronchiolitis (OB) following heterotopic tracheal transplantation during the early stages of tolerance induction by donor specific transfusion and anti-CD154.Methods Tolerance induction was achieved in tracheal allografts from BALB/C to C57BL/6 mice by donor specific transfusion of splenocytes intravenously and intraperitoneal injection of anti-CD154 mAbs.The differential gene expression was detected in tolerant murine heterotopic tracheal allografts by means of gene microarrays.Differential expression of selected genes was confirmed bv real-time fluorescent quantitative reverse transcription ( RT-qPCR).Results Comparative analysis between DST/anti-CD154 protocol-induced tolerant and rejected grafts revealed many genes were specifically downregulated in the tolerant allografts,including RAP1 pathway,CD40/CD40L concerned T cell surface molecules and many profibrotic genes,which were upregulated in the rejected allografts.In the tolerant allografts there was also marked expression of a number of genes for proinflammatory factors.Conclusion These results suggest a vital role for RAP1 pathway,CD40/CD40L concerned T cell surface molecules and many profibrotic genes in inducing airway transplant tolerance in this model. Key words: Lung transplantation; Obliterative bronchiolitis; Immunological tolerance

CD154 blockade alters innate immune cell recruitment and programs alloreactive CD8+ T cells into KLRG-1(high) short-lived effector T cells.

CD154/CD40 blockade combined with donor specific transfusion remains one of the most effective therapies in prolonging allograft survival. Despite this, the mechanisms by which these pathways synergize to prevent rejection are not completely understood. Utilizing a BALB/c (H2-Kd) to B6 (H2-Kb) fully allogeneic skin transplant model system, we performed a detailed longitudinal analysis of the kinetics and magnitude of CD8+ T cell expansion and differentiation in the presence of CD154/CD40 pathway blockade. Results demonstrated that treatment with anti-CD154 vs. DST had distinct and opposing effects on activated CD44high CD62Llow CD8+ T cells in skin graft recipients. Specifically, CD154 blockade delayed alloreactive CD8+ T cell responses, while DST accelerated them. DST inhibited the differentiation of alloreactive CD8+ T cells into multi-cytokine producing effectors, while CD40/CD154 blockade led to the diminution of the KLRG-1low long-lived memory precursor population compared with either untreated or DST treated animals. Moreover, only CD154 blockade effectively inhibited CXCL1 expression and neutrophil recruitment into the graft. When combined, anti-CD154 and DST acted synergistically to profoundly diminish the absolute number of IFN-γ producing alloreactive CD8+ T cells, and intra-graft expression of inflammatory chemokines. These findings demonstrate that the previously described ability of anti-CD154 and DST to result in alloreactive T cell deletion involves both delayed kinetics of T cell expansion and differentiation and inhibited development of KLRG-1low memory precursor cells.

Allograft rejection is restrained by short-lived TIM-3+PD-1+Foxp3+ Tregs

Tregs play a pivotal role in inducing and maintaining donor-specific transplant tolerance. The T cell immunoglobulin and mucin domain-3 protein (TIM-3) is expressed on many fully activated effector T cells. Along with program death 1 (PD-1), TIM-3 is used as a marker for exhausted effector T cells, and interaction with its ligand, galectin-9, leads to selective death of TIM-3+ cells. We report herein the presence of a galectin-9-sensitive CD4+FoxP3+TIM-3+ population of T cells, which arose from CD4+FoxP3+TIM-3- proliferating T cells in vitro and in vivo and were often PD-1+. These cells became very prominent among graft-infiltrating Tregs during allograft response. The frequency and number of TIM-3+ Tregs peaked at the time of graft rejection and declined thereafter. Moreover, these cells also arise in a tolerance-promoting donor-specific transfusion model, representing a pool of proliferating, donor-specific Tregs. Compared with TIM-3- Tregs, TIM-3+ Tregs, which are often PD-1+ as well, exhibited higher in vitro effector function and more robust expression of CD25, CD39, CD73, CTLA-4, IL-10, and TGF-β but not galectin-9. However, these TIM-3+ Tregs did not flourish when passively transferred to newly transplanted hosts. These data suggest that a heretofore unrecognized graft-infiltrating, short-lived subset of Tregs can restrain rejection.

Immunomodulatory Effect of Nuclear Factor-κB Inhibition by Dehydroxymethylepoxyquinomicin in Combination With Donor-Specific Blood Transfusion

Nuclear factor-κB (NF-κB) is a key molecule in alloimmune responses, however, its role in tolerance induction is not clear. We have previously reported that dehydroxymethylepoxyquinomycin (DHMEQ), a novel NF-κB inhibitor, prolongs cardiac allograft survival. In this study, we evaluated the immunomodulatory effects of DHMEQ when combined with a donor-specific blood transfusion (DST), and assessed whether the treatment induces tolerance in a mouse heart transplantation model. DST (20×10 splenocytes) was given intravenously at day -7. DHMEQ (30 mg/kg/day) was administered intraperitoneally for 14 days after DST. Graft survival and histology were evaluated. The underlying mechanisms of immunomodulation by DST and DHMEQ treatments were investigated by assessing alloimmune responses after transplantation. In fully mismatched H2-to-H2 heart transplants, DST alone prolonged allograft median survival time to 15 days, whereas when DST was combined with DHMEQ treatment, the graft median survival time was prolonged to 39.5 days. When the donor-recipient strain combination was reversed, that is, H2-to-H2, heart transplants were accepted (>150 days survival) in more than 60% of recipients treated with a DST and DHMEQ, whereas control allografts were all rejected within 8 days. The combined therapy markedly inhibited immune responses by both the direct and indirect allorecognition pathways mainly attributed to promotion of activation-induced cell death and Treg generation. Our results demonstrate the distinctive ability of NF-κB inhibition in combination with donor alloantigen to promote transplantation tolerance through multiple cellular mechanisms.

Regulatory T-Cell Activation among Patients Who Displayed Operational Tolerance following Intra-portal Administration of Donor-Specific Antigens in Living Donor Liver Transplantation

Immunologic tolerance is the goal for all transplant surgeons. We have reported that repeated donor-specific antigen transfusion (DST) via the portal vein allowed rapid reduction of immunosuppressants with decreased acute cellular rejection episodes among living donor liver transplantations (LDLT). Moreover, we demonstrated that intraportal DST induced macrochimerism of donor type CD56+ T cells in the liver graft. We examined the impact of FoxP3+CD4+CD25+ T cells in recipients who acquired almost tolerance after LDLT with intraportal DST. We defined the amount of immunosuppressants administered less than one time per week as “almost tolerance” after LDLT, which occurred among 14% of DST patients after adult-to-adult LDLT. Two patients (4%) have gotten been we used from immunosuppressants more than 2 years after LDLT 4 years prior. We examined the impact of FoxP3+CD4+CD25+ T cells both in recipients with almost daily immunosuppressants and those who acquired almost tolerance. The proportion of FoxP3+/CD4+CD25+ T cells in the almost tolerance group was significantly higher than that in the almost daily immunosuppressant group (P < .05). The increased proportion of FoxP3+/CD4+CD25+ T cells significantly correlated with time after LRLT (y = 0.0964x + 42.02, R2 = 0.8854). Repeated intraportal DST may be a goot tool to induce immunologic tolerance after LDLT. Both donor type CD56+ T cells and FoxP3+/CD4+CD25+ T cells may act as important regulatory cells for tolerance. The period after LDLT is important for acquiring immunologic tolerance.

Fates of CD4+ T Cells in a Tolerant Environment Depend on Timing and Place of Antigen Exposure

In experimental organ transplantation, tolerance is induced by administration of anti-CD40L mAb in conjunction with donor-specific splenocyte transfusion. Multiple, sometimes conflicting mechanisms of action resulting from this treatment have been reported. To resolve these issues, this study assessed the fates of graft reactive cells at different times and locations in the tolerant environment. Alloantigen-specific CD4(+) T cells transferred at time of tolerance induction (7 days before transplantation) became activated, expressed CD69 and CD44, and proliferated. Importantly, a large subset of this population became Foxp3(+) , more so in the lymph nodes than spleen, indicative of differentiation to a regulatory phenotype. In contrast, graft reactive CD4(+) T cells transferred to tolerogen-treated recipients at the time of transplantation failed either to proliferate or to differentiate, and instead were deleted via apoptosis. In untreated rejecting recipients graft reactive CD4(+) T cells became activated, proliferated and differentiated mainly in the spleen, and many of these cells were eventually deleted. These data resolve many apparent contradictions in the literature by showing that the timing of antigen exposure, the immunologic status of the recipients and secondary lymphoid organ location act together as key factors to determine the fate of graft reactive CD4(+) T cells.

Cigarette Smoke Exposure Hinders Long-Term Allograft Survival by Suppressing Indoleamine 2, 3-Dioxygenase Expression

Cigarette smoke causes cancer and increases the vulnerability of smokers to infections. Epidemiologic studies have shown that smoking is one of major risk factors for late allograft rejection. Despite statistical data that associate smoking with allograft rejection, no any study has been conducted to prove that cigarette smoke directly causes allograft rejection in a cause-effect manner. In particular, investigation into immunologic mechanisms underlying smoke-related allograft rejection is lacking. Here we found that second hand smoke (SHS) hindered long-term islet allograft survival induced by CD154 costimulatory blockade plus donor-specific splenocyte transfusion (DST), although it failed to alter acute islet allograft rejection. SHS did not directly interfere with vigorously alloreactive T-cell proliferation in vivo and in vitro. Neither naturally occurring nor induced CD4+CD25+ Treg cell numbers were significantly reduced by SHS. However, SHS suppressed mRNA and protein expression of indoleamine 2, 3-dioxygenase (IDO) and its activity upon transplantation while IDO overexpression in islet allografts restored their long-term survival induced by CD154 blockade. Therefore, SHS prevents long-term allograft survival by inhibiting IDO expression and activity. Thus, our study for the first time demonstrates that SHS shortens allograft survival in a cause-effect manner and unveils a novel immunologic mechanism underlying smoking-related allograft rejection.

Cell-Based Therapies in the Prevention of Solid Organ Transplant Rejection

Problem statement: Organ transplantation is a life-saving and increasi ngly common procedure, as it often serves as the only treatment available for end-stage organ disease. Although th e constant development of new and more effective immunosuppressive drugs has revolutionized the prevention and treatment of acute graft rejection, these drugs have significant toxicity, greatly incr ease patient susceptibility to neoplasms and infection a nd exert little impact on chronic rejection. Approach: The literature was reviewed to illuminate the mech anisms by which the anti-donor immune response is initiated and how cellular therapies im pact this response. Results: Data show that Donor Specific Transfusion, Apoptotic Cell therapies and Dendritic Cell therapies all function as a source o f alloantigen to suppress the anti-donor T cell respo nse. Conclusion: Cellular therapies hold promise in the prevention of solid organ allograft rejection, but require optimization and study in large animal mode ls before clinical implementation.

Abrogation of Antibodies Improves Outcome of Renal Transplantation

IntroductionAntibodies are known to cause rejection and therefore are detrimental to graft survival. We describe two protocols of clonal stimulation deletion (CSD) pretransplant followed by grafting with no conventional immunosuppression (IS). MethodsCSD was employed in 54 patients of mean age, 28.7 years and mean human leukocyte antigen A/B/DR match, 3.25. The two protocols both employed stimulation with donor-specific transfusions and stem cells with deletion using total lymphoid irradiation in group 1 (n = 29) or bortezomib in group 2 (n = 25). Other adjuvants in both protocols were cyclophosphamide, rabbit antithymocyte globulin, and rituximab. Stimulation and deletion were monitored by lymphocyte crossmatches and detection of donor-specific antibodies (DSA). Posttransplant monitoring included serum creatinine (SCr) measurements and antibody detection at regular intervals. Graft biopsy performed in the event of dysfunction was managed by standard guidelines. Rescue IS was initiated upon a rise in SCr or DSA. ResultsMean follow-up in group 1 is 3.28 years and 2.11 years in group 2. There was 100% graft and patient survivals in both cohorts with 23 patients without IS and stable graft function with an SCr of 1.3 mg/dL. All acute rejection episodes, which occurred among 24.1% of group 1 and 20% of group 2, were rescued with therapy evolving as a SCr of 1.6 to 1.9. The majority of rejections were antibody-combined with T-cell-mediated responses. We did not observe untoward effects of the protocol. ConclusionAbrogation of antibodies improved renal transplant outcomes.

IDO and Regulatory T Cell Support Are Critical for Cytotoxic T Lymphocyte-Associated Ag-4 Ig-Mediated Long-Term Solid Organ Allograft Survival

Costimulatory blockade of CD28-B7 interaction with CTLA4Ig is a well-established strategy to induce transplantation tolerance. Although previous in vitro studies suggest that CTLA4Ig upregulates expression of the immunoregulatory enzyme IDO in dendritic cells, the relationship of CTLA4Ig and IDO in in vivo organ transplantation remains unclear. In this study, we studied whether concerted immunomodulation in vivo by CTLA4Ig depends on IDO. C57BL/6 recipients receiving a fully MHC-mismatched BALB/c heart graft treated with CTLA4Ig + donor-specific transfusion showed indefinite graft survival (>100 d) without signs of chronic rejection or donor specific Ab formation. Recipients with long-term surviving grafts had significantly higher systemic IDO activity as compared with rejectors, which markedly correlated with intragraft IDO and Foxp3 levels. IDO inhibition with 1-methyl-dl-tryptophan, either at transplant or at postoperative day 50, abrogated CTLA4Ig + DST-induced long-term graft survival. Importantly, IDO1 knockout recipients experienced acute rejection and graft survival comparable to controls. In addition, αCD25 mAb-mediated depletion of regulatory T cells (Tregs) resulted in decreased IDO activity and again prevented CTLA4Ig + DST induced indefinite graft survival. Our results suggest that CTLA4Ig-induced tolerance to murine cardiac allografts is critically dependent on synergistic cross-linked interplay of IDO and Tregs. These results have important implications for the clinical development of this costimulatory blocker.

Antigen-specific induced Foxp3+ regulatory T cells are generated following CD40/CD154 blockade.

Blockade of the CD40/CD154 pathway potently attenuates T-cell responses in models of autoimmunity, inflammation, and transplantation. Indeed, CD40 pathway blockade remains one of the most powerful methods of prolonging graft survival in models of transplantation. But despite this effectiveness, the cellular and molecular mechanisms underlying the protective effects of CD40 pathway blockade are incompletely understood. Furthermore, the relative contributions of deletion, anergy, and regulation have not been measured in a model in which donor-reactive CD4(+) and CD8(+) T-cell responses can be assessed simultaneously. To investigate the impact of CD40/CD154 pathway blockade on graft-specific T-cell responses, a transgenic mouse model was used in which recipients containing ovalbumin-specific CD4(+) and CD8(+) TCR transgenic T cells were grafted with skin expressing ovalbumin in the presence or absence of anti-CD154 and donor-specific transfusion. The results indicated that CD154 blockade altered the kinetics of donor-reactive CD8(+) T-cell expansion, delaying differentiation into IFN-γ(+) TNF(+) multifunctional cytokine producers. The eventual differentiation of cytokine-producing effectors in tolerant animals coincided with the emergence of an antigen-specific CD4(+) CD25(hi) Foxp3(+) T-cell population, which did not arise from endogenous natural T(reg) but rather were peripherally generated from naïve Foxp3(-) precursors.

Islet allograft tolerance in the absence of invariant natural killer T cells

The invariant NKT cells are involved in both immunity and immune tolerance. However, their roles in transplant models remain controversial. We studied the role of NKT cells in the allograft response using two different strains of NKT deficient mice (CD1d-/- and Jα18-/- mice), and found that CD1d-/- and Jα18-/- mice rejected islet allografts with a similar kinetics as wild type B6 mice. Treatment of CD1d-/- and Jα18-/- mice with donor specific transfusion and anti-CD154 induced donor specific tolerance, which was identical to similarly treated wt B6 mice. The islet allograft tolerance requires Foxp3(+) Tregs. In the periphery, Foxp3(+) Tregs in CD1d-/-, Jα18-/-, and wt B6 mice were comparable both phenotypically and functionally. In addition, CD1d-/- and Jα18-/- CD4(+) T cells (non-Tregs) could be readily converted to Foxp3(+) Tregs by TGF-β in vitro. Our data suggest that islet allograft tolerance can be successfully established without invariant NKT cells.

Characterization of waves of leukocyte recruitment to the lung allograft and the effect of CTLA4-Ig

MHC-mismatched lung allografts are rapidly rejected by the host immune response. We analyzed cells infiltrating the grafted lung tissue using a collagenase-digestion method. The grafted lung was filled with host-derived leukocytes as early as day 1 post transplantation and the majority of the initial infiltrating cells were granulocytes. This initial influx of granulocytes waned rapidly, followed by a steady increase in lymphocytes, particularly T cells, and then by macrophages. The proportion of CD4 + T cells that express CD25 were increased in the graft the majority of which were activated CD4 + cells. We applied cytotoxic T-lymphocyte-associated antigen 4 (CTLA4)-Ig treatment in combination with donor-specific blood transfusion to the transplantation of lung allograft, which was significantly prolonged by the treatment. To examine the cellular and molecular basis of the inhibition of the graft rejection, we evaluated number and cytokine mRNA expression of the cells infiltrating in the lung allograft using collagenase-digestion method, although we were unable to detect significant effects of the treatment. Taken together, this study demonstrates that single cell suspensions from cellular infiltrates of lung tissue is useful for phenotypical and functional studies on cells infiltrating lung tissue after graft transplantation. ACTA MEDICA NAGASAKIENSIA 56: 27-34, 2011

IL-6 Induced by Staphylococcus aureus Infection Prevents the Induction of Skin Allograft Acceptance in Mice

Clinical correlations between bacterial infections and rejection suggest a hypothesis that innate immune stimulation by bacterial infections results in the production of inflammatory cytokine that facilitate bystander T-cell activation, increased alloreactivity and inhibition of tolerance induction. Previous studies demonstrated that IFNβ produced during an infection with a model bacterium, Listeria monocytogenes, prevented the induction of transplantation tolerance in mice with anti-CD154 and donor-specific transfusion (DST) (1). We investigated the impact of two clinically relevant bacterial infections at the time of transplantation on the ability of anti-CD154 and DST to induce skin allograft acceptance in mice. Staphylococcus aureus (SA) infection prevented skin allograft acceptance whereas maximally tolerated doses of Pseudomonas aeruginosa infection had no effect. SA induced an acute production of IL-6, which was necessary and sufficient for the prevention of skin allograft acceptance. Furthermore, a single pulse of methylprednisolone modulated IL-6 production during SA infection and facilitated skin allograft acceptance in SA-infected recipients. Taken together, our results suggest that bacterial infections elicit specific proinflammatory cytokines signatures that can serve as barriers to tolerance induction, and that inhibiting the production of or neutralizing these inflammatory cytokines can synergize with costimulatory blockade-based therapies to facilitate the development of transplantation tolerance.

Combination of monoclonal antibodies with DST inhibits accelerated rejection mediated by memory T cells to induce long-lived heart allograft acceptance in mice

Donor-reactive memory T cells mediated accelerated rejection is known as a barrier to the survival of transplanted organs. We investigated the combination of different monoclonal antibodies (mAbs) and donor-specific transfusion (DST) in memory T cells-based adoptive mice model. In the presence of donor-reactive memory T cells, the mean survival time (MST) of grafts in the anti-CD40L/LFA-1/DST group was 49.8d. Adding anti-CD44/CD70 mAbs to anti-CD40L/LFA-1/DST treatment. The MST was more than 100d (MST>100d). Compared with anti-CD40L/LFA-1/DST group, anti-CD40L/LFA-1/CD44/CD70/DST group notably reduced the expansion of memory T cells, enhanced the proportion of CD4+Foxp3+ regulatory T cells (Tregs) and suppressed donor-specific responses. Our data suggest that anti-CD40L/LFA-1/CD44/CD70mAbs and DST can synergistically inhibit accelerated rejection mediated by memory T cells to induce long-lived heart allograft acceptance in mice.

Regulatory T cell therapy combined with donor-specific T cell deletion promotes long-term survival of islet allografts (169.32)

Abstract Although Tregs are vital for peripheral tolerance, adoptive transfer of Tregs is insufficient in inducing long-term allograft survival in lymphoreplete hosts without preconditioning or adjunct immunosuppression. We hypothesize that reducing donor-reactive T cells may create a therapeutic window for Tregs to induce allograft tolerance without additional immunosuppression. We preconditioned recipient mice using donor-specific transfusion followed by cyclophosphamide treatment to selectively kill proliferating donor-reactive cells. This approach deleted 70-90% donor-reactive T cells, but failed to prolong islet allograft survival. Addition of Tregs with direct donor reactivity prior to transplantation led to long-term survival of BALB/c islets in 70% of C57BL/6 recipients. Equal numbers of polyclonal Tregs significantly prolonged graft survival, but 90% of the recipients rejected their grafts by 100 days. Increasing the numbers of polyclonal Tregs was able to confer long-term graft protection. Similar strategy of combining donor-reactive T cell deletion and polyclonal Tregs did not protect C3H islets in autoimmune NOD recipients. Further addition of Tregs specific for islet autoantigen was required to prolong graft survival in NOD mice. Together, these results demonstrate a clinically viable approach to harness the tolerogenic property of Tregs for inducing long-term allograft acceptance without additional immunosuppression.

Transfusion sanguine et transplantation

Pretransplant blood transfusion remains a controversial subject and its history can summarize the last 40 years of transplantation. Until 1971, transfusions were widely used in patients awaiting transplantation, especially due to the anemia induced by the chronic renal dysfunction. Then, a noxious effect of preformed anti-HLA antibodies on renal grafts survival was reported and pretransplant transfusions were stopped. Between 1972 and 1977, improvement of renal graft survival in patients who received pretransplant transfusions was noted. Therefore, from 1978 on, a systematic policy of pretransplant transfusions was adopted by almost all centres of transplantation. During the eighties, it was again abandoned for several reasons: absence of graft survival improvement in patients treated by cyclosporine, HLA immunization leading to an increased incidence of acute graft rejection, risk of viral diseases transmission and human recombinant erythropoietin development. The lack of improvement in graft survival for ten years has been leading the transplant community to look for antigen-specific immunosuppressive strategies to achieve transplantation tolerance. Donor-specific transfusion may have clinical benefits, as long-term grafts survival improvement, through modulation of the recipient's cellular immune system and has been recently reconsidered, especially before living donor transplantation. The immunological mechanisms inducing a tolerance-gaining effect of transfusions are still misunderstood, but the recent discovery of immunomodulatory effects of the apoptotic cells present in cellular products could enlighten our comprehension of pretransplant transfusions benefits and could help to develop specific tolerance induction strategies in solid organ transplantation.

Ursolic acid promotes robust tolerance to cardiac allografts in mice

Nuclear factor (NF)-κB is an important molecule in T cell activation. Our previous work has found that T cell-restricted NF-κB super-repressor (IκBαΔN-Tg) mice, expressing an inhibitor of NF-κB restricted to the T cell compartment, can permanently accept fully allogeneic cardiac grafts and secondary donor skin grafts. In this study, we explore if transient NF-κB inhibition by a small molecular inhibitor could induce permanent graft survival. Ursolic acid, a small molecular compound, dose-dependently inhibited T cell receptor (TCR)-triggered NF-κB nuclear translocation and T cell activation in vitro. In vivo, ursolic acid monotherapy prolonged significantly the survival of cardiac allograft in mice. Assisted with donor-specific transfusion (DST) on day 0, ursolic acid promoted 84·6% of first cardiac grafts to survive for more than 150 days. While the mice with long-term surviving grafts (LTS) did not reject the second donor strain hearts for more than 100 days without any treatment, they all promptly rejected the third-party strain hearts within 14 days. Interestingly, this protocol did not result in an increased proportion of CD4(+) CD25(+) forkhead box P3(+) regulatory T cells in splenocytes. That adoptive transfer experiments also did not support regulation was the main mechanism in this model. Splenocytes from LTS showed reduced alloreactivity to donor antigen. However, depletion of CD4(+) CD25(+) regulatory T cells did not alter the donor-reactivity of LTS splenocytes. These data suggest that depletion of donor-reactive T cells may play an important role in this protocol.

Beneficial effects of donor‐specific transfusion on renal allograft outcome

Donor-specific transfusion (DST) is claimed to improve graft survival in living kidney transplantation. The aim of this study was to determine the influence of DST on the incidence of acute rejection (AR), graft function and survival in the early and late post-transplantation period in transfusion-naïve patients. Three patient groups were compared: group 1 received DST (n = 18), group 2 patients received no transfusion prior to surgery (n = 13) and group 3 consisted of 132 randomly transfused patients. The DST protocol consisted of infusion of fresh whole donor blood (3 × 150 mL) at two-wk intervals accompanied by three d of azathioprine. All patients were grafted within one month after the third DST. Triple drug immunosuppression based on cyclosporine A was given to all patients. DST and polytransfused patients experienced significantly less AR compared with group 2 patients. Two-yr graft function was significantly better in patients in groups 1 and 3 compared with group 2. Although similar eight-yr patient and graft survival was found in all the groups, delayed graft function patients had the longest graft half-life. DST imposes a significant beneficial effect on the incidence of AR, DGF and graft function during the first post-transplantation year in transfusion-naïve patients receiving standard immunosuppression therapy.