Xiangdong Zhao

The Presence of Foxp3 Expressing T Cells Within Grafts of Tolerant Human Liver Transplant Recipients

Background. Some experimental transplant-tolerance models have shown that the presence of regulatory T cells within grafts is important for the development of tolerance (Tol). Methods. To determine if the presence of regulatory T cells correlates with graft acceptance in living-donor liver-transplantation tolerance, the expression of Foxp3 mRNA and the presence of CD4+, CD8+, and Foxp3+ cells were quantified in biopsies from tolerant recipients by real-time polymerase chain reaction and by immunohistochemistry and immunofluorescent staining (Gr-Tol). The results were compared with biopsies from the recipients on maintenance immunosuppression (Gr-IS), grafts removed because of chronic rejection (Gr-CR), or normal liver (Gr-NL). Results. The expression of Foxp3 mRNA in Gr-Tol was higher than that in Gr-IS (P=0.07) and Gr-NL (P<0.0001), but equivalent to that in Gr-CR. In Gr-Tol, Foxp3+ cells were detectable within the clustered CD4+ and CD8+ cells in the portal areas. Ninety-two percent of those Foxp3+ cells were CD4+, whereas 8% were CD8+. The number of Foxp3+ cells was significantly increased in Gr-Tol, compared with that in Gr-IS (P<0.05), although the number of CD4+ or CD8+ cells did not differ between the two. Foxp3+ cells were hardly detectable in Gr-CR or -NL. Conclusions. This is the first report showing that CD4+Foxp3+ cells are present within grafts in a subset of tolerant patients after human liver transplantation. A prospective study is needed to elucidate whether the assessment of intragraft expression of Foxp3 protein, but not Foxp3 mRNA, can aid the identification of living-donor liver-transplantation recipients who can successfully withdraw IS.

Requirement of Protocol Biopsy Before and After Complete Cessation of Immunosuppression After Liver Transplantation

Background. Operational tolerance is defined as long-term acceptance of a transplanted organ after complete cessation of immunosuppression (IS), but may not always protect against antigen-dependent changes in graft morphology. Method. IS free patients after living-donor liver transplantation (LDLT) underwent protocol biopsy (tolerance group [Gr-Tol]) and were evaluated for rejection and fibrosis. The degree of fibrosis was compared with those in the patients on maintenance IS group (Gr-IS) and the base line normal liver group (Gr-BS). When bridging fibrosis or progression of fibrosis was observed, IS was reintroduced or increased in Gr-Tol or in the patients in the weaning process. Results. Neither acute nor chronic rejection was observed. The degree of fibrosis, however, was significantly greater in Gr-Tol than those in Gr-IS and Gr-BS. In Gr-Tol, the number of graft infiltrating FOXP3+ cells was significantly increased, the interval between LDLT and biopsy plus the donor age was significantly longer, and recipient age at LDLT was significantly younger, compared with those in Gr-IS. However, none of these three parameters correlated with the degree of fibrosis. In 7 of 11 patients in whom IS was reintroduced or increased, the improvement of fibrosis was observed by the subsequent biopsy. Conclusion. Grafts of operationally tolerant patients after LDLT did not exhibit acute or chronic rejection, but they exhibited fibrosis. It remains elusive whether fibrosis observed in tolerant grafts is antigen dependent. The finding that after the reintroduction or the increase of IS fibrosis was improved supported the possibility that fibrosis in operationally tolerant patients was antigen dependent.

Factors affecting operational tolerance after pediatric living-donor liver transplantation: impact of early post-transplant events and HLA match*

Pediatric recipients of living‐donor liver transplants (LDLT) can often discontinue immunosuppression (IS). We examined factors affecting development of operational tolerance (OT), defined as off IS for >1 year, in this population. A historic cohort analysis was conducted in 134 pediatric primary semi‐allogeneic LDLT. Multivariate logistic regression analysis was used. The frequency of peripheral regulatory T cells (Tregs) was determined at >10 years post‐Tx by FACS analysis. IS was successfully discontinued in 84 tolerant patients (Gr‐tol), but not in 50 intolerant patients (Gr‐intol). The Gr‐intol consisted of 24 patients with rejection (Gr‐rej) and 26 with fibrosis of grafts (Gr‐fib). The absence of early rejection [odds ratio (OR) 2.79, 95% CI 1.11–7.02, P = 0.03], was a positive independent predictor, whereas HLA‐A mismatch (0.18, 0.03–0.91, P = 0.04) was a negative predictor. HLA‐DR mismatches did not affect OT. The Treg frequency was significantly decreased in Gr‐intol (4.9%) compared with Gr‐tol (7.6%) (P = 0.003). There were increased levels of tacrolimus in the first week in Gr‐Tol (P = 0.02). Although HLA‐B mismatch (8.73, 1.09–70.0, P = 0.04) was a positive independent predictor of OT, its clinical significance remains doubtful. In this large cohort of pediatric LDLT recipients, absence of early rejection, HLA‐A match and the later predominance of Tregs are factors associated with OT.

The generation of donor-specific CD4+CD25++CD45RA+ naive regulatory T cells in operationally tolerant patients after pediatric living-donor liver transplantation.

Background. CD4+CD25++CD45RA+ cells (naïve regulatory T cells [naïve-Tregs]) have been identified as a functionally premature form of CD4+CD25+++CD45RA− cells (conventional-Tregs). However, their contribution to transplant tolerance remains to be elucidated. Method. We examined the frequency and the function of conventional and naive-Tregs in the peripheral blood derived from operationally tolerant patients after pediatric living-donor liver transplant (Gr-tol). The data were compared with those of patients who were unable to be weaned off immunosuppression due to rejection (group-intolerance [Gr-intol]), patients in the process of weaning immunosuppression (Gr-weaning) and healthy volunteers (group-healthy volunteers [Gr-vol]). Results. In Gr-tol, the frequency of conventional-Tregs was significantly higher than that in Gr-vol and tended to be higher than that in Gr-intol. The frequency of naive-Tregs was significantly decreased in Gr-intol versus those in Gr-tol, -weaning, and -vol. In mixed lymphocyte reactions, donor-specific hyporesponsiveness of CD4+ cells was observed only in Gr-tol but not in the other groups. Depletion of conventional or naive-Tregs from CD4+ cells demonstrated that the suppressive properties of donor antigen-reactive conventional and naïve-Tregs were upregulated compared with those of third-party antigen-reactive conventional and naïve-Tregs in Gr-tol only. Conclusions. This is the first report providing detailed evidence that donor-specific naïve-Tregs were generated and their suppressive properties were upregulated in the peripheral blood of tolerant patients, whereas their frequency was downregulated in intolerant patients. Therefore, we speculate that not only conventional-Tregs play a role in Tx tolerance but also the role of naïve-Tregs is critical.

Intragraft Vδ1 γδ T cells with a unique T-cell receptor are closely associated with pediatric semiallogeneic liver transplant tolerance.

Background T-cell receptor V&dgr;2 &ggr;&dgr; T cells (V&dgr;2 cells) participate in host defense, whereas V&dgr;1 &ggr;&dgr; T cells (V&dgr;1 cells) may regulate immune responses. V&dgr;1 cells appear to play a role in fetomaternal tolerance and our aim was to examine their role in liver transplant tolerance. Methods To determine whether V&dgr;1 cells increase within accepted grafts after semiallogeneic pediatric liver transplantation, the V&dgr;1/V&dgr;2 ratio was assessed at the transcriptional level and the complementarity-determining region 3 loop of the &dgr; chain of V&dgr;1 cells was sequenced in biopsies from immunosuppression-free (n=6) or almost free (n=3) liver transplant recipients, referred to as group tolerance (Gr-Tol; n=9). The results were compared with biopsies from grafts of recipients on maintenance immunosuppression due to concern of rejection (Gr-IS; n=11). Chronically rejected grafts (Gr-CR; n=6) and normal livers (Gr-NL; n=8) were also examined. Results The V&dgr;1/V&dgr;2 ratio was the highest in Gr-Tol (0.07±0.06) compared with Gr-IS (0.03±0.02; P=0.04), Gr-CR (0.01±0.02; P=0.008), and Gr-NL (0.02±0.04; P=0.01). There was an identical complementarity-determining region 3 sequence (100% homologous) among all recipients in Gr-Tol, which was dominant in six of nine recipients. This sequence was not seen in Gr-IS or Gr-CR, although it was observed in five of six normal livers. Conclusions A unique V&dgr;1-bearing T-cell clone accumulates within accepted human liver grafts. It might be useful as a biomarker of tolerance and the identification of its ligand might aid in the development of a novel strategy for tolerance induction.

Nebulized Phosphodiesterase III Inhibitor During Warm Ischemia Attenuates Pulmonary Ischemia–Reperfusion Injury

BACKGROUND The control of warm ischemia-reperfusion injury is crucial in managing donors after cardiac death for lung transplantation. We focused on transalveolar administration as a drug-delivery route for such donors. Milrinone is a phosphodiesterase 3 inhibitor that inhibits the breakdown of cyclic adenosine monophosphate and selectively relaxes smooth muscle. We hypothesized that nebulized milrinone would mitigate warm ischemia-reperfusion injury of lung. METHODS This study was conducted with an isolated rat lung perfusion model. Lungs were excised, exposed to 55-minute ischemia at 37 degrees C, and reperfused for 60 minutes. During ischemia, nebulized milrinone (n = 6) or saline (n = 6) was inhaled. Lungs were continuously perfused without ischemia as a sham group (n = 6). Airway resistance, pulmonary vascular resistance, pulmonary compliance, weight gain and blood gas were measured. Adenine nucleotide levels and apoptosis were investigated in the reperfused lungs. RESULTS Milrinone nebulization decreased post-ischemic pulmonary vascular resistance (0.98 +/- 0.05 and 1.74 +/- 0.17 cm H(2)O/ml.min at 60 minutes of reperfusion in the milrinone and control groups, respectively [p < 0.01]). It did not alter cyclic adenosine monophosphate levels, but it did elevate adenosine triphosphate levels (9.87 +/- 0.38 and 6.91 +/- 0.45 in the milrinone and control groups, respectively [p < 0.01]) and suppressed apoptosis (3.83 +/- 0.91 and 46.17 +/- 3.39 of mean apoptotic cell numbers in the milrinone and control groups, respectively [p < 0.01]). CONCLUSIONS Milrinone nebulization decreased post-ischemic pulmonary vascular resistance, elevated adenosine triphosphate levels, and suppressed apoptosis. Nebulized milrinone has some protective effects against warm ischemia.

Value of FOXP3 expression in peripheral blood as rejection marker after miniature swine lung transplantation.

BACKGROUND Outcome for highly immunogenic lung transplantation remains unsatisfactory despite the development of potent immunosuppressants. The poor outcome may be the result of a lack of minimally invasive methods to detect early rejection. There is emerging clinical evidence that, paradoxically, expression of forkhead box P3 (FOXP3, a specific marker for the regulatory T cells) is upregulated within rejecting grafts. METHODS Orthotopic lung transplantation was performed using miniature swine without immunosuppression. Rejection was monitored by chest radiography and open lung biopsy. Expressions levels of FOXP3, perforin, Fas-L and IP-10 mRNA were quantified in the peripheral blood. In addition, rescue immunosuppressive therapy (steroid plus tacrolimus) was administered on post-operative day (POD) 4 or 6. RESULTS Early rejection was detected by open lung biopsy, but misdiagnosed by chest radiography on POD 4. Expression of FOXP3 in the peripheral blood reached its highest value as early as POD 4, followed by a decline. Such an increase of FOXP3 was not observed in recipients given high-dose tacrolimus. Neither perforin, Fas-L or IP-10 in the peripheral blood exhibited significant fluctuations in the early phase of rejection. Rescue immunosuppressive therapy from POD 4, when peak FOXP3 was seen, prolonged graft survival (27.2 days, versus 9.1 days without immunosuppression, p < 0.001), in contrast to POD 6, when rejection was suspected by chest radiography (11.5 days, p = not statistically significant [NS]). CONCLUSIONS In a miniature swine lung transplantation model, the FOXP3 mRNA level in the peripheral blood was upregulated at an early phase of rejection. The clinical implication of this finding remains to be elucidated.

Effect of preprocurement ventilation on lungs donated after cardiac death in a canine lung transplantation model.

Background. One method of countering chronic lung donor shortages is the practice of donation after cardiac death (DCD). However, this technique inevitably leads to pulmonary dysfunction related to warm ischemia. One promising method of alleviating this problem is ventilation. However, it can rarely be initiated from the onset of cardiac arrest, particularly in uncontrolled DCD donors. We investigated the protective effect of the last 60 min of ventilation during a 240-min warm ischemic time. Methods. We rendered donor dogs cardiac dead and left them at room temperature. Six dogs received ventilation with 100% oxygen for 60 min starting at 180 min after cardiac arrest (ventilation group). Eight dogs received no ventilation. Lungs were harvested 240 min after cardiac arrest, then transplanted into recipient dogs. At 60 min after reperfusion, the right pulmonary artery was ligated, and the function of the left transplanted lung was evaluated. Results. In the ventilation group, all six animals survived for 240 min after reperfusion, whereas in the nonventilation group, only four of eight survived. The ventilation group demonstrated significantly better pulmonary oxygenation, shunt fraction, and wet-to-dry weight ratio. Furthermore, the ventilation group revealed significantly higher levels of high-energy phosphates in the lung tissues, fewer apoptotic cells, lower levels of tumor necrosis factor-&agr; and interleukin-8 messenger RNA in the lung tissues, and lower levels of interleukin-6 messenger RNA in the serum. Conclusion. Our results suggest that ventilation during the late phase of the preprocurement period may ameliorate ischemia-reperfusion injury in DCD donors.

The effect of β-2 adrenoreceptor agonist inhalation on lungs donated after cardiac death in a canine lung transplantation model

BACKGROUND It is a matter of great importance in a donation after cardiac death to attenuate ischemia-reperfusion injury (IRI) related to the inevitable warm ischemic time. METHODS Donor dogs were rendered cardiac-dead and left at room temperature. The dogs were allocated into 2 groups: the β-2 group (n = 5) received an aerosolized β-2 adrenoreceptor agonist (procaterol, 350 μg) and ventilation with 100% oxygen for 60 minutes starting at 240 minutes after cardiac arrest, and the control group (n = 6) received an aerosolized control solvent with the ventilation. Lungs were recovered 300 minutes after cardiac arrest. Recipient dogs underwent left single-lung transplantation to evaluate the functions of the left transplanted lung for 240 minutes after the reperfusion. RESULTS Oxygenation and dynamic compliance were significantly higher in the β-2 group than in the control group. The β-2 group revealed significantly higher levels of cyclic adenosine monophosphate and high-energy phosphates in the donor lung after the inhalation than before it. Histologic findings revealed that the β-2 group had less edema and fewer inflammatory cells. CONCLUSION Our results suggest that β-2 adrenoreceptor agonist inhalation during the pre-procurement period may ameliorate IRI.

Oral administration of polyamines ameliorates liver ischemia/reperfusion injury and promotes liver regeneration in rats.

Polyamines are essential for cell growth and differentiation. They play important roles in protection from liver damage and promotion of liver regeneration. However, little is known about the effect of oral exogenous polyamine administration on liver damage and regeneration. This study investigated the impact of polyamines (spermidine and spermine) on ischemia/reperfusion injury (IRI) and liver regeneration. We used a rat model in which a 70% hepatectomy after 40 minutes of ischemia was performed to mimic the clinical condition of living donor partial liver transplantation (LT). Male Lewis rats were separated into 2 groups: a polyamine group given polyamines before and after operation as treatment and a vehicle group given distilled water as placebo. The levels of serum aspartate aminotransferase and alanine aminotransferase at 6, 24, and 48 hours after reperfusion were significantly lower in the polyamine group compared with those in the vehicle group. Polyamine treatment reduced the expression of several proinflammatory cytokines and chemokines at 6 hours after reperfusion. Histological analysis showed significantly less necrosis and apoptosis in the polyamine group at 6 hours after reperfusion. Sinusoidal endothelial cells were also well preserved in the polyamine group. In addition, the regeneration of the remnant liver at 24, 48, and 168 hours after reperfusion was significantly accelerated, and the Ki‐67 labeling index and the expressions of proliferating cell nuclear antigen and phosphorylated retinoblastoma protein at 24 hours after reperfusion were significantly higher in the polyamine group compared with those in the vehicle group. In conclusion, perioperative oral polyamine administration attenuates liver IRI and promotes liver regeneration. It might be a new therapeutic option to improve the outcomes of partial LT. Liver Transplantation 22 1231–1244 2016 AASLD