Amphiregulin Stimulates Liver Regeneration After Small‐for‐Size Mouse Liver Transplantation

This study investigated whether amphiregulin (AR), a ligand of the epidermal growth factor receptor (EGFR), improves liver regeneration after small‐for‐size liver transplantation. Livers of male C57BL/6 mice were reduced to ～50% and ～30% of original sizes and transplanted. After transplantation, AR and AR mRNA increased in 50% but not in 30% grafts. 5‐Bromodeoxyuridine (BrdU) labeling, proliferating cell nuclear antigen (PCNA) expression and mitotic index increased substantially in 50% but not 30% grafts. Hyperbilirubinemia and hypoalbuminemia occurred and survival decreased after transplantation of 30% but not 50% grafts. AR neutralizing antibody blunted regeneration in 50% grafts whereas AR injection (5 μg/mouse, iv) stimulated liver regeneration, improved liver function and increased survival after transplantation of 30% grafts. Phosphorylation of EGFR and its downstream signaling molecules Akt, mTOR, p70S6K, ERK and JNK increased markedly in 50% but not 30% grafts. AR stimulated EGFR phosphorylation and its downstream signaling pathways. EGFR inhibitor PD153035 suppressed regeneration of 50% grafts and largely abrogated stimulation of regeneration of 30% grafts by AR. AR also increased cyclin D1 and cyclin E expression in 30% grafts. Together, liver regeneration is suppressed in small‐for‐size grafts, as least in part, due to decreased AR formation. AR supplementation could be a promising therapy to stimulate regeneration of partial liver grafts.     

The Programmed Death (PD)‐1/PD‐Ligand 1 Pathway Regulates Graft‐Versus‐Host‐Reactive CD8 T Cells After Liver Transplantation

Acute graft‐versus‐host disease (aGVHD) is a life‐threatening complication after solid‐organ transplantation, which is mediated by host‐reactive donor T cells emigrating from the allograft. We report on two liver transplant recipients who developed an almost complete donor chimerism in peripheral blood and bone marrow‐infiltrating T cells during aGVHD. By analyzing these T cells directly ex vivo, we found that they died by apoptosis over time without evidence of rejection by host T cells. The host‐versus‐donor reactivity was selectively impaired, as anti‐third‐party and antiviral T cells were still detectable in the host repertoire. These findings support the acquired donor‐specific allotolerance concept previously established in animal transplantation studies. We also observed that the resolution of aGVHD was not accompanied by an expansion of circulating immunosuppressive CD4/CD25/FoxP3‐positive T cells. In fact, graft‐versus‐host‐reactive T cells were controlled by an alternative negative regulatory pathway, executed by the programmed death (PD)‐1 receptor and its ligand PD‐L1. We found high PD‐1 expression on donor CD4 and CD8 T cells. In addition, blocking PD‐L1 on host‐derived cells significantly enhanced alloreactivity by CD8 T cells in vitro. We suggest the interference with the PD‐1/PD‐L1 pathway as a therapeutic strategy to control graft‐versus‐host‐reactive T cells in allograft recipients.     

The Leuven Immunomodulatory Protocol Promotes T‐Regulatory Cells and Substantially Prolongs Survival After First Intestinal Transplantation

Intestinal transplantation (ITx) remains challenged by frequent/severe rejections and immunosuppression‐related complications (infections/malignancies/drug toxicity). We developed the Leuven Immunomodulatory Protocol (LIP) in the lab and translated it to the clinics. LIP consists of experimentally proven maneuvers, destined to promote T‐regulatory (Tregs)‐dependent graft‐protective mechanisms: donor‐specific blood transfusion (DSBT); avoiding high‐dose steroids/calcineurin‐inhibitors; and minimizing reperfusion injury and endotoxin translocation. LIP was tested in 13 consecutive ITx from deceased donors (2000–2014) (observational cohort study). Recipient age was 37 years (2.8–57 years). Five‐year graft/patient survival was 92%. One patient died at 9 months due to aspergillosis, another at 12 years due to nonsteroidal anti‐inflammatory drug–induced enteropathy. Early acute rejection (AR) developed in two (15%); late AR in three (23%); all were reversible. No chronic rejection (CR) occurred. No malignancies developed and estimated glomerular filtration rate remained stable post‐Tx. At last follow‐up (3.5 years [0.5–12.5 years]), no donor‐specific antibodies were detected and 11 survivors were total parenteral nutrition free with a Karnofsky score >90% in 8 recipients (follow‐up >1 years). A high frequency of circulating CD4⁺CD45RA^‐Foxp3^hi memory Tregs was found (1.8% [1.39–2.21]), comparable to tolerant kidney transplant (KTx) recipients and superior to stable immunosuppression (IS)‐KTx, KTx with CR, and healthy volunteers. In this ITx cohort we show that DSBT in a low‐inflammatory/pro‐regulatory environment activates Tregs at levels similar to tolerant‐KTx, without causing sensitization. LIP limits rejection under reduced IS and thereby prolongs long‐term survival to an extent not previously attained after ITx.     

Superagonistic CD28 Antibody Induces Donor‐Specific Tolerance in Rat Renal Allografts

The ultimate goal of organ transplantation is to establish graft tolerance where CD4+CD25+FOXP3+ regulatory T (Treg) cells play an important role. We examined whether a superagonistic monoclonal antibody specific for CD28 (CD28 SA), which expands Treg cells in vivo, would prevent acute rejection and induce tolerance using our established rat acute renal allograft model (Wistar to Lewis). In the untreated or mouse IgG‐treated recipients, graft function significantly deteriorated with marked destruction of renal tissue, and all rats died by 13 days with severe azotemia. In contrast, 90% of recipients treated with CD28 SA survived over 100 days, and 70% survived with well‐preserved graft function until graft recovery at 180 days. Analysis by flow cytometry and immunohistochemistry demonstrated that CD28 SA induced marked infiltration of FOXP3+ Treg cells into the allografts. Furthermore, these long‐surviving recipients showed donor‐specific tolerance, accepting secondary (donor‐matched) Wistar cardiac allografts, but acutely rejecting third‐party BN allografts. We further demonstrated that adoptive transfer of CD4+CD25+ Treg cells, purified from CD28 SA‐treated Lewis rats, significantly prolonged allograft survival and succeeded in inducing donor‐specific tolerance. In conclusion, CD28 SA treatment successfully induces donor‐specific tolerance with the involvement of Treg cells, and thus the therapeutic value of this approach warrants further investigation and preclinical studies.     

Donor Fas Is Not Necessary for T‐Cell‐Mediated Rejection of Mouse Kidney Allografts

It is important to resolve whether T‐cell‐mediated rejection (TCMR) is mediated by contact‐dependent cytotoxicity or by contact‐independent inflammatory mechanisms. We recently showed that the cytotoxic molecules perforin and granzymes A and B are not required for TCMR of mouse kidney transplants. Nevertheless, TCMR could still be mediated by cytotoxicity via Fas on donor cells engaging Fas ligand on host T cells. We examined whether the diagnostic TCMR lesions would be abrogated if donor Fas was absent, particularly in hosts deficient in perforin or granzymes A and B. Kidneys from Fas‐deficient donors transplanted into major histocompatibility complex (MHC)‐ mismatched hosts developed tubulitis and diffuse interstitial infiltration indistinguishable from wild‐type (WT) allografts, even in hosts deficient in perforin and granzymes A and B. Gene expression analysis revealed similar molecular disturbances in Fas‐deficient and WT allografts at day 21 transplanted into WT, perforin and granzyme A/B‐deficient hosts, indicating epithelial injury and dedifferentiation. Thus, donor Fas is not necessary for TCMR diagnostic lesions or molecular changes, even in the absence of perforin–granzyme mechanisms. We propose that in TCMR, interstitial effector T cells mediate parenchymal injury by inflammatory mechanisms that require neither the perforin–granzyme nor the Fas–Fas ligand cytotoxic mechanisms.     

Flt3L‐mobilized dendritic cells bearing H2‐Kbm1 apoptotic cells do not induce cross‐tolerance to CD8+ T cells across a class I MHC mismatched barrier

Tolerization of allogeneic CD8⁺ T cells is still a pending issue in the field of transplantation research to achieve long‐term survival. To test whether dendritic cells (DC) bearing allogeneic major histocompatibility complex (MHC) class I mismatched apoptotic cells could induce cross‐tolerance to alloreactive CD8⁺ T cells, the following experimental strategy was devised. Rag2/γ_c KO B6 mice were treated with Fms‐like tyrosine kinase 3 ligand (Flt3L)‐transduced B16 melanoma cells to drive a rapid expansion and mobilization of DC in vivo. Of all DC populations expanded, splenic CD11c⁺CD103⁺CD8α⁺ DC were selectively involved in the process of antigen clearance of X‐ray irradiated apoptotic thymocytes in vivo. Considering that CD11c⁺CD103⁺CD8α⁺ DC selectively take up apoptotic cells and that they are highly specialized in cross‐presenting antigen to CD8⁺ T cells, we investigated whether B6 mice adoptively transferred with Flt3L‐derived DC loaded with donor‐derived apoptotic thymocytes could induce tolerance to bm1 skin allografts. Our findings on host anti‐donor alloresponse, as revealed by skin allograft survival and cytotoxic T lymphocyte assays, indicated that the administration of syngeneic DC presenting K^bm1 donor‐derived allopeptides through the indirect pathway of antigen presentation was not sufficient to induce cross‐tolerance to alloreactive CD8⁺ T cells responding to bm1 alloantigens in a murine model of skin allograft transplantation across an MHC class I mismatched barrier.     

Disruption of Type‐I IFN Pathway Ameliorates Preservation Damage in Mouse Orthotopic Liver Transplantation via HO‐1 Dependent Mechanism

Ischemia/reperfusion injury (IRI) remains unresolved problem in clinical organ transplantation. We analyzed the role of Type‐I interferon (IFN) pathway in a clinically relevant murine model of extended hepatic cold preservation followed by orthotopic liver transplantation (OLT). Livers from Type‐I IFN receptor (IFNAR) knockout (KO) or wild‐type (WT) mice (C57/BL6) were harvested, preserved at 4°C in UW solution for 20 h and transplanted to groups of syngeneic IFNAR KO or WT recipients. Liver graft but not recipient IFNAR deficiency was required to consistently ameliorate IRI in OLTs. Indeed, disruption of Type‐I IFN signaling decreased serum alanine aminotransferase (sALT) levels (p < 0.001), diminished Suzuki's score of histological OLT damage (p < 0.01) and improved 14‐day survival (from 42%[5/12] in WT to 92%[11/12] in IFNAR KO; p < 0.05). Unlike in WT group, IFNAR deficiency attenuated OLT expression of TNF‐α, IL‐1β, IL‐6, MCP‐1, CXCL‐10, ICAM‐1; diminished infiltration by macrophages/PMNs; and enhanced expression of antioxidant HO‐1/Nrf2. The frequency of TUNEL+ apoptotic cells and caspase‐3 activity/expression selectively decreased in IFNAR KO group. Small interfering (si)RNA‐directed targeting of HO‐1 restored cardinal features of liver IRI in otherwise resistant IFNAR‐deficient OLTs. Thus, intact Type‐I IFN signaling is required for hepatic IRI, whereas HO‐1 is needed for cytoprotection against innate immunity‐dominated organ preservation damage in IFNAR‐deficient liver transplants.     

PD-1/B7-H1 Interaction Contribute to the Spontaneous Acceptance of Mouse Liver Allograft

The programmed death-1 (PD-1)/B7-H1 pathway acts as an important negative regulator of immune responses. We herein investigated the role of the PD-1/B7-H1 pathway in establishing an immunological spontaneous tolerance status in mouse liver allografting. B7-H1 is highly expressed on the donor-derived tissue cells and it is also associated with the apoptosis of infiltrating T cells in the allografts. Strikingly, a blockade of the PD-1/B7-H1 pathway via anti-B7-H1mAb or using B7-H1 knockout mice as a donor led to severe cell infiltration as well as hemorrhaging and necrosis, thus resulting in mortality within 12 days. Furthermore, the expression of the FasL, perforin, granzyme B, iNOS and OPN mRNA in the liver allografts increased in the antibody-treated group in comparison to the controls. Taken together, these data revealed that the B7-H1 upregulation on the tissue cells of liver allografts thus plays an important role in the apoptosis of infiltrating cells, which might play a critical role of the induction of the spontaneous tolerance after hepatic transplantation in mice.     

ATG‐Fresenius Treatment and Low‐Dose Tacrolimus: Results of a Randomized Controlled Trial in Liver Transplantation

We report the results of a prospective randomized controlled trial in liver transplantation assessing the efficacy and safety of antithymocyte globulin (ATG‐Fresenius) plus tacrolimus monotherapy at gradually decreasing doses. Patients were randomized to either: (a) standard‐dose tacrolimus plus steroids;or (b) peritransplant ATG‐Fresenius plus reduced‐dose tacrolimus monotherapy followed by weaning of tacrolimus starting 3 months after transplantation. The primary end‐point was the achievement of very low‐dose tacrolimus (every‐other‐day or once daily dose with <5 ng/mL trough levels) at 12 months after transplantation. Acute rejection occurring during the first 3 months after transplantation was more frequent in the ATG group (52.4% vs. 25%). Moreover, late acute rejection episodes occurred in all recipients in whom weaning was attempted and no recipients reached the primary end‐point. This motivated the premature termination of the trial. Tacrolimus trough levels were lower in the ATG‐Fresenius group but no benefits in terms of improved renal function, lower metabolic complications or increased prevalence of tolerance‐related biomarkers were observed. In conclusion, the use of ATG‐Fresenius and tacrolimus at gradually decreasing doses was associated with a high rate of rejection, did not allow for the administration of very low doses of tacrolimus and failed to provide detectable clinical benefits. ClinicalTrials.gov identifier:NCT00436722.     

Inhibition of Innate Co‐Receptor TREM‐1 Signaling Reduces CD4+ T Cell Activation and Prolongs Cardiac Allograft Survival

The innate receptor “triggering‐receptor‐expressed‐on‐myeloid‐cells‐1” (TREM‐1) enhances downstream signaling of “pattern recognition receptor” (PRR) molecules implicated in inflammatory responses. However the mechanistic role of TREM‐1 in chronic heart rejection has yet to be elucidated. We examined the effect of TREM‐1⁺ antigen‐presenting cells (APC) on alloreactive CD4⁺ lymphocytes. Bm12 donor hearts were transplanted into wild‐type MHC‐class‐II‐mismatched C57BL/6J recipient mice. Progressive allograft rejection of bm12‐donor hearts with decreased organ function, severe vasculopathy and allograft fibrosis was evident within 4 weeks. TREM‐1⁺CD11b⁺MHC‐II⁺F4/80⁺CCR2⁺ APC and IFNγ‐producing CD4⁺ cells were detected during chronic rejection. Peptide inhibition of TREM‐1 attenuated graft vasculopathy, reduced graft‐infiltrating leukocytes and prolonged allograft survival, while being accompanied by sustained low levels of CD4⁺ and CD8⁺ cell infiltration. Remarkably, temporary inhibition of TREM‐1 during early immune activation was sufficient for long‐term allograft survival. Mechanistically, TREM‐1 inhibition leads to reduced differentiation and proliferation of IFNγ‐producing Th1 cells. In conclusion, TREM‐1 influences chronic heart rejection by regulating the infiltration and differentiation of CD4⁺ lymphocytes.     

An objective definition for clinical suspicion of T‐cell‐mediated rejection after liver transplantation

A uniform definition of clinical suspicion of T‐cell‐mediated rejection (TCMR) in liver transplantation (LT) is needed to homogenize clinical decisions, especially within randomized trials. This multicenter study included a total of 470 primary LT recipients. The derivation cohort consisted of 142 patients who had clinically driven liver biopsies at any time after LT. The external validation cohort included 328 patients who underwent protocol biopsies at day 7‐10 after LT. The rates of moderate‐severe histological TCMR were 33.8% in the derivation cohort and 43.6% in the validation cohort. Independent predictors (ie, risk factors) of moderate‐severe TCMR in the derivation cohort were as follows: serum bilirubin >4 mg/dL (OR=5.83; P<.001), rising bilirubin within the 4 days prior to liver biopsy (OR=4.57; P=.003), and blood eosinophils count >0.1×10⁹/L (OR=3.81; P=.004). In the validation cohort, the number of risk factors was an independent predictor of moderate‐severe TCMR (OR=1.74; P=.001), after controlling for hepatitis C status. The number of risk factors paralleled the rates of moderate‐severe TCMR in the derivation and validation cohorts (P<.001 in both comparisons). In conclusion, increased serum bilirubin, rising bilirubin and eosinophilia are validated risk factors for moderate‐severe histological TCMR and could be used as objective criteria to select candidates for liver biopsy.     

Human CMV‐specific T‐cell responses in kidney transplantation; toward changing current risk‐stratification paradigm

Despite the great efficacy of current antiviral preventive strategies, hCMV infection is still a major complication after renal transplantation, significantly challenging patient and graft survival. This issue seems to be explained because of the rather poor immunologic monitoring of the antiviral immune response. An important body of evidence has shown that monitoring the hCMV‐specific T‐cell response, at different time points of the transplant setting, seems to add crucial information for predicting the risk of viral infection, thus potentially helping individualization of therapeutic decision‐making in clinical transplantation. While several immune‐cellular assays have shown its capability for accurately monitoring hCMV‐specific T‐cell responses, only few such as the IFN‐γ ELISPOT and the ELISA based technology assays might be reliable for its application in the clinic. Nonetheless, an important effort has to be made among the transplant community to standardize and validate such immune assays. Noteworthy, large‐scale prospective randomized trials are highly warranted to ultimately introduce them in current clinical practice as a part of the highly desired personalized medicine.     

The Aggressive Phenotype Revisited: Utilization of Higher‐Risk Liver Allografts

Organ shortage has led to increased utilization of higher risk liver allografts. In kidneys, aggressive center‐level use of one type of higher risk graft clustered with aggressive use of other types. In this study, we explored center‐level behavior in liver utilization. We aggregated national liver transplant recipient data between 2005 and 2009 to the center‐level, assigning each center an aggressiveness score based on relative utilization of higher risk livers. Aggressive centers had significantly more patients reaching high MELDs (RR 2.19, 2.33 and 2.28 for number of patients reaching MELD > 20, MELD > 25 and MELD > 30, p < 0.001), a higher organ shortage ratio (RR 1.51, 1.60 and 1.51 for number of patients reaching MELD > 20, MELD > 25 and MELD > 30 divided by number of organs recovered at the OPO, p < 0.04), and were clustered within various geographic regions, particularly regions 2, 3 and 9. Median MELD at transplant was similar between aggressive and nonaggressive centers, but average annual transplant volume was significantly higher at aggressive centers (RR 2.27, 95% CI 1.47–3.51, p < 0.001). In cluster analysis, there were no obvious phenotypic patterns among centers with intermediate levels of aggressiveness. In conclusion, highwaitlist disease severity, geographic differences in organ availability, and transplant volume are the main factors associated with the aggressive utilization of higher risk livers.     

Proliferation of CD8‐Positive T Cells in Blood Vessels of Rat Renal Allografts

It is still disputed in which anatomical compartments of allograft recipients T‐cells proliferate. After experimental renal transplantation, host monocytes and lymphocytes accumulate in the lumina of graft blood vessels. In this study, we test the hypothesis that T lymphocytes proliferate in the vascular bed of the graft. Kidneys were transplanted in the Dark Agouti to Lewis rat strain combination, an established experimental model for acute rejection. Isogeneic transplantation was performed as a control. Cells in the S‐phase of mitosis were detected in situ three days posttransplantation by pulse‐labeling with BrdU and by immunohistochemical detection of the proliferating cell nuclear antigen (PCNA). More than 20% of all T‐cells in the lumina of allograft blood vessels incorporated BrdU and approximately 30% of them expressed PCNA. In the blood vessels of isografts as well as in other organs of allograft recipients, only few BrdU⁺ cells were detected. A majority of the BrdU⁺ cells in graft blood vessels expressed CD8. In conclusion, we demonstrate that CD8⁺ T lymphocytes proliferate in the lumina of the blood vessels of renal allografts during the onset of acute rejection.     

Long‐Term Medical and Psychosocial Outcomes in Living Liver Donors

Due to the enduring organ shortage, living donor liver transplantation has been a valuable treatment strategy for advanced liver disease patients for over 20 years. A variety of reviews have summarized the extensive data now available on medical and psychosocial risks to living donors in the aftermath of donation. However, evidence on donor medical and psychosocial outcomes beyond the first year postdonation has not been synthesized in any previous review. The evidence base on such “long‐term” outcomes has been growing in recent years. A review of this evidence would therefore be timely and could serve as an important resource to assist transplant centers in their efforts to fully educate prospective donors and gain informed consent, as well as develop appropriate postdonation clinical care and surveillance plans. We reviewed recent literature on long‐term donor outcomes, considering (a) medical outcomes, including mortality risk, rates of complications, abnormalities detected in laboratory testing, and the progress of liver regeneration; and (b) donor‐reported psychosocial outcomes reflecting physical, emotional, and interpersonal/socioeconomic well‐being, as well as overall health‐related quality of life. We summarize limitations and gaps in available evidence, and we provide recommendations for future research and clinical care activities focused on long‐term outcomes in liver donors.