Incidence, epidemiology, and outcomes of acute allograft rejection following liver transplantation in Australia.

Acute allograft rejection is a well-known complication of liver transplantation (LT). The incidence, epidemiology, and outcomes of acute rejection have not been well described in Australia. We retrospectively studied consecutive adults who underwent deceased donor LT at a single center between 2010 and 2020. Donor and recipient data at the time of LT and recipient outcomes were collected from a prospective LT database. Liver biopsy reports were reviewed, and only a graft's first instance of biopsy-proven acute rejection was analyzed. During the study period, 796 liver transplants were performed in 770 patients. Biopsy-proven rejection occurred in 34.9% of transplants. There were no significant changes in the incidence of rejection over time (linear trend p =0.11). The median time to the first episode of rejection was 71 days after LT: 2.2% hyperacute, 50.4% early (≤90 d), and 47.5% late rejection (>90 d). Independent risk factors for rejection were younger recipient age at transplant (aHR 0.98 per year increase, 95% CI: 0.97-1.00, p =0.01), and ABO-incompatible grafts (aHR 2.55 vs. ABO-compatible, 95% CI: 1.27-5.09, p <0.01) while simultaneous multiorgan transplants were protective (aHR 0.21 vs. LT only, 95% CI: 0.08-0.58, p <0.01). Development of acute rejection (both early and late) was independently associated with significantly reduced graft (aHR 3.13, 95% CI: 2.21-4.42, p <0.001) and patient survival (aHR 3.42, 95% CI: 2.35-4.98, p <0.001). In this 11-year Australian study, acute LT rejection occurred in 35%, with independent risk factors of younger recipient age and ABO-incompatible transplant, while having a simultaneous multiorgan transplant was protective. Acute rejection was independently associated with reduced graft and patient survival after adjustment for other factors.

Nicotinamide for Skin-Cancer Chemoprevention in Transplant Recipients.

BACKGROUND: Immunosuppressed organ-transplant recipients have an increased incidence of, and mortality from, skin cancer. Nicotinamide (vitamin B3) enhances the repair of ultraviolet (UV) radiation-induced DNA damage, reduces the cutaneous immunosuppressive effects of UV radiation, and reduces the incidence of keratinocyte cancers (including squamous-cell and basal-cell carcinomas) and actinic keratoses among high-risk immunocompetent patients. Whether oral nicotinamide is useful for skin-cancer chemoprevention in organ-transplant recipients is unclear. METHODS: In this phase 3 trial, we randomly assigned, in a 1:1 ratio, organ-transplant recipients who had had at least two keratinocyte cancers in the past 5 years to receive 500 mg of nicotinamide or placebo twice daily for 12 months. Participants were examined for skin lesions by dermatologists at 3-month intervals for 12 months. The primary end point was the number of new keratinocyte cancers during the 12-month intervention period. Secondary end points included the numbers of squamous-cell and basal-cell carcinomas during the 12-month intervention period, the number of actinic keratoses until 6 months after randomization, safety, and quality of life. RESULTS: A total of 158 participants were enrolled, with 79 assigned to the nicotinamide group and 79 to the placebo group. The trial was stopped early owing to poor recruitment. At 12 months, there were 207 new keratinocyte cancers in the nicotinamide group and 210 in the placebo group (rate ratio, 1.0; 95% confidence interval, 0.8 to 1.3; P = 0.96). No significant between-group differences in squamous-cell and basal-cell carcinoma counts, actinic keratosis counts, or quality-of-life scores were observed. Adverse events and changes in blood or urine laboratory variables were similar in the two groups. CONCLUSIONS: In this 12-month, placebo-controlled trial, oral nicotinamide therapy did not lead to lower numbers of keratinocyte cancers or actinic keratoses in immunosuppressed solid-organ transplant recipients. (Funded by the National Health and Medical Research Council; ONTRANS Australian New Zealand Clinical Trials Registry number, ACTRN12617000599370.).

Association between vessels that encapsulate tumour clusters vascular pattern and hepatocellular carcinoma recurrence following liver transplantation.

Background: Vessels that encapsulate tumor clusters (VETC) is a novel vascular pattern seen on hepatocellular carcinoma (HCC) histology which has been shown to independently predict tumor recurrence and survival after liver resection. Its prognostic value in HCC patients receiving liver transplantation (LT) is unclear. Methods: We retrospectively studied consecutive adults who underwent deceased-donor LT with active HCC found on explant between 2010-2019. Tumor tissue was stained for CD34 and quantified for VETC. Primary and secondary endpoints were time to recurrence (TTR) and recurrence-free survival (RFS). Results: During the study period, 158 patients received LT where HCC was present on explant. VETC pattern was seen in 76.5% of explants. Patients with VETC-positive tumors spent longer on the waitlist (6.4 vs. 4.1 months, P=0.048), had higher median tumor numbers (2 vs. 1, P=0.001) and larger tumor sizes (20mm vs. 13mm, P<0.001) on explant pathology compared to those with VETC-negative tumors. Correspondingly, VETC-positive patients were more likely to be outside of accepted LT criteria for HCC. After 56.4 months median follow-up, 8.2% of patients developed HCC recurrence post-LT. On multivariable Cox regression, presence of VETC pattern did not predict TTR or RFS. However, the number of VETC-positive tumors on explant was an independent predictor of TTR (hazard ratio [HR] 1.411, P=0.001) and RFS (HR 1.267, P=0.014) after adjusting for other significant variables. Conclusion: VETC pattern is commonly observed in HCC patients undergoing LT. The number of VETC-positive tumors, but not its presence, is an independent risk factor for TTR and RFS post-LT.

Liver-specific deletion of miR-181ab1 reduces liver tumour progression via upregulation of CBX7.

MiR-181 expression levels increased in hepatocellular carcinoma (HCC) compared to non-cancerous tissues. MiR-181 has been widely reported as a possible driver of tumourigenesis but also acts as a tumour suppressor. In addition, the miR-181 family regulates the development and function of immune and vascular cells, which play vital roles in the progression of tumours. More complicatedly, many genes have been identified as miR-181 targets to mediate the effects of miR-181. However, the role of miR-181 in the development of primary tumours remains largely unexplored. We aimed to examine the function of miR-181 and its vital mediators in the progression of diethylnitrosamine-induced primary liver cancers in mice. The size of liver tumours was significantly reduced by 90% in global (GKO) or liver-specific (LKO) 181ab1 knockout mice but not in hematopoietic and endothelial lineage-specific knockout mice, compared to WT mice. In addition, the number of tumours was significantly reduced by 50% in GKO mice. Whole-genome RNA-seq analysis and immunohistochemistry showed that epithelial-mesenchymal transition was partially reversed in GKO tumours compared to WT tumours. The expression of CBX7, a confirmed miR-181 target, was up-regulated in GKO compared to WT tumours. Stable CBX7 expression was achieved with an AAV/Transposase Hybrid-Vector System and up-regulated CBX7 expression inhibited liver tumour progression in WT mice. Hepatic CBX7 deletion restored the progression of LKO liver tumours. MiR-181a expression was the lowest and CBX7 expression the highest in iClust2 and 3 subclasses of human HCC compared to iClust1. Gene expression profiles of GKO tumours overlapped with low-proliferative peri-portal-type HCCs. Liver-specific loss of miR-181ab1 inhibited primary liver tumour progression via up-regulating CBX7 expression, but tumour induction requires both hepatic and non-hepatic miR-181. Also, miR-181ab1-deficient liver tumours may resemble low-proliferative periportal-type human HCC. miR-181 was increased with liver tumour growth. More miR-181, darker colour and higher shape. CBX7 was very low in pericentral hepatocytes, increased in early liver tumours, but reduced in advanced liver tumours. Its levels were maintained in miR-181 KO liver tumours. In tumours (T), brown (darker is more) represents miR-181, the blue circle (thicker is more) represents CBX7.

The liver contains distinct interconnected networks of CX3CR1+ macrophages, XCR1+ type 1 and CD301a+ type 2 conventional dendritic cells embedded within portal tracts.

Portal tracts are key intrahepatic structures where leukocytes accumulate during immune responses. They contain the blood inflow, which includes portal blood from the gut, and lymphatic and biliary outflow of the liver, and as such represent a key interface for potential pathogen entry to the liver. Myeloid cells residing in the interstitium of the portal tract might play an important role in the surveillance or prevention of pathogen dissemination; however, the exact composition and localization of this population has not been explored fully. Our in-depth characterization of portal tract myeloid cells revealed that in addition to T lymphocytes, portal tracts contain a heterogeneous population of MHCIIhigh myeloid cells with potential antigen presenting cell (APC) function. These include a previously unreported subset of CSF1R-dependent CX3CR1+ macrophages that phenotypically and morphologically resemble liver capsular macrophages, as well as the two main dendritic cell subsets (cDC1 and cDC2). These cells are not randomly distributed, but each subset forms interconnected networks intertwined with specific components of the portal tract. The CX3CR1+ cells were preferentially detected along the outer border of the portal tracts, and also in the portal interstitium adjacent to the portal vein, bile duct, lymphatic vessels and hepatic artery. cDC1s abounded along the lymphatic vessels, while cDC2s mostly surrounded the biliary tree. The specific distributions of these discrete subsets predict that they may serve distinct functions in this compartment. Overall, our findings suggest that portal tracts and their embedded cellular networks of myeloid cells form a distinctive lymphoid compartment in the liver that has the potential to orchestrate immune responses in this organ.

The self-peptide repertoire plays a critical role in transplant tolerance induction.

While direct allorecognition underpins both solid organ allograft rejection and tolerance induction, the specific molecular targets of most directly alloreactive CD8+ T cells have not been defined. In this study, we used a combination of genetically engineered major histocompatibility complex class I (MHC I) constructs, mice with a hepatocyte-specific mutation in the class I antigen-presentation pathway, and immunopeptidomic analysis to provide definitive evidence for the contribution of the peptide cargo of allogeneic MHC I molecules to transplant tolerance induction. We established a systematic approach for the discovery of directly recognized pMHC epitopes and identified 17 strongly immunogenic H-2Kb-associated peptides recognized by CD8+ T cells from B10.BR (H-2k) mice, 13 of which were also recognized by BALB/c (H-2d) mice. As few as 5 different tetramers used together were able to identify a high proportion of alloreactive T cells within a polyclonal population, suggesting that there are immunodominant allogeneic MHC-peptide complexes that can account for a large component of the alloresponse.

Glycolipid-peptide vaccination induces liver-resident memory CD8+ T cells that protect against rodent malaria.

Liver resident-memory CD8+ T cells (TRM cells) can kill liver-stage Plasmodium-infected cells and prevent malaria, but simple vaccines for generating this important immune population are lacking. Here, we report the development of a fully synthetic self-adjuvanting glycolipid-peptide conjugate vaccine designed to efficiently induce liver TRM cells. Upon cleavage in vivo, the glycolipid-peptide conjugate vaccine releases an MHC I-restricted peptide epitope (to stimulate Plasmodium-specific CD8+ T cells) and an adjuvant component, the NKT cell agonist α-galactosylceramide (α-GalCer). A single dose of this vaccine in mice induced substantial numbers of intrahepatic malaria-specific CD8+ T cells expressing canonical markers of liver TRM cells (CD69, CXCR6, and CD101), and these cells could be further increased in number upon vaccine boosting. We show that modifications to the peptide, such as addition of proteasomal-cleavage sequences or epitope-flanking sequences, or the use of alternative conjugation methods to link the peptide to the glycolipid improved liver TRM cell generation and led to the development of a vaccine able to induce sterile protection in C57BL/6 mice against Plasmodium berghei sporozoite challenge after a single dose. Furthermore, this vaccine induced endogenous liver TRM cells that were long-lived (half-life of ~425 days) and were able to maintain >90% sterile protection to day 200. Our findings describe an ideal synthetic vaccine platform for generating large numbers of liver TRM cells for effective control of liver-stage malaria and, potentially, a variety of other hepatotropic infections.

A Natural Peptide Antigen within the Plasmodium Ribosomal Protein RPL6 Confers Liver TRM Cell-Mediated Immunity against Malaria in Mice.

Liver-resident memory CD8+ T (TRM) cells remain in and constantly patrol the liver to elicit rapid immunity upon antigen encounter and can mediate efficient protection against liver-stage Plasmodium infection. This finding has prompted the development of immunization strategies where T cells are activated in the spleen and then trapped in the liver to form TRM cells. Here, we identify PbRPL6120-127, a H2-Kb-restricted epitope from the putative 60S ribosomal protein L6 (RPL6) of Plasmodium berghei ANKA, as an optimal antigen for endogenous liver TRM cell generation and protection against malaria. A single dose vaccination targeting RPL6 provided effective and prolonged sterilizing immunity against high dose sporozoite challenges. Expressed throughout the parasite life cycle, across Plasmodium species, and highly conserved, RPL6 exhibits strong translation potential as a vaccine candidate. This is further advocated by the identification of a broadly conserved, immunogenic HLA-A∗02:01-restricted epitope in P. falciparum RPL6.

CD147 mediates intrahepatic leukocyte aggregation and determines the extent of liver injury.

BACKGROUND: Chronic inflammation is the driver of liver injury and results in progressive fibrosis and eventual cirrhosis with consequences including both liver failure and liver cancer. We have previously described increased expression of the highly multifunctional glycoprotein CD147 in liver injury. This work describes a novel role of CD147 in liver inflammation and the importance of leukocyte aggregates in determining the extent of liver injury. METHODS: Non-diseased, progressive injury, and cirrhotic liver from humans and mice were examined using a mAb targeting CD147. Inflammatory cell subsets were assessed by multiparameter flow cytometry. RESULTS: In liver injury, we observe abundant, intrahepatic leukocyte clusters defined as ≥5 adjacent CD45+ cells which we have termed "leukocyte aggregates". We have shown that these leukocyte aggregates have a significant effect in determining the extent of liver injury. If CD147 is blocked in vivo, these leukocyte aggregates diminish in size and number, together with a marked significant reduction in liver injury including fibrosis. This is accompanied by no change in overall intrahepatic leukocyte numbers. Further, blocking of aggregation formation occurs prior to an appreciable increase in inflammatory markers or fibrosis. Additionally, there were no observed, "off-target" or unpredicted effects in targeting CD147. CONCLUSION: CD147 mediates leukocyte aggregation which is associated with the development of liver injury. This is not a secondary effect, but a cause of injury as aggregate formation proceeds other markers of injury. Leukocyte aggregation has been previously described in inflammation dating back over many decades. Here we demonstrate that leukocyte aggregates determine the extent of liver injury.

Single-Dose Vaccination with a Hepatotropic Adeno-associated Virus Efficiently Localizes T Cell Immunity in the Liver with the Potential To Confer Rapid Protection against Hepatitis C Virus.

Hepatitis C virus (HCV) is a significant contributor to the global disease burden, and development of an effective vaccine is required to eliminate HCV infections worldwide. CD4+ and CD8+ T cell immunity correlates with viral clearance in primary HCV infection, and intrahepatic CD8+ tissue-resident memory T (TRM) cells provide lifelong and rapid protection against hepatotropic pathogens. Consequently, we aimed to develop a vaccine to elicit HCV-specific CD4+ and CD8+ T cells, including CD8+ TRM cells, in the liver, given that HCV primarily infects hepatocytes. To achieve this, we vaccinated wild-type BALB/c mice with a highly immunogenic cytolytic DNA vaccine encoding a model HCV (genotype 3a) nonstructural protein (NS5B) and a mutant perforin (pVAX-NS5B-PRF), as well as a recombinant adeno-associated virus (AAV) encoding NS5B (rAAV-NS5B). A novel fluorescent target array (FTA) was used to map immunodominant CD4+ T helper (TH) cell and cytotoxic CD8+ T cell epitopes of NS5B in vivo, which were subsequently used to design a KdNS5B451-459 tetramer and analyze NS5B-specific T cell responses in vaccinated mice in vivo The data showed that intradermal prime/boost vaccination with pVAX-NS5B-PRF was effective in eliciting TH and cytotoxic CD8+ T cell responses and intrahepatic CD8+ TRM cells, but a single intravenous dose of hepatotropic rAAV-NS5B was significantly more effective. As a T-cell-based vaccine against HCV should ideally result in localized T cell responses in the liver, this study describes primary observations in the context of HCV vaccination that can be used to achieve this goal.IMPORTANCE There are currently at least 71 million individuals with chronic HCV worldwide and almost two million new infections annually. Although the advent of direct-acting antivirals (DAAs) offers highly effective therapy, considerable remaining challenges argue against reliance on DAAs for HCV elimination, including high drug cost, poorly developed health infrastructure, low screening rates, and significant reinfection rates. Accordingly, development of an effective vaccine is crucial to HCV elimination. An HCV vaccine that elicits T cell immunity in the liver will be highly protective for the following reasons: (i) T cell responses against nonstructural proteins of the virus are associated with clearance of primary infection, and (ii) long-lived liver-resident T cells alone can protect against malaria infection of hepatocytes. Thus, in this study we exploit promising vaccination platforms to highlight strategies that can be used to evoke highly functional and long-lived T cell responses in the liver for protection against HCV.

Direct recognition of hepatocyte-expressed MHC class I alloantigens is required for tolerance induction.

Adeno-associated viral vector-mediated (AAV-mediated) expression of allogeneic major histocompatibility complex class I (MHC class I) in recipient liver induces donor-specific tolerance in mouse skin transplant models in which a class I allele (H-2Kb or H-2Kd) is mismatched between donor and recipient. Tolerance can be induced in mice primed by prior rejection of a donor-strain skin graft, as well as in naive recipients. Allogeneic MHC class I may be recognized by recipient T cells as an intact molecule (direct recognition) or may be processed and presented as an allogeneic peptide in the context of self-MHC (indirect recognition). The relative contributions of direct and indirect allorecognition to tolerance induction in this setting are unknown. Using hepatocyte-specific AAV vectors encoding WT allogeneic MHC class I molecules, or class I molecules containing a point mutation (D227K) that impedes direct recognition of intact allogeneic MHC class I by CD8+ T cells without hampering the presentation of processed peptides derived from allogeneic MHC class I, we show here that tolerance induction depends upon recognition of intact MHC class I. Indirect recognition alone yielded a modest prolongation of subsequent skin graft survival, attributable to the generation of CD4+ Tregs, but it was not sufficient to induce tolerance.

Molecular profiling of postreperfusion milieu determines acute kidney injury after liver transplantation: A prospective study.

Acute kidney injury (AKI) after liver transplantation (LT) is a common event, but its pathogenesis remains unclear. The aim of this prospective study is to investigate the potential relationship between postreperfusion gene expression, serum mediators, and the onset of AKI after LT. Sixty-five consecutive patients undergoing LT were included in the study. Reverse transcription polymerase chain reaction (PCR) was performed on liver biopsies. Gene expression of 23 genes involved in ischemia/reperfusion injury (IRI) was evaluated. The serum concentrations of endothelin (ET)-1 and inflammatory cytokines were analyzed. AKI after LT developed in 21 (32%) recipients (AKI group). Reverse transcription PCR of reperfusion biopsy in the AKI group showed higher expression of several genes involved in IRI compared with the non-AKI group. Fold changes in the gene expression of ET-1, interleukin (IL) 18, and tumor necrosis factor α (TNF-α) were associated with creatinine peak value. AKI patients also had significantly higher ET-1, IL18, and TNF-α postoperative serum levels. Multivariate analysis showed that ET-1 (odds ratio [OR], 16.7; 95% confidence interval [CI], 3.34-83.42; P = 0.001) and IL18 (OR, 5.27; 95% CI, 0.99-27.82, P = 0.048) serum levels on postoperative day 1 were independently predictive of AKI. Receiver operating characteristic analysis demonstrated that the combination of biomarkers ET-1+IL18 was highly predictive of AKI (area under the receiver operating characteristic curve, 0.91; 95% CI, 0.83-0.99). Early allograft dysfunction and chronic kidney disease stage ≥ 2 occurred more frequently in AKI patients. These results suggest that the graft itself, rather than intraoperative hemodynamic instability, plays a main role in AKI after LT. These data may have mechanistic and diagnostic implications for AKI after LT. Liver Transplantation 24 922-931 2018 AASLD.

A Liver Capsular Network of Monocyte-Derived Macrophages Restricts Hepatic Dissemination of Intraperitoneal Bacteria by Neutrophil Recruitment.

The liver is positioned at the interface between two routes traversed by pathogens in disseminating infection. Whereas blood-borne pathogens are efficiently cleared in hepatic sinusoids by Kupffer cells (KCs), it is unknown how the liver prevents dissemination of peritoneal pathogens accessing its outer membrane. We report here that the hepatic capsule harbors a contiguous cellular network of liver-resident macrophages phenotypically distinct from KCs. These liver capsular macrophages (LCMs) were replenished in the steady state from blood monocytes, unlike KCs that are embryonically derived and self-renewing. LCM numbers increased after weaning in a microbiota-dependent process. LCMs sensed peritoneal bacteria and promoted neutrophil recruitment to the capsule, and their specific ablation resulted in decreased neutrophil recruitment and increased intrahepatic bacterial burden. Thus, the liver contains two separate and non-overlapping niches occupied by distinct resident macrophage populations mediating immunosurveillance at these two pathogen entry points to the liver.

Patients with non-viral liver disease have a greater tumor burden and less curative treatment options when diagnosed with hepatocellular carcinoma.

AIM: To assess the impact of underlying liver disease etiology on the presenting features and outcomes in a large cohort of patients with hepatocellular carcinoma (HCC). METHODS: A prospective database of all patients with HCC was established from 1998 to March 2012. One thousand and seventy-eight patients were categorized into three groups, based on the etiology of their liver disease: hepatitis B virus (HBV), hepatitis C virus (HCV) and non-viral liver disease (NVLD). Overall survival was determined by Kaplan Meier analysis to time of death or last follow-up. RESULTS: HCC patients with HCV (85%) were more likely to be diagnosed as part of a surveillance program, compared to HBV or NVLD (both 71%) (P < 0.001). Patients with NVLD were more likely to receive best supportive care (29%) compared to those with HBV (21%) or HCV (20%) (P < 0.02). Twelve percent of NVLD and 13% of HBV patients underwent liver transplantation compared to 21% of HCV patients (P = 0.001). Median survival from presentation was lowest in NVLD (1.7 years) when compared to HBV (2.8 years) and HCV (2.6 years) (P < 0.05). In multivariate analysis, independent predictors of survival included Child Turcotte Pugh score, size of dominant lesion, absence of vascular invasion, and management with surgical resection or liver transplantation. Patient age and the etiology of the underlying liver disease were not independent predictors of survival. CONCLUSION: Patients with NVLD and HCC were less likely to be enrolled in a HCC surveillance program and are less likely to have curative therapies such as liver resection and transplantation after diagnosis with HCC, when compared to patients with Hepatitis B and Hepatitis C.

miR-181a mediates TGF-ß-induced hepatocyte EMT and is dysregulated in cirrhosis and hepatocellular cancer.

BACKGROUND & AIMS: Epithelial-mesenchymal transition (EMT) has been implicated in the processes of embryogenesis, tissue fibrosis and carcinogenesis. Transforming growth factor-ß (TGF-ß) has been identified as a key driver of EMT and plays a key role in the pathogenesis of cirrhosis and hepatocellular carcinoma (HCC). The aim was to identify microRNA (miR) expression in TGF-ß-induced hepatocyte EMT. METHODS: We treated a human hepatocyte cell line PH5CH8 with TGF-ß to induce an EMT-like change in phenotype and then identified dysregulated miRs using TaqMan Low Density Arrays. MiR expression was altered using miR-181a mimic and inhibitor in the same system and gene changes were identified using TaqMan gene arrays. MiR-181a gene expression was measured in human and mouse cirrhotic or HCC liver tissue samples. Gene changes were identified in rAAV-miR-181a-expressing mouse livers using TaqMan gene arrays. RESULTS: We identified miR-181a as a miR that was significantly up-regulated in response to TGF-ß treatment. Over-expression of a miR-181a mimic induced an in vitro EMT-like change with a phenotype similar to that seen with TGF-ß treatment alone and was reversed using a miR-181a inhibitor. MiR-181a was shown to be up-regulated in experimental and human cirrhotic and HCC tissue. Mouse livers expressing rAAV-miR-181a showed genetic changes associated with TGF-ß signalling and EMT. CONCLUSIONS: MiR-181a had a direct effect in inducing hepatocyte EMT and was able to replace TGF-ß-induced effects in vitro. MiR-181a was over-expressed in cirrhosis and HCC and is likely to play a role in disease pathogenesis.

Analysis of the intrahepatic ductular reaction and progenitor cell responses in hepatitis C virus recurrence after liver transplantation.

Fibrosis in livers with hepatitis C virus (HCV) recurrence after liver transplantation (LT) can be rapidly progressive, and the mechanisms underlying this process are poorly understood. In livers with HCV infections in the non-LT setting, there is a significant relationship between the development of structures known as the ductular reaction (DR), hepatic progenitor cells (HPCs), and fibrosis. This study characterizes the DR, HPCs, and fibrosis associated with HCV recurrence after LT. Immunohistochemistry and confocal microscopy were used to characterize the DR, HPC, and fibrosis in liver biopsy specimens. Key findings were confirmed in a separate, independent cohort. The initial characterization cohort had 194 biopsy samples from 105 individuals with HCV recurrence after LT. The immunophenotype, morphology, and location of the DR were consistent with an HPC origin. The DR correlated with intrahepatic fibrosis (rs = 0.529, P < 0.001) and the number of activated hepatic stellate cells (HSCs; rs = 0.446, P < 0.001). There was an early occurrence of hepatocyte replicative arrest as well as increased hepatocyte proliferation that correlated with the DR (rs = 0.295, P < 0.001). Replicative arrest preceded hepatocyte proliferation in early-stage injury. Hepatocyte proliferation decreased with advanced fibrosis; in contrast, the extent of the DR and the number of activated HSCs continued to increase. In the second cohort of 37 individuals, the DR and the number of HPCs similarly correlated with fibrosis and inflammation after LT. In conclusion, this is the first characterization of the DR in HCV-associated liver injury after LT. There was a significant correlation between the DR and the development of progressive fibrosis in HCV recurrence. These results suggest a pivotal role for both the DR and the HPC responses in the aggressive fibrosis seen with HCV recurrence after LT.

Intrahepatic activation of naive CD4+ T cells by liver-resident phagocytic cells.

Naive T cell activation is normally restricted to the lymphoid organs, in part because of their limited ability to migrate into the parenchyma of peripheral tissues. The liver vasculature is unique, however, and circulating leukocytes within the hepatic sinusoids have direct access to liver-resident cells, which include an abundant population of Kupffer cells. It is well accepted that recognition of cognate Ag within the liver leads to naive CD8(+) T cell activation in situ, but it is unclear whether the liver also supports naive CD4(+) T cell activation. In this study, we show that naive CD4(+) T cells can be activated to proliferate in the liver when cognate Ag expression is induced in hepatocytes by recombinant adeno-associated viral vectors. Ag-specific retention and activation of naive CD4(+) T cells within the liver are independent of lymphoid tissues but dependent on a clodronate liposome-sensitive population of liver-resident phagocytic cells. To our knowledge, this study provides the first unequivocal evidence that naive CD4(+) T cells can be activated in a nonlymphoid organ. It also gives critical insight into how CD4(+) T cells specific for Ag expressed in the liver are recruited to participate in protective or pathological responses during hepatotropic infections and autoimmune liver disease.

Antigen expression level threshold tunes the fate of CD8 T cells during primary hepatic immune responses.

CD8 T-cell responses to liver-expressed antigens range from deletional tolerance to full effector differentiation resulting in overt hepatotoxicity. The reasons for these heterogeneous outcomes are not well understood. To identify factors that govern the fate of CD8 T cells activated by hepatocyte-expressed antigen, we exploited recombinant adenoassociated viral vectors that enabled us to vary potential parameters determining these outcomes in vivo. Our findings reveal a threshold of antigen expression within the liver as the dominant factor determining T-cell fate, irrespective of T-cell receptor affinity or antigen cross-presentation. Thus, when a low percentage of hepatocytes expressed cognate antigen, high-affinity T cells developed and maintained effector function, whereas, at a high percentage, they became functionally exhausted and silenced. Exhaustion was not irreversibly determined by initial activation, but was maintained by high intrahepatic antigen load during the early phase of the response; cytolytic function was restored when T cells primed under high antigen load conditions were transferred into an environment of low-level antigen expression. Our study reveals a hierarchy of factors dictating the fate of CD8 T cells during hepatic immune responses, and provides an explanation for the different immune outcomes observed in a variety of immune-mediated liver pathologic conditions.