Distinct Immune Imprints of Post–Liver Transplantation Hepatitis C Persist Despite Viral Clearance

Recurrence or de novo infection of hepatitis C virus (HCV) after liver transplantation (LT) has been associated with progressive graft hepatitis that can be improved by treatment with novel direct‐acting antivirals. Cases of rejection episodes have been described during and after HCV treatment. The evolution of innate and adaptive immune response during and after cure of HCV LT is unknown. We studied 74 protein biomarkers in the plasma of LT patients receiving antiviral therapy. In addition, deep immune phenotyping of both the myeloid and lymphoid immune cell subsets in peripheral blood mononuclear cells was performed. We found that LT patients with active HCV infection displayed distinct alterations of inflammatory protein biomarkers, such as C‐X‐Cmotif chemokine 10 (CXCL10), caspase 8, C‐C motif chemokine 20 (CCL20), CCL19, interferon γ, CUB domain‐containing protein 1 (CDCP1), interleukin (IL)‐18R1, CXCL11, CCL3, IL8, IL12B, tumor necrosis factor‐beta, CXCL6, osteoprotegerin, IL10, fms‐related tyrosine kinase 3 ligand, hepatocyte growth factor, urokinase‐type plasminogen activator, neurotrophin‐3, CCL4, IL6, tumornecrosis factor receptor superfamily member 9, programmed death ligand 1, IL18, and monocyte chemotactic protein 1, and enrichment of peripheral immune cell subsets unlike patients without HCV infection who received transplants. Interestingly, patients who cleared HCV after LT did not normalize the altered inflammatory milieu nor did the peripheral immune cell subsets normalize to what would be seen in the absence of HCV recurrence. Overall, these data indicate that HCV‐specific imprints on inflammatory analytes and immune cell subsets after LT are not completely normalized by therapy‐induced HCV elimination. This is in line with the clinical observation that cure of HCV after LT did not trigger rejection episodes in many patients.

ubiquitously observed in almost all patients who are viremic at the time of LT. (2) The disease prognosis following HCV recurrence or de novo infection has been rapid, leading to fibrosis and cirrhosis in less than 5 years and associated with worse outcomes compared with LTs performed for other indications. (3)(4)(5) Fortunately, treatment with recently approved directacting antiviral (DAA) regimens against HCV are shown to be equally effective and safe as in patients who did not receive transplants with almost all posttransplant patients achieving sustained virological response (SVR). (6) Importantly, HCV treatment after LT improved the outcome of graft hepatitis C. (7,8) Still, it is not yet known to what extent a slightly higher risk for disease progression persists despite viral clearance aregay et al. also after LT, as it has been suggested for patients with HCV who do not receive transplants. In addition, a few cases of graft rejection and HCV-associated histological changes have been described in the context of HCV treatment after LT. (9)(10)(11) As HCV has significant imprints on the immune system, (12) there has been some debate as to what the consequence of an HCV cure would be on the immune system. We and others previously reported that several of the innate and adaptive immune responses remain impaired despite the successful clearance of HCV. (13)(14)(15)(16) Nevertheless, some studies suggested that part of the innate and adaptive immune responses may partially recover after HCV elimination. (17,18) Subsequently, there has been a concern that HCV clearance after LT could increase the risk for rejection through revival of the impaired immune responses and possible increase of allo-reactive responses. (19) Limited data discerning the immune profile of graft hepatitis C and the subsequent attainment of therapyinduced SVR on immune responses are available even though this issue has repeatedly been addressed in the pretransplant setting. We previously showed that regulatory T cells (Tregs) with an effector memory (EM) phenotype are enriched in patients with HCV recurrence after LT (HCV + LT) compared with patients who received transplants in the absence of HCV infection, and some Treg phenotypes could differentiate rejection from graft hepatitis C. (20) However, the impact of a posttransplantation HCV cure on the overall immune compartment has not been addressed. Indeed, no other study has probed broad immune cell parameters and inflammatory milieu in the context of post-LT antiviral therapy.
The aim of this study was to comprehensively characterize the immune cell compartment and inflammatory milieu after LT in patients receiving DAA therapy. We set out to identify immune biomarkers that could lead to further areas of investigation in graft hepatitis C. Particularly, we aimed to define potential immune correlates that could possibly lead to a better understanding and treatment/monitoring plan for patients treated for HCV in the posttransplant setting.

patient cOHOrts anD saMples
Overall, 49 patients with available peripheral blood samples (n = 99) at Hannover Medical School were enrolled in this study. This included 25 LT patients who were HCV-RNA positive (HCV + LT) and undergoing DAA therapy, 14 patients who were HCV-RNA negative without the recurrence of the underlying liver disease (non-HCV LT), and 10 healthy individuals. Patients were recruited between 2013 and 2017.
All patients who received a transplant because of HCV-related liver disease (HCV + LT) experienced graft reinfection and received DAA therapy consisting of sofosbuvir + ribavirin, sofosbuvir combined with either ledipasvir or daclatasvir ± ribavirin, or Original article | 889 ombitasvir/ paritaprevir/ritonavir + dasabuvir, and all patients achieved SVR following therapy. The combined mean duration of treatment was 4.6 months. Patients who underwent LT because of end-stage liver diseases other than HCV infection (non-HCV LT) had no recurrence of the initial underlying liver diseases.
Healthy volunteers were recruited at the Hannover Medical School and University Hospital Essen.
All blood samples taken from LT patients were taken at least 6 months after the date of transplantation. Patients received mainly calcineurin inhibitors as the backbone immunosuppressant. None of the LT patients included in this study experienced biopsyproven rejection at the time of blood collection and at follow-up time points.
The baseline characteristics of patients as well as the clinical parameters of the blood samples are provided in Table 1.

isOlatiOn OF peripHeral BlOOD MOnOnUclear cells anD etHylene DiaMine tetraacetic aciD plasMa saMples
Blood samples collected at regular intervals during routine medical examinations were processed within 24 hours of collection. For the HCV + LT group, blood samples were collected at baseline, at the end of treatment, and  after attainment of SVR (4 weeks after SVR, n = 4; 8 weeks after SVR, n = 1; 12 weeks after SVR, n = 14; 24 weeks after SVR, n = 4; and 56 weeks after SVR, n = 2). Peripheral blood mononuclear cells (PBMCs) were isolated with a Ficoll density gradient technique as described previously. (13) Isolated PBMCs were cryopreserved in freezing medium consisting of 60% fetal bovine serum, 30% Roswell Park Memorial Institute (RPMI) 1640 medium, and 10% dimethyl sulfoxide and subsequently stored in liquid nitrogen until further analyses. Paired plasma samples were collected from ethylene diamine tetraacetic acid (EDTA) Becton Dickinson vacutainer tubes (Thermo Fisher Scientific, Waltham, MA) following centrifugation at 2000g for 10 minutes. The isolated plasma samples were stored at −20°C until further analyses.

MUltiparaMetric FlOW cytOMetry anD iMMUne pHenOtyping OF pBMcs
The staining of the PBMCs with fluorochrome-labeled monoclonal antibodies and immune phenotyping were performed as described previously. (21) Dead cells were excluded with fixable viability dyes. For intracellular staining, cells were fixed and permeabilized using forkhead box P3/transcription staining buffer set (eBioscience, San Diego, CA). A list of the fluorochrome-labeled surface and intracellular monoclonal antibodies used can be found in Supporting Table 1. Samples were acquired using LSR Fortessa flow cytometer (BD Biosciences, Franklin Lakes, NJ). Subsequently, the samples were exported, and the data were analyzed with FlowJo software versions 9.6 or 10.5.3 using either conventional gatings or high-dimensional t-distributed stochastic neighbor embedding (t-SNE). Equal event numbers were downsampled in the analyzed samples and concatenated prior to downstream t-SNE analyses with publicly available FlowJo (Becton, Dickinson and Company, Ashland, OR) plugins (Table 2).

QUantiFicatiOn OF sOlUBle inFlaMMatOry MeDiatOrs anD prOteins By prOXiMity eXtensiOn analyses assay
Simultaneous quantification of 92 human protein biomarkers in plasma were conducted by proximity extension analysis assay (Olink, Uppsala, Sweden). (22) The assay was performed as per the manufacturer's instructions. Briefly, frozen EDTA plasma samples were thawed, and 20 µL of each sample was sent to Olink for Proseek inflammation panel analysis as per the manufacturer's instructions. A pair of oligonucleotide-labeled Proseek probes bound to the target protein in the plasma sample were detected through a proximity-dependent DNA polymerization event. This complex was subsequently detected and quantified using standard real-time polymerase chain reaction. The Cq quantitation cycle values from a DNA extension control were subtracted from the measurement of Cq value, an interpolate control was corrected for, and finally a correction factor was subtracted to yield a normalized protein expression value that is log2 transformed. Finally, 74 proteins were detected above the limit of detection and passed the quality control (Supporting Table 2). These protein biomarkers were used for further analyses.

statistical analyses
Data were analyzed using GraphPad prism V8.4.1 (GraphPad Software, La Jolla, CA). Statistical distribution and normality of samples were evaluated by the D'Agostino and Pearson test and Kolmogorov-Smirnov test. For 2-group comparisons, the parametric Student t test or nonparametric Mann-Whitney U test was performed for samples with normal or without normal distributions, respectively. For comparisons with more than 2 groups, a 1-way analysis of variance (ANOVA) Tukey multiple comparison test was performed. Principal component analysis (PCA) and heat maps were performed using Qlucore omics explorer version 3.4.1 (Qlucore, Lund, Sweden). Q values in PCA were kept at <0.2, and fold changes in the heat maps were calculated as 2 to the power of the mean differences between the analyzed groups. The statistical test used for each graph is indicated in the figure legend. In all cases, significance is shown as *P < 0.05, **P < 0.01, and ***P < 0.001.

etHics stateMent
This study was a priori approved by the local ethics committee of Hannover Medical School, Hannover, Germany. Written informed consent was obtained from all patients, and the study was conducted in accordance with the 1975 declaration of Helsinki.

Hcv reinFectiOn aFter lt Distinctly UpregUlates several inFlaMMatOry analytes UnliKe lt WitHOUt active Hcv inFectiOn
HCV reinfection in LT patients may largely alter inflammatory mediators and proteins, subsequently influencing the immune response toward the graft and its survival. (23) In this regard, we compared the inflammatory analytes between patients with repercussions of HCV infection following liver transplantation (HCV + LT) and LT patients who were HCV RNA negative without a recurrence of the underlying liver disease (non-HCV LT). Interestingly, most of the inflammatory mediators and proteins were differentially expressed between the HCV + LT and non-HCV LT groups (Fig. 1A), and both groups distinctly cluster together in PCA model (Fig. 1B). LT patients with recurrent HCV infection displayed significant upregulation of  Particularly, mediators such as C-X-C motif chemokine 10 (CXCL10), caspase 8 (CASP-8), C-C motif chemokine 20 (CCL20), CCL19, interferon (IFN) γ, CDCP1, interleukin (IL) 18R1, CUB domain-containing protein 1 (CDCP1), CCL3, IL8, IL12B, tumor necrosis factor-beta, CXCL6, osteoprotegerin (OPG), IL10, Flt3L, hepatocyte growth factor (HGF), urokinase-type plasminogen activator (uPA), neurotrophin-3, CCL4, IL6, tumornecrosis factor receptor superfamily member 9, programmed death ligand 1 (PD-L1), IL18, and monocyte chemotactic protein (MCP) 1 were all significantly elevated in patients with graft HCV infection compared with those without (Fig. 1C). These data affirm that a multitude of inflammatory mediators and proteins are uniquely upregulated on graft hepatitis C and that this mainly is driven by the active recurrence or reinfection of HCV rather than the LT alone.

pOst-lt Hcv clearance reDUces FeW OF tHe elevateD inFlaMMatOry MeDiatOrs BUt Falls sHOrt OF nOrMaliZing tO levels seen WitHOUt active Hcv reinFectiOn
In light of our observation that the presence of active HCV infection is largely influencing the inflammatory milieu, we further questioned how therapy-induced elimination of HCV would affect this phenomenon. Importantly, HCV clearance led to significant reduction of some of the upregulated mediators, including CXCL10, CASP-8, CCL20, CCL19, CDCP1, IFNγ, IL-18R1, CXCL11, IL10, and PD-L1 ( Fig. 2A,C). However, the remaining mediators did not display any significant alterations following HCV clearance ( Fig. 2A,C). We then directed our investigation to see if the reduced mediators in LT with cleared HCV after SVR (SVR-LT) could be normalized to the level seen in LT patients without active HCV infection. Notably, majority of the reduced mediators following HCV clearance were still upregulated compared with the same mediators in non-HCV LT patients (Fig. 2B,C). Of note, treatment duration had no meaningful effect on the findings (data not shown). In sum, despite the fact that HCV clearance leads to a reduction in some of the upregulated mediators during graft HCV, the virus clearance still falls short to normalize these mediators to what would be seen in the absence of HCV recurrence.

Distinct alteratiOns OF MyelOiD anD lyMpHOiD iMMUne cell sUBsets On Hcv reinFectiOn aFter lt
Following our observation that active HCV infection leads to significant alterations in inflammatory mediators and proteins, we further investigated how this influences peripheral immune cell subsets in the same group of patients. To address this, we employed broad multiparametric flow cytometry panels, and the obtained data were analyzed by conventional gatings (Supporting Fig.  1) as well as t-SNE. We observed a significant enrichment of classic monocytes, myeloid-derived dendritic cells (mDC), and EM clusters of differentiation (CD) 4+ and CD8+ T cells in HCV + LT patients compared with healthy controls (Fig. 3A,B). Meanwhile, mucosalassociated invariant T cells (MAITs), γδ T cells, Tregs, B cells, and naïve CD4+ and CD8+ T cells were significantly reduced in HCV + LT patients compared with healthy controls (Fig. 3A-C). There was no difference in the detailed phenotypes of Tregs and natural killer (NK) cells as well as other immune cell subsets (data not shown). Surprisingly, unlike the broad alterations seen in inflammatory analytes between the HCV + LT and non-HCV LT groups, only minor changes were observed when we compared peripheral immune cell subsets between LT patients with or without HCV infection. Here, the HCV + LT group showed a reduced frequency of MAITs and increased nonclassical monocytes, whereas all other immune cells did not show a significant difference between the HCV + LT and non-HCV LT groups (Fig. 3D). Collectively, these data indicate that HCV reinfection after LT is not only associated with alterations in inflammatory milieu but also distinct alterations in peripheral immune cell subsets.

tHerapy-inDUceD clearance OF Hcv aFter lt DOes nOt signiFicantly alter FreQUency OF BOtH tHe innate anD aDaptive iMMUne cells
We finally asked if therapy-induced elimination of HCV after LT would lead to a reversion of the frequency  of some of the altered peripheral immune cell subsets. This is important because the possible resurrection of some of the immune cells after HCV clearance might well bear the risk of inducing unwanted outcomes including rejection episodes in the long run. Importantly, none of the altered immune cells in the HCV + LT patients were significantly reconstituted despite achieving therapy-induced SVR (Fig. 4A,B). Indeed, some of the immune cells showed a pattern to be decreasing or increasing after HCV clearance, but none reached a significance level (Fig. 4B). In summary, we report that HCV reinfection after LT leads to distinct alterations in the peripheral immune cell subsets as well as the inflammatory milieu that remain largely irreversible.

Discussion
In this study, we discerned upon the impact post-LT HCV reinfection and the clearance thereof has on immune responses and the inflammatory milieu. We identified possible immune biomarkers that could lead to further areas of investigation and aid in the better planning of treatment of HCV in the posttransplant setting. The data set presented here is robust, representing the Original article | 897 broadest immunological study, to our knowledge, performed on LT patients with HCV reinfection during antiviral therapy with properly defined controls. We hereby unveil that (1) HCV reinfection after LT drives distinct and broad alterations in inflammatory milieu as well as subsequent shifts in peripheral immune cell subsets, (2) these alterations are mainly mediated by infection with HCV rather than the LT itself, and (3) therapy-mediated clearance of HCV after LT does not normalize the altered inflammatory analytes nor does it reconstitute the altered peripheral immune cell subsets. The initial and not trivial observation was that, upon the screening of 74 mediators, a multitude of inflammatory mediators and proteins are uniquely altered in the post-LT setting and active HCV reinfection. We show that the observed alterations are mainly driven by the active presence of HCV after LT instead of the LT itself. It is worth mentioning that beyond the recruitment of immune cells, inflammatory mediators including cytokines and chemokines play major roles in the natural history of HCV infection and may serve as prognostic markers for the rapid progression of fibrosis after LT. (24,25) This might be the case in our study for the family of chemokines such as the C-C motif (ie, CCL20, CCL19, CCL3, CCL2 [MCP-1], CCL4, CCL25) and C-X-C motif (such as CXCL10, CXCL11, CXCL6, CXCL9, CX3CL1) chemokines as these might be associated with a bad prognosis of the graft. For instance, CXCL10 (IFN inducible protein-10) has been repeatedly implicated in HCVinduced severe liver fibrosis in both nonimmunosuppressed (14,(26)(27)(28)(29) and immunosuppressed patients. (24) In addition, a family of chemokine and cytokine receptors that were upregulated in the HCV + LT patients in our study were also implicated with a bad prognosis of the liver after HCV infection. (30) Of note, some of the altered inflammatory mediators may also have direct proviral effects for HCV recurrence and could serve as potential therapeutic targets as described previously. (31) Other proteins such as OPG and urokinase-type plasminogen activator (uPA) that we showed to be altered in the HCV + LT patients might also serve as biomarkers for predicting the severity of liver disease as reported previously in the pretransplant setting. (32,33) Importantly, 1 mediator that was uniquely upregulated in the HCV + LT patients compared with all other controls was CASP-8, a proapoptotic protein that may induce apoptosis of the infected hepatocytes. Of note, apoptosis of hepatocytes is 1 of the ways of leading to liver cirrhosis, and HCV proteins could directly be involved in apoptosis. (34) In the context of LT, immunosuppression may facilitate the HCV-mediated apoptosis of hepatocytes and the subsequent liver fibrosis by impairing the immune control of HCV. (35) Most of the inflammatory mediators are known to recruit innate and adaptive immune cells expressing receptors for the ligands on these mediators. The cellular interactions between immune effector cells, monocytes, dendritic cells (DCs), and hepatic stellate cells combined with soluble immune mediators are believed to determine the outcome of liver injury and the development of liver fibrosis. (36) We observed a distinct enrichment of classical and CD16+ monocytes as well as mDCs in the HCV + LT patients. Meanwhile, a reciprocal significant reduction in MAITs, Tregs, B cells, and γδ T cells was observed in this study. We previously showed that immune suppressants such as cyclosporine could impact the phenotype of DC subsets, (37) and DCs may act as regulatory cells after organ transplantation through IL10 secretion and chemotaxis. (38) Classical monocytes were also previously shown to be rapidly recruited during LT, possibly through MCP1. (39) In addition, the observed simultaneous upregulation of macrophage or DC attractants such as myeloid progenitor inhibitory factor 1 and MCP2 further corroborate the distinct enrichments of monocyte and DC subsets in the HCV + LT patients. The overall reduction in MAITs, Tregs, B cells, and γδ T cells in the HCV + LT patients could be associated with immune suppression (38,40) or the impact of HCV infection as described previously. (15,16,41,42) The reduced frequency of Tregs in the circulation of HCV + LT patients in this study might be attributed to a reciprocal infiltration of liver resident Tregs. (43)(44)(45)(46)(47) Our group has previously demonstrated that Treg phenotypes such as the expression of human leukocyte antigen -DR isotope may differentiate rejection from recurrent HCV infection. (20) However, we could not study this further in this study because none of the patients experienced biopsy-proven rejection episodes.
There has been a concern that a possible restoration of some of the immune cells after HCV clearance could also bear the risk of inducing unwanted sequelae, including rejection episodes in the long run. (9,10,48) There is limited literature that addresses the repercussions of HCV elimination on immune response in the posttransplantation setting. Importantly, we showed for the first time in this study that the therapy-induced clearance of HCV after LT does not significantly normalize a larger fraction of the altered inflammatory analytes in HCV + LT patients. Nonetheless, few of the upregulated mediators (such as IL10, CXCL11, and PD-L1) were significantly restored despite majority of the mediators were not normalized to what would be seen in patients who received transplants without HCV recurrence. These findings were not correlated with patients' human leukocyte antigen (HLA) A02 status or HCV genotype (GT; data not shown). In a similar manner, the clearance of HCV did not lead to significant reconstitutions of the analyzed peripheral immune cell subsets. Previously our group also reported the lack of restoration of the adaptive and innate arms of immune responses following HCV clearance in patients who did not receive transplants. (13)(14)(15)(16) These observations are in line with the clinical observation that a cure of HCV after transplantation did not trigger rejection episodes in many patients.
Of note, in addition to HCV infection, some residual inflammation attributed to other viral infections, level of immunosuppression, marked liver fibrosis as well as additional comorbidities could also influence the level of inflammatory analytes observed. It is worth mentioning that none of the patients included in this study experienced additional viral infections such as cytomegalovirus, hepatitis E virus, or Epstein-Barr virus infection during the study period. Serum levels of individual immune suppressants did not differ significantly between patient groups. However, it needs to be mentioned that the HCV + LT patients experienced some additional comorbidities during the study period, including type 2 diabetes mellitus, chronic kidney disease, and ischemic type biliary lesions. The possible impact (or the lack thereof ) of some of these comorbidities on the observed findings is not to be ruled out. Furthermore, the presence of marked liver fibrosis before or after HCV therapy might have influenced the inflammatory milieu. Unfortunately, in most of the HCV + LT patients, standardized biopsy or elastography data were not available, and we could not clearly delineate liver stiffness.
This study has obvious strengths and limitations. It is the broadest study conducted thus far in LT patients with recurrent HCV and in the context of novel antiviral therapies. Quite a broad array of immunological readouts were investigated. However, we acknowledge that the number of investigated patients within each subgroup was limited and patient groups were heterogeneous, although the overall numbers of patients investigated were extensive. We also acknowledge that we could not analyze patients who were chronically HCV infected before transplant and achieved SVR but still required LT as a result of end-stage liver diseases. We did not investigate virus-specific CD8+ T cells and their functionalities in this study because of the limited numbers of cells obtained. Longitudinal follow-up studies should aid in understanding how the altered immune mediators would be associated with the graft prognosis in the long run. Analyses of liver biopsies should follow this study because the relative abundance of the assessed peripheral immune cells might well differ inside the liver.
In summary, we comprehensively probed the immune cell compartment and inflammatory milieu in LT patients with HCV reinfection under antiviral therapy. We identified the potential immune correlates that could trigger additional areas of investigation and aid in the better planning of treatment for HCV infection in the posttransplant setting. Importantly, we unveil a first-hand investigation that imprints of graft hepatitis C on immune cell distributions and inflammatory milieu persist despite achieving therapy-induced SVR.