We included 100 samples of 80 adult LTRs without replicative viral hepatitis who underwent a liver graft biopsy (LBx) within the prospective LBx repository at our center between 2008 and 2019. LTRs were included if they provided a written informed consent and had a plasma sample stored for research use. LT took place between 1990 and 2017. Both patients with stable liver graft function [liver enzymes < 2× upper limit of normal (ULN)] that had svLBx and with clinical T-cell–mediated rejection (cTCMR) that had indication biopsy were included. Plasma samples were collected within 24 h of the liver biopsy and cryoconserved at −80°C.

The current study is based on our previously published cohort from our ongoing prospective LBx biorepository in LTR (16). There was not enough plasma left for some patients from the published cohort, which led to a reduction of the original cohort size. In addition, we also included all patients with available liver biopsy and paired plasma samples that provided a written informed consent and had a histopathological evaluation of graft injury other than cTCMR and subTCMR (IND).

The study was approved by the local Ethics Committee (protocol number 933 for project Z2 of comprehensive research center 738; MHH Ethikkomission, Hannover, Germany). The study was conducted according to the ethical guidelines of the Declarations of Helsinki and Istanbul.

Standard laboratory parameters were derived from patients’ records and were initially measured in clinical routine using high throughput techniques.

Donor-specific antibodies (DSAs) were detected as described recently (17). The incidence of DSA positivity was analyzed using a mean fluorescence intensity threshold of ≥ 1,000.

For the assessment of the overall degree of IS, we calculated a semi-quantitative score as previously published by Vasudev et al. (18) for the kidney transplant recipients and utilized by our group recently in LTRs (6, 19). One point was assigned to each of the following drug dosages: tacrolimus (TAC) of 2 mg, cyclosporine (CsA) of 100 mg, mycophenolate mofetil (MMF) of 500 mg, prednisolone of 5 mg, and azathioprine of 100 mg. In addition, we assigned one score point to 1.5 mg of everolimus (EVR) or sirolimus (SIR) as previously published (6, 19).

Liver biopsies were performed percutaneously with local anesthesia. The biopsy cylinder was fixed in 4% neutral-buffered formalin and embedded in paraffin wax.

Histological examination and scoring for the rejection activity index (RAI) (20), inflammation grade and fibrosis stage (Ishak score) (21), central perivenulitis, portal microvasculitis, ductular reaction (22), fatty liver disease (23), and total liver allograft fibrosis (LAF) score (24) were performed by experienced liver pathologists in a blinded fashion. At least moderate fibrosis was defined as periportal fibrosis (Ishak F) ≥ 2 and/or any LAF score component ≥ 2. SubTCMR and cTCMR were defined as recently published (16). In detail, subTCMR was defined by a Banff RAI ≥ 1 + 1 + 1, and the patients had to have transaminases [aspartate aminotransferase (AST) and alanine aminotransferase (ALT)] and alkaline phosphatase (AP) below two times ULN. Gamma-glutamyl transferase (gGT) had to be stable or declining prior to svLBX, even if elevated above two times ULN, to be classified as subTCMR. cTCMR was defined as Banff RAI ≥ 1 + 1 + 1, and the presence of elevated liver enzymes (AST, ALT, and/or AP) above two times ULN. Biopsies of patients with any evidence of disease recurrence, active viral infection (e.g., viral hepatitis), or bacterial infections were excluded. No histological signs of rejection (NHR) was defined as RAI ≤ 1. There was no relevant inflammation and no relevant fibrosis (Ishak F ≤ 1, each LAF score component ≤ 1) in samples with NHR, and liver enzymes were within the normal range.

Total nucleic acid was extracted from 200 µL of plasma using the NucliSENS easyMAG platform (bioMerieux, France) as recommended by the manufacturer and eluted in 50 µL of an elution buffer. TTV DNA quantitation was performed by an in-house TaqMan real-time PCR using forward primer AMTS: 5′-GTGCCGIAGGTGAGTTTA-3′, reverse primer AMTAS: 5′-AGCCCGGCCAGTCC-3′, and probe AMTPTU: 5′FAM-TCAAGGGGCAATTCGGGCT-3′TAMRA as described previously (25). The highly conserved untranslated region upstream of Open reading frame 1 (ORF1) is the real-time PCR target site allowing detection of all known TTV species (26). The quantitative PCR reactions were performed in a volume of 25 µL using 2× TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA), containing 5 µL of extracted DNA, 400 nM of each primer, and 80 nM of the probe. Thermal cycling was started for 3 min at 50°C, followed by 10 min at 95°C, and then by 45 cycles at 95°C for 15s, at 55°C for 30s, and at 72°C for 30s using the 7300 Real Time PCR System (Applied Biosystems, Foster City, CA, USA). The linear range of TTV quantitation ranges from 2.7 to 10.7 log₁₀ copies/mL as determined using 10-fold dilutions of a plasmid standard. The limit of detection in plasma is 2.7 log₁₀ copies/mL. In each run, positive and negative controls were included. Prior to the extraction, 5 µL of phocine herpesvirus (PhHV) DNA as internal spike-in control was added to all plasma samples. None of the samples showed any signs of PCR inhibition as assessed by quantitation of PhHV DNA (cycle threshold, 30 ± 2.0).

Statistical analyses were performed with SPSS (version 27, SPSS, Inc., Chicago, IL), R Statistical Software (version 4.1.2, R Core Team), and GraphPad Prism (version 9.4.0, GraphPad Software Inc., La Jolla, CA). The Mann–Whitney U-test was used to compare non-parametric continuous variables between two independent groups. The Kruskal–Wallis test was used for the comparison of non-parametric continuous variables between more than two independent groups. Adjustment for multiple comparisons was performed with Dunn’s post-hoc test. The Fisher’s exact test was used to compare categorical variables between two independent groups. Spearman’s rank correlation coefficient was used for correlation analysis and was plotted using the corrplot package (27) in R Statistical Software. Multivariate linear regression analysis was performed using the Tidyverse (28) package in R Statistical Software. Variables for multivariate linear regression were selected depending on their significant differences in univariate analysis and/or their causal relation to TTVv in the literature. f 2 was calculated as follows and published by Cohen (29): f 2 = R 2 1 − R 2 . P-values below 0.05 (two-tailed) were considered statistically significant in all analyses.

Of all 100 samples analyzed, TTV was detectable in the plasma in 97%. Three patients without detectable TTVv were excluded from further analyses. The characteristics of the cohort are summarized in Table 1 . Seventy-five patients had svLbx, whereas 22 patients were indicated for biopsy due to elevated liver enzymes (AST, ALT, and/or AP ≥ 2× ULN). Only one patient had detectable cytomegalovirus (CMV) replication, which precluded further analysis between CMV and TTVv (not shown). TTVv did not correlate with age (Spearman’s Rho: 0.05; p = 0.66, n = 97) and did not differ between male and female LTR [median log₁₀ TTV copies/mL (range), 5.9 (2.5–9.4) vs. 6.0 (3.2–8.2); Mann–Whitney U-test, p = 0.63). Of note, TTV replication did not correlate with any laboratory tests for liver injury (AST, ALT, AP, gGT, and bilirubin) ( Supplementary Figure 1 ). TTVv was lower in patients with autoimmune liver diseases as cause of transplantation when compared to all other liver diseases ( Supplementary Figure 2A ), mainly due to a non-significant trend of lower TTVv in patients with autoimmune liver diseases being transplanted more than 1 year before sampling ( Supplementary Figure 2B ). A more detailed analysis comparing all liver diseases against each other revealed no significant differences ( Supplementary Figures 2C, D ).

TTV levels are known to decline after solid organ transplantation, even within the first year (11, 13). Hence, the correlation between TTVv and time after transplantation was analyzed. TTV replication showed a negative correlation with time post-LT (Spearman’s Rho: −0.63, p < 0.001, n = 97; Figure 1A ) and a decrease from the first year to later time points ( Figure 1B ). However, the degree of IS, as measured by the IS score, decreased correspondingly with time after transplantation ( Figure 1C ). This decrease in TTVv and IS score over time was observed not only in the overall analysis but also longitudinally in directly paired samples ( Supplementary Figure 3A ). The decrease of TTV replication in these patients was accompanied by a decreased degree of IS ( Supplementary Figure 3B ). As a marked decline of TTVv was seen beyond the first year after LT, we stratified patients according to their time after LT for further analyses. The first group includes patients within the first year after LT, and the second group includes patients beyond the first year after LT. Patients after the first year after LT had lower TTVv and IS score and higher modified histological activity index (mHAI), Ishak fibrosis stage, and LAF score ( Supplementary Table 1 ).

As a correlation between low TTV replication and graft injury by cTCMR within the first year after LT has been described in the literature (11), we stratified patients into two groups. The first group consisted of LTR with stable graft function with AST, ALT, and AP < 2× ULN. These patients received a liver biopsy for histological monitoring (svLBx) according to our local protocol (6). The other group consisted of patients with clinical graft hepatitis (elevation of AST, ALT, and/or AP above 2× ULN). There were no differences in TTV replication between patients with svLBx and those with clinically evident graft hepatitis (liver enzymes ≥ 2× ULN), regardless of the findings in the indication biopsy ( Figure 2A , Supplementary Figure 4A ). Likewise, TTVv was not significantly lower in cTCMR compared with svLBx, neither in the first year nor thereafter, or in the overall cohort ( Figure 2B , Supplementary Figure 4B ). Clinically overt TCMR occurred at a median of 8.4 months after LT (range, 2–107 months; n = 13).

TTVv was not different in patients with any histological graft injury compared to patients with no histological signs of rejection ( Figure 2C ). However, in the overall cohort that was not stratified according to time after LT, TTVv was lower in patients with any histological graft injury ( Supplementary Figure 4C ). Next, the correlation of TTVv with different histological scores of graft injury was assessed according to time after LT. TTVv did not correlate with histological scores of liver inflammation and rejection when assessed separately within or after the first year after LT ( Figures 2D, E , Supplementary Figure 4D ) but correlated negatively with mHAI in the overall cohort ( Supplementary Figure 4E ). TTVv correlated negatively with histological staging of graft fibrosis (Ishak fibrosis stage and LAF score) in the first year after LT ( Figures 2F, G ) but not at later time points. The correlation of TTVv with fibrosis remained when the overall cohort was assessed ( Supplementary Figures 4F, G ). In addition, the mere presence or absence of DSA as marker of allosensitization was not associated with TTV replication ( Figure 2H , Supplementary Figure 4H ). TTVv did not predict whether patients met Banff histological criteria for reduction of IS irrespective of the time after LT ( Figure 2I , Supplementary Figure 4I ). Neither the presence of graft injury (87.5% vs. 76.9%, p = 0.28) nor the median mHAI (3 vs. 3, p = 0.87), LAF score (1 vs. 1, p = 0.59), or Ishak fibrosis stage (1 vs. 1, p = 0.88) was different in patients with autoimmune liver disease prior to LT compared to any other etiology of pre-LT liver disease. Graft injury was more frequent and pronounced in patients beyond the first year after LT (92.0% vs. 68.1%, p = 0.004), which had higher median mHAI (3 vs. 2, p = 0.002) and LAF score (2 vs. 1, p < 0.001) ( Supplementary Table 1 ). In conclusion, graft fibrosis was more pronounced in patients with lower TTV replication, suggesting a relationship between both parameters and lower IS, but only in the first year after LT.

Next, the association between graft injury and measures of IS was analyzed. Unexpectedly, the classic measures of IS degree were not associated with histological graft injury. Neither TAC nor CsA trough levels correlated with mHAI, RAI, Ishak F, or LAF score in the first year after LT as well as in the overall cohort ( Table 2 , Supplementary Table 2 ). Histological scores of graft injury also did not differ between LTR with different daily MMF doses (0 g of MMF vs. 0–1 g of MMF vs. > 1 g of MMF) ( Supplementary Figures 5A–D ). Conversely, the median RAI was higher in patients with prednisolone [3 (range, 0–8)] than that in patients without prednisolone [1 (range, 0–6); p = 0.02] ( Supplementary Figure 4F ). The median mHAI, Ishak F, and LAF score did not differ depending on prednisolone intake ( Supplementary Figures 5E, G, H ). However, a semi-quantitative score assessing the overall degree of IS (18, 19) was negatively associated with histological scores for liver allograft fibrosis [LAF score (Spearman’s Rho: −0.34; p = 0.02)] in the first year but not thereafter ( Table 2 , Supplementary Table 2 ). On the contrary, TTVv showed a moderate correlation with both Ishak fibrosis stage and LAF score in the first year after LT as well as in the overall cohort ( Table 2 , Supplementary Table 2 ). Beyond the first year after LT, neither CsA trough levels nor the IS score or TTVv correlated with histological markers of graft injury ( Table 2 ).

Next, we investigated whether TTVv correlated with the overall degree of IS. TTVv did not differ between patients with mono-, dual-, or triple-IS ( Figure 3A , Supplementary Figure 6A ). Regardless of the choice of primary immunosuppressant, TTVv did not correlate with TAC or CsA trough levels, both in the overall cohort and in the subcohorts stratified according to time after LT ( Figures 3B, C , Supplementary Figures 6B, C ). Thus, there was no significant association between the pharmacokinetic and pharmacodynamic effects of the CNIs. In addition, TTVv did not differ significantly between LTR with CsA and TAC as the primary immunosuppressant in the overall cohort ( Supplementary Figure 6D ) but was lower in patients beyond the first year after LT on TAC as compared to CsA ( Figure 3D ). To assess the dependence of TTVv on MMF dose, patients were stratified into three MMF dose groups as mentioned earlier. No differences in TTVv were observed for MMF ( Figure 3E , Supplementary Figure 6E . In the first year after LT, patients without prednisolone had higher TTVv, but this effect was not seen in the overall cohort or beyond the first year after LT ( Figure 3F , Supplementary Figure 6F ). TTVv was moderately correlated with IS score, with higher IS degree associated with higher TTV replication, but only in the first year after LT (SR = 0.44, p = 0.002) and not thereafter (Spearman’s Rho: 0.08, p = 0.56) ( Figure 3G , Supplementary Figure 6G ).

Both the IS score and TTVv were associated with graft fibrosis in our cohort in the first year after LTx. To determine whether any of these two factors from the univariate analysis were independently associated with TTV replication, we performed a multivariate regression analysis of samples taken within the first year after LT. Although age and sex were not associated with TTVv in our cohort, both demographic parameters were included in the multivariate analysis because such associations have been reported in the literature (10). A multiple linear regression model was used to test whether TTVv, IS score, age, and sex significantly predicted LAF score as an LT-specific score of liver graft fibrosis, which demonstrated an association with both IS score and TTVv. The fitted regression model was follows: LAF score = 4.63 − 0.09*(IS score) − 0.35*[TTV (log₁₀)] − 0.09*(male sex) − age*(0.02) (p = 0.02). The overall model significantly predicted LAF score. Only TTVv was significantly associated with the LAF score (ß = −0.35, p = 0.02), whereas age (ß = −0.02, p = 0.23), male sex (ß = −0.09, p = 0.77), and IS score (ß = −0.09, p = 0.39) did not. The effect size, quantified by, was 0.35, indicating a large effect size. TTVv only explained 26% of the variance in LAF score (multiple R-squared: 0.26, adjusted R-squared: 0.18) ( Table 3 ).