Cytometric bead array (CBA) and intracellular cytokine stainings (ICS) were performed to measure cytokine levels. To determine the role of PD-1 and CTLA-4 on T-cell responses during chronic hepatitis E, cells were cultured in vitro after CFSE labeling in the presence of HEV AT9283 research buy peptide pools and by adding antihuman PDL-1 (eBioscience, San Diego, CA) and CTLA-4 (BD PharMingen, Becton Dickinson, Heidelberg, Germany) antibodies separately or in combination at a concentration of 5 μg/mL along with peptide pools. Fluorescence-activated cell sorting (FACS) stainings were performed at day 7

using CD4-PE and CD8-PE Cy7 antibodies. The Mann-Whitney U test was applied for univariate comparison of independent continuous variables and Fisher’s exact t test for discrete variables using Statistica 9.0 software (Statsoft, Tulsa, OK). P < 0.05 was considered significant. A total of 38 subjects were studied including 19 organ transplant recipients and 19 immunocompetent

healthy individuals. Patients with chronic hepatitis E had higher alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels at baseline as compared with resolved subjects (148 U*L−1 versus 23 U*L−1, P < 0.05; 87 U*L−1 versus 27 U*L−1, P < 0.05, respectively). Genotyping was performed Sotrastaurin in six of the seven patients with chronic HEV, which revealed HEV genotype 3 in all patients (Table 1). Individual characteristics of organ transplant recipients are shown in Table 2. Three of the seven patients with chronic infection cleared HEV after reduction of immunosuppression and three other patients became HEV RNA-negative during treatment with ribavirin. One of the four ribavirin-treated patients did not clear the virus. One transplant recipient with resolved hepatitis E (KTxR1) was treated with ribavirin during acute HEV

infection. In this patient (KTxR1) T-cell responses were studied very early after acute hepatitis E. Anti-HEV IgG titers were higher in patients with chronic hepatitis E than in transplanted patients with resolved hepatitis E Phosphoglycerate kinase or seropositive healthy subjects (Supporting Information Fig. 1). HEV-specific T-cell proliferative responses were investigated in all study subjects after stimulating PBMCs in vitro with HEV overlapping peptide pools for 7 days. Representative FACS plots are shown in Fig. 1A,B. Although strong and multispecific HEV-specific proliferative responses were found in most healthy seropositive subjects (7/9), T-cell responses were weaker in transplanted patients (Fig. 1C, Table 3). However, HEV peptide pools were also recognized by the majority of the anti-HEV-positive/HEV-RNA-negative transplanted patients (8/12) without a distinct dominant pattern across the different peptide pools.