Volume 122, Issue 5, Pages 1201-1215 (May 2002) Activated virus-specific T cells are early indicators of anti-CMV immune reactions in liver transplant patients  Christine Benz, Olaf Utermöhlen, Anna Wulf, Brigitte Villmow, Volker Dries, Tobias Goeser, Ulrich Koszinowski, Dirk H. Busch  Gastroenterology  Volume 122, Issue 5, Pages 1201-1215 (May 2002) DOI: 10.1053/gast.2002.33021 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 Ex vivo detection of CMV-pp65–specific T cells using intracellular IFN-γ staining and HLA tetramer reagents of different HLA types. Peripheral blood mononuclear cells from liver transplant patients with different HLA haplotypes (indicated to the left) were analyzed for CMV epitope–specific T-cell populations by intracellular cytokine staining for IFN-γ (left column, no peptide control; middle column, in the presence of relevant peptide) or by staining with HLA tetramers (right column). Human cytomegalovirus (HCMV) serostatus as determined by serology and HLA haplotype are indicated. Gastroenterology 2002 122, 1201-1215DOI: (10.1053/gast.2002.33021) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 CMV-specific T-cell populations readily expand by in vitro peptide stimulation and demonstrate effective cytotoxic activity. PBMCs from HLA-A2–positive, (A and C) CMV-seropositive liver transplantation patients and (B and D) CMV-seronegative healthy donors were stimulated in vitro with pp65495-503 peptide as indicated in Materials and Methods; representative examples are shown. After 21 days in culture, cells were (A and B) stained with HLA-A2/pp65495-503 tetramers and (C and D) tested for pp65-epitope–specific cytotoxicity by chromium release assays. Gastroenterology 2002 122, 1201-1215DOI: (10.1053/gast.2002.33021) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 Elevated frequencies of CMV-specific T cells in liver transplantation patients. Frequencies of CMV (pp65)-specific T cells were measured in 13 HLA-A2–positive liver transplantation patients (6–33 months after transplantation, 3–10 measurements were performed in each patient, and the values were averaged). Single measurements of CMV (pp65)-specific T cells in 19 HLA-A2–positive healthy blood donors served as controls. The median, 10th, 25th, 75th, and 90th percentiles are plotted as vertical boxes with error bars; single dots indicate outliners. Gastroenterology 2002 122, 1201-1215DOI: (10.1053/gast.2002.33021) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 No increase of EBV-specific T cells in liver transplantation patients. Frequencies of CMV(pp65), CMV(gB)-specific, and EBV(BMLF)-specific T-cell populations were measured by MHC tetramer staining in (C) 14 HLA-A2–positive, CMV-positive liver transplant patients and (D) 14 HLA-A2–positive, CMV-positive healthy blood donors. Liver transplantation patient 15 and blood donor 15 are HLA-A2–positive, CMV-negative controls. Liver transplantation patient 16 and blood donor 16 represent HLA-A2–negative, CMV-positive controls. Examples for tetramer stainings from each group are shown in the upper dot plots (A, liver transplantation-patient 1; B, healthy blood donor 13; see also Table 1 [same patients]). Data for all patients are summarized in the graph below (C, liver transplantation-patients; D, healthy blood donors). Note the differences of the y-axes scales. Gastroenterology 2002 122, 1201-1215DOI: (10.1053/gast.2002.33021) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 Case study: liver transplantation patient #2, primary CMV infection. Immunomonitoring of a pretransplant CMV-seronegative patient (#2; see Table 1) who received a CMV-seropositive transplant. Dot plots show the percentage of CD38-expressing pp65495-503/MHC tetramer–positive CD8+ T cells at (A) 12 weeks, (B) 17 weeks, and (C) 41 weeks after transplantation. (D) The kinetics of CD38-expressing pp65495-503/MHC tetramer-positive T cells and the overall frequency of pp65-specific CD8+ T cells as well as changes in CMV-IgM index values, CMV IgG titers (U/mL), and transaminases (ALT, U/L) are summarized; each parameter has a separate y-axis. Liver biopsy (LB) 12 weeks after transplantation revealed a granulomatous hepatitis. Gastroenterology 2002 122, 1201-1215DOI: (10.1053/gast.2002.33021) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 6 Case study: liver transplantation-patient #4, CMV reactivation, PCR-negative. Immunomonitoring of patient #4 (see Table 1) after transplantation of a liver from a seronegative donor. Dot plots show the percentage of CD38-expressing pp65495-503/MHC tetramer–positive CD8+ T cells (A) 3 weeks, (B) 13 weeks, and (C) 27 weeks after transplantation. (D) The kinetics of pp65495-503/MHC tetramer-positive T cells, CD38-expression of pp65-specific CD8+ T cells, CMV IgM index values, CMV IgG titers (U/mL), and transaminase levels (ALT, U/L) are summarized; each parameter has a separate y-axis. Liver biopsy (LB) 8 weeks after transplantation revealed unspecific mesenchymal activation. Gastroenterology 2002 122, 1201-1215DOI: (10.1053/gast.2002.33021) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 7 Case study: liver transplantation-patient #1, CMV reactivation, PCR-positive. Patient #1 (see Table 1) was CMV-seropositive and received a seropositive transplant. Dot plots show the percentage of CD38-expressing pp65495-503/MHC tetramer–positive CD8+ T cells (A) 3 weeks, (B) 7 weeks, and (C) 16 weeks after transplantation. (D) The frequencies of CD38-expressing pp65495-503/MHC tetramer–positive T cells, the overall frequency of pp65-specific CD8+ T cells, and CMV-IgM index values, CMV IgG titers (U/mL), and transaminases (ALT, U/L) over the course of the study are summarized; each parameter has a separate y-axis. Liver biopsy (LB) 4 weeks after transplantation revealed signs of mild to moderate rejection and confirmed active CMV replication by PCR analysis. Gastroenterology 2002 122, 1201-1215DOI: (10.1053/gast.2002.33021) Copyright © 2002 American Gastroenterological Association Terms and Conditions