Volume 117, Issue 6, Pages 1386-1396 (December 1999) Direct ex vivo analysis of hepatitis B virus-specific CD8+ T cells associated with the control of infection  Mala K. Maini, Carolina Boni, Graham S. Ogg, Abigail S. King, Stephanie Reignat, Chun Kyon Lee, Juan R. Larrubia, George J.M. Webster, Andrew J. McMichael, Carlo Ferrari, Roger Williams, Diego Vergani, Antonio Bertoletti  Gastroenterology  Volume 117, Issue 6, Pages 1386-1396 (December 1999) DOI: 10.1016/S0016-5085(99)70289-1 Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 1 Specificity of the HLA-A2 HBV/peptide tetrameric complexes. CTL clones or lines with specificity as indicated were stained with HLA-A2/peptide tetramers folded with c18–27, p575–583, or e335–343 peptides and anti-CD8 MAb and analyzed by flow cytometry. The specific peptide recognition of the different CTL lines was tested in standard chromium release assays using HLA-A2–matched target cells pulsed or unpulsed with the relevant peptide. Specific lysis of pulsed vs. unpulsed targets was 25% for core 18–27 CTL lines, 11% for envelope 335–343 CTL lines, and 15% for polymerase 575–583 CTL lines at an effector to target ratio of 20/1. Gastroenterology 1999 117, 1386-1396DOI: (10.1016/S0016-5085(99)70289-1) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 2 Functional characterization of tetramer-positive cells. PBMCs from an HLA-A2–positive patient with acute HBV were stained with the T c18–27 and anti-CD8 MAb. Double-positive cells were sorted by (A) flow cytometry and (B) expanded in vitro with PHA stimulation. (C) Sorted and expanded cells were analyzed by flow cytometry after staining with T c18-27 and anti-CD8 MAb. These cells were tested for their ability to lyse HLA-A2–positive target cells pulsed with the core 18–27 peptide (D) by a standard chromium release assay or (E) for IFN-γ production after stimulation with peptide-pulsed target cells by intracellular cytokine staining. Gastroenterology 1999 117, 1386-1396DOI: (10.1016/S0016-5085(99)70289-1) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 3 Quantification of tetramer-positive CD8 cells in peripheral blood of patients with acute HBV. (A) PBMCs of patients with acute HBV (14 HLA-A2 positive, 9 HLA-A2 negative) were stained with HBV tetramers (T c18–27, T p575–583, T e335–343) and anti-CD8 MAb immediately after isolation. Five uninfected HLA-A2–positive donors were also tested. The number of tetramer-positive CD8 cells was counted of 50,000 CD8 cells analyzed. Dashed line indicates the cutoff value of 30.5, calculated as the mean (8.3) plus 4 SD (22.2) of a total of 27 determinations of tetramer-positive CD8 cells from the 9 HLA-A2–negative patients with acute HBV analyzed. (B) FACScan dot plots of PBMCs of 2 acute HBV patients (1 HLA-A2 positive, 1 HLA-A2 negative) stained with anti-CD8 MAb and the HBV tetramers indicated. The numbers in the upper-right quadrants indicate the percentage of tetramer-positive cells of total CD8 cells, calculated with CellQuest software. Gastroenterology 1999 117, 1386-1396DOI: (10.1016/S0016-5085(99)70289-1) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 4 Longitudinal quantification of tetramer-positive populations. Serial determinations of the number of tetramer-positive CD8 cells in 4 patients during acute HBV infection. Tetramer-positive CD8 cells were calculated as shown in Figure 3. The time course (in weeks after clinical onset) is shown on the horizontal axis. Serum transaminase levels are represented by bars, and HBV serological status assayed at the same time points is indicated at the bottom of each plot. ○, T c18–27; ▴, T p575–583; 2, e335–343. Gastroenterology 1999 117, 1386-1396DOI: (10.1016/S0016-5085(99)70289-1) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 5 Expansion of T c18–27+ CD8 cells during the course of acute infection and correlation with core 18–27–specific CTL activity. PBMCs purified at different time points were stained directly with T c18–27 and anti-CD8 MAbs (Fresh PBMC panel) or stimulated with core 18–27 peptide. Cells were cultured for 10 days and then analyzed both by flow cytometry (T-cell lines panel) and by testing for core 18–27–specific CTL activity with a chromium release assay at an effector to target ratio of 20:1 (Cytotoxic Assay panel). Dot plots show T c18–27–positive CD8+ cells in lymphocytes within the live gate, with the percentage of tetramer-positive CD8 cells out of total CD8 cells shown in the right upper quadrant of the FACScan dot plot profiles. The lower level of CD8 fluorescence seen at week 14 reflects the use of a different batch of anti-CD8 MAb. The percent lysis is shown for targets in the presence or absence of core 18–27 peptide. Serum transaminase levels and HBV serological status assayed at the same time points are also indicated. Gastroenterology 1999 117, 1386-1396DOI: (10.1016/S0016-5085(99)70289-1) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 6 Phenotypic analysis of T c18–27+ CD8 cells during acute HBV infection. Fresh PBMCs obtained during acute HBV infection were analyzed by 3-color flow cytometry using phycoerythrin-conjugated T c18–27, cy-chrome–conjugated anti-CD8, and the following FITC-conjugated MAbs: anti–HLA-DR, anti-CD38, anti-CD62L, and anti-CD45RA. (A) Dot plots showing data after gating on CD8+ T cells. Percentage of HLA-DR+ or CD38+ cells of T c18–27+ CD8 cells is indicated in the upper right quadrants. (B) Summary of the phenotype of T c18–27+ CD8 cells at different time points. The percentages of HLA-DR+, CD38+, CD62L+, and CD45RA+ in CD8+ T c18–27+ cells were calculated with CellQuest software. Gastroenterology 1999 117, 1386-1396DOI: (10.1016/S0016-5085(99)70289-1) Copyright © 1999 American Gastroenterological Association Terms and Conditions