Synergistic Reversal of Intrahepatic HCV-Specific CD8 T Cell Exhaustion by Combined PD-1/CTLA-4 Blockade
Figure 7
The functional restoration by PD-1/CTLA-4 blockade is CD28-dependent.
(A) Loss of HCV-specific CD8 T-cell IFN-γ response by CD28 depletion. LIL or PBL from 3 HLA-A2-negative patients with chronic (C08, C275) and acute (A36) hepatitis C were depleted of CD4 without or with additional CD28 depletion before in vitro culture for 7 days with HCV NS3-derived overlapping 15mer peptides in the presence of isotype or PD-1/CTLA-4 blockade. Cultured cells were examined for HCV-specific IFN-γ production in a 45 hour IFN-γ ELISPOT assay. (B) CD28 expression in HCV-specific NS3 1073-specific tetramer+ CD8 T-cells relative to PD-1 and CTLA-4 expression ex vivo. (left) Peripheral HCV 1073-specific tetramer+ CD8 T cells from an HLA-A2+ acute HCV patient (A47) display CD28 expression in 28%. (middle) Gated HCV 1073-specific tetramer+ CD8 T cells show the characteristic PD-1 (97.3%) and CTLA-4 (20.5%) expression. (right) Increased CD28 expression in gated PD-1+CTLA-4+ (Red; 50.0%) HCV tetramer+ CD8 T cells compared to PD-1+CTLA-4− (Green; 19.5%) and PD-1−CTLA-4− (Blue; 12.2%) subsets and isotype control (gray shade) in histogram. (C) Effect of CD28-depletion on antigen-specific expansion in the presence of PD-1 and/or CTLA-4 blockade is shown by CFSE-dilution for HCV NS3 1073-specific tetramer+ CD8 T cells from patient A47. CD4 depleted PBL with and without CD28-depletion were CFSE-labeled and stimulated for 7 days in vitro with HCV NS3 1073 peptide in the presence of isotype or blocking antibodies before flow cytometric analysis. Note that HCV tetramer+ CD8 T cells remain detectable with CD28 depletion in the bottom graphs.