Tim-3 expressing HBV-specific T cells are dysfunctional.

<p>PBMC derived from HLA A2+ patients with CHB were stained with a panel of HLA-A2/HBV multimers and then were stimulated overnight with a pool of HBV peptides of matched specificity to the multimers, followed by intracellular staining for IFN-γor TNF-α. (a) Representative histograms showing levels of Tim-3 (black line) or isotype binding (grey shading) on CD8 T cells binding HLA-A2/HBV peptide multimers or producing IFN-γ upon encounter with HBV peptides. (b) Compiled data from 10 patients with CHB. (c) Tim-3 expression on CD8 T cells binding HLA-A2/HBV peptide multimers or producing TNFa upon stimulation with HBV peptides. (d) FACS plots and (e) summary data showing the induction of caspases (FLICA) and 7AAD in CD8 and CD4 T cells with or without the addition of galectin-9. Active caspases, indicating apoptosis, were determined using a fluorescent-labelled inhibitor of polycaspases (FAM-VAD-FMK, FLICA), and death was identified by 7AAD stain. Early apoptotic events are indicated in the lower right quadrant (FLICA+7AAD−), late apoptotic events in the right upper quadrant (FLICA+7AAD+) and necrotic cells in the left upper quadrant (7AAD+FLICA-). ‘Total death’ was estimated by summing events in these 3 quadrants.