After antigenic in vitro stimulation of chronically infected patients,

we observed Sotrastaurin a strong increase of CD8+Pentc18-27+ T-cell frequencies from day 0 (0.02%) to day 21 (0.56%). Blocking CD244 with anti-CD48 (Fig. 7A) or anti-CD244 (Fig. 7B) augmented virus-specific CD8+ T-cell frequencies in 5 of 12 (1.62-fold) (P = 0.9) or 5 of 10 (1.65-fold) (P = 0.6) chronically infected patients, respectively. The dual blockade of CD244 and CD48 increased the frequencies in five of six patients, which indicates a susceptibility of 83.3% (2.1-fold) (P = 0.09) (Fig. 7C). In comparison, blocking PD-1 by PD-L1/2 did enhance the CD8+Pentc18-27+ T-cell frequencies in six of eight patients 2.98-fold, which represents the most significant increase (P = 0.01) (Fig. 7D). Single find more or dual blockade of CD48 and CD244 significantly enhanced T-cell expansion by CD244high expressing CD8+ T-cells (P = 0.01) (Fig. 5B). No increase in T-cell expansion was detectable in acute patients and resolvers stimulated with the HBV

core peptide as well as healthy individuals stimulated with the EBV peptide, as shown (Supporting Fig. 3). Unspecific background reaction was determined by: (1) stimulation of healthy donors with HBV core peptide in the presence of blocking antibodies (n = 8), and (2) stimulation of HBV patients with HBV core peptide in the presence of isotype control (n = 9). Samples of healthy controls and samples stimulated with isotype control did not show unspecific MCE CD8+ T-cell proliferation (data not shown). We confirmed the impact of CD244 blockade on the restoration

of T-cell proliferation in chronically infected patients (n = 7) using CFSE (Fig. 8A). CD244 blockade led to a four-fold, significantly higher proliferation of virus-specific CD8+ T-cells (6.6%) in comparison to antigen stimulation (1.6%) (P = 0.01) (Fig. 8B). PD-L1/2 blockade augmented T-cell proliferation 2.8-fold from 1.6% to 4.5% (P = 0.03) (Fig. 8C), whereas isotype control (mean: 1.8%) did not induce T-cell proliferation (P = 0.8) (Fig. 8A). Representative FACS contour plots are shown (Fig. 8D). CD244 plays a pivotal role in CD8+ T-cell regulation. Early studies demonstrated that CD244 acts as an activating receptor on NK-cells and T-cells in mice and humans. Cross-ligation enhanced lytic activity and IFN-γ secretion.12-15 CD244 is not only known as an activating molecule, as studies using a CD244-knockout mice model highlighted that CD244 can be an inhibitory receptor in NK-cells.