Reversal of NK-Cell Exhaustion in Advanced Melanoma by Tim-3 Blockade

Melanoma donor (MD) NK cells are functionally impaired/exhausted. A, freshly purified NK cells [healthy donors (HD), n = 12; melanoma donors, n = 5] were stimulated with 200 U/mL of IL-2. Expression of IL-2R (α-chain), IFN-γ production, and cytotoxicity (Lamp-1 expression) were monitored every 2 days over 6 days (day 0, 2, 4, and 6) by flow cytometry. B, the percentage of Lamp-1⁺ NK cells from healthy (n = 30) and melanoma donors (n = 12) after a cytotoxic assay is shown using K562 cells as target cells (top left). The percentage of IFN-γ⁺ NK cells from healthy (n = 22) and melanoma donors (n = 9) is shown after 4 hours of stimulation with IL-12 (middle left). The percentage of proliferating NK cells from healthy (n = 10) and melanoma donors (n = 7) is shown after 6 days of culture in the presence of 200 U/mL of IL-2 (bottom left). On the right panel of each graph, plots depicting the expression of Lamp-1, IFN-γ, and CFSE in NK cells purified from a representative healthy donor and a representative melanoma patient are shown. C, graphs representing the MFI of T-bet and Eomes on NK cells purified from healthy (n = 19) and melanoma donors (n = 14). Representative plots are shown (isotype control, black; healthy donors, unfilled; melanoma donors, gray). All experiments were performed in duplicate.

Tim-3 is upregulated in melanoma donor (MD) NK cells. A, graph comparing Tim-3 expression in NK cells from healthy donors [healthy donors (HD), n = 45) and patients with melanoma (melanoma donors, n = 41). Represented as the percentage of Tim-3⁺ cells (left) and the MFI of the Tim-3⁺ population (right). B, the graphs show the percentage (left) and MFI (right) of Tim-3⁺ NK cells from healthy donors (n = 30) and patients with melanoma stage I (n = 47), II (n = 18), and III/IV (n = 18). All experiments were performed in duplicate.

Tim-3 engagement inhibits NK-cell functions. A, the percentage of LAMP-1⁺ (n = 8) and B, the MFI of IFN-γ⁺ cells (n = 6) of NK cells from melanoma donors preincubated with IgG-coated beads or anti–Tim-3-coated beads for 2 hours before evaluating the cytotoxic function or IFN-γ production. C, reverse-ADCC assay using FcR⁺ P815 cells. NK cells from melanoma patients were cocultured with P815 cells and different antibodies were added to the reaction: anti–Tim-3, anti-CD94 (negative control), or anti-CD16 (positive control). Data were normalized to the values obtained for the condition: (A and B) with IgG-coated beads (100%); (C) with no antibody. All experiments were performed in duplicate.

NK-cell exhaustion can be reversed by Tim-3 blockade. A, graph represents the expression of LAMP-1 (n = 15; MFI) in NK cells from melanoma donors incubated with 10 or 20 μg/mL of soluble Tim-3 blocking antibody 1 hour before the cytotoxic assay. B, the percentage of CFSE⁺7-AAD⁺ K562 cells (percentage of killed K562 cells) in the presence of 10 μg/mL of two different Tim-3 blocking antibodies (clone 2E2 or R&D Systems #AF2365) 1 hour before the killing assay (n = 9 melanoma donors). Graphs represent the expression of IFN-γ (C; n = 12; MFI) and the percentage of proliferating cells (D; n = 10; %) in NK cells from melanoma donors incubated with 10 or 20 μg/mL of soluble Tim-3 blocking antibody 1 hour before the functional assays. A, C, and D, the expression of LAMP-1, IFN-γ production, and proliferation with (right) and without (middle) Tim-3 blockade from a representative melanoma patient. Data were normalized to the values obtained for the condition without blocking antibody. All experiments were performed in duplicate.