CD161 blockade enhances T-cell–mediated tumor clearance in a humanized mouse model. (A) Experimental design. NSG mice were injected IV with 2.5 × 105 NY-ESO-1+ Luciferase-positive ZsGreen+ Raji cells. Tumor engraftment was monitored by bioluminescence imaging. Five days after tumor cell injection, mice were randomized to treatment groups to ensure similar tumor burden. On day 6, a total of 1.5 × 106 NY-ESO-1 TCR+ CD8 T cells (4 × 106 total T cells) were injected IV, and mice were treated twice per week with 5 mg/kg of CD161 (KW7.3.7) or isotype control mAbs. (B) T-cell profile before adoptive transfer. NY-ESO-1 TCR expression, CD4/CD8 ratio, and CD161 expression were measured by flow cytometry. (C) Disease progression after T-cell administration was quantified by bioluminescence imaging, and total flux was quantified. (D-E) Impact of CD161 blockade on tumor burden quantified by bioluminescence (D) and overall survival (E). Panels A-E show representative assay of 2 independent repeats. (F-L) Analysis of T cells in the bone marrow on day 13 after tumor cell injection (day 7 after T-cell administration). (F) Percentage of Raji cells (ZsGreen) vs CD3+ T cells in both treatment groups. (G-L) Flow cytometry analysis of total human CD3+ T cells (G), activation markers expressed by CD4+ T cells (H), and CD8+ T cells (J), IFN-γ and tumor necrosis factor TNFα–positive (TNFα)+ CD4 (I) and CD8 (L) T cells, and granzyme B+ CD8 T cells (K). Panels F-L analyses were performed once with 5 mice per group. Each data point represents a biological replicate (individual mouse); Unpaired t test, ∗P < .5; ∗∗P < .01; ∗∗∗P < .001. APC, antigen presenting cells; BLI, bioluminescence.
