Fig. 3.

NK cells are sparsely found within the ONB TIME. (A) Representative photomicrographs at 20x magnification from a low Hyams Grade ONB of merged and single-color immunofluorescence images assessing the presence of NK cells with a validated panel of five biomarkers, CD16 (orange, Opal 620), CD56 (red, Opal 690), CD3 (turquoise, Opal 480), Granzyme B (green, Opal 520), and synaptophysin (white, Opal 780). Multispectral immunofluorescence images are counterstained with DAPI. Co-localization of CD16 and CD56 without CD3 and a decrease size threshold compared to tumor cells identified NK cells. Expression of Granzyme B identified activated NK cells. Expression of synaptophysin identified tumor cells. (B) Quantification of NK cells and Granzyme B⁺ NK cells per mm² in ONB tumor and stroma (n = 38 tumor cores in triplicate). (C) NK cell density when comparing patients who presented with and without dural infiltration, top, and Dulgerov stages, bottom. (D) Granzyme B⁺ NK cell densities when compared by whether the patient had a post-treatment recurrence, top, and whether their cause of death was ONB specific. All lines are graphed to indicate the median value. For paired comparisons, Wilcoxon matched-pairs signed rank test and for unpaired comparisons, Mann-Whitney U tests were used to test for statistical significance. *p ≤ 0.05, non-significant