Figure 3. Tumor-derived IFN-γ and GM-SCF synergistically differentiate immature neutrophils into a subset of APC-like hybrid neutrophils.

(A) Flow cytometric analysis of CD14 and HLA-DR expression on gated live CD11b⁺CD15^hiCD66b⁺ BMNs cultured in the presence of hybrid-inducing TCM under normoxic and hypoxic cell culture conditions.

(B) Flow cytometric analysis of CD14 and HLA-DR expression on gated live CD11b⁺CD15^hiCD66b⁺ BMNs cultured in the presence of different TCMs (top panel) or with IFN-γ and/or GM-CSF (low panel).

(C) The effect of IFN-γ and GM-CSF blocking Abs (5 µg/ml) in blunting the formation of HLA-DR⁺CD14⁺ hybrid neutrophils in vitro (red box).

(D) The expression of CD14 and HLA-DR markers on live CD11b⁺CD15^hiCD66b⁺ BMNs (top panel) and PD-L1 on gated HLA-DR⁺CD14⁺ hybrid neutrophils (low panel) differentiated with GM-CSF (50 pg/ml) and the increasing doses of IFN-γ in vitro. (E and F) Levels of IFN-γ and GM-CSF in supernatants collected from the cell culture of small size tumor digests where APC-like hybrid TANs were or were not previously detected (set-off was >10% among all TANs) (line represents mean ± SEM, n = 10, Mann-Whitney test for unpaired data). Lower panels represent the correlation between the absolute levels of IFN-γ and GM-CSF in the TCM and the frequency of hybrid neutrophils in each tumor showed in top graphs. Non-parametric Spearman test was used to determine the degree of correlation.

Representative dot plots from 1 of 5 experiments are shown in (A–D).

See also Figure S3.