Fig 2: Combination of primary NK cells and doxorubicin increases MCF7 cell death. (A) Primary NK cells from two healthy donors were expanded and characterized for the expression of CD45, CD56, CD3 and CD16 using flow cytometric analysis. (B) The ability of primary NK cells to kill the MCF7 cells in the presence or absence of doxorubicin. Data are presented as the mean ± SEM. NK, natural killer; ns, not significant.

Fig 3: Prolonged stimulation through activating receptors results in functional and proliferative defects in vivo.(A) Schematic representing experimental design: sublethally irradiated NSG mice were injected (i.v.) with HL-60-Luc2 cells and injected (i.v.) with NK cells 3 days later. NK cells had been incubated for 7 days on plates with either isotype IgG (control) or anti-NKp46 and MICA/B as previously described. (B) Fourteen days after NK cell injection, blood was drawn, and NK cells were counted via flow cytometry. NK cells were human CD45+CD56+CD3–. One-way ANOVA was used for comparisons (n = 8). *P ≤ 0.05. (C) HL-60 tumor burden was tracked via bioluminescent imaging (BLI), with day 21 data pictured. One-way ANOVA was used for comparisons (n = 8). ***P < 0.001. (D and E) Fourteen days after NK cell injection, NK cells were restimulated with K-562 leukemia cells for 4 hours as previously described. Ki67, IFN-γ, TNF-α, and CD107a expression was assessed via flow cytometry (D). Paired t tests were used for comparisons (n = 8). *P ≤ 0.05. Representative data depict Ki67 and IFN-γ expression following K-562 restimulation (E).

Fig 4: Gating strategy for major immune cell populations in pancreatic cancer(A) cell populations include tumor-associated macrophages (TAM) (CD45+CD11b+CD68+), myeloid-derived suppressor cells (MDSC) (CD45+CD11b+CD68-CD66b+), B cells (CD45+CD19+), CD4+T cells (CD45+CD3+CD4+), CD8+cytotoxic T cells (CTL) (CD45+CD3+CD8+ ), γδT cells (CD45+CD3+TCRγδ T+), natural killer cells (NK) (CD45+CD3-CD16+CD56+), dendritic cell (DC) (CD45+HLA-DR+CD11c+) and natural killer T cell (NKT) (CD45+CD3+CD16+CD56+).

Fig 5: MARCO expression in human pancreatic cancer(A) Left panel: Analysis of mRNA expression of MARCO in 171 healthy individuals and 150 PDAC samples. Log2, TPM+1 are shown. Right panel: Kaplan-Meier plot of PDAC patients based on low versus high MARCO expression. Log rank statistical analysis was used.(B) Representative hematoxylin and eosin and immunofluorescence staining of snap frozen and paraffin embedded PDAC sections (n = 10). Three different tumors are depicted.(C) Immunofluorescence staining of paraffin-embedded PDAC sections (n = 10) highlighting tumor, MARCO, T cell (left), and NK cell areas (right). MARCO (red), CD68 (green), CD3 or CD56 (blue), and Hoechst (gray) are shown. Quantification of number of (#) T cells and NK cells in MARCO+ and MARCO− macrophage areas in imaged PDAC sections was performed in ImageJ. Every dot represents an average of all imaged areas of one patient. Statistical analyses were performed using student unpaired T-test.