Fig. 5
NK-Macrophage crosstalk. Peripheral Blood M1-M2 differentiation and quantification of final product. M1 (a) and M2 (b) structural phenotype shown. Macrophages were labeled with CD68-PE (Red), Cytoskeleton stained with phalloidin-Alexa Fluor 488 (Green) and nuclei stained with DAPI (Blue). Bar graph of proportions of M1 and M2 as well as NK cells in active (NA) or resting (N0) form as quantified by flow cytometry (c). Naïve/resting NK cells were activated by using IL-2 at the dose rate of 50 ng/ml. Macrophages were differentiated from human PBMCs into M1 and M2 macrophages. Differentiated macrophages (M1 and M2) and NK (NA and N0) cells were co-cultured. All the experiments were executed in triplicate and mean of the three were considered as an individual observation (n = 3–6). Autologous co-cultures of M1/M2 macrophage with NA/N0 NK cells were stained with M1/M2 phenotype markers. Phenotypic characterization of differentiated macrophages (M1 and M2) were done using Flow cytometer. Single live cells were gated and subsequently sorted for macrophages (CD68) and re-gated for the M1 macrophages activation marker Viz. CCR2, CX3CR1 for surface chemokine and IL-12 and TNFa for intracellular cytokines (d-f). In the same autologous co-culture experiment the macrophages were re-gated for CD68 and gated for M2 macrophage activation marker Viz. Arginase 1, Dectin 1 and IL-10 (g-i). Again in the same autologous co-culture experiment single cells were gated for NK cell markers (CD56+ CD3-) and subsequently sorted for intracellular cytokines and multimeric complexes viz. GZB, IFNg, and Perforin (j-l). Statistical significance was determined by using t-test: two samples assuming equal variance. NK cells were co-culture against macrophages at different Effector to Target (E:T) ratios: Resting and activated NK (N0 and NA) cells cytotoxicity against macrophage (M0, M1 and M2) is shown (m). Cytotoxicity was assessed in flow cytometer using CFSE-FITC for alive and 7 Aminoactinomycin D (AAD)-PE for dead cells. AB depict the difference between the groups within a ratio and ab depict the difference (P < 0.05) among the ratios within a group