Fig. 7: Matrix-M enables antigen cross-presentation in BMDCs.
BMDCs were loaded with LysoTracker Red DND-99, washed and imaged using spinning-disk confocal microscopy for 30 minutes. Following the initial pre-bleaching image acquisition, cells were treated with Alexa Fluor 647–labeled rS (0.5 or 1 μg/mL) and BODIPY-Matrix-M (1 or 2.5 μg/mL) and live-cell imaging continued for an additional 120 min. Pearson’s correlation coefficients (PCC) for the channel combination LysoTracker and Alexa Fluor 647 (A), and BODIPY and Alexa Fluor 647 (B) were determined as indicators of lysosome–antigen or Matrix‒M–antigen colocalization, respectively. Data in panels A and B are presented as smoothed averages ± SEM, pooled from at least three experiments, each performed in triplicates. C–G BMDCs were exposed to 0.8 mg/mL ovalbumin (OVA) and indicated concentrations of Matrix-M, Matrix-A, or Matrix-C for 24 hours. Cells were analyzed by flow cytometry for surface markers and for H-2kB bound to SIINFEKL complexes, with the latter indicative of antigen cross-presentation. The percentage of viable BMDCs positive for H-2kB/SIINFEKL complexes is shown as representative contour plots (C) and corresponding graphs (D) for untreated and Matrix-treated groups. E Representative flow cytometry plots and F graphs show the proportion of major subpopulations within BMDC culture, identified as non-DC-like cells (CD11c− MHCII−, dark blue), immature DC-like cells (CD11c+ MHCII−/lo, turquoise) and mature DC-like cells (CD11c+ MHCIIhi, gray). G The percentage of various BMDC subpopulations within cells positive for H-2Kb/SIINFEKL complexes are shown. Data in panels C–E are representative of three independent experiments, while graphs in F and G show pooled data from three independent experiments, presented as the mean ± SD.
