Figure 1.

Pam₃CSK₄- or depleted zymosan-stimulated hematopoietic stem and progenitor cells (HSPCs) generate antigen presenting cells (APCs) with an altered phenotype in signals 1, 2, and 3. (A) Schematic protocol of in vitro HSPC differentiation. Purified Lin⁻ cells from bone marrow of C57BL/6 mice were treated with Pam₃CSK₄, depleted zymosan, or nothing (none) during the first 24 h of culture, washed thoroughly to remove any remaining stimuli and then continued in culture with GM-CSF for a further 6 days to derive APCs. At day 6, adherent cells were recovered from the cultures, counted and plated at equal numbers for 24 h, and then stimulated with Pam₃CSK₄, depleted zymosan, or nothing (unstimulated) for 24 h to assess: (B) the expression of MHCII (signal 1) and costimulatory molecules (CD40, CD80, and CD86; signal 2) on CD11b⁺ CD11c⁺ APCs by flow cytometry (D0, day 0; N, none; P, Pam₃CSK₄; DZ, depleted zymosan), and (C) cytokine production (TNF-α, IL-6, IL-12 p40, and IL-2; signal 3) in the supernatants from the APC cultures by ELISA. Colormap is based on min-max values per row using the Mean Fluorescence Intensity (MFI) values obtained with the specific antibodies and the isotype controls (shown in Appendix A Figure A1C). Cytokine data represent means ± SD of triplicate cultures, * p < 0.05, ** p < 0.01, *** p < 0.001 with respect to day 0 none. Results shown are from one experiment representing three–five independent experiments.