Supplementary Figure 3: Nonspecific bystander inflammation produces ICTN cells that have altered molecular response but retain survival and proliferative ability.
(a) Experimental Schematic; DR mice were given intraperitoneal injection of pbs (ctr) or pIC for two consecutive days, then on the third day spleen and lymph nodes were harvested and purified by magnetic bead enrichment. Cells were then analyzed directly ex vivo or manipulated as described. (b) DR cells were cultured for 16-20 hours with Ova-pulsed APC, cells were fixed and stained for intracellular phospho-AKT (pAKT). For controls, DR cells were cultured with APC with no antigen (not shown). Bar graphs (right panels) depict mean plus SEM of triplicate values of mean florescence intensity (mfi) of pAKT. Data are representative of three independent experiments, n=3 mice per group. (c-d) CFSE-labeled DR cells were cultured for 5 days with OVA-peptide-pulsed APC and cells were assessed by FACS for proliferation by CFSE dilution and survival by viability marker. (b) CFSE dilution of DR cells; representative FACS histogram (left), bar graph depicting mean plus SEM of triplicate values for proliferation index of DR cells as calculated based on CFSE dilution (right). (c) Frequency of viable DR cells by fluorescent viability marker, bar graph depicts mean plus SEM of triplicate values. Data are representative of three independent experiments, n=3 mice per group. P-value by student’s two-tailed t-test, *p<0.01.
