RNA-seq analysis of B cells cultured with LPS and IL-12 and/or IFN gamma
Purpose: We aimed to find genes differentially regulated by IL-12 and IFNg alone, and in combination, over 4 and 24 hours of stimulation. Methods : Spleens were processed and B cell cultures prepared, in triplicate, according to the manuscript. After the indicated times, cells were washed in PBS, resuspended in 350 ul RLT Plus Buffer (Qiagen) with 1% 2-Me, and frozen at -80 °C. Results: At 4 hours, the main effect of IL-12 is to induce IFNg production from B cells, whereas IFNg induces many gene changes. Comparing 24 hours to the 4 hour stimulation, IFNg alone was insufficient to maintain the majority of IFNg-induced 4 hour changes, while adding IL-12 sustained a portion of the 4 hour gene changes. Additionally, in combination IL-12 and IFNg cooperate to induce unique gene changes that are not shared with either the individual cytokines at either timepoint. Overall design: We used recombinant IL-12 and IFNg or anti-IL-12 and anti-IFNg blocking antibodies to assess the contribution of each cytokine alone and in combination.