Figure 1. Clustering of B cell genes, whose expression was up-regulated by interaction with NK cells.
Follicular B cells were sorted by MoFlo cell sorter (BD Biosciences) on the basis of CD23hi and CD21lo (BioLegend) expression, as described previously [19]. The recovered cells were incubated overnight before coculture with purified NK cells that have been propagated for 7 days in IL-2 and subsequently, sort-purified based on NKp46 staining. Twenty hours after culture, the cells were harvested and NK and B cells separated by FACS sorting (bio-NKp46 and FL CD19). High-quality RNA was extracted and amplified into cRNA and subjected to microarray analysis as described in Materials and Methods. (A) Heat map of up-regulated genes revealed in one of the two microarray runs that fall within the Immune Response and Response to Virus clusters, defined by the Gene Ontology Consortium. The column labeled “Response to IFNα” includes the former two sets, as well as additional ISGs, up-regulated in B cells and extracted from various literature sources. Readings from some genes, where identical oligo probes were slotted several times, are also shown. To illustrate the extent of contamination of the B cell signal by high levels of expression in NK cells, signal from hybridization to sequences from killer cell lectin-like receptor subfamily D, member 1 (KLRD1), and killer cell lectin-like receptor subfamily A, member 10 (KLRA10), genes that are not expressed by B cells, are shown. (B) As the exact levels of increase in expression are not visualized easily in the heat maps, the fold increase in expression of B cell genes as a result of NK stimulation (average of two independent microarray experiments), which has also been shown to be increased by IFN-α or in response to virus infection in various cell types, was plotted. Also shown for comparison is the fold increase induced by IL-28 or IL-29 in Raji cells [20] or human hepatocytes [21], respectively.