Figure 2. CD27^lo cells do not predominantly express antigen presentation/co-stimulation molecules or cytokines.

(a) Row-normalized heatmaps of genome-wide transcriptional analysis using Affymetrix U133 2.0 Plus gene arrays (n = 6 subjects). All probes are shown for each gene plotted. With the exception of CD80, most genes for antigen presentation/co-stimulation were expressed at higher levels in undivided cells. A dagger (†) indicates genes with at least one probe that was found by SAM analysis to be significantly different between CD27^lo and CD27^hi at FDR = 0.03 (n = 2746 probes). An asterisk is (*) indicates genes in the comparison of undivided to CD27^lo cells that had at least one probe significantly different at FDR = 0.01 (n = 7500 probes) and two asterisks (**) are next to genes that were still significant (CD27^lo/undivided comparison) at FDR = 0.008 (n = 3093 probes). (b) Quantitative RT-PCR confirmed relative RNA levels of CD40, CD74, CD80, and CD83 (n = 10 subjects, including the same 6 subjects analyzed by gene array) (paired T-test p values in Supplementary Table 2). The point in each violinplot is the median value and the brackets indicate the interquartile range with the addition of the probability density of the data at different values. (c) Flow cytometric analyses of normal human Bc stimulated for 96 hr with CpG+CK confirmed relative protein levels per generation consistent with RNA levels for CD80 and CD83 and a higher percentage of CD27^lo cells in divisions 1 and 2 (n = 3 subjects). The undivided subpopulation expresses higher levels of these genes. (d) Gene array analysis shows messages for IL-6, IL-7, IL-24, TNFSF12 (April), Lymphotoxin A (LTA), produced in larger amounts by undivided cells than the other cell subsets. While TNF and IL1A were significantly different between groups at an FDR = 0.03, they were not significant at lower FDRs (n = 6 subjects). (e) Relative IL-6 RNA levels were confirmed by qRT-PCR. (n = 10, paired T-test p values in Supplementary Table 2). The point in each violinplot is the median value and the brackets indicate the interquartile range with addition of the probability density of the data at different values. Except for IL-15, the undivided cells produce higher amounts of these cytokine messages. IL-15 message was significantly higher in CD27^hi cells than in CD27^lo.