The gene expression profiles of unstimulated CD4+ T cells of all groups were taken as a baseline, to establish whether there is an intrinsic difference between the groups. downregulated at the gene expression level. Rather an increase in expression of Th1- and Th17-associated genes caused the shift in Th subset outcome. Pertussis or whooping cough, caused by the gram-negative bacterium infection4,5,6,7. Moreover, these Th subsets have been shown by both the mice and baboon models to be crucial in the protection against LPS derivative, to an alum-containing aP vaccine skewed the vaccine-induced CD4+ T cell response towards a Th1/Th17 type of CD4+ T cell response at the cytokine level10. Yet, how the Th subset outcome in the aP vaccine-induced antigens activated the antigen Ptx, FHA, and Prn, after which microarray analysis was performed on RNA from isolated CD4+ T cells. The gene expression profiles of unstimulated CD4+ T cells of all groups were taken as a baseline, to establish whether there AZD-5904 is an intrinsic difference between the groups. No significant differentially expressed genes could be identified between these unstimulated samples (criteria: p-value??0.001, fold ratio (FR) 1.5). Nevertheless, to exclude small intrinsic nonsignificant differences, the expression intensities of the antigen-stimulated samples were corrected for the average expression intensities of unstimulated samples of their corresponding group. In total, 1876 differentially expressed genes (antigen-stimulated samples of vaccinated mice with those of control mice, differential expression (FR??1.5) of 384 and 358 genes was identified in the CD4+ T cells of respectively aP- and aP+LpxL1-vaccinated mice. Overlap comparison showed that 247 genes were differentially expressed in CD4+ T cells of both aP- and aP+LpxL1-vaccinated mice, 137 genes were exclusively differentially expressed in CD4+ T cells of aP-vaccinated mice, and 111 genes were exclusively differentially expressed in CD4+ T cells of aP+LpxL1-vaccinated mice (Figs 1 and ?and22). Open in a separate window Figure 1 Visualization of differences in gene expression in CD4+ T cells of control, aP-, and aP+LpxL1-vaccinated mice by principle component analysis.(A) Principal component analysis, based on the differentially expressed genes, showing (dis)similarities in gene expression in samples stimulated with the Ptx, FHA, and Prn combination (dark colors, n?=?5 per group) and medium controls (light colors, n?=?3 per group) in all vaccination groups (PBS (blue), aP (red), aP+LpxL1 (green)) are shown. (B) Venn diagram showing the amount of overlap between up- (red) and downregulated (green) genes in 24 hour antigen-stimulated CD4+ T cells of aP- and aP+LpxL1-vaccinated mice, as compared to control mice, based on averaged normalized gene expression levels of groups. Open in a separate window Figure 2 Gene expression profiles of antigen-stimulated CD4+ T cells of vaccinated compared to AZD-5904 control mice (FR??1.5). (A) 247 genes were differentially expressed in CD4+ T cells of both aP- and aP+LpxL1-vaccinated mice. (B) 137 genes were differentially expressed in CD4+ T cells of Rabbit Polyclonal to OR52E2 exclusively aP-vaccinated mice. (C) 111 genes were differentially expressed in CD4+ T cells of exclusively aP+LpxL1-vaccinated mice. Expression data shown are averages from the samples of 5 mice per group. Over-representation of immune and metabolism related terms after aP- and aP+LpxL1- vaccination To provide more insight in the differentially AZD-5904 expressed genes, functional annotation and over-representation analysis (Benjamini-corrected p-value??0.05) in GO-BP and KEGG databases were performed using DAVID17. Analysis of the overlapping 247 differentially expressed genes in CD4+ T cells of both aP- and aP+LpxL1-vaccinated mice showed that 74?GO-BP terms and 8 KEGG pathways were enriched. Based on exclusion of overlapping terms/pathways and their relevance, a selection of these terms/pathways is shown in Fig. 3A. The enriched terms/pathways are mainly involved in the regulation of the adaptive immune response, as indicated by terms as regulation of lymphocyte activation (GO:0051249), proliferation (GO:0050670), and differentiation (GO:0045597), and cytokine signaling, including chemotaxis (GO:0006935) and Jak-STAT signaling pathway (mmu4630). Moreover, the enrichment of the asthma pathway (mmu05310) indicates the presence of Th2-associated genes. Further, terms involved in metabolic processes are enriched, including positive regulation of macromolecule metabolic process (GO:0010604) and positive.