Figure 2. Transcriptional profiling and functional characterization of neonatal mucosal CD4 T lymphocytes.

Transcriptome analysis of flow cytometry-sorted CD4⁺CD8⁻TCRβ⁺ cells isolated from the indicated organs/age of the animals (days, d). The array was performed in quadruplicates with each group comprising sorted cell populations pooled from one litter of neonate animals (d6 and day 11) and two to three adult mice per sample. Thymus (Th) d6, SI d6, SI d11, mLN d6, mLN d56, LP d28, LP d56 and PP d56. (a) Principal coordinate analysis of the groups based on 10,000 differentially regulated individual genes using multigroup comparison (ANOVA analysis) and a false discovery rate (FDR) of <10⁴. (b) Functional analysis of up- (red) and downregulated (green) genes of d6 SI compared with d6 mLN using two group analysis, FDR=0.05 and a fold-change cutoff of 2. Analysis was performed in PANTHER using the gene ontology term biological process. (c) Transcriptome analysis of signature T-helper genes d6 SI and d56 PP. (d) Fold-change values (normalized to the mean intensity of all samples) of CD44 and CD69 mRNA measured by microarray analysis in CD4 T lymphocytes from thymus (Th) and small intestine (SI) of 6-day-old mice, SI of 11-day-old mice and PPs and LP of 56-day-old mice (left panels) and comparative FACS analysis of the CD44 or CD69 versus CD62L surface expression on CD4 T lymphocytes in PP of 11-day-old mice and PP and LP of 56-day-old mice (right panels; n=4 for left and right panels, mean±s.d.). (e) Intracellular FACS staining for IFNγ and IL-17A in CD4 T cells isolated from PPs from 11- and 56-day-old mice restimulated with PMA/Ionomycin (n=4, representative of three experiments, mean±s.d.). (f) Cytokine detection by Multiplex Bead Array analysis in the supernatant of FACS-sorted CD4 T cells isolated from PPs of 11- and 56-day-old mice and restimulated with PMA/Ionomycin. (n=4, representative of two experiments, mean±s.d.; two-way ANOVA, Bonferroni's post test, ***P<0.001; *P<0.05).