CpG-ODN Induces a Dose-Dependent Enrichment of Immunological Niches in the Spleen and Lungs of Neonatal Chicks That Correlates with the Protective Immunity against Escherichia coli

Figure 1

CpG-ODN-induced a dose-dependent enrichment of lung immunological niches. CpG-ODN treatment resulted in an increase in the number of infiltrating antigen-presenting cells and T lymphocytes in the lung. Flow cytometric analysis of lung cells at 72 hrs post in ovo injections (day 21 embryo) (). First, cells were plotted based on forward vs. side scatter to gate monocyte/macrophage populations. Then, using PE-labeled mouse anti-chicken monocyte/macrophage (clone: KUL01) and AF488-labeled mouse MHC II monoclonal antibodies, the double-positive population was gated to quantify the number of APCs (dot plot panels, a). Bar diagrams (b) show the means of the total percentage of MHC II⁺ APC in various groups. Histogram panels (c) indicate the level of CD40 expression on MHC II⁺ APCs (the MFI is indicated). Black histogram = isotype control, blue = saline, and red = CpG-ODN treated. Bar diagrams (d) show the means of MFI of CD40 expression on MHC II⁺ APC (vertical line and horizontal bar show the standard error of mean-SEM), . Dunnett’s test following ANOVA testing was used to test for significant differences between CpG-ODN doses and the saline control group. Asterisks indicate groups that were significantly different from the control group (). (MFI—geometric mean fluorescence intensity). (e–g) Flow cytometric analysis of lung T-cell populations in each group () at 72 hr post in ovo injections (day 1 hatch). CD4⁺ and CD8⁺ T-cells in the lung (dot plot panels, e) were quantified using PE-labeled mouse anti-chicken CD4 and FITC-labeled mouse anti-chicken CD8 monoclonal antibodies. Bar diagrams show CD4⁺ and CD8⁺ T-cell number in the lungs (f), and the total number of CD4⁺ T-cells and CD8⁺ T-cells combined (g) in various groups. Two-way ANOVA following the Bonferroni posttest was done when CD4⁺ and CD8⁺ cells were compared between CpG-ODN received groups and saline control. Dunnett’s test following ANOVA testing was used to test for significant differences in total T-cells between different CpG-ODN doses and the saline control group. Vertical line and horizontal bar show the standard error of mean-SEM. Asterisks indicate groups that were significantly different from the control group, , , and .

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