Fig 1. Vitamin D inhibits chicken T-cell proliferation and IFN-γ production ex vivo.

Splenocytes were pre-treated for 4hrs with Vitamin D (100 and 10 nM) or vehicle only (DMSO). (A) BrdU incorporation measured by ELISA assay to quantify T cell proliferation after stimulation with different concentrations of Concanavalin A. The results are presented as absorbance at OD₄₅₀ from three independent experiments with 3–4 biological replicates in each experiment. (B, C) The combined results from proliferation from 6 independent experiments for absorbance at OD₄₅₀ (B) and the percentages of inhibition by Vitamin D are shown. (D) The frequency of IFN-γ producing mononuclear cells stimulated with a T-cell stimulation cocktail of PMA and Ion was detected using a chicken IFN-γ ELISPOT assay. The results are presented as spots forming unit (SFU) per 1.0 x 10⁶ cells from five independent experiments with 3–5 biological replicates in each experiment (19 replicates in total). (E) Represents the percentage of inhibition for IFN-γ production by mononuclear cells after Vitamin D₃ pre-treatment from five independent experiments. Each dot represent a biological replicate. Non-parametric Wilcoxon tests (Mann-Whitney) was used to assess normal distribution and test significance. The results are shown as mean ± SD. † (symbol) represents cells treated with Vehicle only (DMSO). * indicates a statistically significant difference (P < 0.05).