H6PDH regulates the differentiation and immunostimulatory potential of spleen CD11c+DCs. (A) CD11c+ DCs were sorted from spleens of H6pdh−/− or H6pdh+/+ littermate control mice and incubated in normal media alone (+0) or in the presence of 10−7 M A or 10−7 M B for 18 hours before pulsing with OVA323-339 peptide (or irrelevant peptide) for 2 hours. DCs were then washed and cells were used at the indicated ratios in stimulation assays with 1 × 105 carboxyfluorescein succinimidyl ester (CFSE) –labeled OT-II CD4 cells for 96 hours. (Top) Flow cytometric histograms from a representative experiment showing CFSE staining of gated T cells incubated with H6pdh+/+ or H6pdh−/− CD11c+ DCs in the presence of vehicle (left), 10−7 M A (middle) or 10−7 M B (right) at a DC:T-cell ratio of 1:4 (n = 3 independent experiments). Values in the upper left corner show mean ± SD for the average number of T-cell divisions calculated for each group. Bar in upper right corner indicates the position of undivided cell sets according to CFSE staining of T cells incubated with DC pulsed with irrelevant peptide. (Bottom) Histograms showing summary data for the average number of T-cell divisions at different DC:T-cell ratios as a percentage of maximum (at a DC:T-cell ratio of 1:2) under each experimental condition. Quantitative data showing absolute numbers of divided T cells per well according to genotype of stimulator DC and exposure to A or B are shown in supplemental Figure 1. (B) Representative histograms for CD86 staining in gated MHC class IIhigh CD11chigh splenic CD8α+ DCs or CD8α– DCs from H6pdh−/− (CD45.2):H6pdh+/+ (CD45.1)→H6pdh+/+ (CD45.1) mixed BM chimeras that were evaluated 12 to 16 weeks after hematopoietic reconstitution and 24 hours after intraperitoneal treatment with nil or CpG. Staining for each DC subset derived from H6pdh−/− (bold black line, open histograms) or H6pdh+/+ (thin black line, solid gray histograms) BM is shown. Summary data from 3 independent experiments are shown to the right of each set of histograms.
