MDCs from ΔdblGATA mice have reduced APC phenotype in vivo but not in vitro. (A-C) BM from WT and ΔdblGATA mice was treated with GM-CSF and IL-4 for 5 days. To induce BMDC maturation, the cultures were treated with LPS for an additional 3 days. Data represent the average of 2 independent experiments. (A) GATA1 expression by BMDCs determined by real-time PCR. (B) Flow cytometry plots showing MHC-II and CD11b expression among CD11b⁺CD11c⁺ BMDCs. (C) Frequency of MHC-II^hi cells among CD11b⁺CD11c⁺ BMDCs. (D) Flow cytometry plots showing MHC-II and CD11b expression among CD11b⁺CD11c⁺ MDCs were generated in vitro from CD45⁺CD11b⁺Ly6G^lo/−Ly6C^hi monocytes purified from the BM of WT and ΔdblGATA mice. Histograms overlays (right) of CD80 and CD86 expression from MHC-II^hi and MHC-II^lo cells (left) are depicted. (E) Proliferation of cocultures of LPS-matured MDCs from panel D with DO11.10 CD4⁺ T cells that were stimulated with OVA_323–339 for 72 hours. Proliferation was measured by [³H]adenine incorporation assay. Error bars are SEM and significance was calculated by unpaired Student t test. (F) WT and ΔdblGATA mice were immunized for EAE induction and sacrificed on day 16 after immunization (n = 14-15 per group). CNS mononuclear cells were isolated by Percoll density gradient centrifugation, and cells from 4 to 5 mice were pooled. Monocytes were then highly enriched with CD11b magnetic beads followed by fluorescence-activated cell sorting for CD11b⁺Ly6C^hiLy6G^lo/− cells. RNA was isolated, and the transcriptomes of WT and ΔdblGATA monocytes were sequenced (bulk RNA sequencing; n = 3 per group). Differentially expressed genes with adjusted P < .05 were used for pathway analysis using DAVID bioinformatics database. The top 5 significantly enriched pathways using gene ontology and KEGG databases are shown. (G) Heatmap showing differentially expressed genes from the top gene ontology term from panel F. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Norm., normal.