Lack of Foxp3 expression by macrophages. (A) WT and DEREG mice were treated on 2 consecutive days with 1μg DT or PBS intraperitoneally. One day later, collagenase/DNase-digested splenocytes were analyzed by flow cytometry. CD11b (M1/70), Foxp3 (FJK-16s) or rat IgG2a/κ isotype staining is shown. (B) BM was isolated from 2-3 weeks old WT or scurfy males and analyzed by flow cytometry. CD11b and Foxp3 expression is shown. (C) BM of PBS- or DT-treated WT and DEREG mice was analyzed by flow cytometry. CD11b expression of CD3ϵ/TCRβ/TCRγδ⁻ cells is plotted against GFP/autofluorescence. (D) BMM were generated as described⁹  from WT or DEREG mice except 100ng/mL DT was added on d0 and d3 of culture where indicated. BMM were harvested using accutase and analyzed by flow cytometry. CD11b vs Foxp3 expression is shown in the top panel. The bottom panel is unstained for Foxp3. (E) B16-OVA tumors were established in WT mice⁵ , harvested, collagenase/DNase-digested and analyzed by flow cytometry. CD11b, Foxp3 or isotype staining is displayed. (F) B16-OVA tumors were established in WT or Rag KO mice, resected and cryopreserved. Five micrometer sections were analyzed by immunofluorescence microscopy after staining for Foxp3 (FJK-16s), F4/80 (BM8), CD3 (N1580) and AlexaFluor488- or AlexaFluor555-labeled secondary antibodies. AxioImager Z1, Axiovision 4.6.3.0 software, and AxioCam MRm were applied for image acquisition and analysis. Foxp3 (red), CD3 (green) and DAPI (blue) signals are displayed in the upper panel; the lower panel shows Foxp3 (red), F4/80 (green) and DAPI (blue) stainings. All images were acquired with 400× magnification and a 50 μm white scale bar is displayed. (A-E) All flow cytometry analyses were performed after Fc receptor blocking with anti-CD16/32 (2.4G2). Cells were acquired on LSRII (BD Biosciences), analyzed by FlowJo (Tristar) and dead cells were excluded by ethidium monoazide (EMA) photolysis. (A-F) All experiments are representative of 2-3 independent experiments.