Fig 1: Lung TNFR2- cDC2 mediate CDG-induced humoral response.A. Flow cytometry analysis of TNFR2 expression on lung CDG-FITC+ cDC2 from WT mice. n=3. B. Lung TNF production in CDG treated (i.n.) MPYS-/- mice adoptively transferred with WT TNFR2+ or TNFR2- lung cDC2. n=3 C. Sorted TNFR2+ and TNFR2- lung cDC2 from WT mice were labelled with CFSE and adoptively transferred into the MPYS-/- mice. Recipient mice were treated with CDG (i.n.) for 16hrs. CD86 and CCR7 expression on CFSE positive lung cells were examined by Flow cytometry. n=3. D. Sorted TNFR2+ and TNFR2- lung cDC2 from WT mice were adoptively transferred into the MPYS-/-PspA or CDG/PspA twice. Serum anti-PspA mice. Recipient mice were immunized (i.n.) with IgG were determined by ELISA. n=3. E. Sorted TNFR2+ and TNFR2- lung cDC2 from WT mice were adoptively transferred into IRF4fl/flCD11ccre mice. Recipient mice were immunized (i.n.) with PspA or CDG/PspA. Serum anti-PspA IgG were determined by ELISA. n=3. F. Lung cells from recipient IRF4fl/flCD11ccre mice were stimulated with 5?g/ml PspA for 4 days in culture. Cytokines were measured in the supernatant by ELISA. G. A cartoon illustrating following CDG administration, TNFR2+ cDC2 activate RelB to induce Th1, Th2 and Th17 responses while TNFR2- cDC2 mediate the antibody response. Graphs represent means ± standard error from three independent experiments. The significance is represented by and asterisk (*) where p<0.05 (unpaired Student’s t test).
Fig 2: Lung cDC2 consist of two distinct subpopulations.A. Flow cytometry analysis of lung TNFR2+ vs TNFR2- cDC2 at steady-state. n=3. B-C. Flow cytometry analysis of IRF4 and IRF8 (B) and Zbtb46 (C) in TNFR2+ and TNFR2- cDC2. n=3. D. Flow cytometry analysis in lungs of IRF4fl/flCD11ccre mice reconstituted with CD45.1+ pre-cDC2. n=3. pre-cDC2 were sorted from the bone marrow of B6.CD45.1 mice and transferred (i.n.) into IRF4fl/flCD11ccremice. n=3. E. Phenotypic analysis of CD45.1 TNFR2+ and TNFR2- cDC2 transferred into IRF4fl/flCD11ccre mice. n=3. F. Flow cytometry analysis of TNFR2 expression on cDC2 subpopulations transferred into MPYS-/- mice treated with CDG (i.n.) for 16hrs. n=3.
Fig 3: cDC2 expression of TNFR2 and RelB is required for Th1 and Th17 responses, but dispensable for CDG-induced antibody response.A. IRF4fl/flCD11ccre mice were adoptively transferred (i.n.) with lung cDC2 sorted from WT or TNFR2-/- mice lung and immunized (i.n.) with PspA or CDG/PspA. Serum anti-PspA IgG and BALF anti-PspA IgA were determined by ELISA. n=3. B. RelBfl/fl and RelBfl/flCD11CCre mice were immunized (i.n) with CDG/PspA or PspA alone as before. Serum anti-PspA IgG and BALF anti-PspA IgA were determined by ELISA. n=3. C. Lung cells from immunized RelBfl/fl and RelBfl/flCD11CCre mice were stimulated with 5?g/ml PspA for 4 days in culture. Cytokines were measured in the supernatant by ELISA. n>3. Graphs represent means ± standard error from three independent experiments. The significance is represented by and asterisk (*) where p<0.05 (unpaired Student’s t test).
Fig 4: TNFR2- cDC2 express mTNF but have few processed antigen.A. WT mice were administered with saline or CDG (5µg) for 16hrs. mTNF expression was determined by Flow cytometry using mouse TNFR2-Fc recombinant protein. n=3. B-C. Flow cytometry analysis of mTNF expression in lung DCs (B) and cDC2 (C) n=3. D. Flow cytometry analysis of antigen uptake and processing in lung CD11b+ DC from WT mice treated (i.n.) with DQ-OVA (20ug) and CDG (5ug) for 16 hours. n=3. Graphs represent means ± standard error from three independent experiments. The significance is represented by and asterisk (*) where p<0.05 (unpaired Student’s t test).
Fig 5: CDG activates TNFR2-/- cDC2 in vivo to produce TNF.A-B. Indicated mice were administered (i.n.) with saline or CDG for 5hrs. TNF production was measured in lung homogenates by ELISA. n>3. C. WT and TNFR2-/- mice were treated with saline or CDG for 16 hours. TNF in lung cDC2 was determined by an intracellular cytokine stain. n=3. D. TBK1fl/fl and TBK1fl/flVavcre mice were administered (i.n.) with saline or CDG for 5hrs. TNF production was measured in lung homogenates by ELISA. n=3. E. Flow cytometry analysis of p-TBK1 expression in lung cDC2 from WT and TNFR2-/- mice treated with saline or CDG for 16 hours. n=3. Graphs represent means ± standard error from three independent experiments. The significance is represented by and asterisk (*) where p<0.05 (unpaired Student’s t test).
Supplier Page from Sino Biological, Inc. for Mouse TNFR2 / CD120b / TNFRSF1B Protein (Fc Tag)