Display Settings:

Items per page
We are sorry, but NCBI web applications do not support your browser and may not function properly. More information

Results: 7

1.
Figure 7

Figure 7. From: IBP (IRF-4 Binding Protein) inhibits IL-17 and IL-21 production by controlling IRF-4 function.

The deregulated IL-17 and IL-21 production observed in the absence of IBP is dependent on IRF-4. A. IL-17 production by wt, IBPtrap/trap, IRF-4−/−, and IRF-4−/−IBPtrap/trap CD4+ T cells. Purified naïve CD4+ T cells were stimulated under TH0 conditions for 4 days. IL-17 levels in culture supernatants were determined by ELISA. B. RORγt expression by wt, IBPtrap/trap, IRF-4−/−, and IRF-4−/−IBPtrap/trap CD4+ T cells. Purified naïve CD4+ T cells were stimulated as above and RORγt gene expression was determined by real-time RT-PCR. C. IL-21 production by wt, IBPtrap/trap, IRF-4−/−, and IRF-4−/−IBPtrap/trap CD4+ T cells. Purified naïve CD4+ T cells were stimulated under TH2 conditions for 7 days and then restimulated for 48 hrs. IL-21 levels in culture supernatants were determined by ELISA. D. CFSE profiles of wt, IBPtrap/trap, IRF-4−/−, and IRF-4−/−IBPtrap/trap CD4+ T cells that were either left unstimulated (red) or stimulated under TH0 conditions for 2 days (blue) or for 4 days (green). Experiments shown are representative of 3 independent experiments.

Qinzhong Chen, et al. Immunity. ;29(6):899-911.
2.
Figure 1

Figure 1. From: IBP (IRF-4 Binding Protein) inhibits IL-17 and IL-21 production by controlling IRF-4 function.

Development of arthritis and large-vessel vasculitis in IBPtrap/trap DO11.10 mice. A. Shown are the wrist and ankle of a normal 16 wks old IBP+/+ DO11.10 (IBP+/+ DO) female mouse (left) and of an affected IBPtrap/trap DO11.10 (IBPtrap/trap DO) female mouse (right) that had developed swelling and erythema of the joints. B. Histopathologic analysis (hematoxylin/eosin staining) of wrist joints of a 12-week-old female IBP+/+ DO11.10 mouse (left) and an age- and sex- matched IBPtrap/trap DO11.10 mouse (right). Light microscopy images (magnification of 40 and 100×, as indicated) are shown. These findings are representative of 6 mice for each group. C. Serological analysis. Sera from IBP+/+ DO11.10 and IBPtrap/trap DO11.10 mice (6–25 weeks old, male and female, n=6–12) were collected and levels of rheumatoid factor (RF), anti-collagen II (CII) antibodies, and anti-cyclic citrullinated peptide (CCP) antibodies were analyzed by ELISA. *p<0.05. D. Histopathologic analysis (hematoxylin/eosin staining) of the root of the aorta of a 16 wk-old IBP+/+ DO11.10 female mouse (left panels) and an age-matched IBPtrap/trap DO11.10 female mouse (right panels). Light microscopy images (magnification of 40× and 100×, as indicated) are shown. These findings are representative of 6 mice for each group.

Qinzhong Chen, et al. Immunity. ;29(6):899-911.
3.
Figure 2

Figure 2. From: IBP (IRF-4 Binding Protein) inhibits IL-17 and IL-21 production by controlling IRF-4 function.

Lack of IBP leads to abnormalities in the responsiveness of DO11.10 CD4+ T cells. A. Proliferative responses to OVA323-339 peptide. Naïve CD4+ T cells (CD44lowCD62Lhigh) were isolated from both IBP+/+ DO11.10 (blue) and IBPtrap/trap DO11.10 (red) mice and stimulated for 3 days with IBP+/+ APCs pulsed with increasing doses (0, 0.1, 1, 10 μM) of OVA323-339 peptide. Cell proliferation was assayed by BrdU ELISA. Experiment shown is representative of 3 independent experiments. B. Spontaneous acquisition of an effector phenotype in vivo in the absence of IBP. FACS analysis of CD44 and CD62L expression on CD4+ splenic T cells from a 6-week-old female IBP+/+ DO11.10 (left) and a sex- and age- matched IBPtrap/trap DO11.10 (right) mouse. C. Spontaneous activation of IBPtrap/trap DO11.10 T cells in vivo. The surface expression level of CD69, CD25, and ICOS on KJ1-26high IBP+/+ DO11.10 (blue) and KJ1-26high IBPtrap/trap DO11.10 (red) was analyzed by FACS and overlaid histograms are shown. Data are representative of three 6 week-old mice/group.

Qinzhong Chen, et al. Immunity. ;29(6):899-911.
4.

Figure 3. From: IBP (IRF-4 Binding Protein) inhibits IL-17 and IL-21 production by controlling IRF-4 function.

Lack of IBP leads to aberrant production of IL-17 and IL-21 in vitro and in vivo. A. Naïve CD4+ T cells derived from 6 wks. old IBP+/+ DO11.10 (white bars) or IBPtrap/trap DO11.10 (black bars) mice were cultured with IBP+/+ APCs pulsed with 1 μM OVA323-339 peptide for the times indicated. The production of IL-17 in the supernatants was measured by ELISA. Experiment shown is representative of 3 independent experiments. B. Naïve CD4+ T cells derived from 6 wks. old IBP+/+ DO11.10 (white bars) or IBPtrap/trap DO11.10 (black bars) mice were cultured as above. The production of IL-21 in the supernatants was measured by ELISA. Experiment shown is representative of 3 independent experiments. C. Naïve CD4+ T cells derived from 6 wks. old IBP+/+ DO11.10 (white bars) or IBPtrap/trap DO11.10 (black bars) mice were cultured as above. The production of IL-2 in the supernatants was measured by ELISA. Experiment shown is representative of 3 independent experiments. D. Naïve CD4+ T cells derived from 6 wks. old IBP+/+ DO11.10 (white bars) or IBPtrap/trap DO11.10 (black bars) mice were cultured for 3 days as described above and the mRNA expression of RORγt (left panel) and IL-22 (right panel) genes was measured by real-time RT-PCR. Experiment shown is representative of 3 independent experiments. E. Serum levels of IL-17 and IL-21 in IBP+/+ DO11.10 and IBPtrap/trap DO11.10 mice. Sera from IBP+/+ DO11.10 and IBPtrap/trap DO11.10 mice (12–25 weeks old, male and female, n=4) were collected and levels of IL-17 and IL-21 analyzed by ELISA. *p<0.05. F. Expression of IL-17 and IL-21 in the joints of arthritic IBPtrap/trap DO11.10 mice. Joints from 3 pairs of IBP+/+ DO11.10 and IBPtrap/trap DO11.10 mice (12–25 weeks old, male and female) were collected and IL-17 and IL-21 gene expression was analyzed by real-time RT-PCR. White bars represent IBP+/+ DO11.10 mice and black bars represent IBPtrap/trap DO11.10 mice. G. Blimp-1 and CD3 staining of spleens of IBPtrap/trap DO11.10 mice. Anti-Blimp-1 (blue) and anti-CD3 (red) staining was performed on spleens from IBP+/+ DO11.10 (left panel) and IBPtrap/trap DO11.10 (right panel) mice. Light microscopy images (magnification of 4×, upper panels, and 40×, lower panels) are shown.

Qinzhong Chen, et al. Immunity. ;29(6):899-911.
5.
Figure 4

Figure 4. From: IBP (IRF-4 Binding Protein) inhibits IL-17 and IL-21 production by controlling IRF-4 function.

IRF-4 is a critical regulator of both IL-17 and IL-21 production. A. IL-17 production by wt and IRF-4−/− CD4+ T cells. Purified naïve CD4+ T cells were stimulated under either TH0 (white bars) or TH17 (black bars) conditions for 4 days. IL-17 levels in culture supernatants were determined by ELISA. The experiment is representative of four independent experiments. B. Intracellular IL-17 and IFN-γ production by wt and IRF-4−/− CD4+ T cells. Cells were cultured under TH17 conditions as above and then stimulated with PMA and ionomycin for 5 hours. Intracellular IFN-γ and IL-17 production was measured by FACS. The experiment is representative of three independent experiments. C. IL-21 production by wt and IRF-4−/− CD4+ T cells cultured under TH17 conditions. Purified naïve CD4+ T cells were stimulated under TH17 conditions for 4 days and then restimulated for either 24 hrs (white bars) or 48 hrs (black bars). IL-21 levels in culture supernatants were determined by ELISA. The experiment is representative of four independent experiments. D. IL-21 production by wt and IRF-4−/−CD4+ T cells cultured under TH0 and TH2 conditions. Purified naïve CD4+ T cells were stimulated under TH0 (white bars) or TH2 (black bars) conditions for 7 days and then restimulated for 48 hrs. IL-21 levels in culture supernatants were determined by ELISA. The experiment is representative of four independent experiments. E. Effects of IRF-4 on the transactivation of IL-21. Jurkat cells that express an empty vector or Jurkat cells expressing IRF-4 were transfected with a luciferase reporter construct driven either by the murine IL-21 promoter (IL-21 LUC), by a murine IL-21 promoter containing mutations within the putative IRF-4 binding site (IL-21 MUT LUC) or with a control luciferase reporter construct (pGL3) in the presence or absence of PMA and ionomycin as indicated. Cotransfection with a renilla-luciferase construct was performed to normalize the transfection efficiency of the different samples. Data are representative of 3 independent experiments.

Qinzhong Chen, et al. Immunity. ;29(6):899-911.
6.
Figure 5

Figure 5. From: IBP (IRF-4 Binding Protein) inhibits IL-17 and IL-21 production by controlling IRF-4 function.

The absence of IBP leads to enhanced targeting of the IL-17 and IL-21 regulatory regions. A. IBP can be found within the nuclear compartment. Purified CD4+ T cells were either left unstimulated or stimulated with anti-CD3 and anti-CD28 Abs for 48 hrs. Nuclear and cytoplasmic extracts were then prepared, resolved by 8% SDS-PAGE, and then analyzed by Western blotting using an anti-IBP antiserum (upper panel). The blot was later stripped and reprobed with an anti-Lamin B (middle panel) or an anti-β-tubulin (lower panel) antibody to assess the purity of the different fractions. B. Nuclear IBP interacts with IRF-4. Purified CD4+ T cells were stimulated with anti-CD3 and anti-CD28 Abs for 48 hrs. Nuclear extracts were then prepared and immunoprecipitated with an anti-IRF-4 antibody (IRF-4 IP) or with a control antibody (Control Ab IP). The immunoprecipitates were resolved by 8% SDS-PAGE, and then analyzed by Western blotting using an anti-IBP antiserum (upper panel). The blot was later stripped and reprobed with an anti-IRF-4 antibody (lower panel). C. Lack of IBP leads to enhanced targeting of the IL-17 and IL-21 promoter by IRF-4 in vivo. CD4+ T cells were purified from either IBP+/+ (wt) or IBPtrap/trap mice, cultured under TH0 conditions for 7 days, and then either left unstimulated (unst) or restimulated for 24 hrs (st) with anti-CD3 and anti-CD28 Abs. Chromatin immunoprecipitation assays were then carried out on these cells with either an anti-IRF-4 antibody or a control Ab, and PCR for the IL-21 and IL-17 promoters was performed as indicated. The total input control is shown on the left. Actin was used as a negative control. D. Lack of IBP leads to enhanced binding of IRF-4 to the IRF-4 binding site within the IL-21 promoter. CD4+ T cells were purified from either IBP+/+ (wt) or IBPtrap/trap mice, cultured as described in C. and subjected to an oligonucleotide precipitation assay (ONP) with a biotin-labelled oligonucleotide corresponding to the IRF-4 binding site within the IL-21 promoter. Precipitates were separated by 8% SDS-PAGE and analyzed by Western blotting with an IRF-4 antibody (top panel). The levels of IRF-4 in the input samples are shown on the right (due to the high levels of IRF-4 in the input extracts a shorter exposure time of the IRF-4 input is shown). The blot was then stripped and reprobed with an anti-IBP antibody (lower panel). The levels of IBP in the input samples are shown on the right.

Qinzhong Chen, et al. Immunity. ;29(6):899-911.
7.

Figure 6. From: IBP (IRF-4 Binding Protein) inhibits IL-17 and IL-21 production by controlling IRF-4 function.

IBP inhibits the DNA binding and transactivating activity of IRF-4. A. The absence of IBP does not alter the expression or localization of IRF-4. CD4+ T cells were purified from either IBP+/+ (wt) or IBPtrap/trap mice, cultured under TH0 conditions for 7 days, and then either left unstimulated (unst) or restimulated for 24 hrs (st) with anti-CD3 and anti-CD28 Abs. Nuclear and cytoplasmic extracts were then prepared, resolved by 8% SDS-PAGE, and then analyzed by Western blotting using an anti-IRF-4 antiserum (upper panel). The blot was later stripped and reprobed with an anti-Lamin B (middle panel) or an anti-β-tubulin (lower panel) antibody to assess the purity of the different fractions. B. Mapping of the IBP domain responsible for the interaction with IRF-4. 293T cells were cotransfected with an IRF-4 expression construct together with either HA-tagged full length IBP or the panel of HA-tagged IBP mutants described in the diagram. Extracts were immunoprecipitated with an anti-HA antibody, precipitates were separated by 8% SDS-PAGE and analyzed by Western blotting with an IRF-4 antibody (top panel). The blot was later stripped and reprobed with an anti-HA antibody (bottom panel). C. IBP can block the binding of IRF-4 to an oligonucleotide containing the IRF-4 binding site within the IL-21 promoter. An oligonucleotide precipitation assay (ONP) was performed on 293T cells that were either mock transfected or transfected with IRF-4 in the presence/absence of HA-tagged FL IBP, HA-tagged IBPΔN, or HA-tagged IBP 1-385. Precipitates were separated by 8% SDS-PAGE and analyzed by Western blotting with an IRF-4 antibody (left panel). The levels of IRF-4 in the input samples are also shown (due to the high levels of IRF-4 in the extracts a shorter exposure time of the IRF-4 input is shown). A nonspecific band detected in the ONP from mock transfected 293 T cells is indicated as ns. The blot was later stripped and reprobed with an anti-HA antibody to assess the levels of the different IBP constructs in the input samples (right panel). D. IBP can inhibit the transactivating activity of IRF-4. Jurkat cells that express an empty vector or Jurkat cells expressing IRF-4 were transfected with a luciferase reporter construct driven by the murine IL-21 promoter (IL-21 LUC) together with an empty vector, HA-tagged FL IBP, HA-tagged IBPΔN, or HA-tagged IBP 1-385 as indicated. Cells were either left unstimulated or stimulated with PMA and ionomycin as indicated. Transfections with a control luciferase reporter construct (pGL3) in the presence/absence of PMA and ionomycin were also carried out. Cotransfection with a renilla-luciferase construct was performed to normalize the transfection efficiency of the different samples. Data are representative of 3 independent experiments.

Qinzhong Chen, et al. Immunity. ;29(6):899-911.

Display Settings:

Items per page

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Write to the Help Desk