Journal: Mucosal immunology

Article Title: Tregs are Dysfunctional in Vivo in a Spontaneous Murine Model of Crohn’s Disease

doi: 10.1038/mi.2012.67

Figure Lengend Snippet: (A) Representative dot plots of Foxp3 and CD25 expression on MLN CD4 + cells showing complete depletion of CD25 + cells in anti-CD25 Ab treated SAMP mice (left panel). (B) The proportion of CD4 + Foxp3 + cells was significantly higher in MLN cells of SAMP versus AKR mice. The total number of CD4 + Foxp3 + cells in SAMP MLN was 3 to5 times more than that observed in AKR MLN (data not shown). Following anti-CD25 Ab treatment for 6 wks, 28.3% of Foxp3 + cells in MLN of SAMP mice were eliminated, compared to 72.0% in AKR mice. (C) CD25 − Foxp3 + cells significantly increased after anti-CD25 Ab treatment in MLN cells of SAMP mice, compared with AKR controls. Data are presented as the mean ± SEM (* P <0.05, ** P <0.01, *** P <0.001). Control mAb SAMP, n=8; anti-CD25 Ab SAMP & control mAb AKR, n=6 each; anti-CD25 AKR, n=4.

Article Snippet: Anti-CD25 Ab (clone PC61) or a control rat IgG (clone HRPN, obtained from Innovative Research, Novi, Michigan) were dialyzed in PBS, and concentrations were determined by spectrophotometry at 280nm.

Techniques: Expressing

Journal: Mucosal immunology

Article Title: Tregs are Dysfunctional in Vivo in a Spontaneous Murine Model of Crohn’s Disease

doi: 10.1038/mi.2012.67

Figure Lengend Snippet: CD4 + cells from anti-CD25 Ab treated SAMP or AKR mice (200µg) were isolated at 0, 1, 2, 3 and 6 weeks (n=4/time point). Mice were euthanized at 12 wks of age. (A) Time course of the proportion of CD25 + Foxp3 + and CD25 − Foxp3 + cells in spleen from SAMP and AKR mice. (B) Time course of Th1 and Th2 cytokines production measured by ELISA in triplicate from 72-hour cultures of SAMP or AKR unfractionated spleen CD4 + T cells (2×10 5 cells/well) stimulated with immobilized anti-CD3 and soluble anti-CD28. Data are expressed as the mean ± SEM (* P <0.05, ** P <0.01, *** P <0.001).

Article Snippet: Anti-CD25 Ab (clone PC61) or a control rat IgG (clone HRPN, obtained from Innovative Research, Novi, Michigan) were dialyzed in PBS, and concentrations were determined by spectrophotometry at 280nm.

Techniques: Isolation, Enzyme-linked Immunosorbent Assay

Journal: Mucosal immunology

Article Title: Tregs are Dysfunctional in Vivo in a Spontaneous Murine Model of Crohn’s Disease

doi: 10.1038/mi.2012.67

Figure Lengend Snippet: (A) Anti-CD25 treated mice developed more severe ileitis with a higher mean total inflammatory score compared with control mAb treated mice; no significant colitis was detected in either group (n=11/group). (B) Representative photomicrographs of H&E stained sections, 10× and 20× original magnification. SAMP mice treated with isotype control Ab display minimal inflammatory changes with preservation of the villi morphology; anti-CD25 treated SAMP show increased infiltration of inflammatory cells and villous distortion. (C) Serum TNF-α and IFN-γ levels measured by ELISA were elevated in anti-CD25 treated SAMP mice compared to controls (n=6/group). (D) Total RNA was extracted from ileal tissues from anti-CD25 Ab or isotype control Ab treated SAMP mice, and mRNA was quantified by real-time RT-PCR. Both Th1 and Th2 cytokines were significantly increased in anti-CD25 treated mice. Data are expressed as the mean ± SEM (* P <0.05, ** P <0.01, *** P <0.001).

Article Snippet: Anti-CD25 Ab (clone PC61) or a control rat IgG (clone HRPN, obtained from Innovative Research, Novi, Michigan) were dialyzed in PBS, and concentrations were determined by spectrophotometry at 280nm.

Techniques: Staining, Preserving, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: Mucosal immunology

Article Title: Tregs are Dysfunctional in Vivo in a Spontaneous Murine Model of Crohn’s Disease

doi: 10.1038/mi.2012.67

Figure Lengend Snippet: 2×10 5 MLN CD4 + cells from anti-CD25 treated or isotype control mAb treated SAMP mice (12wks, n=6/group) were transferred into adult (4–6 wks) MHC-matched SCID mice. Cells were also adoptively transferred into similarly treated AKR control mice (n=4/group). (A) Rectal prolapse occurred in 83.3% of SCID mice that received CD4 + cells for anti-CD25 treated SAMP mice vs. 16.6% that received cells from control mAb treated SAMP mice. (B) Colon length was significantly lower in anti-CD25 treated SAMP mice. (C) Time course of body weight changes after transfer showed significant weight loss in SCID recipients transferred with cells from anti-CD25 treated SAMP compared to the other experimental groups. (D) SCID mice adoptively transferred with CD4 + MLN cells from anti-CD25 treated SAMP mice displayed higher active, chronic, and total inflammatory scores in the colon compared to mice adoptively transferred with cells from treated and untreated AKR donor mice. Data are presented as the mean ± SEM (* P <0.05, ** P <0.01, *** P <0.001). (E) Representative sections from SCID mice adoptively transferred with CD4 + MLN cells from AKR and SAMP mice treated with either anti-CD25 or isotype control Ab. Severe colonic inflammation was observed in SCID mice receiving cells from anti-CD25 Ab SAMP donors.

Article Snippet: Anti-CD25 Ab (clone PC61) or a control rat IgG (clone HRPN, obtained from Innovative Research, Novi, Michigan) were dialyzed in PBS, and concentrations were determined by spectrophotometry at 280nm.

Techniques:

Journal: Mucosal immunology

Article Title: Tregs are Dysfunctional in Vivo in a Spontaneous Murine Model of Crohn’s Disease

doi: 10.1038/mi.2012.67

Figure Lengend Snippet: CD4 + cells were transferred from anti-CD25 treated SAMP mice into SCID mice (6 wks, n=10) resulting in severe colitis. Six weeks later, 5 colitic SCID recipients per group received a second adoptive transfer of MLN CD4 + CD25 + cells (2×10 5 ) from untreated SAMP or AKR control mice (12 wks, n=3/group). The percentage of FoxP3+ cells was 90.8% and 88.3% in SAMP and AKR mice respectively. (A) Time course of body weight changes after transfer showed significant weight gain in SCID mice treated with AKR versus SAMP CD4 + CD25 + cells. (B) Survival analysis of different experimental groups showed 0% survival in SAMP CD4 + CD25 + treated SCID mice versus 60% in AKR CD4 + CD25 + treated SCID mice. (C) Elevated levels of secreted IL-10 and TGF-β measured from 3-day cultures of SAMP (n=4 mice) or AKR (n=6 mice) MLN or spleen CD4 + CD25 + T cells (2×10 5 cells/well) stimulated with immobilized anti-CD3 and soluble anti-CD28. (D) TNF-α, IFN-γ, IL-3, IL-4, IL-5, and IL-6 measured from 3-day cultures were elevated in SAMP (n=4 mice) compared to AKR (n=6 mice) MLN or spleen CD4 + CD25 + T cells (2×10 5 cells/well) stimulated with immobilized anti-CD3 and soluble anti-CD28. Data are expressed as the mean ± SEM (* P <0.05, ** P <0.01, *** P <0.001).

Article Snippet: Anti-CD25 Ab (clone PC61) or a control rat IgG (clone HRPN, obtained from Innovative Research, Novi, Michigan) were dialyzed in PBS, and concentrations were determined by spectrophotometry at 280nm.

Techniques: Adoptive Transfer Assay