Journal:

Article Title: Spontaneous Colitis Occurrence in Transgenic Mice with Altered B7-Mediated Costimulation 1

doi:

Figure Lengend Snippet: Spontaneous colitis in B7-2 Fc Tg mice. A–C, Diffuse inflammation of the whole large intestine (A), splenomegaly (B), and enlarged MLN (arrows in C) in tissues from a B7-2 Fc Tg+ littermate (right) compared with a B7-2 Fc Tg− mouse (left). D, Severe mucosal inflammation in the large intestine (LI) of the B7-2 Fc Tg+ mouse. E, A characteristic enlarged lymphoid aggregate in the large intestine of a B7-2 Fc Tg+ mouse. F, CD19 staining of the organized lymphoid follicles in the large intestine of the B7-2 Fc Tg+ mouse. G, A mild inflammation in the small intestine (SI) of the B7-2 Fc Tg+ mouse.

Article Snippet: One × 10 5 CFSE-labeled T cells were stimulated in the anti-CD3 coated plate for 4 days in the presence or absence of 60 μ g/ml soluble B7-2 Ig Fc or 0.5 μ g/ml anti-CD28 (37.51, BD Biosciences).

Techniques: Staining

Journal:

Article Title: Spontaneous Colitis Occurrence in Transgenic Mice with Altered B7-Mediated Costimulation 1

doi:

Figure Lengend Snippet: Systemic expansion and activation of CD11b+ cells and CD19+ cells in B7-2 Fc Tg mice. A, A representative surface staining for CD11b together with CD19 (left panel) and F4/80 (right panel). Numbers show the percentage of the population gated as a square among total cells from each organ. Cells were isolated from spleen, MLN, PLN, and large intestine of B7-2 Fc Tg+ (left of each panel) and Tg− (right) littermates. B, Total cell numbers in spleen (top left), CD11b+ spleen cells (top right), total MLN cells (bottom left), and CD19+ cells in MLN (bottom right). Bars represent the averages from diseased B7-2 Fc Tg+ (left, n = 7), disease-free B7-2 Fc Tg+ (middle, n = 6), and B7-2 Fc Tg− littermates (right, n = 10). Statistical analyses were performed using a t test. (*, p < 0.05; ns, not significant) (C) I-Ab, B7-1, B7-2 and CD40 staining of splenic B cells (CD19+CD11b−) from diseased B7-2 Fc Tg+ (solid line) and disease-free B7-2 Fc Tg− (gray filled) mice. Stainings with isotype control for diseased B7-2 Fc Tg+ were shown as broken lines. D, Increased serum Ig in B7-2 Fc Tg mice (both diseased and disease-free). The concentrations of serum Ig subclasses as well as total Ig are shown. Each triangle and square represents individual B7-2 Fc Tg+ and Tg− mouse, respectively. The differences were significant (p < 0.05) in all the subclasses (shown as *).

Article Snippet: One × 10 5 CFSE-labeled T cells were stimulated in the anti-CD3 coated plate for 4 days in the presence or absence of 60 μ g/ml soluble B7-2 Ig Fc or 0.5 μ g/ml anti-CD28 (37.51, BD Biosciences).

Techniques: Activation Assay, Staining, Isolation

Journal:

Article Title: Spontaneous Colitis Occurrence in Transgenic Mice with Altered B7-Mediated Costimulation 1

doi:

Figure Lengend Snippet: Accumulation of activated CD4+ T cells in B7-2 Fc Tg mice. A, Ratio of B cells (CD19+) to T cells (TCR+ in the spleen, MLN, PLN, and large intestine). Bars represent ratios in the diseased B7-2 Fc Tg+ (filled) and Tg− (open) littermates (four mice each).*, Significant (p < 0.05) differences. B, Accumulation of CD4+ T cells in the large intestine of the B7-2 Fc Tg mice. CD4 and CD8 staining for gated TCRβ+ cells from 8- (left) and 14-wk-old (right) diseased B7-2 Fc Tg+ mice and disease-free Tg− littermates (left and right of each panel, respectively) (representative data of four mice analyzed in each group). C, Representative CD43 (1B11) staining for the CD4+ (left) and CD8+ (right) TCRβ+ cells isolated from a diseased B7-2 Fc Tg+ mouse (bold lines) and a disease-free B7-2 Fc Tg− littermate (gray filled). Stainings for the cells isolated from spleen, MLN, PLN, and large intestine are shown.

Article Snippet: One × 10 5 CFSE-labeled T cells were stimulated in the anti-CD3 coated plate for 4 days in the presence or absence of 60 μ g/ml soluble B7-2 Ig Fc or 0.5 μ g/ml anti-CD28 (37.51, BD Biosciences).

Techniques: Staining, Isolation

Journal:

Article Title: Spontaneous Colitis Occurrence in Transgenic Mice with Altered B7-Mediated Costimulation 1

doi:

Figure Lengend Snippet: Increased disease severity in B7− B7-2 Fc Tg mice. A, Histological scores of colitis in B7+ (left, circles) and B7− (right, squares) B7-2 Fc Tg+ mice (filled) compared with Tg− littermates (open). (*, p < 0.05 between B7+ and B7− B7-2 Fc Tg+). B, Number of CD11b+ cells in the spleen (left) and CD19+ cells in the MLN (right) of diseased B7+ B7-2 Fc Tg+ mice (●), B7− B7-2 Fc Tg+ mice (■) and B7− B7-2 Fc Tg− littermates (□). C, Serum IgG1, IgG2a and IgM concentrations from the B7-2 Fc Tg+ (filled) and B7-2 Fc Tg− (open) littermates that are B7+ (circles) and B7− (squares).*, Significant (p < 0.05) differences.

Article Snippet: One × 10 5 CFSE-labeled T cells were stimulated in the anti-CD3 coated plate for 4 days in the presence or absence of 60 μ g/ml soluble B7-2 Ig Fc or 0.5 μ g/ml anti-CD28 (37.51, BD Biosciences).

Techniques:

Journal:

Article Title: Spontaneous Colitis Occurrence in Transgenic Mice with Altered B7-Mediated Costimulation 1

doi:

Figure Lengend Snippet: A reduced percentage of Treg contributes to the severe colitis in B7− B7-2 Fc Tg mice. A, Representative stainings for CD25+ Foxp3+ Treg in the spleen (top) and MLN (bottom) from the B7+ (left) and B7− (right) background B7-2 Fc Tg+ (left in each) and B7-2 Fc Tg− (right in each) mice. B, Percentages of Foxp3+ cells averaged from four mice each from the B7+ or B7− background, diseased B7-2 Fc Tg+ or B7-2 Fc Tg− mice.*, Significant (p < 0.05) differences. C, In vitro coculture experiment showing the suppression of T cell proliferation by Treg in the presence or absence of the B7-2 Ig Fc. CFSE dilutions of responder CD4+CD45RBhigh cells (gated as CD45.1−) cocultured without Treg (gray area), with Treg (solid line), and with Treg in the presence of B7-2 Ig Fc (dotted line) are shown. Numbers indicate the proliferation indexes. D, Injection of CD4+CD25+ cells but not CD4+CD25− cells attenuated the severity of colitis in the B7− B7-2 Fc Tg mice. Averaged histological scores from three parts of the large intestines of B7− B7-2 Fc Tg+ mice injected with CD4+CD25− (n = 4) or CD4+CD25+ (n = 4) cells. (*, p < 0.05).

Article Snippet: One × 10 5 CFSE-labeled T cells were stimulated in the anti-CD3 coated plate for 4 days in the presence or absence of 60 μ g/ml soluble B7-2 Ig Fc or 0.5 μ g/ml anti-CD28 (37.51, BD Biosciences).

Techniques: In Vitro, Injection

Journal:

Article Title: Spontaneous Colitis Occurrence in Transgenic Mice with Altered B7-Mediated Costimulation 1

doi:

Figure Lengend Snippet: The soluble B7-2 Ig Fc accelerates colitis independently of Treg. A, CD4+CD45RBhigh cells were injected to RAG1−/− (circles) or B7-2 Fc Tg+ RAG1−/− (squares) recipients. In a separate group of four RAG1−/− recipients, the B7-2 Ig Fc (100 μg) was injected three times a week for 2 wk (○). The average relative body weight of each group is shown. B, Representative histology (×40 magnification) of the large intestine 6 days post transfer of CD4+CD45RBhigh cells into B7-2 Fc Tg− RAG1−/− (left) and B7-2 Fc Tg+ RAG1−/− (right) recipients. Arrows represent infiltration of mononuclear cells into the mucosa.

Article Snippet: One × 10 5 CFSE-labeled T cells were stimulated in the anti-CD3 coated plate for 4 days in the presence or absence of 60 μ g/ml soluble B7-2 Ig Fc or 0.5 μ g/ml anti-CD28 (37.51, BD Biosciences).

Techniques: Injection

Journal:

Article Title: Spontaneous Colitis Occurrence in Transgenic Mice with Altered B7-Mediated Costimulation 1

doi:

Figure Lengend Snippet: Weak agonistic and antagonistic properties of the B7-2 Ig Fc chimera. A and B, Soluble B7-2 Ig Fc. CFSE-labeled naive CD4+ T cells were stimulated for 4 days with 1.0 μg/ml plate-bound anti-CD3 mAb. A, CFSE dilutions in the presence (solid line) or absence (gray-filled) of 60 μg/ml soluble B7-2 Ig Fc (top) or 0.5 μg/ml soluble anti-CD28 mAb (bottom) are shown. B, IL-2 concentration in the supernatant was measured from cultures in Fig. 7A. C and D, Plate-bound B7-2 Ig Fc. CFSE-labeled naive CD4+ T cells were stimulated with plate-bound anti-CD3 or anti-CD3 with B7-2 Ig Fc for 4 days. C, CFSE dilution in the presence (solid line) or absence (gray-filled) of immobilized B7-2 Ig Fc is shown. These data are representative of two independent experiments. D, IL-2 concentration in the supernatant from a culture in the presence (×) or absence (●) of the immobilized B7-2 Ig Fc. E, The B7-2 Ig Fc inhibits Ab binding to CTLA-4 but not to CD28. Pre-activated CD4+ T cells (for CTLA-4) or naive CD4+ T cells (for CD28) were incubated with 100 μg/ml B7-2 Ig Fc (solid line) for 10 min before staining with PE-conjugated anti-CTLA-4 (top) or anti-CD28 (bottom) Ab. Cells preincubated with 100 μg/ml unconjugated anti-CTLA-4 (for CTLA-4) and naive CD4+ T cells isolated from CD28 deficient mice were used as negative controls, respectively (shown as broken lines). CTLA-4 or CD28 expression without pre-incubation is shown by gray-filled area. F, OT-II OVA specific CD4+ T cells were primed and restimulated in the presence of 0.1 μM OVA. B7-2 Ig Fc (left), anti-CTLA-4 (middle), or neither of them (right) was added in both primary and secondary cultures. Intracellular IFN-γ staining after a brief activation with PMA; ionomycin in the presence of Brefeldin A is shown. Data for all experiments in the figure are representative of at least two experiments.

Article Snippet: One × 10 5 CFSE-labeled T cells were stimulated in the anti-CD3 coated plate for 4 days in the presence or absence of 60 μ g/ml soluble B7-2 Ig Fc or 0.5 μ g/ml anti-CD28 (37.51, BD Biosciences).

Techniques: Labeling, Concentration Assay, Binding Assay, Incubation, Staining, Isolation, Expressing, Activation Assay

Journal:

Article Title: Spontaneous Colitis Occurrence in Transgenic Mice with Altered B7-Mediated Costimulation 1

doi:

Figure Lengend Snippet: IFN-γ dependent and independent colitis induction in the B7-2 Fc Tg mice. A, Cells were isolated from spleen, MLN and LI (as indicated) from 8-wk-old diseased B7-2 Fc Tg+ mice (top) or disease-free B7-2 Fc Tg− littermates. Intracellular stainings of CD4+TCRβ+ cells for IFN-γ/IL-17 (left) and IL-4/IL-10 (right) after ex vivo restimulation are shown. Data are representative from eight diseased B7-2 Fc Tg+ and four B7-2 Fc Tg− mice. B, Averaged histological scores from B7+ (top) or B7− (bottom) background IFN-γ−/− B7-2 Fc Tg+ mice. C, Representative H&E stainings from a middle part of large intestine of B7+ (left) and B7− (right) background IFN-γ−/− B7-2 Fc Tg+ mice. Scale bars showing 1 mm are placed at the left bottom corner of each figure.

Article Snippet: One × 10 5 CFSE-labeled T cells were stimulated in the anti-CD3 coated plate for 4 days in the presence or absence of 60 μ g/ml soluble B7-2 Ig Fc or 0.5 μ g/ml anti-CD28 (37.51, BD Biosciences).

Techniques: Isolation, Ex Vivo