Journal: The Journal of Experimental Medicine

Article Title: Subspecialization of Cxcr5 + T Cells

doi:

Figure Lengend Snippet: Effector machinery of CD57 +/ −CD45RA +/ − or CD57 +/ − CXCR5 +/ − CD4 T cells. (A) Intracellular cytokine analyses of TNF-α, IL-2, IL-10, IFN-γ, and IL-4. (B) ELISA of IL-10 and IL-4. Isolated T cells were activated with 50 ng/ml PMA and 1 μg/ml ionomycin for 4 h for intracellular cytokine analyses, or 24 h for ELISA of IL-10 and IL-4. Representatives of at least four independent experiments are shown.

Article Snippet: In brief, acetone-fixed sections were stained with the primary monoclonal antibody to CXCR5 (R&D Systems), followed by incubation in biotinylated goat anti–mouse IgG (Vector Laboratories).

Techniques: Enzyme-linked Immunosorbent Assay, Isolation

Journal: The Journal of Experimental Medicine

Article Title: Subspecialization of Cxcr5 + T Cells

doi:

Figure Lengend Snippet: Identification of CD57 + CXCR5 + T cells and their localization in GCs. (A) Flow analyses of CD57 and CXCR5 expression on tonsil CD4 T cells. (B) Specific localization of CD57 + CD4 T cells in GCs. (C) Localization of CD57 + CXCR5 + CD4 T cells in GCs. Antibodies to CD57 (green), CD4 (blue), and IgD (red; B) or CXCR5 (red; C) were used for in situ immunohistochemistry.

Article Snippet: In brief, acetone-fixed sections were stained with the primary monoclonal antibody to CXCR5 (R&D Systems), followed by incubation in biotinylated goat anti–mouse IgG (Vector Laboratories).

Techniques: Expressing, In Situ, Immunohistochemistry

Journal: The Journal of Experimental Medicine

Article Title: Subspecialization of Cxcr5 + T Cells

doi:

Figure Lengend Snippet: Chemotactic responses (A) and chemokine receptor expression (B) by CD57 +/2 CXCR5 +/ − CD4 T cells. Optimal concentrations of 5 μg/ml BLC, 1 μg/ml ELC, and 100 ng/ml SDF-1 were used for chemotaxis experiments. Freshly isolated tonsil cells were used for chemotaxis and flow analyses of tonsil CD4 T cell subsets. Representatives of three independent experiments are shown.

Article Snippet: In brief, acetone-fixed sections were stained with the primary monoclonal antibody to CXCR5 (R&D Systems), followed by incubation in biotinylated goat anti–mouse IgG (Vector Laboratories).

Techniques: Expressing, Chemotaxis Assay, Isolation

Journal: The Journal of Experimental Medicine

Article Title: Subspecialization of Cxcr5 + T Cells

doi:

Figure Lengend Snippet: Spontaneous B cell helper activity of CD57 +/ − CXCR5 +/ − CD4 T cells. Sorted CD57 + CXCR5 + , CD57 − CXCR5 + , and CXCR5 − CD4 T cells were cocultured with B cells from the same tonsil for 11–13 d in the absence of any stimulatory agents followed by analyses of secreted IgG, IgA, and IgM. Representatives of at least three independent experiments are shown.

Article Snippet: In brief, acetone-fixed sections were stained with the primary monoclonal antibody to CXCR5 (R&D Systems), followed by incubation in biotinylated goat anti–mouse IgG (Vector Laboratories).

Techniques: Activity Assay

Journal: The Journal of Experimental Medicine

Article Title: Subspecialization of Cxcr5 + T Cells

doi:

Figure Lengend Snippet: Phenotype and effector function of circulating CXCR5 + T cells. (A) Surface phenotype of circulating naive or CXCR5 +/ − memory T cells. **Significant differences between CXCR5 + and CXCR5 − memory cells. (B) IL-4/IFN-γ production capabilities of CXCR5 +/ − T cells during repeated T cell receptor activation (each cycle is composed of 4-d activation with anti-CD3 and anti-CD28 followed by 3-d resting in the presence of IL-2). (C) B cell help activity of circulating CXCR5 + T cells after T cell receptor activation. Naive (IgD + ) or memory (IgD − ) B cells (2 × 10 4 ) from peripheral blood were cultured for 14 d in the presence or absence of various numbers (10 3 , 5 × 10 3 , 10 4 , 2.5 × 10 4 , 5 × 10 4 , and 10 5 ) of autologous T cells (CXCR5 − CD45RA + , CXCR5 + CD45RA − , or CXCR5 − CD45RA − ). Concentrations of IgG, IgA, and IgM in the culture supernatants were measured by ELISA. T cell numbers required for peak levels of antibody production varied among donors or experiments. One peak value with the best antibody production in each T cell group is shown. (D) Loss of CXCR5 expression during T cell receptor activation with anti-CD3 and anti-CD28. Error bars indicate SD of results from at least five different experiments (A). Representatives of three independent experiments are shown (B and D), and results from seven different donors (C) are shown.

Article Snippet: In brief, acetone-fixed sections were stained with the primary monoclonal antibody to CXCR5 (R&D Systems), followed by incubation in biotinylated goat anti–mouse IgG (Vector Laboratories).

Techniques: Activation Assay, Activity Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing

Journal:

Article Title: The immunosuppressive drug mycophenolate mofetil impairs the adhesion capacity of gastrointestinal tumour cells

doi: 10.1046/j.1365-2249.2003.02290.x

Figure Lengend Snippet: Fluorescence analysis (relative fluorescence units, RFU) of endothelial cell ICAM-1, VCAM-1, E-selectin and P-selectin surface expression. For maximal stimulation, HUVEC were incubated with IL-1 (100 U/ml) for 12 h to up-regulate ICAM-1, and to induce VCAM-1 and E-selectin. HUVEC were incubated with PGE2 (10−6m) for 10 min to induce P-selectin expression. FL-1H (log) channel histogram analysis; 1 × 104 cells/scan. Control values were set at 100%. Mean ± s.d. of three experiments.

Article Snippet: After blocking, they were incubated overnight with sheep-antihuman P-selectin-antibodies (sheep IgG antibodies; 1 : 500 dilution; R&D Systems).

Techniques: Fluorescence, Expressing, Incubation, Control

Journal:

Article Title: The immunosuppressive drug mycophenolate mofetil impairs the adhesion capacity of gastrointestinal tumour cells

doi: 10.1046/j.1365-2249.2003.02290.x

Figure Lengend Snippet: MMF suppresses IL-1 induced E-selectin mRNA expression. Total RNA was isolated and E-selectin mRNA levels were determined by Northern blot analysis. GAPDH was used to assess equivalent RNA loading C−= unstimulated control cells, C+= IL-1 stimulated control cells. Results of densitometry are shown in the graph below and are expressed as the ratio of E-selectin : GAPDH mRNA, relative to the control (IL-1 activation without MMF), which was assigned a value of 100%. The figure shows one representative experiment from three.

Article Snippet: After blocking, they were incubated overnight with sheep-antihuman P-selectin-antibodies (sheep IgG antibodies; 1 : 500 dilution; R&D Systems).

Techniques: Expressing, Isolation, Northern Blot, Control, Activation Assay

Journal:

Article Title: The immunosuppressive drug mycophenolate mofetil impairs the adhesion capacity of gastrointestinal tumour cells

doi: 10.1046/j.1365-2249.2003.02290.x

Figure Lengend Snippet: Western blot analysis of P-selectin in endothelial cells. HUVEC were stimulated with 10−6m PGE2 and incubated additionally with different concentrations of MMF. Throm = control analysis of platelet P-selectin protein, C-= unstimulated HUVEC without MMF, C+ = PGE2-stimulated HUVEC without MMF. The figure shows one of four separate experiments.

Article Snippet: After blocking, they were incubated overnight with sheep-antihuman P-selectin-antibodies (sheep IgG antibodies; 1 : 500 dilution; R&D Systems).

Techniques: Western Blot, Incubation, Control

Journal:

Article Title: The immunosuppressive drug mycophenolate mofetil impairs the adhesion capacity of gastrointestinal tumour cells

doi: 10.1046/j.1365-2249.2003.02290.x

Figure Lengend Snippet: Adhesion of CD4+ T (a), CD8+ T (b) or WiDr (c) cells to immobilized adhesion receptor globulin chimeras. Purified T cells or WiDr cells were added to culture dishes coated with the extracellular domain of ICAM-1, VCAM-1, E-selectin or P-selectin. The immobilized adhesion proteins were coupled to goat-antihuman IgG. Cells were pretreated with different concentrations of MMF. Untreated cells served as controls (100% value). Adhesion was measured after 30 min incubation and after removal of non-adherent cells, according to the protocol given in Materials and methods. Each point represents the mean ± s.d. of four experiments; s.d. was usually about 20%.

Article Snippet: After blocking, they were incubated overnight with sheep-antihuman P-selectin-antibodies (sheep IgG antibodies; 1 : 500 dilution; R&D Systems).

Techniques: Purification, Incubation